ADVANCES IN FOOD RESEARCH VOLUME 30
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ADVANCES IN FOOD RESEARCH VOLUME 30
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ADVANCES IN FOOD RESEARCH VOLUME 30
Edited by
C. 0. CHICHESTER Univerdy of Rhode Island Kingston, Rhode island
B. S. SCHWEIGERT Universiv of California Davis, California
E. M. MRAK Universiv of California Davis, California Editorial Board
H. MITSUDA D. REYMOND E. SELTZER V. G. SGARBIERI W. M. URBAIN
F. CLYDESDALE E. M. FOSTER S. GOLDBLITH J. HAWTHORNE J. F. KEFFORD S. LEPKOVSKY
1986
ACADEMIC PRESS, INC. Harcourt Brace Jovanovich, Publishers
Orlando San Diego New York Austin London Montreal Sydney Tokyo Toronto
COPYRIGHT 0 1986
BY ACADEMIC PRESS.INC ALL RIGHTS RESERVED NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS. ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY. RECORDING. OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM. WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.
ACADEMIC PRESS, INC Orlando. Florida 32887
United Kingdom Edition published by
ACADEMIC PRESS INC.
(LONDON)
LTD.
24-28 Oval Road. London NWI 7DX
LIBRARY OF C o N c R t s s CATALOG ISBN 0-12-016430-2 PRlNTCD I N 111F LINITtO S l A l k b 0 1 AMFRICA
86 87 88 89
Y X 7 6 5 4 1 ? I
CARD
NUMBER 48-7808
CONTENTS
WILLIAM
VERE CRUESS
vii
Sulfites In Foods: Uses, Analytical Methods, Residues, Fate, Exposure Assessment, Metabolism, Toxicity, and Hypersensitivity Steve L. Taylor, Nancy A. Higley, and Robert K. Bush I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2
11. Uses of and Exposure to Sulfites in Foods . . . . . . . ...................... 111. Safety of Sulfites in Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . , .
4
IV. Possible Substitutes and Their Limitations . . . . . V. Future Research Needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
32 61 63 64
Maillard Reactions: Nonenzymatic Browning in Food Systems with Special Reference to the Development of Flavor James P. Danehy I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11. Chemistry of Browning in Model Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Role of Browning in Specific Food Systems . . . . . . . . . . . . . . . . .
IV. Browning, Nutrition, and Health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .............. V. Trends in Continuing Research References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
71
84 91 120 123
124
Postharvest Changes in Fruit Cell Wall Melford A. John and Prakash M. Dey I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Components of Primary Cell Wall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Structure of Primary Cell Wall
IV. Fruit Development . . . . . . . . . . V. Concluding Remarks References . . . . . . . .
139
140 149
168
.............
178 180
V
vi
C 0N TEN TS
Soy Sauce Biochemistry Tarnotsu Yokotsuka I. Introduction 11. Manufacture
111. IV. V. VI. V11. VIII.
.............................. .............................. logical Advances in Shoyu Manufacture . . . . . . . . . . .
Recent Resear Color of Shoyu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Flavor Evaluation of Koikuchi Shoyu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Volatile Flavor Ingredients of Koikuchi Shoyu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Safety Problem of Shoyu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Research Needs . . . . . . . . . . ........................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
I96 204 209 24 I 257 261 287 30 I 313
New Protein Foods: A Study of a Treatise Harold L. Wilcke, C. E. Bodwell, Daniel T. Hopkins, and Aaron M . Altschul I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. The Energy-Protein Interaction . . . . . . . . . . . . ....................... 111. IV. V. VI. VII.
INDEX
................................................
332 332 334 335 352 354 360 378 38 I
....................................................................
387
Food Supply . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conventional Sources of Protein Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reflections on Foods from Animal S New Protein Foods Based on Plant Sources ................................ Properties of Plant Protein Products .......................
WILLIAM VERE CRUESS 1886-1 968
INTRODUCTION The field of Food Science and Technology is a relatively new one and it is well that its few pioneers not be forgotten. To this end, Dr. Sam Goldblith described in Volume 27 of Advances in Food Research the life and accomplishments of Dr. Samuel C. Prescott, one of the fathers of modem food science and technology, whose life spanned an era from the first use of the term “microbe” to beyond the discovery of DNA in 1953. The life span of another great pioneer in the field, Dr. William V. Cruess, covered the same period, from 1886 to 1968. While Prescott was working in the East, at the Massachusetts Institute of Technology, on problems related to sanitation and food preservation, Cruess’ entire career was spent on the West Coast, most of it at the University of California. Early in his career as a chemist, Cruess worked primarily on improving Cal-‘ vii
...
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WILLIAM VERE CRUESS
ifornia wines. During the time that prohibition made teaching and research in winemaking illegal, he turned his talents to another area, embarking on a program of intensive research and development in the field of food preservation that yielded revolutionary theoretical and practical results. Thanks to Cruess and his co-workers, the sun drying of fruits, for example, was replaced in California by mechanical dehydration, providing better product of uniform quality. He was also a pioneer in developing new products such as canned fruit cocktail and nectars from surplus fruits. Then, too, he is remembered as one of the early great teachers of food science and technology.
EARLY LIFE Cruess was born on August 9, 1886, in a farming area called Indian Valley, near the town of San Miguel in the Central Coastal area of California. The soil yielded reluctantly and it was not easy to make a living in Indian Valley. In his memoirs Cruess mentions that the family subsisted mostly on red beans, salt pork, homemade bread, and once in a great while a little quail or dove. Fruit and vegetables were scarce. Very dry years on the farm were common. In 1888 the rainfall for the crop year was only 2 inches. Cattle died, wells went dry, and the principal food that year was boiled whole wheat brought in from elsewhere. Cruess attended a one-room grammar school with 20 students, about three miles from his home. One teacher taught all classes from kindergarten through the eighth grade. Normally, Cruess walked to school and back, but on rainy days he was allowed to ride horseback or use the family buggy. Although Cruess was eager to join his five fellow grammar school graduates in the Paso Robles High School, he remained out of school for 15 months, working as a farmhand and cutting firewood to earn enough money to pay for his room, board, books, and clothing. Boys in Indian Valley put in long days in those times, often sleeping in the haystacks so they could get to work early the next day. There was no danger of rain because it was dry country, and they were so tired after haying, harvesting, and hauling sacks of wheat or barley to town they had no trouble getting to sleep. They were up at 6 A . M . and worked until about five in the afternoon. During the harvesting season, they often started on the combine harvester about 5 A.M.
Cruess entered Paso Robles High School in the fall of 1902. During the first year he lived with a family in town, working part-time for the landlord to help pay for part of his $15-a-month bill for room and board. In high school he took courses in algebra, geometry, Latin, Spanish, history, chemistry, physics, and English.
WILLIAM VERE CRUESS
ix
In his memoirs Cruess makes some interesting comments about automobiles in those days. A brand new phenomenon, automobile operation was forbidden in his area during daylight because they frightened horses drawing wagons or buggies into running away. Horses had the right-of-way.
TO THE UNIVERSITY All during high school Cruess dreamed of going on to the University of California. A serious difficulty, however, was that he had no money. His father offered to sell one of their best horses and borrow necessary additional funds, but it seemed more sensible to Cruess to keep the horse and work on the farm rather then go into debt for his college expenses. He delayed going to college for a year in order to earn enough money to pursue a higher education. During this period, he worked as a harvest hand in summer, and in the fall he moved to the city of Oakland, where he worked in a car barn. He earned enough in 15 months to cover school expenses and room and board for the first year in college. Late in the summer of 1907, he went to the University of California at Berkeley and enrolled in chemistry. He had intended to enroll in mining engineering, but the dean of the College of Chemistry, Professor Edmund O’Neill, who had been a classmate and close friend of his father in grammar school, persuaded him to major in chemistry because of the demand for chemists. Cruess followed O’Neill’s advice and never regretted it. At the beginning of the second semester Dean O’Neill offered Cruess a parttime job in the chemistry department as assistant to a lecturer. Cruess accepted with pleasure, for the offer included rent-free use of two rooms on the top floor of the chemistry building. As Cruess put it, he was well fixed for living quarters. At night the campus watchman often dropped in to chat with him and other students andeven to play a game of cards with them. The watchman was a Civil War veteran and Cruess learned a great deal from him about that war. As Cruess put it, “This was an interesting association in the Chemistry Department.” Thanks to his job at the University during the school term and to field work in the summers, Cruess was able to finance his education and even to graduate with a few dollars in his pocket. While in college he joined the La Junta Club, a social house club that later became a chapter of the national Sigma Phi fraternity. A fellow member was Earl Warren, who became Governor of California and subsequently Chief Justice of the U.S. Supreme Court. During his senior year, 1910- 1911, Cruess held a part-time job with Professor M. E. Jaffa, who gave several courses in nutrition and was head of the Food and Drug Laboratory of the State Board of Health, then located on the Berkeley
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WILLIAM VERE CRUESS
campus of the University of California. Assisting Jaffa in analyzing feed stuffs such as alfalfa hay and cottonseed meal taught the young chemist a great deal about proteins, fat, sugar, crude fiber, and so on in animal feeds. Twenty years later in 1931, while working full time for the University at Berkeley, he earned his Ph.D. in biochemistry from Stanford University. His thesis was concerned with the chemistry of the bitter principle in olives.
UNIVERSITY APPOINTMENTS Cruess’ first appointment after graduation was in the Division of Viticulture and Enology in the College of Agriculture at Berkeley. He had taken courses under Professors Frederick Bioletti and Hans Holm in zymology, or winemaking. The supposedly temporary appointment as a substitute for Professor Holm who was on a 1-year leave of absence became permanent when Holm resigned before the end of the year to take a position with a university in New England. Cruess’ job included two lectures and two laboratory periods a week in zymology. Instruction covered making culture media, sterilizing Petri dishes, isolating pure cultures of yeast and wine bacteria, yeast spore formation, the fermentation of grape must for wine, and acetic acid fermentation. Cruess wrote that with Professor Bioletti’s advice and assistance he managed “to get by.” Zymology was a long way from chemistry, although his knowledge of chemistry was extremely valuable. Cruess was Assistant in Zymology from 1911 to 1914, Assistant Professor from 1914 to 1918, Associate Professor from 1918 to 1929, and Professor of Food Technology from 1934 to 1955, when he assumed the Emeritus title. Shortly after his appointment in 1911 Cruess was asked by Professor Bioletti to do some work on controlling fermentation in a small California winery about 30 miles from Berkeley. Spending several days a week that year at the winery, he learned the rudiments and operations in commercial wine making, and especially, the use of pure yeast and control of fermentation with SO,. It was an excellent experience for him. The next year he conducted further research at the Swett winery near Martinez, California. The owner was a son of John Swett, founder of California’s school systemand a friend of John Muir, the great naturalist. This afforded Cruess an opportunity to meet Muir, who-told him of the marvels of the high Sierra mountains of California. Muir’s descriptions of them made such an impression on Cruess that in later years he spent a great deal of time walking the trails, fishing the streams and lakes, skiing on the slopes, and climbing the peaks of the Sierras. He loved to “rough it” in the mountains and his lovely wife was always with him; but although she loved the beauty of the Sierras, roughing it in the wilderness did at times reach the limit of endurance.
WILLIAM VERE CRUESS
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The experiments at the Swett winery were concerned with the clarification of fresh grape juice, the use of pure yeast cultures in winemaking, and the recovery of residual wine from pressed red grape pomace by use of a diffusion battery system. Cruess’ experience with wine also included studies made in the laboratory at Berkeley with various yeast strains, SO,, clarification, and other winemaking problems. He made a collection of yeasts and some of them survived in laboratory cultures all through the years of prohibition. It was during his trips to the wineries in the Napa Valley area that Cruess met a lovely young school teacher, Marie Gleason, whom he married in 1917. Marie Cruess became a wonderful partner for the professor. She took a great interest in his students and they deeply appreciated it. She was an accomplished artist and her work was often exhibited. One of her paintings of Cruess now hangs in the lobby of Cruess Hall on the Davis Campus of the University of California.
FOOD TECHNOLOGY The 19I8 Constitutional amendment prohibiting alcoholic beverages put an end to winemaking until its repeal early in Franklin Roosevelt’s administration. Meanwhile, there was a need for instruction and research on the preparation and preservation of unfermented fruit products. Consequently, several years before actual repeal of the 18th Amendment, Cruess initiated a lecture course entitled “Zymology 116,” which was concerned with the canning, sun drying, dehydration, and production of juices and other unfermented products from fruit; and the class was well attended. Research in dehydration resulted in the forced-draft, counter-current-tunnel dehydrator which Cruess collaborated on with A. W. Christie, P. F. Nichols, and E. M. Mrak. Cruess’ early emphasis in departmental research, therefore, was in the preservation and utilization area. California’s agriculture at that time was to a considerable extent tree-fruits oriented, principally toward fresh markets. Only perfect fruits qualified for this type of marketing, leaving behind large quantities of socalled “culls.” Converting those to acceptable consumer products by known processing techniques or by devising new methods was a personal challenge he accepted and pursued throughout his career. Problems encountered in this area could only be solved by the acquisition of new knowledge, so new staff members were appointed to provide the appropriate research specialty. His early leadership in faculty growth prevailed throughout the entire history of the department. This early work was the beginning of food technology in California, though the subject was called “fruit products” in those days and some members of the
xii
WILLIAM VERE CRUESS
faculty of the College of Agriculture felt that it was not a respectable area worthy of teaching and research. At the time, it took a good deal of courage on the part of Cruess to continue in the new field that is known today as “food science and technology.” In those days, it was alright to teach and conduct research on fertilizers and even manure, but not on the food we eat. How times and attitudes have changed, for food science is indeed a respectable field today! It was the courage and perseverance of Cruess and a few others that helped to bring about this change.
DEPARTMENT OF FOOD TECHNOLOGY Shortly after the repeal of prohibition, the Department of Fruit Products was established in Berkeley. The name of the department was soon changed to Food Technology. Cruess was chosen to head and build a good department and this he did. When the Food Products Department was first established, the curriculum was essentially practical, lacking both breadth and a firm theoretical foundation. In strengthening the department, Professor Cruess built a staff of young men welltrained in the basic sciences, some of whom also had the practical outlook of the food plant operator. Under his guidance, the curriculum gradually acquired greater breadth and depth; with greater emphasis being placed on the basic disciplines-chemistry, biochemistry, mathematics, and engineering-the groundwork was laid for what I term today the full-spectrum program in food science and technology. In brief, Professor Cruess shared with Dean Prescott of MIT the honor of having placed the entire field of food technology, as we know it today, on a firm basis-truly, a great achievement. Aside from his notable professional accomplishments, Professor Cruess was distinguished by a great dedication to his students. He was a demanding teacher who expected much of his students; but in turn he gave generously to them of his time and interest. He guided them, both professionally and personally, and in some cases even provided financial assistance to enable them to complete their studies. He and his charming wife often entertained his students in their home, inviting as many as 50 or 100 for barbecues, even dances. A few of his students were even married in his home. The sincere mutual respect and affection between Professor Cruess and his students offers, perhaps, both a lesson and hope to those who are now concerned about faculty-student relations in the highspeed, impersonal environment of the “multiversity. ” After 15 years of prohibition, there were few experienced wine makers in this country but there were plenty of home wine makers, “bathtub gin artists,” so to speak, and people who thought they knew how to make wine commercially but
WILLIAM VERE CRUESS
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did not. They made more vinegar and other undrinkable liquids than potable wine. As a result, Cruess and members of his department, especially Maynard Joslyn and George Marsh, spent much of their time during the first few years after repeal, instructing California wine makers in the basic principles and practices of making sound wines. With Joslyn and Marsh, Cruess also did a great deal of work on the freezing of California fruits and vegetables. They collaborated in early investigations on the preservation of perishable fruits and vegetables by freezing both for home and commercial use. Among his other distinctions, Cruess was the first person in the United States to work on problems relating to the processing of olives. He spent a great deal of time on the bacteriological and chemical aspects of olive processing. Later, Dr. Reese Vaughn joined him in conducting research on olive processing. To learn more about the wine, olive, and food industries in Europe, Cruess took several sabbatical leaves to make observations in Spain, France, Italy, Denmark, Sweden, Norway, England, Ireland, Canada, and even Egypt. His Egyptian visit included two months of lectures. Later he made trips to Hawaii to obtain a firsthand view of problems relating to the preservation of fruits and nuts in that area. These included, in particular, the production and treatment of macadamia nuts.
PUBLICATIONS Through the years Cruess published a great deal, including more than 600 scientific and applied papers and books. His most important book, published in 1923, was Commercial Fruit and Vegetable Products. It was a first and a monument in the area of commercial food practices and was translated into several other languages. Other books were Principles of Wine Making, Methods of Wine Analyses, Laboratory Manual for Fruit and Vegetable Products, Home and Farm Food Preservation, and Technology of Wine Making. The last revision of Commercial Fruit and Vegetable Products was published in 1958 and a new book, Technology of Wine Making, was published in 1960. He also did much to help establish the Fruit Products Journal. He published much in this journal, thus initiating the new area for publication of research in food science. In fact, he was considered the guardian of the journal and the field it covered. Cruess was very active in the development of the Institute of Food Technologists. The first organizational meetings were held at MIT under the guidance of Dean Prescott. When it was decided to make a national organization, Cruess was heavily involved. He eventually became the national president and had
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much to do with the organization of the northern and southern California sections of the Institute.
HONORS Over the years Cruess received many honors. These included the Nicholas Appert and Babcock Awards from the Institute of Food Technologists; Chevalier et officer du Merite Agricole of France; election to the American Academy of Microbiology, New York Academy of Science; and Academia Italian della Vita e del Vino of Italy. He was awarded the LL.D degree by the University of California, Davis, in 1960. The citation read: “Alumnus of the University in the Class of 1911. A member of her faculty for more than forty years, a biochemist in the Experiment Station, for some years department chairman, and now Professor Emeritus, of Food Science and Technology. Holder of the Babcock-Hart Award, of the first Nicholas Appert Medal, an Award given for your outstanding contributions to food technology, and of citations from several branches of the armed services for your work during World War 11. A highly productive research scientist, you have admirably combined the advancement of science with service to California agriculture. In 1952 the new Food Technology building at Davis was named Cruess Hall. He also received the Service Award of the 49’ers of the Canning Industry, and a recognition from the California Farm Bureau Federation, the Food and Container Institute of the U.S. Armed Forces, the Raisin Industry, the Dried Fruit Association of California, and the Fig Institute. In spite of these many honors, Cruess was a modest person with great humility, as his acceptance address of the Appert Award makes clear: ”
“The speaker feels honored far beyond his just due in having been selected to receive the Nicholas Appert Award of the Institute of Food Technologists for 1942; the first of the series of yearly awards established by the Chicago Section of I.F.T. There are many in our organization who are much more worthy of this recognition. Also, it should be stated that an investigator’s reputation often depends not so much on his own accomplishment as on those of his immediate associates. He may, as the titular head of a laboratory, symbolize its achievements and receive the honors that should be shared with his co-workers. The present case is no exception. It is difficult to express adequately in a few words the extent of Professor Cruess’ service to the University of California, to the food industry, to those who
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had the privilege to know him personally, particularly students and fellow workers, and to the larger community of mankind. His career was marked by distinction in all phases; and his modesty, selflessness, and dedication, as well as his professional accomplishments and talents, won him the highest regard of his students, colleagues, and friends. He was also a leader in developing interest and leaders in the field of food technology of promising young scientists, who have done much to advance the field. Some of these are: C. 0. Chichester, A. W. Christi, M. A. Joslyn, G. L. Marsh, E. M. Mrak, P. F. Nichols, H. J. Phaff, J. Irish, andmany, manyothers. He was indeed a great man.
CRUESS, WILLIAM VERE 1886-1968 B.S.-University of California, 1911 Ph.D.-Stanford University, 1931 Assistant in Zymology, 1911-1912 Assistant Professor of Zymology, 1913- 1920 Assistant Professor of Fruit Products, 1920- 1921 Associate Professor of Fruit Products, 1921- 1934 Chemist in the Experiment Station, 1925-1945 Biochemist in the Experiment Station, 1945-1954 Professor of Fruit Technology, 1934- 1945 Professor of Food Technology, 1945- 1954 Emeritus, 1954
EMILM. MRAK
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SULFITES IN FOODS: USES, ANALYTICAL METHODS, RESIDUES, FATE, EXPOSURE ASSESSMENT, METABOLISM, TOXICITY, AND HYPERSENSITIVITY STEVE L. TAYLOR,* NANCY A. HIGLEY,*t AND ROBERT K. BUSH$ *Food Research Institute, University of Wisconsin, Madison, Wisconsin 53706 $William S. Middleton Memorial VeteransHospital, Madison, Wisconsin 53705 #Department of Medicine, University of Wisconsin, Madison, Wisconsin 53706
1. 11.
111.
IV.
V.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Uses of and Exposure to Sulfites in Foods A. Description . . ..... B. Natural Occurrence of Sulfites in Foods . . . . . . . . . . . . . . . . . . . . . . . . C. History of Use of Sulfiting Agents as Food Ingredients D. Current Food Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Methods for Measurement of Sulfite Residue Levels . . . . . . . . . . . . . . F. Chemistry of Sulfites and Fate in Foods . . . G. Treatment Levels versus Residual Levels . . . . . . . . . . . . . . . . . . . . . . . H. Exposure Assessments Safety of Sulfites in Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Metabolism of Sulfites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Human Challenge Trials C. Animal and Cellular Toxi D. Hypersensitivity to Ingested Sulfites . . . . . . . . . . . . . . . . . . . . . . . . . . . Possible Substitutes and Their Limitations A. Control of Enzymatic Browning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Control of Nonenzymatic Browning . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Use as Antioxidants or Reducing Agents D. Use as an Antimicrobial Agent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Use as a Bleaching Agent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future Research Needs . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 4 4 6 7 8 17 21 30 30 32 32 38 39 47 61 61 62 62 62 63 63 64
?Resent address: International Flavors & Fragrances, Research & Development, Union Beach, New Jersey 07735. 1 Copyright 6 1986 by Academic Press, Inc. All rights of reproduction in any form rewrved.
2
STEVE L. TAYLOR ET AL.
I. INTRODUCTION Sulfiting agents have a long history of use as food ingredients. The term sulfiting agents refers to sulfur dioxide (SO,) and several forms of inorganic sulfite that liberate SO, under the conditions of use. In addition, naturally occurring sulfites are present in many foods. The yeast cultures used in the fermentation of wines and beers naturally produce a portion of the sulfites found in these products. Sulfiting agents are added to foods for many important technical purposes, including the control of enzymatic and nonenzymatic browning, antimicrobial action, antioxidant and reducing agent uses, bleaching agent uses, and a variety of processing aid uses. In many products, the sulfites serve more than one purpose. Alternatives to the sulfiting agents are not easy to identify. Possible alternatives usually provide a narrower range of benefits, are often less effective, and are almost always more expensive. Sulfiting agents are currently used in a wide variety of food products. Data on the treatment levels for the sulfites and residual sulfites are not available for some food products, and wide variations in treatment modes and levels for particular products are known to occur in the industry. The analysis of sulfite residues in foods is confused by the rapid reaction between sulfiting agents and various food components. Sulfites react readily with reducing sugars, carbonyls, and proteins to yield a variety of organic combined sulfites. Analytical procedures are available for “free” SO, (SO, and the various inorganic sulfite salts) and “total” SO, (free SO, plus some of the combined forms of sulfite). Processing, storage, and preparation act to lower the available residual levels of sulfites in foods. The actual levels of free and total SO, in a particular food product are dictated by the extent of absorption of the sulfites during treatment, the nature of the processing treatment following sulfite addition, and the conditions of storage. The actual levels of free and total SO, remaining in foods at the point of consumption have received less attention. The fate of sulfites added to specific foods is a largely unexplored area of study. The rapid reaction of sulfites with food components would be expected to leave little free SO, in the product at the point of consumption (Green, 1976; Joslyn and Braverman, 1954; Schroeter, 1966). Recently, the safety of the continued use of sulfites in foods has been questioned on the basis of their alleged role in the initiation of asthmatic reactions in certain sensitive individuals. Numerous cases of sulfite-induced asthma have been reported in the medical literature since 1977 (Baker etal., 1981; Buckley et al., 1985; Bush et al., 1986; Stevenson and Simon, 1981b; Towns and Mellis, 1984; Twarog and Leung, 1982) and additional anecdotal reports have been made to the Food and Drug Administration. These cases of sulfite-induced
SULFITES IN FOODS
3
asthma were confirmed by positive challenges with capsules or solutions containing inorganic sulfites. Only a small subgroup of the asthmatic population has sensitivity to sulfites in capsules. Important questions remain regarding the possibility that sulfited foods might initiate asthmatic reactions in these sensitive individuals. Although some of the described patients report asthmatic reactions to sulfited foods and many of the anecdotal cases involve suspicions of reactions to sulfited foods, only a few controlled challenges with sulfited foods have been performed with sensitive asthmatics (Howland and Simon, 1985; Seyal et af., 1984). We speculate that the degree of hazard posed to sulfite-sensitive asthmatics by sulfited foods may be considerably diminished by the reactions of the sulfites with food components. The majority of the challenges described in the medical literature thus far have involved inorganic sulfites in capsules or in acidic solutions. There can be little doubt that some asthmatics are sensitive to free inorganic sulfites, although this may be related to the conversion of inorganic sulfite salts to SO, at acidic pHs. However, as mentioned, most sulfited foods contain little free inorganic sulfite; sulfited lettuce is an exception (Taylor er af., 1985). These free inorganic sulfite residues would probably induce reactions in a manner similar to sulfites in capsules. The combination of sulfites with food components would drastically lower the free SO, content of most foods, thereby limiting exposure to these free forms of sulfites. Further research is needed to determine the effects of ingestion of combined forms of sulfites on the sulfitesensitive asthmatics. Concerns have also arisen in recent years regarding the possibility that many consumers may be exceeding the Acceptable Daily Intake (ADI) for sulfites, although the recent report of the Ad Hoc Review Group on the Reexamination of the GRAS (Generally Recognized as Safe) Status of Sulfiting Agents indicates that these concerns are probably unwarranted (Life Sciences Research Office, 1985). They estimate that total intake of sulfites as SO, is about 10 mg/capita/day, which is well below the AD1 of 42 mg for a 60-kg person (Life Sciences Research Office, 1985). Again, the same questions arise about the relative contributions of free and combined sulfites to the total sulfite intake. The intake of combined sulfite likely exceeds the intake of free sulfite by many fold. Because of their stabilities, the combined forms of sulfites would likely pose a lower hazard to consumers than free sulfites. More research will be required to firmly establish the relative toxicity of the free and combined sulfites. In this article, the current uses of sulfites in foods will be examined. The critical issue of exposure assessment will be explored in a review of the fate of sulfites in foods, residual levels, and analytical methodology. The questions about the safety of the use of sulfites in foods will be tackled by reviewing the available information on the metabolism of free and combined sulfites, the
4
STEVE L. TAYLOR ET AL.
toxicity of free and combined sulfites, and the hypersensitivity reactions among certain asthmatics. In the final section of this review, the remaining unresolved issues will be highlighted with a discussion of future research needs. In our opinion, further research is necessary before decisions can be made on the future regulatory status of sulfites, although tremendous pressure is being exerted on the Food and Drug Administration to make a decision on the status of sulfites. More information is needed on the responses of sulfite-sensitive asthmatics to sulfited foods, the comparative reactivities of asthmatics to free and combined sulfites, the comparative toxicities of free and combined sulfites, the fate of sulfites in a variety of foods, and the extent of consumer exposure to free and combined sulfites.
II. USES OF AND EXPOSURE TO SULFITES IN FOODS A. DESCRIPTION Sulfur dioxide and several forms of inorganic sulfites that liberate sulfur dioxide under the conditions of use are food additives known collectively as sulfiting agents. Sulfur dioxide (SO,), potassium bisulfite (KHSO,), potassium metabisulfite (K,S,O,), sodium bisulfite (NaHSO,), sodium metabisulfite (Na,S,O,), and sodium sulfite (Na,SO,) are listed in the Code of Federal Regulations (CFR) as GRAS provided that they are not used in meats or other foods recognized as a source of thiamine. However, the GRAS status of these sulfiting agents is currently being reexamined, and changes may be made (Life Sciences Research Office, 1985). In addition, other sections of the CFR specifically allow the use of sulfiting agents in a variety of food-related processes. A list of the CFR sections and the processes covered by each section is provided in Table I. Note that all of the GRAS sulfiting agents are presently allowed for use for certain of these purposes. Potassium sulfite (K,SO,) and sulfurous acid (H,SO,), which are not GRAS substances, are specifically allowed for use only in the processing of caramel. Sulfiting agents are also permitted for use in wine and beer, although the Bureau of Alcohol, Tobacco, and Firearms (BATF) has proposed that the use of sulfites in wine and beer be curtailed to some extent (Anonymous, 1984). Presently, the levels of use of the sulfiting agents in most foods are not strictly limited by regulation. Exceptions are glucose syrup, dextrose monohydrate, and wine where the maximum allowable residual levels of SO, are specified, and food starch bleaching where the treatment level of SO, is controlled to a maximum of 0.05%. It should be noted that the levels of sulfites used in some products such as wines are self-limiting because of organoleptic considerations. The theoretical yield of sulfur dioxide varies for the different forms of the
5
SULFITES IN FOODS
TABLE I CODE O F FEDERAL REGULATIONS SECTIONS PERTAINING TO THE USE OF SULFlTlNG AGENTS IN FOODS AND/OR FOOD INGREDIENTS
CFR section 2lCFR 2lCFR 2lCFR 2lCFR 2lCFR 2lCFR 2lCFR 2lCFR 2lCFR 2lCFR 2 ICFR 2lCFR 2lCFR
182.3616 182.3657 182.3739 182.3766 182.3798 182.3862 73.85(2) 168.111 168.120 172.892 173.31O(c) 177.1200(c) 177.1400
Subject
Sulfiting agents allowed
GRAS status GRAS status GRAS status GRAS status GRAS status GRAS status Caramel Dextrose monohydrate Glucose syrup Food starch bleaching agents Boiler water additives Cellophane Water-soluble, hydroxyethyl cellulose film
KHSO3 K2S205 NaHS03 Na2S205 Na2S03
so2 H2SO3, Na2SO3. K2SO3 SO2 (20 ppm maximum residual)
SO2 (40 ppm maximum residual) SO2 (0.05% maximum)
Na2S205,Na2S03 NaHS03, Na2S03 All GRAS sulfiting agents
sulfiting agents, as outlined in Table 11. Consequently, different treatment levels are required with the various sulfiting agents to yield equivalent doses of the active agent, SO,. For comparative purposes, it is helpful to calculate treatment levels on the basis of percentage of theoretical yield of SO,. However, it must be realized that these theoretical yields would almost never be achieved in food applications because the sulfiting agents react rapidly with food components, can be volatilized into the atmosphere, or can oxidize to sulfate. As will be emphasized later, these reactions are dependent on a number of variables, including pH, temperature, and storage time. TABLE I1 THEORETICAL YIELD AND SOLUBILITY O F GRAS SULFITING AGENTS" ~~~~~~
~
Chemical
Formula
Sulfur dioxide Sodium sulfite, anhydrous Sodium sulfite, heptahydrate Sodium bisulfite Sodium metabisulfite Potassium metabisulfite Potassium bisulfite
Na2S03 Na2S03 . 7H20 NaHS03 Na2S205 K2S205 KHSO3
Z,
From Green (1976).
so2
Theoretical yield of so2 (%)
Approximate solubility @/lo0 ml H20)
100.00 50.82
11 at 20°C 28 at 40°C 24 at 25°C 300 at 20°C 54 at 20°C 25 at 0°C -
25.41 61.56
67.39 57.60 53.32
6
STEVE L. TAYLOR ET AL.
The Food Chemicals Codex supplies specifications for the food grades of four
of the sulfiting agents. In general, food grade sulfiting agents must be at least 90% pure to meet these standards. Some problems arise in the definition of sodium bisulfite, since there is some doubt about the existence of sodium bisulfite in the solid state. It may exist entirely as sodium metabisulfite or as a mixture of bisulfite and metabisulfite (Green, 1976). For that reason, the Food Chemicals Codex defines the purity of sodium bisulfite on the basis of SO, equivalents. B. NATURAL OCCURRENCE OF SULFITES IN FOODS In addition to their use as food additives, it must be remembered that the sulfites can also occur naturally in foods. Foods contain a variety of sulfurcontaining compounds, including the sulfur amino acids, sulfates, sulfites, and sulfides. These sulfur-containing compounds are interconvertible in some food systems that possess the appropriate enzymes. The natural occurrence of sulfites in foods has been most thoroughly studied in alcoholic beverages such as wine and beer (Eschenbruch, 1974). The ability of yeasts to produce sulfite has been known since the end of the last century. Sulfite arises from sulfate via a multienzyme pathway. The sulfite can be converted into methionine and cysteine, but sulfite always exists in the fermentation medium. Sulfite can also be converted into H,S and other sulfides, which are organoleptically undesirable in wines and beers. Most strains of Saccharomyces cerevisiae generate between 10 and 30 ppm SO,, although some strains producing in excess of 100 ppm SO, have been identified (Eschenbruch, 1974). Sulfite serves several functions in wine, including antimicrobial functions, prevention of browning, and binding of acetaldehyde (Eschenbruch, 1974). However, sulfite can be detected organoleptically if the concentration becomes too high; the threshold is thought to be about 50 ppm as free sulfite. Because of its adverse effects on the organoleptic quality of the wine and the potential for abuse of sulfites in making wine from inferior grapes, many countries have imposed strict limitations on the amount of residual SO, allowed in wine. In the Federal Republic of Germany, for example, the limits are 50 ppm of free sulfite and 300 ppm for total sulfite in wines of the Qualitatswein class. In the United States, the upper limit is 350 ppm as total residual SO,, although BATF is proposing an upper limit of from 125 to 175 pprn (Anonymous, 1984). Consequently, the formation of sulfite by yeasts must be critically controlled. The choice of yeast strains is important since they can vary by an order of magnitude in their capacities for sulfite formation (Dott et al., 1976; Eschenbruch, 1974; Rankine and Pocock, 1969). The high sulfite formation by certain yeast strains can be attributed to several metabolic differences related to sulfite
SULFITES IN FOODS
7
metabolism (Dott et al., 1977; Eschenbruch and Bonish, 1976; Heinzel and Truper, 1976). However, both low- and high-sulfite-forming strains are equivalent in their abilities to reduce sulfite to the obnoxious sulfides (Dott and Truper, 1976). The extent of sulfite formation is also related to other factors, including the amount of sulfite-bindingcompounds produced in the fermentation (Rankine and Pocock, 1969; Weeks, 1969). Acetaldehyde, pyruvic acid, and aketoglutaric acid bind SO, and serve to control the quantities of free SO, in the fermentation medium (Burroughs and Sparks, 1973a-c; Rankine and Pocock, 1969; Weeks, 1969). Only the free SO, has antimicrobial properties. The situation is much the same in beer except that lower levels of SO, are produced during beer fermentation (Hysert and Morrison, 1976). The SO, is derived mainly from sulfate and also serves as a precursor for sulfides. Wine and beer cannot be made without formation of sulfites. In beer, residual total SO, levels ranged from 0.2 to 11 ppm in the absence of added SO, in one study (Hysert and Morrison, 1976), although higher natural levels of formation might be expected to occur. Much of the residual sulfite in beer is in the combined state (Chapon et al., 1982). In wine, natural SO, formation can account for 15-125 ppm of residual SO, in the finished product. In other food products fermented by yeasts, we would expect that SO, formation from sulfate would occur naturally, although we are not aware of studies confirming this suspicion. C.
HISTORY OF USE OF SULFITING AGENTS AS FOOD INGREDIENTS
The sulfiting agents have enjoyed a long history of effective use as food and drug ingredients. Ancient Greeks wrote about the use of SO, for the fumigation of houses. The Romans and Egyptians are supposed to have used SO, for the sanitation of wine vessels (Roberts and McWeeny, 1972). Its use as a food preservative dates to at least 1664 when cider was added to flasks while they still contained SO, (Evelyn, 1664). The inorganic sulfites appeared as food additives at a much later date. The first years of use of the various inorganic sulfiting agents in the United States are as follows: sodium bisulfite, 1921; sodium sulfite, 1930; potassium and sodium metabisulfite, 1939 (Subcommittee on Review of the GRAS List, 1972). The sulfiting agents were first used in wine and beer. Among nonalcoholic products, the sulfiting agents were first used on dried fruits and vegetables in all likelihood. However, their use in foods spread rapidly as a consequence of the absence of toxic hazards and their widespread functional effectiveness. In the decade between 1960 and 1970,.a 30-70% increase in the amounts of several sulfiting agents used annually in the United States was observed (Subcommittee on Review of the GRAS List, 1972), a testament to the
8
STEVE L. TAYLOR ET AL.
TABLE I11 FOOD USE OF SULFlTING AGENTS (UNITED STATES, 1976)
Sulfiting agent
Amount produced (Ib)
Sodium bisulfite Sodium metabisulfite Potassium metabisulfite Sulfur dioxide Sodium sulfite
4,900,000 92,000 220,000 2,200,000 15,000
growing utilization of these additives. Table 111 contains data on the production of sulfur dioxide and the inorganic sulfiting agents for use in foods. Foods represent one of the larger uses for these chemicals. SO, and sodium bisulfite are produced and used in far larger quantities than most of the other sulfiting agents.
D. CURRENT FOOD APPLICATIONS 1.
Overview of Current Applications
Sulfiting agents are used in foods for many important purposes: the inhibition of nonenzymatic browning, the inhibition of various enzymatic reactions including enzymatic browning, inhibition and control of microorganisms, an antioxidant and reducing agent including dough conditioning, a bleaching agent, a processing aid, and several secondary uses including pH control agent and stabilizing agent. Each of the general categories will be discussed with a brief explanation of the mechanism of action of the sulfiting agents in effecting these changes. As might be expected for any group of substances that possesses so many useful properties, an enormous number of specific applications have been found for sulfiting agents in foods. Later in this section, we will make an attempt to identify these applications and the treatment levels associated with each application. Other uses for the sulfiting agents have been devised, and these uses will be described, although we are not certain that they are being used in the food industry. Finally, the sulfiting agents provide other benefits in foods beyond those which are readily identified with the sulfiting agents. These benefits will be identified and discussed. Several previous reviews on the applications for sulfiting agents in foods have appeared (Green, 1976; Joslyn and Braverman, 1954; Roberts and McWeeny, 1972; Schroeter, 1966). Based on our current knowledge, these reviews are inadequate and should be considered to be out of date (Joslyn and Braverman,
SULFITES IN FOODS
9
1954; Schroeter, 1966), oriented toward the British food industry (Green, 1976; Roberts and McWeeny, 1972), or incomplete. Many additional applications of the sulfiting agents have now been identified, although the previous reviews do provide much valuable information on some of the major uses of the sulfiting agents in foods. 2 . Inhibition of Nonenzymatic Browning
Nonenzymatic browning is a term used to describe a family of diverse reactions that commonly involve the formation of carbonyl intermediates and brown, polymeric pigments. Examples include the reactions between amino acids and reducing sugars and carmelization of sugars. The chemistry of the reactions involved is complex and not completely understood. An excellent review of the chemistry of nonenzymatic browning and the effects of the sulfites on these reactions was prepared by McWeeny et al. (1974). The sulfites can be used to control nonenzymatic browning because of their ability to react with the carbonyl intermediates. A variety of carbonyl intermediates can be formed during the nonenzymatic browning process, including reducing sugars, simple carbonyls, dicarbonyls, and a,P-unsaturated carbonyls. The sulfites can react with all of these intermediates and thus block formation of the brown pigments. Wedzicha et al. (1984) developed a kinetic model for the inhibition of nonenzymatic browning by sulfites. Reaction of sulfites with the carbonyls generated by nonenzymatic browning accounts for most of the loss of sulfites in dehydrated vegetables (Wedzicha et al., 1984). Some of the sulfite-carbonyl reaction products are more stable than others. The sugar hydroxysulfonates formed between reducing sugars and sulfites are the least stable, although they are quite stable at acid pHs. The sulfonated carbonyls formed on reaction of the sulfites with the a,@-unsaturatedcarbonyls are extremely stable and the reaction is generally considered to be irreversible. The differences in stability of the sulfite addition products can influence the effectiveness of the sulfites in certain food applications. For example, sulfite can almost totally inhibit nonenzymatic browning of glucose-glycine solutions because of the irreversible reaction of the sulfites with the a$-unsaturated carbonyl intermediates of this reaction (Wedzicha and McWeeny, 1974a). The glucose-glycine system typifies the situation that occurs in dehydrated potatoes. A kinetic model for the glucose-glycine reaction and its inhibition by sulfites has been developed by Wedzicha (1984). On the other hand, sulfites can only retard the formation of browning pigments in the ascorbic acid-glycine reaction because the principal intermediates of this reaction are dicarbonyl compounds which react reversibly with the sulfites (Wedzicha and McWeeny, 1974a). The ascorbic acid-glycine system is typical of the situation existing in fruit juices and drinks. Sulfites find wide use as inhibitors of nonenzymatic browning. They have
10
STEVE L. TAYLOR ET AL.
been used for this purpose to control discoloration of wines, dried fruits, dehydrated vegetables, dehydrated potatoes, coconut, pectin, some varieties of vinegar, and white grape juice. Sulfites are also used to control the commercial carmelization process. In warmer climates, sulfites can be used to control nonenzymatic browning in fruit juices and drinks (Joslyn and Braverman, 1954). Sulfites are also used to control juice color formation in the production of beet sugar (McGinnis, 1982).
3. Inhibition of Various Enzymatic Reactions
SO, and sulfites can act as inhibitors of numerous enzymatic reactions, including polyphenoloxidase, ascorbate oxidase, lipoxygenase, peroxidase, and thiamine- dependent enzymes. The actions of the sulfiting agents on oxidizing enzyme systems have been reviewed by Haisman ( 1974). Inhibition of polyphenoloxidase is useful in the control of enzymatic browning. Polyphenoloxidase catalyzes the oxidation of mono- and ortho-diphenols to quinones. The quinones can cyclize, undergo further oxidation, and condense to form brown pigments. The mechanism of action of the sulfites in preventing enzymatic browning is not known, but very likely involves several different types of actions. Sulfites may directly inhibit the enzyme; potassium metabisulfite has recently been shown to inhibit strawberry polyphenoloxidase at 10 mM concentrations (Wesche-Ebeling and Montgomery, 1983). Sulfites may also interact with the intermediates in the enzymatic browning reaction and prevent their participation in the reactions leading to formation of the brown pigments. For example, sulfites may combine with the quinones and prevent their participation in the further oxidation, cyclization, and condensation reactions. Evidence for the formation of quinone-sulfite complexes has been reviewed (Haisman, 1974). Alternatively, the sulfites may simply act as reducing agents promoting the reaction of the quinones back to the original phenols. The level of sulfites necessary to prevent enzymatic browning depends on the nature of the available substrate. When only monophenols such as tyrosine are present, fairly low levels of sulfite are effective. Potatoes are an example of this situation. When diphenols are present, much higher concentrations of sulfites are necessary. An example of this situation would be guacamole. The sulfites do not irreversibly inhibit the enzymatic browning reaction so the required concentrations are also dependent on the length of time that the reaction must be inhibited. Inhibition of enzymatic browning is the primary reason for using sulfites in salad bar items, including cut fruits, lettuce, and guacamole. Sulfites have also been used to prevent enzymatic browning in prepeeled potatoes, sliced potatoes, cut apples and other fruits supplied to the baking industry (Ponting et al., 1971), fresh mushrooms (Komanowsky et al., 1970), and table grapes (Nelson, 1983).
SULFITES IN FOODS
11
A similar reaction occurs in shrimp where enzymatic tyrosine oxidation leads to black spot formation. The reaction is catalyzed by tyrosinase, a type of polyphenoloxidase. Black spot formation in shrimp can be controlled by the addition of sulfites (Fieger, 1951). Sulfites can also prevent the oxidation of ascorbate by ascorbate oxidase and other enzymes. Ascorbate levels decrease very quickly following the maceration of plant tissues due to the action of ascorbate oxidase. Sulfite addition preserves ascorbate and can be used in potato, pumpkin, cauliflower, tomato, and green and red pepper products (Haisman, 1974). Sulfites can also inhibit lipoxygenase, an enzyme known to cause formation of off-flavors during postharvest storage of vegetables such as peas (Haisman, 1974). Treatment with sulfites will prevent formation of these off-flavors, an added benefit to their use in dehydrated peas and other vegetables. Anaerobic bacterial fermentation can be inhibited in grape juice by sulfites. This inhibition is essential to the production of wines. The mechanism of this inhibition is not entirely understood, but it is partially due to the destruction of thiamine, which serves as an essential cofactor for several of the fermentative enzymes (Haisman, 1974). These enzymes are thus inhibited. 4.
Inhibition and Control of Microorganisms
The sulfites play crucial roles in the inhibition of bacteria in several food processes. In winemaking, the sulfites are employed to prevent undesirable bacterial fermentation of the grape or fruit juice. Sulfites are also essential in the corn steeping process used to facilitate removal of the corn starch; the sulfites prevent bacterial growth in the steep liquor (Schroeter, 1966). The application of sulfites to table grapes is critical to prevent bacterial and mold growth (Nelson, 1983; Nelson and Ahmedullah, 1973, 1976). Although not a common practice in the United States, sulfites have been widely used to prevent mold damage in fruits prior to jam production (Roberts and McWeeny, 1972). Sulfites have also found use in the prevention of postharvest deterioration of fruits used for the production of juices (Moms et a f . , 1979). The use of sulfites as antimicrobial agents has been reviewed by Roberts and McWeeny (1972), Joslyn and Braverman (1954), and Ingram (1959). The sulfites are selective antimicrobial agents with more inhibitory effect on acetic acid bacteria, lactic acid bacteria, and various molds than on yeasts (Joslyn and Braverman, 1954). This selectivity enhances their value in the control of undesirable fermentation in winemaking. The mechanism of the antimicrobial action of the sulfites is not well understood. However, several factors are known to control the antimicrobial efficacy of the sulfites. One of the more important factors is pH which controls the form of sulfite present in the food. Apparently, H,SO, is the
12
STEVE L. TAYLOR ET AL.
active form of the sulfites in terms of their antimicrobial actions (Carr et al., 1976; Ingram, 1959), so lower pHs enhance the antimicrobial effect. The combination of sulfites with food components also affects their antimicrobial activity (Ingram, 1959; Joslyn and Braverman, 1954). The sulfite adducts have no antimicrobial activity. Consequently, more sulfite is required to preserve a glucose syrup than a sucrose syrup, since sulfites will combine with glucose but not sucrose (Ingram, 1959). Considerable sulfite must be added to wine because of the binding of the sulfites to fermentation products such as acetaldehyde. The volatilization of SO, from acidic products also affects the level retained for antimicrobial action. Sulfites can have some detrimental effects as a result of their antimicrobial actions. In red wines, high levels of SO, inhibit the desirable malolactic fermentation, which serves to reduce the acidity of wines produced in cool regions (Liu and Gallander, 1983). Although we know of no practical use of this antimicrobial activity, sulfites also inactivate certain types of enteroviruses including poliovirus type I, coxsackievirus type A9, and echovirus type 7 (Salo and Cliver, 1978). 5. Antioxidant and Reducing Agent Uses
The antioxidative effects of the sulfites are partially responsible for their preserving effect on ascorbate and their inhibition of nonenzymatic and enzymatic browning. The ability of the sulfiting agents to promote the reduction of the oxidized quinones to reduced phenols is one of the mechanisms available for the inhibition of these processes by the sulfites. Sulfites also prevent the oxidation of essential oils and carotenoids, which would generate off-flavors (Baloch et al., 1977; Roberts and McWeeny, 1972). A major function of SO, in beer is the inhibition of oxidative changes that are considered undesirable to flavor development (Roberts and McWeeny, 1972; Schroeter, 1966). Sulfites are widely-used as dough conditioners in the baking industry for biscuits, crackers, cookies, and frozen pizza doughs and pie crusts. In these products, sulfites act by breaking the disulfide bonds in the gluten fraction of the dough (Wade, 1972). The sulfites also promote disintegration of the protein matrix during the corn steeping process, which facilitates rapid hydration, softening of the kernel, and extraction of the starch (Schroeter, 1966). The sulfites may exert this action via their ability to reduce disulfide bonds, although we know of no direct proof for this possibility. SO, has also been used to improve the extraction of pectins from various sources through its ability to depolymerize the pectins (Roberts and McWeeny, 1972).
SULFITES IN FOODS
13
6. Bleaching Agent Uses The major application of the bleaching properties of the sulfites is the bleaching of cherries for the production of maraschino cherries and g l a d fruit products (Josyln and Braverman, 1954; Weigand, 1946). The sulfiting agents are also reported to bleach pectins (Roberts and McWeeny, 1972). The uniformity and translucency of color of orange, lemon, grapefruit, and citron peel are improved by storage in a sulfite brine (Cruess and Glickson, 1932). Sulfur dioxide can also be used as a bleaching agent for food starches (Table I). The bleaching of table grapes during sulfite fumigation is considered detrimental to quality (Nelson, 1983).
7.
Use as a Processing Aid
Many of the applications of sulfites fall into the category of processing aids. This particular category of use for sulfiting agents is difficult to define and variations probably exist in its definition within the food industry. Obviously, sulfite residues can originate from the use of sulfited products in the formulation of the end product. Examples would include the use of beet sugar or corn syrup in a variety of products and the use of maraschino cherries in fruit cocktail. Typically, SO, residue levels from such uses would be rather low. Further investigation of the use of sulfiting agents as processing aids will be necessary to obtain a better picture of the extent of such uses and their contribution to consumer exposure.
8. Secondary Uses This category of uses of the sulfiting agents is diverse because these additives have many desirable secondary benefits beyond the primary reasons for their use. Examples would include their facilitation of corn starch extraction (Schroeter, 1966), a secondary benefit to the primary purpose of preventing microbial growth in the corn steep liquor. Another example would be the control of excess alkalinity and the improvement in boiling properties of beet sugar juice, a secondary benefit to the primary purpose of control of color formation in the juice (McGinnis, 1982). Many additional examples could be selected.
9. Specific Applications and Treatment Levels The above discussion clearly shows that sulfiting agents are used in the food industry for a variety of products and for many different reasons. The Federation
14
STEVE L. TAYLOR ET AL.
of American Societies for Experimental Biology (FASEB) panel attempted to identify these applications and the residue levels resulting from each use (Life Sciences Research Office, 1985). This information may not be representative of the entire food industry, since variations exist in the use of sulfiting agents, the type of sulfiting agents employed, the treatment levels, and the means of applying the sulfiting agents to the foods, all of which would affect residual levels. Sulfite uses have been identified in baked goods and baking mixes, alcoholic and nonalcoholic beverages, coffee and tea, condiments and relishes, dairy product analogs, prepared fish and shellfish products, fresh fish and shellfish, fresh fruits and fruit juices, fresh vegetables, gelatins, grain products, gravies and sauces, jams and jellies, nuts and nut products, processed fruits and fruit juices, processed vegetables and vegetable juices, snack foods, soups and soup mixes, sugar, and sweet sauces, toppings, and syrups. These categories correspond to those listed in the CFR (21CFR 170.3). In Table IV, specific uses of the sulfiting agents within each of these categories are identified, and reported residual levels and exposure estimates are given (Life Sciences Research Office, 1985); most of the information in Table IV was obtained from the FASEB compilations (Life Sciences Research Office, 1985). Some of the information in Table IV needs to be verified for accuracy, but it is probably the best and most complete survey of sulfite use ever conducted. Also, the uses and residual levels may not be representative of the entire industry. A major deficiency has been the lack of analyses of sulfited foods at the point of consumption. Storage, processing, and preparation can affect residual sulfite levels in the product prepared for consumption. Further research will be needed to determine, with greater accuracy, actual consumer exposure to sulfites. 10. Other Uses for Sulfiting Agents in Foods
Certain other uses have been developed for the sulfiting agents in foods. We are not certain that these processes are actually being used in the food industry, but they are feasible. Two examples will be cited, although many more appear in the literature. A procedure for improved color retention in canned garbanzo beans that involves a presoak in NaHSO, has been developed (Daoud et al., 1977; Luh et al., 1978). The pink discoloration noted with certain varieties of canned pears can be prevented by use of SO, (Chandler and Clegg, 1970). 11. Additional Benefits of Sulfiting Agents in Foods
Sulfites provide additional benefits in foods beyond those already discussed. To our knowledge, they are not used in foods for these purposes, so these benefits might best be classified as fortuitous or potential uses. The carcinogenic
15
SULFITES IN FOODS
TABLE IV ESTIMATED INTAKE OF SULFITES FROM VARIOUS FOODS"
Category
Subcategory
Baked goods and baking mixes
Cookies Cake with dried carrots Crackers Sheeting doughs Pie dough Pizza ciust Pie crust Tortilla shells Total Cola and pepper Lemon-lime Orange Root beer Ginger ale Grape Juice-containing carbonated beverage Beer Wine Instant tea Liquid concentrated tea Tea leaves Olives Pickles/relishes Salad dressing mix (dry) Vinegar Malt vinegar Wine vinegar Filled milk Dried cod Shrimp Fruit salad Grapes Total Apple concentrate, imported Cheny-beny Grape, red or purple Grape, white, white sparkling, pink sparkling, or red sparkling
Beverages, nonalcoholic
Beverages, alcoholic Coffee and tea
Condiments
Dairy analogs Fish and shellfish Fresh fruit Dried fruitd Fruit juices
Estimated level in product as consumedb (ppm SO2) 5 10 5 intraperitoneal > per 0s. By sonie routes of administration in some species, a dose killing 50% of the animals was not achieved; these doses are reported in Table VI as LD,,,. The LD,, values do not always agree when independent studies are compared such as the LD,,s for intravenous administration of Na,SO, to mice (Table VI). Many factors could explain the discrepancies. In particular, it must be remembered that sulfites are unstable in aqueous solutions, so any storage of the solution before dosing would result in a loss of sulfite and an apparent decrease in toxicity. Cohen et al. (1973) determined that the intraperitoneal LD,, of NaHSO, was 181 mg/kg (1 11 mg SO,/kg) in sulfite oxidase-deficient rats as compared to 473 mg/kg (291 mg SO,/ kg) in normal rats. The acute toxicities of the combined forms of sulfite have received little study. Lewis and Tatken (1979) list an LD,,, of 1220 mg/kg as SO, for oral administration of acetaldehyde hydroxysulfonate in the rabbit. Walker et al. (1983b) could not determine an oral LD,, for DSH in rats or mice and conclude that the oral LD,, for DSH exceeds 5 g/kg. These scattered results would tend to indicate that some of the combined sulfites are less toxic than the inorganic sulfites, but further studies are needed on additional compounds and on other routes of
40
STEVE L. TAYLOR ET AL.
TABLE VI LD,, AND/OR LD,,, OF SULFITING AGENTS
Species
Mouse
Rat
Rabbit
Route"
Chemical
iP iv iv iv iv iP PO Po iP iP iv iv Po Parenteral iP iv
NaHS03 NaHS03 Na2S0 Na2S03 Na2S03 Na2S03 . 7 H 2 0
Po
Hamster Guinea pig Cat Dog Human
sc iv iv iv iv iv sc iv sc iP sc iv
SO26
s02c NaHS03 NaHS03 NaHS03 Na2S03 K2S205
Na2S205 NaHS03 NaHS03 Na2S03 Na2S03 Na2S03 Na2 s 2 O5
NaHS03 Na2S03 Na2S03 Na2S03 Na2S03 Na2S03 NaHS03 Na2S03 Na2S03 . 7 H 2 0
LD5,, (mg/kg)
LD,,, (mg/kg)
675 130 130 155
175 277 1040 2000
650 473 115
I15 1800 500 300 65 65 95 95 -
-
244 -
SO2 equiv. (mglkg) 416 80 66 79 89 70 1040
2000 400 29 1 71 58 1037 337 I85 40 1435 I52 33 129 58 48 102 305 66 1 102 I50 661 189
Reference Wilkins et al. (1968) Hoppe and Goble (1951) Lewis and Tatken (1979) Jaulmes (1970) Hoppe and Goble (195 I ) Nofre er al. (1963) Jaulmes ( 1970) Jaulmes ( 1970) Wilkins et al. (1968) Cohen et al. (1973) Hoppe and Goble (1 95 I ) Lewis and Tatken (1979) Lauteaume er al. ( 1 969) Ezrielev (1968) Wilkins et al. (1968) Hoppe and Goble ( I 95 1) Lewis and Tatken ( 1 979) Lewis and Tatken (1979) Lewis and Tatken (1979) Lewis and Tatken (1979) Hoppe and Goble (1951) Lewis and Tatken (1979) Lewis and Tatken (1979) Lewis and Tatken (1979) Lewis and Tatken (1979) Lewis and Tatken (1979) Wilkins et a/. (1968) Lewis and Tatken (1979) Lewis and Tatken (1979)
ip, Intraperitoneal; iv, intravenous; PO, per 0s; sc, subcutaneous. As a 6.5% aqueous solution. As a 3.5% aqueous solution.
administration before firm conclusions can be drawn. Walker (1984) notes that much less information is available on the toxicity of the combined sulfites than is known about the reactions leading to their formation. 2 . Subchronic and Chronic Toxicity Numerous subchronic and chronic toxicity studies have been conducted on the free inorganic sulfites. For the purposes of this review, the early studies will be
SULFITES IN FOODS
41
ignored because of the distinct possibility that many of the toxic manifestations were the result of thiamine deficiency, since the impact of sulfite on thiamine was not recognized at that time. Some of these studies have been reviewed elsewhere (Cluzan et al., 1965; Ti1 et al., 1972a). The more recent studies of subchronic and chronic toxicity of sulfites generally fall into two categories: those in which the sulfite was administered with the drinking water and those in which the sulfite was administered with the diet. Both of these approaches have disadvantages. Sulfites are unstable in drinking water; Lockett and Natoff (1960) observed a 20% decline in sulfite levels within 48 hr. Some investigators have ignored the stability problems, making their studies difficult to interpret. The drinking water approach has been favored by some investigators because it avoids the problem of thiamine destruction that is inherent with the incorporation of sulfites into the diet. Gunnison et al. 11981a) showed that sulfites do not destroy thiamine systemically, although Gunnison (1981) notes that sulfite ingested with drinking water might destroy some thiamine in the stomach. The incorporation of sulfites into the diet is also fraught with difficulties, since the sulfites are extremely reactive with other dietary components. These reactions can substantially decrease the free sulfite content of the diet and makes interpretation of the results difficult. Many of the recent studies have focused on attempts to confirm the finding reported by Fitzhugh et al. (1946), who administered NaHSO, to rats in their diets for up to 1 year. The diet was often left in the feeder cups unchanged for up to 1 week, which resulted in losses through reaction of up to 75% of the sulfite (Gunnison, 1981). The diets contained 0.05-2.0% NaHSO, (0.08-13 mmol/kg/day) originally. Fitzhugh et al. (1946) noted toxic manifestations at bisulfite levels above 0.1 % that included growth retardation, clinical polyneuritis, “spectacle” eyes, bleached incisor teeth, brown uteri, atrophy of various viscera, calcified renal tubular casts, atrophy of bone marrow and bone, myocardial necrosis and fibrosis, and gastric squamous epithelial hyperplasia. These results have been questioned because of the diminishing levels of sulfite in the diets and the probable destruction of thiamine in the diet. Fitzhugh et al. (1946) attempted to correct the thiamine deficiency through supplementation. Polyneuritis was not observed in the supplemented animals, but the other toxic manifestations persisted. Gunnison (198 1) has questioned whether the thiamine supplementation was sufficient to entirely correct the deficiency. Based on the severity of the manifestations observed in this experiment by comparison to others (see later), we would echo these sentiments and further note that other dietary factors might have been affected by the storage of diet in the feeder cups for prolonged periods which could lead to other deficiencies. Bhagat and Lockett (1964) noted that diet prepared with metabisulfite and stored at room temperature would quickly become deficient in thiamine. On prolonged storage of 3-4 months at room temperature, the diets would cause problems, such as chronic diarrhea, that could not be
42
STEVE L. TAYLOR ET AL.
reversed by thiamine supplementation (Bhagat and Lockett, 1964). This is an indication that other factors in the diet may also be destroyed by sulfite addition and contribute to the toxicological evaluations if diets are not properly prepared and stored. The results of Fitzhugh et al. (1946) have not been corroborated in other chronic toxicity studies. Three of these studies have involved the incorporation of sulfites into the drinking water (Cluzan et al., 1965; Lauteaume ef al., 1965; Lockett and Natoff, 1960). Lockett and Natoff (1960) administered 0, 375, and 750 ppm of SO, as Na,S,O, in the drinking water of rats in a 3-year multigeneration study. They observed no effects of sulfite on growth, food intake, fecal output, fertility, weight of the newborn, growth during lactation, or any of the pathological signs noted earlier by Fitzhugh et al. (1946). The study of Lockett and Natoff (1960) was compromised by the losses of SO, in the drinking water (10% in 24 hr) and the fact that many of their animals developed respiratory ailments during the course of the experiment. Cluzan et al. (1965) conducted a multigeneration study in rats over a 20-month period, administering 700 ppm of SO, as K,S,O,. They found no evidence of toxicity as mortality, growth rate, feed and water consumption, organ weights, hematological values, clinical symptoms, and reproductive capacity were equivalent to controls. Cluzan et al. (1965) did not provide any evidence for the stability of sulfites in their experiment. Lauteaume et al. (1965) administered sulfites by gastric intubation to rats over a 2-year period at a rate of 3 m1/100 g body weight/day. The rats were divided into three groups that received (1) water and 450 ppm SO,, (2) red wine with 110 pprn SO,, or (3) red wine with 450 ppm SO,. The sulfites did not affect growth rates, reproduction, or the development of macroscopic or microscopic lesions. The most thorough evaluations of the chronic toxicity of sulfites were performed by Ti1 et al. (1972a,b) using incorporation of Na,S,O, into the diets of rats and pigs. Losses of sulfite through reactions with other dietary components were minimized by frequent diet preparation and frozen storage. The amount of sulfite loss was measured and the data were reported using the corrected values. Thiamine was added to the diets 'in sufficient quantities to overcome any thiamine destruction by sulfite. On a percentage basis, sulfite losses were greatest at low sulfite concentrations, while thiamine losses were highest at high sulfite concentrations. Sulfite losses ranged from 4.5 to 22%, while thiamine losses ranged from 1.7 to 15.4%. In the rat study (Ti1 ef al., 1972a), the added levels of Na,S,O, were 0.125, 0.25,0.50, 1.O, and 2.0%. The study was conducted over a period of 2 years and involved three generations. The 2.0% Na,S,O, diet caused slight growth retardation in the F, and F, generations, but had no effect on the F, generation. Part of this effect is explained on the lower birth weights in the F, and F, generation,
SULFITES IN FOODS
43
although other reproductive effects were absent. Occult blood was observed in the feces of rats receiving the 1.0% and 2.0% Na,S,O, diets. Kidney weights were slightly increased with the 2% diet in the F, females only, and this change was not accompanied by any functional or histopathological changes in the kidneys. Histopathological observations were largely normal except for the existence of hyperplasia in the fore and glandular stomachs of the rats receiving 1% and 2% Na,S,O, diets. This hyperplasia was noted in all three generations and was observed to a lesser extent in the forestomachs only of some rats on 0.5% Na2S,0, diets. Beems et al. (1983) have further examined this hyperplastic response and concluded that it involves chief cells, but the mechanism of the response remains unknown. The no-effect level from the rat study was 0.25% Na,S,O, in the diet, which is equivalent to 72 mg SO,/kg/day after conversion and correction for sulfite losses. The Joint FAO/WHO Expert Committee on Food Additives (1974) used the results of this experiment to establish the AD1 of 0.7 mg SO,/kg by simply applying a 100-fold safety factor to the no-effect level obtained by Ti1 er al. (1972a). Although this experiment is the most carefully controlled study of the chronic toxicity of sulfites in existence, it has been criticized. Hickey er al. (1976) point out that the levels of sulfite oxidase in humans are much lower than the levels in normal rats, so a study of sulfite toxicity using ,normal rats is not justified. Subchronic toxicity studies with sulfite oxidase-deficient rats clearly demonstrate that such animals are more susceptible to the toxic effects of sulfites (Gunnison er al., 1981b). However, the 100-fold safety factor is intended partly to correct for such differences in detoxification pathways. The study of Ti1 et al. (1972a) should be recognized as a study of the toxicity of total sulfite rather than free sulfite, however. Ti1 er al. (1972a) analyzed for sulfite residues in their diets by the method of Reith and Willems (1968), which detects total sulfite levels. Therefore, some of the Na,S,O, added to the diet may have reacted with dietary components but would be recovered as SO, during the analytical procedure. In all likelihood, Ti1 er al. (1972a) underestimated the degree of free sulfite loss by reaction with dietary components. Ti1 et al. (1972b) also conducted a chronic toxicity study in pigs. The techniques were identical to those used in the rat study (Ti1 et al., 1972a). A 48-week feeding period was employed. The results varied somewhat, however. Some growth retardation was noted in diets having 0.83 and 1.72% residual sulfite, although this was due to diminished food intake, as a later paired feeding trial did not demonstrate any differences in growth rates or food conversion. Organ to body weight ratios were increased at the 0.83% and 1.72% levels for liver, kidney, heart, and spleen, although this is ascribed to the lower body weights. In contrast to the rat study, no occult blood was observed in the feces. Histopathological examinations were normal except for mild inflammation and hyper-
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STEVE L. TAYLOR ET AL.
plasia in the stomach at the 0.83% and 1.72% levels at both the 15-week and 48week observation periods. Subchronic toxicity studies were also conducted by Ti1 et al. (1972a,b) on both rats and pigs. In rats (Ti1 et al., 1972a), high sulfite levels (0-8%) were fed in the diet for 10-56 days. Diets containing 6% sulfite caused marked growth depression, reduced food intake, and lowered food conversion efficiency. Severe anemia, increased spleen weights, and slightly elevated leukocyte counts were also observed. The hyperplasia of the forestomach was found with 1% sulfite or more, while glandular stomach hyperplasia, hemorrhagic erosions, necrosis, and inflammation were found with 4% sulfite or more. Forestomach ulcers and papillomatous elevations occurred at 6 and 8% sulfite. All of these effects were reversible. In pigs (Ti1 e f al., 1972b), the changes observed after 15 weeks of feeding were similar to those encountered after 48 weeks of feeding. Bhagat and Lockett (1964) observed diminished growth rates in rats fed 0.6% Na,S,O, in the diet over a 5- to 7-week period, but this effect could be reversed by supplementation with thiamine. Gunnison et al. (198 la) confirmed the observation of anemia in rats and attributed it to the interaction of sulfite with dietary factors, perhaps vitamin B12. Gunnison et al. (1981a) conducted their experimeots with sulfite oxidase-deficient rats and showed elevated excretion of S-sulfonates can occur after administration of low levels of sulfite (0-3.5 mmol/kg/day). Obviously, sulfite oxidase-deficient rats are more susceptible to the toxic effects of oral sulfite, and Gunnison (1981) has suggested their use in sulfite toxicity studies. Few experiments have been conducted on the chronic and subchronic toxicities of combined forms of sulfite. Dietary studies such as those by Ti1 et al. (1972a,b) are probably tests of the toxicity of some mixture of free and combined sulfites. Gibson and Strong (1973) used glucose hydroxysulfonate in some of their metabolism studies. Glucose hydroxysulfonate is likely to be stable to stomach acid, but likely decomposes to free sulfite in the neutral pH of the small intestine. They found no histological abnormalities in the livers and kidney of rats dosed with glucose hydroxysulfonate for 30 days. Walker et al. (1983b) did not observe any adverse effects after oral administration of DSH to rats for 14 days.
3. Carcinogenicity Tumorigenic effects were not encountered in any of the chronic toxicity tests described above. In addition, Tanaka et al. (1979) failed to find any tumors in a carcinogenicity test of K,S20, in mice; 0, 1, and 2% K,S,OS was administered in the drinking water. Gunnison et al. (1981a) noted a small incidence (4/ 149) of mammary adenocarcinoma in sulfite oxidase-deficient rats as compared to O / 143
SULFITES IN FOODS
45
in controls after 5 months of feeding of tungsten, but the effect was not statistically significant. 4.
Mutagenicity
The mutagenicity of free inorganic sulfites has been extensively studied. The subject has been reviewed in detail elsewhere (Gunnison, 1981; Shapiro, 1977), and no attempt will be made here to provide such detail. The reactions of sulfite with nucleic acids were covered in Section II,F,7. The mutagenicity of the sulfites is thought to originate from the deamination of cytosine to uracil. The involvement of sulfite-induced deamination of 5-methylcytosine to thymine in the mutagenic process has also been considered, but the cytosine-to-uracil conversions are thought to be quantitatively more important (Wang and Ehrlich, 1980; Wang ef al., 1980). Sulfites are capable of inducing mutations in vitro in several mutagenicity test systems, including E. coli, y phage, T4 phage,yeast, and Vicia faba root meristems (Chambers et al., 1973; Dorange and Dupuy, 1972; Hayatsu and Miura, 1970; Mukai et al., 1970; Njagi and Gopalan, 1982; Summers and Drake, 1971). However, these experiments required high concentrations of sulfite and acid pHs in the vicinity of pH 5 . When incubations were performed at neutral pH, no measurable mutagenic response was observed (Mukai et al., 1970). MacRae and Stich (1979) found that sulfite induces dose-related sister chromatid exchange in Chinese hamster ovary cells, but the potency of this induction was relatively weak. Sulfites can also cause chromosome damage when incubated in v i m with oocytes from mice, cows, or sheep (Jagiello et al., 1975). However, they could not induce chromosome aberrations in mouse oocytes cultured in vitro after an intravenous injection of sulfite. Despite the evidence for mutagenicity of sulfite in the systems described above, there is no evidence for sulfite-induced mutagenesis in other systems. The Food and Drug Administration contracted for mutagenicity studies in a variety of systems, and the results of these tests are reported in the 1976 GRAS evaluation document (Life Sciences Research Office, 1976). Sodium bisulfite was not mutagenic in the host-mediated assay in mice, the dominant lethal assay in rats, the in vivo cytogenetic assay in rats, and human tissue culture cells in vitro (Life Sciences Research Office, 1976). Sodium sulfite and potassium metabisulfite were not mutagenic in vitro in the Ames Salrnonellalmammalian microsome test (Life Sciences Research Office, 1976). Sodium metabisulfite did cause mitotic inhibition and damage to anaphase cells when added to human embryonic lung cells in culture (Life Sciences Research Office, 1976). However, sodium metabisulfite was not mutagenic in the host-mediated assay, the dominant lethal assay, or in vivo cytogenetic assays (Life Sciences Research Office, 1976).
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Generoso et al. (1978) showed that sulfite was negative in the dominant lethal assay in mice after intraperitoneal injections. Drosophila ingesting a 0.08 M solution of NaHSO, (5120 ppm SO,) displayed a mutation rate that was not significantly different from controls (Valencia er al., 1972). Renner and Wever (1983) were unable to induce cytogenetic damage as monitored by sister chromatid exchange, chromosome aberration, and the micronucleus test in sulfite oxidase-deficient mice and Chinese hamsters after intragastric administration of one or two doses of Na,S,O, (330 or 660 mg/kg) in aqueous solutions or fruit juice. Bisulfite in aqueous solutions or in fruit or vegetable juices was not mutagenic to Salmonella typhirnuriurn strains TA 1535, TA 1538, TA 100, or TA 98, but an increase in revertants was obtained with strain his-G46 (Munzer, 1980). In this strain, more revertants were obtained with sulfited fruit or vegetable juices than aqueous solutions of sulfite (Munzer, 1980). Some evidence also exists for a comutagenic effect of sulfites (Mallon and Rossman, 1981). Enhanced ultraviolet mutagenicity was observed in Chinese hamster V79 cells if they were exposed to 10 mM sulfite either during or immediately following irradiation. A twofold increase in mutagenicity was observed by comparison to irradiated controls not exposed to sulfite. With E. coli, 100 mM sulfite caused an eightfold increase in mutagenicity. Mallon and Rossman (198 1) obtained evidence implying that sulfite was inhibiting excision repair. Sulfite can also be an antimutagen. Sulfite at 200 ppm is able to inhibit the mutagenic effect of coffee in the Salmonellalmammalian microsome system and the induction of prophage A (Suwa et al., 1982). Sulfite also suppressed the mutagenicities of the 1,2-dicarbonyls, diacetyl and glyoxal (Suwa et al., 1982). Almost no information is available on the mutagenicity of the various combined forms of sulfite. Walker et ul. (1983b) demonstrated that DSH is not mutagenic in the Ames Salmonellulmammalian microsome assay. 5.
Teratogeniciry
Teratogenicity studies on NaHSO,, Na,S,O,, and K,S,O, have been conducted in several species on behalf of the Food and Drug Administration; the results of these evaluations were reviewed in the 1976 GRAS evaluation document (Life Sciences Research Office, 1976). These sulfites were administered orally to rats and mice on a daily basis on day 6 through day 15 of gestation and similarly in hamsters except on day 6 through day 10 of gestation. The doses (in mg/kg) for mice, rats, and hamsters ranged up to 150, 110, and 120, respectively, for NaHSO,; up to 160, 110, and 120, respectively, for Na,S,O,; and up to 125 and 155 in mice and rats, respectively, for K,S,O,. The incidence of teratogenic effects was unchanged from control animals. Maternal and fetal survival were also not affected by these sulfites.
SULFITES IN FOODS
47
Dulak et al. (1984) investigated the reproductive toxicology of sulfite in sulfite oxidase-deficient rats. Exposure to sulfites from 3 weeks before mating until day 20 of gestation revealed no reproductive hazards for sulfite. Mating and pregnancy rates, gestational weight gain, preimplantation loss, resorbed and dead fetuses, litter size, fetal weights, and malformations were unaffected by sulfite treatment.
6. Studies in Cell Cultures Sulfites have a variety of effects on cultured cells. Sulfites are cytotoxic to mouse fibroblasts, mouse liver cells, HeLa cells, Chorella pyrenoidosa cells, and human lymphocytes in culture (Das and Runeckles, 1974; Thompson and Pace, 1962; Timson, 1973). This cytotoxic effect was observed in the 0.1-20 mM range, although the minimum inhibitory concentrations varied among the different cultures. DNA synthesis can be inhibited in chick embryo fibroblasts by 0.1-1 .O mM sulfite (Chin et al., 1977). Sulfite can also prevent the adhesion of Chinese hamster cells to the substratum (Kudo et al., 1980). Kikigawa and Iizuka (1972) showed that 7.5 mM sulfite inhibited the ADP- and collageninduced aggregation of rabbit platelets. D.
HYPERSENSITIVITY TO INGESTED SULFITES
1 . History of Asthma and Other Adverse Reactions to Sulfites
Recently, sulfiting agents have been reported to induce asthma when administered to certain asthmatics (Baker et al., 1981; Freedman, 1977; Kochen, 1976; Stevenson and Simon, 1981b). The first reports, generated by Kochen (1976) and Freedman (1977), did not immediately attract much attention. However, the simultaneous reports by Allen and Collett (198 1) and Stevenson and Simon (1981a) at the American Academy of Allegy meetings, which linked sulfite ingestion in foods and drugs with asthmatic episodes in several patients, sparked considerable interest and additional research. The evidence linking ingestion of sulfiting agents with exacerbation of asthma in a segment of the asthmatic population is now compelling, although the role of sulfited foods in the initiation of these reactions has not been clearly established, as will be indicated later. Additionally, sulfiting agents have been implicated in a few rare instances with other types of hypersensitivity reactions, including anaphylactoid reactions, hypotension, and contact sensitivity (Fisher, 1975; Prenner and Stevens, 1976; Rudzki, 1979; Schwartz, 1983), indicating that asthma is not the only adverse reaction to sulfiting agents. However, asthma is very likely to be the most common adverse reaction to the sulfites. In this section, each of the published studies on adverse reactions to ingestion of sulfiting agents will be reviewed, with particular empha-
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sis on its contribution toward evaluating the degree of hazard posed by the use of sulfiting agents in foods. Studies pertaining directly to respiratory exposure to SO, will not be reviewed in detail because SO, is a well-documented hazard to virtually all asthmatics and others when inhaled (Boushey, 1982;Koenig et al., 1980;Linn et al., 1983;Nadel et al., 1965;Sheppard et al., 1980), and tolerance levels have been established for exposure to SO, in the workplace and the ambient air. However, the inhalation route of exposure may have some relevance to the discussion because such exposures might occur from inhaling the air released during the opening of a bag of dried fruit (Werth, 1982) or during ingestion of an acidic beverage (Delohery et al., 1984a).
2 . The Earliest Reports Kochen (1976)reported the case of a child with mild asthma who experienced acute transient episodes of asthma after the consumption of sulfited foods. Confirmatory sulfite challenges were not conducted. This report was considered to be an isolated, unique, and not fully substantiated case until the later reports began to appear. The pioneering study of the induction of asthma by ingested sulfites was published by Freedman (1977).Freedman interviewed 272 asthmatic patients and queried each of these patients about their asthmatic experiences following ingestion of a particular type of orange drink. This type of orange drink, which contains orange juice, sweetener, tartrazine, sodium benzoate, sulfur dioxide, stabilizers, and artificial flavorings, is not available in the United States. Of the 272 patients, 30 (1 1%) reported experiencing asthma soon after ingestion of such orange drinks. Of these 30 patients, 14 volunteered for oral challenges with sulfur dioxide, sodium benzoate, and tartrazine. The challenges were administered to the patients following an 8-hr period of abstinence from bronchodilators or cromoglycate and a 3-hrperiod of abstinence from food. Sodium metabisulfite was dissolved in a citric acid-water solution so that the challenge dose was 250 ml containing 100 ppm SO,. This would be equivalent to a dose of 25 mg of SO,. With the addition of citric acid, the pH of the solution was acid, and therefore most of the SO, probably existed as HSO, and H,SO,. Of the 14 patients, 8 showed a decrease in lung function as determined by a drop in their forced expiratory volume in 1 sec (FEV,) as measured by spirometry. Any decrease in FEV, exceeding 12% was considered positive. The group included 5 females and 3 males, and 3 of these patients also developed asthma when challenged with sodium benzoate. On challenge with SO,, the maximal drop in FEV, occurred by 1 1 min (a range of 2-25 min) with measurable decreases often occurring within 1-2 min. The maximal depression in FEV, ranged from 12 to 57%, with an average of 31%. Three patients had decreases in FEV, of less than
SULFITES IN FOODS
49
20%. One of these patients, who had a marginal drop in FEV, of 12% on administration of 25 mg of SO,, was challenged with 75 mg of SO, and experienced a decrease in FEV, of 37%. Prior administration of sodium cromoglycate protected 4 of 4 patients from the effects of ingested SO,. Several features of Freedman’s study are subject to criticism and possible misinterpretation. The study is sometimes quoted as being an evaluation of the sensitivity of 272 asthmatics to sulfiting agents. In fact, only 14 patients were actually challenged with sulfites. Freedman used a drop of 12% in FEV, as an indication of a positive response. This is an extremely conservative approach. Most pulmonary specialists would consider a 12% drop as only marginal and would require either a 15 or 20% drop to indicate a positive response. At the 20% level, the number of responders to the 25-mg challenge would drop from 8 to 5. Freedman did not conduct the challenges in either a placebo-controlled or double-blind manner. Placebo control of such challenges is considered to be the minimal safeguard against biased results and double-blind confirmation of my reactions is preferred (Bush er al., 1986). The pH of the challenge solution may have contributed greatly to the acquired results. SO, will be evolved from an aqueous solution only if the pH is below 4.0. Freedman does not state the pH of his challenge solutions. However, he prepared the solution by dissolving 0.75 g sodium metabisulfite and 0.75 mg citric acid in 1 liter of water and then diluting by a factor of 5. In our hands, such a solution has a pH of 2.94. At this acidic pH, most of the free SO, would be in the HSO, form, with about 10% as H,SO, (Green, 1976; Joslyn and Braveman, 1954). About 6% of the added metabisulfite would be evolved as gaseous SO, at this pH. This would be equivalent to 1.5 mg of SO,, a dose sufficient to induce bronchoconstrictionin asthmatics if inhaled. Therefore, Freedman’s study may simply represent another demonstration of the ability of gaseous SO, to induce asthma. Freedman also made some rather intriguing observations which need to be resolved with the subsequent results of Stevenson and Simon (1981b). Freedman observed rather rapid decreases in FEV,, with 6 of the 8 patients reaching maximal loss of lung function within 10 min or less. By contrast, Stevenson and Simon (1981b) measured FEV, at 30-min intervals and observed a slower response of 15-30 min. The difference may be due to the fact that Freedman used a beverage vehicle, while Stevenson er af. used capsules. The beverage vehicle allowed exposure of the sublingual and buccal mucosa in addition to the gastrointestinal tract. The rapidity of the response suggested to Freedman that the route of absorption of the sulfite was by inhalation of SO, vaporizing from the solution or absorption of the sulfite through the sublingual and/or buccal mucosa. Based on the pH of his challenge solutions, the most likely possibility is that SO, was vaporized from these solutions and inhaled by the sensitive patients. Variable inhalation of SO, from acidic solutions has now been demonstrated to be the
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STEVE L. TAYLOR ET AL.
mechanism of reaction to these solutions (Delohery et a l . , 1984a,b). Another intriguing aspect of Freedman’s work was the blockage of the response by prior administration of sodium cromoglycate. Since cromoglycate acts by stabilizing the mast cell membrane, thereby preventing release of histamine and other mediators of the allergic response, the inhibitory action of cromoglycate would possibly suggest that mediator release plays a role in the mechanism of the response to ingested sulfiting agents. However, cromoglycate is known to have other actions, including phosphodlesterase inhibition, reduction of mucosal hyperactivity, and inhibition of neurological reflexes, so blockage of histamine release may not be the only explanation for the actions of cromoglycate.
3. 1981 American Academy of Allergy and Immunology Reports The earliest reports by Allen and Collett (1981) and Stevenson and Simon (1981a) were brief abstracts of presentations made at the 1981 American Academy of Allergy and Immunology meeting. Allen and Collett (1981) reported 2 patients with sensitivity to sodium metabisulfite. One of these patients had had asthmatic reactions elicited by sulfites in foods, while the other patient had experienced asthma following administration of drugs containing sulfites. The sensitivity to sulfites was confirmed by double-blind challenges with capsules containing 500 mg of sodium metabisulfite. The 500-mg challenge dose is rather high by comparison to the amounts used by Freedman (1977), Stevenson and Simon (1981a,b), and the levels presently being used by the Australian group (Baker and Allen, 1982; Delohery et a l . , 1984a,b). The patients described here were also sensitive to tartrazine, aspirin, and sodium benzoate. Stevenson and Simon (1981a) identified 4 asthmatic patients with sulfite sensitivity. They also employed capsule challenges, but used potassium metabisulfite. The threshold doses for decreases in lung function ranged from 10 to 50 mg. These patients were not found to be sensitive to sodium benzoate, aspirin, tartrazine, or monosodium glutamate. 4 . Further Reports from Australia
A later report by Allen’s group describes in detail the cases of 2 sulfitesensitive patients (Baker et a l . , 1981). It is not clear if these patients are identical to the ones described in the earlier abstract. The first case was a 67-year-old female who had experienced asthma after ingesting a crabmeat salad prepared with vinegar dressing. A subsequent challenge of this patient with capsules of sodium metabisulfite confirmed the existence of an asthmatic reaction related to the consumption of sulfites. The second case was a 23-year-old female whose asthmatic symptoms worsened on ingestion of wine. A subsequent challenge
SULFITES IN FOODS
51
with a capsule containing 500 mg of sodium metabisulfite confirmed the existence of the asthmatic reaction to sulfites. The challenges were done double blind, with lactose as the negative control. A third report from the Australian group, also in abstract form, details their experiences with metabisulfite challenges through early 1982 (Baker and Allen, 1982). By this time, they had identified 8 patients with asthmatic sensitivity to oral challenge with metabisulfite (presumably the sodium salt). Of the 8 patients, 3 were also sensitive to aspirin and other food additives, including tartrazine and benzoate. The challenge protocol had been modified to include administration of graded doses starting at 10 mg and progressing through 300 mg, with lung function evaluations at 0.5-hr intervals. Curiously, 4 of the 8 sensitive patients did not react to a 300-mg capsule challenge, but did react to a 25-mg challenge of metabisulfite dissolved in 50 ml of 0.5% citric acid. This mode of administration is quite similar to that of Freedman (1977). The reactions to acidic sulfite solutions occurred within 1-5 min, while positive capsule challenges showed a 20- to 30-min lag period. They conclude that the response to acidic sulfite solutions is due to inhalation of vaporized SO,. More recent reports from the Australian group (Delohery et af., 1984a,b) delve more deeply into the comparative responses to capsule versus beverage challenges. Acidic solutions of metabisulfite were able to provoke asthma in 60% of all asthmatics, a much higher percentage than found with capsule challenges (Delohery et al., 1984a). A comparison of sulfite reactors with asthmatics not reactive to sulfites revealed that both groups were equally sensitive to inhaled SO,, but that the sulfite reactors were the only group responsive to ingestion of sulfited acidic beverages. The sulfite reactors responded to a mouthwash with a sulfite solution, but not to sulfite solution administered directly into the stomach via a nasogastric tube. It must be assumed that this group of reactive asthmatics does not have any capsule reactors because they would be predicted to respond to any direct gastric challenge. Delohery et al. (1984a.b) conclude that the beverage reactors are inhaling SO, as they swallow, while nonreactors can swallow without inhalation. Allen and Delohery (1985) revealed that these asthmatics do not respond to sulfited acidic beverages if they take a deep breath and hold it before using a sulfite mouthwash. The existence of such a high percentage of beverage reactors is somewhat surprising, although all asthmatics respond to inhaled SO,. However, the practical significance of this type of sulfite sensitivity is uncertain. Delohery et af. (1984a,b) used challenges of 50 mg of metabisulfite in a citric acid solution. It is unlikely that asthmatics would routinely encounter such levels of free sulfite in most beverages. Wine might easily contain 50 mg of total sulfite per serving, but the majority of this sulfite would be in the form of combined sulfites. Still, this type of sensitivity may explain the common complaints of asthmatics about adverse reactions to the ingestion of wines.
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Allen and Delohery ( 1985) also investigated the mechanism involved in reactions to sulfite in capsules. After ingestion of 25- to 50-mg capsules of metabisulfite, 4-50 ppm of SO, could be detected in the stomach via a nasogastric tube. They speculate that SO, is evolved from metabisulfite by the action of stomach acid and that the SO, can be inhaled following eructation. Unfortunately, they did not measure SO, concentrations in the nasopharynx after capsule ingestion. 5 . Further Reports from the La Jolla Group
Stevenson and Simon (1981b) also published a more detailed account of their initial findings. Descriptions of 5 sulfite-sensitive patients are provided in this report. Four of the patients were identical to the ones described in their earlier abstract. The challenges were performed with capsules of potassium metabisulfite. Graded doses starting at l mg were employed, with the doses increasing to 5, 10, 25, and 50 mg of K,S,O, until an asthmatic response was noted. The 5 patients had asthmatic reactions beginning at 15-30 min after administration of the threshold dose. The threshold dose was 10 mg for 2 of the patients, 25 mg for another 2 patients, and 50 mg for the fifth patient. Since several doses were administered at 30-min intervals, it is possible, though unlikely, that the patients were reacting to an accumulated dose rather than the last dose administered. Falls in FEV, ranged from 23 to 49%. The challenges were placebo controlled, reproducible, and blinded to some.extent. This experimental design was imperative, since all of these patients were severe asthmatics who required steroids for control. Such asthmatics would be predicted to be unstable, so repeat challenges and blinded challenges were necessary. These patients were not sensitive to aspirin, tartrazine, or monosodium glutamate. Stevenson and Simon (1981b) attempted unsuccessfully to define the mechanism of action of potassium metabisulfite in these patients. Evidence for an IgEmediated reaction could not be found. In fact, no evidence could be found that mediator release is involved in the reaction. Cutaneous testing with 0.02 mg of K,S,O, given intradermally was negative in the 4 tested patients. Incubation of peripheral basophils with K,S,O, in concentrations up to 0.01 M failed to induce histamine release. These tests would be positive in reactions involving mediator release whether IgE-mediated or not. Despite the lack of evidence for an IgEmediated reaction among the patients studied by Stevenson and Simon (1981b), systemic sensitivity beyond altered lung function was noted in all of their patients. The systemic symptoms were flushing, weakness, and hypotension. These symptoms can be involved in IgE-mediated reactions or other reactions involving mediator release. Stevenson and Simon (1981b) hypothesize that potassium metabisulfite acts via stimulation of the cholinergic reflex arc. This
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stimulation would account for some of the observed symptoms, including bronchoconstriction. However, it is difficult to explain hypotension on this basis. The therapeutic effectiveness of atropine is also consistent with this mechanism. Some evidence suggests that inhaled SO, activates irritant receptors in the bronchial tubes and that these receptors may activate the cholinergic reflex arc (Boushey, 1982; Nadel et al., 1965). However, other theories of the actions of inhaled SO, also exist (Boushey, 1982). Further proof will be needed before cholinergic stimulation will be accepted as the mode of action of ingested sulfites. In a 1981 abstract from the American Academy of Allergy and Immunology meeting, Simon et al. (1982) presented the first indication of the prevalence of sensitivity to ingested sulfites among asthmatics. A total of 61 asthmatics chosen randomly were challenged with potassium metabisulfite capsules containing 10, 25, 50, 100, and 200 mg K,S,O, at 30-min intervals. A positive reaction was defined as a fall in FEV, of at least 25%. Challenges were placebo controlled and single blind, with repetition of any positive response in a second challenge. Of the 61 patients, 5 (8.2%) reacted to K,S,O,. The reactions were milder than those encountered in their earlier studies (Stevenson and Simon, 1981a,b), and the threshold doses tended to be higher. This study would suggest that the prevalence of sulfite sensitivity among asthmatics is rather high. However, we question whether the population of asthmatics used in this survey was truly random. Many of the asthmatics used in this survey had severe asthma, and the study group was probably not a true cross section of the entire asthmatic population. The La Jolla group presented three abstracts at the 1984 American Academy of Allergy and Immunology meeting (Goldfarb and Simon, 1984; Jacobsen et al., 1984; Simon et al., 1984). Goldfarb and Simon (1984) evaluated the comparative sensitivities of sulfite-sensitive asthmatics (SSA) as a function of the route of exposure. Six SSA were used in this study; all 6 SSA had reacted to capsule challenges with 10-50 mg of sulfite, with a fall in FEV, of >25%. The minimum provoking dose for a beverage challenge was approximately one-half that of the capsule challenge. Inhalation of nebulized sulfite solutions provoked reactions at one-tenth to one-one hundredth of the capsule challenge dose. None of these SSA reacted to subcutaneous administration of sulfites at doses up to 10 times higher than their provoking capsule dose. Obviously, inhalant exposures are the most hazardous to SSA. Inhalant exposures could be encountered through the use of bronchodilator solutions preserved with sulfites (Koepke et al., 1983). Usually, the bronchodilating effect of the active ingredient would overwhelm the bronchoconstricting effect of sulfite, although a few patients seem to suffer paradoxical bronchoconstriction when treated with sulfited bronchodilators (Koepke et al., 1984a; Simon, 1985).
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Jacobsen et af.(1984) focused their efforts on elucidation of the mechanism of sulfite sensitivity in asthmatics. Using skin biopsies from SSA, Jacobsen et al. ( 1984) cultured skin fibroblasts and demonstrated that these cells had diminished levels of sulfite oxidase by comparison to cells cultured from normal individuals and asthmatics without sulfite sensitivity. The depressed levels of sulfite oxidase may indicate that these individuals are heterozygous for a deficiency of the enzyme. The diminished sulfite oxidase levels could compromise the detoxification of sulfite in these individuals (see Section IILA), but further studies will be needed to define the full implications of this finding. Jacobsen et af. (1984) were also able to show that cyanocobalamin (vitamin B,,) can protect SSA from the effects of ingested sulfite. Up to 50 mg of vitamin B,, orally was necessary to block the reaction to ingestion of a 50-mg capsule of K,S,O,. The vitamin B,, action was catalytic, as demonstrated by the observation that 5 mg would protect against 50 mg of K,S,O,. The B,, effect is probably associated with the known ability of the cobalamins to catalyze the oxidation of sulfite to sulfate (see Section II,F,6). The effective dose of vitamin B,, is far in excess of the recommended dietary allowance for this vitamin. It is even in excess of the levels of vitamin B,, used to treat pernicious anemia. However, such pharmacological doses of vitamin B,, may provide a convenient means of prophylaxis for SSA. R. A. Simon (personal communication) is now counseling his SSA patients to take 5 mg of vitamin B,, before eating a restaurant meal. Simon et al. (1984) evaluated the effectiveness of a variety of possible blocking agents on sulfite-induced asthma among SSA. Vitamin B,,, atropine, cromolyn, and doxepin were all effective blocking agents. These agents were effective irrespective of the route of administration of the sulfite. The effectiveness of all four agents is rather surprising, since they have different modes of action. Atropine is an anticholinergic agent, cromolyn is a mast cell membrane stabilizer and calcium channel blocker, doxepin is a broad spectrum antihistamine, and vitamin B,, catalyzes sulfite oxidation. Rather high doses of these blocking agents were necessary, and it is possible that at such high doses these agents could have additional effects beyond those just mentioned. This experiment does not provide many clues to the mechanism of action of sulfites in provoking asthma in these subjects. The equivalent effectiveness of these agents toward ingested versus inhaled sulfite is also rather surprising, since the mechanisms of the two routes of exposure are almost certain to be different. At the 1985 American Academy of Allergy and Immunology meeting, this group presented two additional abstracts on sulfite sensitivity (Howland and Simon, 1985; Simon, 1985). One of these reports involved the description of two cases of paradoxical bronchoconstriction in sulfite-sensitive asthmatics after administration of a sulfited bronchodilator (Simon, 1985). Howland and Simon
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(1985) challenged 5 sulfite-sensitive asthmatics with 3 oz of sulfited lettuce containing 80-90 mg of bisulfite (calculated by assessing the amount of sulfite solution not recovered after drainage). All 5 patients experienced pronounced decreases in lung function; the mean FEV, decrease was 44%, with a range of 31-64%. Untreated lettuce had no effect. This challenge study demonstrates conclusively that sulfited lettuce can elicit asthmatic reactions. Whether other sulfited foods will elicit these reactions is not known. Sulfited lettuce contains appreciable quantities of free SO, (Taylor et al., 1985), which may enhance the likelihood that lettuce will initiate asthmatic reactions by comparison to other sulfited foods.
6. Other Reports of Sulfite-Induced Asthma Several additional studies on the prevalence of sulfite sensitivity were conducted after Simon et al. (1982) reported that 8.2% of all asthmatics might be affected. Bush et al. (1985) showed that sensitivity to encapsulated sulfites was an appreciable risk only for those patients who require steroids for the control of their symptoms. Among 83 steroid-dependent or severe asthmatics, the prevalence of sulfite sensitivity was 8.4%. Among 120 mild or nonsteroid-dependent asthmatics, the prevalence of sulfite sensitivity was only 0.8%. Mild asthmatics make up about 80% of all asthmatics, so the overall prevalence for the total population of asthmatics is estimated to be 1.8% from this study. Buckley et al. (1985) selected 134 patients from a total clinic population of 1073 asthmatic subjects; 50/134 or 37% reacted to oral challenges with capsules of K,S,O,. This suggests a minimal prevalence of 4.6% (50/1073). However, as with the population examined by Simon et al. (1982), there is no indication that the patient population evaluated by Buckley et al. (1985) is representative of the overall asthmatic population. Towns and Mellis (1984) performed oral sulfite challenges with both capsules and citric acid solutions of Na,S,O,. None of the children developed asthma after challenge with capsules, but 19 of 29 (66%) experienced a significant decrease in FEV, after challenge with an acidic sulfite solution. This confirms previous suggestions that many more asthmatics are sensitive to acidic solutions of sulfite by comparison to encapsulated sulfites (Delohery et al., 1984a,b). Other case reports of asthmatic sensitivity to sulfites have also appeared (Sprenger et al., 1985; Altman et al., 1985; Schwartz and Chester, 1984; Koepke et al., 1984; Yang et al., 1985; Werth, 1982; Twarog and Leung, 1982). One was a patient with a history of asthma that worsened with ingestion of certain foods, particularly dried apricots and Catawba grape juice (Werth, 1982). The patient also experienced flushing during these episodes. Both of these foods are sulfited. Occasional asthmatic attacks were experienced following ingestion
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of wine, beer, cheese, blueberries, apples, and strawbenies. Of these foods, only wine, beer, and possibly freshly cut fruits would be expected to contain residual SO,. Symptoms of asthma were produced in the patient by sniffing a freshly opened bag of dried apricots. Oral challenge with capsules of potassium metabisulfite at doses up to 50 mg were negative. Inhalation of nebulized K,S,05 in water induced a rapid decline in FEV, . Apparently, this patient is another example of an individual who responds to inhaled SO, but not to ingested sulfites. He constitutes further proof for our suggestion that two groups of sulfite-sensitive asthmatics exist. Another case was reported by Twarog and h u n g (1982). This patient had perennial asthma and had experienced several adverse reactions to drugs that contained sodium bisulfite or sodium metabisulfite. Oral challenge of this patient with sodium metabisulfite in water revealed that a 5-mg dose caused a 52% drop in FEV,. The reaction to a 5-mg dose makes this patient the most sensitive described so far. Flushing was also noted. In addition, this patient may be unique, since evidence of mediator release in response to the sulfites was obtained in her case. Skin testing with sodium bisulfite at 0.1 mg/ml resulted in a definite wheal and flare reaction. Sodium bisulfite at concentrations of lop310- M also caused release of histamine from this patient’s leukocytes. For both skin testing and leukocyte histamine release, control tests on other individuals were negative. These findings do not constitute proof for the existence of an IgEmediated or type I reaction, however, because no evidence for the existence of a specific antibody was obtained. However, this patient seems to be unique, since Stevenson and Simon (198 lb) found no evidence of mediator release in 4 of their sulfite-sensitive patients. This patient probably represents a small subgroup of sulfite-sensitive patients. Apparently, the majority do not react via mediator release, but obviously some patients may mount such responses. This patient was challenged with sodium metabisulfite in water, a slightly acidic solution. It is difficult to determine if her response was due to inhalation of SO, or ingestion of sulfites. The ingestion route would seem most probable, since a 10-min lapse occurred between administration of the dose and the fall in FEV,. Also, SO, would not be evolved from a water solution, which would have a pH of greater than 4.0. Altman et al. (1985) and Sprenger et al. (1985) provide some additional evidence for the possibility of mediator release in the pathogenesis of sulfiteinduced asthma. Sprenger et al. (1985) describe a single patient with sensitivity to both inhaled SO, and aqueous solutions of K,S,O, (the pH was not specified). In this patient, an increase in the level of neutrophil chemotactic activity (NCA) in the serum was observed 2 hr after the maximal decline in FEV, . Altman et al. (1985) identified 3 additional patients with similar patterns of sensitivity along with increased serum NCA. NCA can be released from mast cells with appropri-
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ate antigen challenges. However, these findings are somewhat confusing. The increase in NCA in serum did not correspond in time to the decreased lung function. Also, the pH of the sulfite solutions is not provided, so it is impossible to know if these patients fall in the small group with sensitivities to encapsulated sulfites or the large group with sensitivities to ingestion of acidic sulfited beverages. The data from Sprenger et al. (1985) suggest that patients with sensitivities to ingested sulfites would also display inhaled sulfite sensitivity. Koepke et al. (1984b) performed inhalation challenges on 3 sulfite-sensitive (by capsule challenge) and 10 nonsulfite-sensitive asthmatics. All 3 sulfite-sensitive asthmatics and 4 of the 10 others had declines in FEV, of 20% or greater. The remaining 6 asthmatics had diminished lung function also, but it had not reached a 20% decrease at the administered levels of sulfite. Again, these data suggest that all patients with reactions to ingested sulfites will respond to inhaled sulfites. Schwartz and Chester (1984) obtained some conflicting information. Six asthmatics who developed airway obstruction after ingesting solutions of K,S,O, were subjected to inhalation challenge. Only 3 of the 6 patients responded to both ingestion and inhalation challenges with sulfite. These data suggest that a positive oral sulfite challenge is usually but not invariably accompanied by a positive aerosol challenge. Yang et al. (1985) identified 3 sulfite-sensitive asthmatics using oral challenges with K,S,O, capsules. Two of the patients had positive intradermal skin tests to 1 mg/ml solutions of K,S,O,. Passive transfer was also demonstrated with unheated serum from one of these patients. They conclude that IgE mechanisms may play a role in a subset of sulfite-sensitive asthmatics. Several reviews on asthmatic reactions to sulfites have appeared (Bush et al., 1986; Schwartz, 1984; Simon, 1984; Stevenson and Simon, 1984; Twarog, 1983). 7.
Other Adverse Reactions to Sulfites
Asthma has not been the only adverse reaction associated with ingestion of sulfites, although it appears to be the most common. Prenner and Stevens (1976) reported a patient who experienced urticaria and pruritis, swelling of the tongue, difficulty in swallowing, and tightness in the chest after ingestion of a sulfited restaurant salad. The patient had a positive scratch test to 0.2 mg of sodium bisulfite. An oral challenge with 10 mg of sodium bisulfite produced itching, nausea, flushing, cough, tightness in the throat, and erythema. Passive transfer testing was also positive. The passive transfer test indicates the presence of a serum factor involved in this patient’s response to sulfites. However, even in this case, this cannot be construed as definite evidence of an IgE-mediated reaction,
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although it is suggestive of such a reaction. Prenner and Stevens (1976) mentioned in their report that several food handlers had described instances of contact sensitivity from handling sulfite solutions. Fisher (1975) had previously reported a case of eczema in a food handler, which had been attributed to bisulfite exposure. Rudzki (1979) recently identified sulfites as contact allergens as well. Several other cases of urticaria and angioedema attributed to sulfites have been described (Allen et al., 1984; Habernicht et al., 1983; Huang and Fraser, 1984). Habernicht et al. (1983) described two women who reported urticaria and angioedema after ingestion of sulfited foods. One of these patients developed urticaria and burning of the scalp within 15 min following challenge with 25 mg K,S,O, in a capsule. Allen et al. (1984) note that urticaria can be induced by sulfite challenges, but that larger doses are usually required than those used in challenges of asthmatic subjects. Huang and Fraser (1984) suggested that subcutaneous administration of sulfites could provoke urticaria, angioedema, and laryngeal edema in sensitive individuals. Subcutaneous injection of 1.8 ml of lidocaine, which contains 0.5 mg of NaHSO,, produced palmar pruritis in a patient. No controlled challenge was administered. Yang et al. (1985) described a single patient with urticaria and angioedema after oral challenge with K,S,O, capsules. Very recently, another type of adverse reaction to sulfites has been described (Schwartz, 1983). Two patients were identified with anaphylactic-like reactions possibly associated with restaurant meals. The first patient had experienced an episode of clammy skin, weakness, headache, chest tightness, tachycardia, and a feeling of dissociation from his body commencing 10 min after eating a restaurant salad. The second patient had developed dizziness, nausea, palpitations, hives, dysphagia, chest tightness, and dyspnea after a restaurant meal of shellfish and salad. Both patients were administered single-blind, placebo-controlledchallenges with metabisulfite (Na or K salt not specified). With increasing doses in the range of 10-50 mg, progressively worsening hypotension was observed in both patients. Abdominal distress, nausea, dizziness, and weakness were also noted. Not all of the symptoms from the restaurant episodes were seen in the challenges, but this may have been due to the exposure to lower doses of sulfites in the challenges. Neither patient experienced asthma and neither had positive skin tests, so these reactions were not IgE mediated. These cases are the first reports of hypotension without asthma following challenge with sulfites. Stevenson and Simon (1981b) noted hypotension in some of their patients who also experienced asthma on challenge with sulfites. The frequency of the hypotensive response to sulfites is unknown. Sulfiting agents may also cause problems when administered as a component of a drug formulation. The most common manifestation is asthma, as described previously. The use of bisulfite in epidural anesthetics has recently been associ-
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ated with paralysis of the lower extremities (Wang et al., 1984). This rare reaction occurs when the anesthetic is accidentally injected into the subarachnoid space. The paralytic condition was duplicated in rabbits by injecting 1.2-2.4 mg of sodium bisulfite into the lumbar subarachnoid space. Flaherty et al. (1985) described an unusual case of sulfite sensitivity in a patient with underlying liver disease (sclerosing cholangitis) and ulcerative colitis. This patient’s liver condition was observed to worsen after ingestion of home-preserved juices and restaurant salads. These episodes were often accompanied by palmer and plantar erythema with pruritis. The liver function tests in this patient improved on a sulfite-free diet. An increase in serum levels of liver enzymes was noted after challenge with 500 mg of metabisulfite. This increase could be blocked by prior administration of 3 mg of vitamin B,*. Sulfites have been evaluated for their possible role in other conditions as well. Sonin and Patterson (1985) failed to trigger episodes of idiopathic anaphylaxis in 12 patients using oral challenges with Na,S,O, in lemonade. Similarly, Meggs et al. (1985) could find no role for sulfites in the elicitation of idiopathic anaphylaxis in challenges of 25 patients with capsules of NaHSO,. However, plasma histamine levels were elevated twofold in 23 of the 25 patients following bisulfite challenge. Eight patients with systemic mastocytosis were subjected to similar challenges, and no evidence was found to implicate sulfites in this condition (Meggs et al., 1985). Like the patients with idiopathic anaphylaxis, plasma histamine levels were elevated twofold in 7 of the 8 patients with systemic mastocytosis after bisulfite challenge.
8. Sensitivity to Suljited Foods Many of the reported sulfite-sensitive asthmatics provide a history of asthmatic reactions to foods that are suspected to contain sulfite residues. Their sensitivities to free inorganic sulfites have been documented through capsule and/or beverage challenges. However, there are only two reports of controlled challenges to a sulfited food or beverage unless one wants to count the challenges performed with sulfites in citric acid solutions or lemonade. We do not believe that the challenges with citric acid solutions are representative of the situation that exists with most foods, since most sulfited foods have pHs above 4.0 and would not spontaneously liberate SO,. The first study of the sensitivity of asthmatics to sulfited foods or beverages was conducted by Seyal et al. (1984) with wine. The subjects were asked to drink 4 oz of white wine containing 140 mg/liter of SO,. Only 1/25 asthmatics and 0/25 controls developed asthma following the challenge. Unfortunately, Seyal et al. (1984) did not prescreen their asthmatic population for sulfite senstivity, so the number of sulfite-sensitive asthmatics in their group is unknown and probably small. The study would have
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been strengthened considerably if it had been conducted on a group of sulfitesensitive asthmatics. Also, Seyal et al. (1984) considered a drop in FEV,of 12% or greater as a positive response. As noted previously, most pulmonary specialists would require a drop of at least 15-20% to signal a positive response. Therefore, the single responder in this study may be questionable. The other challenge study of sulfited food was conducted by Howland and Simon (1985) with sulfited lettuce; the results were described earlier. As discussed earlier, SO, and the inorganic sulfites react rapidly with food components. The rate, completeness, and products formed by these reactions are dependent on the pH, temperature, sulfite concentrations, type and concentration of various food components, and other factors. The primary products in many foods are the hydroxysulfonates of aldehydes, ketones, and reducing sugars. In vitro experiments have shown that these sulfite addition compounds are rather stable in dilute acid at room temperature (Adachi et al., 1979; Burroughs and Sparks, 1973; Green, 1976; Joslyn and Braverman, 1954). Therefore, they would not be expected to liberate SO, in the stomach under its acidic conditions. Some release of sulfites from sugar hydroxysulfonates might be predicted to occur in the neutral pH conditions of the small intestine. However, other hydroxysulfonates would be stable even under these conditions. Very recent work indicates that one hydroxysulfonate is not metabolized at all in rats or mice after feeding in the diet (Walker et af., 1983a). Sulfite addition compounds can also be formed with amino acids and proteins (Green, 1976; Schroeter, 1966). The stability of these adducts in gastric acid is not known, but they are probably more stable than many of the hydroxysulfonates. The question then centers on the role of the sulfite addition products in the induction of the asthmatic response. The answer to that question is not known. Some added SO, and inorganic sulfites remain in the food product in the uncombined state. This free SO, would probably react much like the sulfites ingested in capsules. Only if the food was below pH 4.0 would the gas, SO,, be evolved from the food in the oral cavity. In other foods, free SO, would exist primarily as HSO, and SO:-. Gaseous SO,, if it exists in the food, would likely pose a hazard to asthmatics who might inhale it during consumption of the food (Delohery et al., 1984a,b; Towns and Mellis, 1984). Other forms of free SO, would pose a possible hazard only to those individuals with sensitivites to sulfites in capsules. The combination of sulfites with food components would drastically lower the free SO, content of most sulfited foods (lettuce is an exception), thereby limiting exposure to these free forms of the sulfiting agents. Even with the free sulfites, the food matrix may diminish the degree of sensitivity by slowing rates of absorption and access to the sites of action. The degree of hazard posed to sulfite-sensitive asthmatics by sulfited foods can only be established with actual food challenges of sensitive patients. Since
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foods vary in the nature of their combined sulfites and in the amount of residual free sulfites, such challenges will need to be performed with a variety of sulfited foods. We expect, on the basis of chemical considerations, that most sulfited foods will be much less hazardous than equivalent amounts of sulfites in capsules. Again, sulfited lettuce may be an exception, since it contains a high proportion of free SO, (Taylor et al., 1985).
IV. POSSIBLE SUBSTITUTES AND THEIR LIMITATIONS If the current GRAS review leads to some limitation on the continued use of sulfites, it will be necessary to consider alternatives. This section is designed to present some possible alternatives and their limitations. A complete substitute for the sulfiting agents which would possess all of the desirable properties of this group of food additives will be virtually impossible to find. Replacements for each of the individual benefits provided by the sulfiting agents might be identified. However, in many foods, sulfiting agents are used for more than one purpose, e.g., the use in white wines for both its antimicrobial and antibrowning properties. The potential substitutes are also less effective and more costly in most cases. Many of the suggestions presented in this section were obtained from the review by Roberts and McWeeny (1972). A. CONTROL OF ENZYMATIC BROWNING Enzymatic browning will be inhibited by any process that destroys or inactivates the enzyme. Blanching would obviously work, but is impractical for use on fresh fruits and vegetables. Acid denaturation of the enzyme is also feasible, e.g., with application of lemon juice or vinegar. Since polyphenoloxidase is dependent on cupric ions for activity, the removal of these metallic ions may inhibit the process. EDTA would serve this purpose. Citric acid and tartaric acid also work in this manner. The activity of the enzyme can be slowed by lowering the pH through the addition of acids or fermentation. Lowering the water activity of the food can also diminish this reaction, but again dehydration is often not practical. Removal of oxygen works quite well, since oxygen is a required substrate for polyphenoloxidase. Reducing agents can be effective in converting the quinones back to the diphenols. Ascorbate and cysteine have been used in this manner. On cut surfaces, the sulfites have the advantage of being able to penetrate quickly into the cellular matrix, a property not shared by their substitutes. Hence, the substitutes are less effective. Several alternatives using combinations of the above materials for control of enzymatic browning have been developed. One procedure involves ascorbate
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and CaCl,. Another alternative is a combination of phosphate, citric acid, dextrose, aluminum sulfate, and sodium erythrobate. Both of these alternative methods work primarily on the basis of acidification with reducing activity. Ponting er al. (1971) pioneered use of ascorbate and calcium in the preservation of sliced apples. Montgomery (1983) recently noted that enzymatic browning of pear juice concentrate can be prevented by cysteine. Cysteine does not work well on cut surfaces because of its lack of cellular penetration. B. CONTROL OF NONENZYMATIC BROWNING Nonenzymatic browning can be controlled by (1) elimination of the active compounds, (2) lowering pH, (3) separation of the active species, or (4) behydration to low water activities. The removal of sugars can be be effected by fermentation, glucose oxidase, or leaching, as is done with potatoes. Theoretically, cysteine could be effective in competing for reaction with reducing sugars, but this has never been evaluated as a practical alternative. The browning reaction can also be slowed by the addition of acids such as lactic, tartaric, citric, acetic, or ascorbic acids, or aluminum sulfate. Physical separation of the active species can occasionally work if, e.g., the sugar is in the sauce and the amino acid is in the entree. Dehydration to less than 4% water will also inhibit nonenzymatic browning, but is not economically feasible. The replacement of sulfites in the control of nonenzymatic browning will be difficult because none of the above treatments is as effective or universally applicable. C. USE AS ANTIOXIDANTS OR REDUCING AGENTS Ascorbic acid can replace sulfites as antioxidants in beer, but naturally occurring levels of SO, in beer may make replacement unnecessary. Sulfites have the added advantage of controlling nitrosamine formation in the malt (Lukes et al., 1980). As a reducing agent, it may be possible to replace sulfites with cysteine or other mercaptans, although these substitutes have undesirable organoleptic properties and undesirable color and texture. D.
USE AS AN ANTIMICROBIAL AGENT
For wines and corn steep liquors, alternative agents will be difficult to find. Other antimicrobial agents will inhibit undesirable fermentations, but all have drawbacks in terms of expense, stability, specificity, or objectionable off-flavors. Agents such as lactic acid or sorbic acid may be useful in lowering the necessary levels of SO,. In table grapes, the gaseous nature of SO, is indispensable and a substitute will be difficult to identify.
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USE AS A BLEACHING AGENT
For the bleaching of cherries in the production of maraschino cherries, SO, has no equal. Sodium chlorite is useful for secondary bleaching (Beavers and Payne, 1968), but is far inferior for primary bleaching.
V.
FUTURE RESEARCH NEEDS
Despite their long history of use as food additives, much remains to be learned about sulfites which would be helpful to the present concerns about their safety. Better information is needed on the issues of consumer exposure assessment and the toxicity and hypersensitivity reactions to sulfited foods. For the purpose of improving consumer exposure assessments, better analytical methods and more analytical data are needed. The methods should emphasize determination of both free and combined sulfites. In particular, better methods are needed for the determination of combined or total sulfites. The combined sulfites appear to be less toxic than the free sulfites, so analytical data for both free and combined (or total) sulfite are needed. The analytical data should emphasize samples taken from typical points of consumption so that the losses on storage and preparation can be taken into account. As part of this effort, further investigations into the fate of sulfites in specific foods are needed. Emphasis should be placed on the identification of combined forms of sulfite so that their toxicity might be evaluated. From the viewpoint of toxicity assessment, further work is especially needed on the assessment of the toxicity of the combined sulfites. Since the bulk of sulfite ingestion is in the form of combined sulfites, the general lack of such information makes hazard evaluation virtually impossible. The toxicological studies should probably be focused on chronic and subchronic toxicity and should emphasize the oral route of administration. Further toxicological comparisons are needed in sulfite oxidase-deficient versus normal animals. On the hypersensitivity issue, a variety of unknowns remain. The major issue will be the determination of the responsiveness of sulfite-sensitive asthmatics to sulfited foods in controlled challenge trials. Only through the use of such challenges will the tolerance of these asthmatics for sulfited foods become available. In all likelihood, many sulfited foods will contain such low residual levels that they will not elicit asthma in these patients. The issue of the incidence of sulfite sensitivity in the asthmatic population remains to be answered as well. The incidence among mild asthmatics is unknown, although it appears as though the severe asthmatics are most likely to be sulfite reactors. The existence of more than one type of sulfite-sensitive asthmatic, acidic beverage reactors versus
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capsule reactors, seems likely from present data, but more studies are needed to establish the mechanisms (e.g., site of exposure) responsible for the existence of these two groups. The mechanism of action of encapsulated sulfites in inducing asthma in some asthmatics remains a mystery. Effective treatment may depend on the elucidation of this mechanism. Lastly, the existence of other types of hypersensitivity responses to sulfites has been established, but more studies are needed to establish the prevalence of such reactions.
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MAILLARD REACTIONS: NONENZYMATIC BROWNING IN FOOD SYSTEMS WITH SPECIAL REFERENCE TO THE DEVELOPMENT OF FLAVOR JAMES P. DANEHY Department of Chemistry, Universdy of Notre Dame, Notre Dame, Indiana 46556
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Introduction . .......... A. Historical ...................................... B. Maillard a nition of Browning Phenomena ain Research Stream . . . . . . . . . . . . . . . . . . . . . C. An Overview o Chemistry of Browning in Model Systems . . . . . . . . . . . . . . . . . . . . . . . . . . A. Preliminary Considerations B. First Steps in the Sequence of Maillard Reactions . . . . . . . . . . . . . . . . C. Some Informative Model Studies D. Empirical Relations between Reactants and Aromas . . . . . . . . . . . . . . Role of Browning in Specific Food Systems . . . . . . . . . . . . . . . . . . . . . . . . . A. Chocolate and Cocoa B. Bread and Other Bake ........................ C. Meat Flavors: Natural and Artificial . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Other Food Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Important Compounds in Browning Flavors . . . . . . . . . . . . . . . . . . . . . Browning, Nutrition, and Health A. Limited Loss of Nutrients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Possible Development of Mutagenicity . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . Trends in Continuing Research . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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INTRODUCTION
HISTORICAL BACKGROUND
For the individual, especially a civilized person, the selection of foods is determined largely by flavor. Concern for physical well-being, and the nutri77 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
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JAMES P. DANEHY
tional values involved, come far behind flavor as a factor determining human dietary choice. The concept of flavor is intrinsically complex, since it comprises the qualitative summation of the three distinctly different sensations involved in the normal process of eating: aroma, taste, and touch.' Actually, the additional senses of sight and sound make a substantial contribution to the anticipation and appreciation of flavor: red or brown (not green) meat, green (not yellow) broccoli, and crunchy (not limp) celery and potato chips. Fortunately for our ancestors, the appreciation of flavors, either natural ones or those produced by the empirical achievements of culinary skill, did not have to await the belated arrival of chemistry. But the latter does have an important contribution to make, both in understanding what determines all three components of flavor and in improving them, individually and collectively. Rohan (1972) has pointed out ". . . that, whereas much is known about the flavor of chemicals, there is very little known of the basic principles governing the chemistry of flavor." While little is as yet known regarding the interaction of specific molecules in foods with the equally specific taste buds or olfactory receptors, a great deal of tedious and painstaking work has gradually provided the identification of key compounds primarily responsible for well-known flavors, e.g., vanilla, clove, and cinnamon.
vanillin (vanilla)
eugenol (clove)
cinnamaldehyde (cinnamon)
On the other hand, chemistry has demonstrated that some very important aromas, including those of coffee and cocoa, cheeses, meats, baked products, and others, are not produced as a result of the presence of a unique characterizing 'Aromas (or odors) are the signals perceived by the olfactory organs, tastes are the signals perceived by the lingual organs, and flavors are the simultaneous perceptions of the other two.
MAILLARD REACTIONS
79
compound. Rather, the aroma is the result of a reproducible blend of a very large number of components in proper balance, no one of which alone would even suggest the familiar aroma. It is useful to make a distinction between native flavors and developed flavors. The first, characteristic of fruits and flowers, is determined by the specific molecules produced by the plants as secondary metabolities. The second is illustrated very strikingly by the familiar and unmistakable flavor of maple. The fresh sap of the sugar maple contains no hint of the familiar flavor, nor is it a matter of dilution, for freshly freeze-dried sap, though it tastes sweet, is colorless and lacking in aroma. The boiling process, however, serves not only to remove water, but to induce the chemical reactions that produce the familiar colored and aromatic products. Very different, but equally valid examples are the transformations of “green” beans into roasted coffee and cocoa and the change of a comparatively bland carcass into roasted turkey. B.
MAILLARD AND THE RECOGNITION OF BROWNING PHENOMENA
These and many other examples of developed flavors are of ancient origin. The commencement of the scientific studies of this general flavor problem probably was initiated by Louis-Camille Maillard (1912a,b). In a misguided attempt to determine the biological synthesis of proteins, he heated concentrated solutions or semidry mixtures of D-glucose with amino acids and observed a gradual darkening, a frothing, and the development of odors somewhat reminiscent of the baking of bread or the roasting of animal or vegetable products. This work attracted sufficient attention to persuade many others to continue the study of what came to be called the Maillard reaction. Eventually it became apparent that the work being done and reported on the browning reaction during this early period was distributed among three major categories, as follows: 1. Controlled studies of model systems designed to provide an experimental basis for the determination of the actual compounds formed and the elucidation of the mechanisms by which these compounds arise; 2. Attempts to inhibit the browning reaction in those systems in which its occurrence renders certain food products inedible; and 3. Attempts to exploit browning, either by producing products which can be used as flavoring agents or by providing conditions such that the browning reaction takes place during the processing of foods so as to maximize development of desirable flavors.
80
JAMES P. DANEHY
Both the volume of the work published and the recognition of its potential importance to the food industry led to the appearance in 1951 of the first general review of the subject (Danehy and Pigman, 1951). Only 2 years later a second review, limited largely to a consideration of model systems and mechanistic interpretations of their reactions, was published (Hodge, 1953). To date more than a dozen general reviews have been published, as have many more reviews directed to special areas in one or another branch of the food industry. Recently two international symposia have been devoted to the Maillard reaction (Uddevalla, Sweden, and Las Vegas, Nevada) (Eriksson, 1981; Waller and Feather, 1983). It is important to emphasize two important characteristics of the browning reactions: First, they can be harmful as well as valuable, and second, high temperatures are not always necessary for the development of browning reaction products. The classic example exemplifying these characteristics is the dried-egg problem of World War 11. In the early 1940s the U.S. Army’s Quartermaster Corps found that dehydrated, but not bone-dry eggs became all but inedible when distributed to their field stations, particularly in the South Pacific. It was, however, clearly demonstrated that a slow browning reaction between glucose and nitrogenous constituents of the broken eggs, at ambient temperatures, was responsible; the problem was definitely solved by treating the eggs with glucose oxidase before dehydration to remove the free glucose and so prevent the browning reaction from taking place. In 1953, Hodge concluded his review with the statement that “. . . the control of browning reactions to produce only wanted flavors and odors is an intriguing possibility. Control of browning to do man’s will is the ultimate goal of browning research, but progress toward this goal can be made only as the reaction mechanisms are better understood” (p. 941). Not the least part of that understanding has been achieved by Hodge and his colleagues at the U.S. Department of Agriculture’s (USDA) Northern Utilization Laboratory in Peoria, Illinois. As early as 1947, Barnes and Kaufman published a succinct summary of what had developed in the area of browning reactions since Maillard had published his papers in 1912. While the thrust of their summary is negative (browning is responsible for deteriorative changes in food products), Barnes and Kaufman did recognize that the Maillard reaction may also be the contributing factor in the development of many of our characteristic food flavors. Although no evidence was as yet available, there was reason to suspect that the distinctive flavor differences in such items as breakfast’foods, the crust of baked bread, and roasted coffee may be attributed to chemical combinations brought about during the heat treatment operations.
MAILLARD REACTIONS
81
C. AN OVERVIEW OF THE MAIN RESEARCH STREAM The many investigations that have been reported during the past 35 years in journal articles, books, and patents can be divided for the most part between three categories: 1. Detailed, organic chemical studies of the firsr stages of reaction between reducing sugars and various nitrogenous compounds, including amino acids; 2. Pyrolytic studies of mixtures of reducing sugars and various nitrogenous compounds, including amino acids; and 3. Identification of the volatile products formed during certain major types of food processing, and the correlation of these compounds, both with flavors and with the hypothetical reactions by which these products and flavors may be formed. The first group, organic chemical studies of the first stages of reaction, established as a first step the formation of N-glycosides by the reaction of reducing sugars with basic nitrogen compounds under very mild conditions and the spontaneous conversion of the N-glycosides into isomeric forms (the Amadori rearrangement). These studies were reviewed in great detail by Reynolds (1963) and earlier by Hodge (1953). A consensus has long since been reached as to the nature of the main sequences of chemical reactions involved in the browning of foods, known collectively as the Maillard reactions. These chemical sequences are summarized in Figs. 1 and 2. During the past decade Hayashi and Namiki have used electron spin resonance (ESR) spectroscopy to study browning reactions in model systems consisting of reducing sugars and alkylamines heated to 98°C in aqueous or alcoholic solutions (3 M in each reactant) (see Waller and Feather, 1983, pp. 21-46, for a summary of this work which cites all earlier references). They demonstrated the formation of free radical products at a very early stage and, from the analysis of the spectra, proposed that the radical products are N,N-disubstituted pyrazine cation radicals, assumed to be formed by bimolecular condensation of a two-carbon enaminol. This assumption was supported by the isolation and identification of glyoxalcyclohexylimine. To the extent that this scheme has any relevance in food systems or even in model systems with amino acids (some data on amino acids are included), it should probably be thought of as a concomitant pathway rather than as a revision of the established one. The present review is concerned specifically with the role of Maillard reactions in developing flavors during the cooking of certain foods. It seems best,
82
JAMES P. DANEHY
A
R
R \N/
H
H
/R
\
- F 1b
H-C-OH
‘
I
HO-C-H
I
H -C-OH
C
JN:
H -C-
OH
I
HO-C-H I
H-C-OH
I H - C-OH
I
H2COH I H2COH
-N-alkylarnino-D-glucoside a H2COH
0-91 ucose = R
,R
N/ H
H\,
I y 2
I
HO - C -
C
H C-OH
c=o H
R
/ N
H
\
\
H
y H;-C-
I
+
HO - C - H I H-C-OH
I
I
H -C-OH
H - C -OH
I H - C-OH
I
HzCOH
+/
A
H
OH
I HO-C-H
I (-H+
H -C-
OH
I
H-C-OH I H2COH
I H2COH
1-deoxy-1-N-a1 kyl ami no-
--D-fructose-
FIG. 1. An overview of reactions involved in the nonenzymatic browning of foods. (A) Aldoseamine condensation followed by Amadori rearrangement. (B) Reaction products derived from the aminoglycosides. (C) Oxidative degradation of a-amino acids by reductones: Strecker degradation.
83
MAILLARD REACTIONS
B
H
4
R \N/
c=o
I HO-C-H
I H-C-OH
H-$-OH
1 $-H,O H E L \c4 \ R
H-C-OH I
'I
(1,4-elimination)
!1
1,4-el imination
H
I H
\4\ C
I1
OH
I
C
/\
H-$-OH
O=C
i H
C
p\
OH
+H20 O +RNH2
I C
H
\
\/
H \ / C
C
OH
1 H-$-OH
, H
H-$-OH I
H O
\c4
C
I
I
c=o
I
Methyl a - d i ca r b o n y l compound
c=o
I
-H20
r"'
H-5-OH
1
H\
'C Further reactions, with and w i t h o u t amines
3- deoxyhexosone
FIG. 1B.
84
JAMES P. DANEHY
HO, C.:";.,
R
,R'
N=C, *'
+H20
HO
>
)c=c
R'
R
'
+
'
o=c
R," H '
H
R I'
C 'C
H'
'
R
\
NH2
FIG. IC.
however, to include a brief summary of the studies of model systems, since the results of these studies have provided a basis for interpreting what goes on during the far more complex processes of roasting and baking natural foods.
II. CHEMISTRY OF BROWNING IN MODEL SYSTEMS A.
PRELIMINARY CONSIDERATIONS
The gradual recognition that complex, presumably nonenzymatic transformations which take place in certain foods, responsible for deterioration in some cases and for the development of traditionally valued flavors in others, have a common chemical basis led to the carrying out of experiments in relatively simple model systems. It is often difficult to make meaningful comparisons of the results of different
MAILLARD REACTIONS
85
FIG. 2. Hodge’s view of the pathways by which browning products are formed. From Hodge ( 1953).
studies, even when the reactants are the same, because of the wide differences in reaction conditions, particularly with regard to concentrations of reactants, temperatures, and pH values. In attempting to make such comparisons, it is well to keep in mind three major sets of conditions and the directive influences they have in transforming food products: 1. Low moisture-high temperatures for relatively short periods of time. These are the conditions which develop flavors and colors that otherwise would not appear at all, e.g., in the roasting of coffee beans, cocoa beans, nuts, grains, and meats. Presumably the earliest stages of the browning sequence (Fig. 1A and
86
JAMES P. DANEHY
B) take place rapidly to furnish the intermediates which undergo the final transformations (Figs. 1C and 2). 2. Low moisture-moderate temperatures for relatively long periods of time. These are the conditions that produce deterioration, i.e., off-flavors and unwanted colors on storage of foods where it is desired to retain the properties as originally packaged. The classic case, referred to previously and studied by Kline and Stewart (1948), is the deterioration of dried eggs. 3. High moisture (i.e., solutions) over a wide range of temperatures (-20110°C) and times (hours to weeks). This set of conditions is the least relevant to food conditions, except for beverages, where browning is not usually important, either for good or evil. Yet these are the conditions that have been employed for most of the simple model studies, since they are suitable for the kinds of measurements that were made, i.e., development of color, decrease in concentration of reactants, and appearance of products. These studies have made it possible to determine the relative activities of specific carbonyl compounds and trivalent nitrogen compounds in various combinations, usually by measurement of absorption in the visible range and by determination of free amino groups. These studies have also demonstrated unequivocally the molecular structure of the first intermediates and the conditions under which they form. Further experiments with these isolated intermediates do furnish justification for the reaction sequences shown in Figs. 1B and C and 2. The model systems which have been studied largely parallel the food systems, though in simplified form, i.e., mixtures of amino acids and sugars, either in aqueous solutions or in the semisolid state. The bulk of the work has been done with glucose, as might have been expected, since it is cheap and readily available and, more importantly, because it is the most widespread and abundant reducing sugar in foodstuffs. Xylose is the runner-up, and fructose is a weak third. It is common knowledge that reduction products (glycerol, sorbitol, etc.) and oxidation products (gluconic acid, tartaric acid, etc.) do not contribute to browning. Indeed, it has been claimed that the latter inhibit browning, probably nonspecifically, by lowering the pH value of the system. However, Nafisi and Markakis (1983) have shown that aspartic and glutamic acids quantitatively inhibit browning in buffered aqueous solutions where the only difference is the presence or absence of the amino acid anions. B. FIRST STEPS IN THE SEQUENCE OF MAILLARD REACTIONS The reaction initiating the sequence between a carbonyl group and a trivalent nitrogen atom is the most thoroughly investigated and best understood of all the reactions. As early as 1963 Reynolds published a review with 140 references,
MAILLARD REACTIONS
87
limited largely to the studies of reactions of aldoses with amines, the determination of the structures and properties of the first product of reaction (a glycosylamine), and the rearrangement of the latter to a more stable ketoseamine. Typically, an aldose reacts spontaneously and reversibly with an amine to form an aldosylamine (an N-alkyl glycoside). Much of the earlier work was done with aromatic amines in order to facilitate isolation of the products, but a wide variety of primary and secondary aliphatic amines have also been used. In particular, the esters of amino acids readily yield crystalline glucosylamines. In neutral or alkaline solution these glycosylamines exhibit no reducing properties, as would be expected, since they are the nitrogen analogs of the 0-glycosides. Like the latter, the glycosylamines are readily hydrolyzed by acids to the parent aldose and amine. The stability of the glycosylamines is limited. Even in the dry or nearly dry state at 25”C, they rearrange spontaneously to 1-amino-1-deoxy-2-ketoses, the so-called Amadori rearrangement (Gottschalk, 1952; Hodge, 1955). The importance of these Amadori compounds for Maillard-type browning was demonstrated by Hodge and Rist (1953). They found that D-glucosylpiperidine was slowly transformed during storage at room temperature into a dark, tarry substance from which 1Wpiperidino- 1-deoxy-D-fructose (the Amadori compound) could be isolated in yields of up to 50%. But 2-O-methyl-~-glucosylpiperidine, in which the hydroxyl group on C-2 is blocked, remained colorless and stable after storage for 2 years at 25°C. Thus, preventing the Amadori rearrangement of an N-substituted glycosylamine inhibited subsequent browning. When the carbonyl compound is a ketose (fructose, for example) rather than an aldose, the initially formed fructosylamine (an N-alkyl fructoside) rearranges in an exactly analogous manner to form a 2-alkylamino-2-deoxy-D-glucose (the Heyns rearrangement) (see Fig. 3) (Heyns and Noack, 1962). These Heyns compounds also are precursors of the browning phenomena. Subsequent to the formation of the Amadori products (and presumably of the Heyns products), alternative pathways become available for the next stage in the browning sequence, depending upon whether the enolization of the 2-keto compound (for example) involves the C-1 atom or the C-3 atom (Fig. 1B). The reductones* shown in Fig. 1 B have been isolated and identified (Hodge et *Reductone is the trivial name for 3-hydroxy-2-ketopropanal.By extension, “reductones” are vicinal dicarbonyl compounds capable of some degree of enolization.
88
JAMES P. DANEHY
CH20H
I c=o
+ RNH2
I HO-C-H
-
CH20H I .#/R HO-C-N. I ‘H HO-C-H
-H,O L
I
I
I
H-C-OH
H-C-OH
H-:-OH
t
H
I
I
I
I
/OH
H\
I ../R H-C-N 1 H‘ HO-C-H I
H-C-OH
\
CH20H I /R C=N: 1 HO-C-H
CH20H
II ../ I H‘
‘
C-N HO-C-H
I
H-?-OH
R ~
-Ht
I +/R
C=N, I HO-C-H
H
I
H-C-OH
I
FIG. 3. The Heyns rearrangement: from a ketose and an amine to a 2-aklylamino-2-deoxyaldose.
al., 1963). Moreover, their further thermal decomposition, both alone and in the presence of amino compounds, produces compounds identified in food systems. It must be remembered that in food systems the thermal degradation of carbohydrates alone (Fagerson, 1969) takes place simultaneously with Maillard reactions and provides an independent source of conjugated compounds (reductones, furan derivatives, pyran derivatives, cyclopentene derivatives, and unique compounds), some of which are further intermediates and others of which are terminal products which contribute to aroma, flavor, and color as well (caramels). Insofar as Maillard reactions are concerned, the best understood of the reactions subsequent to the thermal fission of the Amadori and Heyns compounds are the Strecker reactions (Fig. lC), which produce aldehydes and new nitrogencontaining compounds. The former are major contributors to aromas and the latter are intermediates for producing additional flavorants. Further information and discussion on the compounds contributing to the aroma, flavor, and color of browned foods will be presented in Section 111.
MAILLARD REACTIONS
C.
89
SOME INFORMATIVE MODEL STUDIES
The study carried out by Rooney ef al. (1967) on model systems is outstanding; well conceived, comprehensive, and lucidly written, it is worthwhile summarizing it in some detail. Two model systems were used: (1) 0.2 M (equimolar) aqueous solutions of a sugar and an amino acid, pH 5.5,95"C, 12 hr; and (2) 100 g wheat starch mixed with 65 ml of a solution 0.02 M in both sugar and amino acid, pH 5.5, rolled and baked at 425°F (218°C) for 30 min. The carbonyl compounds produced were separated and determined quantitatively by paper chromatographic methods. The results from both systems were mutually complementary. The aldehyde produced is controlled mainly by the amino acid, while the amount of aldehyde is determined mostly by the type of sugar. Alanine, isoleucine, leucine, methionine, phenylalanine, and valine produced predominantly those aldehydes that would have been expected from Strecker degradations. In addition, smaller quantities of acetone, formaldehyde, and other carbonyl compounds were also found. Lysine, arginine, histidine, and tryptophan caused rapid and intense browning, but did not produce significant quantities of specific carbonyl compounds. Glutamic acid and proline caused relatively little browning and a small production of carbonyl compounds. Both with regard to color formation and production of carbonyl compounds, xylose was most reactive, maltose was least reactive, and glucose was intermediate. Isoleucine, leucine, and lysine produced pleasing aromas, while methionine and phenylalanine gave unpleasant aromas. Somewhat earlier, Rothe (1960) and Rothe and Voight (1963) conducted a rather similar, though less comprehensive investigation. Generally, both teams are in agreement, though there are some discrepancies in intensities of browning. Rooney et al. found that lysine, arginine, histidine, and tryptophan caused intense browning; Rothe and Voight agreed that lysine caused intense browning, but reported that arginine and histidine browned weakly, and tryptophan, scarcely at all. Using xylose only, Rothe and Voight recognized an inverse relation between browning and quantity of aldehyde produced and suggested that this might be accounted for if the aldehydes formed via Strecker degradation were consumed in subsequent pigment formation before they could be swept out. At a very early stage in the serious study of the Maillard reaction (19501952), Lea and his colleagues made a remarkably comprehensive and quantitative study of the reaction between glucose and the polar functional groups of casein (Lea and Hannan, 1950a-c; Lea ef al., 1951). Since they focused sharply on the problems of Maillard chemistry, established several points which are still valid today, and influenced strongly other workers in the field, it is worthwhile summarizing their work. Sodium caseinate (69 g) and glucose (31 g) were
90
JAMES P. DANEHY
dissolved in water, adjusted to pH 6.3, lyophilized, and stored at 37°C and 70% relative humidity. After 5 days, about two-thirds of the free amino groups had reacted and the product was still colorless and water-soluble. The powerful reducing properties of the product ". . . and the further observation that glucose cannot be regenerated from it by treatment with acid, indicates that the product . . . is not a simple N-glycoside, although such a substance may well be first formed and immediately undergo isomerization by some intramolecular change such as the Amadori rearrangement" (Lea and Hannan, 1950a, p. 528). After 30 days when the product had become brown and poorly soluble over a wide pH range, about 90% of the lysine, 70% of the arginine, 30% of the histidine, 50% of the methionine, and 30% of the tyrosine had reacted. Acid hydrolysis liberated all of the combined methionine, most of the tyrosine, and 70% of the lysine, but none of the arginine or histidine. There was no demonstrable loss of tryptophan or of total acidic or amide groups. When the remaining free glucose was removed by dialysis from the caseinglucose system which had been stored 5 days at 37°C and 70% relative humidity and the sample relyophilized and stored again at 37°C and 70% relative humidity, the complex browned rapidly, at a rate which indicated that ". . . decomposition of carbohydrate attached to the protein amino groups could account for most of the darkening of a casein-glucose mixture at 37"" (loc. cit.). Fully acetylated casein, stored with glucose in the same manner, browned only very slowly at 37"C, but at 60°C the acetylated glucose underwent changes in color, solubility, and amino acid content 20 times faster than the casein-glucose system. Since a free hydroxyl group on the C-2 atom of an aldose is essential for an Amadori rearrangement, one would expect that a 2-deoxyaldose would not initiate or support Maillard browning. Lea and Rhodes (1952) found that whereas galactose reacted with the free amino groups of casein at a rate very similar to that previously observed with glucose, 2-deoxygalactose reacted with the amino groups considerably more slowly. The development of a brown discoloration, however, was very much more rapid with the modified than with the normal sugar. Here, then, are two experimental anomalies which have never been explained: (1) Acetylated casein-glucose at 60°C browns 20 times faster than caseinglucose; and (2) casein- and 2-deoxygalactose browns much more rapidly than does casein-galactose. These three studies, then, early on gave a sound experimental basis for inferring the sequence of chemical reactions responsible for the colors and the flavors, both desirable and undesirable, which are formed during browning in relatively low-moisture (-2-40%) food systems.
91
MAILLARD REACTIONS
D. EMPIRICAL RELATIONS BETWEEN REACTANTS AND AROMAS In what was perhaps the earliest report to describe a deliberate attempt to produce aromas useful in foods via the Maillard reaction (Kiely et al., 1960), 7 sugars were heated individually with 20 amino acids in the presence of 15-50% water, at pH of 4.0,5.0,6.0, and 8.0, at 50, 100, and 150°C until a golden color had been reached. While details are not given, it was stated that “Although a very careful comparison was made of the eight sugars in the reactions, significant differences in the production of bread aromatics between the sugars was not apparent; there were some differences, however, in the rates of reaction. In view of the fact that flavor, and to a lesser extent, color, is what Maillard browning is all about, it is remarkable how few model studies give even a hint as to the aroma or flavor of the products obtained. There are at least two reasons for this situation. First, many chemists have been content to study the chemistry per se without regard to practical aspects. Second, while it is relatively easy to present data on color, at least in terms of absorbance at specific wavelengths, it is not so easy to describe aromas. Even when specific molecules of known odor are identified, this in itself gives no true picture of the overall aroma of the complex product. We shall have a good deal more to say about flavors in conjunction with specific food systems, and we shall present some correlation of flavors and aromas with classes of organic compounds and specific members thereof. In Table I are summarized those observations reported on aroma in studies of the reaction of one specific amino acid with one specific sugar system. ”
111.
ROLE OF BROWNING IN SPECIFIC FOOD SYSTEMS
Many bland or even downright unpleasant-tasting substances are transformed into some of the most desirable flavors and popular foods by roasting. Thus, those foods representing such different tastes and aromas as chocolate, bread, roast beef, coffee, and toasted nuts have in common the fact that they are products of the Maillard browning reaction. The enormous variety in flavor is due almost entirely to the large number of permutations from the interactions of a relatively few primary reactants and to the importance of balance between the components finally present. The reproducibility obtained in these seemingly chaotic and certainly random systems is as remarkable as the sensitive discrimination of the mammalian olfactory-gustatory system.
92
JAMES P. DANEHY
TABLE 1 AROMAS AND SPECIFIC VOLATILE COMPOUNDS ARISING FROM MAILLARD REACTIONS
OF L-AMINO ACIDSO
Amino acid Alanine
a-Aminobutyric acid
Arginine Aspartic acid Cysteine
Cystine Glutamic acid Glycine
Histidine Hydroxyproline
Isoleucine
Leucine
Lysine Methionine
Aroma or compounds produced Acetaldehyde(Rooney et al.. 1967; Rothe, 1960; Rothe and Voight, 19631 Roasted barley (Rothe and Voight, 1963) Caramel (Wiseblatt and Zoumut, 1963) Propionaldehyde (Rothe and Voight, 1963) Walnuts (Rothe and Voight, 1963) Breadlike (Kiely er al.. 1960) Very weak (Lea and Hannan, 1950b) Very weak (Wiseblatt and Zoumut, 1963) Meaty (Kiely et al.. 1960) Thiol, H2S (Wiseblatt and Zoumut, 1963) Overboiled egg (Arroyo and Lillard, 1970) Cooked meat (Led1 and Severin, 1973, 1974) Burned horn (Rothe and Voight, 1963) Meaty (Kiely et al.. 1960) Chicken broth (Wiseblatt and Zoumut, 1963) Caramel (Kiely et al.. 1960) Baked potato (Wiseblatt and Zoumut, 1963) 2.5-Dimethylpyrazine, trimethylpyrazine (Dawes and Edwards, 1966) Breadlike (Kiely et al., 1960) Very weak (Wiseblatt and Zoumut, 1963) Potato (Kiely et al., 1960) Weak (Wiseblatt and Zoumut, 1963) Cookie- or mushroom-like (Dawes and Edwards, 1966) Fruity (Kiely et al., 1960) 2-Methylbutanal (Rooney et al.. 1967; Rothe, 1960, Rothe and Voight, 1963) Crust (Wiseblatt and Zoumut, 1963) Furfural (Rothe and Voight, 1963) Pleasant (Rooney et al.. 1967) Breadlike (Kiely et al.. 1960) 3-Methylbutanal (Rooney et al.. 1967; Rothe, 1960; Rothe and Voight, 1963) Cheesy (Wiseblatt and Zoumut, 1963) Baked potato (Wiseblatt and Zoumut, 1963) Pleasant (Rooney et al., 1967) Furfural (Rothe and Voight, 1963) Roasted barley (Rothe and Voight, 1963) Dark corn syrup (Wiseblatt and Zoumut, 1963) Pleasant (Rooney et al., 1967) Methional (Rooney et al., 1967; Rothe, 1960; Rothe and Voight, 1963) Baked potato (Wiseblatt and Zoumut, 1963) Furfural (Rothe and Voight, 1963) Unpleasant (Rooney et al., 1967)
MAILLARD REACTIONS
93
TABLE I (Conrinued) Aroma or compounds produced
Amino acid
Phenylalanine
Proline
Serine Threonine Valine
Boiled potato (Arroyo and Lillard, 1970) Objectionable (Arroyo and Lillard, 1970) Cabbage (Rothe and Voight, 1963; Lindsay and Lau, 1972) Floral, rose (Kiely et 01.. 1960; Rothe and Voight, 1963) Phenylacetaldehyde (Rooney er al., 1967; Rothe, 1960; Rothe and Voight, 1963) Strong hyacinth (Wiseblatt and Zoumut, 1963) 2.5-Dimethylpyrazine (Dawes and Edwards, 1966) Unpleasant (Rooney er al., 1967) Cornlike (Kiely et al., 1960) Crackers, toast (Wiseblatt and Zoumut, 1963) Cracker odor (Hunter er al., 1969) Strongly browned flour (Rothe and Voight, 1963) Weak breadlike (Wiseblatt and Zoumut, 1963) Very weak (Wiseblatt and Zoumut, 1963) Fruity (Kiely et al., 1960) 2-Methylpropanal (Rooney er al.. 1967; Rothe, 1960; Rothe and Voight, 1963) Yeasty, protein hydrolyzate (Wiseblatt and Zoumut, 1963) Furfural (Rothe and Voight, 1963) Roasted barley (Rothe and Voight, 1963)
The carbonyl compound used in each case was ( I ) eight different sugars (no significant influence on flavors produced by different amino acids) (Kiely el al., 1960); (2) dihydroxyacetone (Wiseblatt and Zoumut, 1963; Hunter et al., 1969); (3) fructose (Dawes and Edwards, 1966); (4) glucose (Rothe, 1960; Kobayashi and Fujimaki, 1965; Arroyo and Lillard, 1970; Lindsay and Lau, 1972); (5) xylose (Rooney et al., 1967; Rothe, 1960; Rothe and Voight, 1963; Led1 and Severin, 1973, 1974); and (6) maltose (Rooney et al., 1967).
A.
CHOCOLATE AND COCOA
, ~ determined One of the world's most popular flavors, chocolate and C O C O ~ is by a physical-chemical composition which starts with the seeds of the plant Theobroma cacao and continues with an empirical process discovered and perfected by the Aztecs, or by an earlier society from whom the Aztecs received it. Two entirely separate stages are essential for the development of this flavor: the fermentation of the beans (seeds) in their mucilaginous pulp enclosure when the pod is opened, and the roasting of the dried, fermented beans. It has long
Thocolate is the unctuous, extremely bitter low-melting substance (called chocolate liquor in the industry) obtained by the crushing and milling of roasted cacao beans. Cocoa is the free-flowing powder obtained by the partial defatting of chocolate liquor.
94
JAMES P. DANEHY
been known that neither aroma nor aroma precursors are present in unfermented cacao beans which, when roasted, develop an odor reminiscent of broad beans (Rohan, 1963). Substantially the only sugar present in unfermented beans is sucrose, but a mixture of fructose and glucose accounts for most of the sugar in fermented beans (Rohan, 1964; Reineccius er al., 1972a). Also, in going from unfermented to fermented beans, the concentration of free amino acids increases between 3- and 10-fold (Rohan, 1964; Rohan and Stewart, 1965). The justification for the preceding summary statement is found in the reports of a well-conceived and carefully executed research program carried out by Rohan. Starting with the variables involved in fermentation techniques used on West African farms, he did the following (Rohan, 1958a,b; Holden, 1959): 1. Determined the amino acid and sugar contents of unfermented and fermented beans; 2. Prepared aqueous methanolic extracts of both kinds of beans; 3. Showed that roasting of the dehydrated extract of the second (but not the first) kind of bean produced the characteristic cocoa aroma; and 4. Determined the changes in sugar and amino acid contents of the dehydrated extract brought about by fermenting and roasting (Rohan, 1963, 1965, 1964, 1967; Rohan and Stewart, 1965, 1966a,b, 1967a-c). TABLE I1 FREE AMINO ACIDS IN THE DRIED EXTRACT
OF FERMENTED AND UNFERMENTED CACAO BEANS"
Amino acid (g)/IOO g dry substance of extract Amino acid
Fermented
Unfermented
Leucine Lysine Phenylalanine Threonine Valine Arginine Glycine Alanine Isoleucine Proline Serine Tyrosine Glutamic acid Histidine
4.75
0.45
0.56 3.36 0.84 2.60 0.35 0.35 3.61 1.68 1.97 1.99 1.21 1.71 0.04
0.08 0.56
a
Based on data of Rohan (1964).
0.14 0.57
0.08 0.09 I .04
Ratio of fermented to unfermented 10 7
6 6 5 4 4
0.57 1.02
3.5 3 3 2 2 I .5
0.08
0.5
0.56 0.72
0.88
MAILLARD REACTIONS
95
Table I1 presents a compilation of Rohan’s data for the increase in free amino acids in the extract brought about by fermentation. Subsequently, Rohan and Stewart (1966a,b) presented data graphically for the gradual destruction of total amino acids and sugars during a roast at 182- 183°C of dehydrated extracts of fermented beans. Within 30 min, almost half of the amino acids had disappeared, and only 10% of the reducing sugars were left. Mohr et af. (1971) started with an aqueous methanolic extract of defatted, ground Ghana cacao beans prepared just as Rohan (1964) had prepared his extract. But Mohr passed the deep brown extract through a “polyamide” column to adsorb polyphenolic and quinonoid substances before lyophilizing the almost colorless extract. Mohr extended Rohan’s study by determining both free and peptide-bound amino acids, both before and after roasting. Mohr heated his reaction mixtures only to 121”C,which was reached in 8 min, since he found that at that temperature a thin layer of the dried extract began to brown rapidly and to give off a typical cocoa aroma. Mohr’s data are presented in Table 111. The amino acids are arranged in the descending order of free amino acids present before roasting. Cumulative data for changes in amino acids and carbohydrates are presented in Table IV. Several conclusions can be drawn from these data. First, without exception, free amino acids are much more sensitive to destruction in this system than are the peptide-bound amino acids. This conclusion might have been inferred from Rohan’s observation (1964) that only the extract from fermented beans gave rise to cocoa aroma upon roasting, but the fermentation produces reducing sugars from sucrose as well as amino acids from polypeptides, so that conceivably the reducing sugars might have produced aroma at the expense of peptides. Mohr’s data show that this unlikely hypothesis is not tenable. Second, differences in the stability of amino acids under these conditions are not all that great: from 25% for isoleucine to 68.5% for lysine, over a relatively short period of time. In this system the reducing sugars must be the limiting factor, since the glucose and fructose are completely destroyed or removed. Third, neither cystine nor cysteine is reported to be present, and the only other sulfur-containing amino acid, methionine, is present at a much lower concentration than any other amino acid. Clearly, as we shall see later, cocoa would probably have a considerably different flavor if cysteine or cystine were present in the fermented beans. Although Rohan (1964) had suggested that the operative reaction in the development of chocolate aroma might be a Strecker degradation of the amino acid fraction, in none of his reports does he give any data on the composition of cocoa volatiles. Bailey et al. (1962) followed the Strecker lead. Using gas chromatography and mass spectral analysis, they determined that the volatiles from a typical sample of roasted, ground Bahia cocoa contained, in the mole ratio
TABLE 111 CHANGES IN THE FREE AND PEPTIDE-BOUND AMINO ACID CONTENT OF THE EXTRACT OF AROMA PRECURSORS UPON ROASTING AT 121°C" ~
~
Before roasting Amino acid Leu Ala Phe Glu Ser Val TYr Thr NH3
pro Ileu LYS ASP
Arg GlY His Met a
Free amino acid (mmollkg)
Peptide-bound amino acid (mmollkg)
102.1 84.3 73.6 63.5 63.3 50.3 41.9 38.0 37.0 33.7 30.0 26.6 24.9 23.5 14.4 7.5 1.9
108.1 144.2 83.9 302.4 113.0 158.5 41.5 91.2 150.0 81.3 84.0 63.0 302.7 55.5 139.9 16.7 7.1
Loss upon roasting
After roasting
Fb
1.1
I .7 I .4 4.7 1.7 3.1 0.9 2.4 4.0 2.4 2.8 2.4 12.2 2.3 9.7 2.2 3.7
Based on data of Mohr et al. (1971). F, Peptide-bound amino acid/free amino acid.
Free amino acid (mmollkg)
Peptide-bound amino acid (mmol/kg)
51.5 53. I 15.2 26.9 34.7 32.3 16.6 15.1 15.5 21.6 22.5 8.0 16.0 13.6 7.9 3.0 0.9
90.9 130.7 73.1 301.4 115.8 143.0 43.0 94.5 150.0 97.9 68.0 36.9 243.5 49.1 129.6 10.9 7.3
Free amino acid
Peptide-bound amino acid
Fb
(%)
(%)
I .8 2.4 I .9 11.2 3.3 4.4 2.6 6.2 9.7 4.5 3.0 4.6 15.2 3.6 16.4 3.6 8.1
49.7 37.0 65.7 57.7 45.2 35.8 60.5 60.3 58.0 35.9 25.0 68.5 35.8 42.2 45.2 60.0 52.6
16.4 9.4 13.0 0 2 9.8 0 0 0 0 19.2 41.5 19.6 11.6 7.4 34.8 0
97
MAILLARD REACTIONS
TABLE IV CHANGES IN THE COMPONENTS OF THE EXTRACT OF AROMA PRECURSORS UPON ROASTING AT 120°C'~b
Before roasting (mmol/kg)
After roasting (mrnol/kg)
Decrease
Component
Total free amino acids Total peptide-bound amino acids Glucose Fructose sucrose Citric acid
717 1868 167 556 32 378
364 1789
49.2 4.2 100
0 14
30 367
(a)
97.5 6.0 3 .O
Dry substance of this extract amounted to -5% of the weight of the shelled cacao beans (fermented and air dried). Based on the data of Mohr et al. (1971).
shown in parentheses, isovaleraldehyde (42.0), isobutyraldehyde ( 15.4). propionaldehyde (13.0), methanol (9. I), acetaldehyde (7.0), methyl acetate (6.3), butyraldehyde (3.0), diacetyl (2.8), and at least eight other assorted compounds in lesser amounts, none of them containing nitrogen. The first, second, and fifth most prominent compounds identified could be related to leucine, valine, and alanine as their precursors. Obviously, however, while a synthetic mixture corresponding to the above would be fragrant, it would certainly not suggest the aroma of cocoa, based on the work reported by many others. What then is the chemical basis for the aroma and flavor characteristic of cocoa and chocolate products? During the period of 1964-1976, more than a dozen reports addressed themselves to this problem. They ranged from the herculean labors of the Firmenich group, which carried out a classical fractionation of 750 kg of Arriba (Venezuelan) cocoa, which confirmed the presence of 43 compounds previously reported by others and identified 29 compounds not previously reported (Dietrich et al., 1964), to the powerfully instrumented investigations (high-resolution gas-liquid chromatography, infrared spectroscopy, and mass spectrometry) which, to date, have claimed the identification of more than 300 constituents of cocoa volatiles. The methods of extraction have commonly employed aqueous ethanol (from 70 to 92°C v/v) (Dietrich et a f . , 1964), steam distillation (Darsley and Quesnel, 1972), codistillation with 1,2-propanediol (Flament and Stoll, 1967) or with ethanol (van der Wal et al., 1968, 1971), and supercritical carbon dioxide (Vitzthum et al., 1975). In some cases the extracts were first fractionated into neutral, acidic, and basic fractions (Dietrich et al., 1964; Rizzi, 1967; van F'raag et al., 1968). In other cases an acidic extraction was employed in order to give only a basic fraction (Stoll et al., 1967a; Reineccius et al., 1972).
98
JAMES P. DANEHY
The cumulative result of all this effort is the reasonably sure establishment that at least 350 organic molecules are present in cocoa volatiles in at least detectable amounts and that a goodly, though indeterminable number of them are final products of the Maillard reactions. Few, if any, of these molecules would be odorless. But how they combine in intensity and specificity to produce the instantly recognizable aroma and flavor of chocolate is still unknown. In 1964, Dietrich et al. suggested that their failure to reconstitute the aroma of chocolate from the 72 components known to them at that time could be attributed to the fact that other components had escaped them. Seven years later van der Wal et al. (1971), with semiquantitative data on 181 compounds, made an attempt to duplicate the aroma concentrate using the gas chromatogram as a guide to estimate the proportions and amounts of the constituents involved. Although this synthetic mixture was reminiscent of cocoa, it lacked the pronounced aroma of the extract and was easily distinguishable from it. They concluded from this that probably important aroma components still await detection. In view of the fact that holding large numbers of organic compounds of diverse functionality in a homogenous system at room temperature, much less at about 100°C, is conducive to chemical reaction, we should consider at least two other reasons for the lack of success in attempts to reconstitute the aroma of cocoa. First, some of the compounds actually contributing to the aroma of cocoa may have decomposed and are no longer present in the extract analyzed. Second, some of the compounds identified in the extract may be artifacts, not actually present in the cocoa, but synthesized during the extraction and working-up process. Under the circumstances, we shall not list all of the approximately 350 compounds which have been claimed to be present in cocoa volatiles. They are listed in overlapping tables in the following papers: Dietrich et al. (1964); Marion et al. (1967); Flament et al. (1967); van der Wal et al. (1968); Vitzthum et al. (1975); Stoll et al. (1967b); Reineccius ef al. (1972); Rizzi (1967). In Table V are listed the classes of compounds, minimum number for each class, identified in the analysis of cocoa volatiles. In almost all cases the specification of a compound is purely qualitative, with no information whatsoever as to what fraction of the cocoa is accounted for by that compound. An important and notable exception is the report of Flament et al. (1967), who recorded a large number of substances as "%" of fraction A, which in itself is a 10.0-g concentrate from 204 kg of ground, roasted cacao beans (chocolate liquor). In Table VI are listed those compounds making up the largest part, but not necessarily the most important part, of fraction A, and the percentage of the original chocolate liquor for which they account. These data support the commonly held opinion that carbonyl compounds and pyrazines are
MAILLARD REACTIONS
TABLE V CLASSES OF COMPOUNDS IDENTIFIEDIN THE ANALYSIS
OF COCOA VOLATILES"
Hydrocarbons Aliphatic (8) Terpene (6) Aromatic (17) Ketones Aliphatic (12) T e v m (5) Aromatic (6) Phenols (5) Disulfides (5) Furans ( 15) Pyrazines (34) Oxazoles (4)
Alcohols Aliphatic (4) Terpene (9) Aromatic ( 5 ) Carboxylic acids Aliphatic (15) Aromatic (14)
Aldehydes Aliphatic (10) Terpene (2) Aromatic (2) Esters Aliphatic (30) Aromatic (7)
Esters and acetals (12) Trisulfides (2) Other 0-heterocycles (6) Nitriles (4)
Sulfides (4) Other sulfur compounds (6) Pyrroles (9) Pyridines (9)
Number indicates the minimum number of compounds identified.
major contributors to the aroma of cocoa and show that they are effective in the range of parts per 10 million. Flavor is aroma plus taste, and it is important to remember that cocoa itself is quite bitter. Three diketopiperazines,cyclo(-Asn-Pro-), cycle(-Ala-Gly), and cycld-Asn-me-), have been isolated from roasted, but not unroasted, cacao TABLE
VI
PRINCIPAL COMPONENTS OF THE FRACllON A, FROM A CODISTILLATION
OF THE VOLATILES OF A CHOCOLATE LIQUOR WITH I .2-PROPANEDIOL'
Compound Trimethylpyrazim Tetramethylpyrazine 2.5- + 2,bDimethylpyrazine 2,5-Dimethyl-3-ethylpyrazi~ Acetophemme 2-Methylbutanal 3-Methylbutanal 2-Phenylethyl acetate
2,5-Dimethyl-3-isoamylpyrazi~ 3-Hydroxy-2-butanoW Ethyl caprate 0
b
Percentage of A, 21.0 20.3 7.3 5.6 3.7 3.5 2.5 2. I I .4 I .4 1.1
Percentage
of a chocolate liquorb 10.5 10.15 3.65 2.8 1.85 1.75 1.25 1.05 7.0 7.0 5.5
(10-5) (10-5) (10-5) (10-5) (10-5)
(10-5) (10-5)
(10-9 (10-9 (10-9
Based on the data of R a n t et 01. (1967). Percentage of Al X (10/204.oOO) = Percentage of A1 (0.5)(10-5).
100
JAMES P. DANEHY
beans. Carefully planned and carried out experiments indicate that the bitter taste of cocoa is due to the simultaneous presence of 30-50 ppm of a diketopiperazine and 100 ppm of theobromine, the characteristic xanthine of cacao beans. Diketopiperazines containing the phenylalanyl residue resemble the bitter principle of cocoa most closely (Pickenhagen et al., 1975). Mohr et al. (1976) have isolated several peptides from fermented cacao beans, and they have found that when these peptides, along with the amino acids prominent in fermented cacao beans, are pyrolyzed with fructose, the resulting aroma is much closer to that of roasted cacao beans than when peptides or amino acids alone are pyrolyzed with fructose. Many of the major classes of compounds and even specific compounds found in cocoa volatiles are also found in the volatiles of other browned food products. We shall deal with these flavor compounds later. B.
BREAD AND OTHER BAKED CEREAL PRODUCTS
Browning plays an essential role in the development of flavor in bread, one of man’s most important foods. As early as 1910 the occurrence of maltol and isomaltol in bread as natural flavorants was reported. Not until more than 40 years later did research on the flavor of bread enter a sustained phase, which, lasted about 15 years (1953-1969). Some of the important model studies were carried out by investigators primarily interested in bread baking and allied problems (Rooney et al., 1967; Rothe, 1960; Kiely et al., 1960; Wiseblatt and Zoumut, 1963; Johnson and Miller, 1961). Baker et al. (1953) demonstrated that both fermentation and the formation of a brown crust are essential for satisfactory flavor. Bertram (1953) addressed himself to an immediate practical problem: Why did the flour from a certain strain of low-protein Dutch wheat, upon baking, give a crust with a gray color? He showed that the addition of either dried egg white or wheat gluten to the flour gave crusts with normal brown color. The results prompted him to carry out some model experiments in which mixtures of wheat starch, different sugars, and dried egg white or amino acids were heated, using bicarbonate rather than yeast as a leavening agent. The results are the first unequivocal demonstration of the importance of the Maillard reaction in crust color. Aldehyde formation received a great deal of attention in these studies of bread aroma (Rothe, 1960; Rothe and Thomas, 1959, 1963; Wiseblatt and Kohn, 1960; Wiseblatt, 1960a), since they are prominent and easy to identify semiquantitatively by trapping them as 2,4-dinitrophenylhydrazones,followed by chromatography. There was an early consensus that while they are formed in the crust by Strecker degradation of amino acids they are withdrawn into the crumb upon cooling and storage of the bread. Thomas and Rothe (1957) emphasized the
MAILLARD REACTIONS
-
101
importance of furfural, which is not formed by a Strecker degradation, and showed that addition of 0.7% of xylose to the flour increases the total aldehyde content of the bread volatiles 4-fold and the furfural content 10-fold; arabinose, sorbose, fructose, and glucose were less effective. But it was soon recognized that aldehydes, other carbonyls, and ethanol are not the whole story in terms of flavor. Wiseblatt and Kohn (1960) found that neither the actual distillate containing these compounds nor several synthetic blends of them have proved of any value in enhancing the palatability of a bland chemically leavened bread. Gradually the authenticated list of compounds found in preferments, dough, oven vapors, and bread (Wiseblatt, 1960b, 1961; Wick et al., 1964; Johnson et al., 1966) has grown to include more than 70, including alcohols, aldehydes, ketones, carboxylic acids, esters, and a very few miscellaneous compounds: methyl mercaptan, hydrogen sulfide, maltol, and isomaltol. Is the small number of miscellaneous compounds realistic or have significant compounds been missed by inadequate analytical methods? On the basis of the evidence available, the aroma constituents of bread appear to differ qualitatively from those of cocoa, roasted nuts, and cooked meat most strikingly in the complete absence of nitrogenous constituents, particularly pyrazines. However, Mulders et al. (1972a,b; Mulders and Dhont, 1972; Mulders, 1973a) made a determined gas chromatographic study of the constituents in the vapor above fresh white bread and their odor values. They found 52 compounds, of which 42 had not been reported previously. Several of them were pyrazines, lactones, and derivatives of furan or pyrrole. While great differences existed in the quantities of components between individual loaves, although the baking protocol had been rigorously standardized, the odor was quite similar for all. An aqueous synthetic mixture, prepared in such a way that the chromatogram of its vapor was identical to the average chromatogram of bread vapors, had an odor which scarcely resembled that of bread; it was rather doughlike. Therefore, the components detected in a normal vapor sample cannot account for the characteristic odor of fresh white bread. The odor of the synthetic mixture changed from doughlike to breadlike upon addition of a particular gas chromatographic fraction of a white bread extract. The work of Hodge and Moser (1961) confirms the contribution of maltol and isomaltol to bread aroma 50 years after the demonstration of their presence. It has been shown repeatedly that L-proline is particularly important as a precursor of bread aroma constituents (Wick et al., 1964; Morimoto and Johnson, 1966). There have been a number of investigations of the effect on bread aroma of adding sugars or amino acids to a dough before baking (Thomas and Rothe, 1957; Linko et al., 1962, 1963). The most recent report (Salem et al., 1967) is the most comprehensive and summarizes the situation very well. Systematically, 0.02 mol of an amino acid and 0.02 mol of either glucose or xylose were added
102
JAMES P. DANEHY
to a dough containing 700 g flour, processed and baked according to a “straight dough” procedure. Reflectance data on the top and bottom crusts showed that addition of amino acids increased the intensity of the crust color in all cases. Methionine and arginine, with added glucose, produced the darkest color. Proline had little effect on the crust color. Generally, xylose-amino acid mixtures gave darker crusts than glucose-amino acid mixtures, probably because xylose is nonfermentable and more reactive in browning. Analytical data indicate that the composition of carbonyl compounds in crust and crumb vary with the amino acid added, with glucose as the sugar added, with the effect much more pronounced in the crust than in the crumb. The contents of furfural and 5-hydroxymethylfurfural (HMF) were less than the control in all cases. This is not surprising, since these compounds serve as intermediates and undergo further condensation with free amino groups, which are more abundant with the deliberate addition of amino acids. Alanine and valine increased the yield of acetone as well as the expected Strecker aldehydes. Addition of leucine and isoleucine doubled the presence of the Strecker aldehydes. Lysine increased the concentration of all carbonyl compounds three- to fourfold. Histidine increased both acetone and aldehyde three- to fourfold and isobutyraldehyde and isovaleraldehyde twofold. Interestingly enough, proline, which everyone agrees has a definite positive effect on the aroma, had a modest increasing effect only on acetone; for other carbonyl compounds the content was either the same or lower than in the control. Parallel experiments in which xylose rather than glucose was the sugar gave generally comparable results. But xylose definitely gave lower levels of aldehydes than did glucose. The most plausible explanation of this anomaly is that xylose is so reactive that most of the carbonyl compounds formed in the early stages of baking were volatilized or reacted quickly with free amino groups to give melanoidins, consistent with the dark color of the crusts. Although aldehydes are produced during fermentation of the dough, they are volatilized during the later stages of fermentation and the early stages of baking. Addition of isovaleraldehyde to the dough did not increase the isovaleraldehyde content of the crust or crumb. But addition of leucine to the dough produced a two- to threefold increase in the isovaleraldehyde content of the crust. While the sugar added (xylose or glucose) had no effect on the aroma, the addition of the following amino acids did produce these significant effects: leucine and isoleucine, cheeselike; phenylalanine, floral; methionine, obnoxious; other amino acids, subtle indescribable aromas. From these results it appears possible to alter bread aroma and flavor by the addition of amino acids to bread formulas. The possibilities for enhancing the aroma of toasted bread by amino acid addition appear promising.
MAILLARD REACTIONS
103
C. MEAT FLAVORS: NATURAL AND ARTIFICIAL Meat is the muscular tissue of common domestic animals, which are considered and used as food for human consumption. Meat is at least a three-phase system consisting of (1) a hydrophilic, but water-insoluble fibrous protein network, (2) a hydrophobic fat deposit held together by membranes, and (3) an aqueous solution containing many soluble low-molecular-weight compounds. Raw meat has very little aroma at room temperature, although it is usually possible to distinguish beef, pork, lamb, and chicken by sniffing. The taste of raw meat, which is not at all palatable to most human beings, can be described as somewhat salty and metallic. Raw meat must be cooked in some fashion in order to develop any organoleptically acceptable odor and flavor. Clearly, then, the unheated tissues must contain precursors which undergo thermally induced chemical reactions; these reactions produce both volatile compounds with desirable aromas and nonvolatile compounds which influence the taste; the combination of these two categories determines the flavor. As we proceed, striking analogies between the development of flavors in the cooking of meat and in the two food systems we have already discussed (cocoa and baked cereal products) will become apparent.
I.
Precursors of Flavor in Meat
In a series of investigations (Hornstein and Crowe, 1960; Wood, 1961; Macy et al., 1964a,b; Wasserman and Gray, 1965; Landmann and Batzer, 1966; Zaika et al., 1968; Wasserman, 1972; Wasserman and Spinelli, 1970; Jarboe and Malbrouk, 1974), it was definitely established that the extraction of minced lean meat (beef, pork, and lamb) with cold water gave solutions that contained salt, lactic acid, glycoproteins, inosinic acid, taurine, glutamine, asparagine, glucose, and some amino acids. Aging, particularly in the case of beef, is important in the development of the various flavor precursors (Wasserman, 1972). Thus, glycogen undergoes glycolysis to lactic acid almost completely within 24 hr after slaughter. Partial autolysis of the proteins and nucleic acids gives an assortment of peptides and amino acids from the former, and a mixture of inosinic acid and its fragments (inosine, hypoxanthine, ribose-5-phosphate, and ribose) from the latter. When the filtered aqueous extract of ground beef is heated, a sequence of aromas is developed, beginning with a faint, bloodlike aroma in the barely warm solution, passing through a phase in the boiling solution which gives off the aroma of boiled beef, and terminating in the hot, dried, brown residue (100150°C) with an aroma resembling broiled steak. When this original filtered
104
JAMES P. DANEHY
aqueous extract was subjected to dialysis against water in cellulosic sausage casings and the low-molecular-weightfraction that passed through the membrane was lyophilized to a white powder and then pyrolyzed, it underwent the typical Maillard browning to produce a strong aroma of broiled meat. This aroma was substantially the same, whether the original lean meat was beef or pork. This may result from the fact that the amino acid contents of beef, pork, and lamb are semiquantitatively rather similar. It appears, then, that there is a general meaty aroma common to beef, pork, and lamb (and probably poultry), attributable to the pyrolysis of the mixture of low-molecular-weight nitrogenous and carbonyl compounds extracted from the lean meat by cold water. But the aromas of roast beef, roast pork, roast lamb, and roast chicken are unmistakably different. The chemical compositions of the muscular fat deposits of these animals differ appreciably, and it is to these lipid components that we must look to account for the specific flavor differences. Heating the carefully separated fat alone does not give a meaty aroma at all, much less an animal-specific one. It is the subsequent reactions of pyrolysis products of nonlipid and lipid components that give the characteristic aromas and flavors of roasted meats (Wasserman and Spinelli, 1972). Since it is precisely at the surface of roasting meat that water concentrations are lowest and temperatures are highest, it is at the meat surface that the flavorand color-generating activities during roasting are most prominent. This situation is analogous to the formation of crust and aroma in bread and other baked cereal products. The same facts also account for the significant difference between the flavor of roasted and boiled meats. 2.
Compounds Associated with the Flavors and Aromas of Cooked Meat
Paralleling the studies of the volatile products of roasted cacao beans and of baked cereal products and using the same techniques, a great deal of effort has gone into the determination of the compounds present in the volatile fractions of cooked meat. Most of these have been concerned only with beef, either roasted or boiled, but chicken has also received appreciable attention (Wilson and Katz, 1972). Several lists of compounds isolated from the volatiles of cooked beef have been published (Hen and Chang, 1970; MacLeod and Coppock, 1976; Chang and Peterson, 1977), both cumulative and newly isolated ones. The totals for chicken (as of 1972) and for beef (as of 1977) are more than 200 each. It must be emphasized again that these are qualitative identifications, not quantitative accountings. These cumulative tables for cooked meat volatiles are very difficult to distinguish from those published somewhat earlier for cocoa volatiles. Indeed, the larger cumulative tables (Wilson and Katz, 1972; MacLeod and Coppock, 1976)
MAILLARD REACTIONS
105
resemble somewhat abridged versions of the Aldrich and Eastman Kodak catalogs of organic chemicals. Meaningful comparisons are hindered by two quite different facts. First, there is usually no hint as to the fraction of the meat or even the fraction of the volatiles that is comprised by a given compound. Second, we are probably getting a great deal of noise with the signals; i.e., there must be many compounds which. even though they have odors, would not be missed if they were absent. We simply cannot believe that more than 200 compounds are required to produce any one of the distinctive roasted food aromas. But the human nose has no difficulty in distinguishing chocolate from roast beef, and the flavor chemist is trying to catch up with this degree of discrimination. Chang and Peterson (1977) have suggested that lactones, acyclic sulfur compounds, nonaromatic heterocyclic compounds containing ring S, N, or 0 atoms, and aromatic heterocyclic compounds containing ring S , N, or 0 atoms may be important contributors to meat flavor, even though none of them alone tastes anything like cooked meat. Wilson er al. (1973) identified 46 sulfur-containing compounds from the volatiles of lean beef pressure-cooked with water at 163 and 182°C. Most of these compounds were thiophene or thiazole derivatives, but acyclic thiols, methyl sulfide, and four disulfides were also present. In addition, as we shall see later, sulfur compounds (especially cysteine) play a key role in manufacturing artificial meat flavors. Tonsbeek er al. (1968, 1969) have isolated 4-hydroxy-5-methyl-2,3-dihydrofuran-3-one and 4-hydroxy-2,5-dimethyl-2,3dihydrofuran-3-one, particularly pungent compounds, from cooked beef. Pyrazines are a particularly important class of flavor compounds, but it was not until 1971 that their presence in beef volatiles was reported (Flament and Ohloff, 1971). and by 1973 a total of 33 had been identified (Mussinan er al., 1973). Recently, Flament er al. (1977) identified several pyrrolo[l ,2-a]pyrazines. Several other papers have contributed additional data on compounds isolated from volatiles of cooked beef (Watanabe and Sato, 1972; Brinkman er al., 1972; Schutte and Koenders, 1972; Shibamoto, 1980a; Hartman er al., 1983; Galt and MacLeod, 1984). Despite the large amount of qualitative if not quantitative data on the chemical composition of the volatiles from cooked meat, no one has yet claimed anything like a duplication of a meat aroma by the combination of the pure chemicals identified in meat aromas. Once more, the parallel with cocoa and baked products is striking. Just one example points up the elusive relationship between chemical compounds and food flavors. Hydrogen sulfide has the odor which does characterize rotten eggs, yet it appears to be a necessary component of meat aromas. Its odor threshold is 10 ppb, but its concentration in freshly cooked chicken is 20 to 100 times greater. It is generally agreed that the aroma of a food is the sensed perception of an extremely complex interaction of many compo-
106
JAMES P. DANEHY
nents, but one reads between the lines the disappointment of some who report new compounds and note that they do not have a meatlike aroma. Chang and Peterson (1977) suggest the justifiable fear that some components may have been decomposed or missed, but their hope is less justifiable that a unique component may still be found which alone or in combination will have a characteristic beef aroma.
3. Artificial Meat Flavors In the light of what has just been presented regarding the chemical origin of the natural flavors of cooked meat, it is not surprising that the heating of semidry mixtures of hydrolyzed vegetable proteins (HVPs) with reducing sugars gives rise to an aroma and flavor somewhat similar to that of cooked meat. HVPs are produced on an industrial scale by hydrolyzing soy protein, wheat gluten, or corn gluten in hot aqueous hydrochloric acid, neutralizing the excess acid with sodium hydroxide, and evaporating to dryness, which yields a mixture of amino acids and sodium chloride (HVPs). The shelf item packets of soup mixes and gravy mixes found in all groceries are practical examples of Maillard technology as testified by the food ingredient disclosures on their labels: HVPs, usually cysteine or cystine, glucose, sometimes arabinose, inosinates or guanylates, and less important adjuncts. The key involvement of organic sulfur compounds in development of meatlike flavors was announced simultaneously in 1960 by several investigators. In what was the earliest paper to describe deliberate attempts to produce aromas useful in foods via Maillard reactions, Kiely et al. (1960) noted that both cysteine and cystine gave meaty odors when heated with reducing sugars. May et al. received several equivalent patents (May and Akroyd, 1959a,b; May and Morton, 1960; May, 1960; Morton et al., 1960) in which they claimed that heating cysteine or cystine with furan or substituted furans, pentoses, or glyceraldehyde gave a meatlike flavor. Hsieh et al. (1980a,b) have experimented with the development of a synthetic meat flavor mixture by using “surface response methodology.” It is precisely to the production of meatlike flavors that the great majority of patents based on the Maillard reaction have been directed. Most of the latter indicate cysteine or cystine as the essential sulfur-containing compound. Other patents claim alternative sources for sulfur, e.g., derivatives of mercaptoacetaldehyde (Broderick and Linteris, 1960), mercaptoalkyl amines (Ohwa, 1972), Sacetylmercaptosuccinic acid (Mosher, 1973), 2,2‘- bis(thieny1)tetrasulfide(Katz er al., 1972), a sulfide (Heyland and Cerise, 1979), and hydrogen sulfide (heated with aqueous xylose without any amino acid (Gunther, 1972). Several patents (Bidmead et al., 1968; Giacino, 1968) claim the contribution
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to meatlike flavors made by thiamine when it is present in the standard pyrolytic mixture. Arnold et al. (1969) have reported on the volatile flavor compounds produced by the thermal degradation of thiamine alone. It is generally agreed that the presence of methionine, the other sulfur-containing amino acid in the flavordeveloping mixture, produces negative and/or undesirable results. Two patents claim that the addition of a ypyrone to meat itself before cooking prevents the development of ‘‘warmed-over’’ flavors (Sato and Hegarty, 1974, 1976). Since the great majority of the patents dealing with the application of Maillard technology to the production of artificial flavors are concerned specifically with meatlike flavors, it is appropriate here to comment on the significance of patents covering “reaction flavors,” as they are known in the trade. During the past 30 years several hundred patents have been granted worldwide for processes and products based on nonenzymatic browning technology. But Chemical Abstracts has not abstracted many more than 100 of them, since they abstract only the first issued of several equivalent patents and list the later ones in a patent concordance. There appear to be about 45 “standard” patents, i.e., patents which specify mixing one or more amino acids with one or more carbonyl compounds and heating, with some or all of the operating conditions given, i.e., temperature, time, water content, pH, and sometimes additives. Although slight changes in initial composition and reaction conditions produce appreciable changes in the flavor and aroma of the reaction products, not one of these patents gives a rigid, controlled specification. The wide ranges of operating conditions and the numerous alternatives offered produce such a complete overlap between these patents that not even an expert chemist and a wily lawyer could distinguish one from the other. These patents surely have very little value, either from the standpoint of the patent holders or from the standpoint of those who might hope to leam by studying them. All the standard patents say substantially the same thing, and they contain little, if anything, that was not fully disclosed in earlier model studies. It is very doubtful if any one of them could be upheld in court in view of the prior published art. Nor is it likely that a holder of one of these patents could sue successfully for infringement, for two quite different reasons. First, the extreme complexity of the composition of the reaction products would make it impossible to determine by examination how they were made. Second, in view of the redundancy of the patents, it would be overwhelmingly difficult to determine whose patent was being infringed. In order to get reproducible results, it is essential to exercise the most precise control at every stage of the process. The basis for this is not provided by any of the patents that characteristically give broad ranges. Maillard reaction products
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arc being manufactured commercially today by detailed proprietary processes that are not described by any patent. The reactions of sugars with amines and ammonia to form glycosylamines and Schiff s bases (Ellis and Honeyman, 1959) and to form nitrogen-containing heterocyclic compounds (Grimmet, 1965; Kort, 1970) have been known for more than a century. Recently, Shibamoto and Bernhard (1976, 1977a) wrote that a systematic investigation of reaction parameters for controlling the composition of pyrazine products and maximizing the yields in the sugar-amine model system could be a key element to understanding the mechanism of pyrazine formation and consequently the characteristics of smoky or roasted flavors of foods. They did reinvestigate the glucose-ammonia-water system. Holding glucose at molar concentration, they systematically varied the concentration of ammonia from 0.1 to 15 M , the temperature from -5 to 160°C (mostly 100"C), and the reaction time from 15 min to 30 days (mostly 2 hr). At lOO"C, increasing the concentration of ammonia increased pyrazine formation up to 8 M NH,, beyond which the pyrazine level remained approximately constant (at -I%, based on glucose). The distribution pattern of the pyrazines was independent of reaction conditions. The principal products, in decreasing order, were 2-methyl-, 2,6-dimethyl-, 2,5-dimethyl-, unsubstituted, 2,3-dimethyl-, and trimethylpyrazine. Based on this systematic study of glucose, Shibamoto and Bernhard (1977b) investigated the heating at 100°C for 2 hr of molar solutions of mannose, galactose, rhamnose, fructose, 2-deoxyglucose, xylose, arabinose, glyceraldehyde, dihydroxyacetone, sorbitol, and glycerol in 8 M aqueous ammonia. Mannose, galactose, and fructose gave -1% total pyrazines (based on sugar), as had glucose. Both pentoses gave slightly higher yields (- 1.2%), but rhamnose gave a surprising 12.5%. 2-Deoxyglucose gave only a 0.5% yield, probably by reason of the blocking of the Amadori rearrangement early in the reaction sequence. Glyceraldehyde gave 0.6% and dihydroxyacetone a 1.2% yield. Sorbitol and glycerol, as might have been expected, gave no pyrazines. It was explicitly stated that no imidazoles or piperazines were detected. Recently, Shibamoto et al. (1979) have given a sobering lesson on the importance of experimental details in determining the outcome of an investigation. In the earlier studies, the cooled solutions had been extracted with four 50-ml portions of H,CCI,. In this study, however, continuous extraction of a heated glucose-ammonia-water system with H,CCI, for 16 hr gave an extract from which 54 compounds were isolated and determined quantitatively. In addition to the pyrazines already reported, pyrroles and imidazoles, including 2-methylimidazole, which makes up 72.5% of the total area of the chromatographic peaks, were found.
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Several studies on model systems have been focused directly on the production of meatlike flavors. Two of these have reported the 41 sulfur-containing compounds and 27 non-sulfur-containing compounds identified when cysteine and xylose are heated together (Ledl and Severin, 1974; Ledl et al., 1973). Another patent reports not only on heating cysteine with xylose, but on heating of a cysteine-xylose-HVP system as well and lists the 24 sulfur-containing compounds identified in the reaction mixture (Mussinan and Katz, 1973). Shibamoto and Russell ( 1976) heated an aqueous glucose-ammonia-hydrogen sulfide solution at 100°C for 2 hr. Of the 34 major components identified, 2methylthiophene accounted for 24.9% of the area of the chromatographic peaks; ethyl sulfide, thiophene, furfural, and 2-acetylfuran each accounted for 10-1 1%; methyl sulfide and 2,5dimethylthiophene, -7% each. The reaction mixture as a whole was deemed by sensory panel evaluation to have a cooked beef odor. Once more, although the distributions are expressed quantitatively, there is no information on the yields of those interesting compounds based on glucose, ammonia, and hydrogen sulfide. However, we noted earlier that in their studies of the systems, carbohydrates-ammonia, Shibamoto and Russell found that the amounts of total pyrazines produced, based on the sugars, were in the range of 1-2%. Wilson’s review (1975) of thermally produced imitation meat flavors, though more than 10 years old, is still well worthwhile consulting. Shibamoto (1980a) lists 161 heterocyclic compounds alone which have been found in cooked meats.
D. OTHER FOOD SYSTEMS 1. CofSee
The great importance of coffee has prompted a large amount of research and development involving all aspects of coffee aroma and flavor, including the determination of the aroma constituents and the formulation of concentrated coffee flavors. When Stoll ef af. (1967b; Goldman ef al., 1967) published the results of their monumental study in 1967, they identified 240 volatile constituents, 174 of them for the first time, but they cited almost 150 previously published reports. The roasting of the green inedible coffee beans to produce a fragrant brown product invites comparison with cocoa, but the differences are more prominent than the similarities. The cacao beans are imbedded in a mucilaginous pulp and undergo a spontaneous fermentation as soon as the pod is open. The coffee bean is really a berry, and fermentation plays no significant role in its processing. While chocolate and the cocoa derived from the cacao bean are highly nutritious
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product^,^ the solubles extracted in a cup of coffee, while very flavorful, are devoid of nutritive value. Both cacao beans and green coffee beans contain relatively small amounts of reducing sugars, but appreciable amounts of sucrose which have quite different fates in the two different beans. During the fermentation that follows the opening of the cacao pods (see Section III,A), the rise in temperature kills the seeds so that the invertase within the seeds transforms the sucrose almost completely into glucose and fructose. Feldman et al. (1969) noted that during the roasting of coffee, the sucrose is quickly pyrolyzed, falling from 4.6% (dry basis) for Colombian green beans or 5.5% for Santos to 0.2-0.3% for a medium roast and less than 0.1% for a dark roast. Feldman also states that the major water-soluble polysaccharide of green coffee beans is arabinogalactan, and that arabinose practically disappears during the roasting of the water-soluble fraction. It may well be that the arabinose residues of this polymer are responsible for Maillard reactions that generate aroma constituents during the roasting of coffee, for evidence from other systems indicates clearly that sucrose is noncontributory (cf. the cocoa and baking systems as well as the model studies). Despite the probability that Maillard reactions do play a role in coffee flavor, they have attracted remarkably little attention from the investigators of coffee flavor (Pokorny et al., 1974, 1975). For this reason we must necessarily devote little space to coffee in this report. But much effort is doubtless continuing in the field of coffee research. Efforts are being made to improve extracts and to improve coffee flavors, and new compounds are still being identified in the the stavolatile fraction. Kung (1974) has isolated 3-hydroxy-3-pentene-2-one, ble, enol form of 2,3-pentanedione, which has a buttery, caramel aroma. It has been known for a long time, of course, that phenolics are important constituents of coffee, e.g., up to 7.5% of chlorogenic acid in green beans, almost half of which survives in roasted coffee and is extracted into the brew. 0 II
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46