ANTIMICROBIALS IN FOOD
Third Edition

FOOD SCIENCE AND TECHNOLOGY A Series of Monographs, Textbooks, and Reference Books
Editorial Advisory Board
Gustavo V. Barbosa-Cánovas Washington State University–Pullman P. Michael Davidson University of Tennessee–Knoxville Mark Dreher McNeil Nutritionals, New Brunswick, NJ Richard W. Hartel University of Wisconsin–Madison Y. H. Hui Science Technology System, West Sacramento, CA Lekh R. Juneja Taiyo Kagaku Company, Japan Marcus Karel Massachusetts Institute of Technology Ronald G. Labbe University of Massachusetts–Amherst Daryl B. Lund University of Wisconsin–Madison David B. Min The Ohio State University Leo M. L. Nollet Hogeschool Gent, Belgium Seppo Salminen University of Turku, Finland James L. Steele University of Wisconsin–Madison John H. Thorngate III Allied Domecq Technical Services, Napa, CA Pieter Walstra Wageningen University, The Netherlands John R. Whitaker University of California–Davis Rickey Y. Yada University of Guelph, Canada

76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91.

Food Chemistry: Third Edition, edited by Owen R. Fennema Handbook of Food Analysis: Volumes 1 and 2, edited by Leo M. L. Nollet Computerized Control Systems in the Food Industry, edited by Gauri S. Mittal Techniques for Analyzing Food Aroma, edited by Ray Marsili Food Proteins and Their Applications, edited by Srinivasan Damodaran and Alain Paraf Food Emulsions: Third Edition, Revised and Expanded, edited by Stig E. Friberg and Kåre Larsson Nonthermal Preservation of Foods, Gustavo V. Barbosa-Cánovas, Usha R. Pothakamury, Enrique Palou, and Barry G. Swanson Milk and Dairy Product Technology, Edgar Spreer Applied Dairy Microbiology, edited by Elmer H. Marth and James L. Steele Lactic Acid Bacteria: Microbiology and Functional Aspects, Second Edition, Revised and Expanded, edited by Seppo Salminen and Atte von Wright Handbook of Vegetable Science and Technology: Production, Composition, Storage, and Processing, edited by D. K. Salunkhe and S. S. Kadam Polysaccharide Association Structures in Food, edited by Reginald H. Walter Food Lipids: Chemistry, Nutrition, and Biotechnology, edited by Casimir C. Akoh and David B. Min Spice Science and Technology, Kenji Hirasa and Mitsuo Takemasa Dairy Technology: Principles of Milk Properties and Processes, P. Walstra, T. J. Geurts, A. Noomen, A. Jellema, and M. A. J. S. van Boekel Coloring of Food, Drugs, and Cosmetics, Gisbert Otterstätter

92. Listeria, Listeriosis, and Food Safety: Second Edition, Revised and Expanded, edited by Elliot T. Ryser and Elmer H. Marth 93. Complex Carbohydrates in Foods, edited by Susan Sungsoo Cho, Leon Prosky, and Mark Dreher 94. Handbook of Food Preservation, edited by M. Shafiur Rahman 95. International Food Safety Handbook: Science, International Regulation, and Control, edited by Kees van der Heijden, Maged Younes, Lawrence Fishbein, and Sanford Miller 96. Fatty Acids in Foods and Their Health Implications: Second Edition, Revised and Expanded, edited by Ching Kuang Chow 97. Seafood Enzymes: Utilization and Influence on Postharvest Seafood Quality, edited by Norman F. Haard and Benjamin K. Simpson 98. Safe Handling of Foods, edited by Jeffrey M. Farber and Ewen C. D. Todd 99. Handbook of Cereal Science and Technology: Second Edition, Revised and Expanded, edited by Karel Kulp and Joseph G. Ponte, Jr. 100. Food Analysis by HPLC: Second Edition, Revised and Expanded, edited by Leo M. L. Nollet 101. Surimi and Surimi Seafood, edited by Jae W. Park 102. Drug Residues in Foods: Pharmacology, Food Safety, and Analysis, Nickos A. Botsoglou and Dimitrios J. Fletouris 103. Seafood and Freshwater Toxins: Pharmacology, Physiology, and Detection, edited by Luis M. Botana 104. Handbook of Nutrition and Diet, Babasaheb B. Desai 105. Nondestructive Food Evaluation: Techniques to Analyze Properties and Quality, edited by Sundaram Gunasekaran 106. Green Tea: Health Benefits and Applications, Yukihiko Hara 107. Food Processing Operations Modeling: Design and Analysis, edited by Joseph Irudayaraj 108. Wine Microbiology: Science and Technology, Claudio Delfini and Joseph V. Formica 109. Handbook of Microwave Technology for Food Applications, edited by Ashim K. Datta and Ramaswamy C. Anantheswaran 110. Applied Dairy Microbiology: Second Edition, Revised and Expanded, edited by Elmer H. Marth and James L. Steele 111. Transport Properties of Foods, George D. Saravacos and Zacharias B. Maroulis 112. Alternative Sweeteners: Third Edition, Revised and Expanded, edited by Lyn O’Brien Nabors 113. Handbook of Dietary Fiber, edited by Susan Sungsoo Cho and Mark L. Dreher 114. Control of Foodborne Microorganisms, edited by Vijay K. Juneja and John N. Sofos 115. Flavor, Fragrance, and Odor Analysis, edited by Ray Marsili 116. Food Additives: Second Edition, Revised and Expanded, edited by A. Larry Branen, P. Michael Davidson, Seppo Salminen, and John H. Thorngate, III 117. Food Lipids: Chemistry, Nutrition, and Biotechnology: Second Edition, Revised and Expanded, edited by Casimir C. Akoh and David B. Min 118. Food Protein Analysis: Quantitative Effects on Processing, R. K. Owusu- Apenten

119. Handbook of Food Toxicology, S. S. Deshpande 120. Food Plant Sanitation, edited by Y. H. Hui, Bernard L. Bruinsma, J. Richard Gorham, Wai-Kit Nip, Phillip S. Tong, and Phil Ventresca 121. Physical Chemistry of Foods, Pieter Walstra

122. Handbook of Food Enzymology, edited by John R. Whitaker, Alphons G. J. Voragen, and Dominic W. S. Wong 123. Postharvest Physiology and Pathology of Vegetables: Second Edition, Revised and Expanded, edited by Jerry A. Bartz and Jeffrey K. Brecht 124. Characterization of Cereals and Flours: Properties, Analysis, and Applications, edited by Gönül Kaletunç and Kenneth J. Breslauer 125. International Handbook of Foodborne Pathogens, edited by Marianne D. Miliotis and Jeffrey W. Bier 126. Food Process Design, Zacharias B. Maroulis and George D. Saravacos 127. Handbook of Dough Fermentations, edited by Karel Kulp and Klaus Lorenz 128. Extraction Optimization in Food Engineering, edited by Constantina Tzia and George Liadakis 129. Physical Properties of Food Preservation: Second Edition, Revised and Expanded, Marcus Karel and Daryl B. Lund 130. Handbook of Vegetable Preservation and Processing, edited by Y. H. Hui, Sue Ghazala, Dee M. Graham, K. D. Murrell, and Wai-Kit Nip 131. Handbook of Flavor Characterization: Sensory Analysis, Chemistry, and Physiology, edited by Kathryn Deibler and Jeannine Delwiche 132. Food Emulsions: Fourth Edition, Revised and Expanded, edited by Stig E. Friberg, Kare Larsson, and Johan Sjoblom 133. Handbook of Frozen Foods, edited by Y. H. Hui, Paul Cornillon, Isabel Guerrero Legarret, Miang H. Lim, K. D. Murrell, and Wai-Kit Nip 134. Handbook of Food and Beverage Fermentation Technology, edited by Y. H. Hui, Lisbeth Meunier-Goddik, Ase Solvejg Hansen, Jytte Josephsen, Wai-Kit Nip, Peggy S. Stanfield, and Fidel Toldrá 135. Genetic Variation in Taste Sensitivity, edited by John Prescott and Beverly J. Tepper 136. Industrialization of Indigenous Fermented Foods: Second Edition, Revised and Expanded, edited by Keith H. Steinkraus 137. Vitamin E: Food Chemistry, Composition, and Analysis, Ronald Eitenmiller and Junsoo Lee 138. Handbook of Food Analysis: Second Edition, Revised and Expanded, Volumes 1, 2, and 3, edited by Leo M. L. Nollet 139. Lactic Acid Bacteria: Microbiological and Functional Aspects: Third Edition, Revised and Expanded, edited by Seppo Salminen, Atte von Wright, and Arthur Ouwehand 140. Fat Crystal Networks, Alejandro G. Marangoni 141. Novel Food Processing Technologies, edited by Gustavo V. Barbosa-Cánovas, M. Soledad Tapia, and M. Pilar Cano 142. Surimi and Surimi Seafood: Second Edition, edited by Jae W. Park 143. Food Plant Design, edited by Antonio Lopez-Gomez; Gustavo V. Barbosa-Cánovas 144. Engineering Properties of Foods: Third Edition, edited by M. A. Rao, Syed S.H. Rizvi, and Ashim K. Datta 145. Antimicrobials in Food: Third Edition, edited by P. Michael Davidson, John N. Sofos, and A. L. Branen

ANTIMICROBIALS IN FOOD
Third Edition

Edited by

P. Michael Davidson John N. Sofos A. L. Branen

Boca Raton London New York Singapore

A CRC title, part of the Taylor & Francis imprint, a member of the Taylor & Francis Group, the academic division of T&F Informa plc.

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Preface
The consequences of unintended microbial growth in foods are hazards due to the presence of pathogenic microorganisms or economic losses due to spoilage microorganisms. Preservation technologies are designed to protect foods from the effects of microorganisms and inherent deterioration. Microorganisms in foods may be inhibited or inactivated by physical methods (e.g., heat, cold, reduced water activity) or through application of antimicrobial compounds. Food antimicrobials, including chemical sanitizers, may be broadly defined as chemical compounds present in or added to foods, food packaging, food contact surfaces, or food processing environments that inhibit the growth of, or inactivate, pathogenic or spoilage microorganisms. Historically, the primary function of food antimicrobials has been to prolong shelf life and preserve quality through the inhibition of spoilage microorganisms. In the past 10 to 15 years, however, antimicrobials have been increasingly utilized as a primary intervention for the inhibition or inactivation of pathogenic microorganisms in foods. This function becomes increasingly important as food processors search for more and better tools to improve food safety. Antimicrobials continue to be one of the most important classes of food additives. Research on antimicrobials, especially naturally occurring compounds, has increased dramatically in the past 10 to 15 years. The primary incentive for searching for effective antimicrobials among naturally occurring compounds is to expand the spectrum of antimicrobial activity over that of the traditional, regulatory-approved substances. Most of the traditional food antimicrobials have limited application due to pH or food component interactions. Interest in natural antimicrobials is also driven by the fact that international regulatory agencies are generally very strict as to requirements for toxicological evaluation of novel direct food antimicrobials. An argument often used to justify natural antimicrobials is that they will produce “green” labels (i.e., with few or no “synthetic” additives in the ingredient list). However, this justification may lead consumers to the mistaken belief that antimicrobial food additives currently in use are potentially toxic and should be avoided. In short, natural antimicrobials have excellent potential but likely will not produce miracles. This has not stopped researchers from continuing to look for the “perfect” food antimicrobial. However, a single compound that is effective against all microorganisms in all storage situations and in all foods likely does not exist. More research is needed on the effectiveness of antimicrobial combinations and antimicrobials in combination with physical methods (e.g., hurdle technology) that are effective against different groups of microorganisms. Combinations could well be the ideal antimicrobial for which everyone is searching. It has been approximately 12 years since the second edition of Antimicrobials in Foods was published. In that time, many changes have taken place in the field of food microbiology and the research area of food antimicrobials. At the time of the second edition, major outbreaks of Escherichia coli O157:H7 and Listeria monocytogenes had not occurred, consumer and regulatory demands for improved food safety were only beginning, and use of naturally occurring antimicrobials was in its infancy. At the time of the second edition, lysozyme, lactoferrin, ozone, and several other compounds were not approved for use in or on foods in the United States. Since the time of the second edition, a great deal of progress has been made on determining the spectrum of action, environmental effects on activity, and mechanisms of action of a number of naturally occurring antimicrobials. Because of the many changes since the second edition of Antimicrobials in Foods, considerable revisions have been made for this third edition. As previously, one thing that has not changed is the excellent international reputations of the authors of each chapter. Some of the authors are new,

but, as in the second edition, all are well-recognized experts on their topics. The format for each chapter varies somewhat depending on the areas each author wishes to emphasize. In general, chapters on specific antimicrobials include information on spectrum of activity, application to foods, mechanisms or potential mechanisms of action, regulations, toxicological aspects, and assay in foods. In the third edition, five new chapters have been added. New chapters on specific antimicrobials include those on lysozymes, naturally occurring antimicrobials from animal sources, and naturally occurring antimicrobials from plant sources. In addition, chapters have been added on hurdle technology and mechanisms of action, resistance, and stress adaptation. All existing chapters were extensively revised to reflect research that has taken place since 1993, the publication date of the second edition. The editors would like to thank the chapter authors for their dedication and hard work in producing their excellent works. We sincerely hope that these discussions will continue to be useful for industrial, academic, and regulatory research scientists, technical advisors, industry consultants, regulators, consumers, and consumer advocates regarding the potential value of antimicrobials in the food supply. P. Michael Davidson John N. Sofos Larry Branen

Editors
P. Michael Davidson is a professor of food microbiology at the University of Tennessee, Knoxville. Serving on the editorial board of several journals and as scientific coeditor of the Journal of Food Protection, Dr. Davidson is the author or coauthor of more than 150 journal articles, book chapters, and abstracts. He is a Fellow of the American Academy of Microbiology and the Institute of Food Technologists (IFT) and is a member of the American Society for Microbiology (ASM), the Society for Applied Microbiology, and the International Association of Food Protection. He served as chair of the food microbiology divisions of both the ASM and IFT. Dr. Davidson received the B.S. degree (1972) from the University of Idaho, Moscow, the M.S. degree (1977) from the University of Minnesota, Minneapolis-St. Paul, and the Ph.D. degree (1979) in food science and technology from Washington State University, Pullman. John N. Sofos, Ph.D., is professor of food microbiology at Colorado State University, Fort Collins. Serving on the U.S. National Advisory Committee on Microbiological Criteria for Foods and as a scientific coeditor of the Journal of Food Protection, Dr. Sofos has authored or coauthored more than 220 referred papers, book chapters, books, and more than 250 abstracts. He is a Fellow of the American Academy of Microbiology and of the Institute of Food Technologists. He also has received research awards from the American Meat Science Association and the American Society of Animal Science, the Educator Award from the International Association for Food Protection, and the United States Department of Agriculture Secretary’s Honor Award for Superior Service for studies on bacterial pathogen control. Dr. Sofos received a B.S. degree from the Aristotle University of Thessaloniki, Greece, and M.S. and Ph.D. degrees from the University of Minnesota. Alfred Larry Branen is associate vice president for research and outreach and associate director of the Center for Advanced Microelectronic and Biomolecular Research (CAMBR) at the University of Idaho Research Park in Post Falls. Formerly dean of the College of Agricultural and Life Sciences at the University of Idaho and head of the Department of Food Science and Technology at the University of Nebraska, Lincoln, Dr. Branen is the author or coauthor of more than 50 professional papers and coeditor, with P. Michael Davidson, John Thorngate, and Seppo Salminen, of Food Additives. A member of the Institute of Food Technologists and the former chair of its Toxicology Division, Dr. Branen received the B.S. degree (1967) in food science from the University of Idaho, Moscow, and the Ph.D. degree (1970) in food science from Purdue University, West Lafayette, Indiana.

Contributors
Stella M. Alzamora Departamento de Industrias Ciudad Universitaria Universidad de Buenos Aires Buenos Aires, Argentina A.L. Branen College of Agriculture and Life Sciences University of Idaho-Coeur d’Alene Coeur d’Alene, Idaho Scott L. Burnett Ecolab Inc. Research Center Saint Paul, Minnesota Francis F. Busta Department Food Science and Nutrition University of Minnesota St. Paul, Minnesota Haiqiang Chen Department of Animal and Food Sciences University of Delaware Newark, Delaware John R. Chipley Research and Development Division U.S. Tobacco Company Nashville, Tennessee Bruce R. Cords Ecolab Inc. Research Center Saint Paul, Minnesota P. Michael Davidson Department of Food Science and Technology University of Tennessee Knoxville, Tennessee Joss Delves-Broughton Aplin and Barrett, Danisco Cultor United Kingdom Craig H. Doan Heinz Frozen Food Company Ontario, Oregon Stephanie Doores Department of Food Science Pennsylvania State University University Park, Pennsylvania Matthew Finley Ecolab Inc. Research Center Saint Paul, Minnesota David A. Golden Department of Food Science and Technology University of Tennessee Knoxville, Tennessee Grahame W. Gould University of Leeds Bedford, United Kingdom John Hilgren Ecolab Inc. Research Center Saint Paul, Minnesota Dallas G. Hoover Department of Animal and Food Sciences University of Delaware Newark, Delaware Eric A. Johnson Food Research Institute University of Wisconsin Madison, Wisconsin Jon J. Kabara Med-Chem Labs Longboat Key, Florida

Ann E. Larson Food Research Institute University of Wisconsin Madison, Wisconsin Stanley E. Katz Department of Biochemistry and Microbiology Cook College Rutgers-The State University of New Jersey New Brunswick, New Jersey Lothar Leistner Federal Centre for Meat Research Kulmbach, Germany Aurelio López-Malo Vigil Departamento de Ingeniería Quimica Universidad de las Américas Puebla Escuela de Ingeniería Cholula, Puebla, Mexico Douglas L. Marshall Department of Food Science & Technology Mississippi State University Mississippi State, Mississippi Joshua Magnuson Ecolab Inc. Research Center Saint Paul, Minnesota Cornelius S. Ough University of California (retired) Davis, California Enrique Palou Departamento de Ingenieria Quimica Universidad de las Americas Puebla Escuela de Ingenieria Cholula, Puebla, Mexico Mickey E. Parish Citrus Research and Education Center University of Florida Lake Alfred, Florida A.D. Russell School of Pharmacy Cardiff University Cardiff, Wales, United Kingdom

Julie Seiter Oakland Community College Farmington Hills, Michigan Leora A. Shelef Department of Nutrition and Food Science Wayne State University Detroit, Michigan Panos N. Skandamis Department of Animal Science Colorado State University Fort Collins, Colorado John N. Sofos Department of Animal Science Colorado State University Ft. Collins, Colorado Jarret D. Stopforth Department of Animal Science Colorado State University Fort Collins, Colorado Linda V. Thomas Aplin and Barrett, Danisco Cultor United Kingdom R. Bruce Tompkin Product Development Laboratory ConAgra Refrigerated Prepared Foods (Retired) Downers Grove, Illinois Paula Marie L. Ward Department of Biochemistry and Microbiology Cook College Rutgers-The State Univ. of New Jersey New Brunswick, New Jersey Lilian Were Department of Food Science and Nutrition Chapman University Orange, California Randy W. Worobo Department of Food Science and Technology Cornell University Geneva, New York

Contents
Chapter 1 Food Antimicrobials — An Introduction...........................................................................................1 P. Michael Davidson and A.L. Branen Chapter 2 Sodium Benzoate and Benzoic Acid ...............................................................................................11 John R. Chipley Chapter 3 Sorbic Acid and Sorbates.................................................................................................................49 Jarret D. Stopforth, John N. Sofos, and Francis F. Busta Chapter 4 Organic Acids...................................................................................................................................91 Stephanie Doores Chapter 5 Sulfur Dioxide and Sulfites ...........................................................................................................143 Cornelius S. Ough and Lilian Were Chapter 6 Nitrite .............................................................................................................................................169 R. Bruce Tompkin Chapter 7 Nisin ...............................................................................................................................................237 Linda V. Thomas and Joss Delves-Broughton Chapter 8 Natamycin ......................................................................................................................................275 Joss Delves-Broughton, Linda V. Thomas, Craig H. Doan, and P. Michael Davidson Chapter 9 Parabens..........................................................................................................................................291 P. Michael Davidson Chapter 10 Dimethyl Dicarbonate and Diethyl Dicarbonate ...........................................................................305 David A. Golden, Randy W. Worobo, and Cornelius S. Ough

............................................. Marshall Chapter 12 Lysozyme ........ P...................................................... Burnett. Sofos Chapter 16 Sanitizers: Halogens.................................................Chapter 11 Medium-Chain Fatty Acids and Esters........... Ward Chapter 19 Update on Hurdle Technology Approaches to Food Preservation.................... Alzamora Chapter 15 Naturally Occuring Compounds — Animal Sources.... Parish.......... and Stella M.. and John N... Scott L.................................. Shelef and Julie Seiter Chapter 18 Antibiotic Residues in Foods and Their Significance.. Katz and Paula Marie L... Gould Chapter 20 Mechanisms of Action.............. Cords...633 A.... and Stress Adaptation ..................................... Resistance...573 Leora A............... and Peroxides ............................................................................ and P.................................... Panos N.......... Russell Chapter 21 Methods for Activity Assay and Evaluation of Results ........................................ Larson Chapter 13 Bacteriocins with Potential for Use in Foods .......................................389 Dallas G.........D........... Kabara and Douglas L.... Johnson and Ann E............. Enrique Palou................ Skandamis........................... Matthew Finley.................................................453 Jarret D.....................621 Lothar Leistner and Grahame W...................................507 Bruce R.................... John Hilgren........................................... and Joshua Magnuson Chapter 17 Indirect and Miscellaneous Antimicrobials .........................659 Aurelio López-Malo Vigil....... Enrique Palou.599 Stanley E............................................................. Mickey E. Hoover and Haiqiang Chen Chapter 14 Naturally Occurring Compounds — Plant Sources ........... Michael Davidson............361 Eric A....327 Jon J.............. Stopforth............................................429 Aurelio López-Malo Vigil........ Michael Davidson Index ........................ Surface-Active Agents...681 ..........

................................................................................ 2002)........ Responsibility for determining if risks outweigh benefits for any particular additive is 1 .............................. 2002)....................................... The focus of this book is food additives that contribute to preservation.........................................................7 Toxicologic Safety ........................ and it is doubtful that many additional ones will be approved in the future................................... or processing aids (Branen and Haggerty..............................4 Physicochemical Properties of the Antimicrobial .................................................................5 Process Factors............................8 Sensory Effects ........................... In very few cases are additives totally devoid of risk........2 Selection of Antimicrobials ....................................7 Sanitation .............................8 Resistance Development ....9 References ............ addition or replacement of flavor................................................ For example........................................................ Balancing the risks versus the benefits is not easy and requires extensive research about the usefulness and toxicologic safety of the additives in question................. Few new additives have been approved by regulatory agencies in recent years.....................................................................10 ROLE OF ADDITIVES IN FOOD Food additives may be classified by one of six primary functions they serve: preservation.............................................................................................................................................. addition or replacement of color............ it is apparent that such risk must be balanced against the benefits of the use of such additives.......................L.......................... improvement in texture.......................................... reduction in the number of food poisoning cases or reduced food loss resulting from spoilage are benefits provided by antimicrobial additives......... Although there is no question that the risk from an additive must be minimal.................... although some food antimicrobials also contribute to color stability (sulfites.................................................................................. an assessment of the degree of acceptable risk is often required............................................ nitrites) or flavor (nitrites and certain organic acids)................ Readers are referred to Food Additives for a more complete treatise of all additives (Branen et al......................................................................................................................................7 Labeling .........8 Activity Validation Methods ...............................................................................1 Definition and Function of Chemical Food Preservatives ........... Despite the recognized requirement for food additives............................................................3 Antimicrobial Spectrum............................................................................................................................................................................................................... Branen Role of Additives in Food......1 CONTENTS Food Antimicrobials — An Introduction P..................................................................................................6 Considerations in the Use of Food Antimicrobials .........................8 Economics of Use..................... improvement in nutritional value........9 Future of Antimicrobials...... thus.... their toxicologic safety continues to be evaluated and is often questioned......................................................................5 Food-Related Factors ......................................... Michael Davidson and A....

and animal sources. to prevent enzymatic and nonenzymatic browning. plant. In addition. Examples include nitrite inhibition of Clostridium botulinum in cured meats and. products seldom are grown and sold locally as in the past. One of the reasons for increased use of chemical preservatives has been the change in the ways foods are produced and marketed. legislators. and heating have always been the primary methods used to prolong the shelf life of food products. and lactate and diacetate to inactivate Listeria monocytogenes in processed meats (65FR17128. such as nisin. few food antimicrobials have been used exclusively to control the growth of specific foodborne pathogens. more recently. because food antimicrobials are generally bacteriostatic or fungistatic.S.1 and 1. when added to food. The former are approved for use in foods by many international regulatory agencies (Tables 1. lactoferrin.. Those preservatives used to prevent chemical deterioration include antioxidants. Antimicrobials may be classified as traditional or naturally occurring (Davidson. Food antimicrobials are classified as “preservatives. nisin and lysozyme against Clostridium botulinum in pasteurized processed cheese. Today. To accomplish the long-term shelf life necessary for such a marketing system. most others have seen extensive use only recently. or chemicals applied for their insecticidal or herbicidal properties. nitrites. and vitamins. have been in use for many years.” The traditional function of food antimicrobials is to prolong shelf life and preserve quality through inhibition of spoilage microorganisms.” The FDA defines antimicrobial agents (21CFR 170. 21CFR 101. and lysozyme. such as salt. lipids. 2001). Although some improvements have been made using packaging and processing systems to preserve foods without chemicals. to be “free” of foodborne pathogens. 2002). Several months or years may elapse from the time food is produced until it is consumed. food antimicrobials are not able to conceal spoilage of a food product (i. to prevent texture changes. but does not include common salt. mold and rope inhibitors. fermenting. preservatives are used to prevent or retard both chemical and biological deterioration of foods. Naturally occurring antimicrobials include compounds from microbial. antimicrobial preservatives play a significant role in protecting the food supply (Davidson et al. for the most part. However. and sulfites. foods produced in one area are often shipped to another area for processing and to several other areas for distribution. including fungistats. to prevent autoxidation of pigments. They are. chemicals have been added to preserve freshly harvested foods for later use. antibrowning compounds. with rare exceptions.e. sugars. It is essential that all involved in the decision process be acutely aware of the risks and benefits of all additives. In addition. food processors. Whereas some chemical food preservatives. It is important to note that. selected organic acids as spray sanitizers against pathogens on beef carcasses. substances added to food by direct exposure thereof to wood smoke. because of changes in the marketing for foods to a more global system. they will not preserve a food indefinitely.2 Antimicrobials in Food with scientists. spices. Today. cooling.2). natamycin.” Therefore. or oils extracted from spices. multiple effective means of preservation are often required. 2003).” Chemical preservatives are defined by the U. Surprisingly. are approved by regulatory agencies in some countries for application . DEFINITION AND FUNCTION OF CHEMICAL FOOD PRESERVATIVES Since prehistoric times. the food remains wholesome during its extended shelf life). only proposed for use in foods. Those additives used to prevent biological deterioration are termed “antimicrobials. and antistaling compounds. A few. flavors.. vinegars. Drying.22(a)(5)) as “any chemical that. consumers expect foods to be readily available yearround. March 31. antimicrobials have been used increasingly as a primary intervention for inhibition or inactivation of pathogenic microorganisms in foods (Davidson and Zivanovic.3(o)(2)) as “substances used to preserve food by preventing growth of microorganisms and subsequent spoilage. and consumers. Food and Drug Administration (FDA. and to have a reasonably long shelf life. tends to prevent or retard deterioration thereof. regulatory personnel. 2000).

lactates Lactoferrin Lysozyme Microbial Target Yeasts.1768 GRAS Notice No. SELECTION OF ANTIMICROBIALS It is not an easy process to select the appropriate preservation system for a particular food product. potato products. cooked meat.1670. wines Fruits. 182.3795 Various For meat products.3225. fermented foods Meats Cheese. Section 424. 2000). and then the possible preservation systems must be evaluated via model studies and studies in the food product in question. (1998). 184. Food and Drug Administration) Food Antimicrobialsa (Title 21 of the Code of Federal Regulations) Compound(s) Acetic acid.1754. . GRN 000065 Nitrite. fruit products. beverages. other bacteria 172. methyl.133 184.S.1185. bacteria Primary Food Applications Baked goods.6197. Source: Davidson and Harrison.160.1 and 1.177 184. sorbates Sulfites a Clostridium botulinum Yeasts. other bacteria Natamycin Nisin Molds Clostridium botulinum. 184. 184. 184. acetates. Title 9. molds Yeasts Bacteria Bacteria Clostridium botulinum.22. GRAS Notice No. wines 184. dry sausage CFR Designation 184. benzoates Dimethyl dicarbonate Lactic acid. heptyl) of p-hydroxybenzoic acid) Propionic acid. fruit products. 184. 2002 to foods (Tables 1. and Davidson and Zivanovic (2003). The target pathogen or spoilage microorganisms must be identified first. bacteria Yeasts.21 and 424. 172. Sofos et al.175.145 Molds Yeasts.1081. molds.155 184.1721.1784 182. 182. fats/oils. 184. and poultry products Cured meats Beverages. confections. 172.1207. 182. 184. Generally. baked goods. 172.1021. casings for frankfurters. molds Bakery products. cooked meat. margarine Beverages Meats.1061.1005. dairy products Most foods. nitrate Parabens (alkyl esters (propyl. 184. propionates Sorbic acid.130 184. 172. GRN 000064 Yeasts. meats. dairy products.1221. food antimicrobials permitted by USDA Food Safety and Inspection Service are listed in the Code of Federal Regulations. 184.1490. dehydroacetic acid Benzoic acid.1538.170.1550. and poultry products Cheese Cheese. molds.1639.3089.1733 172.2). bacteria (Gram positive) 172. diacetates.Food Antimicrobials — An Introduction 3 TABLE 1. a combination of chemical preservatives and other preservation methods is needed (Leistner. casings for frankfurters. condiments. GRN 000067 184. Extensive reviews on natural antimicrobials may be found in Dillon and Board (1994). 172. GRAS Notice No.1 Traditional or Regulatory-Approved (U. 184. Naidu (2000). syrups.3640. 182. sauces Beverages.

although desired. species. including the spectrum of antimicrobial activity. Few chemicals have the ability to inhibit several different types.. Source: Verbruggen. Quite often.4 Antimicrobials in Food TABLE 1. which have the ability to screen the antimicrobials because of the outer membrane lipopolysaccharide layer. a broad spectrum of activity.g. and Gram reaction (positive vs. The antimicrobial spectrum should involve an evaluation of the compound against various types of microorganisms (e. “Methods for Activity of Assay and Evaluation of Results. yeasts. the chemical properties of the antimicrobial. 2002. ANTIMICROBIAL SPECTRUM The initial selection of the antimicrobial is normally based on an assessment of the overall microbial spectrum of the chemical in question. is not easy to achieve. molds) and forms of those microorganisms (vegetative cells vs. negative) can have dramatic influences on apparent activity. Even species. strain. and the type of preservation or processing and storage systems used. The antimicrobial spectrum of a compound is generally determined by following the growth of organisms in the presence of various concentrations of the antimicrobial. For example. however. the physicochemical properties and composition of the food product in question. Selection of the proper antimicrobial depends on several primary factors. Each of these factors is discussed in detail. or strains of microorganism.2 Regulatory-Approved Food Antimicrobial Designations in the European Union (E Numbers) and in Codex Alimentarius (INS Numbers) Compound Sorbic acid K sorbate Ca sorbate Benzoic acid Na benzoate K benzoate Ca benzoate Ethyl paraben Na ethyl paraben Propyl paraben Na propyl paraben Methyl paraben Na methyl paraben Sulfur dioxide Na sulfite Na hydrogen sulfite Na metabisulfite K metabisulfite Ca sulfite Ca hydrogen sulfite K hydrogen sulfite Nisin a E or INS Number 200 202 203 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 226 227 228 234 Compound Natamycin DMDCa K nitrite Na nitrite Na nitrate K nitrate Acetic acid K acetate Na acetate Na diacetate Ca acetate Lactic acid Propionic acid Na propionate Ca propionate K propionate Boric acid Na tetraborate Na lactate K lactate Ca lactate E or INS Number 235 242 249 250 251 252 260 261 262 262 263 270 280 281 282 283 284 285 325 326 327 Dimethyl dicarbonate. does growth in a synthetic . bacteria.” Seldom. spores). the activity of very hydrophobic antimicrobials may be limited against Gram-negative bacteria. Appropriate methods for in vitro evaluation of the activity of food antimicrobials are described in Chapter 21.

the mode of action. However. -odors.. which contributes to an off-flavor in a food product. flavor. Hydrophilic properties appear necessary to allow water solubility where microbial growth occurs. are vaporized. Interaction with lipids probably results in the greatest interference with antimicrobial activity. Other factors leading to reduced effectiveness among food antimicrobials are food component interactions. The boiling point of a compound can also directly influence the activity of an antimicrobial. and other food additives can result in an overall decrease in the activity of the antimicrobial compound. and -colors. however. and significant losses occur during a cooking process. The balance needed in a synthetic medium. Thus. for example. owing to polarity of the food components. Most food antimicrobials are amphiphilic. Certain phenolic compounds. The final confirmatory test for the antimicrobial spectrum and activity must be carried out in a food product because food components and properties can dramatically alter the overall spectrum and activity of the antimicrobial. like emulsifiers. a highly volatile compound can be lost.. which has a kerosene off-odor.3 pentadiene. it is important to know the conditions under which any antimicrobial spectrum was determined before projections are made regarding the usefulness of an antimicrobial. Highly active antimicrobial compounds that are hydrophobic tend to partition . inactivation of essential enzymes. can also result in the formation of off-flavors. they can solubilize in or be bound by lipids or hydrophobic proteins in foods.Food Antimicrobials — An Introduction 5 microbiological medium parallel that in a food product. or inhibition of protein synthesis. Probably the best method for determining what type of food antimicrobial to use would be based on its mechanism of action and/or target in the cell. lipophilic characteristics appear to be required to allow the antimicrobial to react with the membrane of the microorganisms. Chemical reactions. where microbial growth occurs (Robach. or texture of a food product. 2002). Mechanisms of action of food antimicrobials generally are classified as reaction with the cell membrane. in addition to decreasing antimicrobial activity. combinations of antimicrobials with different mechanisms could be used against the microorganisms in the food product (Davidson et al. especially its carry-through properties. 1980). PHYSICOCHEMICAL PROPERTIES OF THE ANTIMICROBIAL The overall microbial spectrum. Reaction with lipids. High volatility can also result in a noticeable odor. antimicrobials acting on the hydrophobic cell membrane appear to require some lipophilic properties (Branen et al. interference with genetic mechanisms. have actually been fully elucidated. At the same time. for example. proteins. 1980). antimicrobials appear to require a specific hydrophile–lipophile balance for optimal activity. causing permeability changes or interference with uptake and transport. even for the regulatory-approved food antimicrobials such as organic acids. carbohydrates. A sensory evaluation is often needed to assure that antimicrobials do not directly or indirectly through chemical reaction alter the color. Unfortunately. If a food is heated during processing. As a rule. making them less available to inhibit microorganisms in the food product. As such. The polarity of a compound is probably the most important physical property. one must be wary of an antimicrobial spectrum determined only in a synthetic medium. however. Thus. may differ significantly from that needed for a food product. few targets. the exact mechanisms through which antimicrobials affect microbial growth are complex and difficult to determine. and the efficacy of compounds are largely dependent on the chemical and physical properties of the antimicrobial. Sorbic acid. can be degraded by certain Penicillium species isolated from cheese to produce 1. FOOD-RELATED FACTORS The chemical reactivity of the antimicrobial with other food components can significantly affect activity. Water solubility or hydrophilic properties appear to be necessary to assure that the antimicrobial is soluble in the water phase. If the mechanism of the compound is known.

All regulatory-approved organic acids used as antimicrobials have pKa values less than 5. Packaging can directly alter the environment of the food and thus influence the overall growth pattern and type of organisms in the food. Rico-Munoz Davidson. Although the undissociated form of a weak acid has most of the antimicrobial activity. In addition. thus requiring an antimicrobial capable of limiting the growth and activity of these organisms.6 Antimicrobials in Food TABLE 1. Refrigeration generally selects for psychrotrophic Gram-negative microorganisms. They expand the activity of certain antimicrobials (e. A lowering of the water activity can select for those organisms that have the ability to survive and/or grow at lower water activity. the acid dissociates because the cell interior has a higher pH than the exterior. nisin. Protons generated from intracellular dissociation of the organic acid acidify the cytoplasm and must be extruded to the exterior. thus indicating the need for a different antimicrobial.3 pKa of Regulatory-Approved Organic Acids Compound or Group of Compounds Acetic acid Benzoic acid Lactic acid Propionic acid Sorbic acid pKa 4. It is theorized that chelators may destabilize the LPS layer of the outer cell membrane of the Gram-negative bacteria. Chelating compounds such as ethylenediamine tetraacetic acid (EDTA) or its salts have a potentiating effect on some antimicrobials. 1995. which are not normally inhibited by the compounds alone (Cutter and Siragusa.75 into the lipid areas of the food and away from the water phase. the constant influx of these protons will eventually deplete cellular energy. Bacteria maintain internal pH near neutrality to prevent conformational changes to the cell structural proteins. For food products with a pH of 5. enzymes. PROCESS FACTORS The type of preservation process used in conjunction with antimicrobials has a significant influence on the type and level of antimicrobial needed. organic acids function at low concentrations only in high-acid foods (generally less than pH 4. organic acids can penetrate the cell membrane lipid bilayer more easily. Generally. For example. Eklund (1983) demonstrated that the anion does contribute slightly to antimicrobial activity.79 4. 2004). This is because the most effective antimicrobial form is the undissociated acid. Vacuum or modified atmosphere packaging results in . lysozyme) to include Gram-negative bacteria. 1983).5 or greater. In the undissociated form.g. Once inside the cell. where microbial growth occurs (Branen et al..3).75 4.0 (Table 1. some Gram-positive bacteria are more susceptible to certain antimicrobials in the presence of chelators including EDTA. Branen and Davidson.87 4.5 to 4. which exists in the majority only at a pH below the pKa of the compound. there are very few compounds that are effective at low concentrations.6). Certain preservation processes may result in the need to control sporeformers that have the ability to survive the heating process. and phospholipids. 1980. allowing the antimicrobials access to the inner cell membrane.. Because protons generated by the organic acid inside the cell must be extruded using energy in the form of adenosine triphosphate (ATP). which means their maximum activity will be in high-acid foods. pH of the food can result in ionization of an antimicrobial and a change in activity.19 3. molds survive and yeasts can grow at a lower water activity than bacteria. nucleic acids.

It is also important that the antimicrobial be metabolized and excreted by the body. because of the unknown problems that may result from an increased consumption of such compounds in combination with other additives and food components. The latter may be problematic even for some . although food antimicrobials will extend the lag phase or inactivate low numbers of microorganisms. significantly higher quantities of an antimicrobial may be needed. low temperature). the ability of an antimicrobial to contribute to the prevention of foodborne illness must be taken into account when assessing the overall safety of the individual antimicrobial. The majority of the antimicrobials now being used in food products have been extensively tested for toxicologic safety. the microbial flora may have the ability to metabolize the antimicrobial and thus. Few. 1995. an evaluation of the risks versus the benefits indicates that these antimicrobials are acceptable. Certainly. and although questions will continue to be asked. In some cases.g. it is unlikely that the current marketing system could exist without the use of antimicrobials. render it ineffective. This is not always true. Ultimately. their effects can be overcome. Although attempts have been made to use other preservation systems or to produce additive-free foods. If the number of microorganisms contaminating a food product is high. an antimicrobial must be nontoxic to test animals and humans. however. of course. A possible total or partial shift to naturally occurring antimicrobials is being investigated by many researchers. regulatory-approved antimicrobials are able to preserve a product that is grossly contaminated. It is obviously essential that an additive for use as a food antimicrobial be safe for human consumption. 2000). CONSIDERATIONS IN THE USE OF FOOD ANTIMICROBIALS SANITATION An antimicrobial is never a substitute for good sanitation in a food processing plant. In addition.. food antimicrobial compounds are primary contributors to a combination of inhibitors and inhibitory conditions (e. A naturally occurring antimicrobial must be shown to be nontoxic either by animal testing or by its continuous consumption by consumers as a food over a long period. these antimicrobials can be volatilized or directly react with other food components. The food industry will always be reluctant to expand the use of food preservatives. which inhibits growth of molds and several bacteria but allows certain facultative anaerobic microorganisms such as lactic acid bacteria to grow. The compound or its breakdown products should also not result in buildup of residues in body tissues. based on several studies. Because they occur in nature. however. it is often thought that naturally occurring antimicrobials are less toxic than synthetic compounds. Leistner. TOXICOLOGIC SAFETY Perhaps the most important aspect of any compound proposed for use as a food additive would be its toxicologic characteristics. The stringent requirements for toxicologic safety testing can result in costs in the millions of dollars before approval for a new additive can be obtained. This would allow inhibition of spoilage or pathogenic microorganisms at the surface of a packaged product. if any. Requirements for toxicologic safety will limit the ability of the industry to develop new antimicrobials.Food Antimicrobials — An Introduction 7 low oxygen tension. The ability of certain antimicrobials to inhibit microorganisms can be overcome on extended storage. Depending on the time and temperature of storage. A significant amount of research has been done on the addition or incorporation of food antimicrobials to packaging materials. Generally. which has led to limitations on their use. The safety of some food antimicrobials has been questioned over the years. Valid assay methods for the antimicrobial are also a necessity so that levels can be easily followed. and low microbial loads must always be sought. This is sometimes termed “hurdle technology” (Leistner and Gorris. low pH. after an extended time.

In addition to lack of toxicity. naturally occurring compounds must be able to be metabolized and excreted so as to not lead to residue buildup. 2003). would defeat the purpose of using a natural compound. 21CFR 184.8 Antimicrobials in Food common potential natural antimicrobials such as spice extracts. it would be unacceptable for a food antimicrobial to mask spoilage because spoilage may protect consumers from ingesting foodborne pathogens. A potential problem with natural antimicrobials is that if they are highly purified. This is because. In addition. LABELING One of the alleged attractions of naturally occurring antimicrobials is their reduced negative impact on the labeling of foods. odor. compounds that negatively affect flavor and odor or contribute inappropriate flavors and odors would be unacceptable. it is known that peptides. If a product is simply an “extract of” a commonly consumed plant or animal food product. less purification may be better.S. assays need to be developed that evaluate the activity of these compounds against the pathogen they are designed to kill. The reason for these assays is that various conditions of process or storage could reduce the effectiveness of the compound. This is only possible if the product from which the extract is taken is known to be nontoxic. Therefore. ECONOMICS OF USE A food antimicrobial will not be useful to the food industry unless it is inexpensive enough and has the ability to pay for itself based on reducing spoilage and minimizing foodborne illness. For example. they may need to be approved as food additives. In many cases. Code of Federal Regulations. The efficiency of these compounds can be extremely important in determining the overall economics of their use. of course. 21CFR 184. If antimicrobials are to be used exclusively as inhibitors of pathogens in food products. Extensive studies at a pilot-plant level are necessary on any food additive to prove its overall usefulness. there are few standardized methods for validation of the activity of regulatory-approved food antimicrobials. . although spices have been consumed for centuries. In the United States. Finally. ACTIVITY VALIDATION METHODS Currently. so should there be validation for the activity of food antimicrobials. it is much less likely to require complex regulatory approval for use (Davidson and Zivanovic. there are no other activity assays specified or required for food antimicrobials. they should be nonallergenic or bind or destroy important nutrients in a food product (Harlander. the compound would probably have to be listed using a chemical name on a food label. or texture. Many antimicrobials must be used at high concentrations to achieve activity against target microorganisms. 1993). This. such as nisin. In addition to adverse effects on flavor.S. Consumers are reportedly concerned about the presence of synthetic chemicals in their foods and would prefer natural compounds. SENSORY EFFECTS Another major factor that needs to be addressed when applying antimicrobials is their potential impact on the sensory characteristics of a food. just as thermal processes need validation. an additional 2 or 3 days of shelf life can significantly help to offset the cost of using an antimicrobial. they are not normally consumed in the concentrations necessary to achieve antimicrobial activity.1538). there are methods for the determination of the activity of lysozyme (U.1550) and nisin (U. Obviously. Code of Federal Regulations. are susceptible to inactivation by enzymes in foods. For that reason. This would involve very expensive and time-consuming toxicologic testing.

and allyl isothiocyanate (AIT). Attaining synergistic activity with antimicrobial combinations requires that the components have different mechanisms. preservatives will be needed. If foods are to arrive in the condition expected. This will also require the development of uniform worldwide regulations regarding the use of these chemicals in food products.Food Antimicrobials — An Introduction 9 RESISTANCE DEVELOPMENT Because the activity spectra are often different for each antimicrobial. and consumers will be better able to determine whether the risks outweigh the benefits for selected additives now available and for some compounds of potential future value. Because of their specificity. 2002). not to select an antimicrobial solely according to its ability to control the predominant microorganism present. 1999).. Appropriate or compatible use would involve using these compounds in foods in which they add to the positive sensory characteristics of the product in addition to improving food safety or increasing shelf life. but because of the reduced activity against Gram-negative bacteria. it has not been shown to occur in an actual food processing system (Davidson and Harrison. it has been demonstrated that some bacterial pathogens may develop a tolerance or adaptation to organic acids following prior exposure to low pH. 1999). favorable conditions can be created for growth and spoilage by these latter organisms. Buchanan and Edelson. The global economy in which we live results in foods being transported throughout the world. The information should also serve as a basis for selection of any new antimicrobial developed in the future.g. First is the expansion of information on the antimicrobial spectrum of natural antimicrobials. In addition. cold. natural antimicrobials will be increasingly looked on as adjuncts in hurdle technology and used with milder nonsterilizing. One should be cautious. the microflora contaminating a food product significantly influences the choice of the antimicrobial needed. There has been much interest in the effect of environmental stress factors (e. Microorganisms exposed to a stress may become more resistant and have enhanced survival to subsequent stresses (Foster. . regulators. nonthermal processing methods such as high hydrostatic pressure or pulsed electric fields (Smid and Gorris. low pH/organic acids) on developed resistance of microorganisms to subsequent stressors. 1995. selecting antimicrobials that control some genera but not others may result in selecting for and creating favorable conditions for growth of other organisms. carvacrol. A second major area of research involves use of antimicrobials in combinations with each other and with traditional or novel processing methods. Although this increased resistance may be a problem in application of organic acids for controlling pathogens. To more effectively apply antimicrobials so that synergistic activity is possible will require knowledge of the mechanisms of action of the compounds. For example. This research will be more focused on the appropriate use of natural antimicrobials or utilization of compounds in situations in which they are compatible. such as thymol. certain compounds. The future of research in the area of food antimicrobials will likely be on two fronts. however. Potential food antimicrobials should not contribute to the development of resistant strains nor alter the environment of the food in such a way that growth of another pathogen is selected. heat. It is hoped through the information presented in the following chapters that food scientists. are not compatible with certain foods. phenolic compounds may inhibit certain Gram-positive food-poisoning bacteria. For example. For example. FUTURE OF ANTIMICROBIALS Antimicrobials will undoubtedly continue to be needed to provide the food supply that will be demanded in the future. starvation.

in Food Additives. and Board. 1983. Dillon.A. Inc.K. San Diego..J. 58:977–983.. L.. Marcel Dekker. and Montville. Leistner. 62:211–218. Rhaman.J. A.. Zeuthen. Food Prot. Robach. Davidson. and Siragusa.H.. Food Technol.. 1980. 1994. L. 54:383–389... The use of natural antimicrobials.. B. J. Davidson.. 593–627.G. A. L. Antimicrobial agents. D. 1995. Foster. Eds...M. Intl.M. A. 34(10):81. Doyle. Washington.. 2000. G. 1993. Rico-Munoz. and other process controls.. 48:1284–1288.. E. P.G. Branen. J.M. Eds.. P. and Branen. 34(5):42. Regulatory aspects of bacteriocin use. Food Technol.L. Resistance and adaptation to food antimicrobials. Branen. pH-Dependent stationary-phase acid resistance response of enterohemorrhagic Escherichia coli in the presence of various acidulants..L.10 Antimicrobials in Food REFERENCES Branen. 132. U. 56(11):69–78. Microbiol.. Ed. Davidson.G. and Edelson. P.. 2nd ed. 103 pp.. and Haggerty. S. L. Marcel Dekker. R. Davidson. M.. Davidson.H. The effect of corn oil and casein on the antimicrobial activity of phenolic antioxidants. Chemical preservatives and natural antimicrobial compounds.R. Harlander.R. Branen. L. Eds. LLC.H. C. 2nd ed. E. Salminen. 2000. 2nd ed. Branen. R. A. p. sanitizers. S. Davidson. p. Eds. Antimicrobial properties of phenolic antioxidants and lipids. Intl. Rev. Eds. Food Technol. S.. Task Force Report No.. and Gorris. 2001.J. and Gorris. P. 2002.R. A. Population reductions of gram-negative pathogens following treatments with nisin and chelators under various conditions.W.. J.S.C. and Thorngate. V. Naidu. 563–620. J. Hoover. 2004. and Thorngate. L. Ames. 1983.M. J.K.. Natural antimicrobials for food preservation...M. and Davidson.L. Eds.K. T.A. Food Microbiol. 1980. J. Natural Food Antimicrobial Systems. 1999. M. M. J. Marcel Dekker. P. Basic aspects of food preservation by hurdle technology.. Davidson. Leistner. S. P. CRC Press. Ed.R. J. Introduction to food additives...M. V. Food Sci. Use of preservatives to control microorganisms in food. L. Appl. 1998.K. Cutter. Smid.M.M. P.. 1999. in Food Additives. L.K. . Trends Food Sci. Davidson. Food Microbiol.. Academic Press.L. D. CAB Intl..P. P.. in Food Additives. and Thorngate. M. Enhancement of nisin.M.G. Food Additives. Crit.. in Food Preservation Techniques. Low pH adaptation and the acid tolerance response of Salmonella typhimurium. 2002.H. and Davidson. Naturally Occurring Antimicrobials in Food. P. R.L.M.. Food Prot. in Handbook of Food Preservation.. New York. Council for Agricultural Science and Technology. and Thorngate. The antimicrobial effect of dissociated and undissociated sorbic acid at different pH levels. and Steenson.C. Eds. Buchanan. and monolaurin antimicrobial activities by ethylenediaminetetraacetic acid and lactoferrin.L. Boca Raton.. 1995. A. Oxon. J. Natural Antimicrobial Systems and Food Preservation.N. American Society for Microbiology. Branen.L. Food preservation by hurdle technology. P. 21:215–237. New York. J. Branen. S. J. in Bacteriocins of Lactic Acid Bacteria. Davidson. 55:181–186. E. 2nd ed. 2nd ed. and Bogh-Sorensen. R. and Harrison. 1–9. FL. in Food Microbiology: Fundamentals and Frontiers.M. 6:41–46. S.N. Beuchat. Salminen. 2003. and Zivanovic. Wallingford. 2002. Marcel Dekker. Davidson.G.S. Marcel Dekker. P. New York.. J. Eklund. Verbruggen.M. A.... J. P. Food additive regulations in the European community. P. 2002. Bacteriol.. Salminen. Juneja.M. Cambridge. Beuchat.. lysozyme. T. 1995.M.M. J. p. 2002. U. Sofos. and Katz. New York. Salminen.. and Johnson. Technol. S. New York. 90:63–74. IA. Woodhead Publishing Ltd.

........................................29 Applications .......................................12 Antimicrobial Activity ........................................ 1992)................................ ease of incorporation into products........................................2 CONTENTS Sodium Benzoate and Benzoic Acid John R.......................29 Use as a Postharvest Fungicide ..................... lack of color....................................................................... 11 ............................ and food industries........................... several articles concerning various aspects of food additives and preservatives (e................. Chipley Introduction and Historical Background ..... 1995)....................................................................17 Influence of Other Chemicals and Physical Environment ....................32 Other Applications .................................................................... 1980).................29 Use in Various Food Systems.......17 Spectrum of Action........ Sodium benzoate was the first chemical preservative approved for use in foods by the U...................... General evaluations of the use of these compounds in foods (Vogel............................................................... and in consumer attitudes toward the use of preservatives (Jager... drug..22 Microbial Metabolism of Benzoic Acid ......28 Regulatory Status ..........12 Mechanism of Action.............38 INTRODUCTION AND HISTORICAL BACKGROUND Benzoic acid is one of the oldest chemical preservatives used in the cosmetic......... Its preservative action appears to have been first described in 1875................................................................................... it was not introduced for food preservation until around 1900 (Lueck................................................................................................................................................................................34 Assay ................. 2000)...................................................................................... in meat products (Gerhardt...... 1995)....................... Food and Drug Administration (FDA) (Jay.......... During the last 10 years.................................. in prevention of microbial spoilage (Giese........................ and relatively low toxicity subsequently caused benzoic acid to become one of the most widely used preservatives in the world (Davidson...............20 Acquired Resistance to Benzoic Acid ...............37 Acknowledgments ........................................................... 1994)............................ benzoic acid) have been published... The advantages of its low cost..................................S.. Because benzoic acid could not initially be produced synthetically in large quantities......................................................11 Physical and Chemical Properties and Natural Occurrences..........................................................................26 Reaction with Food Constituents.......................................................................................................................g.........................34 Toxicology................................................................................. when a relationship was established between the action of benzoic acid and that of phenol (Lueck......... 1994) serve as examples................................ in beverage manufacture (Giese........................................................................................................................................................................................................................ 1980)..............................................................38 References ........ 2001)......33 Storage and Handling ................................................................................................

Orange.. apple. 1999). Benzoic acid (molecular weight 122. Sieber et al. Gabel (1921) was one of the first to demonstrate that benzoic acid was effective against bacteria in acid media at a level of 0.1) is a white granular or crystalline powder. 1990). and a mixture of these.2 g dissolves in 100 ml water at 4°C. Yogurts have been found to contain natural levels of benzoic acid (Stijve and Hischenhuber. occurs in pure form as colorless or white needles or leaflets. It accounted for approximately 16% of the growth inhibition of Saccharomyces bayanus and Pseudomonas fluorescens from ethanolic extracts of cranberries (Marwan and Nagel. 20°C. Potassium benzoate and calcium benzoate have also been approved for use. It is much more soluble in water than benzoic acid (62. This may be of significance in countries like Switzerland. O OH Benzoic Acid O O− Na+ Sodium Benzoate FIGURE 2.2 to 2.. where benzoic acid has not been approved as a food additive (Sieber et al.1% and in neutral media at 0.. 0. and 74.3).1). .. Teuber. Benzoic acid has also been identified as a natural by-product in culture filtrates of Lactobacillus plantarum (Niku-Paavola et al.2 g dissolves in 100 ml water at 0°C. and 75°C. For this reason. also called phenylformic acid or benzenecarboxylic acid.1. 66.1 Structures of benzoic acid and sodium benzoate. Sodium benzoate (molecular weight 144. MECHANISM OF ACTION It is the undissociated molecule of benzoic acid that is responsible for antimicrobial activity (Table 2. 18°C. Several new types of low-molecular-mass compounds were also identified. Naturally occurring nonflavonoid phenolic compounds. was found to have antimicrobial properties. 1991). were used to characterize commercial fruit juices (Fernandez de Simon et al. It is soluble to a limited extent in water (0. may also contribute to benzoic acid generation. and grape juices were analyzed to establish phenolic profiles unique to each type of juice. p-hydroxybenzoic acid was detected at 0.12 Antimicrobials in Food PHYSICAL AND CHEMICAL PROPERTIES AND NATURAL OCCURRENCES Benzoic acid (C6H5COOH) and sodium benzoate (C6H5COONa) have the structural formulas shown in Figure 2.. In an extensive review. although their solubility in water is less than that of the sodium salt. of mushrooms.6 mg/l. Benzoic acid occurs naturally in several foods and commodities (Table 2. pineapple.18. pear. and 2. respectively). peach. including derivatives of benzoic acid. 1929). depending on the variety (Abdullah et al.2).1).2% but inactive in alkaline media.27. 1992). and of fresh tomatoes (Marlatt et al. A third pathway. 1989. 1995). 1984. it is preferred for use in many cases.2). 1992).0.8. oxidation of benzaldehyde. In four of the seven juices. and 100°C. apricot. along with benzoic acid. 1994). Benzoic acid has also been identified as a major constituent in extracts of blackberries (Humpf and Schreier. Its presence appears to occur as a by-product of the microbial degradation of either hippuric acid or phenylalanine in these products (Figure 2. 1986a). (1995) surveyed and analyzed many types of cultured dairy products and cheeses for the natural occurrence of benzoic acid (Table 2. respectively). Similar results were reported for fungi and yeasts (Cruess and Richert.

ripe Licorice Coffee beans Honey Mushrooms Teas.1 Natural Occurrence of Benzoic Acid Category Fruits and berries Product Apples Apricots Berries (i. non-smear-ripened Cheese.2 Benzoic Acid Content of Cultured Dairy Products Product Yogurts Yogurt. TABLE 2. fruit Sour cream Buttermilk Cottage cheese Cheese Cheese. greengage Prunes Strawberries Tomatoes Beers Dairy. (1995). black Wines Cinnamon Cloves.. green Tobacco Fermented products Spices and flavors Others Note: Levels of benzoic acid vary (10 to 1000 mg/kg) depending on the food.Sodium Benzoate and Benzoic Acid 13 TABLE 2.e. Source: Chipley (1993). smear-ripened Source: Adapted from Sieber et al. blackberries) Blueberries Cherries Cranberries Grapes Plums. Fenaroli (1996). Range in Concentration (mg/kg) 9–56 5–39 10–18 10–19 2–18 0–200 0–41 0–622 . cultured Teas.

and that the toxicity of sodium benzoate in solution was a result of the undissociated benzoic acid molecule.19 Source: From Baird-Parker (1980). Saturation was reached in about 2 minutes and remained constant thereafter.5 59. The strong dependence of uptake on pH was because of the relative distribution of undissociated and dissociated forms in solution. sucrose. . The effect of temperature on the uptake was similar to that observed in enzymatic reactions. NaCl. that only the undissociated acid was antimicrobial. and sorbic and benzoic acids on the growth of 30 strains of food spoilage yeasts have been reported (Praphailong and Fleet.44 0. Macris (1975) observed a rapid uptake of benzoic acid in Saccharomyces cerevisiae. Source: Adapted from Sieber et al. The effects of pH.144 4. Zygosaccharomyces bailii and Yarrowia lipolytica were the most resistant to the two preservatives at pH 5.3 Effect of pH on the Dissociation of Benzoic Acid pH 3 4 5 6 7 pK Undissociated Acid (%) 93. Rahn and Conn (1944) reported that the antimicrobial effect of benzoic acid was nearly 100 times as efficient in strongly acidic solutions as in neutral solutions. increasing the pH of the culture medium decreased the effectiveness of this preservative. not to the pH itself.3 12.2 Biosynthesis of benzoic acid by lactic acid bacteria in fermented dairy products.4. Sorbate was more inhibitory than benzoate on the strains that were tested. TABLE 2. The combined effects of pH and benzoate on the growth of these strains are presented in Table 2. (1995).8 1. The undissociated form was the only one taken up by cells.14 Antimicrobials in Food O OH N H O Hippuric Acid O S-CoA Benzoyl-CoA (a) O OH NH2 O OH Hydrocinnamic Acid (b) O OH Benzoic Acid O OH Cinnamic Acid O OH Benzoic Acid Phenylalanine O H Benzaldehyde (c) O2 (microbial and/or chemical) O OH Benzoic Acid FIGURE 2. 1997). As expected. Proteinaceous material was apparently involved in the uptake of this preservative.0.

the glucose-induced switch from gluconeogenesis to glycolysis was prevented.Sodium Benzoate and Benzoic Acid 15 TABLE 2. in general. 1994). Source: Adapted from Praphailong and Fleet (1997). cerevisiae with benzoate or sorbate at an extracellular pH of 6. of Strains Tested 3 1 5 1 2 2 4 4 6 2 pH 2 NG — — — — NG NG — NG NG pH 3 — — 250 — — — — — 250 — pH 5 500 250 1200 500 750 750 500 750 1200 750 pH 7 1200 1200 1200 1200 1200 1200 1200 1200 NG 1200 Note: NG. then added glucose. (1993) preincubated cells of S. The authors concluded that the metabolic effects of benzoate or sorbate were only marginally pH dependent. The effects of benzoic acid were studied in the preservative-resistant yeast Z. 1991b).8. Inhibition depends more on the preservative concentration within cells rather than on undissociated acid concentration per se. Growth rates were substantially reduced by both preservatives even at an extracellular pH of 6. cerevisiae (Cheng and Piper. adenosine triphosphate (ATP) levels declined. Sorbic and benzoic acids both affected the heat shock response and thermotolerance of S. At concentrations up to 4 mM.8. bailii was to cause a general energy loss (ATP depletion). — no growth in the presence of 250 mg of benzoic acid/L. cerevisiae). The authors reported that these preservatives were among the first compounds shown to act as selective inhibitors of heat-induced protein expression in yeasts. Inhibition of several glycolytic enzymes was not observed. membranaefaciens Saccharomyces cerevisiae Kluyveromyces marxianus Kloeckera apiculata Zygosaccharomyces bailii Z. and benzoate was accumulated to higher levels. . Two modeling studies to predict the inhibitory effects of organic acid preservatives have been reported. Fermentation was inhibited as a result of high ATP usage rather than of lowered intracellular pH (Warth. In yeast (S. fermentation was inhibited. Lambert and Stratford (1999) demonstrated the following: 1. rouxii No. Microbial cells can pump protons out during extended lag phase and raise internal pH despite further influx of preservative. The authors stated that. The resulting metabolic effects included reduced glucose consumption and suppression or reduction of glucose-triggered reactions within cells. Near the minimum inhibitory concentration (MIC) (10 mM).4 Effect of Benzoic Acid on the Growth of Some Important Food Spoilage Yeasts at Different pH Values Maximum Concentration (mg/L) Allowing Growth Yeast Debaroymyces hansenii Yarrowia lipolytica Pichia anomala P. 1991a). Burlini et al. These effects were dependent on the pH of the culture medium. fermentation was stimulated and low levels of benzoate were accumulated. 2. The author concluded that the primary action of benzoic acid in Z. no growth in the absence of benzoic acid. bailii (Warth.

Inhibition of transport in turn results from the destruction of the proton-motive force caused by the continuous shuttle of protons into cells by benzoic acid (Davidson.. and the effectiveness of undissociated acids as preservatives has been noted for many years (Ingram et al. At pH 6..3).0) for 1 hour (Buazzi and Marth. Reduction . 1980). A pH-related increase in protein production was observed when cultures of E. such as benzoic acid. In addition to these mechanisms. They also stated that although both the undissociated and dissociated forms of these acids cause the intracellular pH to fall. phosphofructokinase activity was inhibited (Francois et al. coli were grown in liquid media with or without 20 mM benzoate (Lambert et al. 1973).. fluorescens was completely inhibited by sodium benzoate (Andersson et al. yielding protons that acidify the alkaline interior of the cell.. 1984). benzoate addition resulted in an increased production of 33 proteins. 1989). Similar effects have been found using a variety of microorganisms. Synthesis of messenger ribonucleic acid (mRNA) was critical for restoration of salt tolerance.0 as well as pH 6. 1975.. enzymes involved in acetic acid metabolism and oxidative phosphorylation can be inhibited (Bosund. and the ability of benzoic acid molecules to delocalize the negative charge of ions within the interior of cells and thus increase their membrane mobility. as food preservatives is related to their capacity specifically to reduce the intracellular pH. Only those organic acids that are lipophilic. Hsiao and Siebert (1999) constructed a model based on physical and chemical properties of organic acids and principal components analysis. Metabolic injury was evident by the inability of cells to tolerate 6% NaCl in tryptose agar and the ability to grow on this medium with no added salt. 1982). Davidson. 1976. 1962). B. 50% of the trimethylamineN-oxide reductase activity was inhibited by 1.16 Antimicrobials in Food 3. 1956. Eklund.. In the citric acid cycle.. 1992). Production of 12 of these was induced at pH 8. Gould. coli have been reported (Salmond et al. 1997).5.2 mM benzoic acid (Kruk and Lee. In yeast cells. and Pseudomonas aeruginosa (Freese et al. 1978). 1986). Theoretical ATP consumption for proton pumping can be directly correlated with reduction in cell yield. membrane transport of amino acids is inhibited.5% sodium benzoate (pH 7.. show antimicrobial activity. 1980. 1980). MICs were determined for each acid and used with principal components to produce models with good correlations. The effects of organic acid preservatives on the growth and intracellular pH of E. Chipley and Uraih. α-ketoglutarate and succinate dehydrogenases appear to be quite sensitive (Bosund. E. 1983. The authors suggested that the potency of weak acids.. 1960. 1989. benzoic acid or sodium benzoate can also inhibit specific enzyme systems within cells (Webb.5. causing uncoupling of both substrate transport and oxidative phosphorylation from the electron transport system (Freese et al. 1973. resulting in nutritional starvation of cells (Gould et al. 1977. coli. Eklund. 1962). 1980). Corlett and Brown. 1997). In resting cells of E. its pK value (Table 2. such as benzoic. The production of lipase by P. Uraih et al. in many bacteria and yeasts. 1966). coli. Duration of the lag phase can be predicted from the model. In Bacillus subtilis (Freese. subtilis. The inhibitory potency of this acid can be determined by its lipid/water partition coefficient. Acid-susceptible bacteria (Bacillus and Alicyclobacillus) and acid-resistant bacteria (Lactobacillus and Escherichia coli) were grown in media containing organic acids. In these studies it was suggested that the undissociated form of benzoic acid may diffuse freely through the cell membrane and then ionize in the cell. For example. 1973). growth inhibition is the result predominantly of the undissociated acid. One hypothesis is that they inhibit or kill microorganisms by interfering with the permeability of the microbial cell membrane. 4. Increased permeability of cell membranes during the course of benzoate injury was not observed.. Aflatoxin production by a toxigenic strain of Aspergillus flavus was greatly reduced by the presence of these compounds in synthetic media (Uraih and Chipley. More than 99% of the viable cells of Listeria monocytogenes were injured after exposure to a solution of 8. Sheu et al. Benzoate also inhibits amino acid uptake in Penicillium chrysogenum (Hunter and Segel. 2001).

and most yeasts and fungi are inhibited by 0. the use of benzoic acid or sodium benzoate as a food preservative has been limited to those products that are acid in nature. but many spoilage bacteria are much more resistant. as a weak inhibitor of poly(ADP ribose) polymerase (Oikawa et al. The authors concluded that subinhibitory concentrations of these compounds may stimulate toxin production in some cases. including pH. The average accumulation of mycelial dry weight was greater in the presence of benzoate than in its absence. composition of the growth medium. 1977. yeasts. .. temperature. MICs for some of the bacteria.. 1979). these compounds are used primarily as antimycotic agents. 1987). Several factors interact to determine the MIC. coli and Salmonella Enteritidis (Chipley. benzoic acid cannot be relied on to effectively preserve foods capable of supporting bacterial growth (Chichester and Tanner.. Similar results were observed with 2. and sodium benzoate was determined (Manganelli and Casolari. as an inhibitor of passive anion transport (Lucas-Heron and Fontenaille. In benzoic acid-supplemented cultures. 1972. and environment from which the microorganism was originally isolated. This might result in inhibition of membrane-bound enzymes as a secondary effect. and as an inhibitor of nitrosamine formation (Sung et al. Benzoate may also serve as a scavenger for free radicals (Harvath.1979).5 and 2. The sensitivity of 42 yeast cultures to sorbic and benzoic acids. Currently. 1974). and fungi involved in food poisoning and food spoilage are given in Tables 2. Food-poisoning and spore-forming bacteria are generally inhibited by 0. Yagi et al. 1980). genus and species of the microorganism in question.6. ANTIMICROBIAL ACTIVITY SPECTRUM OF ACTION Because the quantity of undissociated acid decreases with increasing pH (Table 2. Baird-Parker. In liquid media. potassium sorbate. benzoic acid might also change the permeability of the microbial cell membranes by possibly causing a conformational change in the lip moieties of these structures. With the previously mentioned results in mind.Sodium Benzoate and Benzoic Acid 17 was accompanied by the appearance of a yellow pigment that could be converted into aflatoxin B1 by cell-free extracts. The scattering of MIC values was lower with potassium sorbate and benzoic acid and higher with sorbic acid and sodium benzoate. 1983). In general.3% sodium benzoate. aflatoxin B1 production was higher than in controls. Sodium benzoate was found to control both growth and aflatoxin production by Aspergillus parasiticus in liquid media (El-Gazzar and Marth. flavus have been reported (Bauer et al. Therefore. prior exposure to the preservative. Increasing the concentration of sodium benzoate increased the percentage of inhibition at the end of incubation (10 days). all treated cultures produced measurable levels of toxin 3 to 7 days later than controls. 1988). as an inhibitor of Damino acid oxidases (Quay and Massay. 1980).02% undissociated acid. 4-dinitrophenol and cell envelope-associated enzymes of E. Different concentrations of benzoic acid were tested to determine the effective levels capable of reducing the mycelial growth of six Fusarium and eight Penicillium species by 50% (Thompson.05% to 0. The effects of several preservatives and antimicrobial agents on aflatoxin B1 production by A. 1992). The results suggested that these preservatives blocked an enzymatic step late in the biosynthetic pathway of aflatoxin B1. Conversion could be prevented by addition of benzoic acid or sodium benzoate. with the greatest increase occurring when the medium contained 0. cerevisiae decreased the biomass and increased the specific oxygen uptake rate of cells (Verduyn et al. 1981).1% of the undissociated acid. Fusarium species were more sensitive to benzoic acid (210 to 420 µg/ml) than were Penicillium species (250 to 3000 µg/ml). 1997). Addition of benzoate to chemostat cultures of S.. 1979). 1980). It has been reported to have a flower-inducing effect in plants (Watanabe and Takimoto.01% to 0..3). however.

0 MICa Bacillus cereus Escherichia coli Klebsiella pneumoniae Lactobacillus sp. cerevisiae Torulopsis sp. (1999). Pseudomonas sp. Penicillium citrinum Penicillium glaucum Rhizopus nigricans a 1500 20–300 >4000 2000 500 1000 100 30–120 30–280 2000 400–500 30–120 Minimum inhibitory concentration in µg/ml (ppm).0 4. 500 50–120 100–200 100–200 300–1800 3000 2000 50–100 200–480 200–500 50–100 200–400 Sporogenic yeasts Asporogenic yeasts Candida krusei Debaryomyces hansenii Hansenula sp.3 5.5 4.6–5. Davidson and Juneja (1990).0 6.6–4. Aspergillus parasiticus Aspergillus niger Byssochlamys nivea Chaetomonium globosum Cladosporium herbarum Mucor racemosus Penicillium sp.0 6. Hansenula subpelliculosa Oospora lactis Pichia membranefaciens P.2–5. Zygosaccharomyces bailii Z. Pseudomonas aeruginosa Staphylococcus aureus Streptococcus sp.2–5.8 4. Source: Adapted from Chipley (1983.0 4. and Fungi Microorganisms pH Bacteria 6.6 (4°C) 5. Russell (1991). .1 5.6 (21°C) 5.0 20–200 70–150 300–700 500 180 200–300 300 700 300 100–200 330 600 200–500 4500 1200 500–1100 1000 Alternaria solani Aspergillus sp.5–5.3 5. Saccharomyces bayanus S.0 5.0 4.18 Antimicrobials in Food TABLE 2.5 5.0 5.6 6. pastori Rhodotorula sp.0 3.0 5.0 4.8 Fungi 5. Listeria monocytogenes Micrococcus sp.0 5.0–5.0 5. 1993).0 5.0 2.8 4.0 5. and Steels et al. Yeasts.0 4. rouxii 4.5 Antimicrobial Spectrum of Benzoic Acid against Selected Bacteria.0 3.6 Yeasts 2.0–5.0 4. lentus Z.6 6.0 6.3–6.

1995). were tested against both the opaque and translucent morphotypes of Vibrio vulnificus (Sun and Oliver. Beuchat (1980) reported that sodium benzoate (300 µg/ml) inhibited the growth of Vibrio parahaemolyticus in laboratory media and enhanced the rate of thermal inactivation of this organism at slightly higher concentrations. Incubation of cells in minimal media caused injury that depended on the temperature of incubation but not on the presence of benzoic acid. Sodium benzoate has been suggested as an inhibitor of cellulose-decomposing bacteria and fungi (Sauer. according to the results of a study involving several strains of Trichophyton and Microsporum (Pelayo.5) without addition of benzoic acid.. Most were isolated from spoiled foods that had contained preservative. selected organic acids promoted growth and toxin production when added to the media. including benzoic (Rusul and Marth.. Growth and aflatoxin production by toxigenic strains of Aspergillus were partially or completely inhibited by the undissociated form of six organic acid preservatives. bacteriostatic and bactericidal properties of benzoic acid can also be demonstrated. The authors questioned the suitability of benzoic acid alone to control this pathogen in foods. Salts. Growth of this fungus was accompanied by production of fumitremorgin mycotoxins. Fungal growth was reduced by lowering the pH of laboratory media from 7. but relatively modest bactericidal. Small amounts (75 mg/L) of potassium sorbate or sodium benzoate completely inhibited germination of ascospores and subsequent outgrowth. Eight of these had a lethal effect on both morphotypes of this bacterium.6 Minimum Inhibitory Concentration of Benzoic Acid for Food Spoilage Yeasts Isolatea Kluveromyces fragilis Kloeckera apiculata Pichia ohmeri Hansenula anomala Saccharomyces cerevisiae Zygosaccharomyces rouxii Zygosaccharomyces bisporus Candida krusei Saccharomycodes ludwigii Schizosaccharomyces pombe Zygosaccharomyces bailii a MIC (ppm) 173 188 200 223 170–450 242–330 200–350 440 500–600 500–567 600–1300 A total of 23 isolates were tested.0 to 2. 1979). Source: Adapted from Warth (1989c). enhanced aflatoxin production when present at low levels but became inhibitory at higher levels. activities against cells in a liquid minimal medium.5. 1988. Neosartorya fischeri is one of the most frequently isolated heat-resistant fungi causing spoilage of fruit juices and other heat-processed fruit-based products (Nielsen et al. Both fungistatic and fungicidal properties have been attributed to benzoic acid. Ten generally regarded as safe (GRAS) substances.Sodium Benzoate and Benzoic Acid 19 TABLE 2. 1988). it was found that benzoic acid at concentrations of approximately 1000 to 3000 ppm had strong bacteriostatic. In a series of studies involving L. and its survival . Under appropriate conditions. Isolates were grown at 25°C in yeast extract medium containing 5% glucose (pH 3. such as sodium and potassium chlorides and sodium nitrate. 1989). This organism was isolated from milk. 1989). 1977). monocytogenes (El-Shenawy and Marth. Yousef et al. including benzoic acid.

0) were used and required lower levels of sodium benzoate. especially when used at pH <6. pH. cereus. At <25°C. coli at all temperature/pH/NaCl combinations. Individual and synergistic effects of pH. 1998). bayanus and Hansenula species. A typical dose-response effect may be observed. Chichester and Tanner. 1963). or sucrose (Rehm. Infinite inhibition concentrations could be affected by the growth medium. In theory. This method was based on finding the relative effectiveness of an inhibitor. 1993).. they were very effective inhibitory agents for heatdamaged spores (Lopez et al. and three preservatives on the growth of three foodborne bacterial pathogens were studied using gradient gel plates (Thomas et al. Combining glycerol monolaurate with benzoic acid gave greater inhibition of L.3). It should then be possible to achieve (1) a broader spectrum of action or (2) an increased antimicrobial action by using this combination (Lueck. and antagonistic effects reported in several early studies have been extensively reviewed by Lueck (1980) for benzoic acid as well as for other preservatives. sorbate was more effective than sodium benzoate against S. For example. respectively. aureus when used with higher concentrations of NaCl. 1960. A plot of the inhibitor concentration versus the reciprocal of relative effectiveness was linear (Figure 2. Effectiveness increased as the pH of the recovery media decreased to 5. Sodium nitrite was the least effective preservative tested. bailii were determined and expressed in polynomial equations (Cole and Keenan. Potassium sorbate and sodium benzoate did not have any significant effects on the heat resistance of four strains of B. 1986b).. 1996). 1972). aureus. benzoate was the most effective preservative against S. 1989). At 35°C. The effects of temperature. The growth patterns of S. Additive. It was also the most effective against B. The infinite inhibition concentrations for S. The anionic form of benzoic acid was shown to have a distinct effect on doubling time. when these organisms were grown in Trypticase® soy broth.4. boric acid.20 Antimicrobials in Food and growth were determined in media supplemented with organic acids and sodium benzoate (ElShenawy and Marth. However. Increasing the acidity and/or NaCl generally improved the effect of all the preservatives. An interesting method to determine concentrations of antimicrobial agents that provide infinite microbial stability has been developed (Marwan and Nagel. synergistic. Synergistic effects have also been reported using combinations of benzoate and sulfur dioxide. at various concentrations.0. 1988). one should be able to combine various antimicrobials having different modes of action to compensate for this deficiency. sodium chloride. The relative effectiveness values of benzoic acid were established for S. The x-axis intercept was the concentration of the inhibitor that gave infinite microbial inhibition. monocytogenes than when each preservative was tested alone (Oh and Marshall. Potassium sorbate was completely effective against Vero cytotoxigenic E. sodium chloride. carbon dioxide. benzoic acid. stearothermophilus spores (Lopez et al. inactivation or inhibition of growth occurred in inoculated media when a lower incubation temperature (13°C) and pH (5. The influence of combinations of chemical antimicrobials on the growth of food-poisoning and spoilage microorganisms has been reported (Banks et al.. In general. bayanus in the presence of different concentrations of benzoic acid are shown in Figure 2. Synergism between benzoic and sorbic acids was pH dependent. 1980). combinations of sorbic acid and benzoic acid inhibit several bacterial strains better than either antimicrobial alone (Rushing and Senn. . and sorbic acid on the growth rate of the yeast Z.. INFLUENCE OF OTHER CHEMICALS AND PHYSICAL ENVIRONMENT No antimicrobial is completely effective against all microorganisms present in a given foodstuff. when these preservatives were added to recovery media. bayanus and Hansenula species were 330 and 180 ppm benzoic acid. 1994). 1986). like benzoic acid.

Sodium benzoate prevented the growth of Staphylococcus aureus in an intermediate-moisture food (Boylan et al. However. an experimental bottled beverage made from papaw fruit was reported to have a shelf life of 80 weeks at 10°C and 30°C when pasteurized and supplemented with sodium benzoate (1250 ppm and pH 3. 30°C. benzoic acid can be solubilized with a surfactant to hold it in solution in its undissociated form (Lewis and Payne.Sodium Benzoate and Benzoic Acid 21 1.8 0. Growth at 21°C. The combined effects of preservatives and incubation temperature on biomass and patulin production by Byssochlamys nivea in apple juice have been determined (Roland and Beuchat. or drying. 1990). it took the treated populations to reach a density of 70 Klett units divided by the time it took the control populations to reach the same value). and 37°C over a 25-day incubation period was significantly retarded by 75 ppm sulfur dioxide. Sulfur dioxide was apparently antagonistic when used in combination with any of the other antimicrobial agents (including benzoate) tested in this study. irradiation. relative effectiveness (the time. For example. 1958). In addition to combining several preservatives together.4 0.2 0.. the antimicrobial activity of benzoic acid is reduced by nonionic surfactants (de Navarre and Bailey. 1984). respectively. Source: Marwan and Nagel (1986b). The effects of pH and benzoate on the growth of yeasts in an experimental grape drink containing 1. refrigeration. 150 ppm potassium sorbate. 1975). Antagonistic as well as synergistic effects were observed when a wine yeast strain of S. .0. Closed circles: yeast nitrogen base. followed by potassium sorbate and sodium benzoate. such as heating.. A combination of benzoic acid and low temperatures was used effectively to reduce the numbers of yeasts in grape juice (Pederson et al.9) (Okoli and Ezenweke. Sulfur dioxide had the most significant effect on the rate of biomass and patulin production by this organism.0 50 100 150 ppm 200 250 300 350 FIGURE 2. cerevisiae was inoculated in a laboratory medium supplemented with several combinations of preservatives (Parish and Carroll. 1988). 1961). 1976). 1956). Open circles: Trypticase® soy broth.3 Effect of the medium on the inhibition of Saccharomyces bayanus by benzoic acid at 30°C and pH 4. and 500 ppm sodium benzoate.6 1/RE 0.b). The presence of other chemicals may be beneficial or detrimental to the action of the preservative.5 volumes of CO2 have been reported (Kelly. Synergistic effects on yeasts and fungi using a combination of benzoic acid and heat have been reviewed by Lueck (1980) and have also been reported by Beuchat and Jones (1979) and Beuchat (1981a. Likewise. it may also be advantageous to combine preservatives with physical methods of food preservation. depending on the chemical in question.0 0. RE. in hours.

2002).02% sodium benzoate. recent studies imply that this may no longer be the case. Adding 1% quinic acid to media almost completely eliminated the inhibitory effect of 0.4 Inhibition of Saccharomyces bayanus by benzoic acid in Trypticase® soy broth at 30°C and pH 4. . Source: Marwan and Nagel (1986b).005%) and 0. In a German study of 1959. but mold growth was accelerated in the presence of quinic acid.5 (Sagoo et al. cited by Lueck (1980).025% sodium benzoate acted synergistically against three food spoilage yeasts suspended in saline solutions at pH 6. coli after 14 passages in media containing benzoic acid. They had been supplemented with the maximum levels of benzoic acid permitted for use in these products.2 and 4. Growth of yeasts was unaltered. The effects of quinic acid on yeasts and molds were investigated (Kallio et al.0. At these pH values.22 Antimicrobials in Food 300 0 200 100 150 100 90 80 70 60 50 40 30 200 250 Klett Units 20 10 20 40 60 Hours 80 100 120 FIGURE 2. the authors proposed that chitosan is highly polycationic and could react with the anionic components of the microbial surface. The concentrations of benzoic acid tested (in parts per million) are indicated. no resistance could be observed in cells of E. However.01% to 0. The resistance of a wide variety of yeasts to benzoic acid has been reported in several studies. ACQUIRED RESISTANCE TO BENZOIC ACID There have been conflicting reports in the literature concerning the possibility of acquired resistance to benzoic acid by microorganisms. 1985). this might enhance the antimicrobial effects of benzoate.. A combination of the polysaccharide chitosan (0. Carbonated and noncarbonated citrus drinks and fruit juices were involved. This acid was antagonistic to the antifungal effects of both potassium sorbate and sodium benzoate..

and lethality was enhanced as the pH of the heating medium was reduced.4 to 3. 1988) it was determined that energy was required for the reduction in cytoplasmic benzoate concentration and for the maintenance of internal pH. Because this system required energy. Fumaric.. Source: Adapted from Warth (1988). 1987). flavus.7). such as glucose.0 and 4. A strain of S.Sodium Benzoate and Benzoic Acid 23 An isolate of Pichia membranaefaciens from spoiled mango fruit was resistant to sodium benzoate (1500 and 3000 ppm) when grown in laboratory media at pH 4. Thus. were tested for tolerance to several organic acids during and after heating (Beuchat. 1990). . and in some cases. TABLE 2. respectively (Ethiraj and Suresh. cerevisiae was found to have multiple resistances to both physical treatment and chemical preservatives (Guerzoni et al. was present. However.25 mM benzoic acid (first five species) or 2 mM benzoic acid (last four species). Three strains of T. heat tolerance appeared to be related to the morphology of the ascospores. The authors observed that preservative resistance may be strain specific. Warth (1977) reported that cells of Z. 1988). The heat-resistant yeast Talaromyces flavus was isolated from fruit juice concentrates involved in spoilage of packaged reconstituted fruit juice (King and Halbrook. cells were resistant only when a sufficient energy source. this isolate was less resistant to potassium sorbate. the energy source was unavailable for growth. 1988). In a later study (Warth. sorbic. Resistance resulted primarily from an inducible energy-requiring system that transported benzoate from the cell.19). The effects of sorbic and benzoic acids on the viability of ascospores varied depending on the strain and were influenced by other constituents in the heating medium. and benzoic acids were the most effective. resulting in reduced growth yields and rates of cell production by yeasts. with the required concentrations lower when the pH of the growth media was reduced from 5. Several species of yeasts grown in the presence of benzoic acid tolerated 40% to 100% higher benzoic acid concentrations than yeasts grown in its absence (Table 2. In an inoculated peach-based system. isolated from spoiled fruit products. bailii grew in the presence of high concentrations (600 mg/L) of benzoic acid at pH values below the pK of this acid (4.5. Heat resistance increased with age. Potassium sorbate and sodium benzoate prevented outgrowth.7 Resistance of Yeast Species to Benzoic Acid MIC of Benzoic Acid (mg/L) Isolate Saccharomyces cerevisiae Kluveromyces fragilis Kloeckera apiculata Hansenula anomala Saccharomyces cerevisiae Candida krusei Saccharomycodes ludwigii Schizosaccharomyces pombe Zygosaccharomyces bailii a Cells Grown without Benzoic Acid 100 125 125 140 175 300 300 325 600 Cells Grown with Benzoic Acida 170 173 188 223 285 440 650 567 1250 Adapted to benzoic acid by overnight growth in the presence of 0.5. the effectiveness of benzoic acid on this strain decreased as the water activity decreased and only a slight synergistic action with thermal treatments was observed.

Five strains of this yeast grew over a wide range of temperature (4°C to 25°C) and pH (2.2 to 7.. 1989c). Growth in the presence of weak acid preservatives appeared to involve a common resistance mechanism.0).24 Antimicrobials in Food The genus Zygosaccharomyces is often regarded as being synonymous with food spoilage as a result of the remarkable resistance of its species to commonly used food preservatives such as sorbic. and most can grow at very low pH. 1988). 1996). It was preserved using benzoic acid (1. Benzoic acid was taken up 27 times faster by cells than propanoic acid. Resistance to dimethyldicarbonate was greater than that of Z. bailii were proportional to the concentration of undissociated acid (Warth. 1989b). The principal mechanism of uptake appeared to be diffusion of undissociated acid. Growth at 4°C was significant. It is unlikely that removal of weak acids by metabolism could occur fast enough to alter the initial impact of these substances on spoilage yeasts. cerevisiae. growth of 22 isolates of 11 yeast species in the presence of benzoic acid enhanced their subsequent resistance to benzoic acid and other weak acid preservatives. These media were also evaluated for enumerating two of these strains grown in blueberry syrup with or without sodium benzoate (0. or legally. and all strains were osmotolerant. Growth of yeasts in the presence of benzoic acid reduced their subsequent permeability to this acid. is a common and major determinant of the preservative tolerance of yeast species (Table 2. bailii strain that was resistant to both benzoic and sorbic acids. Spoilage resulted from a Z. the capacity of the cell to remove it. and acetic acids and ethanol (Thomas and Davenport. 1999). Uptake of weak acid preservatives by passive diffusion is quite rapid. Uptake rates of benzoic and propanoic acids in cells of Z. Z. The Zygosaccharomyces genus also contains some of the most osmotolerant species known. Adaptation to higher preservative concentrations was demonstrated with benzoic acid. A new osmophilic. Resistant species may be less permeable than sensitive ones to undissociated benzoic acid (Warth. Differences in resistance among yeast species could be the result of differences in the rate of penetration of the cell by benzoic acid. They were resistant to sorbic acid (≤400 mg/l) and benzoic acid (≤900 mg/l) at pH 4. bailii can grow in concentrations of food preservatives in great excess of those normally. The authors concluded that Z. The authors stated that microorganisms resist or adapt to weak acid preservatives (e. bailii and S. The nonselective medium supported higher recovery of all nine strains from the six food products compared to the three selective media. Glucose stimulated the rates of uptake and was also required for continuous elimination of benzoate from cells. requiring only 5 to 15 minutes to reach equilibrium.5 g/kg) and packaged in glass jars. Three selective media were compared for detecting and enumerating nine strains of Z. Mustard spoilage by Z. bailii in the spoilage of refrigerated foods. However. 1985). and 600 µg/ml). In another study (Warth. Foods at particular risk are acidic and contain relatively high levels of sugar. benzoic. The minimum pH for growth was not related to this acquired resistance.. in which benzoic acid is continuously removed from the cell. Zygosaccharomyces lentus. Zygosaccharomyces and Saccharomyces are able to adapt to changing levels of preservatives such as benzoate..g. 1999). benzoate) by one of the following three methods: 1. Removal or metabolism. . Some yeasts are intrinsically resistant to relatively high concentrations of preservatives (Stratford and Lambert. Eight tons of table mustard were exported to the United States. 1989c). Adaptation can result in rapid growth in moderate levels or slow growth in previously inhibitory levels. 1989a). allowed in beverages and can adapt to concentrations even higher. bailii has been described (Buchta et al. 300. or an intrinsic sensitivity to benzoic acid or its anion (Warth. bailii grown in six commercial food products (Makdesi and Beuchat. 1996). preservative-resistant spoilage yeast. has been described (Steels et al. Cells grown in syrup without sodium benzoate were more sensitive to selective media than were cells grown in syrup containing the preservative. results also suggested that the resistance mechanism.6) (Warth.0. lentus was likely to be more significant than Z.

Cells of E. 1997. 1998). 1985). The authors proposed that in yeasts. coli O157:H7 and L. which is freely permeable across the plasma membrane and is thus able to enter the cell. were isolated and found to be resistant to benzoic acid. efflux of protons and anions by cells would not create a “futile cycle” if there was a concurrent reduction in the ability of weak acids to diffuse across the cell membrane and enter the cytosol. When G. The net result is that the preservative diffuses into the cell until equilibrium is reached in accordance with the pH gradient across the membrane. However. which cannot cross the plasma membrane. Whether efflux pumps are involved is open to debate. if damage is caused by low intracellular pH. simply pumping preservative anions out of cells could create a so-called “futile cycle” where the anions would reassociate at the lower external pH and reenter the cell. several workers have proposed that the actual inhibitory action of weak acid preservatives (benzoate and sorbate) could be the result of the induction of a stress response that attempts to restore homeostasis but results in the reduction of available energy pools needed for growth and other essential metabolic reactions (Warth 1991a. Therefore. 1996..8. MICs of sorbic and benzoic acids for Gluconobacter oxydans were 1000 and 900 mg/L. undissociated state of the molecule. they cited reports that adapted yeasts reduced the diffusion coefficient of preservatives across their plasma membrane so that transport of weak acids into cells was indeed reduced.0 survived exposures to inorganic acid or to acid pH plus organic acid that prevented subsequent growth of cells grown at pH 7. which can infect apple tissues. 1989).. Several microbial-resistance mechanisms induced by weak organic acid preservatives were also reviewed by Brul and Coote (1999). in media at pH 3. For example. coli initially grown in media at pH 5. (1998) demonstrated the existence of a multidrug-resistance pump that actively extruded preservative anions from cells of this yeast. This favors the uncharged. oxydans was grown in the presence of sublethal concentrations of either compound before the MIC was determined. cerevisiae were able to extrude benzoic acid by an energy-dependent mechanism. Holyoak et al. This appears unlikely because uptake occurs rapidly by passive diffusion. Piper et al. the MIC of both antimicrobials increased substantially within 1 hour. Bracey et al. inhibition of microbial cell growth occurs because various key metabolic reactions are inhibited. Brul and Coote (1999) described the modes of action of several types of preservative agents for foods and the microbial resistance mechanisms induced by these compounds. monocytogenes and the role of membrane ATPase in the resistance of food spoilage yeasts. Acquired resistance to benzoic acid has been reported for other microorganisms. When this happens. resulting in the release of charged anions and protons. Mutants of the fungus Nectria galligena. Benzoic acid occurs naturally as a phytoalexin of immature apples (Seng et al. They included the acid tolerance responses of E. respectively. The pathogenicity of this fungus could be increased by growth on a medium supplemented with benzoic acid before inoculation into fruit.3 reduced the MIC of both antimicrobials by about 300 mg/L (Eyles and Warth.. as cited previously (see “Mechanism of Action”). adaptation could involve enhanced removal of protons using H+ATPase. The inhibitory action is classically believed to occur when the molecule encounters the higher pH inside the cell and then dissociates. In an extensive review. Adaptation to or repair of damage. In yeasts.. Weak organic acid preservatives have maximum inhibitory activity at low pH. However.0 (Goodson . They assumed that any rate of diffusion across the plasma membrane remained unchanged and that cells would not alter their membrane composition or structure to reduce uptake of the toxic compound. Several causes of damage by weak acid preservatives have been proposed. The authors proposed that some yeast adaptation to weak acid preservatives might simply be the result of accumulation of anions. Anions and protons accumulate inside the cell. earlier studies by Henriques et al. and adaptive mechanisms have been suggested. b. 3. Reducing the pH of the media to 3. Henriques et al. however.Sodium Benzoate and Benzoic Acid 25 2. (1997) found that cells of S. Prevention of uptake.

However. They also found that once induced. Eleven E. acid-resistance systems remained active for prolonged periods of cold storage at 4°C. When challenged at pH 2. including benzoic. Russell (1991) noted that choosing a suitable preservative or a combination of preservatives might be the most effective. The enzymes of the pathway are inducible. MICROBIAL METABOLISM OF BENZOIC ACID Johnson and Stanier (1971) stated that aerobic metabolism of benzoate by bacteria frequently proceeded through the β-ketoadipate pathway (Figure 2. He stated that there were two types of bacterial resistance to preservatives. When considering ways of overcoming resistance or of preventing it.. Resistance to benzoic and sorbic acids is well documented in yeasts (Warth 1977. Russell (1991) examined mechanisms whereby bacteria resist the effects of preservatives and nonantibiotic antibacterial agents. Resistance to preservatives caused by chromosomal gene mutation is an example. benzoate could be metabolized through the β-ketoadipate pathway. In a taxonomic study.0. Both of these systems were effective in protecting E. Moscoso-Vizcarra and Popoff (1977) found that Enterobacteriaceae could be divided into two groups with respect to benzoic acid metabolism. The authors proposed that acid-adapted organisms might survive in acid foods.. coli in the gastrointestinal tract. In the first group. monocytogenes against lethal chemicals after adaptation to sublethal environmental stress (Lou and Yousef. Source: Adapted from Zeyer and Kearney (1984). 1997). coli O157:H7 strains and four commensal strains of E. the second group consisted of those organisms that did not metabolize benzoate.5 Proposed pathway for the aerobic microbial metabolism of benzoic acid. However. Six organic acids. . this mechanism has not yet been reported for bacteria. coli against the bactericidal effects of a variety of weak acids. Second. the arginine-dependent system provided more protection to the O157:H7 strains than to commensal strains.5). the glutamate-dependent system provided better protection than the arginine system and appeared equally effective in all strains. acquired resistance could result from genetic changes in a cell either by mutation or by acquisition of genetic material from another cell (usually by a plasmid). intrinsic resistance could result from a natural chromosomally controlled property of a bacterial cell. including benzoic acid. Examples of this type would include basic cell wall structure/organization and the possession of constitutive enzymes that enable bacteria to metabolize preservatives. The authors suggested that several acid-resistance systems might contribute to the survival of pathogenic E. were evaluated. 1988). Thayer and Wheelis (1976) characterized an inducible benzoate permease system involved in the uptake of benzoate by cells of Pseudomonas putida. coli O157:H7 (Lin et al. Others include the induction of acid resistance in E. 1996). cis-Muconic Acid CO2H CO2H O β-Ketoadipic Acid Succinic Acid + CoA-SH Acetyl-CoA FIGURE 2. This is the result of an enhanced ability of adapted cells to eliminate these acids by energy-dependent extrusion. Three previously reported acid-resistance systems were tested. Metabolism of benzoate occurred by the O OH OH OH Benzoic Acid Catechol CO2H CO2H cis. They included an acid-induced oxidative system. coli were tested for their ability to survive extreme acid exposures (Lin et al. First. an acid-induced arginine-dependent system. none of these acids inhibited the subsequent growth of acid-adapted cells. 1996) and protection of L. 1989). and their synthesis is elicited by primary substrates or metabolic intermediates in the pathway.26 Antimicrobials in Food and Rowbury. and a glutamate-dependent system.

Benzoate was converted to catechol by the strains able to grow on this substrate. 1993. and with Micrococcus (Haribabu et al. Nitrate. 1982). benzoate and salicylate are metabolized via the protocatechuate and β-ketoadipate pathways. cis. The enzymes of these pathways appeared to be under coordinate control and were repressed in the presence of a primary substrate. Pseudomonas solanacearum. A mutant incapable of growth on benzoate was isolated and characterized. Hugo (1991) described the ability of nine species of Pseudomonas and Vibrio to degrade 18 aromatic compounds used as preservatives in medicines. and sulfate can be used as electron acceptors during metabolism (Colberg and Young. Compared to relatively well-characterized aerobic mechanisms for metabolism of benzoate and other aromatic compounds. and β-ketoadipate were identified as intermediates of benzoate metabolism (Figure 2. 1995). including Pseudomonas. a commonly occurring soil bacterium. such as glucose. putida (Jeffrey et al. Further metabolism proceeded via the catechol branch of the β-ketoadipate pathway with the occurrence of high specific activities of catechol 1.6. gentisate. cis-muconate. and (5) a β-oxidation sequence. 1988). respectively (Shailubhai et al. Seventeen strains of aerobic bacteria were tested for their ability to degrade 15 aromatic substrates (Lang. Benzoate was metabolized by 16 of these strains when added as the sole carbon source to liquid mineral media. Harwood and Gibson (1997) stated that this pathway could be divided into several phases: (1) formation of CoA thioesters. with Rhodococcus (Janke et al. 1995).2-dioxygenase. 1994). Harwood and Gibson (1986) stated that aerobic degradation of benzoate by various Gramnegative bacteria.. and cosmetics. In the presence of benzoate.. Anaerobic metabolism can also occur but involves reductive saturation of the nucleus. (3) introduction of a carbonyl group.5). Similar results have been reported with Bacillus stearothermophilus (Adams and Ribbons. 4-dioxygenase was induced. In Alcaligenes eutrophus. end product of the β-ketoadipate pathway (Figure 2. Outlines of the new aerobic pathway were deduced. Both chromosomal and plasmid-encoded pathways have been described for the catabolism of benzoate in P. The proposed intermediates included benzoyl-CoA. was also capable of metabolizing benzoate (Kumari and Mahadevan. and fumarate + pyruvate. A variant of aerobic benzoate degradation has been found in a denitrifying bacterium (Pseudomonas) in which benzoyl-coenzyme A (CoA) is the first intermediate (Altenschmidt et al. fumarylpyruvate.5) (Ampe et al. A permease-like protein involved in benzoate transport and degradation has now been isolated from cells of Acinetobacter (Collier et al... Niemetz et al. foods.2-dioxygenase for ring fission. 3-hydroxybenzoyl-CoA. One pathway proposed for the anaerobic microbial metabolism of benzoate is shown in Figure 2. 1988). 1984). 1984).. which are central intermediates in the anaerobic metabolism of many aromatic compounds (Elder and Kelly. 1997). protocatechuate 3. iron.. 1982). 1997). Active transport of benzoate may occur in this organism (Thayer and Wheelis. one of the causal agents of wilt in banana plants. Catechol. These results were confirmed by assaying key enzymes from benzoate-grown cells. This isolate was able to oxidize benzoate completely to carbon dioxide. maleylpyruvate. whereas salicylate induced catechol 1.Sodium Benzoate and Benzoic Acid 27 β-ketoadipate pathway.. 1997). Several actinomycetes were tested for the degradation of benzoate by growth in a liquid medium containing sodium benzoate (1 to 3 g/L) as the principal carbon source (Grund et al. involves monooxygenase and dioxygenase enzymes that catalyze the incorporation of molecular oxygen into the aromatic nucleus to form dihydroxylated intermediates (see also Heider and Fuchs. . This is followed by conversion of the remainder of the molecule to acetyl-CoA. 1992). Kinetic models have been derived for the metabolism of [14C]-benzoate by Pseudomonas species (Simkins and Alexander.. In Aspergillus niger. 1990). He reported that benzoic acid was easily metabolized by all nine bacterial species tested.. 1996). (2) ring reduction. The enzymes responsible for ring cleavage were induced depending on the substrate used for growth. both aerobically and anaerobically. 1984). the rate of benzoate metabolism may be regulated by the presence of succinate. (4) ring opening. anaerobic pathways have been far less studied and reported.

coli. This enzyme cleaves hippuric acid into the constituent products glycine and benzoic acid. making the models unacceptable under these conditions. Source: Adapted from Koch et al. antilisterial activity was much lower than predicted. Sodium benzoate has been shown to inhibit the growth of some of the microorganisms used for analyzing the vitamin content of foods (Voigt et al. 1988). Ganzfried and McFeeters (1976) developed a model system to evaluate binding of benzoic acid by lipids and proteins. Solid arrows indicate intermediates detected in benzoate-grown cultures. jejuni could be developed. Nocardia asteroides and several fungi. most of which were fungi. However. has become recognized as a leading cause of human gastroenteritis (Hani and Chan. In foods with high protein or lipid content. 1996). .28 Antimicrobials in Food O OH CoA-SH Benzoic Acid O S-CoA 2H Benzoyl-CoA O S-CoA 2H O S-CoA H2O Cyclohex-1-ene carboxyl-CoA 2H OH O S-CoA H2O S-CoA 6-Hydroxycyclohex-1-ene carboxyl-CoA O S-CoA −2H Cyclohex-1. the hippuricase assay is the only reliable biochemical assay that can distinguish C. In the genus Campylobacter. 5-diene carboxyl-CoA O S-CoA H2O S-CoA COOH Pimelyl-CoA HO O O S-CoA COOH Acetyl-CoA + CO2 O 2-Ketocyclohexane carboxyl-CoA 3-Hydroxypimelyl-CoA FIGURE 2. 1995). the authors concluded that it may not be limited to a small number of microorganisms. Both bound benzoic acid to approximately the same extent. including species of Rhizopus and Mucor. jejuni from C.. The N-benzoyl-glycine amidohydrolase (hippuricase) test is of paramount importance to differentiate C. REACTION WITH FOOD CONSTITUENTS The variability of the levels of benzoic acid in samples of commercial food products may indicate a preferential binding of this compound by certain food constituents. C.. one species. An equation was obtained to estimate the ratio of bound to unbound acid in food products based on their lipid and protein content. They also showed that the gene was absent in other Campylobacter species. monocytogenes by benzoic acid and other compounds (Ramos-Nino et al. Dashed arrows indicate proposed intermediates. The authors isolated the gene responsible for the expression of hippuricase from 12 strains of C. the authors proposed that a species-specific diagnostic DNA probe for human gastrointestinal infection caused by C. 1979). jejuni. Harwood and Gibson (1997). coli. Currently. Microbial reduction of benzoate to benzyl alcohol under aerobic conditions has been reported (Kato et al. Heintze (1978) reported that benzoic acid accumulated in the lipid phase of herring salad. although stability was not adversely affected with time. For example. jejuni and demonstrated that this gene was not present in 17 isolates of C. Arfmann and Abraham (1993) found that benzoic acid as well as three hydroxyl derivatives could not be reduced to their respective alcohols when screened with a set of 20 microorganisms.6 Proposed pathway for the anaerobic microbial metabolism of benzoic acid. Based on these results. were found to have this capability. Although this appears to be a unique reaction occurring in microbial cells under aerobic conditions. Models were tested in several food systems to determine the inhibition of 18 strains of L. Note: This sequence of reactions is compiled from several studies.. jejuni (hippuricase positive) from other species. (1993). 4-diene carboxyl-CoA O OH 2-Hydroxycyclohexane carboxyl-CoA Cyclohex-1.

1%. An ADI (average daily intake) value of 0 to 5 mg/kg of body weight has been specified for benzoic acid (Pollard. Potassium benzoate is now commercially available and apparently evolved in response to consumers’ interest in reduced sodium intake. Product information for both salts is available (Miles Laboratories.15% and 0. the authors recommended that the combination of ascorbic acid and sodium benzoate in foods and beverages be evaluated more carefully. Benzoate does not control the growth of high levels of microorganisms and therefore cannot be used to compensate for poor ingredients or processing. Regulatory agencies consider it to be GRAS to the same extent that sodium benzoate is GRAS as a preservative with the limitation of 0. the off-flavor they may impart to the foods they are to preserve. They may also include foods from which benzoate is excluded in the United States. and lack of color. Lueck (1980).). Similar results were reported by McNeal et al. 1990). although its solubility in water is much less than the sodium salt (Pollard.4 g per 100 ml solution) than the sodium salt. applications.5 or that can be brought into this range by acidification. . Subsequent studies indicated that soft drinks containing both ascorbic acid and sodium benzoate could produce very low levels of detectable benzene. and limitations of preservatives and antimicrobial agents. and their toxicologic properties compared to some other preservatives have all contributed to recent efforts by food processors to replace benzoic acid and sodium benzoate with other preservatives with better characteristics. 1977/1988. Approximately 1.1%. Inc. their main advantages include low price. bakery products.Sodium Benzoate and Benzoic Acid 29 In the early 1990s it was discovered that the combination of ascorbic acid and sodium or potassium benzoate in beverages could. generate detectable levels of benzene. 1990). Kimble (1977). USE IN VARIOUS FOOD SYSTEMS Benzoic acid and sodium benzoate have been widely used to preserve beverages. In one case (a fruit juice–mineral water combination).25%. ease of incorporation into products. fruit products. including benzoic acid. 184.1021 and 184. Although the detectable yield was extremely low (<1 ppb). 1993). However. 1994). As food preservatives. the use of benzoic acid and other chemicals in the processing and freezing of shrimp has been reviewed (Chandrasekaran. In most other countries.1 times more potassium salt than sodium salt is required to obtain the same level of benzoic acid in solution and the same level of antimicrobial effect. Secs. the maximum permissible quantities generally range between 0.8 and 2. Gardner and Lawrence (1993) demonstrated that low levels of detectable benzene could be produced in aqueous solutions of sodium benzoate and ascorbic acid in the presence of the transition metal catalysts iron (III) or copper (II).1733) up to a maximum permitted level of 0. under certain conditions. REGULATORY STATUS In the United States. (1993) when they analyzed several foods and fruit-flavored soft drinks for benzene. A Food Technology special report (1986) reviewed the characteristics. In addition. APPLICATIONS Benzoic acid and sodium benzoate are most suitable for foods and beverages that naturally are in a pH range below 4. and Chipley (1983. the narrow pH range in which they are effective. benzoic acid and sodium benzoate are GRAS preservatives (Code of Federal Regulations. Title 21. benzene levels higher than the permissible levels for mineral waters prompted a recall in one state. and other foods in several countries (Tables 2. Applications of these preservatives to foods have been reviewed by Chichester and Tanner (1972). Calcium benzoate has also been approved for use. They may also be used in certain standardized foods.9). The potassium salt is less water soluble (42.

30 Antimicrobials in Food TABLE 2. b Soft drinks for consumption after dilution. The effectiveness of sodium benzoate and potassium sorbate. Propionic acid (0. . c Soft drinks for consumption without dilution. niger. and Penicillium sp.06%) or potassium sorbate (0. Recent reports indicate that these preservatives continue to be of benefit in a variety of products. Several additives and preservatives were evaluated as inhibitors of melanosis (black spot) and microbial spoilage in prawns in chilled storage (Montero et al. separately or combined. a maximum level of 1000 ppm benzoic acid or its salts may be used for all products listed. semipreserved Fruit juice Fruit pulp Jam. A combination of sodium benzoate and kojic acid was effective in inhibiting melanosis. carbonated a Canada 1000 1000 1000 1000 1000 Denmark Norway Sweden U.K. Source: Adapted from Chipley (1993). was evaluated against the growth of Z. 2001). separately or combined. Fresh catfish were dipped in various concentrations of sodium benzoate or potassium sorbate and smoked. Sorbate treatment was more effective than benzoate and increased the shelf life by 8 days. 1994). jellies Liquid egg white. A.. Orange juice not for manufacturing may not contain a preservative (federal standards of identity). However. However. 4-hexylresorcinol was the most effective inhibitor of both melanosis and microbial spoilage in prawns.04%) inhibited growth of all three fungi. ketchup Soft drinks containing fruit juice Soft drinks. bailii. salad dressings Sauces. Potassium sorbate was more effective than sodium benzoate. flavus was most sensitive to potassium sorbate. Halotolerant fungi were isolated from salted dried fish (Chakrabarti and Varma.3% sodium benzoate was three times longer than that of the controls (Ponce de Leon et al. The shelf life of sardines stored in an acid brine containing 0. was most sensitive to propionic acid and sodium benzoate. yolk or whole Margarine. relishes Salads.a 1000 200 500 500 5000 1500 1500 1500 5000 2000 1000 1000 1000 10. were dominant.S.02%) or sodium benzoate (0. but A. salted Mayonnaise Mustard Pickles. A maximum level of 2000 ppm may be used in orange juice for manufacturing. d Good manufacturing practice. bailii in an inoculated salsa mayonnaise stored at room temperature (Wind and Restaino.. prevented spoilage of the product by Z. then evaluated during shelf life (Efiuvwevwere and Ajiboye. Penicillium sp. neither preservative. U. 1996).000 2000 2000 2000 2000 2000 2000 1000 1000 800 800 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 200 200 3000 3000 1500 1500 1500 1500 500 250 250 250 800b 160c 1000 1000 1000 1000 1000 1000 1000 GMPd In the United States. 2000). Aspergillus flavus.8 Maximum Permitted Levels (ppm) for Benzoic Acid and Its Salts in Foods in Selected Countries Food Fish products Fish. 1995).

The minimum temperature that allowed growth was 8°C. condiments. cooked Low-sugar jams. Comparable rates were noted when each preservative or the combination of both was used in mango juice heated to 85°C. sodium benzoate. and calcium salts are all permitted for use (E210–E 213) (Pollard. 1995). In the EU. or whole egg Liquid dietary food supplements Dietetic foods (excluding foods for infants and young children) Level for Use 150 200 200 2000 600 2000 200 2000 500 1000 2000 1500 500 500 500 1000 1000 300 1500 150 1000 5000 2000 1500 Fish Fruits and vegetables Sauces Other a The European Union (EU) is currently composed of 12 member nations. The minimum temperature that allowed growth of the E.. 1990). In both studies. unfermented Liquid tea concentrates Semi-preserved fish products (including fish roe) Salted. Growth and control of four Salmonella serotypes in a soft Hispanic type cheese were evaluated (Kasrazadeh and Genigeorgis. potassium. yolk. brine. (1995). dried fish Shrimp.Sodium Benzoate and Benzoic Acid 31 TABLE 2. Source: Adapted from Anon. The levels of some nutrients were reduced when tomato juice was treated with dimethyl dicarbonate. bailii. 1996. fischeri were evaluated in fruit juices (Rajashekhara et al.. acetic acid. The effects of sodium benzoate and potassium sorbate on the thermal death rates of ascospores from the heat-resistant mold N. 1997). Tomato concentrate could be kept for up to 12 months at room temperature with the addition of salt. 1998).9 Maximum Permitted Levels (ppm) for Benzoic Acid and Its Salts in Foods in the European Uniona Commodity Beverages Food Nonalcoholic flavored drinks (excluding dairy-based drinks) Spirits with <15% alcohol by volume Alcohol-free beer in keg Grape juice. mustard Liquid egg white. or oil Prepared salads Olives and olive-based preparations Aspic Emulsified sauces with fat content ≥60% Emulsified sauces with fat content <60% Nonemulsified sauces Nonheat-treated dairy-based desserts Confectionery (excluding chocolate) Chewing gum Seasonings. benzoic acid and its sodium. models were developed relating lag time and specific growth rate to . 1995). marmalades. No viable microorganisms.. and potassium sorbate (Uboldi Eiroa et al.. including Z. In grape juice. 1994. and other fruit-based spreads Candied fruits and vegetables Vegetables in vinegar. coli O157:H7 were also evaluated in this cheese (Kasrazadeh and Genigeorgis. were observed in any samples containing preservatives. The microbial stability and quality of tomato juice and fermented cucumbers and carrots were improved by addition of a combination of sorbate and benzoate or benzoate alone (Bizri and Wahem. potassium sorbate was more effective than sodium benzoate or their combination. Growth and control of two strains of E. 1994). coli strains was 10°C. Montano et al. Fleming et al. jellies.

monocytogenes. milk. C. USE AS A POSTHARVEST FUNGICIDE Because of the long and successful use of benzoic acid in the processed food industry. 1989).. Unpasteurized apple cider has been implicated in several foodborne disease outbreaks caused by pathogenic microorganisms. and ethanol were very effective bactericidal treatments for L. However. but benzoate was about two to eight times more effective than sorbate (Splittstoesser et al. At 8°C. several factors may influence the effectiveness of benzoic acid in the treatment of fresh .. A study was conducted to evaluate aqueous dipping solutions of organic acids or salts to control L. monocytogenes usually attach following cooking and during slicing and packaging (Farber and Peterkin.32 Antimicrobials in Food temperature. 1998). jejuni. vacuum-packaged bologna stored at 4°C for up to 120 days. Most research efforts have been directed toward E. 2000). These solutions could be used for dips to sanitize chickens intended for frying before presentation to consumers (Hathcox et al. 2001). Combinations of organic acids. Listeria monocytogenes is recognized as a foodborne pathogen of major concern to humans. Tompkin et al. A preservative treatment was developed that was capable of achieving the FDA mandate for a 5-log reduction of E.9 with propionic acid.5% lactic acid combined with 0. or S. especially in younger children and the elderly. Control strains of E. Application of antimicrobials as sprays or mists to meats following processing may be more effective than their addition in the meat formulation. Sorbates and benzoates are currently approved in various countries for use as dipping solutions to prevent fungal growth in dry sausages (Sofos.. Benzoate was one of the most effective compounds tested in the presence or absence of ethanol. 2001).3%) to cheese (pH 6. 1996. aureus.3% or 0... coli O157:H7 in apple cider (Comes and Beelman.. Ethanol-enhanced killing correlated with damage to the bacterial cytoplasmic membrane. No viable cells from any of the four pathogens were detected on skin washed with lactic acid/benzoate solutions and stored for 8 days at 4°C. the combination of sorbate and benzoate was more effective than either preservative alone (Zhao et al. Washing the skin with solutions of either 0.3%) to cheese (pH 6. monocytogenes on sliced. cheeses. the growth of some O157:H7 strains in apple cider was not affected by the presence of either preservative (Miller and Kaspar. 2001). growth was inhibited by benzoate or sorbate (Koodie and Dhople. Chicken skin was inoculated with Salmonella spp. Large outbreaks of infection have been linked to consumption of contaminated coleslaw. coli O157:H7 in apple juice and apple cider with or without potassium sorbate or sodium benzoate has been investigated. 1999). 1993). this antimicrobial was evaluated for control of postharvest diseases of various fruits and vegetables. These strains grew well in unpasteurized and pasteurized apple juice.15% fumaric acid and 0. The antimicrobial can be applied directly onto the product surface where cells of L. Dock et al. L.5% or 5% acetic acid. Resistance to these preservatives was greater at 4°C than at 25°C (Fisher and Golden.05% sodium benzoate followed by holding at 25°C for 6 hours and at 4°C for 24 hours.0) made from milk acidified to pH 5. hot dogs. 1995).05% sodium benzoate reduced the numbers of these pathogens compared to washing with water. However. coli O157:H7. 1995). 2002). or 5% potassium benzoate for 120 days (Samelis et al.. monocytogenes (Barker and Park. and luncheon meats. Growth was either prevented or delayed by adding sodium benzoate (0. The treatment that was successful included addition of 0.6) or adding potassium sorbate (0. Both preservatives reduced the heat resistance of E. 1999. The same preservatives added to cider resulted in a greater than 5-log reduction in less than 5 and 2 hours when held at 25°C and 35°C. No significant increase in the numbers of this pathogen occurred on bologna slices treated with 2. 5% sodium diacetate. Survival of E. coli O157:H7 in cider because of the severity of illness this pathogen causes. 1994). coli failed to grow in either type of apple juice. low pH. respectively. and wash solutions were evaluated for effectiveness in decontaminating the skin (Hwang and Beuchat. Contamination of many of these foods frequently occurs after processing.

A benzoic acid-based polymer coating for apples has been developed (Ivanov et al. following official methods of analysis (Zani et al.. insoles for shoes. and treatment for microbial infections in trees. respectively. 1989). Patents have also been issued for the use of benzoic acid or sodium benzoate as an industrial fungicide in several products. Significant antifungal properties were demonstrated against food spoilage fungi. A mixture of fruit-coating polymers and potassium sorbate or sodium benzoate completely inhibited postharvest fungal growth on bananas (Al Zaemey et al.1% and 0. especially against toxigenic strains of A. 1994). 1981).. Methylcellulose was mixed with chitosan and 4% sodium benzoate or potassium sorbate to produce a food-grade film (Chen et al. and as a preservative in certain brands of cough syrup (Chen et al.. 1980). textiles. A sodium benzoate–sorbic acid combination used in a pharmaceutical product for treating ulcers was effective against a wild strain of Pseudomonas cepacia. wood preservatives.. varnishes. The chilling-induced production of ethylene in cucumbers was inhibited by the addition of sodium benzoate (Wang and Adams. Sodium benzoate has also been used to inhibit postharvest changes in fruits and vegetables. this preservative system was ineffective against an adaptiveresistant strain. modified starch adhesives. used in cosmetics has been published (Bach et al. The shelf life of sliced apples and potatoes was extended by 1 week when they were dipped in a polysaccharide/protein edible coating containing sodium benzoate or potassium sorbate (Baldwin et al. rubber manufacturing. These include animal feedstuffs. 1968). 1990). the cosmetic applications for benzoic acid have largely been taken over by more potent antimicrobial agents and compounds that are active over a wider pH range (Manowitz.. as a disinfectant for artificial kidneys (Kolmos. Currently.1%. possible areas for specialized applications still exist — for example. Sodium benzoate did not inhibit growth of any Gram-positive cocci.05% to 0. Hewala. in peanuts (Uraih and Offonry. 1997). lubricants.. flavus. concentrations of 0. their concentrations were high enough to inhibit growth of periodontal pathogens. It is currently used in animal feeds and in some tobacco products as a fungicide at levels of up to 0. OTHER APPLICATIONS Benzoic acid is one of the oldest preservatives in the cosmetic and pharmaceutical industries. liquid coolants. 1988. . with perhaps the most pertinent consideration the pH of the superficial tissues (Eckert. 1968). adhesives for paper. The minimum inhibitory concentrations of sodium benzoate and dichlorobenzyl alcohol for 115 strains of dental plaque microorganisms were determined (Ostergaard. For example.1% to 0. have been used to preserve cosmetic formulations. Wang and Baker (1979) found that chilling injury to cucumber and sweet pepper fruits could be reduced by addition of 10 mM sodium benzoate as a 5-minute dip before chilling. A single oral rinse with a mouth rinse containing these antimicrobials did not affect plaque removal (Danielsen et al. benzoic acid was used for oral preparations in concentrations of 0. 1996). In the pharmaceutical industry. A summary of the antimicrobial activities of 30 preservatives.. 1994). as a control for dentureborne Candida albicans (Lambert and Kolstad. A p-hydroxybenzoate-based system was effective in protecting the product against a variety of strains of P.025%. cepacia grown under different conditions. However. 1986). saliva samples from volunteers who used a dentifrice containing these antimicrobials indicated that for 5 to 10 minutes after toothbrushing. 1996)..5%. 1967). Benzoic acid and its derivatives have been proposed for use as fungicides. and leather. 1996). Generally. However.Sodium Benzoate and Benzoic Acid 33 fruits and vegetables. 1994). including benzoic acid. incorporated as sodium benzoate. 1976). sealing gaskets for food containers. Although its use in pharmaceuticals has largely been displaced by other compounds (Grundy.

0 g per 60–100 days 5–10 g for several days (as sodium benzoate) 40 mg/kg body weight per day 40 mg/kg body weight per day 5% of diet (as sodium benzoate) 1. dry place. Route of administration was per os. cat. dog Mouse Mouse Mouse Human Human Human Human Mouse Rat Rat Rat Rat 3 months 5 days 3 months 3 months 14 days 60–100 days Several days 17 months 18 months 2 weeks 2 weeks Time Period Level 1.10.. 1989). There is little danger of its being a toxicant or fire hazard under normal conditions of use. Hippuric acid. is then excreted in the urine (White et al.b rabbit. . The overall reaction sequence proceeds as shown in Figure 2. TOXICOLOGY Several toxicologic studies involving different animal species have been conducted using both benzoic acid and sodium benzoate. However. Its formation can be increased greatly by administration of benzoic acid. Source: Adapted from Lueck (1980). The first reaction is catalyzed by a synthetase enzyme. In addition. and intraperitoneal routes (Sax and Lewis. Benzoate does not appear to be accumulated in the body. This product is not corrosive. Excretion of benzoic TABLE 2.10 Toxicity of Benzoic Acid and Sodium Benzoate by Exposure Categorya Category Acute Species Tested Rat Guinea pig..3–4.11).4–2. Extensive human feeding trials conducted early in the twentieth century led Chittenden et al.34 Antimicrobials in Food STORAGE AND HANDLING Sodium benzoate should be stored in a cool. intramuscular.0 g/kg body weight Results 50% mortality 100% mortality Subchronic Chronic 80 mg/kg body weight 3% of diet 4% of diet (as sodium benzoate) 1 g/day 12 g per 14 days 0. The apparent reason for this involves a detoxifying mechanism whereby benzoate is absorbed from the intestine and “activated” by linkage with CoA to yield benzoyl coenzyme A. (1909) and Dakin (1909) to conclude that sodium benzoate was not deleterious to human health.7–4. Containers should be kept closed as much as possible. synthesized in the liver. These have been summarized by exposure category in Table 2. the second is catalyzed by an acyltransferase enzyme. 1964). Sax and Lewis (1989) reported data for several animal species based on the route of administration (Table 2.0 g/kg body weight 1. both benzoic acid and sodium benzoate are moderately toxic by ingestive.5% of diet 1% of diet (as sodium benzoate) Mortality rate increase 50% mortality No effect No effect No effect No effect No effect Growth disturbance Growth disturbance 100% mortality Decreased growth rate No effect a b Route of administration was peroral. 1964).7 (White et al.

11 Toxicity of Benzoic Acid and Sodium Benzoate by Route of Administration Benzoic Acid Species Tested Human Human Rabbit Rabbit Rabbit Rabbit Dog Cat Rat Mouse Mouse Guinea pig Guinea pig a Sodium Benzoate Toxicity Resultsb TDLo LDLo MLD SEV LDLo LDLo LD50 LD50 LD50 LD50 LD50 LDLo LDLo Level mg/kg 6 500 500 mgc 100 mgc 2000 2000 2000 2000 2530 1940 1460 2000 1400 Species Tested Rabbit Rabbit Dog Rat Rat Rat Mouse Guinea pig Route of Administrationa Oral Scu Oral Oral Oral Ipr Ims Ipr Toxicity Resultsb LDLo LDLo LDLo TDLo LD50 TDLo LD50 LDLo Level mg/kg 1994 2000 2018 44000d 4070 3000e 2306 1400 Route of Administrationa Skin Oral Skin Eye Oral Scu Oral Oral Oral Oral Ipr Oral Ipr Scu.Sodium Benzoate and Benzoic Acid TABLE 2. subcutaneous. mild irritation effects. Ipr. SEV. c Total dosage. e Total dosage for 3 days to pregnant rats. LD50. lowest published toxic dose. d Total dosage for 22 days to pregnant rats. lethal dose 50 percent kill. 35 . b Source: Adapted from Sax and Lewis (1989). severe irritation effects. intramuscular. MLD. lowest published lethal dose. TDLo. intraperitoneal. Ims. LDLo.

Sodium benzoate inhibited the synthesis of glucose from lactate and generation of urea from ammonia when added to suspensions of rat hepatocytes (Cyr et al. S. see Chipley. The authors reported that both the biotransformation of benzoic acid to hippuric acid and the urinary excretion of hippuric acid followed first-order reaction rates.9%) of the absorbed benzoic acid was conjugated with glycine to form hippuric acid. Acceleration of the conversion of benzoyl CoA to hippurate by the addition of glycine restored the levels of free CoA and acetyl CoA and the rates of gluconeogenesis and ureagenesis. In guinea pigs. He also suggested that the remaining portion of the benzoate not excreted as hippuric acid may have been detoxified by conjugation with glucuronic acid and excreted in the urine by this route. 1991). The author proposed that large-scale consumption of these preservatives in the human diet might generate oxidative stress within the epithelia of the gastrointestinal tract. Cell sensitivity to these acids was enhanced by aerobic rather than anaerobic growth conditions. only a small portion (6. However. . the route of administration significantly affected the efficiency of benzoic acid metabolism (Nathan et al. Akira et al. 1993). The food preservatives were shown to have a strong prooxidant effect on aerobically grown yeast cells. These authors analyzed plasma concentrations of benzoic and hippuric acids and urinary levels of hippuric acid after oral doses of sodium benzoate (40. Inhibition was caused by accumulation of benzoyl CoA with a resultant depletion of free CoA and acetyl CoA. benzoic acid has been used in the treatment of patients with congenital errors of urea synthesis to develop an alternative pathway of nitrogen waste excretion (Kubota and Ishizaki. 1991). Since the late 1970s.7 Metabolism of benzoic acid by mammals. Different animal species differ in their ability to metabolize benzoic acid. acid by this particular pathway was first reported by Dakin (1909). (1994) used deuterated benzoic acid and nuclear magnetic resonance spectroscopy to monitor metabolism in a healthy male volunteer. Source: White et al. 80. The amount of sodium benzoate absorbed by human subjects may be estimated by determining the amount of hippuric acid excreted in the urine (Fujii et al. or 160 mg/kg) were given at least 1 week apart to 6 healthy male volunteers. When applied topically.36 Antimicrobials in Food O OH + ATP + HS-CoA Benzoic Acid O S-CoA Benzoyl CoA + H2N COOH O S-CoA + AMP + PPi Benzoyl CoA O NHCH2COOH + HS-CoA Hippuric Acid Glycine FIGURE 2. (1964). A single oral dose of 250 mg of benzoic acid was quantitatively metabolized to hippuric acid and excreted in the urine within 4 hours. 1999). 1990). it was excreted almost completely as hippuric acid after systemic administration. These results have been confirmed in several animal studies. cerevisiae was used as a model system for testing antioxidant or prooxidant properties of benzoic acid and other weak organic acid food preservatives (Piper. This may be related to differences in the ability of liver and kidney cells to carry out glycine and glucuronic acid conjugation (for review.. Griffith (1929) found that this mechanism accounted for 66% to 95% of benzoic acid fed in studies in which the quantities were far in excess of those that might be ingested from foods preserved with permitted levels of benzoate. 1991).. They were also mutagenic to the yeast mitochondrial genome but only in the presence of oxygen..

Sax and Lewis (1989) list benzoic acid as a human skin and eye irritant. 1991). Nishiyama et al. on bacteriuria and pyuria in elderly women (Avorn et al. a natural source of benzoic acid (Table 2.. ASSAY Because benzoic acid is volatile in steam. extraction with organic solvents may be advisable (Lueck. In an oral provocation test with 29 patients. Occasionally. For further information.1). The concentrations of several preservatives were determined and daily intakes were estimated in a survey of Japanese foods (Ishiwata et al. 1996) and two Spanish reviews of benzoic and sorbic acids (Frias et al. These authors also described a procedure known as COMPACT (computer-optimized molecular parametric analysis of chemical toxicity). A novel impedance-splitting method was evaluated for the microbial analysis of benzoic and sorbic acids in selected foods (Kroyer and Futschik. 1991). Several excellent reviews of the toxicologic and safety aspects of food additives used as antimicrobials or antioxidants have been written.. The authors stated that beliefs about the effects of cranberry juice on the urinary tract might have microbiological justification. Benzoic acid and its salts have been given a recommended average daily intake level of 0 to 5 mg/kg of body weight (Pollard. Microbiological assays for benzoic acid have also been reported. Reversed-phase. They also cited data that indicated that sodium benzoate may be both an experimental teratogen and a mutagen. Consumption of nonalcoholic beverages containing benzoic acid accounted for about 87% of the daily intake.. In addition to occasional hypersensitivity reactions. Parke and Lewis (1992) stated that the main toxic effects of benzoic and sorbic acids were various allergic responses in humans. the reader should also consult the proceedings of a World Health Organization conference (Anon. At the end of the study. it can be quantitatively isolated by acid steam distillation from foods. One procedure used two different genera of yeasts. 1980. high-performance liquid chromatography was used in the second study to quantitate sodium benzoate in three types of soda beverages (Woodward et al. The findings suggested that use of a cranberry beverage reduced the frequency of bacteriuria with pyuria in older women. steady increases in the incidences of bacterial food poisoning in Europe during the previous decade along with the emergence of “new” bacterial pathogens indicated that misplaced concern for potential toxicity of preservatives had resulted in increasing rather than decreasing health risks to consumers.. 1995). 1996). Subjects were randomly assigned to consume 300 ml/day of a standard cranberry beverage or a placebo drink for 6 months. 1996). bacteriuria with pyuria was found in 28% of urine samples in the placebo group but in only 15% of the group randomized to the cranberry beverage. This procedure predicted if food additives were potential substrates of the cytochrome P450 family of enzymes and therefore susceptible to becoming potential carcinogens. 1990). and quantitative analysis of these preservatives in various food extracts . the release of histamine and prostaglandin from mucosa was significantly increased by sodium benzoate exposure. Benzoic acid in human plasma and urine has been quantitated using a gas–liquid chromatographic technique (Sioufi and Pommier. and the minimum detectable concentration of benzoic acid was approximately 100 ppm (Ellerman. E.. 1980). In their opinion. A medical study was conducted to determine the effect of regular intake of cranberry juice. Both benzoic and sorbic acids had COMPACT ratios that were moderate. parameters were optimized. Both methods were adopted as official first action. 1977). 1979).Sodium Benzoate and Benzoic Acid 37 Some adverse reactions to benzoate ingestion have been reported (Schaubschlager et al.. 1994). For example. The first involved spectrophotometric determination of benzoate in ground beef (Maxstadt and Pollman. 1997). The estimated daily intake of benzoic acid was 11 mg/person based on the consumption of foods analyzed in this survey. severe anaphylaxis may occur (Michils et al.. Concentrations of 10 ng/ml could be detected in both substances. 1980).. coli was used as the test organism. Two early collaborative studies should be noted.

Advantages of the method were noted. GLC/TLC. fruit juices. D. 17:231. Biotechnol. ultraviolet. 1994. NMR. 1996). Agric. nuclear magnetic resonance. (1991) Kakemoto (1992) Khan et al. high-performance liquid chromatography. The metabolism of aromatic compounds by thermophilic bacilli. REFERENCES Abdullah.12. Appl.. Inhibitory effects were strongly affected by pH. inoculum amount. to Sharon McGinness for her patience in typing the manuscript. and it was recommended for screening purposes. The use of impedance microbiology to study effects of environmental factors on fungi has been reported by Nielsen (1991). J. orange juice (as adulterant) Yogurt Various Beverages Various Various beverages and foods Orange juice (as adulterant) Packaged vegetables Various beverages and foods Various Orange juice (as adulterant) Various Various Topical ointments Reference Boland (1991. a biosensor prepared from tyrosinase-containing mushroom tissues has been developed and used to assay for the presence of benzoic acid (Wang et al. highperformance thin-layer chromatography. gas chromatography-mass spectrometry. James and John. D. J. and to Dr. 1988. W. UV. (1995) Pant and Trenerry (1995) Mannino and Cosio (1996) Vogels et al. and temperature. E. 2002). to Barbara Borrelli for library searches. Adams. I dedicate this review to my sons. (1995) Lee (1995) Montano et al. Supercritical fluid extraction of carboxylic and fatty acids from Agaricus spp. Three isolates of the heat-resistant fungus Neosartorya fischeri were tested for their resistance to sodium benzoate. selected ion monitoring. I. 1999) Serrano-Olea et al. gas–liquid chromatography/thin-layer chromatography.. (1994) Chen and Fu (1995) de Luca et al. Procedures for both qualitative and quantitative determination of benzoic acid and sodium benzoate can also be found in the Association of Official Analytical Chemists’ Official Methods of Analysis (AOAC.38 Antimicrobials in Food TABLE 2. D. GC-MS. (1990) HPLC. citrus juices. mushrooms. Food Chem. ACKNOWLEDGMENTS Sincere appreciation is given to Miriam Chipley for compiling the reference citations. was performed.12 Recent Developments in Detection and Quantitation Methodologies for Benzoic Acid Methoda Various analytical methods HPLC GC-MS HPTLC Reversed-phase HPLC SIM/GC-MS Liquid chromatography Reversed-phase HPLC Capillary chromatography HPLC NMR spectroscopy Capillary electrophoresis Spectrophotometric/GLC/TLC UV spectrophotometry a Food Fruits. HPTLC. In addition. . SIM. C. 42:718. Recent developments in detection and quantitation methodologies for several food products are outlined in Table 2. Tod Miller for figure preparation. Biochem.. M. Young. and Ribbons. and Games. (1996) Waldron and Li (1996) Fazio (1999) Mahalanobis et al.

Bosund. J. Med. Bach. J. L. Cairo. p. and Park... Herrmann. A. W. 7:373. 1. 9:151.. Mikrobiol. Effects of food preservatives and antioxidants on colony formation by heated conidia of Aspergillus flavus. and Hagenmaier. AOAC (Association of Analytical Chemists International). E. K. L. 41:472.Sodium Benzoate and Benzoic Acid 39 Akira. 1980. Steiner. Association of Analytical Chemists International. H. EC Directive 95/EC. 97:1463. E. J. 1999. Food additives other than colours and sweeteners. N. and Abraham.. R.. Anal. Food Microbiol. G. J. National Research Center. 1980. L. Acta Aliment. B. 175:4851. 1996. Technol... Appl. M.. R. Anonymous. J. 1979. I. Altenschmidt.. Barker. Seifen. 1981a. 2001. sodium benzoate. Morgan. 37:155. Postharvest Biol.. B. C. M.. and Jones. Environ. Chen. R. R. Organic acids. The influence of combinations of chemical preservatives on the growth of food poisoning and spoilage microorganisms. Naguib. Acad.A. 46:771. J. Ed. Beuchat. New York. Sensitization of Listeria monocytogenes to low pH. and glycerol and sucrose esters of fatty acids. Microbiol. 1988. D. Naguib. Beuchat. G. 1991. Baldwin. Microbial reduction of aromatic carboxylic acids.. 1993. Aflatoxin B1 production in the presence of preservatives and antimicrobial agents. H. A. Oele Fette Wachse 116:345. N. B. M. 74:143.. Silliker.. and Pohland. Combined effects of solutes and food preservatives on rates of inactivation of colony formation by heated spores and vegetative cells of molds. R. G. Microbiol. O. Fruits and fruit products. Improving storage life of cut apple and potato with edible coating. Pohl. Eur. H. 48:52. 471. In Microbial Ecology of Foods. 271:751. Amer. Off. Bacteriol. Food Sci. B. In Official Methods of Analysis of AOAC International. Benzoate degradation via the ortho pathway in Alcaligenes eutrophus is perturbed by succinate. Microbiol. Sci. Bodin. Comm. Fruits and fruit products. Microbiol.-L.. W. F. Beuchat. Boland.. Studies on the effect of fruit-coating polymers and organic acids on growth of Collectotrichem musae in vitro and on post-harvest control of anthracnose of bananas. M. Gaithersburg. Baird-Parker. Lebensm. pp. J. J. and Wahem. Nisperos. X. J. I. AOAC International. and Stringer. organic acids.. Z. A.. Ser. F. S. Reduction of bacteriuria and pyuria after ingestion of cranberry juice. Cunniff. Food Sci. 16th ed. Res. Oswald. W. A.. 2. R. L. Res. Preservatives and their practical application in cosmetics. F. Banks.. Academic Press. I. A.. Bizri. E. R. S.. Toxicologic evaluation of certain food additives. F. Environ. Choodnovsky. F. 1980. 67:1594. Arfmann. Chem. and osmotic stress by ethanol. K. Mycolog. Assoc.aoac. L. 63:2765. and Waider. R.. H.htm. B. 1995. 1988. M. Klein. 1990. Farrant. Biochem.. D. P... Monane. K. Lindon. Chapter 37. H. Gurwitz. Aragao. 39:1178. MD. Scholtyssek. Camden Food Preserv. p. 126. 1994... Domsch. Park. Eds. F.. Technical memorandum. Al Zaemey. Vol. 1960.R. Bauer. Appl. C. and Lipsitz. Uribelarrea. The effect of some preservatives on growth. E. E. Hung. T. A.coenzyme A in a denitrifying Pseudomonas sp.K. 249. Boland. Citric acid and antimicrobials affect microbiological stability and quality of tomato juice. V. Influence of organic acids on heat resistance characteristics of Talaromyces flavus ascospores. A. Enrivon. Glynn. and Thompson A.. Andersson. G.org/index. Beuchat. Chem. Anonymous. 37–1. U.. J. and Gedek. J. Magan. Nicholls. 6:97 Beuchat. A. Environ. 59:130. 2002. WHO Food Addit. J. 13:793.. Anal. Ed. J. K. and Lindley.. L... D.. Ampe. Int. D. In Proceedings of the International Symposium on Mycotoxin... Naturfosch. A. Appl. S. Gaithersburg. Siemer. J. M. and Snygg. Plant. Influence of pH on the total uptake of benzoic acid by Proteus vulgaris and baker’s yeast. L. 221:297. . Technol. M. Official Methods of Analysis of AOAC International... High-field deuterium nuclear magnetic resonance spectroscopic monitoring of the pharmacokinetics of selectively deuterated benzoic acid in man. 1994. L. 1996. Available at: http://www. 1994. 1981b. and Fuchs. Avorn. Hoelzl. L61:48. Assoc. N. M.. and Nicholson. 6:161. Montgelas. Off. 1997. J. 1994. F. Appl.. lipase production and lipase activity of Pseudomonas fluorescens. G.. Assoc...Comparison of anti-Vibrio activities of potassium sorbate. C. E.. J. New aerobic benzoate oxidation pathway via benzoyl-coenzyme A and 3-hydroxybenzoyl. Caddick. R. J. Physiol. The bacteriostatic action of benzoic and salicylic acids. H. R. E. 1981. Vol. MD. G. Synergistic effects of potassium sorbate and sodium benzoate on thermal inactivation of yeasts. 1993.

The action of benzoic and salicylic acids on metabolism of microorganisms. M. 1977/88. J. Chromatogr. Y. L. 1993. P. M.H. R.C. M. Int. and Cerniglia.. P. 2002.. Vadkartiova. Buazzi.H. sweeteners and antioxidants in foods by paired-ion liquid chromatography. In Handbook of Food Additives. Simultaneous determination of preservatives. R. R. 1962.. Boylan. J. V. A. 31:113. N. and Chiang. p. and Tanner. J. Eds. and Coote. Collier. pH and Acidity. Microbiol. and Young. Acott. and Davidson. J. Chen.. 1995. Ed. J... Food Process. Food and Drugs.A. 1972. Chromatogr. Agric. D. Chichester. P. 2nd ed. 65:476. M. High-pressure liquid chromatographic assay of dextromethorphan hydrobromide. 179(18):5943–5946. S. p. p. L. Burlini. Chakrabarti. M. Chemical Rubber Publishing. Sci. Bacteriol. L.. J. Chipley. Branen. Yeh. E. New York. E. Food Res.. P. U. p. Government Printing Office. R. Sodium benzoate and benzoic acid. 159:220. Nebe-von Caron. p.. Pellegrini.. J. M. P. R. Microbios 70:199. 1974. Bracey. Food Sci. M. C. Eds. N.. B. Cleveland.. Tortora. Metabolic effects of benzoate and sorbate in the yeast Saccharomyces cerevisiae at neutral pH. Wiley-Liss. 92. and Coote. Title 21. J. H. G. Preservative agents in foods. Chipley. P. J. strain ADP1. 1909.. E. Food Microbiol. R. Food Prot. and Varma. Addition of fumaric acid and sodium benzoate as an alternative method to achieve a 5-log reduction of Escherichia coli O157:H7 populations in apple cider. S. M. T. C.. Chen. Food Microbiol. and Marth. 307. Technol. 1996. Chandrasekaran. 50:1.4-dinitrophenol and N.. E.. Y. R. Dept.40 Antimicrobials in Food Bosund. S. T. 1980. G. Antimicrobial food additives. New York. Long.. Appl. 37:72. 20:379. Aspergillus niger and Penicillium sp. L. 1994. 1. and Daly. C. 1995. 1999. Meth. P. Alt. A.. Sodium benzoate and benzoic acid. 1976. International Commission on Microbiological Specifications for Foods. Comes. D. 1996. Preserv. Eds. Weak acid preservatives block the heat shock response and heat-shockelement-directed lacZ expression of low pH Saccharomyces cerevisiae cultures. Characteristics of sodium benzoate injury of Listeria monocytogenes. B. Mode of action and microbial resistance mechanisms. 1993. 11:331. Bull. Holyoak. In Microbial Ecology of Foods. In Antimicrobials in Foods. R. J. N. New York. Microbios 10:115. and Keenan. 115. and Uraih. Yeast 2:93. Brul. N’-dicyclohexylcarbodiimide on cell envelopeassociated enzymes of Escherichia coli and Salmonella enteritidis. and Jilek. Food Sci. R. India 31:441. Corlett. L. E. Jr. 1994. U. and Labuza. 41:43. A. Chipley. guaifenesin. H... New York. and Neidle. Adv.. Methods for preprocessing and freezing of shrimps: a critical evaluation. 1986. J. sorbate and benzoate.S. and Beelman. S. and Guerritore. E. 40:352. Inhibition of Aspergillus growth and aflatoxin release by derivatives of benzoic acid... 88 Code of Federal Regulations. benK encodes a hydrophobic permease-like protein involved in benzoate degradation by Acinetobacter sp.. A.. Chittenden. H. R. or sulfate reduction. P. J. Cheng. 1998. Technol. P. J. Microbiol. Washington. B. F. C. 26:636. N. S. R. H. and Herter. Staphylococcus aureus challenge study in an intermediate moisture food. C. W. 13:133. and Brown. 11. A. Zygosaccharomyces bailii as a potential spoiler of mustard. Slavikova. 1988. J. J. Marcel Dekker. Arch. 140:1085. D. I. Furia. Environ. H. 1997. Pacifieo. Eds. K.. to propionate. Chen. In Microbial Transformation and Degradation of Toxic Chemicals.. Determination of the intracellular pH (pHI) of growing cells of Saccharomyces cerevisiae: the effect of reduced-expression of the membrane H+ATPase. J.L. T. E. . Chipley.. and Piper. 1992. 1983. Microbiol. The sensitivity of halotolerant Aspergillus flavus.. Facheris. Synergistic effects of weak-acid preservatives and pH on the growth of Zygosaccharomyces bailii. S.. Academic Press. B. 41:918. Marcel Dekker. M. R. Young. H. Parts 100-199. 2000. D. M. P. and Fu. Colberg. iron. D. Nichols. W. Effects of 2. 1980. Anaerobic degradation of nonhalogenated homocyclic aromatic compounds coupled with nitrate. J. Branen. Cole. F.. In Antimicrobials in Foods. and Davidson.S. J. C. 11. L. Buchta. P. Microbiol. Food Sci. Antimicrobial and physicochemical properties of methylcellulose and chitosan films containing a preservative. Vol. J. L. and sodium benzoate in an expectorant syrup.. an inhibitory action partially relieved by respiratory deficiency.

1989. 2. Int. Food Prot. Appl. Control of microbiological quality and shelf-life of catfish (Clarias gariepinus) by chemical preservatives and smoking. E. a means toward product stability. and Kelly. A. R. J. J. Ellerman. Floros. L. H.. J. 8:335.. A.. J. 1996. 47–1. 1980. A. 1988. 1999. A. New York. Doyle. 1929. Chem. J. Simultaneous determination of sorbic acid. M. 1996. Vol. Brini. J. Technol. Ed. Farber. Burdock.Sodium Benzoate and Benzoic Acid 41 Cruess. Academic Press. Assay of sorbate and benzoate by the disc plate method.... and McFeeters. V. J. R. Food Microbiol.. J. Fazio. Chem. Int. Food Prot. B. Marth. 52:771. and sodium benzoate. E. Eklund. G. M. Fejerskkov. D. Soc. 1977. and Tremblay. L. 1999.. G. and Marth. benzoic acid and parabens in foods: a new gas chromatography–mass spectrometry technique adopted in a survey on Italian foods and beverages. E. Cunniff. Fleming. R. The bacterial degradation of benzoic acid and benzenoid compounds under anaerobic conditions: unifying trends and new perspectives. Food Sci. J. and Golden. Bacteriol. Food Sci. 593. Food Technol. Bacteriol. D.C. Egan. 80:465.. E.. Pichia membranaefaciens: a benzoate-resistant yeast from spoiled mango pulp. The effects of a mouthrinse containing sodium benzoate and alcohol on the quantity and metabolism of dental plaque. 1. In Food Microbiology Fundamentals and Frontiers.. E. Special report. 2000. A. Dock. M. Effect of hydrogen ion concentration on the toxicity of sodium benzoate to microorganisms. de Luca. Inhibition or inactivation of Listeria monocytogenes by sodium benzoate together with some organic acids. and Salminen. 1994. J. J. 1991. Eds. C. 2002. Washington. C. R. J. J. Eckert. Efiuvwevwere. M. 51:525.1988. H. 40:104. P. On the mechanism of inhibition of gluconeogenesis and ureagenesis by sodium benzoate. 48:423. 83.. Food Additives Contamin. 17:363. M. Sodium benzoate in the control of growth and aflatoxin production by Aspergillus parasiticus. Sodium benzoate inhibits growth of or inactivates Listeria monocytogenes. M. M. 1967. T. Boca Raton. 7:103. sodium benzoate. Ethiraj. Organic acids and esters. FL.16th ed. W. p. P.. Elder. 63: 1026. L. 7:427. II. AOAC International. Bacteriol. and Petgerkin. S. 12:1. Francois.. S. Food additives: Direct. J. p. 1996.. V. G. 1989. H. Dakin. New York. G. Cosmet. W. Vol. J. and Richert. 63:904. H. E. Incidence and behavior of Listeria monocytogenes in meat products. R. F. T. H.. ASM Press. D. Eyles. M.. P. Elsevier Applied Science. H. El-Shenawy. Appl.. L. and Junja. Marcel Dekker. p. T. Eklund.. D. Chemical preservatives and natural antimicrobial compounds. 505. J. Cyr. L. Fenaroli’s Handbook of Flavor Ingredients. 154:141. Marcel Dekker. Food Prot.. 42:3. India 25:63.. Antimicrobial agents. Chapter 47. Davidson.C.. Eur. E. Survival of Escherichia coli O157:H7 in apple cider as affected by dimethyl dicarbonate. J. Rev. 161. E.. Dent. Davidson.. sodium bisulfite. Dairy Technol. . and Warth. D. In Mechanisms of Action of Food Preservation Procedures. O. FEMS Microbiol.. G. The response of Gluconobacter oxydans to sorbic acid and benzoic acids. Food Prot. Food Sci. D. Heat inactivation of Escherichia coli O157:H7 in apple cider containing malic acid. El-Gazzar.. G. Ed. M.. T. P. Ed. Davidson. S. Biol. 1987. H. J.. Torgeson. A. Fenaroli. J. 32:13. Ed. In Fungicides. Eds. Effect of benzoate on the metabolism of fructose 2. 46:91. 61:832. W. p. F. S. 1990. Food Technology. Branen. J. J. Gould. H. J. In Food Additives. CRC Press. In Listeria. and Montville. London. In Official Methods of Analysis of AOAC International. 50:305. Antimicrobial agents.6biphosphate in yeast.. E. Gaithersburg. L. and Ajiboye. El-Shenawy. J. E. 4th ed. G. Biochem. and potassium sorbate. J. and Suresh. Van Scjaftingen. The interference of non-ionic emulsifiers with preservatives. New York. T. Ed. and Marth. and Linton. MD. Aust. Assuring microbial and textural stability of fermented cucumbers by pH adjustment and sodium benzoate addition. M. De Navarre. 1998. 1956. Pharmacol. 13:441. M. J. D. and Quattrucci. and Hers. 2nd Ed. A. 1989. 287. Passi. P. p.. A. O. Biochem.. C. Beuchat.. P. I. B. D. and Scheie. Thompson. Listeriosis and Food Safety. J. H. Danielsen. K.. Inhibition of growth and uptake processes in bacteria by some chemical food preservatives. A. 1995. Application and use of post harvest fungicides. 1986. M. and Marth. 1909. 1986. P. 2001. and Bailey. B. Fisher.. P. The fate of sodium benzoate in the human organism.. J.

Bacteriol. 1991. Antimicrobials: Assuring food safety. Appl. Giese. Ed. and Beuchat.. Roberts. 566. E. L. Agric. I. K. J. E. M. I. Soc. Resistance of acid-habituated Escherichia coli to organic acids and its medical and applied significance. Gardini. T. B. S. A. 60:604. C.. and Estrella. Bacteriol. Resurreccion. 1994. Henriques. S. J. 1993. R. Infect. C. Anal. Gabel. J. Food Technol. 1978. I. Nature 241:321. V. IL. FEMS Microbiol.. H. Assoc.123. Urinary excretion of hippuric acid after administration of sodium benzoate: (Biological monitoring 1). Consumer evaluation of chicken after washing in trisodium phosphate or lactic acid/sodium benzoate solutions. E. and Fletcher. A. Environ. 1976. Wiss.. Binding of benzoic acid by lipids and proteins in acidic foods. A. Harwood. 49:63. 1995. 1990. Technol. Function of lipophilic acids as antimicrobial food additives. and McFeeters. Gardner. D. Alimentaria 27:109. Lea and Febiger. 1973. Omori. J.. J. Pharm. W. Hathcox. American Oil Chemists’ Society. Hewala. Importance of phenolic compounds for the characterization of fruit juices. 1979. and Chan. J. F. A. Sheu. Catabolism of benzoate and monohydroxylated benzoates by Amycolatopsis and Streptomyces spp. guaiphenesin. Fujii. Lett. Guerzoni. Gerhardt. E. Brown.. 1990. C. C. J. 1996. U. Jpn. Quintas. Harvath. R. 1989.42 Antimicrobials in Food Freese. Extrusion of benzoic acid in Saccharomyces cerevisiae by an energy-dependent mechanism. 177:2396. Benzene production from decarboxylation of benzoic acid in the presence of ascorbic acid and a transition-metal catalyst. Am. 1995. 1929... G. 32:177. Frias.. R. M. Heintze. 1989. and Duan.. Fernandez de Simon. Lett.. A. Mechanism of growth inhibition by lipophilic acids. and Block. T. and Preservation. and Eichenlauv.. sodium benzoate and oxomezanine in cough syrup. E. Lawrence. J. W. A. J. Kamath.... 10:767. 1986. Sierra. Academic Press.. K. L. C. Mechanisms of action of food preservation procedures. Microbiol. C.. 1983. V. 56:1459. New York and London.. Interaction between inhibition factors on microbial stability of fruit-based systems. J. and Loureiro-Dias.. R. Hani. Benzoylated amino acids in the animal organism. H. Expression and characterization of Campylobacter jejuni benzoylglycine amidohydrolase (hippuricase) gene in Escherichia coli. Preservative effect and stability of benzoic acid in herring salad. In The Pharmacological Effect of Lipids. M. 9:171. C. L. Knorr. Gould. K. W. Gould. Agric.. 10:1. Alvarez. A method for the investigation of the origin of glycine. p. 1997. F. A. Lebensm. and Rowbury. and Ogata. and Gibson. Microbial anaerobic aromatic metabolism. J. C. S. F. Technol.. Kabara J. Stability-indicating HPLC assay for paracetamol. Chem. W. 1921. In Disinfection. Wiss. Bacteriol. Griffith. J. London. 1978. Giese. Mechanisms of Action of Food Preservation Procedures. IV. Food Technol. E. Harwood. 143:1877.. A. p. Philadelphia. and Hardisson. Eds. J. Ed.1995. 25:473.. B. Uptake of benzoate by Rhodopseudomonas palustris grown anaerobically in light.. Preserving agents in meat products – a real necessity? Fleischerei 46:6. 67. J. C.. M. 48:101. Freese. J. Taguchi. Grundy.. Microbiol. G. A. A. 82:415. G.. 165:504. 1997. L. B. J. Anaerobe 3:1. W. Elsevier Applied Science. 1994. p. Food Chem. Bromatological and toxicological aspects of the preservatives benzoic and sorbic acids. M. Food Chem. Food Microbiol.. Ganzfried.. Eds. 1995. Developments in beverage additives. Perez-Ilzarbe. C. Gomez-Cordoves. 8:211. Antimicrobial preservatives in pharmaceuticals. 1997. I. 179 (2):301–309. Lett. 1984. J. S. and Gibson. Champaign. M. F. 11:147. T. and Galliers. 27:71–93 . E. Food Hyg. Sterilization.. and Vaidyanathan. Leensm. In Food Microbiology: Advances and Prospects. M. 21:97. S. and Lawrence. and Fuchs. Hwang. L. Haribabu. C. Degradation of substituted benzoic acids by a Micrococcus species. C. Shedding light on anaerobic benzene ring degradation: a process unique to prokaryotes? J. Enhancement of granulocyte chemiluminescence with hydroxyl radical scavengers. V. J. G. Food Sci. Int.. 1968. Biol. K. 41:693. and Skinner. Goodson. Immun. T. Grund. Microbiol. The relative action of preservatives in pharmaceutical preparations. R. 1992. 40:1531. Heider. Appl.

S. Basic Microbiol. D. Y. J. Food Prot. Brown. Koch. Potential growth of Escherichia coli 0157:H7 in soft Hispanic type cheese. D.. J. 27:185. B. Quantitative high performance thin layer chromatographic determination of organic acid preservatives in beverages. and Beuchat. A. S. 2001. S. 1999. M. R. Storage of polymer-coated apples. C. 1989.. I.. H. F. 834. Ed. D. M. 594:253. F. Initial enzyme reactions involved in catabolism of aniline. J. . Jeffrey. A. Jay. McMullin. Effect of weak acids on amino acid transport by Penicillium chrysogenum: evidence of a proton or charge gradient as the driving force. Koodie. and Schreier. 174:4986. S.. Sterilization. Lea and Febiger. Kallio. Food Sci. F. Eur. J. Food Microbiol. M.. 22:127. 1988. 1992. Janke. Resnick. Activity of the plasma-membrane H+-ATPase. and Odashima. and Sakazawa. R.. Ind. Cuskey.. M. Chem. Acta..) 42:1154. S. D. Microbiol.. C.. Chem. P.. and Coote. M. Hunter. and Puchs. Kolmos. 1993. Aspen Publishers. Kakemotto. I. 1995. S. Kasrazadeh. C. 17:855. 1971.-P. J. Simultaneous determination of sorbic acid. Int. Ascospore heat resistant and control measures for Talaromyces flavus isolated from fruit juice concentrate. 1977. and Dhople. Pathol. Liquid Chromatogr. L. T. Appl.-A. Products of enzymatic reduction of benzoyl-CoA. The effect of sodium benzoate as a preservative for carbonated beverages. J. Crimmins.. Cole. G. Microbial reduction of benzoate to benzyl alcohol. W. Khan. Murawski. 48:337. Modern Food Microbiology. M. 1994. 39:1830.. Food Microbiol. The preservative action of acid substances in food. C.. Chromatogr. Food Microbiol. 52:1252.. J.. J. C. S.. Uda. (Lond. Inter. Pischevaya Promyshlennost USSR 3:66. W. P. 28:509. A. A food preservatives analytical method. J. Gaithersburg. and Preservation. and Genigeorgis. 1994. 1987. Microbios 104:167. Efficacy of selected chemicals for killing pathogenic and spoilage microorganisms on chicken skin. Kato. The bactericidal action of benzoic acid and sodium acetate on the gram-negative flora of dialysis fluid.. Res. H. a key reaction in anaerobic aromatic metabolism. 2000. Kasrazadeh. Stratford. Bacteriol. In Disinfection. Food Prot. P... and Stanier. 1988. 1994. D. 2nd ed. M. and Siebert. J. J. H. 1977. J. Inc. Chemical food preservatives.. W. A. K. and Prauser. Scand. K. dehydroacetic acid and benzoic acid by gas chromatography-mass spectrometry. Regulation of the β-ketoadipate pathway in Alcaligenes eutrophus. 47:189. An 117 and An 213. Massayuki. Jager. A. 107:476. Ottoway. S.Sodium Benzoate and Benzoic Acid 43 Holyoak. Washington. J. J. Bound aroma compounds from the fruit and the leaves of blackberry (Rubus laciniata L.. phenol and benzoate by Rhodococcus sp. 1996. J. K. L.. Schumann. H. J. Biodeterior. H. U. Environ. 6th ed. 211:649. Potential growth and control of Salmonella in Hispanic type soft cheese. and Sherma. Technol. S. J. A. C. Agric. 113:1184. 1973. 1976. Chromosome tests with 134 compounds on Chinese hamster cells in vitro: a screening for chemical carcinogens. Ingram. Biol.. S. Hugo. I.. M. King. Bacher. Hsiao. J. 48:327. M. P. S. and Halbrook. 1985. H. Effect of quinic acid on the growth of wild yeasts and molds..). 25:289. p. Eisenreich. Proceedings of the 22nd Annual Meeting of Soft Drink Technologists. Chapman. Modeling the inhibitory effects of organic acids on bacteria.. Straube. J.. Ivanov.C. Ahtonen. H. E. U. and Seel. B. 62:3158. J.. and Sarimo. and Olsen. Kimble. Johnson. 1992. Hwang. Int. 1991. N. A. M. 1956. L. 1991 The degradation of preservatives by microorganisms. Acid tolerance of Escherichia coli 0157:H7 and its survival in apple juice.. and optimal glycolytic flux are required for rapid adaptation and growth in the presence of the weak acid preservative sorbic acid. J. J.. M. B 84:259. Mutat. Int. Ishidata.. M. Bacteriol. J. Humpf. H. and Coppock. and Genigeorgis. F. G. 8:4. C. and Rogozhnik.. M. J. Konishi. 52:1855 Kelly. R. Microbiol. 58:19. Bacteriol. Characterization of Pseudomonas putida mutants unable to catabolize benzoate: cloning and characterization of Pseudomonas genes involved in benzoate catabolism and isolation of a chromosomal DNA fragment able to substitute for xylS in activation of the TOL lower-pathway promoter. W. Critical steps in degradation of chloroaromatics by rhodococci. R. Dorofeev. L.. Jr. Block. 1975. P.. Food Chem. MD. Philadelphia. Biochem. Are preservatives still necessary? Food Market. P. B. 1995. Al-Mofarji. B. J. Agric..

R. Food Prot. 1997. 1998. Microbiol. C. P.. 1986a. T. I. Adaptation to sublethal environmental stresses protects Listeria monocytogenes against lethal preservation factors. Maxstadt. Assoc. Appl. Microbiol. Degradation of benzoate by Pseudomonas solanacearum. 179:7975. 1996. K. Cosmetic preservatives. 76:1213. Lambert. Smith. K. M. and Yousef. M. Mechanism of benzoic acid uptake by Saccharomyces cerevisiae. Dent. and Casolari. and Fontenaille. 51:1009. Manganelli. Anal. and Block. Environ. 1958. K. Appl. Pharm. 1975. C. L. Lambert... A. and Cosio. M. P. and Ishizaki. and Nagel. T. In Disinfection. G. G. K. 26. Gonzalez. C. and Nagel. Lee. Hoct..769. Microbiol. Marwan. Microbiol. 1980.. 8:311. Microbial inhibitors of cranberries. 1984. K. C. Food Chem. Gonzalez. H. Lin. J. J. 1982. Mechanisms of acid resistance in enterohemorrhagic Escherichia coli. E. D. 1996. Chem. and Stratford. Martin. Microbiol. Appl.. and Chien. Environ. 41:363.. 1996. A. Determination of benzoic and sorbic acids in food by microdialysis sampling coupled with HPLC and UV detection. Kruk. Inhibition of Escherichia coli trimethylamine-N-oxide reductase by food preservatives. J. Marwan. A.. Springer-Verlag. S. A. Benzoic acid as a fungicide.. S. Eds. 55:699. 45:241. sorbates. 1968. J. 1979. 1990. Thermal resistance of Bacillus stearothermophilus spores in different heating systems containing some approved food additives. A. 1992.. and Hollifield. G. J. Performance of selective media for enumerating Zygosaccharomyces bailii in acid foods and beverages. British Patent 794. 1993.. Mahalanobis. K. Microbiol. Anal. C. W. 78:80. New York. and Payne. Mannino. P.. Sensitivity of yeasts to sorbic and benzoic acids and their salts. Sensitization of thermally injured spores of Bacillus stearothermophilus to sodium benzoate and potassium sorbate. p. Food Sci. Clin. S. M. Effect of a benzoic acid-detergent germicide on denture-born Candida albicans.. 1995. Ital. benzoates. E. 1997. W. Off. and Pollman. J. Anal. Acta. . Mazas. Lopez. Lett. M. Lawrence. J. E. C. Appl. Manowitz. E. J. L. and Mahadevan. 23:187. A. Lewis. 1996. R. D. A. J. Application of a new impedance-splitting method for the microbiological analysis of food preservatives. Abshire. C. S. J. 1991. and Kolstad. Lambert. Assoc. 553:284. Prosthet. 1996. Biophys. G. and Roy. Appl. Lou. J. Microbiol. Int. J. ascorbates and added blood in ground beef: collaborative study. 1986b.. Baik. Diachenko. 27:331.. 91:383. 59:652. 40(2): 249–252. 63:1252. Marlatt. J. 1996. Antimicrobial Food Additives. Studies of aroma constituents bound as glycosides in tomato. P.. Food Sci. R. Urate transport in human blood cells: activation by ATP. Weak-acid preservatives: modelling microbial inhibition and response. J. J. Bennett. R. J. Chem. p. K. Off. Appl. S. Kubota. M. M. 62:3094. and Bernardo. Lea and Febiger.. Eur. IFT annual meeting: Book of Abstracts.. 52:98. Spectrophotometric determination of sulfites. Bacteriol. and Lee. Assoc. Lueck. J. Blankenhorn. B. D. Lett. Lopez.. A. A. L.. A. J. C. H. Makdesi. 51:559. Nyman. H. and Foster. and Futschik. Chem. Lucas-Heron B. 86:57. Diversity of bacterial capabilities in utilizing alkylated benzenes and other aromatic compounds. Agr. particularly for citrus fruit.. Survey of benzene in foods by using headspace concentration techniques and capillary gas chromatography. B. Protein induced in Escherichia coli by benzoic acid.. Sterilization.. Environ. Pfanzenkr. R. Chapin. A. 23:257. J. M. Liquid chromatographic determination of benzoic acid in orange juice: interlaboratory study. M. W. Dose-dependent pharmacokinetics of benzoic acid following oral administration of sodium benzoate to humans. 1980. G. and Slonczewski. Quantitative determination of infinite inhibition concentrations of antimicrobial agents. Biochim. M. 63:667. Kumari. Appl. J. Int. J. Food Prot. T. J. Ind.. Philadelphia. Microbiol. J. Lang. S. J. S.44 Antimicrobials in Food Kroyer. A. Conserve 58:23. Pfanschutz. Basu. 555. and Bernardo. Y. S. J. W. Pharmacol. A. R. Simultaneous spectrophotometric determination of benzoic acid and salicylic acid contents in ointment by least squares method in matrix form. Sci. Ind. and Preservation. McNeal. Martinez. N. 1983. Off. J. Macris. Gonzalez. 30:503. Appl. and Beuchat. 1999.. J. 1986.

W. Acta. C.. Benzoyl-coenzyme-A-monooxygenase.. C. 1990.. E. Food Sci. and Sugmura. and Carrol. 52:335.-H. 1988. N. Ann. F. M. Matsumura. A.. L. J.. R. L. Kurita. 46:495. A. and Ezenweke. A. J. Pharmaceut. S. 35:83. K. V. 2001... Parke. Rev. D. J. Oh. 66:1201.. Altenschmidt. K. S. 1990. 7:1147. P. A. Rejano. New York. Appl... and Kaneda. Microbiol. and Fuchs. and benzocaine in the hairless guinea pig. 1995.. Niku-Paavola. Contamin. Evaluation of the antimicrobial effects of sodium benzoate and dichlorobenzyl alcohol against dental plaque microorganisms. A. J. Pollard. 1995. Food Sci.. R. V. Safety aspects of food preservatives. and Trenerry. A. and Haikaara. Pelayo. N. R... Olea-Serrano. Lancet 337:1424. P. A. A. 1999.. 1977. Duchateau. J.. 1980. Egner.. M. Ostergaard. Analysis of preservatives in foods by using steam distillation with ammonium sulfate saturated solution. Niemetz. Parish. and Gould. High performance chromatography determination of chemical preservatives in yogurt. Medic. EMBO J. M. and Frisvad. 227:161. 9:162. and Perez-Mateos. Nielson.. U. The growth of yeasts in grape juice stored at low temperature. . Miller. 31:121. Appl. J. R. E. Inhibitors of poly (ADP ribose) polymerase induce sister chromatid exchanges.. R. 1991. C. P. 1995. 1961. S. 17:4257. Free Radic. M. M. F. J. C. 25:706. I. J. L. benzoic acid. Commun. 57:460. 1994. and Poppoff. D. Determination of benzoic acid in packaged vegetable products. Mahe. and Marshall. 53:219. U.. Lichtin. and Kuchler... Sanchez. L. P. H. M. O. G. Neosartorya fischeri as measured by an impedimetric method. H. 59:1258. G. a flavin-dependent hydrolyase.. 1989 Growth and fumitremorgin production by Neosartorya fischeri as affected by food preservatives and organic acids. Res. Food Prot. Effects of combined antimicrobial agents on fermentation initiation by Saccharomyces cerevisiae in a model broth system.. J.. Fungistatic effect of organic acids. Y. An in vitro study. and Rejano.. In vitro skin absorption and metabolism of benzoic acid. Eur. and Bronaugh. Food Hyg. M.. 97:1311. 1994. Liquid Chromatogr. V. Anaphylaxis with sodium benzoate. Food Chem. Microbiol. E. Int. Kurozuka. Trop. Oukuni. N. 1995. Technol. p. V. D. and Yernault. J. 14:709.. and Christensen. Piper. Analyst 120:2483. Formulation and shelf life of a bottled pawpaw juice beverage. The determination of sorbic acid and benzoic acid in a variety of beverages and foods by micellar electrokinetic capillary chromatography. L. Odontol. 86:29. L. Cubana Med. New types of antimicrobial compounds produced by Lactobacillus plantarum. Nielsen. Sanchez-Lopez. some properties and its role in aerobic benzoate oxidation via gentisate in a denitrifying bacterium. I. 1990. Microbiol. H. Purification. T. 56:1735. 9:561. Albury. Nishiyama. Montano. D. Okoli. M. 1999. 1994. J. Montero.Sodium Benzoate and Benzoic Acid 45 Michils. M. Sanchez. D. and Levis D. and Noguera-Revilla. Brucker. The effect of inhibitors and high pressure treatment to prevent melanosis and microbial growth on chilled prawns (Penaeus japanicus). The in vitro efficacy of salicylic acid. R.. Miwa. 1979... Preservative and temperature effect on growth of three varieties of the heat-resistant mold. J.. A. Mattila-Sandholm. Tohda.. 1991. IV. W. Soc. and Kaspar.. J. Pandjaitan. Pant. A. J. Food Sci.. J. O. A. J. Biochem. Holyoak. T. Lopez-Caballero. M. J. Thompson. and deCastro. W. Sakr. 27:1219. E. Food Addit. Jpn. Oikawa.. Enhanced inhibition of Listeria monocytogenes by glycerol monolaurate with organic acids.. C. In Food Preservatives.. Pederson. G. 235. Piper. L. Scand. P. Food Sci. 1997. L.. M. C.. Coote. Beuchat. 53:240. J. Food Sci. Escherichia coli O157:H7 acid tolerance and survival in apple cider.. S. C. N. A. Y. Yeast superoxide dismutase mutants reveal a pro-oxidant action of weak organic acid food preservatives. E. Moscoso-Vizcarra. 1991. Bacteriol.. V. Appl.. Pathways of benzoic acid dissimilation in Enterobacteriaceae. p-aminobenzoic acid. 1992. Vandermoten. (Paris) 128A:199. Morioka. Food Microbiol. Int. Res. Van Nostrand Reinhold. D. L. Awano. Kanai. Laitila. Russell. Muhbauer. Legislative aspects. boric acid and diiodate thymol solutions on several species of dermatophytes. 66:197. Biophys. 1998. Eds.. Montano. M. G. Biochem. G. The Pdr12 ATP-binding cassette ABC is required for the development of weak acid resistance in Saccharomyces cerevisiae. Biol.. Nathan.. Processing and storage of lye-treated carrots fermented by a mixed starter culture.

Appl. Thermal death rate of ascospores of Neosartorya fischeri ATCC 200957 in the presence of organic acids and preservatives in fruit juices. G. O.. Environ. J. J. and Bompeix. Appl. C. 47:1299. U.. S. R. State Hort.. J. D. and Freese. R..46 Antimicrobials in Food Ponce de Leon. 7th ed. Van Nostrand Reinhold. Food Sci. A. J. Hyg. Allergy Appl. M. Splittstoesser.. L. Eng. O. Sreevalsan. 1984 Models for mineralization kinetics with the variables of substrate concentration and population density. A. 36:185. Zabel. J. J. M. Kimura.. C in brine containing sodium benzoate. Arch. Rajashekhara. D. Gen. Benzoic acid as a natural compound in cultured dairy products and cheese. E. Quay. and Senn. 31:66. Praphailong. 2001. 1996. Kain. Fla.. Food Microbiol. Agents Chemother. Biomed. The effect of pH. R. Biochemistry 16:3348. V. H. B. K. J. E. and Modi. . J. and Ruegg. 1980.. 1997. Sofos. J. Chromatogr. Bul.-M. L. D. J. Indian J.. Induction of Nectria galligena mutants resistant to benzoic acid and study of their aggressiveness towards immature apples.. R. and Adams. Bosset. Scanga. Control of yeasts and molds with preservatives. W. and Churey. Release of mediators from human gastric mucosa and blood in adverse reactions to benzoate. N. Inst. Mclellan. N. Mitteil. 49:402. Effect of pH on the interaction of benzoate and D-amino acid oxidase. 71:191. 1990.. Boca Raton. Shailubhai. J. P. Sardine storage at 15 deg. E. sorbic. 181:161. CRC Press.. 34:168... E. Microbiol. G. 1984. Biomass and patulin production by Byssochlamys fulva in apple juice as affected by sorbate. 45:19. 1988. J. M. Rusul. Sorbate Food Preservatives. Butikofer.. 20:166. and Conn. P. I. Food Prot. Vol. S. 14:459. Appl. Food Prot. and Fleet. sucrose. S. S. Proc. R. Degradation of benzoate and salicylate by Aspergillus niger. A.. V. Immunol. Organic acids and their salts as dipping solutions to control Listeria monocytogenes inoculated following processing of sliced pork bologna stored at 4°C in vacuum packages. J. J. Geb. R. J. Food Technol. cinnamic acid and benzaldehyde on Listeria monocytogenes.. Salmond. Antimicrob. L. F. Ind. B. Sieber.. The effect of benzoic. Sax. and Lewis R. 61:1358. O. 1991. Appl. New York. 2002. U. Mycopathologia 101:13. N. 59:226. Sofos... T. F. Sr. M. Salomon. 131:1863. 1984. Bacteriol.. N. Samelis. R. W. J. N. Clifford. Chem. Lebensm. and dehydroacetic acids on the growth of citrus products spoilage organisms.. H. J.. U. 1989. and Massay. 1989. and Booth. 1977. 1995. J. J.. I.. Pasteur. 1944. M.. Benzoic acid as a natural compound of foodsreview. 1985. Lett. N. H. Becker. Effect of increase in acidity on antiseptic efficiency. W. J. Occurrence of benzoic acid in fermented milk products and cheese. The effect of food preservatives on pH homoeostasis in Escherichia coli. Simmons. and Marth. Butikofer. Saindrenan. F. Rahn. Belk.. 1977. Butikofer. R. Simpkins. 1960. and Bosset.. 5:227. C. 1998.. 1996.. Dairy J. S. Sheu. benzoate. Board. 96:97. sodium chloride.. R. and Roller. N. A study on the antimicrobial activity of mixtures of agents used for the preservation of liquids. Inhibitory effects of lipophilic acids and related compounds on bacteria and mammalian cells. G. and Alexander. Microbiol. and benzoate on the growth of food spoilage yeasts. Int. Baumann. K. 2.. R. Suresh. Ann. R. 130:2845. and Ethiraj. 7:349. Rehm. Hokkaido Univ. and Shinano. Mechanisms of bacterial resistance to non-antibiotics: food additives and food pharmaceutical preservatives. 80:345. Sieber. M.. Int. SO2 and temperature. Quantitative structure activity relationship for the effect of benzoic acid. U. Sieber. Gas chromatographic determination of low concentrations of benzoic acid in human plasma and urine. O. 80:303. and Bosset. 1963. Gen. T. V. Faculty Fisheries. Sioufi. 81:484. M. Sauer. O. Roland. H. Biol. W. N. Appl. Chitosan potentiates the antimicrobial action of sodium benzoate on spoilage yeasts. Russell. 11:217.. J. Kroll. 1982. Bacteriol. 1991. 1989. Dangerous Properties of Industrial Materials. E. 11:1772. M. and Beuchat. sorbate. C... Schaubschlager. G. W. Ramos-Nino. Exp. Sagoo. Rushing. M. Food Prot. Schade. FL. Food additives and plant components control growth and aflatoxin production by toxigenic aspergilli: a review. 76:271. and Pommier. and Smith. Hyg. Microbiol. Geb. Heat resistance of Escherichia coli O157:H7 in apple juice. C. S. E. K. J. Microbiol.. Rao.. Soc. 1975.. Seng. Lebensm. J. 1994. J.. J. Mitteil. Inoue. V. G. and Schlaak.

1999.. J. Waldron. 1999. W. Dukel.. C. E. A. D.. E. 1976. F. R.. J. Fleischerei 44:175. 87:520. Terwel.. Food Nutr.:43. N-nitrosamines in Korean ordinary soy sauce. Bacteriol. D. Teuber. Ubodi Eiroa. Effect of three preservatives on the growth of Bacillus cereus. and Li.. P. M. 44:723. N..Sodium Benzoate and Benzoic Acid 47 Steels. Biotechnol. G. H. R. 43:215. Food Prot. K. Weak-acid preservatives: mechanisms of adaptation and resistance by yeasts. 6:6. 683:47. van den Berg. Appl. 1996. F. J. 1997. Food Environ. Y. .. T. R. 34:51. 11:549. Uraih. 1980.. H. Vogel. R. Food Microbiol. D. P. Plant Cell Physiol. Jardim. Roberts./Feb. and Oliver. L. R. The influence of fermentation on the nutritional quality of dairy products.. T. 1984. J.. R. and Chipley.. and Davis. O. Wang. Can. M. R. Effect of two free radical scavengers and intermit tent warming on chilling injury and polar lipid composition of cucumber and sweet pepper fruits. Jan. 60:1262. Technol. J. R. 110:37. 2:157. and Baker. Effect of phenolic compounds on mycelial growth of Fusarium and Penicillium species. W. Stijve.. Uraih. Thayer. and van den Greef. W. Appl. A. Stratford. Active transport of benzoate in Pseudomonas putida. Smyth. 66:841. 17:125. and Rossi. Dtsch. and Davenport. P.. Runschau.. preservative-resistant spoilage yeast. Verduyn. Kane. K. James. High performance liquid chromatographic determination of low levels of benzoic acid and sorbic acid in yogurts. 128:1749. and Rogers. Vitamin analysis by microbial and protozoan organisms: response to food preservatives and neutralization salts. Postma. Jie Liu. J. Thompson. Thomas. Y. sorbic and other weak acids used as food preservatives. Y.17:289. J. J. R. Facts and fiction. Plant Physiol. 1995. H. Thayer. J. S. Zygosaccharomyces bailii — a profile of characteristics and spoilage activities. W. Scheffers. 1996. and Van Dijken. and Stratford. and Lee. Partial characterization of the mode of action of benzoic acid on aflatoxin biosynthesis. J. Bernard. T. 1992. M. 19:551. and Lambert. N. 1988. and Wheelis. Inhibition of aflatoxin production in groundnut with benzoic acid derivatives and possible toxic effects of their aromatic residues. 1995. Sung.. Antimicrobial action of some GRAS compounds against Vibrio vulnificus. Ciencia e Technologia de Alimentos 15:128. Food Market.. D. Warth. S. Food Sci. Appl. and Gombas. N. 1981. Korean Soc. N. World Ingred. M. D. J. J. and Hischenguber. 1976. O. A. Chromatogr. O. G. A. Biomed. W. J. S. R. A. N. Microbiol. I. Zygosaccharomyces lentus: a significant new osmophilic. 1985. 20:243.. 1979. J. Investigation of a pulsed-laser thermo-optical absorbance detector for the determination of food preservatives by capillary electrophoresis. Sveum. Vogels.. J. J. Effects of various acids and salts on growth and aflatoxin production by Aspergillus flavus. N. Arch. Lebensm. Food Australia 51:26. Sanit. and Offonry. N. C. Microbiol. 1982. Mechanism of resistance of Saccharomyces bailii to benzoic. Uraih. C. B. Thomas. vero cytotoxigenic Escherichia coli and Staphylococcus aureus. M. Effect of benzoic acid on metabolic fluxes in yeasts: a continuous-culture study on the regulation of respiration and alcoholic fermentation. Microbios 31:93. 23:1580. 1977. N. Tompkin. Microbiol. A. B. D. R. V. 1999. L. M. J. V. Microbial stability of tomato concentrate preserved by hurdle technic and stored at room temperature.. S. J. 1992. Wang. 80:81. Eitenmiller.. Nutritional and practical significance of food additives. Wang. L. W. Hwang. E. J. and Wheelis.. Ethylene production by chilled cucumbers (Cucumis sativus). K. and Adams. 1996. 1995. C. on plates with gradients of pH and sodium chloride concentration. L. Detection of adulteration in orange juices by a new screening method using proton NMR spectroscopy in combination with pattern recognition techniques. Microbiol.. Guidelines to prevent post-processing contamination from Listeria monocytogenes.. R. capable of growth at low temperature. C. 1977. Yeast 8:501. J. 1993. C. Scott. Wimpenny. 1979. T. J. Food Microbiol. J. J... R. Cassity. M. E. M. Tas. Mushroom tissue-based biosensor for inhibitor monitoring. Food Additives Contamin. Microbios 17:51.. and Ware.. Characteristics of a benzoate permease mutant of Pseudomonas putida. H. Food Technol. R. Sun. Gen. S. Voigt. Int. R. R. and Chipley.

97:370. Environ. Microbiol. 1988. A. D. Heffelfiinger.. Commun. Appl. Chem. Antimicrobial effectiveness of potassium sorbate and sodium benzoate against Zygosaccharomyces bailii in a salsa mayonnaise. High pressure liquid chromatographic determination of sodium saccharin. Principles of Biochemistry. F. B. M. and Kearney. R. A. Yagi. Warth.. and Besser. McGraw-Hill. D. 1989a.. 20:847. Academic Press.. Effect of benzoic acid on glycolytic metabolite levels and intracellular pH in Saccharomyces cerevisiae. Wind. 1984. 54:2091. Flower-inducing effects of benzoic acid and some related compounds in Lemna paucicostata. the use of a wild strain of Pseudomonas cepacia. Degradation of o-nitrophenol and m-nitrophenol by Pseudomonas putida. Relationships among cell size. Handler. Warth. 1989b. Inactivation and injury of Listeria monocytogenes in a minimal medium as affected by benzoic acid and incubation temperature. M. 1995. Appl. propanoic and benzoic acids and to methyl paraben and pH. P. Fate of enterohemorrhagic Escherichia coli O157:H7 in apple cider with and without preservatives. P. G. E. Watanabe. Effect of benzoic acid on growth yield of yeasts differing in their resistance to preservatives. Appl. Environ... J.. J. Zeyer. and intracellular pH. P. L. A. 8:343. E. L. P. A. Warth. 1993. p. D. C. and preservative resistance in yeast species. . Environ. Food Microbiol. A... Food Sci. 1966. J. New York. Appl. A. 135:1383. J. Microbiol. 1989c. T. A.. 1997. Mechanism of action of benzoic acid on Zygosaccharomyces bailii: effects of glycolytic metabolite levels. 1991b. A. Microbiol. D. D. 1979. Microbiol. Yousef. P. Appl. 2. 1991a. and Takimoto. I. Zhao. D. membrane permeability. Off. L. New York. Zani. 54:560. Evaluation of preservative effectiveness in pharmaceutical products. K. El-Shenawy. Woodward. 1980 Spectroscopic demonstration of an initial stage of the complex of D-amino acid oxidase and its substrate D-∝-aminobutyric acid. Vol. Environ. Microbiol. 59:2526. P. Food Chem. energy reduction. Maggi. R. 83:322.. and Ruggles. A. Webb. 62:1011. 1979. 55:2995. Plant Cell Physiol. and Smith. Transport of benzoic and propanoic acids by Zygosaccharomyces bailii.48 Antimicrobials in Food Warth. and Mazza. E. 58:1257. J.. Biophys. 1964.. Int. 3rd ed. Warth. Appl. J. K. A. and Marth. sodium benzoate and caffeine in soda beverages: collaborative study. Warth. D. 32:238. White. 1989. and Douzou. Lang. Res. Santi. Enzyme and Metabolic Inhibitors. J. Microbiol. J. E. Relationships between the resistance of yeasts to acetic. Doyle.. Anal. Assoc. Minutello. and Restaino. 57:3415. Agric. P. H. 57:3410. C. Microbiol. B. L. Gen. Environ.. Biochem. J. A. Food Prot. 245.

......................... Inc...................... 1981)................................................................................................................... Stopforth....50 Antimicrobial Activity .................. M.............. by E......... Studies of that period also examined the biological properties and established guidelines for the safety of the compound..............................................................................73 Regulatory Status .............................................................................. 1976........................ Sofos.............................................................................................................54 Inhibition ............................................... Miller and C.............................. Research efforts during the late 1950s and in the 1960s dealt mostly with the mechanism of sorbic acid 49 ........................ 1989).................. 1989)..........................................................................3 CONTENTS Sorbic Acid and Sorbates Jarret D.................... The first U..................................... 1980... 1980.............61 Vegetable Products................ respectively (Lück...................................................................................................................................................................... M......... The compound was first isolated as naturally occurring from the oil of unripened rowanberries (sorb apple or mountain ash tree) by the German chemist A...............57 Applications ..........................................................................................67 Mechanisms of Action .......................................................................................................... patent on sorbic acid was awarded in 1945 to C...........................63 Fruit Products. 1945)................................................................................................71 Detection and Analysis .............. both in Germany and in the United States..................................................... Sofos....................74 References ................................................ Gooding in the late 1930s and early 1940s........ W........61 Dairy Products ................................... John N.....................................................................................................................................64 Bakery Products ........................68 Toxicology and Safety ............................................... These developments resulted in the extensive use of sorbic acid and its salts as preservatives of foods and other materials throughout the world (Lück....................... Busta Introduction ..67 Other Applications ................ von Hoffmann in 1859 in London (Sofos................................... for their discovery that the compound was a good fungistatic agent for application to foods and food wrappers (Gooding......................... 1976.............................................................................................................49 Chemistry ........................ and Francis F.57 Interactions.................................. Sorbic acid became available commercially during the late 1940s and early 1950s when testing as a preservative agent increased.......65 Miscellaneous Food Products ... 1981............. Gooding and Best Foods.............................. Sofos et al......75 INTRODUCTION The antimicrobial and preservative properties of sorbic acid were first discovered............................................................ 1979a... Sofos and Busta...............................65 Meat Products .54 Selective Action ..........................................................................................................................................................................................................S..........................................................................................55 Degradation .......................................................... Sofos and Busta................................................

and it constitutes the most soluble form of sorbate (Table 3. however. but it increases with temperature. The extensive use of sorbates as preservatives is based on their ability to inhibit or delay growth of numerous microorganisms. The sodium salt has a water solubility of 32% (wt/vol). Inhibition of microbial metabolic function is probably the result of morphologic alterations in the structure of the cells. are collectively known as sorbates and are used widely throughout the world as preservatives for various foods. especially in combination with reduced nitrite levels for a reduction in nitrosamine formation (Sofos et al.22. 1985. 1989). Currently. Sofos et al. 1982). depending on differences in microbial types. probably through action on spore membranes or protease enzymes involved in germination (Blocher and Busta. Sorbic acid forms colorless flakes or needles when crystallized. especially potassium sorbate. 1989. Robach and Sofos. The solubility of sorbic acid is also higher in alcohols. some microbial strains are resistant to inhibition by sorbate or even metabolize the compound. stability. and environmental factors.2% and is insoluble in fats. species and strains. potassium sorbate) of sorbic acid may find more frequent applications in foods because of their greater solubility in water. it is available as a white freeflowing powder or as granules.50 Antimicrobials in Food activity against microbial growth and the potential application of the compound in additional food products (Sofos and Busta. has an antimicrobial potency of 74%. The salts (e. Sodium sorbate is a white fluffy powder and is commercially available as an aqueous solution that is sensitive to oxidation and stable for only a few weeks (Lück. with a molecular weight of 112. sorbic acid and its more water-soluble salts. and . Robach and Sofos. while the health effects of sorbate were reexamined (Sofos.2% w/v at 20°C).g. sodium. There is variation. Sofos. substrate properties. 1989. The mechanisms of antimicrobial activity of sorbates are not fully defined (Sofos and Busta. Inhibition of bacterial spore germination (Sofos et al. 1986.. 1979d) by sorbate is believed to occur at the connecting reactions of the germination process. In general. sorbates are considered effective food preservatives when used under sanitary conditions and in products processed using good manufacturing practices. which makes it valuable as a delayed-release form of sorbic acid (Sofos.1).2). alterations in cell membranes.13 (Table 3. and potassium sorbates. or both. 1982). and bacteria. changes in the genetic material... 1989). and inhibition of enzymes or transport functions (Sofos. Sofos. in inhibition of microorganisms by sorbates..15 g per 100 ml.. however. The conjugated double bonds of sorbic acid are also reactive and can be influential in its antimicrobial activity as well as on the quality and safety of food products (Wedzicha and Brook. CHEMISTRY Sorbic acid is a straight-chain. 1981. especially ethanol. as animal feeds.4-hexadienoic acid. and in other industrial applications (Sofos. The solubility of sorbic acid (Table 3. 1981. The calcium salt is formed as a white odorless and tasteless powder. CH3CH=CH-CH=CH-COOH). During the 1970s extensive research was performed on the potential for using sorbic acid and its salts as antibotulinal agents in meat products. 1979a. 1980). 1989. and it has a weak but characteristic acrid odor and acid taste. including yeasts. as pharmaceuticals and cosmetics.2) in water at room temperature is only 0. In food systems the solubility of the compound is estimated as higher than 50%. The volatility of the compound in steam is useful in its isolation for quantitative detection in foods or other materials. 1986). molds. trans-trans unsaturated fatty acid (2. Sofos. on an equivalent weight basis with the acid. Commercially available salts include calcium. Sofos et al. The carboxyl group of sorbic acid is highly reactive and results in formation of various salts and esters. Under certain conditions. the pH of the solution. The molecular weight of potassium sorbate is 150. Good solubility (58. 1981). Potassium sorbate may be manufactured as a powder or granules and. 1989). and in glacial acetic acid. 1989). 1981. 1992). Calcium sorbate has a water solubility of 1.

the partitioning of sorbic acid between the water and fat phases of foods depends on the pH of the food. °C Heat of combustion at 25°C. Sofos. at 20°C 120°C 140°C Boiling point.5 10 3 0. malonic acid. 1995). maximum% Source: From Sofos (1989). Btu/Lb Alkalinity/acidity Vapor pressure. maximum% Heavy metal content. Sorbic Acid 2. It is proposed that EDTA-Fe2+ complexes catalyze sorbic acid autoxidation. maximum ppm Arsenic content.. increasing the production of carbonyls. 1989). 1989). °C (COC ASTM D-92) Ionization constant at 25°C Density at 20°C g/ml Melting range. Thus.22 — — 1. and β-carboxylactolein (Arya and Thakur. Ethylenediamine tetraacetic acid (EDTA) enhances sorbate degradation probably as a result of iron scavenging from the packaging material (glass or polypropylene) through EDTA complexation. °C 760 mm Hg 50 mm Hg 10 mm Hg Purity. The partition quotient is increased from 3. The rate of oxidation in aqueous solutions is increased at lower pH values by the presence of light and acids or increased temperatures. acetaldehyde. and other ingredients present (Sofos. appropriate packaging materials.927 — Potassium Sorbate 2.13 126 1.0 10 3 — <0.1 g — — — — — — >98 1. the higher iron content in the EDTA-containing systems might be responsible for the higher rates of sorbic acid degradation as well as for the increase in nonenzymatic browning observed (Campos et al. 1989.1 N HCL per 1.36 Decomposes higher than 270°C — ml 0. Oxidation of sorbic acid yields various carbonyl compounds. mm Hg. 1981). 1988.2 ease of manufacture make potassium sorbate the most widely used form in food systems (Sofos. 1992). whereas in the pure dry form they are stable. malonaldehyde.0 to 7.. which take part in nonenzymatic browning. 1996).4-Hexadienoic acid potassium salt CH3-CH=CH-CH=CH=COOK 150.Sorbic Acid and Sorbates 51 TABLE 3. Aqueous solutions of sorbates are unstable and degrade through oxidation. % Water content.8 ml 0. Oxidation and losses of sorbic acid are inhibited by antioxidants. maximum% Ash. such as crotonaldehyde.73 ∞ 10-5 — 132–137 11. The amount of sorbic acid in the aqueous phase is reduced in food systems of higher lipid content because the solubility of sorbic acid in fat is approximately three times that in water (Gooding et al. In general. formic acid.0 with increased levels (50%) of soluble food components.1 Physical and Chemical Properties of Sorbic Acid and Potassium Sorbate Property Chemical name Molecular formula Molecular weight Flash point. 1992). . acrolein.001 10 43 Decomposes 143 119 >98 0. such as sugars and salts (Sofos and Busta. and anaerobic conditions (Sofos.1 N NaOH to 0. the type and amount of fat.4-Hexadienoic acid CH3-CH=CH-CH=CH-COOH 112.

00–15.00 0.30 6. packaging material. processing conditions.28 1. 100°C Corn oil.30 Potassium Sorbate 58.00 34.00–5. 10% solution Sucrose. 50°C Water. 20°C Methanol. Sodium chloride at 3. 5% solution Sodium chloride.60 45.03 — 0. 1984.50 12.02 0. amino acids.10 16. 20°C Sucrose. 85.04 11.00 64.10 0.14 24.00 55.9) Water.16 0. metal ions. 1994). 20°C Cyclohexane.29 — 12.30 2.26 0. 20°C Carbon tetrachloride. 5% Ethanol. 50°C Pentane.00 12.30 0. 20°C Acetone.00 20.00 45.4) Water.00 0. 60% solution Sodium chloride. 75°C Ethyl ether. 20°C Corn oil.08 2.1) Water.52 Antimicrobials in Food TABLE 3.52 1...00 47. 20°C Propylene glycol.20 — — 61. 1983.01 — — 0. 20°C Source: From Sofos (1989). 20% Ethanol.0% enhanced sorbic acid destruction. antioxidants. 50°C Propylene glycol.02 0.50–12.80 0. 1990). 15% solution Acetic acid.5% Citric acid.60–14. 50°C Benzene.15 0.b. glacial Lactic acid. Gerschenson et al.25 0.00 48. but at 13. 1986a. oxidative degradation of . Vidyasagar and Arya. 20°C Cottonseed oil.50 0.01 58. 1987. 20°C (pH 5. 20°C Glycerol.5% and 8. 1989.22 1.07 0.00 — <0. 95% Ethanol. 100% Propylene glycol. Sofos. 50% Phosphoric acid.00 0. 10% solution Sodium chloride.80 2.60 1.15 0.20 12.31 9.11 0. Sorbic Acid 0. 1980.90 0.00 28. 50% Ethanol.34 8.08 0.0% it had a protective effect (Guerrero et al. 40% solution Sucrose.05 5.90–14.55 4.01 Losses of sorbic acid during storage of foods depend on sorbate levels.15 0. 100°C Pentane. 20°C (pH 3. and storage temperature and time (Bolin et al.20 0. 1992.2 Solubility (%) of Sorbic Acid and Potassium Sorbate Solvent Water.01 0.00 5.00 0.00 — — — <0.12 0. 50°C Soybean oil.10 0. types of food materials present.40 54. In general. moisture content.. other additives present. 75°C Benzene.00 — — — — 57. light. the nature and pH of the food.50 2. 20°C (pH 4. 25°C Benzene. 85% Ethanol. 25°C Pentane.

the water-soluble (6. broadly tested. and sorbic anhydride. Sofos and Busta. The lipid and protein fractions that appear to bind sorbic acid or its degradation products were identified with the help of 14C-sorbic acid. A mixed anhydride of sorbic and palmitic acids called sorboyl palmitate has found some application in yeast-leavened bakery products. some of these compounds are sorboyl palmitate. and sorbic aldehydes were also effective antimicrobials (Troller and Olsen. which are available as powders. Commercial application of such derivatives. 1963). moisture.3%). however. and the acetic acid-soluble components (0. water-soluble formulations of sorbic acid in the form of epoxidized polymers of polyvinylpyrrolidone (PVP) containing covalently bonded sorbic acid (polymeric esters of sorbic acid) and complexes of PVP with hydrogen-bonded sorbic acid have been shown to be more effective fungicidal agents than sorbic acid polymeric esters (Tzatzarakis et al. salt-. and widely used as antimicrobial agents. Sofos. 1992). 1989). 1989. and acetic acid-soluble fractions suggested that 14C activity tends to be associated with the low-molecular-weight components of these fractions. but commercial production of sorbic acid has mostly involved the methods of oxidation of . strong offflavors and odors.6 to 9. amine salts.. 1989). a significant amount of sorbic acid is not recovered by extraction with methanol. is hindered by disadvantages.2) (Dudman. salt-.5±0. 1995). In addition. and water activity (Giannakopoulos and Guilbert. A proportion of the binding is with the low-molecular-weight components of these fractions (Jideani and Wedzicha. the salt-soluble (3. the ethanol-soluble (4. Although the salts of sorbic acid have been commercially developed. When wheat flour doughs are treated with sorbic acid and heated. Troller and Olsen. alcohols. Fractionation of the lipids and proteins from fura showed the 14C to be associated with the petroleum spirit crude fat extract (28±5%). fura. 2000. and it is influenced by the nature of the food and its components and by processing. and potential health problems (Lück. was an effective mold inhibitor over a wide pH range (3..5±0. structure.7%). 1976). it is important to be able to predict or to control migration of sorbic acid during processing and storage of foods because it can influence product preservation (Sofos. 1981). and dark storage areas.9±0.6%). 1992). to a lesser extent.7%). Specifically.b. Commercial applications of sorbates include use of the free acid or its potassium and. Jideani and Wedzicha (1994) showed that when sorbic acid is incorporated into millet dough a significant proportion (approximately 40%) of the additive becomes unavailable on extraction with methanol. 1980.8. and amide derivatives. 2001). The results show that sorbic acid binds with the lipid components. sorbohydroxamic acid. These include esters. New controlled-release. suspensions. with a dissociation constant (pKa value) of 8. they are shipped and stored in fiber drums with a moisture barrier in the carton wall. and storage conditions (Sofos. these products have low toxicity and are effective in a wide range of concentrations and thus may be considered for food application or feed protection. ethyl sorbate. moisture. other derivatives of sorbic acid have also been examined. physical state. sorbamide. Charvalos et al. the water-. 1976). and ethanol-soluble components of fura from pearl millet. and light. Several procedures have been patented for the manufacture of sorbates (Sofos. such as low solubility in water. handling. These cartons should be kept closed in cool. granules. calcium or sodium (salts). or solutions. methyl sorbate.2 ± 0. 1990). In general. Dialysis of the ethanol-. Because the compounds are sensitive to heat.. 1963. dry. Vojdani and Torres. Uptake and diffusion of sorbate into foods should be influenced by properties of the substrate. Sorbamide was 1000 times more inhibitory than sorbic acid against yeast alcohol dehydrogenase (Martoadiprawito and Whitaker. and a further loss is apparent after the dough is cooked to give the traditional West African food. This suggests that sorbic acid is indeed reacting with thiol groups in the flour protein.. sorbohydroxamic acid. 1994).Sorbic Acid and Sorbates 53 sorbic acid in foods is less extensive than in aqueous solutions. such as composition. It is suggested that decomposition of the adducts requires the sulfur atom at position 5 of the substituted 3-hexanoic acid to be protonated and that a proportion of sorbic acid present in flour-based products may be reversibly bound to components of the food (Khandelwal et al. aldehydes. and the acidlabile nature of this binding is the result of the instability of the thiol-sorbic acid monoadducts under acid conditions (Khandelwal et al. 1986a.

and sorbic acid. Monilia. 1988). 1998. were able to depress mycelial weight and aflatoxin production by Aspergillus flavus or Aspergillus parasiticus cultured in yeast extract-sucrose (YES) broth (Fan .. 1970.. Pullularia. Bracey et al. Aspergillus.b. Gliocladium. 1984. 1984. Hansenula. 1999).30%. Tsai et al. CO2 atmospheres. Mold species inhibited by sorbates belong to the genera Alternaria. reaction conditions. 1992. Ulocladium. Penicillium. 1970.. The inhibitory action of sorbate against yeasts was first documented in the 1950s in fermented vegetable products. potassium sorbate used at 150 ppm resulted in final counts. use of a hurdle approach resulted in a synergistic effect (El Halouat and Debevere. Ascochyta. Geotrichum. candy. 1972). Tong and Draughon. ANTIMICROBIAL ACTIVITY INHIBITION Sorbic acid is active against yeasts and molds. 1983. Melnick et al. 1996. syrups. Torulaspora. 1989). Mucor. Cryptococccus. 1954a.80 to 0. neither sorbate nor propionate. Rosellinia. 1984. Heterosporium.. Candida. Pepularia.b.. Trichoderma. Torulopsis. tomato products. 1999. Rhizoctonia. Melnick and Luckmann. 1954a. Cephalosporium. 1993. Sorbates inhibit the formation of mycotoxins by various molds in culture media and in foods (Bullerman. 1954a. Effective antimicrobial concentrations of sorbates in most foods are in the range of 0. Under an 80% CO2 atmosphere. salad dressings. 1998. Chaetomium. 1983. 1989). Garza et al.54 Antimicrobials in Food 2. 1985. Bhattacharya and Majumdar. jellies. The effectiveness of sorbates against yeasts has been documented by numerous studies (Emard and Vaughn. Liewen and Marth. the inhibitory amounts of sorbic acid were reduced by 40% to 50%. sausages. Matamoras-León. and other suitable salts in the presence of necessary additives. and Zygosaccharomyces (Sofos. Lennox and McElory. Skirdal and Eklund. Huang and Armstrong. Curvularia. whereas no growth was observed when the preservative concentration was increased to 220 ppm (El Halouat and Debevere. Although. Alkaline salts of sorbic acid are formed by its neutralization with alkali metal carbonates. Huang and Armstrong. fruits and juices. 1985a. Yeasts inhibited by sorbates include species of the genera Brettanomyces. 1985a). after 21 days. cakes. Gourama and Bullerman. Endomycopsis. 1959. Cladosporium. Baldock et al. Saccharomyces. and purification procedures follow the second method. 1996). sorbates inhibit yeasts in fruit juices. 1998). Truncatella. Zygosaccharomyces rouxii is considered resistant to sorbate treatments. and smoked fish (Liewen and Marth. Pederson et al. Helminthosporium. Smith and Rollin.. applied at 10 mg/ml. Rhizopus. Sporotrichum. cottage cheese. Kaul et al. bread. which is most widely used. Fan and Chen. 1957. 1993..b. A major application of sorbates in food products is their use for inhibition of molds in cheeses (Chichester and Tanner. Sofos. 2000). dried fruits. 1954a.4-hexadienal and the condensation reaction of ketene and crotonaldehyde. Extensive research during the 1950s demonstrated the impressive effectiveness of sorbates against yeasts and molds and resulted in the extensive use of the compounds as fungistatic agents in many foods. lower than the inoculum level (103 CFU/g).. Kloeckera. Liewen and Marth. hydroxides. Ferguson and Powrie. Numerous patents describing catalysts.. Fusarium. Sorbates also inhibit molds in butter.02% to 0. 1961. Humicola. jams. 1952. Pichia. wines. Debaryomyces. Ascosphaera. 1982. Phoma. Colletotrichum. as well as against many bacteria (Sofos.. 1984. 1986. 1979. Botrytis. Roland and Beuchat. Under this atmosphere and at aw values in the studied range (0. and others. Rhodotorula. In addition to their effectiveness in fermented vegetables. 1996). 1984.90). Marshall and Bullerman. Sporobolomyces. such as carbonated beverages.b.. Use of sorbates for inhibition of yeasts is especially important in low-pH and/or intermediate water activity (aw) products. Cunninghamella. Piper et al. Pestalotiopsis. Hurdles used in combination included aw. 1979. Deuel et al. 1984. El Halouat et al. and meat and fish products. and chocolate syrup (Restaino et al. 1985. However. Numerous studies have also documented the effectiveness of sorbates against molds (Emard and Vaughn. Aly. grains. Kivanç. 1952. Geminder.

Arthrobacter. which could have a protective effect on E.04% to 0. Micrococcus. 1980a. Sofos.. Seward et al. Potassium sorbate sensitized cells of L. Koodie and Dhople. Alteromonas. Rusul and Marth. potassium sorbate slightly decreased L. sorbates can inhibit Gram-positive and Gramnegative. Enterobacter. additives present. In other studies (Kouassi and Shelef. The combination of 0. Salmonella. and mesophilic and psychrotrophic microorganisms. 1989). Proteus. under certain conditions. and some may even metabolize the compound (Sofos. Sanchis et al. 1985. Monnet et al. strains. SELECTIVE ACTION Microbial inhibition by sorbate is variable and depends on species. Stimulation of mycotoxin formation by low levels of sorbate depends on species and strains of molds. Moraxella.1% sodium benzoate substantially decreased Escherichia coli O157:H7 contamination and suppressed the growth of yeasts and molds. composition of substrate.. Serratia. 1985a. could extend shelf life and increase safety (Kouassi and Shelef. Bacterial species inhibited by sorbate belong to the genera Acetobacter. however. coli O157:H7 in apple cider (Koodie and Dhople. monocytogenes presence in two commercial cheese brines and thus could be used as an antilisterial agent in commercial brines. Liewen and Marth. 1995a. storage temperature.b. Addition of 0. 2000.. 1995. pH. 2001). increasing its survival. Pediococcus. which inhibited growth.Sorbic Acid and Sorbates 55 and Chen.. the addition of sorbic acid (0. Escherichia. Achromobacter. Inhibition of bacteria by sorbate appears to cause an extension of the lag phase. with a lesser influence on rate and extent of growth (Larocco and Martin. coli O157:H7. 1986. Palou et al. At the relatively high level of 1%. 1987a. Klebsiella. 1993). 1999). 1984. subinhibitory levels of sorbate may simulate the production of mycotoxins (Bullerman and Olivigni. Gareis et al. Smoot and Pierson. temperature of storage. Greer. however. aw. Staphylococcus aureus producing staphylococcal thermonuclease (TNase) retained its full activity and was not inhibited even after exposure to chilling and refrigeration temperatures when sorbic acid was applied at 0. Chung and Lee. monocytogenes and Zygosaccharomyces bailii to inactivation by high hydrostatic pressure (Mackey et al.. food-processing treatments. 1999).2% to 0. sorbic acid is considered as a more effective inhibitor of yeasts and molds than bacteria (Skirdal and Eklund. coli O157:H7 during storage of apple cider (Miller and Kaspar.1% potassium sorbate and 0. and other factors (Sofos. 1982.05% sorbic acid was found to inhibit growth of E. Bacillus.4%) to HPMC did not substantially enhance bactericidal activity (Zhuang et al.b). Kouassi and Shelef. 1997). 1981. 1989). 1988).5% (Kumar et al.b. Zhao et al. Staphylococcus. 2000). however. 1988) and in a coldpack cheese food (Ryser and Marth. 1987. 1988). type . although sorbate did not affect growth of L. 1995a.. outgrowth. Sofos.. it has also been shown that potassium sorbate or sodium benzoate did not affect survival of E. 1987). 1993. Blocher and Busta... Certain bacterial strains are not inhibited by sorbate. Aeromonas. 2000). It was concluded that combinations of sorbate with propionate or lactate. Overall.b. 1979a–d. as well as spoilage and pathogenic bacteria. Lund et al.. Clostridium. sorbate inhibited or inactivated Listeria monocytogenes in a broth substrate (El-Shenawy and Marth. 1974. However. it suppressed cysteine activation of listeriolysin. The effect of sorbate on spore-forming bacteria may be exerted on spore germination. Vibrio. A hydroxypropyl methylcellulose (HPMC) coating significantly reduced the number of viable Salmonella Montevideo cells on the surface of tomatoes. Zamora and Zaritzky. but the cost effectiveness of adding high levels (1%) of substrate is questionable. Depending on pH and concentration. 1988. Yersinia. however.. and their long-term stability in the high-salt and low pH environment of brines is not well documented (Larson et al. and others (El-Shenawy and Marth.. Tsay and Chou. Sofos.. aerobic and anaerobic. 1981. monocytogenes in broth. 1982. gas atmosphere. 1989). and/or vegetative cell division (Sofos et al. In fact. Mycobacterium. 1995a. Acinetobacter. Campylobacter. 1993. Alcaligenes. 2001). 1982. Pseudomonas. Lactobacillus. catalase-positive and catalase-negative.b. 1988. 1994). 1996). Overall. and result in spoilage of the product (Zhao et al. 1981. 1989).

Mihyar et al. 1982. which occurs during adaptation to sorbic acid. York and Vaughn.. Splittstoesser et al. 1989). and Triganopsis (Warth.. Yeast strains resistant to sorbate belong to the genera Zygosaccharomyces.. 1982. 1985. Early studies indicated that sorbate could be used as a selective agent for catalase-negative lactic acid-producing bacteria and clostridia because it was highly inhibitory against catalasepositive organisms (Phillips and Mundt. confers resistance to the inhibitory effects of sorbic acid (de Nobel et al. 1987). Blocher et al. 1977).. Functional categories of genes that are induced by sorbic acid stress included cell stress (particularly oxidative stress). Potassium sorbate or sodium benzoate resulted in complete inhibition of Z. under certain conditions some species and strains of yeasts and molds are resistant to inhibition by sorbate. 1996. 1998). mating response. and previous exposure of the organism to low levels of sorbate (Sofos. various species and strains of microorganisms exhibit different sensitivities to inhibition by sorbate. Lenovich et al. Kondaiah et al. 2001).. Exposure of Saccharomyces cerevisiae to sorbic acid caused strong induction of two plasma membrane proteins.9 mM sorbic acid at pH 4. Resistance of osmotolerant yeasts to inhibition by sorbate was acquired by preconditioning the yeast to sorbate (Bills et al.. and concentration of sorbate.. 1981. Growth of Gluconobacter oxydans in the presence of sublethal concentrations of sorbic acid before determination of the minimal inhibitory concentration (MIC) resulted in a substantial increase in the MIC within 1 hour (Eyles and Warth. 1952.. 1985). Hansen and Appleman. In general.. transposon function. Blocher and Busta. bailii in salsa mayonnaise more than sodium benzoate (Wind and Restaino. a heat shock protein of S. 1984. Variations and resistance to inhibition by sorbate may lead to failures in preservation and defective food products (Sofos. bailii tolerated 800 ppm of sorbic acid (Neves et al.. and energy generation. such as polyols (Bills et al. 1982. Restaino et al. 2001). 1951. 1997). other studies have reported either no effect or inhibition of lactics and clostridia by sorbate (Costilow et al. Varying sensitivities of bacterial species and strains to sorbate may lead to shifts in the microbial flora during storage of foods (Chung and Lee. Other proposed mechanisms of yeast resistance to sorbate at reduced aw have been related to yeast cell shrinkage and decreases in membrane pore size. 1982. 1998). 40% tolerated 500 ppm. 1994).. subsequent exposure of the cells shows little effect of solute type on sorbate resistance (Lenovich et al. However. 1983). potassium sorbate suppressed growth of Z. rouxii in high moisture prunes (El Halouat et al. cerevisiae to 0.. inoculum level.56 Antimicrobials in Food of packaging. 1983. retarding the flow of sorbate into the cell (Restaino et al. 1977. 1950. 1955). or protection of enzyme systems from inhibition by sorbate through production of compatible solutes. Another bacterium that appears to be more resistant to inhibition by sorbate than other spore formers is Sporolactobacillus (Botha and Holzapfel. cerevisiae. The induction of Hsp26. 1987.. . In addition to bacteria. Hamdan et al. Emard and Vaughn. Lynch and Potter. Lahellec et al. Piper et al. When the yeast cells have been previously adapted to sorbate in media containing glucose or sucrose. Exposure of S. however. Cole et al. most tolerated 150 ppm sorbic acid. 1985. In contrast. storage temperature. 1989). 1955.5 resulted in the increased transcription and translation (upregulation) of genes encoding 10 different proteins and the downregulation of three proteins (de Nobel et al. Bills et al. 1981.. which explains the usefulness of the compound as a preservative in vegetable fermentations.. 1982). 1982). Resistance of yeasts to inhibition by sorbate depends on species and strains. 1995). Saccharomyces. the inhibitory action against lactics by sorbate is less than that against yeasts.. 1983. pH.... one of which was identified as adenosine triphosphate (ATP)-binding cassette transporter (Pdr12). sorbate concentration.. 1988... 1971). 1954. 1955. Candida. 1988). Of 100 yeast strains isolated from spoiled foods and beverages. Torulopsis. which is essential for the adaptation of yeast to growth under weak acid stress and confers weak acid resistance by mediating energy-dependent extrusion of water soluble carboxylate ions (Holyoak et al. 1989). Vaughn and Emard. energy-requiring system that transports the preservative out of the cell (Warth. McMeekin et al. Overall. Brettanomyces. and two strains of Z. 1982. 1978. One proposed mechanism of resistance of osmotolerant yeasts has involved an inducible.

using it as a carbon source. packaging. 1989). 1975. however. dehydration of cultures. The MIC of sorbic acid increases with the size of the inoculum.6 kcal/g.. 1980).b). Mucor. In general. In general. 1989). These factors can act synergistically or be antagonistic and either enhance or negate the antimicrobial activity of sorbate (Sofos.. Liewen and Marth. coli O157:H7 from 36 to 5. prior exposure to subinhibitory levels of sorbate. such as concentration.. coli O157:H7 during storage . certain strains are resistant and can metabolize the compound. 1966. When sorbate levels are high. substantial metabolism of sorbic acid. It should be noted that 0. and other additives (Sofos and Busta. a geranium-like odor is usually associated with wines treated with sorbate and contaminated with high microbial loads. The study showed that the resistance of Z. 1977. 1992). 2000).5-diene (Edinger and Splittstoesser. Finol et al. 1982). as a fatty acid through β-oxidation. 1-ethoxyhexa-2. inoculum size. bailii to sorbic acid was largely the result of the presence of a small fraction of resistant cells and was not heritable or the result of the existence of a mixed culture (Steels et al. 1985a).. which is a volatile compound formed through a decarboxylation reaction and has a kerosene-like. temperature. 2000). Sofos. Degradation of sorbate by lactic acid bacteria has been associated with geranium-type off-odors in wines and fermented vegetables. and Geotrichum (Sofos.b. sorbate yields 6. Sofos. and type of substrate (Sofos. about a 7-fold increase in lethality (Splittstoesser et al. but an inoculum effect that may be caused by the diversity of the cells in inocula or initial contamination as regards to sorbic acid resistance (Steels et al. and environmental factors.. In addition to certain molds. some bacterial strains may also degrade sorbate under appropriate conditions. 2000). Liewen and Marth. 1989. Degradation of sorbate by molds depends on species and strains. Sorbic acid (0. microbial flora. A study found a pronounced positive inoculum effect of Z. It appears. 1954b. Other strains of molds that may degrade sorbate belong to the genera Aspergillus. pH. or binding of sorbic acid to dead cells. although many molds are sensitive to inhibition by sorbate.. Sofos. This metabolism is mostly associated with lactic acid-producing bacterial strains present as high inocula in sublethal concentrations of sorbate (Crowell and Guymon. level of inoculum. Like caproic and butyric acids.. 1985a..3-pentadiene. large inocula at high cell concentrations therefore require considerably higher concentrations of the inhibitor to prevent growth than do dilute cell suspensions. Some mold strains can grow and metabolize sorbate under certain conditions as detected in cheeses and fruit products (Melnick et al. other ingredients. Products of sorbate metabolism by molds include 1. plastic paint. Because it is metabolized like other fatty acids. caused by ethyl sorbate.1%) reduced the D50°C of E. 1995). under certain conditions. Fusarium..18% to 1. Mold strains of the genus Penicillium isolated from cheese treated with sorbate were able to grow and metabolize high (0. bailii resistance to sorbic acid activity. that there is no apparent relationship between sorbate resistance and the toxigenic properties of molds (Tsai et al. or hydrocarbon-like odor (Marth et al. 1966. 1985). of which 50% is biologically usable.1% sorbate is usually sufficient to inhibit sensitive molds (Liewen and Marth. Bullerman. 1986a. 1988). amount of sorbate present. gas atmosphere. which was not an artifact caused by insufficient growth time. Horwood et al.2 min.4-diene. 1981. 1989. 1981. sorbate is completely oxidized to carbon dioxide and water. processing. under normal conditions of alimentation. Increasing the concentration of sorbic acid from 200 to 1000 mg/L in apple cider decreased the D50°C value of E... Steels et al. there is also evidence of some ω-oxidation (Deuel et al. 1989). Lück. 1954b. 1989).20%) sorbate levels (Marth et al. 1985a–c). INTERACTIONS The antimicrobial activity of sorbates is influenced by compositional.Sorbic Acid and Sorbates 57 DEGRADATION Animals and certain microorganisms can metabolize sorbate. It appears that selection may occur in sorbate-treated cheeses for certain molds tolerant to the compound (Schroeder and Bullerman. and 2-ethoxyhexa-3. aw. 4hexenoic acid.

5%) reduced the inhibition of Clostridium botulinum by sorbate in a nutrient broth (Wagner and Busta. 5.. salt and sugars) also reduce the concentration of sorbate in the aqueous phase (Gooding et al. Sofos. 1983).. Interactions of sorbate with heat may affect the rate and extent of microbial destruction during heating. and it may inhibit the repair and growth of . 1993). however (Sofos. however. 1976. 1988). Sofos and Busta. 1976.. Robach and Stateler. and sodium chloride. however.1% sorbate (Bills et al. In certain instances.5 for propionate and benzoate. 1993).1%) enhanced destruction of E. Sofos and Busta. In general. Thus. 1955. Lück.g.. 1982. In environments of pH higher than 6.. 1941.0 to 4. it is believed that both undissociated and dissociated sorbic acids have antimicrobial activity (Skirdal and Eklund. Sofos et al. have reduced the synergistic effect of sorbate and heat on thermal inactivation of microorganisms (Beuchat. Cerruti et al. Studies have indicated that the dissociated sorbic acid also had antimicrobial activity. 1981. Acott et al. Liewen and Marth. Use of artificial saltwater prevented dissociation of the 1% sorbic acid. coli O157:H7 cells at pH 3. might not be effective owing to their increased solubility in fat. 1981). 1980. where it is needed for microbial control (Oka. Sofos and Busta. Sofos and Busta. approaching its dissociation constant (pKa = 4. 1976.. as well as dormancy and recovery of heated microorganisms (Sofos.. 1944.64 min (Splittstoesser et al. 1988). 1989).b).. Cerruti et al. 1981.0 (Raevuori. 1982. Hoffman et al. Nevertheless. 1976. 1956.76) (Cowles. 1983). Sodium chloride (1. 1981). Gooding et al. 1959. 1981a–c. 1985a). sorbates have the advantage of being effective at pH values as high as 6. solutes should increase the inhibitory activity of sorbate by reducing the aw of the substrate (Sofos. 1981. 1984. but increased levels of microbial contamination reduce antimicrobial activity. Sucrose. 1990). The increased antimicrobial activity of sorbate at lower pH values has been attributed to the increased amount of undissociated acid present.. 1993).4 (Liu et al.5 is advantageous because it allows for their use in foods of higher pH values in which preservatives. Statham and McMeekin. Sheneman and Costilow. 1979a. more than 50% of the inhibition was the result of dissociated sorbic acid (Eklund. Beuchat. 1990). which exhibited favorable antimicrobial properties against Vibrio vulnificus.5 (Bell et al.58 Antimicrobials in Food of apple juice from 18 to 5. Sorbic acid (0.. 1981c). Skirdal and Eklund. glucose. 1957. Chung and Lee. but it was 10 to 600 times less inhibitory than the dissociated acid (Eklund. act synergistically to enhance the antimicrobial activity of sorbate (Costilow et al.. certain studies have indicated antimicrobial activity by sorbate at pH values as high as 7. 1954a. This popular theory has been questioned. the maximum pH for antimicrobial activity by most other common food preservatives is lower — for example. 1980. Rahn and Conn. The increased activity of sorbates at pH values higher than 5. 1944. Increased amounts of fat in a product reduce the concentration of sorbate in the water phase. In contrast. 1982). 1983. Sorbate may enhance heat activation and destruction of spores. 1980. 1989. 1989). respectively (Sofos and Busta. sorbates should be used to preserve foods processed using good manufacturing practices. Preconditioning of Saccharomyces rouxii cells in 60% sucrose + 0. 1960).. such as parabens. 1988).. Other food ingredients (e. 1985a.0 to 5. not as a substitute for appropriate sanitation and hygienic practices. 1987. 1994). Statham and McMeekin. The antimicrobial action of sorbate is pH dependent and increases as the pH of the substrate decreases. Lund et al. 1989. sorbates can partially or totally replace benzoate even in foods of lower pH to avoid possible off-flavors caused by the higher benzoate levels needed for inhibition and to extend the range of microbial groups inhibited compared to benzoate or propionate used singly (Melnick et al. which is believed to be the effective antimicrobial form (Lück.25 and 2. whereas benzoic acid reduced it to 0.5 and 4. 1981. Lück. 1955. Sugar and salt. supporting previous evidence that sorbic acid is effective in its undissociated form (Sun and Oliver.1% sorbate rendered the cells more sensitive to inhibition by sorbate than preconditioning in 0% sucrose + 0. 1996).. Although activity is greater at low pH values. 1955. Cerruti et al. Not only are certain strains of microorganisms resistant to inhibition by sorbate. 1997).4 but not at pH 6. however... 1980. 1955.. Lowering the aw enhanced the resistance of the same organism to increasing concentrations of sorbate (Restaino et al. however.0.2 minutes.

1982. 1982. 1984. Lusher et al. The antimicrobial activity of sorbate is usually enhanced under vacuum or modified gas atmosphere storage conditions. Sofos. Specific effects. they can enhance its antimicrobial activity by increasing the concentration of undissociated sorbic acid. 1999)... rouxii to sorbate was enhanced in cells preconditioned to elevated sorbate concentrations.. 1989). Sorbic acid (0.1% had little effect on the thermal resistance of ascospores of Neosartorya fischeri.. 1980.1% potassium sorbate did not have any significant effects on the heat resistance of Bacillus stearothermophilus spores in distilled water. Elliot et al. 1985. but the effect of sorbate on thermal inactivation and recovery of injured microorganisms is variable among species and strains.. 1989). Tuncan and Martin. 1979.. 1982. tertiary butyl hydroquinone (TBHQ). 1981.. Oloyede et al.1% sorbate. 1996. 1989). vary with substrates.. 1989). Sofos. and particularly in the presence of sucrose as opposed to glucose. 1981a–d. 1983.. 1996). Davidson .2% to 0. 1983. 1982. 1989). 1981. 1999). Roland et al. Another report also indicated that potassium sorbate inhibited the heat-resistant N. 1983). sorbate inhibited induction of thermotolerance by sublethal heat shock. 1976. a true aerophilic mold involved in the spoilage of grape preserves (Splittstoesser et al. microorganisms. 1984. It is of particular interest that 0. Park and Marth. especially in media containing 0. Concentrations of sorbate as high as 0. McMeekin et al. Inhibition of microbial growth by sorbate is more effective as storage temperature decreases.. 2000). Tolerance of Z. Roberts et al. 1981. the specific anion itself may contribute antimicrobial activity (Juven. and as such would therefore be important only in improving microbiological stability through inhibitory effects on germination and/or outgrowth of heat-damaged spores (López et al. 1985).. Lopez et al.Sorbic Acid and Sorbates 59 thermally injured organisms (Beuchat. butylated hydroxytoluene (BHT). The finding that plasmolysis increased in cells grown in sucrose.. 1980. cerevisiae (Cheng et al.. Sorbic acid was found to induce thermotolerance without inducing the heat shock response through accumulation of trehalose in S. cerevisiae depending on pH (Cheng and Piper.. Huhtanen et al. Although food acids may reduce the water solubility of sorbate. Heat resistance increased substantially when cells were grown in media containing sorbate. Banks et al... 1980. 1996). Combinations of acidification and treatment with sorbate may enhance the storage stability of fruit juices even at temperatures higher than normal refrigeration (5°C) (Alli and Kermasha.as opposed to glucose-containing media provides evidence that cell morphology influences heat resistance (Golden and Beuchat. Park et al. 1992).. 1995). At low pH. 1961. 1973. Cells grown in sucrose-supplemented media tolerated higher concentrations of sorbate than did those grown in glucose-supplemented media. but growth of surviving spores that had been exposed to high temperatures was greatly inhibited by sorbate concentrations as low as 0. had increased antimicrobial activity compared with individual components (Klindworth et al. Sorbate may also eliminate the protective effect of sucrose against the thermal inactivation of yeasts and molds (Sofos. indicating that the compound should be more useful as a preservative in refrigerated foods (Pederson et al. but at pH 5. 1970. Sorbic and benzoic acids affected the thermotolerance and heat shock response of S. Low concentrations of sorbic and fumaric acids in the heating medium had little effect on the heat resistance of Eurotium herbariorum... Statham et al. Combinations of carbon dioxide and sorbate have also been reported as effective inhibitors of microbial growth (Danzinger et al.. as indicated with several food items. and types of acids (Restaino et al.007% (Splittstoesser and Churey.. 1994. Gray et al. Elliot and Gray. 1989). did not enhance the bactericidal activity of cellulose-based edible films against Salmonella Montevideo (Zhuang et al. Combinations of sorbate with antioxidants. and propyl gallate (PG). Reductions of 5 to 10 log-units of E. 1982. In addition.4%). such as butylated hydroxyanisole (BHA). Robach. 1984) and fish products (Bremmer and Statham. McMeekin et al. 1994..5 it acted as a powerful inducer of thermotolerance in the absence of sublethal heat treatment. Roland and Beuchat. 1972. fischeri (Nielsen et al. including meat (Wagner et al. however. 1984. 1984. Myers et al.. coli O157:H7 or Salmonella typhimurium DT104 in apple cider were achieved through freeze-thaw treatments in the presence of sorbic acid (Uljas and Ingham. 1988. 1984).. Splittstoesser et al.. Huhtanen and Feinberg. 1985). however.

..5..95 (Deak and Beuchat. fatty acids. 1960.93. Poerschke and Cunningham. Sorbate was completely effective against E.06%) combined with aw values lower than 0. at aw of 0. .. botulinum nonproteolytic type B to grow in the presence of sorbic acid was at least as great at 20°C as at 30°C (Lund et al. 1989). and mild heat (Guerrero et al. Sofos. Morad et al.5 in the absence acid being 0 and –1 under the same conditions but in the presence of a calculated undissociated sorbic acid concentration of 200 mg/L... interactions between two or more of these factors resulted in marked inhibition with the inhibitory effect of potassium sorbate occurring at the highest concentration (0. 1993. in a medium containing 0. Mendonca et al. 1984. 1979b. 1983. 1986a. 1978. 1995). Numerous publications and patents describe interactions of sorbate with many other factors. 1988). or at 10°C (Deak and Beuchat. bailii is highly resistant to individual inhibitory factors.. Bacillus coagulans. molds.8. Nelson et al. Seward et al.. Amano et al.. The effect of temperature. Robach et al. Increase in the acidity and/or the NaCl concentration improved the effect of all the preservatives.4 and addition of 250 ppm ascorbic acid. 1985). Clostridium pasteurianum. Kadam et al. sulfur dioxide. 100 ppm potassium sorbate. 1983. the probability of growth was approximately –6 (Lund et al. ascorbate. equivalent to a total sorbic acid concentration of 1300 mg/L..06% potassium sorbate. 1999). As indicated.. mixtures of sorbate with various antibiotics have demonstrated increased antimicrobial activity (Schmidt. including sorbate. pH 3. Penicillium glabrum was considered as the most resistant Penicillium mold tested and was found to be inhibited by the combination of 500 ppm of vanillin and 300 ppm of potassium sorbate for at least 1 month at 25°C. 1981. Robach. sorbate was more effective than benzoate against S. Variations existed. and S. These combinations offer the advantage of simultaneous inhibition of microbial growth and development of rancidity. 1987. Z. 1989.. sucrose fatty acid esters. however.. 1981. A study found that growth was not inhibited at pH 3.. Wagner and Busta. and aw 0. Lahellec et al.. propylene glycol. 1994).. and potassium sorbate was evaluated against growth of three foodborne bacterial pathogens (Bacillus cereus. antioxidants. Thomas and Wagner. and other compounds (Ferguson and Powrie. pH. The ability of C.98. these concentrations of chemicals represented a greater than 50% reduction of the amounts needed to suppress growth when used individually (Matamoras-León et al. 1993). Miller and Brown. After 14 days at 30°C in the presence of 280 mg undissociated sorbic acid/L. as well as multifactorial interactions (Sofos. aureus.. Gourama and Bullerman. 1990). Potassium sorbate was found more inhibitory against yeasts than hydroxycinnamic acid (Stead. and Clostridium butyricum by adjustment of aw to 0. coli at all temperature/pH/NaCl combinations and was the most effective preservative tested against B. In addition. with types of microorganisms. 1957. Deak and Beuchat. 1985. Bell and De Lacy. 1984. sodium chloride concentration. the log probability of growth of a single vegetative cell is approximately –4. The synergistic effect observed when vanillin and potassium sorbate were used in combination could be considered for use to reduce the amounts needed to inhibit mold growth (MatamorasLeón et al. 400 ppm sodium bisulfite. Guerrrero et al. 1993). the log probability of growth of a single bacterium at 12°C after 60 d at pH 5. 1988). 1968. and substrates. aureus when used with higher concentrations of NaCl (Thomas et al. At <25°C. Several studies have also indicated increased antimicrobial effects when sorbate was combined with various phosphates (Ivey and Robach. certain amino acids. Thomas et al. cereus. At temperatures of 12°C and below. 1986.. sorbic acid resulted in marked inhibition. 1960. 1994. 1982. 1994). 1982. TellezGiron et al. verocytotoxigenic Escherichia coli. 1990).60 Antimicrobials in Food et al. 1999). Marshall and Bullerman. Kabara. 1981.. glucose oxidase. aureus) using gradient gel plates (Thomas et al. 1994). Combinations of sorbate with benzoate or propionate may be used to expand the range of microorganisms inhibited with reduced concentrations of each preservative.97 and pH to 3. 1987. Ough and Ingraham. Interactions and increased antimicrobial effects have also been observed in combinations of sorbates with propionate. The microbial stability of shelf-stable banana puree was enhanced through inhibition of inoculated osmophilic and nonosmophilic yeasts. except nitrite when used against S. However. 1994).

APPLICATIONS Sorbates are widely used food preservatives throughout the world. dusting with a powder. purees. packaging materials. juices. dry sausages Miscellaneous: dry sausage casings. jams. They should be considered as adjuncts to proper sanitation and hygiene. cheese spreads. In the United States. olives. presence of other ingredients and preservatives. . 1992). sorbates have found wide application in various foods. salad dressings Meat and fish products: smoked and salted fish. pies.30 0.3% are tolerated.05–0. confectionary Source: From Sofos (1989). cheese dips. toppings. Application methods for sorbates include direct addition in the formulation. Amounts of sorbate used in foods are in the range of 0. In general.10 0.02–0.4). fudges. cheese spreads. concentrates Beverages: still wines. amounts of 0. equipment available. sanitation and contamination. In the United States.05–0. fresh salads Fruit products: dried fruit. sorbates may be used in any food product that allows generally recognized as safe (GRAS) food additives and in approximately 80 additional food products that have federal standards of identity (Sofos. jellies.05–0. product processing. syrups.30 In general.05–0.F. objectives to be accomplished. pharmaceuticals.3). but levels as low as 0. DAIRY PRODUCTS Cheese preservation is one of the most common applications of sorbates. higher levels may cause undesirable changes in the taste of most foods. including dairy products. federal standards of identity permit the use of sorbates as preservatives in more than 40 types of cheese. packaging.02–0. cosmetic products. low-calorie drinks Food emulsions: mayonnaise. relishes. and technical preparations that come in contact with foods or the human body. yogurt Bakery products: cakes. cake mixes. carbonated and noncarbonated beverages. and cheese food [21 C.1% may be detectable in some foods. fruit drinks. fruit and vegetable products. semimoist pet foods. as is the case with all food preservatives.20 0. The compounds find application in human foods of all types.02% to 0. doughnuts Vegetable products: fermented. and convenience (Sofos. fruit salads. animal feeds.30 0.1% to 0.3%. cottage cheese. sour cream. Although these concentrations have no major impact on food quality. margarine.03 0. However.03–0. 1989). Selection of the most appropriate method depends on processing procedures. certain meat and fish products. pickles. the extent of the antimicrobial activity of sorbate under commercial conditions is difficult to predict because it is influenced by a variety of factors. edible fat emulsion products. type of food. Levels (%) 0.Sorbic Acid and Sorbates 61 TABLE 3. Part 133 (2001)]. Sofos. 1989). or addition in a coating or packaging material (Table 3. icings. processed cheeses. These factors include product pH and composition (fat and moisture). 1989) (Table 3. 1989. As yeast and mold inhibitors.25 0. mixes. fillings. bakery items.10 0. sorbates should not be considered as substitutes for good sanitation or agents to be used to improve the quality of partially spoiled or degraded foods.R.02–0.3 Common Applications of Sorbates as Antimicrobial Agents Products Dairy products: aged cheeses. and storage temperature (Sofos and Busta. The most commonly used forms include sorbic acid and the potassium salt. spraying or immersing the food material in a solution. and sugar and confectionery items (Sofos. gas atmosphere. 1981.

1981).05–0. the high activity of sorbates against molds. but they also protect human health by preventing the formation of toxic mold metabolites (mycotoxins). It is obvious. slaw.05–0. 1980.1–0. depending on the technique used to treat the cheese (i.06 0.3 0. In certain products. available facilities and equipment..1–0.2 0. Kivanç (1992) found that Penicillium was the major genus found on cheddar cheese.06 0. pasta filata.1–0. 1996).15 0.05–0.1–0.05–0.05–0.4 0.1 0. Gouda Dry sausage or casings Dairy: Swiss.g. and distribution.15 0.05–0. and regulations.3 0.03–0. Emmentaler Dairy: cheddar.62 Antimicrobials in Food TABLE 3. in general. vegetable. Muenster. where mold development usually occurs.1–0.25 0.3 0. however. Caciocarallo. however (e.1 0. Monterey Jack. pickles.1 0.025–0.1 0.15 0. Addition of sorbate does not significantly affect the organoleptic properties of mozzarella cheese.3 0.3 0. sorbic acid may be incorporated into the cheese curd. toppings.e. cottage cheese).3 Spraying Immersion or spraying Dry mixing Direct addition Dry mixing or direct addition Source: From Sofos (1989). fudges Vegetables: fresh salads (potato.02–0.05–0. For application on . sauerkraut) Fruits: syrups. preservation objectives. and domestic and imported cheeses. Edam. 1976. that sorbates should not be used in mold-ripened cheeses.1 0. and the improved action of sorbates at high pH values compared to other common preservatives (Lück.3 0. is limited to the surface of cheese. there may develop a slight objectionable flavor (Aly.5–5 5 Application Level (%) 0.5–5 5 2.075 0. aging. relishes.1–0. fillings. Swiss cheese. which may vary among countries (Lück. mozzarella Dried fruits: Prunes Raisins Figs Smoked and salted fish Bakery: Cheesecake Angel food cake Fruitcake Cake mixes Doughnut mixes Pie crust dough Dairy: processed cheese.. 1980). Sorbates are widely used in preserving cheese owing to the susceptibility of all types of cheese to microbial deterioration.4 Methods of Application of Sorbates Methods Immersion Products Dairy: provolone. beverages. The method of application varies with the type of cheese.02–0. Gruyere. especially surface mold growth during storage.01–0.075 0.075–0. such as Roquefort or blue cheese and Camembert cheese. Sorbate application.04 0. Sofos and Busta.05–0.5–10 20–40 20–40 2.1 0. 1976. drinks Still wines Other: Margarine Semimoist pet food Bakery: pound cake Devil’s food cake Chocolate cake Solution (%) 20–40 2.05–0. spreads Cottage cheese Bakery: icings. dipping or in brine salting). Siciliano.3 0. in which they can interfere with the desirable molds involved in the manufacture of these cheeses. olives.1 0. In these applications sorbates not only prevent cheese spoilage by mold.05–0.05–0. however. blue. Colby.1–0.

ripened cheeses. thermophilus and Lactobacillus delbrueckii var. whereas when applied to packaging films. 1995). 1980.1 to 0. The appearance of Penicillium verrucosum var. 1989). 1980). when applied to the surface of hard. monocytogenes (Piccinin and Shelef. and pickled vegetables (Sofos. initial numbers of yeasts were low and relatively low concentrations of potassium sorbate and sodium benzoate (<150 and <250 mg/kg. These concentrations are in agreement with use of these preservatives in other dairy products. 1989. one of the following procedures may be followed: (1) potassium sorbate in solution or in a brine. or calcium sorbate impregnated into the packaging material or other coatings (e.30% (Lück.. 1995). respectively) to maintain stability when kept at 5°C for 7 or 14 days. potassium. respectively) extended the shelf life by 14 days. Growth of Salmonella in soft cheese was inhibited if the cheese was prepared from milk that was acidified with propionic acid to pH 5.6°C) temperatures (Kasrazadeh and Genigeorgis. especially at lower (≤6. and 25°C after 30 days (Kivanç. Addition of potassium sorbate (≤6%) to mozzarella cheese inhibited growth of Streptococcus salivarius var. 1996). potassium sorbate was added to the cheese (pH 6. 1997). monocytogenes survival in two commercial cheese brines (Larson et al. The use of sorbates in the processing of vegetables was one of the first applications.g. 1996). soft cheese (Kasrazadeh and Genigeorgis. however. acetic. In addition to reducing cheese spoilage and the possibility of aflatoxin formation.05% to 0. and (3) sorbic acid. use of sorbates in aged cheeses also reduces labor and other costs through reduction of the need for cheese washing and trimming. 15°C. wax) used in cheese storage and marketing (Han et al. VEGETABLE PRODUCTS The water-soluble salts of sorbic acid are used widely in the preservation of a variety of plant products. (2) a sorbic acid powder for dusting.0) at levels of ≥0. respectively (Aly. 1999)..9. Levels of 0. fermented. which can be used to dip. including fresh. 1994). potassium sorbate inhibited L. Inclusion of sorbic acid in cottage cheese was effective as a preservative but not against L. addition of potassium sorbate may increase the meltability and improve the fat leakage of certain cheeses (Aly. or propionic acid significantly increased the effectiveness of potassium sorbate. In another study. Treatment of mozzarella cheese with potassium sorbate in the kneading water or brine may be more effective than dipping (Aly. Calcium sorbate may be preferred in cheese preservation over other forms of sorbate owing to its solubility properties. Labaneh with higher initial yeast counts needed higher concentrations of preservative (>300 and >400 mg/kg for K-sorbate and Na-benzoate.Sorbic Acid and Sorbates 63 the surface of the cheese. bulgaricus. 1998. 1994). cyclopium mycelia and spores isolated from cheeses and inoculated as suspensions into YES broth containing 1000 µg of potassium sorbate/ml was not detected at 5°C. Therefore. In labaneh (a yogurt product) produced under strict hygienic processing measures and effectively chilled. 1981). amounts of 2 to 4 g/m2 are used (Lück. 1996). and it was initiated through extensive research performed during the early stages of sorbate testing. Sofos. or wash. potassium sorbate (0.07% sorbic acid are generally used in direct addition to cheese. and the cheese was stored at ≤30°C (Kasrazadeh and Genigeorgis.0 with hydrochloric.. and was especially effective against mold and yeast contamination by Penicillium roqueforti and Mucor miehi.. coli O157:H7 in queso fresco. 1976).3%) showed a significant effect in delaying growth of E. Sofos and Busta.05% to 0. Absorption of the compound from the surface into the cheese is dependent on the porosity of the surface and the amount and distribution of fat in the cheese. at the relatively high level of 1%. 1997).3 g/dm2. such as cottage cheese and yogurt (Mihyar et al.3%) or sodium benzoate (0. Amounts of sorbic acid permitted and applied in cheese preservation range from 0. 1992). The compound is insoluble in fat and only slightly soluble in water.3%. Milk acidification to pH 6. This research demonstrated the effectiveness of sorbates against undesirable organisms without interfering with the desirable . Furthermore. Weng and Chen. spray. Concentrations applied to cheese surfaces range from 0. most of the preservative remains on the surface of the cheese without migrating inside the cheese or dissolving in the water on the shelves during the long maturation period of these cheeses (Lück.

Mold and yeast spoilage is also prevented by sorbates in pickled vegetable products (e. enzymatic. 1988a. but it makes them susceptible to mold and yeast spoilage. depends on products and specific fermentation conditions (e. 1994).20% inhibits growth of organisms not desired in vegetable fermentations. 1980).05% and as such may be an option that processors have to reduce contamination in their products (Koodie and Dhople. Potassium sorbate is preferred because of its solubility.. smaller quantities of sorbic acid are adequate for preservation because of the synergistic action of sorbate with sugar (Lück. sorbate is either added directly into the product or applied to the surface of the product or packaging material.10% may be used to preserve refrigerated fresh salads. The higher moisture content in these products is preferred by the consumers. prunes. Concentrations as low as 0. beverages. 1972.g. coli O157:H7 in apple cider. Potassium sorbate was found more effective than chitosan in inhibiting growth of Aspergillus niger on lowsugar candied “kumquat” (Fang et al. FRUIT PRODUCTS Fruit products preserved with sorbates throughout the world include dried fruits. Inclusion of sorbate at the initial stage of the process results in a “clean” fermentation and prevents turbidity development in the final product (Jones and Harper. and putrefactive bacteria. Because potassium may result in potassium bitartrate precipitation in some wines. sorbate is more effective than benzoate owing to the pH of these products.02% to 0. molds.g. sorbates are used mostly at the preprocessing stage. Potassium sorbate and sodium benzoate were highly effective in reducing yeast and mold populations in tomato juice (Bizri and Wahem.g. Depending on the particular product. sorbates are used to prevent refermentation (Parish and Carroll. cucumbers and olives). In sweet cucumber packs. 1980).. Sorbate concentrations as low as 0. which may influence the survival of E.g.05% sorbate to a 15% to 20% brine retards yeast scum formation.. raisins.1% sodium benzoate and 0.. molds. however. coli O157:H7 and result in spoilage of the product (Zhao et al. Other vegetable products preserved with sorbates include tomato products and fermented Asian sauces (Chichester and Tanner.1% sorbate and 0.02% to 0. in high-sugar products (e.g. and wines. In fruit juice and drink processing. dried fruits). Combinations of sorbic acid and sulfur dioxide are also used in the preservation of high-pulp fruit juices. In wine processing. 1989). Lück. salt concentration)..02% sorbic acid in combination with . Use of sorbates at levels between 0. such as yeasts. fruit cocktails. In sweet relishes. Addition of 0.. Also.02% to 0.b). Levels of 0. At these concentrations the selective action of sorbic acid allows growth of the desirable lactic acidproducing organisms.. fruit juices and syrups. the pH values of fermented green olives favor the use of 0. 1955.. the lower is the sorbate level needed for preservation. 2001).64 Antimicrobials in Food fermentations. fermentation). 1994).05% sorbates in the fermenting and holding tanks. while additionally suppressing the growth of yeasts and molds. preserves.05% and 0. In these products sorbic acid acts as the microbial inhibitor and sulfur dioxide prevents oxidation and enzymatic spoilage (Lück. Another study showed that E.g. 1993). 1952.5% acetic acid completely inhibit microbial spoilage at sucrose concentrations ranging from 2% to 40%. Good wine preservation is achieved with concentrations of 0. together with sulfur dioxide and pasteurization. 0. jams. sodium sorbate may be used to avoid the problem. jams and jellies). Selection of precise levels of sorbate. Lück. coli O157:H7 growth was inhibited in both tryptic soy broth and in apple cider by sorbic acid applied at 0. or bacteria. In these products. to inhibit chemical. Sorbate levels in the range of 0.10% are adequate in improving the preservation of soft drinks.02% to 0. 1980). sorbate acts as an inhibitor of yeasts.1% potassium sorbate substantially inhibited E.05% are adequate for preserving high-moisture dried fruits (e. Sofos.05% to 0.. however.10% sorbate reduce the number of bloaters and poor seed cavities. Also. Other fruit products that may be preserved with sorbates include maraschino cherries and strawberry puree. figs). or microbiological deterioration (e. jellies. The lower the moisture content of the product (e. 1980.02% to 0. Use of the combination of 0. Costilow et al.

Concentrations of 0. 1980). and marmalades). 1954). Sorbate acts as the inhibitor of yeast refermentation during storage. sorbates are used in combination with benzoate in preserving fruit juices and drinks. 1989). 2000). Often. unless the amount of yeast and the leavening time are increased. Sorbates. To avoid the effect of sorbates on desirable yeasts in yeast-leavened products. in general. upon heating in the baking process. including cakes and confectionery.30% sorbic acid are adequate for preserving bakery products. sorbates are more common meat preservatives in other countries. Sorbate levels above 0. toppings. however. Yeast-leavened products may be protected with sorbate spraying after baking. Tompkin et al.002% to 0. sorbates may also be useful in preventing aflatoxin formation (Lück.g. which is especially important in preservation of juices. Potassium sorbate increased the shelf life of acidified tortillas. considering the permitted use of potassium sorbate as a mold inhibitor in dry sausages and reports suggesting that sorbic acid was promoting growth of clostridia (Emard and Vaughn. pizza shells and toppings. Sofos. and fillings. However.03% are not recommended in wine preservation because they may impart an off-flavor to the delicate taste of wine. may also be used. and doughnut mixes (Sofos. In such applications sorbate may be used in the coating oil or the pan grease (e. the compound is chemically degraded and releases sorbic acid after the yeast-leavened dough is raised. In dried products with an irregular surface conformation (e. 1980). 1980). aureus... 1989). examined its activity against various pathogenic bacteria in an uncured. 1969. Sorbic acid and calcium propionate have been shown to be significantly more effective preservatives than salt and sugar when used to prolong the shelf life of bread (Doulia et al. In addition to preventing mold spoilage. BAKERY PRODUCTS Although propionic acid-based preservatives are the most widely used compounds in preserving bakery goods.g. the dipping method of application is more effective (Sofos.. refrigerated dough products. muffins. 1952.10% sorbate inhibit the spoilage of cake icings. such as Japan and Korea (Sofos. In addition to undesirable molds.004% sulfur dioxide.03% to 0. and sulfur dioxide protects against enzymatic and oxidative changes and bacterial fermentation. Sorbates are also used in the preservation of pie crusts and fillings. jellies. such as S. York and Vaughn. soft drinks. In 1974. cooked sausage product. Sorbates should be valuable in baking powder-raised products. 1989..Sorbic Acid and Sorbates 65 0. Lück. Tompkin et al.. In fruit preserves (e. which is a mixed anhydride of sorbic and palmitic acids that has no direct antimicrobial activity. and other beverages. 1988). brown-and-serve products). 1999).05% to 0. sorbates are also valuable because they are active at higher pH values and effective against molds (Chichester and Tanner.. (1974) . may be more suitable for use in fruit products than other common preservatives because of their milder organoleptic properties and their neutral taste. jams. 1989). in cream pies (Schmidt et al.. 1976. Use of this compound has the advantage of preserving yeast-leavened bread without interfering with the fermentation process (Lück. especially when used in combination with calcium propionate (Tellez-Giron et al. 1972. 1989). It has also been used in champagne preservation (Sofos. Sorbate can also be added before bottling the wine and is very valuable in the preservation of sweet wines. sorbate also inhibits rope-forming bacteria and pathogens. MEAT PRODUCTS The only approved use of sorbates in meat products in the United States is to suppress mold growth on the surface of dry sausages during the drying period. A few sporadic studies in the 1950s and 1960s also indicated its potential as a preservative for meat products (Sofos.g. mold inhibition may be achieved by sprinkling sorbate on the surface of the product. Concentrations of 0. figs). Use of sorbates must be avoided in products raised by yeasts. the derivative sorboyl palmitate. For this purpose a solution of up to 10% potassium sorbate may be used to protect unrefrigerated dry sausages. 1989).

1982. 1984). mesophiles. Paquette et al. In addition to C. Bacillus cereus.. Huhtanen and Feinberg. 1978. Sofos et al. 1983. 1974) was followed by extensive testing that examined the action of sorbates against clostridia and other pathogens in a variety of fresh. and safety were examined. Sofos et. 1979. Rice and Pierson..... pork slurries (Roberts et al. 1983). To and Robach.. Tompkin et al. 1980. 1982.c. Draughon et al. Wagner and Gehrke. Bushway et al. Extensive studies published in the 1970s and the 1980s established the antibotulinal and overall antimicrobial activity of sorbates in various cured and uncured meat and poultry products. 1978. 1984. and it can react with amines to form carcinogenic nitrosamines during meat product processing and cooking (Sofos et al. Sofos. McMeekin et al.. beef steaks (Unda et al. (1979a). 1982. Elliot et al. low pH.. 1983. 1979b.. Petaja et al. 1979.. uncured cooked sausage (Tompkin et al. 2000). Products in which these microorganisms are inhibited include bacon and other cured meats.. 1982. These studies have been thoroughly reviewed by Sofos and Busta (1980). Clostridium sporogenes. Chang et al. Ivey et al.b). 1979. 1979. 1980b). Salmonella. 1978. 1987a. and the National Academy of Sciences (1981. Berry et al. 1985. Nelson et al. 1980c. 1989).. Sofos et al. Zamora and Zaritzky. 1981. U. Wagner and Busta. 1980a.. cured poultry products.. Morad et al. 1981. 1989) and cooked pork chops (Prabhu et al. 1979a. 1989). Robach. however. Greer. Gray et al. Wagner et al.b. poultry products (Robach et al. 1979a. Vareltzis et al. botulinum in cured meat products. 1989. sorbate was proposed as a means of reducing nitrite and nitrosamine levels in bacon while maintaining antimicrobial activity and inhibition of C.. Escherichia coli. 1981. In addition. Robach and Sofos.. Perry et al. raw (Mendonca et al. Robach and Ivey.. botulinum. 1982. comminuted pork products (Ivey and Robach. 1982).. Robach and Sofos (1982)... did not take effect following a report that consumption of experimental bacon resulted in allergic-type reactions in certain individuals (Berry and Blumer. 1982). sodium chloride. 1961.. 1979a. and hamburger beef (Yamamura et al. uncured. the effect of sorbates on overall product shelf life. 1981a. 1982. U. Based on the results of the various studies. Hall and Maurer. 1990). 1979b. Yersinia enterocolitica. 1980b. 1983). Pierson et al... botulinum. and increased carbon dioxide atmospheres (Sofos.. Lactobacillus. aureus. 1982. 1984.. low storage temperature..b. Bacillus licheniformis. Berry and Blumer. and uncured poultry meat and carcasses.. The reason for extensive testing of the antimicrobial properties of sorbate in meat products was the perceived need for an alternative to sodium nitrite as an inhibitor of C. sliced bologna (Chang et al. Kemp et al.S. Department of Agriculture.. 1989) and that a National Academy of Sciences committee concluded that although the flavor of bacon treated with combinations of sorbate and nitrite was not the same as the flavor . Pseudomonas. 1964.. aureus and delayed growth and toxin production by C... low oxygen. National Academy of Sciences 1982). 1986b. Vareltzis and Buck. Sofos (1981. Clostridium perfringens. Robach.. eating quality. National Academy of Sciences. Hallerbach and Potter. Nitrite was reported to be a potential carcinogen.. 1981. 1980. other pathogenic and spoilage bacteria inhibited by sorbate in various meat products include S. phosphates.. Amundson et al. 1981. 1977.66 Antimicrobials in Food showed that sorbate retarded growth of Salmonella species and S. 1983. 1981). 1988). 1974). Robach et al. The antimicrobial activity of sorbate in these products has been enhanced when combined with nitrite. Sofos et al.. 1981.S. 1978. This work (Tompkin et al. Robach et al. beef and pork frankfurters (Sofos et al. Sorbate in meat products (<0. 1983. It should be noted. 1980a.. Bauermann. that no other studies reported undesirable flavors or allergic reactions (Sofos. 1982. and processed cured meat products (Sofos. This proposal. 1979b). inclusion of sorbate and reduction of nitrite levels in bacon-pumping brine solutions resulted in reduced levels of nitrosamines in the fried product (Ivey et al. Brochothrix thermosphacta. antioxidants. Cunningham.3%) had no major adverse effects on sensory qualities. 1979. 1980b)... 1979b. 1980. however.. acids.. nitrosamine formation. psychrotrophs. Myers et al. 1981). raw beef and pork. 1978. 1984. al. 1978.. Serratia liquefaciens. 1983). Chambers et al. botulinum spores during temperature abuse of the product. such as color and flavor (Frank. The antimicrobial activity of sorbate was demonstrated in bacon (Ivey et al. In addition. Department of Agriculture. 1974. Huhtanen et al.b. and lipolytic organisms (Kaloyereas et al.

depending on moisture content. at which they prevent growth of molds and osmophilic yeasts in items such as fillings for chocolate and pralines (Chichester and Tanner. Debevere and Voets.. delicatessen items. Bremner et al.20%.10%. 1988). 1959. 1995). 1982. tomato juice. mayonnaise.05% to 0. In certain instances and for better preservation. 1983. however. diastatic powers. Application of potassium sorbate with bifidobacteria extended the shelf life of whole and peeled shrimp by 3 days (Al-Dagal and Bazaraa. Lück. orange peels. MISCELLANEOUS FOOD PRODUCTS Sorbates are commonly used in the preservation of oil-in-water or water-in-oil emulsion-based products. shredded cabbage. as well as fermented plant foods (Chichester and Tanner. tangerine sherbet base. 1980. sour cream. 1982.1%)–benzoate (0. This optimal concentration of sorbic acid coincided with the lethal dosage for most of the associated fungal microorganisms during malting. and animal feeds (Sofos. and cold and hot water extracts of the malt samples. 1972. Shaw et al. especially those of the emulsion type.. Bremner and Statham. 1978. A sorbate (0. sorbic acid may be mixed with its potassium salt or with other preservatives. 1989). In Asian countries sorbates are commonly used in combination with other preservatives to preserve fish sausages and similarly processed fish and meat products. such as margarine. Sofos. are also effective in preventing mold growth on animal feeds during storage. pineapples. cosmetic products. Regenstein. 1972. potato chips. salad dressings. 1976. 1978. 1972. strawberry puree. Sorbates are also used to prevent mold growth on the surface of dried and smoked fish (e. Sharp et al. 1983. and similar products. the flavors of both types of bacon were equally desirable (National Academy of Sciences. cut squash. Sofos and Busta. indicated that toxin production by C. Fey and Regenstein.5% did not adversely affect the germination energies and germination capacities of sorghum grain and did not significantly affect malting loss. Lynch and Potter.Sorbic Acid and Sorbates 67 of bacon treated only with nitrite. ethyl and propyl) may find applications in cosmetics and pharmaceuticals.. 1999).g. In addition. 1981). in which the emulsifiers may be inactivated by nonionic compounds (Chichester and Tanner. 1989).1%) was used (Fletcher et al. Mayer and Hillebrandt (1997) established that chemical preservation with sorbic acid was a possible way of preventing potato pulp from spoiling for subsequent practical application (feed). 1991). 1981. such as benzoates. 1989). One study. inhibiting their growth.. Samples treated with sorbic acid lost biological activity within a few days of aerobic storage (confirmed by aerobic . 1980. Robach and Sofos.. which is longer than the preservation (31 days) achieved by the most effective bacteriocin (nisin) tested (Einarsson and Lauzon. Lück. Sorbates may be particularly important in intermediate-moisture (25% moisture content) pet foods. botulinum in shucked scallops developed slightly more rapidly at 27°C in vacuum packages when sorbate (0. 1976). Chung and Lee. the esters of sorbic acid (e. it may be desirable to apply sorbic acid during malting (Ogundiwin et al.03% to 0. and pancake batter (Sofos. 1989). 1986). dried cod). Sorbic acid levels in the range of 0. Mexican-type hot sauces... garlic oil. The literature contains reports on several other food products that may be preserved with sorbates (Sofos. Several studies have reported improved preservation of various types of fish with sorbate (Geminder. cucumbers. Robach and Hickey.30% can be effective mold inhibitors. This should assure mycotoxin-free animal feeds and thus eliminate any hazards associated with mycotoxin-contaminated feed.g. in which levels of up to 0. 1976. 1983. OTHER APPLICATIONS Other items that may be preserved by sorbic acid and its salts include pharmaceuticals. Statham and Bremner. They include high-moisture marshmallow candy. 1983. Lück. 1982).1%) solution preserved brined shrimp for 59 days. 1982. 1972. Sorbic acid at a concentration of 0. thus. Other food-related applications of sorbates include sugar-based and confectionery products at concentrations of 0. prepeeled carrots.

Sofos.. 1997). 1973. and the type of food processing. when present. when used individually or in combination as preservatives of an artificial diet for leafcutting bees.68 Antimicrobials in Food degradation of starch and triglycerides). with bulbous formation and defective division (Seward et al. Other proposed mechanisms of inhibition of microbial growth by sorbate include alterations in the morphology. reduced ensiling losses and increased the feed value characteristics of citrus pulp silages (Filya et al. 1989). Several mechanisms of inhibition of metabolic function by sorbate have been proposed. 1989). Blocher and Busta. and the inner cell wall appeared to be thickened. Although the significance of such alterations is unknown. therefore. 1988). Sofos.b). and sorbate concentration (Sofos. irregular nuclei. but the exact mechanisms of antimicrobial activity are not well defined (Freese et al.. such as sorbate. 1989). they may be the result of the incorporation of sorbate into specific cell structures and alteration of biosynthetic processes in the cell (Sofos et al. 1989). Other reported uses of sorbates include preservation of tobacco products. 1985).. 1981. 2001). and it was thus concluded that it inhibits spore commitment to germination. there was no detection of pectinolytic activities (Mayer and Hillebrandt. 1986.. 1989). Septation. sporogenes were usually filamentous and nonseptate but with distorted shapes characterized by numerous bends and bulges. Sorbate-treated cells of C. 1996). Sofos et al. Treatment of Alteromonas putrefaciens with sorbate at pH 7. molds.. the outer cell wall was absent in many areas (Ronning and Frank. and glues (Lück. Inhibition has involved various species of bacteria in laboratory culture media and in foods and has been influenced by species. 1982. Seward et al.. including types and species of microorganisms. Although the presence of enzyme activities was shown. It was also postulated that sorbate probably inhibits a postgerminant binding step in the process of germination. integrity. 1986). 1989). and function of cell membranes and inhibition of transport functions and metabolic activity (Sofos.. resulted in minicells. 1976. the surviving microorganisms may resume growth and spoil the food. 1988. 1979d. 1985). 1986. Smoot and Pierson. Death of microorganisms exposed to high concentrations of preservatives. MECHANISMS OF ACTION Sorbate concentrations used in foods (<0. type of substrate. Another study reported that sorbate inhibited spores triggered to germinate or after germinant binding (Blocher and Busta. strains. and vacuoles.1% sorbic acid to citrus pulp were effective in inhibiting yeasts.3%) usually inhibit microorganisms. botulinum were long.0 increased cell hydrophobicity and cell wall lysis on exposure to lysozyme. Ronning and Frank. Inhibition of the not well-defined connecting reactions of spore germination may be taking place through the interaction of sorbate with spore membranes and through increases in their fluidity (Blocher and Busta. Sofos. environmental conditions. Sorbate inhibits cell growth and multiplication as well as germination and outgrowth of spore-forming bacteria. enterobacteria. Such changes have been observed in yeast cells as dense phosphoprotein granules. 1986. Sofos. Wagner and Busta. Freese and Levin. 1989. and it may be possible that several of them may be functional under various conditions. 1982. Consequently. Published data have suggested that sorbate acts as a competitive and reversible inhibitor of amino acid-induced germination (Smoot and Pierson. 1981). 1985a. Sofos et al. and not triggering of germination. 1985. 1989). Cells of C. when the sorbate hurdle is reduced or removed. pH. increased numbers and variable sizes of mitochondria.. Blanching (80°C) and the application of 0. which could be overcome to some extent by addition of magnesium ions. There was also evidence of other membrane damage in sorbate-treated cells (Statham and McMeekin. 1978. adhesives. The preservatives sorbic acid and methyl paraben. Under certain conditions sorbates have changed the morphology and appearance of microbial cells (Statham and McMeekin. has been attributed to generation of holes in the cell membrane . did not influence the susceptibility of the bees to the fungus Ascosphaera aggregate (Goettel and Duke. whereas higher amounts may result in death. Several studies have indicated that sorbate inhibits bacterial spore germination (Sofos et al. and clostridia common in the spoilage of silage and.

inhibition of the electron transport system. and inhibition of metabolic energy utilization by the amino acid transport systems (Sofos. and acetaldehyde (York and Vaughn. Sorbate is known to inhibit the in vitro activity of many enzymes. and ficin (Melnick et al. the suggestion that inhibition of the membrane function is the primary site of action for sorbic acid is strengthened by an observation of damage to the outer cell membranes by sorbate ions at pH 7. 1972. such as sorbate. 1992). 1965)..b). 1959. Binding and inhibition of CoA should result in inhibition of oxygen uptake and microbial growth (Sofos.. 1973. These peroxides then would inactivate catalase (Troller.. especially sulfhydrylcontaining enzymes (Kouassi and Shelef. Harada et al. Inhibition of cell metabolism by sorbate in these studies may have been the result of inhibition of enzymes.Sorbic Acid and Sorbates 69 (Freese and Levin. inhibition of synthesis or depletion of ATP. 1995a. aureus cells has clearly shown that the acid acts in its dissociated anionic form primarily on the substrate transport systems creating a starvation condition in the cells. It was also proposed (Whitaker.0 in Alteromonas putrefaciens (Sayeed and Sankaran. 1985). may interfere with substrate and electron transport mechanisms. Azukas et al. 1992). Martoadiprawito and Whitaker. 1989). a membrane-active compound. York and Vaughn. nutrient uptake. or a specific inhibitor of metabolism (Azukas et al. Harada et al. The highly reproducible and characteristic thermograms of S. Sheu et al. Thus. where it may cause steric disorganization of active membrane transport proteins (Sofos. acetate. aspartase. 1989). 1981).. Some reports. Sheu and Freese. Sorbic acid could conceivably inhibit microorganisms as a weak-acid preservative. 1991). Enzymes inhibited by sorbate include alcohol dehydrogenase. however. Secondary events such as partial inhibition of the electron transport and inhibition of the activity or synthesis of catalase enzyme may have resulted in cell death (Sayeed and Sankaran. 1970. 1961. or unsaturated fatty acid. 1961).. succinate. Sorbate inhibits the activity of several enzyme systems.and/or β-carbon of sorbate (Martoadiprawito and Whitaker. 1973). Sofos and Busta. enolase. 1978). 1960. Additionally. 1968.2%) for use in foods. Tuncan and Martin. microcalorimetric measurement of the effects of sorbic acid on S. pyruvate. . 1991). 1968. catalase. Troller. 1964. Whitaker. 1973. Inhibition of catalase by sorbate was attributed to the formation of sorbyl peroxides through the autoxidation of sorbic acid. and replication. 1978). malate dehydrogenase. 1964. 1992). 1963. Another postulation has indicated that sorbate may act competitively with acetate at the site of acetyl coenzyme A (CoA) formation (Wakil and Hubscher. Sorbate has decreased the assimilation of carbon from several substrates. 1954b. 1972. 1963. Both the peak heat and total heat dissipation profiles were affected by 50% of the common concentration (0. Lipophilic acid food preservatives. inhibition of transport enzymes. 1965). cell metabolism. Inhibition of substrate transport into the cell by uncoupling it from the electron transport system results in cell starvation (Deak and Novak. into the cell membrane. Sofos. 1963). Inhibition of nutrient uptake may be the result of neutralization of the proton-motive force (PMF) needed for substrate uptake. including glucose. 1959) that sorbate inhibits the sulfhydryl enzymes through the formation of a thiohexenoic acid derivative (CH3-CH=CH=RSCH-CH2CO2H). fumarase. aureus metabolism were significantly affected by sorbic acid in a concentration-dependent manner (Sayeed and Sankaran. 1989. ethanol. Sorbic acid has inhibited the uptake of glucose (Yousef and Marth. α-ketoglutarate. 1983) and amino acids (Hunter and Segel. α-ketoglutarate dehydrogenase.. 1975. which may lead to disruption of vital processes involved in transport functions. as well as the electron transport system (Anderson and Costilow. growth.. Inhibition of yeast alcohol dehydrogenase has been attributed to formation of a covalent bond with the sulfhydryl or ZnOH group of the enzyme and the α. have indicated no inhibition of enzymatic activity by sorbate (Sofos. succinic dehydrogenase. This effect may be occurring through incorporation of sorbic acid. Freese et al. oxaloacetate. 1991). There is evidence to suggest that protein denaturation or enhanced protein turnover is a further consequence of weak acid stress resulting from the expression of many proteins. or various transport systems. Freese and Levin. Inhibition of sulfhydryl enzymes by sorbate has been attributed to binding the compound with sulfhydryl groups and decreasing the number of such active sites on the enzyme. lactate.

In lower pH environments. This could then discharge the pH gradient required for ATP formation according to the chemostatic theory of oxidative phosphorylation (Sofos. 2000).. Conversely. 1989. 1998). This was partly attributed to the activation of protective mechanisms. the available evidence suggested that the inhibitory action of sorbic acid was the result of the induction of an energetically expensive protective mechanism that compensated for any disruption of pHi homeostasis but resulted in less available energy for normal growth.. Correlation of sorbic acid resistance with ethanol tolerance and the partition coefficient strongly suggest an inhibitory role for sorbic acid as a membrane-active compound (Stratford and Anslow. Indeed. 1980. found no decreases in ATP in the presence of sorbic acid (Sofos. Specific inhibition of metabolism is also unlikely because inhibitory activity is shared by several small carbon compounds. as indicated. Similar degrees of inhibition by sorbic alcohol and sorbic aldehyde suggest that sorbic acid does not act as a weak-acid preservative. Cells of Z. cerevisiae yeast cells in the presence of benzoate or sorbate at an extracellular pH value of 6.. 1989). little correlation was found between reduced growth on exposure to sorbic acid and reduction of intracellular pH (pHi) (Bracey et al. Evidence also suggests that this energy-demanding stress could also be the result of the induction of ATP-driven plasma membrane pumps for active extrusion of weak acids from the cell (Henriques et al. 2000). Previous studies with bacteria. 2000). Ronning and Frank. Preincubation of S. A stringent response involves readjustments occurring in bacteria when amino acids become limiting or their specific ratios are disturbed (Sofos. which ensured that pHi did not decline when cells were exposed to sorbic acid (Henriques et al. it has been shown that sorbic acid acts at high pH where weak-acid preservatives are not expected to be active (Stratford and Anslow.70 Antimicrobials in Food which act as molecular chaperones (de Nobel et al. resulting in inhibition of growth but in maintenance of cell viability (Sofos. 1984. which is one of the components of the PMF (Sofos. a pump recently identified as the Pdr12 protein is induced by sorbic acid in S. a proposed mechanism of ATP depletion includes hydrolysis of ATP by the primary sodium/hydrogen pump in an attempt to maintain ion balance in the cell (Przybylski and Bullerman. which decreases the intracellular pH and dissipates the PMF of the membrane that energizes transport of compounds such as amino and keto acids. 1998). undissociated sorbic acid acts as a protonophore. 1973). 2000). 1996). 1992. 2000). In fact. Salmond et al. This accumulation of hydrogen ions inside the cell acts as an inhibitor by interfering with metabolic processes and causing a dissipation of the transmembrane proton gradient. Therefore. 1983. 1968. Sofos. 1994). however. such as increased proton pumping by the membrane H+-ATPase. 1980). 1987. According to this theory.8 elicited a set of metabolic effects on sugar metabolism.. 1988. 1985. 1997). the difference across the cell membrane is large. Hunter and Segal. weak-acid preservatives such as sorbic acid and lead to starvation of cells from compounds that are transported actively by the PMF (Eklund. it is believed that the inhibitory action of sorbic acid is the result of excessive consumption of cellular energy that occurs as a consequence of the cell eliciting a stress response that attempts to maintain pHi homeostasis such that the available energy for growth and cell division is drastically reduced (Bracey et al.. rouxii cultured in media supplemented with sorbate contained higher percentages of C18:1 fatty acids than cells cultured in media without sorbate (Golden et al.. Inhibition of uptake of such components is believed to induce a stringent-type regulatory response in the cells. As the hydrogen influx exceeds the pumped efflux. In another study using a novel method to measure pH in growing cells. Thus. and the amount of acid entering and dissociating in the cytoplasm is higher. 1998). Because inhibition of microbial growth by sorbate has been associated with decreased levels of ATP (Harada et al. growth inhibition correlated with an increase in the intracellular adenosine diphosphate (ADP)/ATP ratio as a result of increased ATP consumption by the cells. 1997).. Inhibition of microbial growth by sorbate may be based on neutralization of the PMF that exists across the cell membranes by lipophilic. a shift in charge may potentially take place and lead to a decrease in the net negative intercellular charge. cerevisiae (Piper et al. 2001).. 1998). Sorbic acid in its MIC releases far fewer protons than other weak-acid preservatives. which became ..

the LD50 for common salt (NaCl) is 5 g/kg body weight. but not of cytoplasmic malate dehydrogenase (Burlini et al. as well as influence on metabolism.6-bisphosphate. Sorbic acid has been shown to inhibit microbial growth. Lück. it is believed to be adequate to energize the uptake of substances needed for cell maintenance and growth. it is believed that neutralization of the pH difference across the cell membrane is not the only mechanism of microbial inhibition by sorbate (Stratford and Anslow. however. 1980). Overall. is smaller. sorbate is considered one of the least harmful preservatives in use. 1992). its effect on the other component of the PMF. which is the difference in electrical potential. and (5) block of catabolite inactivation of fructose-1. the World Health Organization has set the acceptable daily intake for sorbate at 25 mg/kg body weight per day. 1989. The increase in liver weight has not been associated with histopathologic changes and has been interpreted as functional hypertrophy as a result of the caloric utilization of sorbic acid (Deuel et al. For comparison. 1993). this pattern resulted in prevention of glucose-induced switch of metabolism from a gluconeogenic to a glycolytic state. in such situations. carcinogenicity. Thus. (4) substantial reduction of glucose-triggered peak of fructose 2. and teratogenicity after short. 2000). 1973.. 1948. unlike former assumptions.and fructose-phosphorylating activities. Acute toxicity studies with rats have determined LD50 (mean lethal dose) values for sorbates in the range of 4. Sofos. TOXICOLOGY AND SAFETY In the United States. The relative nontoxicity of sorbates was established early in their testing as food preservatives. Demaree et al.. 1993). (3) suppression of glucose-triggered peak of hexose monophosphates. these studies demonstrated the relative harmlessness of sorbates and their relative superiority in safety compared to other chemical additives. Bracey et al. 1954a). 1954a. and environmental conditions (Sofos.6-bisphosphatase and phosphoenolpyruvate carboxy-kinase. Reasons for this conclusion include the following: 1. 1980. the mechanisms involved in inhibition of microbial metabolism and proliferation by sorbic and other similar lipophilic acid preservatives appear to be different. . Rat feeding studies have indicated that a dosage of 10% sorbic acid in the diet could be tolerated for 40 days (Lück.2 to 10. intracellular acidification is not likely to mediate the bulk of metabolic effects of benzoate and sorbate because under the described working conditions intracellular pH was kept close to neutrality (Burlini et al. substrates. However. These effects can be summarized as follows: (1) reduced glucose consumption. On the whole. 1976). The compounds were fed to various animal species for determination of acute toxicity. Overall. uptake of the compound based on pH difference across the cell membrane would not be adequate to explain the observed antimicrobial activity. 1955.. Additional studies with rats and dogs have shown no damage with feeding 5% sorbic acid for 90 days. Deuel et al. 2.. 1998. and because it is a metabolizable fatty acid... Neutralization of the PMF is probably not involved in growth inhibition in the presence of carbohydrates because they do not depend on this force for their transport (Sofos. On extending the feeding period to 120 days. the growth rate and liver weight of the animals increased (Demaree et al. (2) inhibition of glucose.5 g/kg body weight (Smyth and Carpenter.. 3.Sorbic Acid and Sorbates 71 apparent after the subsequent addition of glucose. Thus. 2000).. sorbate is considered a GRAS compound. Food and Drug Research Laboratories. 1954a. even at pH values near neutrality. although less efficiently. based on acute toxicity. but 8% sorbic acid resulted in an increase in liver weight (Deuel et al. depending on the type of microorganism. 1998.or longterm exposure. 1989). Sofos. Thus. Lück. Although dissociation of the acid inside the cell may eliminate the pH difference across the membrane. 1955).

. Walker (1990) concluded that sorbate use. no direct relation could be proved between symptoms and specific ingredients used in formulating the bacon (U. 1993. 1977. Some taste panelists reported certain “allergic-type” symptoms after tasting uninoculated experimental bacon (U. 1999). 1975. 1981.b). 1992). 1974. 1982. 1980.. Osawa et al. 1982). in general.5. Shtenberg and Ignat’ev.. No abnormalities have been observed. 1975. 1982). 1979b). 1974. which is the average pH of most meat products. Some sensitive individuals. Under conditions typical of .. 2000a. This. sorbates have been extensively examined for skin tolerance (Lück. which makes it susceptible to nucleophilic attack.72 Antimicrobials in Food Chronic toxicity studies have involved feeding rats and mice through the life span of one or two generations with sorbic acid concentrations as high as 90 mg/kg body weight or diets containing 10% sorbic acid. sometimes giving mutagenic products (Ferrand et al. Sofos and Busta.0. In addition. does not rule out the possibility that high concentrations of sorbate may act as irritants to certain susceptible individuals (Sofos. Sorbic acid has a conjugated system of double bonds. probably because the acid environment of the stomach leads to the breakdown of the sorbic-protein adducts (Quattrucci and Masci. Considering the average use levels of 0. 1982. 1981. may undergo nonenzymatic browning reactions when stored with sorbic acid at a favorable range of aw and temperature (Quattrucci and Masci.. cysteine. leading to decreased amino acid availability (Quattrucci and Masci. it may apply also to more complex systems such as foods. 1979. The literature is contradictory. 1983. does not appear to create toxicologic problems. 1979. 1992). 1981. and some very sensitive individuals may show irritations even at lower levels (Lück. The products of such reactions have the potential of being mutagenic (Sofos. 1980. Kito et al. Litton Biometrics. National Academy of Sciences. of course. and Robach and Adam (1980) were able to induce such symptoms from panelists consuming commercial bacon manufactured without sorbate. Average sorbic acid concentrations for skin irritations are in the range of 1%. and (3) mutagen formation is inhibited by such ingredients as ascorbic acid. Lück.30% in food processing. (2) formation of the c-nitroso mutagens requires the presence of excess nitrite.S. As preservatives for cosmetics and pharmaceutical products. Department of Agriculture. Berry and Blumer. botulinum. Robach et al. apparently because of an increased caloric intake resulting from the metabolizable sorbic acid. 1981). 1980a. 1976. Sofos. it may react with nitrite in products such as cured meats. Although it was implied that sorbate might have been involved in producing those symptoms. 1970. and in some instances the growth rate of sorbate-fed animals increased significantly. 1981. 1989). An allergic-type response has been reported for sorbic acid in one study in which bacon samples with sorbate–nitrite combinations were tested against C. 2001). but it indicates that most people are not affected by sorbic acid applied to the skin. not at 6.10% to 0.. Food and Drug Research Laboratories. 1980. 1979b. 1992). however. Sorbates were found to react easily with proteins. 1980) and because sorbic acid is an unsaturated fatty acid. Department of Agriculture. 1981. In general. 1976. No carcinogenic or mutagenic effects have been observed with sorbate alone (Dickens et al. no such symptoms were observed by other individuals who tasted sorbate–nitrate experimental bacon from other studies. 1968. Khoudokormoff.S. and vegetable juices and is inactivated by heat (Kada. the requirements for the reactions to proceed and the instability of the products make it unlikely for mutagens to be formed at detectable or harmful concentrations in foods treated with nitrite and sorbate (Sofos. show skin irritations when exposed to sorbic acid (National Academy of Sciences. including lysine and glutamate. however.. 1978. This reaction was demonstrated in model systems but. Gaunt et al. the potential for such irritations in commercial products is minor. Hendy et al. 1989). Osawa and Namiki. 1994). Because there is evidence of reactions between nitrite and fatty acids (Benedict. 1986.. 1989).. It has been regarded that amino-compounds. forming high-molecular-weight products that do not seem to result in a nutritional loss. Thus. various studies have indicated the following: (1) nitrite reacts with sorbic acid to form mutagens optimally at pH 3. Patrizi et al. 1976). Namiki. There has been some concern about possible sorbate–nitrite reactions when added to the same substrate (Sofos. to some extent. Dejobert et al.

including parabens. . Rao et al.. Sofos. dialysis. Overall. that certain studies have reported inhibition by sorbate of carcinogenic nitrosamine formation in model systems (Tanaka et al. dichloromethane. After development by Melnick and Luckman (1954a). however. Stafford. 1972.. 1974. 1980). 1962) and spectrophotometric (Melnick and Luckman. It should be noted. The derivatives of sorbic acid are also relatively nontoxic. and solvent extension using ethyl or petroleum ether.. 1989). in cyclic interaction products. Lathia and Schellhoeh. 1976.31).. Larsson. absorption) procedure has also been used in many foods. 1989). and isooctane. Extraction by steam distillation has been used extensively. polarography.. 1983. method 975. however. 1981. AOAC Int. and sausage products (Maxstadt and Karasz. although chromatographic methods have gained acceptance in recent years. however. Chung.Sorbic Acid and Sorbates 73 food processing (50°C to 80°C) cyclic derivatives resulting from a double addition reaction between sorbic acid and various amines were analyzed. methods 971. to cyclic derivatives resulting from double addition (Ferrand et al. did not protect against the toxic effects of other substances (Daoud and Griffin.. Extraction methods include acid-steam distillation. No synergism in acute toxicity has been detected for combinations of sorbic acid with various other additives. have been colorimetric (Schmidt. Mutagenesis studies. 2000). 1999. 1973. successive extractions with ether. 2000). In some foods. 1962. Sofos. the method was modified by Alderton and Lewis (1958).b). Mandrou et al.. filtration. dialysis and solvent extraction. involving the Ames test and genotoxicity studies with HeLa cells and on plasmid DNA. 1981a..15 and 975. 1960. selective gas diffusion. Such combinations have involved extraction under acid conditions from the steam distillate and reextraction with sodium hydroxide. 2000a. 1989). colorimetry. bakery items.. bromometry. and double distillation and ether extraction (Tjan and Konter... Potential injury in cell hepatocytes induced by potassium sorbate was prevented by antioxidants (Sugihara et al. Noda et al. and chromatography. and propionate (Sofos. showed that none of the products studied presented mutagenic or genotoxic activities (Ferrand et al. Sorbic acid. sodium hydroxide. at 20°C. but it is time consuming and the compounds present in the food or generated by decomposition of lipid materials may interfere with colorimetric or spectrophotometric detection of sorbic acid (Harrington et al.. 1989). Sofos. Sofos. cheese (AOAC Int. 1983.10) by the color reaction with αthiobarbituric acid (Roy et al. 1976. The spectrophotometric (ultraviolet/UV. 1997). 1980). had no effect on nitrosamine formation from aminopyrine and sodium nitrite in animal stomachs (Kawanishi et al. to linear monoadducts and.b). 1995. Sorbate. Sofos. it involves measurement of absorbance at 260 nm (250 to 290 nm). 1995. benzoate. sorbates appear to be one of the safest food preservatives available.b. and methylene chloride. 1980). 1978. The formation of new products by addition reactions using ethyl sorbate and various amines led. enzymatic. The method is simple and usually very specific (Lück. 2000a. or direct analysis has been used (Sofos.. Massey et al. cheese. Montano et al. 1988). wine. 1989). The colorimetric detection of sorbic acid at an absorbance of 532 nm is an official method for quantitative determination in foods and beverages (AOAC Int. 1982. however. 1981.. Special extraction and purification steps have been proposed in the literature to reduce interference problems (Sofos. method 975. whereas combinations of various treatments have also been used to improve extraction and reduce interference (Sofos. at 50°C. Holley and Millard. 1989). including fruit products. 1954a). Puttermans et al. 1982. spectrophotometry. The most widely used methods... 1955. DETECTION AND ANALYSIS Analytic methods used or tested for qualitative and quantitative detection of sorbates in foods include acidimetry. Detection methods require quantitative extraction and separation of sorbic acid from the food material without food ingredient interference (DeLuca et al. 1972. 1989). and wine (AOAC Int.22). Several modifications have been proposed as useful in avoiding interference and in improving the accuracy of the steam distillation procedure (Luckmann and Melnick. Yamamoto et al. Wilamowski.

and this status has been reaffirmed (U. The method is based on sorbic acid being converted to sorbyl CoA with acyl CoA synthetase in the presence of coenzyme A and adenosine-5’-triphosphate. 1989).74 Antimicrobials in Food 1989).. Microdialysis was used to extract sorbic acid and benzoic acid from food to be separated and detected by HPLC (Mannino and Cosio. and reliable and is thus well suited for routine determinations. and theophylline and as such may be a beneficial alternative to conventional HPLC. 2000). such inhibition may be caused by a number of other inhibitors. Inhibition of microorganisms has also been evaluated as a procedure for the qualitative detection of sorbate (Ellerman. method 980. Others. It is obvious. and micellar electrokinetic capillary chromatography (MECC) and ion chromatography. ion chromatography was effective in simultaneously determining artificial sweeteners. precise.. sorbates in the United States . Solid extraction has been used as a cleanup procedure for the determination of sorbic acid by liquid chromatography in fruit products (Mandrou et al. 1996). 2002). 2000). 1978) by a select committee of the U. Ion chromatography has been shown to be in good agreement with HPLC in determinations of sorbic acid in food and pharmaceutical preparations (Chen and Wang. 1989). method 974. the scope of application of the proposed method for the determination of sorbic acid has been extended to cover juice and drink samples that cannot be analyzed by the AOAC procedure (Fung and Luk.. that in addition to sorbate.16) for sorbic and benzoic acids in foods (AOAC Int. caffeine. and advantages over other preservatives. Dobiasova et al.08) and dairy products (AOAC Int.10) (AOAC Int. wine. 2001). method 974. The method was found to be fast. In addition. paper chromatography. 1999). In addition. rapid method with high reproducibility for determining sorbic acid and other additives in fruit juices (Zou et al.. The use of reversed-phase highperformance liquid chromatography (RP-HPLC) was found to be a simple.. preservatives. A polarographic method using differential-pulse voltammetry with the use of a hanging mercury drop electrode (HMDE) was developed for the determination of sorbic acid in fruit juices and drinks. 2000).S. and other substrates (Cancalon. 1990). Department of Agriculture. thin-layer chromatography.. 2000).17) and final action procedure for wines (AOAC Int. however. In the United States. REGULATORY STATUS Several forms of sorbate are allowed for use in a variety of foods throughout the world (Sofos. Moreover. low toxicity. considering their extensive testing. 1998). Food and Drug Administration. In addition.. 2000. The method is an official first action procedure for ground beef (AOAC Int. 1977). This major use may expand in the future. 2000). method 983. it can be used for the analysis of intensely colored juice samples. An enzymatic method used to determine sorbic acid based on the spectrophotometric measurement of sorbyl CoA at 300 nm has been developed (Hofer and Jenewein. Castineira et al. theobromine. have reported that cleanup procedures did not improve determination by liquid chromatography (Benassi and Cecchi. highperformance liquid chromatography (HPLC). 2001).S. which is proportional to sorbic acid levels in the sample. especially for high sample throughputs (Hofer and Jenewein. sorbic acid and potassium sorbate are GRAS. where the absorbance is measured at 300 nm and is specific for sorbyl CoA. A rapid method for the identification and quantitation of sorbic and benzoic acids in beverages and foods by MECC has been reported (Pant and Trenerry. 1999. 1998. however. 1995). Mercier et al. Capillary electrophoresis was also applied for detection in citrus juices. HPLC was found to be adequate in obtaining accurate stability data for sorbic acid in creams (de Villiers and Bergh. They include gas chromatography. The reaction is quantified by irreversibly hydrolyzing pyrophosphate to phosphate in the presence of inorganic pyrophosphatase. The procedure is simple and specific and is not subject to interference from many substances commonly found in soft drinks such as ethanol and benzoic acid. The literature contains numerous reports of chromatographic methods used or evaluated for determination of sorbate in food products (Sofos. A gas chromatographic method is a first action procedure (AOAC Int. Thus.

J. and Kawabata. Shibasaki. Appl. Graham. furylfuramide and sorbic acid. M. 1989.. R. 43:877–891. Frank. Preservation of fish sausage with tylosin. 62:51–56.Sorbic Acid and Sorbates 75 are sanctioned in more than 80 food products designated by standards of identity. 1980. Al-Dagal. T. Berry. 1998. Effect of belly composition on sorbate-cured bacon. sodium benzoate. Potassium sorbate as a fungistatic agent in country ham processing. A. F. Food Sci. 63:14. AOAC International. F. R. and cooking characteristics of bacon processed with varying levels of sodium nitrite and potassium sorbate. sorbic acid and monolaurin as preservatives in pasteurized cured meat products. 1979.. 1980. 1988.. Use of acidification. Sensory. J. M. T. Determination of sorbic acid and its disappearance from pickled brines. Bell. Etchells. E. Rust. Chromatogr. Site of inhibition of yeast respiration by sorbic acid. The use of sorbates may be requested for any food product that allows preservatives. 46:321–327.” “to extend shelf life. 23:338. I. Food Microbiol. Costilow. Food Sci. B. J. M. M. Anderson. potassium sorbate or sorbic acid) should be listed on the product label and its function should be indicated by an explanatory description (e. Alli. L. pH and organic acids. 1958. G. Shelflife characteristics of bacon processed with various levels of sodium nitrite and potassium sorbate. J. R. Alderton. 77:573–580. Kraft. 1988. M.. H. AOAC Int. Benassi. A. F. K. and Blumer. Rust. and Labuza. A. Quick. J. C. and Bazaraa. C. Berry. Kraft.g. T.. 46:596–600. Influence of sorbic acid on the growth of certain species of bacteria.. N. Microbiol. J. M. H. and Robach. According to the Code of Federal Regulations. its common name (e. J. phenolic compounds. and Ivey. Amundson. 1981. 1963. E. Bacteriol. 1996.” or “as a preservative”). Z. L. K. Liq. 2000. L. P.. M. 47:218–221. Maryland. J.. N.g. K.. 1999. Environ. 47:222–225. frozen. T. 81:189–194. M. and Sadoff. G. J. S. J.. 39:1178–1182. Inhibition of alcoholic fermentation by sorbic acid. 42:780–783. Amundson. R. Food Res. Bauermann. Wiss.. 41:541–546. B. R.. REFERENCES Acott. Prolongation of the keeping quality of Mozzarella cheese by treatment with sorbate. C. C. prerigor) and smoking delay on regular and sorbate-cured bacon. Biochemical basis for nitrite-inhibition of Clostridium botulinum in cured meat. G.. R.. Benedict. S. C. 29:41–49. and Lewis. and sorbic acid in white wines. and Kermasha. N. 1981. Wagner. I. Gaithersburg. P. A.. E. low temperature. Bell.. B. J. M. 1961. Azukas. N.. Official Methods of Analysis. Food Prot. Extension of shelf life of whole and peeled shrimp with organic acid salts and bifidobacteria. 33:42–43. 1959. J. A. 4:277–283. . R. and glycerol and sucrose esters of fatty acids. W. Evaluation of antimicrobial agents in a microbial challenge study for an intermediate moisture dog food. and Cecchi. Degradation products of sorbic acid in aqueous solutions. J. Inhibition of heated Bacillus spores by combinations of potassium sorbate. L. E.. A. Food Prot. K. Bacteriol. 1987.. 21:491–501. W. physical. Lebensm. G. S. T. J. and sorbates for storage of orange juice. 54:674–678. I. Effect of belly handling (chilled. G. Method development for the simultaneous determination of carboxylic acids. E. M. and Borg. and De Lacy. 22:881–885. 1968. Rel. J. and Stringer. M. Beuchat. Aly. J.. W. Food Technol. Bacteriol. T.. activities of potassium sorbate.. 1976. and Robach. 1981b. J. Douglass. J. Comparison of anti-Vibrio.. R. Amano. A. Technol. J. Sebranek. Technol.. J. L. Proc. Food Prot. Die Nahrung 40:194–200. J. F. Arya. Food. Sebranek. A. K... D. Food Technol. Wagner. J. and Thakur. Prot. “to maintain freshness. P. Food Sci.. Yokoseki.. when a food preservative is used in a food product. R. M. Banks. J. Processing of poultry products with and without sodium nitrite. M.. 17th ed. and Costilow. Food Chem. A. C. sodium benzoate. 21:250–255. Sloan. Morgan. S. P. W. M. Food Sci. Food Prot. and Tennent. The efficacy of nisin. 1981a. Baldock.. yeasts and filamentous fungi. 1979.

P. 1984. Influence of potassium sorbate and pH on ten strains of type A and B Clostridium botulinum.. Influence of potassium sorbate and reduced pH on the growth of vegetative cells of four strains of type A and B Clostridium botulinum.. A. Effects of potassium sorbate on growth and ochratoxin production by Aspergillus ochraceus and Penicillium species.... B. M. Growth response of an osmotolerant sorbate-resistant yeast. L. Bullerman. A. I. 1955. 31:435–446. 1983.. F. L. and Stafford. J. J. W. A. 1999. 1983. 159:220–224. J. Food Sci.. J. Beuchat. Boyd. N. Synergistic effects of potassium sorbate and sodium benzoate on thermal inactivation of yeasts. Annu. Pellegrini. 47:312–316. Botha. 1981a. Bushway. Chrom. and Olivigni. and Busta. Busta. P. B. L. Effect of nitrite and sorbate on total number of aerobic microorganisms in chicken white and dark meat patties. Food Sci. L. 59:130–134. Food Sci. Int. L. J. Citric acid and antimicrobials affect microbiological stability and quality of tomato juice. Food Prot. and Wahem. A. King. Cancalon. Environ.. A. L. R. Holyoak. 1974. 1985. Bullerman. J. Nutr. J. L. D. J. Bolin. J. Sorbic acid loss from high moisture prunes. F. F. G. H. J.. C. and Tarr. C. 1981c. Herrero. 59:469–478. 1984. HRC-J. Resistance of Sporolactobacillus to potassium sorbate and sodium nitrite. Bracey. F. H. J. Fickner. 1977. C. Microbiol. 48:574–575. J. Blocher. J. 1987. Saccharomyces rouxii. and Sofos. J. R. . and Coote. R. Beuchat. C. A. 29:259–264. J. 82:95–106. Blocher. J. Food Sci. J.76 Antimicrobials in Food Beuchat. Facheris. R. 1978. L.. Blocher. at different sucrose and sorbate levels. 1994. Simultaneous determination of organic acids in wine samples by capillary electrophoresis and UV detection: optimization with five different background electrolytes. Bullerman. 45:1120–1124. Food Microbiol. N. J. Bacteriol. J. B. Taste panel assessment of textural properties of fish minces from Australian species. Food Sci. J. 13:307–318. Appl. 45:1434–1435. 5:331–336. A. J. 85:1056–1066.. F. Effects of potassium sorbate on growth and patulin production by Penicillium roqueforti and Penicillium patulum. B.. R. 1982. C. A. 863. Studies of the effect of ethylene diamine tetraacetic acid (EDTA) on sorbic acid degradation. Bullerman. 1993.. N. Analytical monitoring of citrus juices by using capillary electrophoresis. Campos. Combined effects of solutes and food preservatives on rates of inactivation of and colony formation by heated spores and vegetative cells of molds. Food Prot. Technol. Tortora. 1983. H. J. F. R. Mycotoxin producing-potential of molds isolated from cheddar cheese. Microbiol. 2000. Appl. Bills. A. 44:765–769. N. Comparison of the inhibitory effect of sorbic acid and amphotericin B on Saccharomyces cerevisiae: is growth inhibition dependent on reduced intracellular pH? J. M. L. and Holzapfel. Food Technol. 1982. C. Laslett. Food. 1128. 23:647–652. J. 1996. R.. Effect of some antifungal compounds on growth of Penicillium citrinum and citrinin production. 580. 46:940.. Bhattacharya.. Metabolic effects of benzoate and sorbate in the yeast Saccharomyces cerevisiae at neutral pH. Effects of potassium sorbate and sodium benzoate on inactivating yeasts heated in broths containing sodium chloride and sucrose. 9:411–412. AOAC Int. 1981b. J.. Bremner. J. Castineira. Thermal inactivation of yeasts in fruit juices supplemented with food preservatives and sucrose. High Res. 47:2028–2032. 47:858–860. H. Pena. 21:38–40.. A. L. Effects of potassium sorbate on growth and aflatoxin production by Aspergillus parasiticus and Aspergillus flavus. Effect of potassium sorbate on refrigerated storage of vacuum packed scallops. J. S. M. F. 41:472–477. M. 1982. A. Food Technol. 39:1166–1168. S. 48:1042–1047.. A. W. Food Sci. and Statham. Jr. A. L. W. Inhibition of mold and yeast development in fish products. and Jen. S. D. D. L. J. and Garcia-Martin. and Lenovich. J. J.. Prot. Food Prot. Food Sci. Restaino. Aliment. H. Bremner. Microbiol. Appl. B. N. Arch. Burlini. Bullerman. Incidence and control of mycotoxin producing molds in domestic and imported cheeses. J. and Majumdar. P. Food Sci. S. Food Sci. 46:771–777. 47:1679–1682. 1982. Bizri. Rojas.... Food Res. Multiple modes of inhibition of spore germination and outgrowth by reduced pH and sorbate. and Guerritore. 48:162. F. P. Intl. E. L. and Gerschenson. D. Food Sci. Beuchat. and Busta. 1998. and Olley. K. 1980. M.. Food Prot. 1985.

Costilow. Brekke. butylated hydroxytoluene. Chen. and Guymon.. Chambers. Robins. Food Sci. Coughlin. and Ray. 1981. C. Sensory attributes and instron measurements of reduced-nitrite poultry frankfurters with sorbic acid or potassium sorbate. Bowers. Appl. Costilow. and Nelson.. Weak acid preservatives block the heat shock response and heat-shockelement directed lacZ expression of low pH Saccharomyces cerevisiae cultures. G. IV. J. W. 1956. Costilow. Inhibition of microbial growth in English sole (Parophrys retulus). S. and Wang. 1988.. and identification of 2-ethoxyhexa-3.. Chayon Kwahak Pyon 13:177.. Title 21.. A. Part 133.Sorbic Acid and Sorbates 77 Cerruti.. C. Cheng. G.S.. T. K. not displayed by the more preservative-resistant Zygosaccharomyces bailii. R. Effect of retinoic acid. 1982. M. J. P. Food Sci. J. W. I. pp. Cheng. and Piper. J. L. and Branen. D. and Craig.. Am. S.. Sorbic acid as a selective agent in cucumber fermentations.. and Lee.. Potassium sorbate inhibition of microorganisms isolated from seafood. Chem. Appl. Microbiol. S. W. F. E. H. Controlled release of water-soluble polymeric complexes of sorbic acid with antifungal activities. E. Robach. 1999. Walker. 1955. B. J. Deak. 1970. 1981. E. Moghraby. C. A study of the acid-forming bacteria from cucumber fermentations in Michigan. 5:373–379. Food Prot. T. Furia. M. Tsatsakis. Effect of sorbic acid on the yeast and lactic acid fermentations in brined cucumbers. J.. Acta Microbiologica 17:257. and Petrikkos. Effect of sorbic acid on microorganisms associated with cucumber fermentations. 1990. M. 1980. and Piper. S. J. 1981. 1981.. M. 44:863–864. E. and Lee. P. and Tanner. Simultaneous determination of artificial sweeteners. M. 1987. and Chirife. A. A. J. S. Chang. Am. Ed. Biol Med. J. and Novak. Tzatzarakis. E. Chung. 46:314–316. Daoud. Inhibition of nitrosamine formation in vitro by sorbic acid. moisture content and sorbate on safe storage for wet corn. Chung. A. D. 45:1310. . 48:861–864. FL. C. J. potassium sorbate or lactic acid starter in culture for control of Clostridium sporogenes (PA 3679) growth in sliced bologna. 94:12081. Eng. Packaging film permeability in conjunction with sodium nitrite. Food Res. 2001. T. M. Enol.5-diene as a source of geranium-like off-odor. W. and Griffin. M. W. D. R. 47:207–209. M. F. CRC Press. theobromine and theophylline in food and pharmaceutical preparations by ion chromatography. H. 44:66–68. Cancer Lett.. Microbiol.. Chichester. F. Sorbic acid as a selective agent in cucumber fermentations. S. E. J. M. J. 26:97–102. and Galloway. 13:571. J. E. I. 21:27–33. 16:679–682. H. Government Printing Office. C. M. P... Y. 2001. Yale J. J. FEMS Microbiol. Vitic. 937:57–64.. Cowles. F. P. 57:770–775.. “Cheeses and related cheese products. J. Ferguson. H. Biotech. Microbiology 140:1085–1096. Shelf-life and quality characteristics of poultry parts dipped in potassium sorbate.. J. Code of Federal Regulations. Coughlin. and Rasheb. B. Chrom. Food Sci.. and Hsu. Effect of CO2. A. and potassium sorbate in combination. E. Davidson. 9:299–304. J. A multi-parameter approach to control the growth and Saccharomyces cerevisiae in laboratory media.P. J. Chung. 1983. Trans. Boca Raton. 1972. preservatives. K. Soc... Antimicrobial food additives.-W. T. 1973.. J. Food Prot. K. tertiary butylhydroquinone. 1979.. W. and Keenan. L. 1994. L.” U. Sebranek. Franklin. 1957. N. Y. and Chirife. G. F. The germicidal action of the hydrogen ion and of the lower fatty acids. Effect of potassium sorbate and sodium bisulfite on thermal inactivation of Saccharomyces cerevisiae in media or lowered water activity. E. Abstr. N. 53:1911–1912. caffeine. 4:115–119. Probability of growth of the spoilage yeast Zygosaccharomyces bailii in a model fruit drink system. Prusa. 1975. selenium and sorbic acid on azo-dye hepatocarcinogenesis. 3:341–345. Food Sci. Microbiol. Yongnam Taehakkyo Nonmunjip. Food Microbiol.. Wine constituents arising from sorbic acid addition. Lett. In Handbook of Food Additives. J. M. Weak organic acid treatment causes a trehalose accumulation in low-pH cultures of Saccharomyces cerevisiae. 1941. K.. Crowell. M. Agric. Effect of sorbic acid on the growth of yeasts on various carbohydrates. J. Alzamora. 115–184. A. F. N. Alzamora. II. 2001. Antimicrobial activity of butylated hydroxyanisole. Danzinger. an inhibitory action partially relieved by respiratory deficiency. 55:837–840. Cole.. Cunningham. Appl. P. Q.. A. R. 170:89–95. P. A. L. Food Sci. Steinberg. Cerruti. J. Charvalos. Food Sci.. J.

H. Assoc. F. G. E. R.. Katsinis. J. Effects of modified atmosphere packaging and preservatives on the shelf-life of high moisture prunes and raisins. Anisfeld. Assay of sorbate and benzoate by the disc-plate method. I. 26:539–547. A. Food Addit. Vitic. 23:263–267. Alfin-Slater.. Microbiol. The antimicrobial effect of dissociated and undissociated sorbic acid at different pH levels. Piette.. D.. Biopreservation of brined shrimp (Pandalus borealis) by bacteriocins from lactic acid bacteria. 1986a. F. Microbiol. J. and Splittstoesser. Sci. H. P. J.78 Antimicrobials in Food Deak. H. C. El Halouat. 50:1360–1363. H. Food Microbiol. and Waynforth. 2000. P.. Debevere. Doulia. Food Microbiol. B. F. Pharm. J. Metabolism of α-unsaturated fatty acids with emphasis on sorbic acid. Gen. J. Pazourek. and Havel. 51:1077–1078. J. 1995. 1972. 33:219–229. 35:351–356... and Bergh. H.. Appl. Food Res. and Gray. with special consideration of Zygosaccharomyces bailii. Simultaneous determination of sorbic acid. 1981. Edinger. Influence of modified atmosphere and preservatives on the growth of Zygosaccharomyces rouxii isolated from dried fruits. 1954b. R. R. M. the precursor of the geranium odor compound. R.. Elliott. The effect of sorbic acid and esters of p-hydroxybenzoic acid on the protonmotive force in Escherichia coli membrane vesicles. Sorbate tolerance by lactic acid bacteria associated with grapes and wine. Dairy Technol. and Quattrucci. 1982.. J. and Voets. 1980. M. Microbiol. J.. Elliott. P. I.. J. 32:13–14. T. Sorbic hydroxamic acid. D. H. J. R. 1968. J. W. F. Bacteriol. B. E. Int. Gourama. T. J. Jr. J. C. P. and Gray. J. Phar. J.. 54:383–389. McKeehan. H. Eklund. 23:405–417. Contam. J. H. J. Dudman. 1985. J. Delaporte. Use of indirect conductimetry to predict the growth of spoilage yeasts. Weil. H. Food Prot. 45:291.. Bacteriol. Z. Dejobert. P. de Villiers. J. F. Inhibition and inactivation of Staphylococcus aureus in a sorbate/modified atmosphere combination system. 2001.. Enol. A. H. J. and Gray. and Beuchat. Am. Int. T. D. Dickens. 1963. J.. Environ. T.. J. McCashland. F. Contact Derm. Bacteriol... Calbert. W. Food Sci. DeLuca. Sung.. R. Jones. Int. H. F. Food Res. Food Prot. 61:669–676. Drug Develop.. Appl. E. Effect of the parabens with and without nitrite on Clostridium botulinum and toxin production in canned pork slurry. A. Further tests on the carcinogenicity of sorbic acid in the rat. J. 1998.. Food Microbiol. 1996. C. J. 131:7376. Elliott. and Blunden. D. 1955. S. Ed. Harmlessness of sorbic acid as a dietary component. W. Edinger. Ellerman.. Deuel. D. C.. F. 2000.. and Davidson. Aust.. Mount. Appl.. Tomlins. Uyttendaele. Appl. Br. Preliminary studies on the effect of feeding sorbic acid upon the growth reproduction. and Cosgrove. Food Prot.. Comparing HPLC and UV spectrophotometric analysis methods for determining the stability of sorbic acid in nonionic creams containing lactic acid. 1994. Rigas. 1985. II.. H. Influence of some preservatives on the quality of prepacked cod fillets in relation to the oxygen permeability of the film. Salmonella sensitivity in a sorbate/modified atmosphere combination system. 1954a. Jr. Sjogren. E. 2002. H. Int. M. P. Cancer 22:762–768. 11:362–364. Y. Electrophor. Indus. 112:1–7. Vesicular eczema and systemic contact dermatitis from sorbic acid. G. J. Passi. 41:177–184. H. G. M. Sorbic acid as a fungistatic agent for foods. J. . 1986b. D. 47:1635–1637.. 1983. Control of microbial spoilage on fresh poultry using a combination potassium sorbate/carbon dioxide packaging system. H.. Food Sci. and Lauzon. M. 44:619–621. Jr. 44:903. F. L. Appl. J. 1995. Demaree. L. Eklund. and cellular metabolism of albino rats. 37:34–38. L. H. Dobiasova. 19:13–19. Food Sci. and Debevere. J. and Debevere. S. El Halouat. Am. and Smyth. an antifungal agent effective over a wide pH range. 19:1–12. Einarsson. J. R.. Production by lactic acid bacteria of sorbic alcohol.. Multiparametric investigation of mould-free shelf life of bread via factorial design.. M. 48:423–432. S. and Splittstoesser. R. Sorbic acid as a fungistatic agent for foods. 3:363–374. P. and Thomas. W. E. Deuel. 1982.. Inhibition of growth and uptake processes in bacteria by some chemical food preservatives. benzoic acid and parabens in foods: a new gas chromatography-mass spectrophotometry technique adopted in a survey on Italian foods and beverages. J. I. H. W.1642. 45:1112–1116. Tomlins. M. D. J. Simultaneous determination of trans-resveratrol and sorbic acid in wine by capillary zone electrophoresis. J. Draughon. A. 1977. Eklund.

Studies on the preservation of fresh apple juice with sorbic acid. 53:349–352. and Gangolli. Use of potassium and sodium sorbate in extending shelf-life of smoked fish. 358. 51:842–847. Food and Drug Research Laboratories. H. B. I.. von Montegelas. Hardy. 1959..S. S. M. 207. S. Antifungal activity of chitosan and its preservative effect on low-sugar candied kumquat. T. 17:895–901. M. Li. Karabulut. and Chirife. Viñas.. and Warth. Teratologic evaluation of potassium sorbate-sorbistat in mice and rats. Turkish J. L. J. and de Saint Blanquat. H. and Regenstein. S. J. 1973. Alzamora. 47:1048–1054.. Ferrand. C. 1989. Virginia Polytechnic Institute and State University. J. P. S. C. Inc. J. Polarographic determination of sorbic acid in fruit juices and soft drinks.. Society for Industrial Microbiology. Chemical and toxicological studies of products resulting from sorbic acid and methylamine interaction in food conditions. 1987. Gerchenson. Ed. N. Food and Drug Research Laboratories. M. M. Food Technol.. D. G. J. J. R. D. 8:335–342. 1982. P. W. D. Washington. Canela. Department of Commerce National Technical Information Service Publication PB-245. Stimulation of aflatoxin B1 and T-2 toxin production by sorbic acid. Alzamora. Inhibition of aflatoxin-producing fungi by Welsh onion extracts. Alzamora. Finol. C. H. Gaunt. pp. The response of Gluconobacter oxydans to sorbic and benzoic acids. J. Environ. Freese. Food Sci. 13:459–461. Fletcher. and Levin.. J. Nature (London) 241:321–325. Filya. Blacksburg. Eyles. J. Action mechanisms of preservatives and antiseptics. Canbolat. S. 409. J. Degirmencioglu. Appl. Ferrand.. F. Effects of potassium sorbate on growth and penicillic acid production by Aspergillus ochraceus and Penicillium aurantiogriseum. M. J. C.. E. Technol. Extending shelf-life of fresh wet red hake and salmon using carbon dioxide and oxygen modified atmosphere and potassium sorbate ice at 1°C. Vet. Freese. O. J. R. J. 5:41–43. and de Saint Blanquat. Food Prot.. 25:939–945. M. 1994.. Fritsch.C.. Frank. and Galliers. P... Marc. Lebensm. K. Food Add. L.. Fang. 1986b. E. A. B. Inc. G.. and Gedek. Zentralbl. 47:416–418. N. Analyst 115:1219–1221. Underkofler.. Food Sci. Gerchenson.. and Lindsay. H. Packaging of scallops with sorbate: an assessment of the hazard from Clostridium botulinum. Y. J. Int. Shao W. M. J.. G. C. 1975..-S. W. L.. and Powrie. G.. 63:487–494. thesis... Function of lipophilic acids as antimicrobial food additives. Report prepared for the Food and Drug Administration. A. Technical note: kinetics of sorbic acid loss during storage of peaches preserved by combined factors. D. Fritsch. J. Selectivity of sorbic acid media for the catalase negative lactic acid bacteria and clostridia.. Butterworth. and Vaughn. 1982. E.. J. Marth. A. L. Garza. and Kalkan. Scientific Literature Reviews of Generally Recognized as Safe (GRAS) Food Ingredients — Sorbic Acid and Derivatives. 1993. Stewart. Food Prot. J. 57:136–140. 1977. S. Improving of conservation and feed value characteristics of citrus pulps. 52:414–417. Toxicol.. and Sanchis.. Stability of sorbic acid in model food systems of reduced water activity: sugar solutions. M. I. and Luk. Food Cosmet. Statham. F.. Sheu. 1978. W. Gareis. . Food Prot.. E.. 148:343–350. V. Wiss. Washington. Anim.. M. Potassium sorbate as a fungistatic agent in country ham processing. C. Contam.. 51:1028–1031. Food Microbiol. Washington. A. H. and Chirife. Cassand. Bacteriol.Sorbic Acid and Sorbates 79 El-Shenawy. O. L. U.. J. 13:31–45. E.. Food Prot. Food Technol. Gerchenson. Emard. 1957. In Developments in Industrial Microbiology. 1975. 1952. Marc. A. R. 2000a.S. Sci. W. VA. J. H.C. R. L. Mikrobiol. 45:398–404. and Bremner. A. Chin F. 1984. 1988. 21:517–519. C. Effect of sodium chloride and glycerol on the stability of sorbic acid solutions of reduced water activity. R. 2001. N. Fung. 2000b. J. Murrell. Geminder.-F. Depletion of sorbate from different media during growth of Penicillium species. J. J. and Chen. 20:98–99. Ferguson. Inhibition and inactivation of Listeria monocytogenes by sorbic acid. M.. Appl.. Fey. Amino Acids 18:251–263. J. 1973. Fan. F. D.. 1988. and Shih. Microbiol.. 1990. and Chirife. E. Food Sci. I. P. Mutagenicity and genotoxicity of sorbic acid-amine reaction products. Cassand. Microbiol. D. P. 1986a. Bauer. Long-term toxicity of sorbic acid in the rat. B. Y. and Marth. A.C.. D. 1999. S.

and Luckmann. J.. Toxicol. and Hitchcock. Effects of nitrite and sorbate on bacterial populations in frankfurters and thuringer cervelat. Lawrence. K. Microbiol.. Giannakopoulos. Environ. Sorbic acid diffusivity in relation to the composition of high intermediate moisture model gels and foods. Inhibition of the respiration in yeast. 1962. P. and Butterworth. 22:107–122. 32:940. J. K. Appl.. Effects of nitrite. Enzymatic determination of sorbic acid.. W. Lloyd. C. Elliot. Hendy. Anne. Food Sci. J. Chem. Harrington. S. 1994. I. Gray. D. S.. Preserv. 1976. L. Hallerbach. and Guilbert. S. C.. K. J. L. 1986a. J. S. J. G. and Floros. Y. 211:72–76. and Stark. Beuchat. J. R. Gooding. M. Jr. Food Microbiol. Food Prot. 1980.. Eur. potassium sorbate and incubation temperature. 27:15–19. I. Food Process.. 62:3158–3164. Harada. 4:539–545. Golden. 21:339–353. Hansen.379. The effect of microbial inhibitors on the storage life of cottage cheese exposed to ambient temperatures. Determination of sorbic acid diffusivity in model food gels. D. H.. Food Technol. Int. D.. Sorbic acid as a fungistatic agent for foods. D. J. A. and Guilbert.. Food Prot. ascorbate. propionic and caproic acids on the growth of certain clostridia. P.. D. O. 1955. A. Long-term toxicity studies of sorbic acid in mice. J. S. R. H. 2000. J. M. Can. Technol. and Maurer. 179. and Appleman. Deane. R. 21:293–303. N. 1996.294. U. Patent 2.. 21:477–485. Mechanism of beef shelf life extension by sorbate.. R. An off-flavor associated with the use of the sorbic acid during feta cheese maturation. Microbiol. The effect of sorbic. 1992. C. Food Res. Can. J. IX. and Kunsman. 1994. Effects of potassium sorbate on growth patterns. P. F. Guerrero. G. Agric. L. Development of a shelf-stable banana puree by combined factors: Microbial stability. 1986b. Kiss. C. L.. J. Anal. Food Prot.. J. and Utsumi. and Potter. Physicochemical considerations in using sorbic acid to protect foods. and Gerchenson. Food Cosmet. N. and Hills. Henriques. E. 1984. J. Crimmins. R. 14:381–386. A. Activity of the plasma membrane H super (+)-TPase and optimal glycolytic flux are required for rapid adaptation and growth of Saccharomyces cerevisiae in the presence of the weak-acid preservative sorbic acid. Golden. J. Process of inhibiting growth of molds. J. N. Simulating diffusion model and determining diffusivity of potassium sorbate through plastics to develop antimicrobial packaging films. M. 1988. Hofer.80 Antimicrobials in Food Giannakopoulos. I. 1968. 44:341–346. C. and Gerschenson. Hall.. Huang. Food Res... morphology.. D. S. and heat resistance of Zygosaccharomyces rouxii at reduced water activity. I. 59:1616–1617. Z. Food Technol. 49:142–145. Biol. R. H. Tech.. N. 57:902–907. D. B. Schweitzer. and Tomlins. Stability of sorbic acid in aqueous solutions of sodium chloride. F. S. Gourama. M. A. G. .. J... J. Melnick. H. G. Hoffman. J. T. 1997. Milk Food Technol. L.. I. Control of two major pathogens on fresh poultry using a combination potassium sorbate/carbon dioxide packaging treatment. J. Chem.. M.. Extrusion of benzoic acid in Saccharomyces cerevisiae by an energy-dependent mechanism. and Armstrong. and Bullerman.. R. Effect of potassium sorbate on yogurt cultures. 20:639–648. 20:92–96.. J. Holley. Food Sci.. E. 1982. and Louriero-Dias. Gaunt. Alzamora. Food Sci. J. J. Holyoak. C. M. Quintas. R. G.. M.. and Millard. Ultraviolet spectrophotometric determination of sorbic acid in dry fermented sausage. D. McMullin. 38:1252–1259. Ramshaw. Stratford. Diary Technol. L. Food Res. Spectrophotometric determination of sorbic acid in apple cider. A. Horwood.. Changes in fatty acid composition of various lipid components of Zygosaccharomyces rouxii as influenced by solutes. M. Studies on sorbic acid. and Beuchat.. Poultry Sci. R. M. 51:139–144. 1980. 1955. 34:307–311. L. 63:1332–1335. K. H. J. Aust. C. 143:1877–1883. Inst. Technol. Food Res. H. E. 1990. S. B. G. D. Brown. and Coote. F. 1971. Han. and sorbate on Clostridium botulinum in turkey frankfurter slurries. M. 1981. R. Lebensm. Hizuchi. Greer. 1981. Guerrero. Fungistatic properties of the fatty acids and possible biochemical significance. J. T. Food Prot. A. H. 1970. Gooding. 45:82–83. 3:157–161. A. W. Microbiol. 36:38–40. Off. 23:271–273. J. M. H. Hamdan. Part IV. J. and Jenewein.S. E. D. A. Wiss. J. Assoc. Alzamora. Cole. and Dalby. Effects of potassium sorbate and natamycin on growth and penicillic acid production by Aspergillus ochraceus. Hardy. 1998.. 1945. 1944.

1995. B. K. C. A. L. 1978. U. Food Safety 6:197–201. Y. 1981. J. Food Add. Takahashi.. Inhibition of Clostridium perfringens by butylated hydroxyanisole. J. 12:17–30. N. Huhtanen. and Genigeorgis. M. V.. T. Use of sorbic acid and carboxymethyl cellulose as a surface coating in the preservation of apple jam.. Rep. 1992. J. Quality of boneless dry-cultured ham produced with or without nitrate. I. B. Bacterial spoilage of citric products of pH lower than 3. Effect of potassium sorbate on the microbiological quality of butter... Milk Food Technol. L. F.. A new N-nitropyrrole. 22:372–373... coli. A. Hepatotoxicity by the simultaneous administration of several drugs and nitrite. Acid tolerance of Escherichia coli O157:H7 and its survival in apple juice.. Reaction of sorbic acid in wheat flour doughs: reaction with thiols. faecalis and Cl. Crown. S. and Branen. Food Technol. and Phillips. 43:656–657. Talley. 44:914–915. Kemp. B. Toxicol. Juven. J. Feinberg. 1984.. C. Y. Food Add. Toxicol. S. P. Effect of weak acids on amino acid transport by Penicillium chrysogenum: evidence for a proton or charge gradient as the driving force. K. 46:807–810. N. and Omori. Studies on nitrosamine formation by the interaction between drugs and nitrite.. 41:621–625. C. N. B.. Christiansen. Food Prot. 1983. 2001. Khoudokormoff. Hunter. A. 11:539–548. Solomon. Ivey.. N. 1961. Kito. L. 1979. H. 6:271. J. J. K. D... D. Kadam. J. Food Prot.. R. 113:1184–1192. and McCleskey. J. F. Brekke. M. M. Food Sci. Natl. 1976. and Wedzicha. H. Food Cosmet. J. and Tompkin.. and Harper. 14C-sorbic acid distribution in the aqueous and non-aqueous extracts of cooked millet dough (fura). Thomas. 1952.. D. Food Microbiol. Zeuthen. K. Kaloyereas. B..Sorbic Acid and Sorbates 81 Huhtanen. and Fox.. Y. Microbios 104:167–175. J. and Wedzicha. Singh. 9:493–497. Ohno. Food Technol. J. Koodie. J.. Experiments on preservation with a new ice containing glycol diformate and sorbic acid. Khandelwal. 15:361–364. Measurement of the amount of nitrosamine formed in rat and guinea pig stomachs. 25:289–300.. Contam.. Flavor and antibotulinal evaluation of sorbic acid-containing bacon. 1974. F. Kivanç. A. A. Kasuya. Food Sci. Food Sci. B. 48:1709–1714. Evaluation of the mutagenicity of sorbic acid-sodium nitrite reaction products. T. Kawanishi. J. I. E.5. D. Effect of chemical dips on unchilled fresh beef inoculated with E. Toxicol. L. L. M. L. J. netting or potassium sorbate. J. 1992.. J. Y. Genet. 1. Food Sci. J. 1978. Food Sci. M. V. Takahashi. Klindworth. J. J. Feinberg. Sci. . 46:1796–1800. Namiki. L. Inhibition of Staphylococcus aureus in a model agar-meat system by monolaurin: a research note.. 1978. P. Y. Jones. A. B. Ohno. Food Add. 6:261–270. K. 1979. A. and Feinberg. Y. and Wedzicha. 44:564–567. 1973. A. Effect of potassium sorbate on toxogenesis by Clostridium botulinum in bacon. D. B. 36:578–583. Kawanishi. J. Sorbic acid inhibition of Clostridium botulinum in nitrite-free poultry frankfurters. Int. Studies on nitrosamine formation by the interaction between drugs and nitrite. and Dhople. N. Davidson. Fungal contamination of Kahar cheese in Turkey. C. and Robach. Technol... Potential growth and control of Escherichia coli O157:H7 in soft Hispanic type cheese. Ivey. Shaver. E. Contam.. J. P.. 1981a.. Kemp. 1981b. Die Nahrung. DNA-damaging products from reaction between sodium nitrite and sorbic acid. M. B. J.. C. C. R. S. G. 1983. Kabara. J. Tanimura. J. 12:161–166. J. Kada. 1979. G. Jpn. formed by the reaction of sorbic acid with sodium nitrite. T. J. Y. J. Rimmer.. Tetrahedron 34:505–508. Kaul. Y. Contam. Huhtanen. J.. M. 1985. I. 1981. and Fox. G. Reaction of sorbic acid in millet and sorghum doughs: reaction with thiols. A preliminary study of factors affecting the quality of pickles on the Canadian market. A. S. 39:819–822. 24:43–44... Onoda.. R. M.. II. 19:405–406. Ishiwata. and Tsuji. C. Acid enhancement of Clostridium botulinum inhibition in ham and bacon prepared with potassium sorbate and sorbic acid. R. Food Sci. and Omori. and Phillips. Food Sci. Jideani. Sunouchi. J. J. 1994. perfringens and stored at 30°C and 20°C. Langlois. Bacteriol. Meat Sci. Sci. and Kuila. Langlois. 43:1782–1785.4-dinitro-2-methylpyrrole... G. J. J. J. 6:304–308. Kasrazadeh. M. and Segel.. Effect of sorbic acid and sodium nitrite on Clostridium botulinum outgrowth and toxin production in canned comminuted pork. Kondaiah. Food Prot. and Ingle. D.. Effect of potassium sorbate and vacuum packaging on the quality and microflora of dry-cured intact and boneless hams. Trenchard. B. Jideani. 1985. Inst. G. and Jul. Kasuya. M. J. S. 1980. 45:453–457. J. 1995. H.

Department of Commerce. Growth of sorbate-resistant and -sensitive strains of Penicillium roqueforti in the presence of sorbate. George. L. Washington. U. J. 1984. E... D. 82:1860–1868. J. N. Sharma. and McElroy. D. H. J. F. and Martin. J.. Denyer. M. L. Food Prot. Lennox. X... B. B. Mazas. Appl. Kouassi. M. Spectrophotometric determination of sorbic acid in foods in general. Department of Commerce. and Franklin.. Uses. P. 45:824–828. J. Liu. J. L. L. Food Sci. J. Food Prot. Effect of solute type on sorbate resistance in Zygosaccharomyces rouxii. 1983. K. Lück. and Chism. Inhibition of growth and patulin synthesis in Penicillium expansum by potassium sorbate and sodium propionate in culture. Sorbic acid as a food preservative. 1995. Antimicrobial Food Additives. 59:935–941. J. E. J.. Forestiere. Listeriolysin O secretion by Listeria monocytogenes in broth containing salts of organic acids. Microbiol. Kumar. F. Inhibition of penicillia and aspergilli by potassium sorbate. J. 48:525–529. 58:1314–1319. A note on the effect of dilution and temperature on the bactericidal activity of potassium sorbate. and Kulkarni. Dairy Sci. B. Microbiol. and Isaacs. 48:1031–1033. Production of mycotoxins by sorbate-resistant molds. Recent Adv. A. A. Effect of preservation techniques and food additives on staphylococcal thermonuclease. S. 44:531–534. 1983. Inhibition of nitrosamine formation in vitro in presence of both ascorbic acid and sorbic acid. H. Graham. Food Prot. Food Saf. Effects of potassium sorbate on normal flora and on Staphylococcus aureus added to minced cod. B. Gonzalez. 47:554–556. and Shelef. B. Litton Biometrics. Appl. M. D. M. 48:364–375. P. X. S. Mackey. W. Fung. Nutr.. 66:66. 1977. 43:914–916. K. F. B. H. E. Larson. M. type B Clostridium botulinum. pH and sorbic acid on the probability of growth of non-proteolytic. E.127.. Appl.. M. Food Prot. 46:568–570. 20:649–654. Luckman. Effects of potassium sorbate alone and in combination with sodium chloride on the growth of Salmonella typhimurium 7136. 1996. Yousef. D. Springer-Verlag. Mutagenic evaluation of calcium sorbate. Liewen. Growth effect of sorbate and selected antioxidants on toxigenic strains of Staphylococcus aureus. A. N. Litton Biometrics. G. 1976. J. Liewen. Environ. Factors affecting the resistance of Listeria monocytogenes to high hydrostatic pressure. H. Microbiol. W. B. J. Int. and Melnick. Responses of conidia of Penicillium roqueforti to sorbic acid. Growth and inhibition of microorganisms in the presence of sorbic acid: a review. Listeriolysin O secretion by Listeria monocytogenes in the presence of cysteine and sorbate. 1988. 1995a. K. 1985c. Lett. M.C. E. E. and Potter. Johnson. 1981. Sorbic acid as a fungistatic agent for foods. and Brown. 1999. 69:481–492. J. Food Prot. J. Y. D. B. 16:287–299. Lathia. Germany.. Buchanan.82 Antimicrobials in Food Kouassi. Clin. H. 1:189–190. Liewen. Publication PB-245–434. E. Flavors Food Addit. E. Liewen. Inhibition of type A and type B (proteolytic) Clostridium botulinum by sorbic acid. and Marth. Appl. 1995b. and Marth. 1982. and Nelson. and Hugo. and Restaino. M. Environ. and Bernardo. Larocco. Food Prot.. Lahellec. Lett. and Cunningham.. and Marth. P. A. Lopez... . S. Thermal resistance of Bacillus stearothermophilus spores in different heating systems containing some approved food additives. Larsson. E. J.. E. D.. Lusher. A. Inactivation of Escherichia coli O157:H7 by the combination of organic acids and pulsed electric field. 1987. B. M. M. Survival of Listeria monocytogenes in commercial cheese brines. 1981.C. S. C. The combined effect of incubation temperature. 48:156–157. B. National Technical Information Service. Lynch. K.. J. Food Res. 1984. Food Biotech. Publication PB-266–894. A. and Shelef. R. N. D. E. Mutagenic evaluation of potassium sorbate. 7:122–124.. J. A. J.S. Microbiol. U. A. 2000. National Technical Information Service. E. 23:187–191. and Marth. M. George. 1990. E.. and Marth. Anal.S. Liewen. Characteristics. I. Y. J. 9:1–11. 66:775–780. 1955. J. 1985b. A. 20:295–299. L. 1984. Assoc.. 57:179–181. J. Berlin. Inc. M. Bacteriol. R. H. H. Lück.. Appl. Dairy Sci. Effects. 1997. M.. Food Sci. Die Nahrung. Off. G. N. J. Washington. J. K. 1980. Food Prot. Chem. and Schellhoeh. Gonzalez. 44:272–275. 1981. Inc. Lund. Appl. Y. Worley. B. 1974.. Lund. Lenovich. L. Gas–liquid chromatographic determination of benzoic acid and sorbic acid in foods: Nordic Committee on Food Analysis collaborative study. Bacteriol. 1985a.

Food Prot. and Walker. M. T. 1979. 1982. Food Res. D. L. Maxstadt... Effect of sucrose esters in combination with selected mold inhibitors in growth and aflatoxin production by Aspergillus parasiticus. Nolleau. Vahlteich. D. 1997. Mendonca. Morad. O.. J. Escherichia coli O157:H7 acid tolerance and survival in apple cider. and Brekke. Melnick.. V. G. Assoc. J. D. J. Luckmann. and McWeeny. Monnet. R. 1997.. Chromatogr. and Rejano.. D. A.. Sorbic acid as a fungistatic agent for foods. 57:460–464.. L. Microbiol. IV.. presented at the ACS/CSF Chemical Congress. J.. J. 1963. J. R. 1954a. B. Forsythe.-O. and Al-Sa’Ed. Mihyar. Comparative evaluation of methods. Morin. Effectiveness of sorbic acid in protecting cakes. Sanchez. W. Appl. Sorbic acid as a fungistatic agent for foods. F.. H. R. V. A.. 49:378–382. I. Martoadiprawito. F. Antibacterial effects developed by the reaction of sorbic acid with sodium nitrite. sodium acetate. T.. Rel. 1972. and Hussang. W. 1982. 1986. H. R. Sorbic acid as a fungistatic agent for foods. A. physical modification. C. Food Res. K. Migration of sorbic acid from wrapper into cheese. Offic. 54:2167–2173. Technol. J.. XI. Degradation of potassium sorbate by Penicillium species. Molins. Montano. R. Myers. 19:33–43. Mayer. and Thomas. Food Res. 1994. Antimicrobial activity of butylated hydroxyanisole and potassium sorbate against natural microflora in raw turkey meat and Salmonella typhimurium in cooked turkey meat. Food Sci. Chromatogr. 21:133–146. Spectrophotometric determination of sorbic acid in cheese wrappers. Food Safety 6:261–270. Anderson. Branen. Potassium sorbate and recovery of pectinolytic psychrotrophs from vacuum-packaged pork. 19:20–27. P.. and Brown. L. Food Res. H. 1983. M. . F.. 19:28–32. III. E. T. L. Honolulu. A. and Bullerman. Jackson. H.. and Cosio. D. 1956. A. Edmondson. J.. A. J.. L. VI. F. E. and Marshall. and Luckmann. Analyst 120:2483–2487.. Biotechnol. and Tambute. Solid phase extraction as a clean up procedure for the liquid chromatographic determination of benzoic and sorbic acids in fruit derived products. and Luckmann. 1995... J. 1984. Potassium sorbate inhibition of yeast alcohol dehydrogenase. Environ. Potato pulp: microbiological characterization. 388. and Whitaker. C. and Namiki. and application of this agricultural waste product. Determination of benzoic and sorbic acids in food by microdialysis sampling coupled with HPLC and UV detection. S. Dairy Sci. 1989. A.. McMeekin. Resistance of sorbic acid in cheese to oxidative deterioration.. A. P. and Gooding. Food Res. 19:44–58. C.. Osawa. 49:188–191. C. F. D. M. 1984. S. D. M. D. R. and Fabre. C. P. N. H. HI. L. Marth. A. H. and Karasz. B. Rapid spectrophotometric method for the determination of sorbic acid in fresh dairy products. Luckmann. Liq. Food Agric. J. The effects of ascorbic acid and sorbic acid on N-nitrosamine formation in a heterogeneous model system. 1954b. 45:1038–1043. Melnick. A. Massey. Chaimbault. Food Sci. E. and Creach. and Hackett. G. S.. Gastaldi. J. Acta 77:536. Yamani. Miller. and Kaspar.Sorbic Acid and Sorbates 83 Mandrou. C. A.. H. Food Prot. Determination of benzoic and sorbic acids in packaged vegetable products. Marshall.. Effect of potassium sorbate on the microbiology of vacuum-packed poultry. C. J... Italian J. D. A. Melnick. Metabolic degradation of sorbic acid in cheese by molds and the mechanism of mold inhibition. Biochim. Hasenzahl. J. phosphates and sodium chloride alone or in combination on shelf life of vacuum packaged pork chops. Vidal. Mannino.. 1954b. Kraft.. W. J. W. and Hillebrandt. Melnick. J. D. W. A. A. 1998. I.. Identification of phosphoric acids by capillary electrophoresis-ion spray mass spectrometry. Sorbic acid as a fungistatic agent for foods. 33:294–298. M. Sorbic acid as a fungistatic agent for foods. Mercier. 49:1197–1205.. Melnick. K. M. Miller. 46:499. 80:2304–2309. Dreux. 55:7–8. M.. R. 21:829–842. Food Prot. M. 48:435–440. Namiki. Microbiol. Appl. E. J. H. Chem. V. 1954a. 8:311–316. and Gooding. 54:302–306. H. J. Food Sci. Anal. April 1–6. F. Resistance of yeast flora of labaneh to potassium sorbate and sodium benzoate. J. Effects of potassium sorbate. Influence of metabolic and physical factors on production of diacetoxyscirpenol by Fusarium sambucinum fuckel. Capp.. 825:71–80. J. Sci. 1996. H. 1999. J. Effectiveness of chlortetracycline in combination with potassium sorbate or tetrasodium ethylenediamine-tetraacetate for preservation of vacuum packed rockfish fillets. M. J. Food Prot. Dairy Sci. 1966. J. B. Biophys.. 1988. M. F. Pennington. P.

K. Chemical aspects of mutagen formation by sorbic acid-sodium nitrite reaction. 1995. 1997. Accumulation of acid antiseptics in yeast cells. E. Tsuji. I.. M. G. Ishibashi. 1972.. N. Neves. Biotechnol. E. Tsuji. and Trenerry. F. K. Am. Inactivation of Salmonella typhimurium by sorbic acid. 1981. M. Ogundiwin.. Oswa. Nielsen. 1988b. Osawa. J. Soc. Welti-Chanes. F. 1980.. National Academy Press. Resistance of food spoilage yeasts to sorbic acid. M. and Marth. M. and Busta. J. 19:8–11. Namiki. Kada. Osawa. L. G.. Bacteriol. Microbiol. and Kada. Osawa.. B. 1970. Effects of various concentrations of sodium nitrite and potassium sorbate on color and organoleptic qualities of commercially prepared bacon. Appl. Effects of combined antimicrobial agents on fermentation initiation by Saccharomyces cerevisiae in a model broth system. Inhibition of Bacillus spores by combinations of heat. Pant. Transfer of antiseptics to microbes and their toxic effect. C. Osawa. and Swanson. Paquette. T. H.. 45:1293–1296. . M. N. Y. T. E. Appl. 1980. E. J. T. National Academy of Sciences. B. 1960. D. and Frisvad. 46:3105–3108. S. K. Alternatives to the Current Use of Nitrite in Foods. V.C. Agric.. 1988a. 66:197–207. Appl. Pampulha. and Ingraham. Kada.. T. Milk Food Technol. O. and Tsuji. Nelson. K. Res. 1982. K. Oswa. C. M. Park. Food Prot. Namiki. 53:237–239. K. Ough. Food Chem. J. and Carroll.. Fessehatzion.. 62:855–857. Lopez-Malo.. K. High hydrostatic pressure as a hurdle for Zygosaccharomyces bailii inactivation.. E. Tetrahedron 45:4399–4402. 1979. and Namiki.. 11:117. 10:579–589. S. N. Carcinogens Mutagens Environ.. T. E. Namiki.. M.. Osawa. K. A new furoxan derivative and its precursors formed by the reaction of sorbic acid with sodium nitrite. J. Vitic. Nitrite and N-nitroso Compounds.. D. Jpn. 1983. J. and Scholefield. J. B. Minimum inhibitory concentration studies on antimicrobic combinations against Saccharomyces cerevisiae in a model broth system... 53:240–242.. Mutagen formation in the nitrite-piperic acid reaction. J. NaCl and pH. Biol. Effect of polyphosphates in combination with nitrite-sorbate or sorbate on Clostridium botulinum growth and toxin production in chicken frankfurter emulsions. Namiki. Bacteriol. E. Kenjo. Namiki. Mutat. 1980. Jpn. S. and Namiki. Enol. O. 24:59–65. Food Chem. sorbic acid and sodium nitrite. Formation of mutagens by sorbic acid-nitrite reaction: effects of reaction conditions on biological activities. J. J. M. Chem. Beuchat. Biophys. H. National Academy Press. potassium sorbate. 1994.. Determination of sorbic acid in cheese.. H. Osawa.. Palou. T. Noda. T. 46:846–850. Washington.84 Antimicrobials in Food Namiki.. Ishibashi.. Survival of Salmonella typhimurium in cold-pack cheese food during refrigerated storage. Washington. Chem. N. H.. Food Sci. G. Biol. Commun. 1981. Kito. F. Desmutagenic action of food components on mutagens formed by the sorbic acid/nitrite reaction. C. 1994. T. and Tsuji. T. C. and Takahashi. Park. Biol. J. M. Robach. J.. J. Soc. Namiki.. Parish. H. Agric. 14:253–257. V. 1989.... L. 33:383–388. Growth and fumitremorgin production by Neosartorya fischeri as affected by food preservatives and organic acids.C. Sofos. T. Agric. O.. 1960. Biochem. Namiki. M.. V. Sofos. W. 3:109 National Academy of Sciences. 1983. Formation of C-nitro and C-nitroso mutagens by the reaction of nitrite with sorbic acid and its analogues and their inactivation with food constituents. M. and Carroll. I. 1982. 1973. 46:2299–2304. Lett.. 35:532–539. O. A. and Loureiro-Dias. J. J. Mutagen formation in the reaction of nitrite with the food components analogous to sorbic acid... K.. D. R. M. Chem. 53:219–236. T. 50:1971–1977.. Busta. H. A. 29:407–411. J. J. The determination of sorbic acid and benzoic acid in a variety of beverages and foods by micellar electrokinetic capillary chromatography. Desmutagenic actions of ascorbic acid and cysteine on a new pyrrole mutagen formed by the reaction between food additives.. 71:139–143. F. J. Bull. 1982. Ilori. F. 95:835–841. A.. and Olson.. Milk Food Technol. Food Sci. S. and Olajuyigbe. Food Sci. E. Food Hyg. 1986. 73:21–28. Udaka. 1991.. Babalola. T. O. World J. H. The Health Effects of Nitrate. D. M. Microbiol. Food Sci. Marth. Res. C. and Kada. K.. T. S.. M. Effect of chemical treatments on the microorganisms associated with malting of sorghum grains and sorghum malt. and Namiki. K. Agric. Chem. M. Use of sorbic acid and sulfur dioxide in sweet table wines.. Agric. P. J. Oloyede. Parish. and Wagner. Oka. Barbosa-Canovas. M. L. and Tsuji. Ishibashi.

M. S. and Adatia. A.. Thompson. 1992. C. 1944. Holyoak. L. Rao.. refrigerated pork chops. Simultaneous determination of synthetic dyes. L. 1982. 38:846–849. Extraction of organic acids by ion-pair formation with tri-n-octylamine. J. and Massart. J. 1964. P. H. St. M. M. Extension of shelf-life of fresh. 938. Poulanne. 1982. 10:52. O. C. G. Off. Survival of Listeria monocytogenes in cottage cheese. G. 1979. R. Pederson.Sorbic Acid and Sorbates 85 Patrizi. E. Drug-nitrite interactions in human saliva effects of food constituents on carcinogenic N-nitrosamine formation. 1961. R. R. Restaino.. L. D. Lenovich. and saccharin in soft drinks and lemonade syrups. J. Appl. J. L. Perry. Growth characteristics of Saccharomyces rouxii isolated from chocolate syrup. 1984. . Robach. L. Appl. 18:891–897. Food Prot. Puttermans.. Food Technol. C. Chem. Contact Dermat. J. Robach. J. M. 47:134–138. Food Qual.. C. M. K. The effect of potassium sorbate and sodium nitrite on the organoleptic properties. Y. E. Meat Res. and growth of Bacillus cereus and Clostridium perfringens in cooked sausage. M. Sorbic acid as an inhibitor of scum yeast in cucumber fermentations. C. K. J... M. Preliminary observations on human reactions related to sensory evaluation of cooked. and Smoot. Proc. 4:291–293. D. Contam. 9:515–525. Petaja. and Bills. Work 25:917. and Masci. and Kuchler. 1983. and Conn. The growth of yeasts in grape juice stored at low temperature. A. A. sorbic acid. and Mundt. In 39th Ann. Phillips. M. Microbiol. and Shelef. 8:113–129. L. Environ. Extension of poultry shelf-life by processing with sorbic acid. and Syracuse. C. J. 1979a. 1980. Rice.. J. Przybylski. D. 1985. J. Ind. Mtg. Albury. Environ. K. The Pdr12 ABC transporter is required for the development of weak organic acid resistance in yeast. Egner.. F. Tscherneff. and Adam. M. Food Sci. Appl. D. Anal. M. Influence of sorbic acid on viability and ATP content of conidia of Aspergillus parasiticus.) Piper. B. A. 61:768–771. 385. 1999. Van Tassel. L. Effect of acids and sorbate combinations on the growth of four osmophilic yeasts. Eng. Poerschke. Microbiol. 45:742–743. J. Effects of acids on potassium sorbate inhibition of food-related microorganisms in culture media. Sebranek. Robach. Pierson. N. 1976... Dryon. Fungistatic effects of organic acids. J. J. D... Effect of heat treatment and selected antimicrobials on the shelf-life and safety of cooked.. Bills... K. vacuum-packaged. C. 36:185–187. Food Prot. 2:205–213. 43:933–935. and Melnick. potassium sorbate and temperature on the outgrowth of Clostridium sporogenes PA 3679 spores in a pre-reduced medium. G. D. J. Effect of increase in acidity on antiseptic efficiency. Sodium nitrite and potassium sorbate inhibition of Clostridium botulinum as influenced by storage temperature variation. Quattrucci. M. Interaction of salt. F. 45:1614–1621. A. Food Prot. 58:128–131. K. R. 42:855–857. Komatsu. C. J. Orlandi. and Cunningham. IV. Inst. F. Food Sci. L. commercial bacon.. Res. Food Sci. 47:1615–1617.... and Christensen. Robach. 373. G. 1979b. Restaino. Microbiol. Prabhu. EMBO J. S. 5:285–300. 1950. L.. Louis. K. S. Rahn. J.. Mahe. R. E... Muehlbauer. 45:375–376. Food Sci. J. (Abstract. L. 53:1270–1274. W.. Appl. S. A. Raevuori. Eur. O. M. 1979. W. and Lenovich. R. 45:1138–1142. stability. and Hill. Food Prot.. Eur. 17:4257–4265. Robach. and Bullerman... Molins. Piccinin. Regenstein. P. benzoic acid. A. Effect of processing variables on the outgrowth of Clostridium sporogenes PA 3679 spores in comminuted meat cured with sorbic acid and sodium nitrite. P. Restaino. whole broilers using a potassium sorbate dip. M. V. Nutritional aspects of food preservatives. J. Food Technol. Food Technol. K. Food Sci. Vincenzi. and Pierson.. and Bardazzi. K. Effect of sorbic acid and potassium sorbate on growth of Bacillus cereus and Bacillus subtilis in rice filling Karelian pasty. L. J.. Inhibition of Salmonella by sodium nitrite and potassium sorbate in frankfurters. p. 1988. Allergic contact dermatitis caused by sorbic acid: rare occurrence. M. L. 9:162–167.. R. J. Pandjaitan. S.. Food Qual. Meet. E. 1998. Coote. 1995. A. Dent. J. 1982. Raevuori. Food Add.. Lawrence. 1981.. J. Osborn. V.. 1983.. Am. E. and Walker. C. M.. The shelf-life extension of haddock in carbon dioxide oxygen atmosphere with and without potassium sorbate. M. M. Microbiol. Chem. M. M.. G. Influence of potassium sorbate and selected antioxidants on growth of Salmonella senftenberg. MO. Assoc. 1980. Kraft. 67:880–885. 1980.

Biomass and patulin production by Byssochlamys nivea in apple juice as affected by sorbate. Food Prot. C. G. and Marth. Robach. and Robinson. I.. J. and Sankaran. 18:237–240. M. A. C.. 1987. Ronning. M. hydrochloric acid. 45:1280–1284. Chemical preservatives to inhibit the growth of Staphylococcus aureus in synthetic cream pies acidified to pH 4. 1988. Schmidt. Appl.. Ronning. 47:237–241. Robach. Automated analysis of sorbic acid in food products. F. Food Technol. and Canela... and Kier. Inhibition of Staphylococcus aureus by potassium sorbate in combination with sodium chloride. C. J. Microbiol. 41:372–374. E. J. H. and Beuchat. and Weiser. J. 1980a. J. J.. M.. and to noninhibitory levels of sorbic acid. E. 1984. E. G. R. 58:1. 51:289–292. J.. and Frank. Food Sci.0. Beuchat. Food Sci. Adam. Robach. Jr. Food Technol. M. Growth and aflatoxin production by Aspergillus parasiticus NRRL 2999 in the presence of potassium benzoate or potassium sorbate and at different initial pH values. K. C. 41:699–702. C. B. fresh poultry and poultry products: a review. Vinas. Z. E.. T. and Ivey. I. M.. I. Environ. 49:402–406. 29:628–630. V. Gibson. R. Food Prot. Morphological changes in putrefactive anaerobe 3679 (Clostridium sporogenes) induced by sorbate.. M. J. J. Food Sci. and Cook. S.. L. Antimicrobial efficacy of a potassium sorbate dip on freshly processed poultry. S. 130:2845–2850. 1980b. Growth inhibition of putrefactive anaerobe 3679 caused by stringenttype response induced by protonophoric activity of sorbic acid. Owens. Schmidt. Robach.86 Antimicrobials in Food Robach. Food Prot.. Factors controlling the growth of Clostridium botulinum types A and B in pasteurized. 53:1020–1027. M. W. Food Prot. Kroll. 1980c. H. H. benzoate. E. and Hickey. Roland.. L. Prot. Biol.. R. J. Toxicol. V. F. The effect of potassium sorbate. Food Prot. H. J. Gould. Rusul. 45:374–383. C. C. 1969. cured meats. Indian J. O. A. Ryser.. 1960. 51:651. C. S. Robach. Survival of Listeria monocytogenes in cold-pack cheese food during refrigerated storage. Can. H. A. 51:615–621. and To. 1988. J. R. C... I. Viladrich. J. and Frank.. Effect of sorbates on microbiological growth in cooked turkey products. Hazelnuts as possible substrate for aflatoxin production. M. The effect of food preservatives on pH homeostasis in Escherichia coli. To.. 1984. J. A specific method for the colorimetric determination of sorbic acid (in non-alcoholic foods). 36:210–211.5 to 5. Schmidt. G. Roberts. Food Prot. 1978. and Booth. Roland. Sahn. tertiary butylhydroquinone. Robach. Use of sorbates in meat products. Quilez. 1978. Exp. Microbiol. Anal. and nitrite). L. Robach. Effects of various concentrations of sodium nitrite and potassium sorbate on nitrosamine formation in commercially prepared bacon. Colorimetric determination of sorbic acid in wine.. 1976. Appl.. R. and Marth. E. H. W. DiFate. and Stateler. S. A. M. Sayeed. Chem. butylated hydroxyanisole or ethylenediamine tetraacetic acid. F.. N. 17:307–326. C.. R. Meydav. Action of sorbic acid on Staphylococcus metabolism: a microcalorimetric investigation. 45:638–640.. A. Food Prot.. J. A. 1980. 1991. A. E.. M. 1984. Ivey. and Hickey. Food. N. M. Ronning. J. Environ. J. H. C. Paquette. Food Prot.. Evaluation of the mutagenicity of sorbic acid-sodium nitrite reaction products produced in bacon-curing brines. H. 178:173–193. Robach. sulfur dioxide and temperature on growth and patulin production by Byssochlamys nivea in grape juice. J. M. L. L. Lebensm. Growth response of putrefactive anaerobe 3679 to combinations of potassium sorbate and some common curing ingredients (sucrose. . and Frank. Sanchis. E. 35:388–398. Microbiol. J. 1988. 1982. C. L. E. and Busta. F. D. 1978. Salmond. R. salt.. A. and Sofos. O. benzoate. 23:1197–1220. Roy. I. Food Safety 3:89–98. J. C. Hickey. C. III. J. 1962. E. H. Dtsch. W. Effects of sorbate. Food Sci. S. Food Cosmet. 1981. 43:208–211. A.. F. 1987. System for evaluation of clostridial inhibition in cured meat products. M. and Conetta. Comparison of antimicrobial actions on monolaurin and sorbic acid. 1989. Inhibition of Vibrio parahaemolyticus by sorbic acid in crab meat and flounder homogenates.. M. Gen Microbiol. and Hitchcock. J. V. R. J. R. L. and nitrite. 41:284–288. 50:820–825. 1982. Rundschau. Worthington. C. SO2 and temperature. C. C. J. Sofos. T.

Eds. Proc. p. 1955. and Ignat’ev. 111:525. FL. 1970. Sheu. Salomon. C. 19:59–65. Effect of potassium sorbates and other antibotulinal agents on germination and outgrowth of Clostridium botulinum type E spores in microcultures.. Food Technol. Ed. 34:104–120. mode of action. Appl. Effectiveness of sorbic acid in protecting cheese. 42:477–483. J. Bacteriol.. 51:16–19. J. N. N. A. 1960. Rec. J. Appl. Shtenberg. Ed. Antimicrobial agents. 1954a. W. C. Appl. J. sorbic acid and potassium sorbate on weight losses and gas production in emulsified meat products. Boca Raton. F. T. Bacteriol. 1972. Sorbic acid as a preservative for sweet cucumber pickles. Potential for development of tolerance by Penicillium digitatum and Penicillium italicum after repeated exposure to potassium sorbate. C. N. N. Smith. J. Sofos.. A. J.. J. Environ. 111:516. M. Use of phosphates in low-sodium meat products. M. Meat Res. T. D. . J. L. Sharp. 1983. N. and Costilow. 1994. and Busta. Norback. Sorbic acid. IL. 115:869–875. R. W. Inc. In Food Additive Toxicology. Deibel. P. Further experience with the range finding test in the industrial toxicological laboratory. Arch. Can. J. 1986. Sofos. Schroeder. Microbiol. J. D.. D. J. Sorbate Food Preservatives. Marcel Dekker. 1981. Food Prot. B. J. Food Technol. L.. Sorbic acid as a fungistatic agent for foods. P. Effects of fatty acids on growth and envelope proteins of Bacillus subtilis. 637–659.. Using a new measure to define shelf life of fresh whitefish. In Natural Food Antimicrobial Systems. 23. M. R. Food Cosmet. Antimicrob. C. 1986c. Smith... J. Lederberg. Sofos. 50:919–923. J. N. 34:244–251. H. N. Sheneman. and Bullerman. R. Microculture model studies on the effect of sorbic acid on Penicillium chrysogenum.. Sofos. J. Maga. A. Environ. Food Sci. 3:186–189. Sorbic acid as a fungistatic agent for foods. J. J. Bacteriol. J. Inc. 16:237–241. Effect of potassium sorbate application on shelf-life of Atlantic cod (Gadus morhua). A. Toxicological evaluation of some combinations of food preservatives. A. F. F. N. 1989. 1982. F. Meat Conf. 11:225. Sofos. Sofos. Sheu. L. P. Toxicol.. Sofos. Need for efficacy in protecting packaged cheese. Inhibitory effect of lipophilic acids and related compounds on bacteria and mammalian cells. 1992.. 1975. C. N. In Encyclopedia of Microbiology. and Rollin.. J. sorbate and pH interaction in cured meat products. Inst. Influence of sodium chloride. A. Lebensmitt. N. Technol. Hyg. J. and Lindsay. N. J. Lipopolysaccharide layer protection of Gram-negative bacteria against inhibition by long-chain fatty acids. E. Skirdal. Jr. A.. Sheu. 1973. 8:133–135. 1981. C. Effects of acetate and other short-chain fatty acids on sugar and amino acid uptake of Bacillus subtilis. San Diego. A.. E. Boca Raton... FL. Food Res. N. 44:1212–1221. 1980. D. Food Sci. Smyth. R. CRC Press. Bacteriol. and Freese. Antimicrobial activity and functionality of reduced sodium chloride and potassium sorbate in uncured poultry products. N. Work 32:303. J. L. W. pp. New York. J. 74:191–195. and Busta. S. F. 501. Ind. Sheu. Microbiol. J. and Freese. 44:614–622. Antimicrobial activity of sorbate. VIII. 1993. Seward. 30:63–70. 51:936–939. Sreevalsan. C. The effect of aureomycin combinations with salts and acids on various kinds of bacteria. and Woyewoda.. 1986b. D. 1981. J. Konings. C. and Freese. 1954b. Meet. Naidu. Nitrite. H. Sofos.. W. Proc. W. 1972. and Stuiber. Shaw. Alternatives to the use of nitrite as an antibotulinal agent. 43–52.Sorbic Acid and Sorbates 87 Schmidt. Smoot. pp. Food Technol. W. and Freese. VII.. Sofos. Mechanism of sorbate inhibition of Bacillus cereus T and Clostridium botulinum 62A spore germination. Microbiol. E. 1985. Sorbic acid. Microbiol. D. 40:52–69. L. Eur. E. Cladosporium cladosporioides and Ulocladium atrum at different pH levels. Academic Press. Sofos. CA. Environ. E. Bligh. 7:349–363. 8:369–380. and Rollin. Agents Chemother. American Meat Science Association. O. Appl. Simmons. T. Hyg. Food Sci. J. Chicago. Toxicol. N. and Pierson.. F. Appl. 2000. D. Volume 4. and Tu. J. and Eklund. and Carpenter. 1986a. 1948. J. A. S.. P. I. CRC Press.

Sun. Food Sci.... R. F. 1985... A. 1996. 1989. 46:1084–1091. Am. 65:469–476. Lett. F.. Inhibition of nitrosamine formation in vitro by sorbic acid. Cytotoxicity of food preservatives in cultured rat hepatocytes loaded with linolenic acid. M. Oxford.. A. 1988. Sofos. J. pp. Steels.. Allen. E. N. Eds. Splittstoesser. M. J. . 1980a. H. D. Storage of Morwong (Nemadactylus macropterus Bloch and Schneider) in combinations of polyphosphate. and Busta. Sofos. M. J. J. D. Davidson. Int. Stafford. J. and Churey. Busta. J. J. F. and Quarmby. J.. and Bremner.. Food Sci. J. T. J. C. Contam. Statham. C. 1988.. 44:1662–1671. 3:1–17. Busta. C. E. L. Antimicrobial action of some GRAS compounds against Vibrio vulnificus. potassium sorbate and carbon dioxide at 4°C. F. Vanderzant. a yeast resistant to sorbic acid. J. 1995. Food Prot. Sodium nitrite and sorbic acid effects on Clostridium botulinum spore germination and total microbial growth in chicken frankfurter emulsions during temperature abuse. Sodium nitrite and sorbic acid effects on Clostridium botulinum toxin formation in chicken frankfurter-type emulsions. M. E. 24:894–895. Rapid analysis of potassium sorbate in dried prunes of ultraviolet on colorimetric procedures.. Yeast 16:1173–1183.. F... C. F. W. D. and Mattick. The effect of potassium sorbate on the structural integrity of Alteromonas putrefaciens. Influence of pH on Clostridium botulinum control by sodium nitrite and sorbic acid in chicken emulsions. D. A. M. and Busta. 1979b. C. A. Microbiol. Microbiological characteristics and shelf life of corn tortillas with and without antimicrobial agents. Sofos.. D. Eds. and Furuno.. Blackwell Scientific. 11:549–558. 1979c. J. Food Prot. Food Add. and Branen. I. Sofos. D. L. J. K. In Principles and Practice of Disinfection. Heat resistance of Eurothium herbariorum. P. F. H. Busta. 78:82–87. and Allen. and Anslow. D. and Allen. Food Sci.. Sofos. Food Prot. H.. J. and Alyiffe.. Effect of potassium sorbate on spoilage of blue grenadier (Macruronus novaezelandiae) as assessed by microbiology and sensory profiles.. Hugo.. Busta. Chung.. Acuff. E. Effect of low concentrations of sorbic acid on the heat resistance and viable recovery of Neosartorya fischeri ascospores. J. 1998. Statham. and Allen.. Bacteriol. J. 1980b. 49–94. 45:1285–1292. D. C. W.. K.. 29:272–276. Sorbic acid resistance: the inoculum effect. J. F. F. W. 16:209–215.. Splittstoesser. Busta. Toxicology 120:29–36. A. 1587.. F. P. Food Prot. and Allen. Appl. J. C. J. R. Evidence that sorbic acid does not inhibit yeast as a classic weak acid preservative. J.. J. C. 1997. Clostridium botulinum control by sodium nitrite and sorbic acid in various meat and soy protein formulations.. K. Inc.. F. 1978. F.. E. Growth of Saccharomyces bisporus var. Y.. A. A. 1993. N. J. James. Food Sci. and Busta. 37:1103–1109. J. Enol. E. A. Mode of action of sorbic acid on bacterial cells and spores. Sofos. M. J. Microbiol. G. 1989. J. 54:683–685. D. 1978. Downing. and Kada. Vitic. 45:7–12. 44:668–672. N. K. N.. Splittstoesser. J. Busta. J. Statham. Appl. Chemical food preservatives. and Paquette. New York. A. T. In Antimicrobials in Food. Russell. A. 2000.. J. 1982. Hayatsu. Lammers. J. Food Microbiol. F.. A. The effect of hydroxycinnamic acids and potassium sorbate on the growth of 11 strains of spoilage yeasts. 1986.. Tellez-Giron. J. Sofos. Food Sci. F. Splittstoesser. R. 1976. S. Bacteriol. F. Sugihara. Heat resistance of Escherichia coli O157:H7 in apple juice. B. F. Effects of various concentrations of sodium nitrite and potassium sorbate on Clostridium botulinum toxin production in commercially prepared bacon. 1979d. J. and Oliver. Toxicol. N. J. 307.. 59:226–229. 51:945–948. A. Rooney. Roberts. and Stratford. 52:821–822. Bhothipaksa. J. McLellan. D. Shimomichi. D. J. L.. a xerophilic mold. 1979a. 50:1580–1584.. Stead. 675. F. T. A. Botulism control by nitrite and sorbate in cured meats: a review. N. M.. Sofos. bisporus. J.. and Allen. Marcel Dekker. M. Blocher. N. F. Bhothipaksa.. Appl. and Churey. Stratford. E. J. 27:203–206. N. 1983. K. pp. F. Queale. Food Sci. and Waniska. J..88 Antimicrobials in Food Sofos. F. and McMeekin. F. and Churey. G. Food Prot. C. 42:739–770. Tanaka. N.. R. Appl. N. Pierson. Environ. 1994. F.. Agric. Sorbic acid and sorbates. Robach. J.. N. R. A. C. Food Cosmet. Preservation and Sterilization. L. Bremner. K. H. Food Chem.

S. J. Final Report July 23. J. Effectiveness of a betalain/potassium sorbate system versus sodium nitrite for color development and control of total aerobes. Inhibition by antimicrobial food additives of ochratoxin A production by Aspergillus sulfureus and Penicillium viridicatum. 1988. 59:726–730.Sorbic Acid and Sorbates 89 Thomas. and Olsen. sp. Tong. 47:532–536. P. 51:457–462. S. Effect of three preservatives on the growth of Bacillus cereus.. M. Department of Agriculture. K 1987. and Walker. and Bullerman. J. 1972. and Draughon. J. 49:86–90. 1974. Food Microbiol. Appl. V. Effect of potassium sorbate on salmonellae. Influence of potassium sorbate on the growth of Yersinia enterocolitica. J. A. R.. Clostridium perfringens and Clostridium botulinum in cooked. Agric. 55:323–326. and Torres. G... T. K. Degradation of sorbic acid in fruit squashed and fish paste. Microbiol. Vero cytotoxigenic Escherichia coli and Staphylococcus aureus. Christiansen. B. Department and Agriculture. 800. Vidyasagar. Proposal to affirm potassium sorbate and sorbic acid as GRAS. Tuncan. C. M. B. and Wagner. on plates with gradients of pH and sodium chloride concentration. J. F. sensory.. To. Microbiol.-Y. Potassium sorbate permeability of methylcellulose and hydroxypropyl methylcellulose coatings: effect of fatty acids. W. Food Sci. 1965. Off. 1990. and Bullerman. F. J. Vaughn. Effect of sodium acid pyrophosphate and/or potassium sorbate on Staphylococcus aureus FRI-100 growth and toxin production. Food Prot. E. Tsai. N.. and Emard. J. Food Technol. Microbiol. H. M. J. Food Add. uncured. Food Technol. Shtilman. A. W. A. J. and Robach. R. and potassium sorbate on amino acid uptake in Salmonella typhimurium 7136. vacuum-packaged turkey products. S. E.S. W. S. Buck... A. Can. C. and Arya. Reg. Toxicity and sorbate sensitivity of molds isolated from surplus commodity cheeses. Food Sci. 52:793–794. Food Safety and Quality Service four-plant study to investigate the use of 40 ppm sodium nitrite and 0. 1984. and Arya. J. temperature. H. Vojdani. Appl. Environ. J. Effects of sorbate and propionate on growth and aflatoxin production by sublethally injured Aspergillus parasiticus. 1978. Vareltzis. M.. Derivatives of sorbic acid as food preservatives. Tsai. A. M. Color stability and sensory attributes of chicken frankfurters made with betalains and potassium sorbate versus sodium nitrite. 55:841–846. 55:1223–1225. 1989. Food Prot. K. To. Unda. sporulating anaerobes. J. C. Tjan. Appl. and Buck. 52:723–726. Uljas. 1980b. O. Food Chem. A. Assoc. Tompkin. M. Appl. 15:543–547. Radicuscucumerinum. Anal. 28:262–264. cooking and physical characteristics of bacon cured with varying levels of sodium nitrite and potassium sorbate. Effect of novel water-soluble polymeric forms of sorbic acid against Fusarium oxysporum f. 1985. Shao.. J. K.. Tzatzarakis. H. L. Food Sci. Int. I. 65:1924–1929. U. B. M. W. Environ. C. B. Inhibition of potential food poisoning microorganisms by sorbic acid in cooked. Poultry Sci. Vidyasagar. Troller. L. 47:41–45. Environ. T. R. K. U. Effect of pH.. Wimpenny. C. 1984. 1985. S. 19:447–454.. sodium chloride and sodium acetate. Contam. E. J. Troller. H. 1999. Microbiological and some physical and chemical changes in vacuum-packaged beef steaks treated with combinations of potassium sorbate. 1951. G. A. B. . Final report: shelf-life. 51:38. J. E. Microbiol. M. 1980a.. Vareltzis. Food Prot. J. C. N. and Ingham. J.. 31:1262–1264. E. Thomas. 32:228–231. J. Proc. J. 1967. and Bolin. 49:1407–1411. Selectivity of sorbic acid media for catalase-negative lactic acid bacteria and obligate. E. phosphate. P. C. M. G.-P. and Martin. K. 1984. M. Fed. J. J. J.. Shaparis.S. U. 1990. Bacteriol. Thin-layer chromatographic identification of preservatives in food. Combinations of intervention treatments resulting in 5-log10-unit reductions in numbers of Escherichia coli O157:H7 and Salmonella typhimurium DT104 organisms in apple cider. R. W. Potassium sorbate dip as a method of extending shelf-life and inhibiting the growth of Salmonella and Staphylococcus aureus on fresh. A. S. July 23.26% potassium sorbate in bacon. E. C. Food Sci. uncured sausage. 1979b. and Robach. L. 43:9823. Food Sci. 17:289–301. H. Tsay. whole broilers.S. 49:505–508. Department of Agriculture. D. 2000. R. 1984. I. J. Stability of sorbic acid in orange squash. D. Food Prot. Tsatsakis. M. and Konter. Microbiol. R. Lotter. L. Clostridium perfringens and Clostridium sporogenes in chicken frankfurters. Liewen. 1983. 11:611–617. H.. L. and Labbe. and Davis. 17:965–971. Staphylococcus aureus. Chem. and Chou. Molins. U. Catalase inhibition as a possible mechanism of the fungistatic action of sorbic acid. J. 1979a. and Vakalounakis. 1993.

1983. G. R. M. 1960. and Huang. J. A. Food Sci. F. Appl. 49:1588–1594. M. J. Sebranek. Resistance of Clostridium parabotulinum to sorbic acid. H 1954. Tanimura. 1964. S. Y-W. E. Zou. K. R. H. 57:675–677. 1977. J. Weng. Simultaneous determination of 8 food additives in fruit juices by reversed phase high performance liquid chromatography. C. Anal. Potassium sorbate inhibition of microorganisms growing on refrigerated packaged beef. J. J. J. E. D. F. Determination of coenzyme A by sorbyl coenzyme A formation. 235:1554–1558. Bacteriol. 1982. 1974. J. Food Chem. 1995. and Besser. Food Sci. Microbiol. J. S. R. K. K. A. M. R. 58:1257–1259. Zamora. 52:1449–1454. R. A. K. Yamamura. and Amundson. and Vaughn. and Marth. G. J. and Zaritzky. G. H. 5:289–298.. K. J. 46:204–208. Antimicrobial activity of undissociated sorbic acid in vacuum packaged beef. and Vaughn. Effect of packaging film on vacuum level on regular and sorbatecured bacon. Takamatsu. Food Prot. Food Res. Zhuang. 18:103–108. and Hubscher. sorbic and other weak acids used as food preservatives.. 1984. Chinnan. 1987b. Food Sci. York. M. Incorporation of [14C] acetate into aflatoxin by resting cultures of Aspergillus parasiticus in the presence of antifungal agents. Wilamowski. F.. J. Health Sci. W. Food Prot. F. Yamada. G. 1985a. Wiss. Wagner. W. Bacteriol. Effect of sodium acid pyrophosphate in combination with sodium nitrite or sodium nitrite/potassium sorbate on Clostridium botulinum growth and toxin production in beef/pork frankfurter emulsions. 59:808–812. R. 59:2526–2530. 1982.. R. T. M. H. L. Food Add.90 Antimicrobials in Food Wagner. K. J. C. E. 30:485–487. Fate of enterohemorrhagic Escherichia coli O157:H7 in apple cider with and without preservatives. Effect of food components and additives on the formation of nitrosamides. Chinese J. F. Warth. 48:990–991. Zhao. R. 1990. 1988. H. H. 43:215–230. 53:257–262. Food Add. 1985. J. 1983.. and Restaino. Food Prot. Appl. R. . 1955. J. Yamamoto. and Watabe. Biol Chem. T. Influence of minimal change in pH on germination of Clostridium botulinum 52A in media containing sodium acid pyrophosphate and potassium sorbate. Contam. Wind. A. M. J. Effect of packaging films and vacuum on the microbiology of bacon cured with or without potassium sorbate. Anal. M. 2001. 48:564–569. Food Res. Eur J. 1993. M. A. 24:37–43.. K. 1997. Inhibitory effects of various salts and/or ionic strengths on growth from Clostridium botulinum 52A spores or vegetative cells. and Busta. Food Sci.. E. Off. 45:854–858.. J. Wedzicha. Zamora. J. D. 29:1192–1195.. Leben. Wakil. Kraft. and Busta. 1987a. E. and Vaughn. Antimicrobial effectiveness of potassium sorbate and sodium benzoate against Zygosaccharomyces bailii in a salsa mayonnaise. Chem. Bacteriol. Mechanism of resistance of Saccharomyces bailii to benzoic. M. K. Chen. D. Sorbic anhydride as antimycotic additive in polyethylene food packaging films. Walker. Toxicology of sorbic acid and sorbates. Food Prot. Inactivation of Salmonella montevideo on tomatoes by applying cellulose-based edible films. and Brook. J. 31:29–40. I. Biotechnol. 993. G. and Shao. Food Sci. Wagner. Food Prot. 2000. Food Prot. K. Assoc.. Beuchat.. Doyle. Appl. and Chen. and Busta. 7:671–676. Food Sci... Chem. and Zaritzky. R. Yoshihira... G. Wagner. and Tomita. and Busta. 1996. B.. J. Reaction of sorbic acid with nucleophiles: preliminary studies. and Gehrke. J. A. P. K. F. Antimicrobial effect of chemical preservatives on enterohemorrhagic Escherichia coli O157:H7. L. Murai.. Resistance of yeast species to benzoic and sorbic acids and to sulfur dioxide. Collaborative study of spectrophotometric method for the determination of sorbic acid in fresh dairy products. 88:411. M. M. J. M. Inhibition of sulfhydryl enzymes with sorbic acid. 47:355. Wagner. 48:421–428. N. E. F. Yousef. H. 1989. Rust. Warth. K. Tech. York. A. Microbiol. Mechanisms in the inhibition of microorganisms by sorbic acid. L. C. J.. A.. York. F. 68:739–744. Contam. M. Inhibition of Clostridium botulinum growth from spore inocula in media containing sodium acid pyrophosphate and potassium sorbate with or without added sodium chloride. Use of sorbic acid enrichment media for species of Clostridium.. 20:60. Whitaker. E. C. Y. D. M. J. 1985b. 1959. M. N. J. L. 48:693–696. Environ. Shewfelt. Wagner. A.

.........................................................................................................................................................................................................................................105 Toxicology .......................................................................................................................................................................................................................................................................116 Application and Regulatory Status .................................................................................................................................................................................................110 Application and Regulatory Status .............................................................................................111 Application and Regulatory Status ........................................................105 Adipic Acid .......................................................116 91 .........................................................................................................................................................................102 Application and Regulatory Status .....................106 Toxicology .105 Antimicrobial Properties .......................................................................................................................................................................111 Antimicrobial Properties .................................................................................................94 Acetic Acid ...........................4 CONTENTS Organic Acids Stephanie Doores Effective Use ........................................................................................................................................................................................................................................................................................................................................................105 Application and Regulatory Status ...................................................................................104 Dehydroacetic Acid...........94 Toxicology ............107 Antimicrobial Properties .................................................104 Antimicrobial Properties ...................................................................104 Application and Regulatory Status ............107 Toxicology .........................................................................101 Acetates ..............103 Sodium Diacetate ....................................................................106 Caprylic Acid .......................................................................................................................................................................................................................................................................................................................................99 Application and Regulatory Status .....106 Application and Regulatory Status .......................................................................104 Toxicology .....................................................................................................................................................................................................................................................................................106 Citric Acid.......................................110 Fumaric Acid...................................................................................................................106 Antimicrobial Properties ..112 Toxicology ....103 Toxicology .....................................................................................................................103 Antimicrobial Properties ..............101 Toxicology ....................111 Toxicology ...................................................................................................................................................................................................................93 The Acids .............................105 Application and Regulatory Status ...................................112 Antimicrobial Properties .......................................................................................................................................................................112 Lactic Acid ..............................................................................................94 Antimicrobial Properties ...........................................................................................................................................................................................................................................................................................101 Antimicrobial Properties .....................................................................................

..........................................................................................................5).......123 Succinic Acid ...... and curing agents in conjunction with acids serve to further decrease processing ...................123 Toxicology ............................................................................ the proper use of an acid in a culture medium can select for a particular group........ In mixed flora..............................................................................120 Application and Regulatory Status ................................................................................................................................................................................. All microorganisms have minimum.120 Toxicology ...................................... thereby creating an unfavorable environment............................................................122 Application and Regulatory Status ..............................5 to 7.................................. and any preexisting conditions in the food that could enhance inhibition......... Success in limiting the numbers of microorganisms will depend on the species of microorganism.....124 Antimicrobial Properties ........................................................................ and seafoods with a pH range of 5..........................................120 Toxicology ................................... sugar..............126 Assay ...................... The continued presence of acid can effectively inhibit germination and outgrowth of spores that survive the thermal process..................................................................................................... the buffering capacity of the food.................................. bacteria primarily spoil proteinaceous foods such as dairy........................................120 Application and Regulatory Status ............................................................................................. Adding an acidulant to the food or enhancing natural fermentation to develop acidity changes the pH of the food.................. poultry................. Some acids................................................120 Malic Acid.................. the lactic acid bacteria are not only tolerant of weak lipophilic acids but also produce them as a by-product of their metabolism................................124 Mode of Action ...........................120 Propionic Acid .................. and altering the hydrogen ion concentration influences the growth or inhibition of an organism.......................................5................................... meat..................................... One effective means of limiting growth is to increase the acidity of a food....5 to 6..............................124 Application and Regulatory Status ................................................................................................................................................. These actions tend to be microbiostatic rather than microbiocidal...... but they will tolerate a pH range of 4 to 9. Yeasts are more tolerant of lower pH values than bacteria............................................ The incorporation of acids into a food can shorten sterilization times for heat treatment owing to the lowered heat resistance of microorganisms in foods with increased acidity............................. and optimum pH levels for growth.............................. Molds have the widest range of acceptable pH.................................123 Tartaric Acid............................................................ For example....................... the type and concentration of the acidulant...................... maximum........................120 Antimicrobial Properties ........116 Antimicrobial Properties ....5 can support the growth of both yeasts and molds..................................................................... Yeasts and molds more commonly proliferate on fruits and vegetables with inherently lower pH values and little buffering capacity.....................................120 Antimicrobial Properties .......................... foods with a pH below 3.......124 Toxicology .......92 Antimicrobials in Food Lactates ........... The acidity or pH of a food can affect the type and number of microorganisms present in a product. and the pH selects the species or group of microorganisms that will predominate in unaltered food products........................ such as acetic acid...................................................................... For example............................................ Salt............................... are critical to the metabolism of the lactobacilli but inhibitory to bacilli.......... time of exposure..................................................124 Acid Adaptation and Resistance ............................ In general...............................123 Antimicrobial Properties ............................................................ Microorganisms display varied tolerances to acids.... bacteria are more fastidious and prefer to grow at a pH near neutrality (pH 6..127 References ................ Tolerance of organisms to widely differing pH levels varies naturally.............................................................................123 Application and Regulatory Status ..................127 Many parameters govern the survival and growth of microorganisms in food.............................

gaseous atmosphere. Acids contribute to the taste and tartness of a product. Because the undissociated portion of the molecule is believed to be responsible for the antimicrobial effect. The use of the multiple-barrier concept results in lowering the concentrations of acidulants or inhibitors. This concept is based on the premise that foods can be preserved using several inhibitors concurrently rather than relying on a single factor.44 5.Organic Acids 93 TABLE 4. and degree of branching to inhibit or kill a wide variety of microorganisms. Because some experiments express concentration in percent.77 4. Effective use of an acidulant depends on the dissociation constant (pKa) or the pH at which 50% of the total acid is dissociated.27 4. (1939) observed that acids with fewer than 7 carbons were more effective at lower pH. Not only would this interaction ensure commercial sterility of the food.40 4. normality. water activity. 1980). concentration.14 3.1 Dissociation Constants of Organic Acids in Aqueous Solutions Acids Acetic acid Dehydroacetic acid Sodium diacetate Adipic acid Caprylic acid Citric acid Fumaric acid Lactic acid Malic acid Propionic acid Succinic acid Tartaric acid Source: From Lide (1991).61 4. This primary concern limits the use of acidulants to foods with pH values of less than 5.98 pK2 pK3 5. adjustments in pH levels can be coupled with changes in temperature. prevent browning. and acids with 8 to 12 carbons were more effective at neutrality and above.87 4. aid in the inversion of sucrose.89 3. organic acids are usually more effective as antimycotic agents. pK1 4. The inhibitory effect of acids has been compared based on pH. Hoffman et al. They can control pectin gel formation. EFFECTIVE USE Various approaches have been used to study the efficiency of antimicrobial effects on cells.75 5.16 2. it would be advantageous to use the acids near these values. Some can be used . and chemical analyses can be found in the references by Ash and Ash (1995) and Doores (2002. physical parameters.41 4. 2003). The multiple-barrier concept (“hurdle” concept) has gained popularity for use in a variety of foods in recent years. type.34 6. and protect color.0. the choice of an acidulant may depend on secondary effects. and other inhibitory compounds. Comprehensive texts describing applications.43 4. but also the decreased processing time would aid in preserving the palatability of the product (Corlett and Brown. In addition to any antimicrobial effects an acid possesses.39 times. or molarity to achieve final pH. Therefore.08 3. chain length.75 4.11 5. Given that this value lies at the lower limits of growth for many bacteria.1). The pKa of most organic acids lies between pH 3 and 5 (Table 4.03 3. it is somewhat difficult to compare acids or make broad statements concerning the choice of the appropriate acid for a particular effect in a category of foods. They can create a synergistic relationship with antioxidants by chelating metal ions.

These acids. however.0 than lactic acid bacteria. 1975b). Most of the acids appear safe for use in food products. sauerkraut. 1966) The toxicology and safety of the acids must be considered when selecting an acid for use.9).7.0. The succession typically begins with growth of coliforms and natural microflora followed by Leuconostoc mesenteroides (heterolactic). many of these studies used high levels of the concentrated acids at low pH values. Although these acid mixtures might be expected to occur naturally during fermentations by heterolactic bacteria. Several animal studies have indicated possible safety hazards. and caprylic acid. . a more acid-tolerant organism. all of the groups were similarly affected (Woolford. are relatively nonpolar compounds. such as pickles. mesenteroides ceased when the internal pH of the cells reached 5. 1939) inhibited Bacillus cereus (pH 4. application. and regulatory status is presented here for each acid. as such. this apparent synergy has not been demonstrated in other foods. which do not.6 to 4. Adams and Hall (1988) showed a weakly synergistic inhibitory effect between acetic and lactic acids with Salmonella enteritidis and Escherichia coli. These organisms are found naturally associated with fermented products. however. as the name implies. would further indicate that hazards are unlikely. the same hazards would not be expected. The long-term consumption of these acids. and vinegar.0). although they could also serve other functions in foods. THE ACIDS Information on the toxicology. Concentrations of acid lower than those needed to inhibit Saccharomyces cerevisiae (pH 3. McDonald et al. This association could explain the apparent stability that occurs in natural fermentations. all these acids can be metabolized by the body primarily through lipid oxidation and via the tricarboxylic acid cycle. and Staphylococcus aureus (pH 5. In addition.94 Antimicrobials in Food as chemical leavening agents. L. (1990) found that growth of L. Owen (1946) suggested that acetic acid inhibited Gram-positive organisms. yeasts. as the pH decreased to 4. and molds.0. antimicrobial properties. which exist naturally in many foods. In some instances. then by Lactobacillus plantarum (homolactic). plantarum maintained its pH gradient in the presence of either 160 mM sodium acetate (SA) or sodium lactate down to an external pH of 3. whereas growth stopped in L.4 to 5. The salts of the acids are important in regulating the acidity of the foods (Gardner. At the levels and pH values in which these acids are normally used in foods. are more tolerant to this acid than homofermentative lactic acid bacteria (homolactics). The lipophilic acids include acetic acid and acetic acid derivatives.9) and Aspergillus niger (pH 4.1) (Levine and Fellers. The carboxylic acids are more polar than the lipophilic acids and are traditionally used in foods for secondary effects rather than for their ability to inhibit microbial growth. the information appears separately for each acid. Bacillus and Clostridium species and Gram-negative bacteria were more inhibited at pH 6. Gram-positive bacteria. The results of these studies are usually presented in the section describing the acid that gave the most antimicrobial effects.8. propionic acid. Microorganisms vary in their susceptibility to acetic acid. Sauerkraut manufacture is a fermentation process brought about by a natural succession of microbes that inhabit the surface of cabbage leaves. such as sauerkraut. They are usually added to foods for their antimicrobial properties. Numerous studies included in this chapter compare several organic acids for their antimicrobial effects.9). plantarum at pH 4. ACETIC ACID Antimicrobial Properties Acetobacter and heterofermentative lactic acid bacteria (heterolactics) produce acetic acid as a byproduct of their metabolism and. Salmonella Aertrycke (pH 4.

8% reduced growth of Micrococcus and Bacillus species. and exposed to 40°C became injured. L.0% lactic.0. 1970). Reduced-calorie mayonnaise (RCM) manufactured with 0.0. aureus in 12 hours (Minor and Marth. and caused 50% inhibition of spore germination at pH 4. coli O157:H7 was inoculated into tryptic soy broth (TSB) containing acetic.Organic Acids 95 S.7% acetic acid. lactic. Both studies concluded that the antimicrobial effect was the result of the undissociated molecule.3% and 0. but no injury occurred in cells not exposed to acidic conditions before heating (Smith et al.7% acetic acid with reduction to undetectable levels in 1 and 2 weeks with 0. Acetic acid exerted the most inhibition but had the lowest dissociation constant.0 with scant growth in TSB containing acetic acid. other ingredients can interact with acid to influence the degree of inhibitory action. citric. When E. aureus.9°C.7% acetic acid (Glass and Doyle. in the aqueous phase of CFM and RCM would still produce a microbiologically sound product. Streptococcus faecalis. pH 4. and hydrochloric acids (Nunheimer and Fabian. It is unlikely that these products would contain such high initial levels of either of these organisms. most likely because of the absence of egg yolks in CFM and the presence of egg white. A concentration of 0. Salmonella Blockley.0 (Conner and Kotrola. cereus was inhibited at pH 5. and tartaric acid permitted growth at pH 4. E. 22°C) containing 1. Gram-negative aerobic bacteria. pH 4.0 with 0.0.4 to 4. 1995.0 with hydrochloric acid was compared to cholesterol-free (egg yolk-free) reduced-calorie mayonnaise (CFM) manufactured with 0. whereas tartaric acid had one of the highest dissociation constants yet exerted the weakest inhibitory action.3. Acetic acid caused greater inactivation of L.. aureus was most sensitive to acetic. citric.1%. aureus preconditioned in acetate buffer (pH 4. therefore the standard use of 0. pH 4. L. and tartaric acids. lactic. 1997). E.6.5 (Noda et al. coli than unbuffered systems.4% to 0. tartaric. malic. and HCl. malic..6. respectively. Both formulations were inoculated with approximately 108 CFU/ml of an 8-strain mixture of Salmonella or a 6-strain cocktail of Listeria monocytogenes and held at 23. 1940). This finding is important in the manufacture of medium acid foods containing more than one organic acid. was reduced by 4 logs in both mayonnaises within 72 hours in formulations containing 0. Salmonella were inactivated within 48 hours in both RCM and CFM formulated with 0.0 buffer.5 for all acidified media at 10°C except for acetic acid. monocytogenes was also reduced to undetectable levels in RCM within 10 days and in CFM within 14 days compared to 2 days for Salmonella. monocytogenes.6) at 20°C shifted to pH 7. 0. At 25°C and 37°C. and Enterobacteriaceae in Japanese fermented soy sauce (Hayashi et al. monocytogenes than lactic and citric acids and increased inhibition as the temperature of incubation . Conner et al. 1984).8. Buffered acidulant systems (brain heart infusion [BHI] broth. Destruction of Salmonella occurred more quickly in CFM than RCM. Similar results were noted when various acids were compared at the pH level required for a 90% and 99% reduction in numbers of S. which has recognized antimicrobial properties.0% acetic acid and 1. or 0. and E.1 and 5. pH 4. In addition. aureus.7 and 4. 1982).15 M acetic acid and with 0. such as mayonnaise (Debevere.7% acetic acid.3%. especially with the use of pasteurized egg products. respectively. followed by lactic.5%. 1988). The effect of temperature-acid interactions is noteworthy for the psychrotrophic pathogens.9 and 4. pH 4. An increased toxic effect of acetic acid against Saccharomyces rouxii and Torulopsis versatilis was demonstrated in brine fermentation of soy sauce as the pH decreased from 5.2 and 5. B.6 and 4. coli O157:H7 is considered more acid tolerant than strains of E.3% acetic acid formulations. malic.33 M lactic acid (Wong and Chen. 0. followed by citric. added or developed through fermentation. L. Acetic acid was effective at pH 5.. A 0. respectively. 1979).1-M concentration of acetate and lactate completely inhibited multiplication at pH 6. respectively.5% and 0. growth occurred at pH 5. 1991). although more resistant than Salmonella. some strains of S.. tartaric.5. or citric acid were far more effective in reducing numbers of S. 1988). S.2 and 4. coli O157:H7 grew in all media at pH 5. monocytogenes and Yersinia enterocolitica. Malic acid allowed growth at pH 4. coli.5 to 3. Traditionally acidic foods contain a single or multiple acids.5.7% acetic acid in the aqueous phase and adjusted to pH 4.

but L. or 50%) for 15 or 30 minutes. 25-g samples. A 90% reduction of the population required exposure to 0.2 N for 4 min..9 log reduction for the nontreated seeds (Weissinger et al. citric acid was more bactericidal than other acids at 25°C and 35°C. a mesophilic organism. 40%. enterocolitica was not detectable after exposure to 2% and 5% acetic acid or 40% and 50% vinegar for 30 minutes (Karapinar and Gönül. inoculated with approximately 107 CFU/g Y. 1990. these foods can be the carriers of psychrotrophic pathogens. A variety of organic acid treatments has been used to control microbial loads associated with meat carcasses and products (Dickson and Anderson. Pork carcasses inoculated with 106 CFU/ml S.33 and 5. containing from 0. and hydrochloric acids. Raw produce is now being subjected to acid rinses in some production practices as a means of reducing high levels of spoilage organisms and foodborne pathogens. and temperature. 1998. In addition. but resulted in changes in sensory characteristics.0 for acetic acid. 1992a. Little et al. but malic acid was more bacteriostatic than other acids at 10°C.10 N for 5 min. Typhimurium. 1999).. monocytogenes when compared to citric. Typhimurium and E. E. 1973). monocytogenes was isolated from 2 of 10. 1990). Smulders and Greer. such as Y. lactic. Treatments spanned varying amounts of time with concomitant decrease in microbial loads up to 3 logs. 1997. Karapinar and Gönül. The greatest antimicrobial effect occurred at 35°C with the greatest survival at 10°C. Y.5 to 3. but acetic acid was less effective. seed germination was effected (Weissinger and Beuchat. Numerous organic acid combinations. 1997. 1989.. and beef carcasses or meat tissue. or 5%) or vinegar (30%.5 min. citric. When comparisons were based on equal molar concentrations. 1992). 1981). Depending on the treatments. citric acid caused greater injury than either lactic or acetic acids and injured organisms survived longer at low temperatures (Ahamad and Marth. and no reduction in microbial . but recovery required the presence of amino acids or peptones (Blankenship. a twofold reduction at pH 2. 1992b).8 to 5. Mung bean seeds inoculated with 3 to 5 logs of S. acidulants. 1991. respectively. pH 4.5 or 2. and 0..3% inhibited L. Conversely.5. Brocklehurst and Lund. enterocolitica at lower pH levels as the temperature increased. Total plate counts were reduced by 4 logs at pH 1. and off-flavors. The minimum inhibitory pH level was pH 4. vapor treatment also reduced levels of resident microflora on seed but did not affect seed germination rates (Delaquis et al. 0.4 to 4. monocytogenes irrespective of temperature.0 have been used to rinse pork. 1989). Dorsa et al. lamb.05 N for 8. Acid injury did not appear to involve damage to ribosomes or other nucleic acid material.. there can be adverse sensory effects including discoloration of meat. Because produce is typically refrigerated with only minimal processing. malic. 0. 1992. enterocolitica. 2001). and L.0 for 30 or 60 seconds (Biemuller et al. 1998).7 log without affecting germination of seeds.72 logs. Dorsa.. 2%.0% acetic acid at pH levels from 1. Acetic acid was more effective than lactic or citric acids in producing this effect (Adams et al. 2000). injury and death rates were not affected by refrigeration temperatures for Salmonella Bareilly. Sorrells and Enigl (1990) also demonstrated interactive effects between sodium chloride. Immersion of alfalfa seeds in mixtures containing 5% lactic or citric acid for 10 minutes resulted in several log reductions. monocytogenes were exposed to acetic acid at a concentration of 242 µL/L of air for 12 hours at 45°C. Parsley. Acetic acid applied as a volatile compound (1000 mg/L of air) for 7 hours at 60°C achieved >3 log reduction of six Salmonella strains inoculated onto alfalfa seeds compared to 1.6% to 3. Acetic acid displayed the greatest antimicrobial action against L.0. Exposure at 50°C for 12 hours with lower levels (100 and 300 mg/L of air) reduced the population >1.4 for malic. Similar growth inhibition was demonstrated for Y. coli O157:H7 to nondetectable levels by enrichment methods. and hydrochloric acids at equal pH values and at all incubation times and temperatures (Sorrells et al.01 N for 75 min. was dipped in acetic acid solutions (1%. Enteritidis were sprayed with acetic acid at pH 1. 1 log unit at pH 2. coli O157:H7. concentration ≥0. however.6 for lactic acid.96 Antimicrobials in Food decreased from 35°C to 13°C. and pH 4. enterocolitica. This treatment reduced S.. In addition. However. Treatment at low temperature (4 days at 10°C) with 200 and 500 mg/L of air was effective in reducing Salmonella by 2. off-odors.5.

0. Enteritidis to 1 of 6 samples at pH 1. Incorporation of pulsed power electricity and a 2% acetic acid spray led to improved reduction of inoculated E. resulted in less effective reduction of S. 1994. anaerobic. or lactic acid led to significant decreases in aerobic plate and coliform counts for acetic and citric acid washes after 14 days of storage at 2°C to 4°C. organic acids can be used in preevisceration rinse systems consisting of a water rinse followed by an organic acid rinse..8 log CFU/cm2. 1994). Typhimurium. Application of a 3% concentration of acetic acid as a spray on beef carcasses was not significantly better than water washes in reducing levels of E...0. Department of Agriculture Food Safety Inspection Service (FSIS). however.8 to 4.. An 18% acetic acid or 12% lactic acid spray significantly reduced bacterial counts on lamb carcasses. and stored for 28 days at 2°C in a high-oxygen barrier film. 1995). coli O157:H7.. 1989b).. Application of a 2% lactic or 2% acetic acid spray to beef strip loins and stored at 1°C resulted in significantly lower aerobic and lactic acid bacteria counts over 112 days of storage (Goddard et al. coli O157:H7 and S. but beef treated with the mixture of acids did not differ in flavor from untreated beef (Bell et al. the pH level decreased to 2. Dickson (1992) contaminated lean and beef tissue surfaces with S. (1980). Meat was sprayed. 1995). As part of recently established guidelines by the U. 1997. some discoloration occurred (Cacciarelli et al. (1991) inoculated beef roasts by injection and on the . Reductions in microbial counts depend on the water temperature..2% acetic acid or 0. 1994.. 1994)..Organic Acids 97 numbers at pH 3. Typhimurium on beefsteaks (Tinney et al. or manure slurry. (1977) reduced bacterial counts by 99. Typhimurium reductions were greater when the bacteria were attached to fatty tissue.. Typhimurium was reduced by 0. At this concentration. 1996).1 and 1. (1987) used intermittent sprays of 1% acetic acid or lactic acids.5 to 0. Cutter and Siragusa.5 and 11 of 72 for pH 2. Quartey-Papafio et al.4°C (Anderson et al. 1997). possibly because this tissue retained less moisture (20%) than lean tissue (75%) and the reduced water activity could have enhanced the antimicrobial effects. and lactic acid bacteria counts.. Increasing the concentration to 2% acetic acid or using 200-ppm hypochlorite solutions before vacuum packaging and storage for 28 days at 4°C significantly lowered aerobic.6% acetic and 0. 1974). Microbial loads on beef carcasses subjected to machine washing and sanitization with 1. and bleaching of the carcasses occurred (Ockerman et al. They found that acetic acid was a superior sanitizer compared to hypochlorite and had a greater residual effect.. L. Temperature played a greater role in reducing numbers than did the acid concentration. dirt. The treatment effectively reduced recovery of S. such as rumen fluid. S. coli. An application of 2% acetic acid reduced the incidence of Salmonella on pork cheek meat in addition to significantly reducing aerobic plate and coliform counts (Frederick et al..S. S. A 3% concentration of acetic acid was quite effective in reducing counts of Enterobacteriaceae in vacuum-packaged pork stored for 6 weeks at 2°C to 4°C (Mendonca et al. a final application of acid washes could provide some residual effect during storage (Brackett et al. Lactobacillus species predominated as storage progressed. Although washing of pork carcasses with 1.5% acetic. 1986). It was noted that the use of acetic acid as a rinse for beef tissue led to sublethal injury of bacterial cells.046% formic acids for 10 seconds caused discoloration from both treatments.6% on meat using a 3% concentration of acetic acid before washing. 1987). however.. S. however. Dorsa et al. this was not significantly different from the controls. citric. type of chemicals. Anderson et al. An increase in organic material. 1997. Typhimurium. which resulted in significant reductions in aerobic plate count (1. monocytogenes was detected in 69% of loins and 33% of chops. suggesting that acids had limited effectiveness (Fu et al. Further work by Anderson and Marshall (1989) demonstrated that application of acetic acid at 70°C was most effective in sanitizing beef semitendinous muscle inoculated with E. Cutter et al. and application rate and sequence in the process (Gorman et al.5% acetic acid were reduced more when treatments were conducted at 52°C than at 14. Beef cubes treated with 1. vacuum packaged. Hamby et al. Unda et al. Hardin et al. found that a 1% formic and 1% acetic acid combination exposed to beef for up to 20 minutes was effective in reducing a variety of bacterial species. respectively.3 log/cm2). 1983). or manure.5. Typhimurium followed by treatment with 2% acetic acid.

citric. succinic. aerobic plate counts were unaffected by the treatments.05%.2% to 0.. 1986). lactic. Other studies continued to explore the use of acetic acid in chill water. For pearl onions. monocytogenes. citric.. greater than a 4% concentration of acid was needed to reduce levels by 2 logs (Tamblyn and Conner. Acidification of the chill water at the end of processing delivers a second intervention step to reduce microbial loads and to increase the shelf life of poultry.86 log MPN/ml for the 0.6% acetic acid-treated carcasses was darkened or yellowed (Dickens et al.0 on inhibition of L.98 Antimicrobials in Food surface with approximately 103 CFU of Clostridium sporogenes and L. (1985) found that mushrooms reached an equilibrium pH of 4.5% concentrations of acetic.35 log MPN/ml for the 0. Stroup et al. Again. 1 mM perillaldehyde—or 0.. a 0. Nisin (an approved bacteriocin for some foods. 1994). further poultry processing can reintroduce microorganisms onto carcasses..0% acetic acid compared to 2 days for 0..05% and 0. Typhimurium using a model system. and 30°C (Oscroft et al. 1987). and 0. Adjustment of the chill water to pH 2.5 mM. Intervention steps incorporated into poultry processing have been effective in reducing microbial contamination.5 to 1.5% acetic acid reduced Enterobacteriaceae counts by 2.9% acetic acid did not support growth or toxin formation of Clostridium botulinum (Ito et al. but changes in pH and concentration of acid were more successful.71 log most probable number (MPN)/ml (Dickens et al. Products are now being preserved by a number of antimicrobial compounds using a multiplebarrier approach to preservation. Rubin (1978) found that acetic and lactic acid acted synergistically to retard growth of S. of the same components—with 2% salt to inhibit growth of contaminating microorganisms for more than 20 days at 27°C. As the incubation temperature to recover spores was lowered. Poultry scald water containing 0. Typhimurium and Campylobacter jejuni by 0.35% solution required 7 days for equilibration for acetic and citric acid but 1 day for 0. germination and outgrowth of Bacillus .6 depending on the concentration of acid in can brine within 1 day with 1.3% acid and 2.. El-Shenawy and Marth (1989b) investigated the effect of acetic. Acetic and tartaric acids were more inhibitory than lactic and citric acids. Expanding on their work.5 logs (Okrend et al.6 or 5. fumaric.0) did not result in a significant reduction of aerobic plate counts but did significantly reduce levels of Enterobacteriaceae by 0. Dickens and Whittemore (1994) exposed broilers to 0. Levels of 0. although the skin of the 0. Air injection did not affect reduction of these counts. and tartaric acid adjusted to pH 5. see Chapter 7) coupled with 0. lactic. but Enterobacteriaceae counts were significantly reduced by 0. acetic acid was the most destructive acid at all combinations of parameters.1% acetic acid at 52°C decreased levels of S. and lactic (Mountney and O’Malley.7% citric acid. with and without the use of air injection to agitate the chill water. There was no significant difference in texture or sensory characteristics between the treatments. 1994). Addition of 1. Immersion of broilers for 10 minutes in 0. 20°C.5 with acetic acid was the most effective antimicrobial treatment followed by other acids in descending order of effectiveness: adipic.0% acetic acid caused instantaneous death. 0. The acid level could be reduced when used in combination with brines and sugar. Kurita and Koike (1983) combined 3% ethanol. 1965).24 log (Lillard et al.6% acetic solution acid (pH 3. 1976). acetic acid caused the skin of the poultry to be hard and leathery. Because of the possible attachment of Salmonella to broiler chicken skin. 1994). Although each individual acid influenced the heat resistance of Bacillus spores differently. Whole pickles preserved with brine at 12° Brix and 0. Despite the reduction in microbial numbers at this pH. Acidified media showed greater inhibitory activity at 35°C than at 13°C. Despite these treatments.6% acetic acid under the same conditions. respectively.7% acetic and 4 days for the same concentration of citric acid.3% and 0. 0. 1997). The acetic acid in the brine inhibited anaerobic and aerobic bacteria. Water pockets occurred under the skin of chicken carcasses with air-agitated samples (Dickens and Whittemore.05% acetic acid.5%. monocytogenes. or citric acids or glucono-delta lactone were studied using Bacillus spores heated at pasteurization temperatures of 65°C and 95°C and recovered at 12°C. 1990).6% acid solutions.

001 M accelerated spindle activity and retarded anaphase in the mitotic cycle of the root tips of the onion. lactic.Organic Acids 99 spores were more restricted. Other forms of acetic acid have antimicrobial uses.8 and 0. it was not possible to determine the actual quantity of acid consumed. and Rhizopus nigrificans at pH 3.4% acetic acid at pH 5.48 mg/m3 altered sensitivity to light. 1976). Rats fed 10% acetic acid and cats fed 20% acid developed ulcers in the gastric mucosa (Okabe et al. Levels of 0. Takhirov (1969) suggested limits of 0. however. in combination with organic acids. respectively (Buchanan and Ayres. interference with blood coagulation (Fin'ko. Rats exposed to vapor levels of acetic acid showed muscle imbalance at 0.2 or 5 mg/m3 but not at 0. Spore-forming bacteria are killed on surfaces at a concentration of 0. and coliforms. are especially susceptible to spoilage because of indigenous microbial populations.01 mg/m3. 1969). Mori (1952) fed rats acetic acid ranging from 10 to 50 cm3 acid/kg of rice. 1968). Rats consuming acetic acid in drinking water in concentrations of 0.2% (0.0001 and 40% relative humidity within 10 minutes (Portner and Hoffman. On contact with organic substrates. however. but a concentration greater than 4% was needed when the pH was at 7. 1968).04 M acetic acid at pH 3 to 4 showed chromosomal and chromatid breaks. and citric acids when temperatures were reduced to 65°C for 60 minutes. Foods. and potatoes than peroxyacetic (80 ppm) acid alone (Hilgren and Salverda. 1969).0. 1937).06 mg/m3.2). Nisin. Cruess and Irish (1932) showed that apple juice containing 0. displayed synergistic effects in increasing the destruction of the organism. aerobic bacteria. followed by glucono-delta lactone. renal failure (Paar et al. Damage from acetic acid resembled that from ionizing radiation or radiometric chemical damage (Manna and Mukherjee.0001 and 0. Studies on nonmammalian species showed that acetic acid at a concentration of 0.01% to 0. 1958). When used as a surface application. Populations of Pseudomonas aeruginosa were reduced by 99. A. pneumonia (Gerhartz. Peracetic acid is formed by an oxygen molecule bound to the carboxyl carbon atom of acetic acid. 1921).. glucono-delta lactone.8% partially inhibited growth and decreased toxin formation by 70% and 90%. particularly precooked. 1932). Allium cepa (Makinen..5 completely inhibited growth and aflatoxin production by Aspergillus parasiticus.0% concentration of acetic acid. Although acetic acid is generally used as an inhibitor of bacteria and yeasts.5 inhibited Saccharomyces ellipsoideus and Penicillium glaucum.2 g/kg body weight) did not show any adverse effects.6% or 0. Lower concentrations of 0.0% acetic acid adjusted to pH 3. Aspergillus fumigatus required 0. and death. 1971). and citric acid when spores were heated at 95°C for 15 minutes and acetic. Grasshoppers treated with 0. All rats showed mucosa damage owing to the feeding method. gastric erosion (O’Keane et al. chilled. a 1% concentration of acetic acid at pH 4. A combination of peroxyacetic (64 ppm)/octanoic (53 ppm) acid solution was more effective as an antifungal agent in flume water for vegetables such as celery. Johnson (1968) noted that rat gastric mucosa responded to a 100-mM concentration of acetic acid by releasing histamine into the interstitial fluid.. This treatment could also be used in recycling of water to reduce levels of yeasts and molds. ready-to-eat products. A combination of these parameters could be used to extend the shelf life of these products.29 mg/m3 caused changes in electroencephalograms.0 and below to inhibit growth (Kirby et al. suggesting its use as a decontamination agent for equipment (Hedberg and Miller. niger. 2000)..5. lactic. Human olfactory thresholds to acetic acid vapor were 0. Ingestion of acetic acid by humans has caused burned lips (Palmer. Toxicology Acute toxicity data for acetic acid in mice and rats indicate varied tolerance levels by different routes of administration (Table 4. and 0. 1971).2% acid at pH 5. it is effective against the black bread molds.8% to 1.5% level. It was found that acetic acid was the most effective acid.60 mg/m3. 1966).999% after exposure to a 1. cabbage. Parmeggiani and Sassi (1954) reported that workers .6% of their body weight (Sollman. At the 0. rats decreased their food intake level and lost 2. it decomposes to yield oxygen and acetic acid. 1949).

cold sensitivities (Wiseman and Adler.2 to 0.310–3.430–4. 1956). Men having 7 to 25 years of exposure displayed blood abnormalities (Cesaro and Granata. (1950) Horn et al. and tartaric acids.700 11. and lungs with an air concentration of 0. (1971) Rat Citrate.430 600 5.000 1. teeth. No toxicologic data were available for peracetic acid. (1971) Gruber and Halbeisen (1948) Gruber and Halbeisen (1948) Lactic Propionic Propionate. (1957) Acetate.500 330 44 1. lactic.380–3.530 380 1. calcium Propionate.200 485 Reference Spector (1956) Orö and Wretland (1961) Spector (1956) Spector (1956) Spencer et al. In rare instances acetic acid.810 625 3. 1955).2 Available Acute Toxicity Data (LD50) for Various Acids Acid Acetic Animal Mouse Rat Mouse Rat Mouse Route of Administration Oral Intravenous Oral Intravenous Oral Oral Intravenous Intraperitoneal Oral Intravenous Intravenous Oral Intravenous Intravenous Intravenous Intraperitoneal Subcutaneous Oral Intraperitoneal Intraperitoneal Subcutaneous Intravenous Intravenous Intraperitoneal Intraperitoneal Intravenous Oral Oral Intravenous Oral Intravenous Oral Intravenous Intravenous LD50 (mg/kg body weight) 4. pharynx. and epidermal reactions (Weil and Rogers.100 1.960 525 3.020 5. sodium Rabbit Mouse Rat Rabbit Rat Guinea pig Mouse Rat Rat Mouse Gruber and Halbeisen (1948) Yokotani et al. 1956). . (1963) Hara et al. as well as citric.860 2. caused allergic reactions by producing canker sores (Tuft and Ettelson.900 680 275 2.340 580–1. (1963) Horn et al.460 1.790 940–960 42 203 961 2. (1971) Gruber and Halbeisen (1948) Horn et al. 1951).040–5.65 mg/L. sodium Dehydroacetic Adipic Rabbit Caprylic Citric Mouse Mouse Enders (1941) Orö and Wretland (1961) Yokotani et al.210 338 3. (1957) exposed for 7 to 12 years to acetic acid vapors in the production of acetyl cellulose displayed corrosion of the hands. although it is assumed that this derivative would be assimilated in a manner similar to that for acetic acid.700 725 884 5. sodium Tartaric Spector (1956) Orö and Wretland (1961) Hara et al. eyes. (1957) Gruber and Halbeisen (1948) Yokotani et al.730 1.100 Antimicrobials in Food TABLE 4.

It is the principal component of vinegars and as such is primarily used for its flavoring abilities.3. Ca+. however. K+. it has been added to infant feeding formulas to replace lactic acid (Seymour et al. 1954). the malic acid content of malic acid should not exceed 0. The salt form. catsup. The acceptable daily intakes for humans for acetic acid and its derivatives are listed in Table 4. Na+ Tartaric Tartrate. NH4+.1005). Na+ Malic DL-malic Propionic Propionate. salad dressings. K+. It is highly soluble in water. 1973) (1973) (1965) (1966) (1963) (1974) (1965) (1965) (1973) (1965) (1966) (1965) (1973) (1973) (1973) 0–5 Not limited Not limited 0–6 Not limited 0–100b Not limited Not limited 0–100c Not limited Not limited 0–30 0–30 Naturally occurring substances. Na+ a Unconditional Not limited Not limited 0–15 Conditional Reference FAO FAO FAO FAO FAO FAO FAO FAO FAO FAO FAO FAO FAO FAO FAO FAO (1965 (1963. and mayonnaise and is found in pickled products such as sausages and pigs’ feet. b Content of DL-lactic acid. It is used in condiments such as mustard. (1989a) used a dip of SA in combination with potassium sorbate (10%) and phosphates (10%) to extend the shelf life of pork chops.05%. 1939). which limits its use. The sodium (SA) and calcium salts are sometimes used in foods and would be expected to have the same antimicrobial properties as acetic acid at the same pH values (Hoffman et al. Mendonca et al. Na+ Sodium diacetate Adipic Citrica Citrate. The data are scarce for the use of peracetic acid in food products. Na+ Fumaric Lactic DL-lactic Lactate. Ca+.3 Acceptable Daily Intake for Humans Limitations (mg/kg body weight) Acid Acetic Acetate.. Ca+.. the estimated acceptable daily intakes listed here do not include amounts occurring naturally. It has primarily been used as a surface disinfectant and is used for preventing fruit flies from laying eggs in damaged tomatoes (Ayres et al. Ca+. K+. K+. Because of cost and antimicrobial action. requires different handling and use procedures than the acid. ACETATES Antimicrobial Properties Several derivatives of acetic acid are currently in use as antimicrobial agents.Organic Acids 101 TABLE 4. . c Content of DL-malic acid.. Acetic acid is generally regarded as safe (GRAS) for miscellaneous and general-purpose usage (21 CFR 184. K+. Application and Regulatory Status Acetic acid is a monocarboxylic acid with a pungent odor and taste. 1980).

1. potassium sorbate (1.5% potassium sorbate. 1994). and held at 4°C for 12 days contained lower population levels of psychrotrophic and anaerobes than untreated samples (Zhuang et al. Color remained acceptable. SA in combination with potassium sorbate and Lactococcus lactis ssp.44% SA showed decreased appetite. A combination of B. The combination of 0. SA at concentrations of 0. monocytogenes in vacuum-packaged turkey bologna during storage at 4°C and was similar to 0.5% SA.0% concentration of SA at pH 4.25% potassium sorbate. 0.5% trisodium citrate with or without B.5 to 1. growth and toxin formation were decreased 70% and 90%. SA alone or potassium sorbate with bifidobacteria retarded growth of psychrotrophs compared with other treatments within 6 days and provided the lowest counts with the potassium sorbate-bifidobacteria mixture. No toxicologic data were available for calcium acetate (Food and Agricultural Organization. and 2.0% SA alone inhibited growth for more than 6 days at 4°C and counts increased only by 1. The combination of 0. Chickens fed diets supplemented with 5. although it is assumed that this derivative would be assimilated in a manner similar to that for acetic acid. sodium lactate (3%). 1963).6 to 0. B. Whole and peeled shrimp were immersed for 2 minutes in solutions containing 10% SA.2. 1951).. Treatment with SA alone or in combination with bifidobacteria extended shelf life at 4°C by 3 days (Kim et al. and increased mortality. but bifidobacteria did not grow within 9 days. Waterhouse and Scott (1962) attributed the toxic effect to the sodium ion rather than the acid moiety.. 1997). 1995b). parasiticus was inhibited by a 1. vacuum packaged. Histologic findings showed hypertrophy of the kidneys and ureters. cremoris culture added to catfish fillets was an effective treatment against growth of Gram-negative spoilage bacteria at 4°C (Kim and Hearnsberger. over 6 days. untreated samples reached the spoilage mark within 10 days. SA alone or SA-MKP-treated fillets were not significantly different in odor from fresh controls up to 9 days.4% monopotassium phosphate (MKP) more effectively prolonged shelf life to 12 days compared with SA alone. 1995a).25% acetate and 2. although a mild acetic acid odor was detected. depressed growth rate. whereas potassium sorbate increased shelf life to 9 days. Catfish fillets treated with 2% SA.3% to 0.to 6-week storage life (Blom et al.26% potassium sorbate or 2% sodium citrate in its efficacy (Wederquist et al. respectively (Buchanan and Ayres.5%). 1996). A combination of 0. For peeled shrimp.6% or 0.75% and 1% significantly reduced the initial aerobic plate counts of catfish fillets by 0. breve and SA exhibited an additive effect that extended shelf life still further. 1999). but SA provided better sensory characteristics and more controlled growth of psychrotrophs than potassium sorbate with bifidobacteria..102 Antimicrobials in Food SA (10%). and trisodium citrate extended shelf life to 6 days. breve. the indicator of spoilage. A 22-year-old woman with sensitivity to acetic anhydride was not allergic to SA (Weil and Rogers. breve culture and stored at 4°C for 9 days (Al-Dagal and Bazaraa. and 1.5% lactic cultures or 0. but they did appear brownish and watery (Kim et al.8%.3 or 1. Psychrotrophic counts from untreated whole shrimp increased to >107/g... or trisodium citrate were used alone or in combination with Bifidobacterium breve (5%) as a dip for fresh camel meat (Al-Sheddy et al.5% lactate was effective as an antimicrobial treatment in sliced servelat sausage over a 4. At lower acid concentrations of 0.7 logs when stored at 4°C compared with the nontreated fillets and increased shelf life by 6 days. 5% sodium lactate.7% SA and 0. although MKP alone had no effect. sodium lactate.5.. Toxicology The LD50 (mean lethal dose) for SA in mice is presented in Table 4.2 logs by day 12. 1999). 1994). 1976). .5%) was also effective in reducing populations of L. SA extended shelf life to 12 days at 4°C. Growth and aflatoxin production of A. SA (0.

L. and 35°C (Shelef and Addala. and the inhibitory effect was attributed to the diacetate moiety rather than pH alone. monocytogenes grown in BHI broth adjusted to pH 5.2% sodium diacetate and 2.5% sodium lactate or combinations of the acids and held at 5°C or 10°C.55. Calcium acetate is classified as a stabilizer when migrating from food packaging materials (21 CFR 181. monocytogenes and S. 0. or S.2% sodium diacetate. Y. L.1% to 2. cereus with no effect against Pseudomonas fragi.2% sodium diacetate.4.25.5 inhibited Aspergillus flavus. 1981).8% and 2.Organic Acids 103 Application and Regulatory Status Sodium (21 CFR 184.1% and 0. monocytogenes increased to approximately 106/g within 3 weeks.3 and incubated at 5°C. Cooked-in-bag ham was formulated with additions of 0. 20°C.1% and 0. aureus (Shelef and Addala.2% sodium diacetate or 1% buffered sodium citrate and within 3 and 5 weeks for products containing 2. and 6. Lactobacillus fermentans. curvatus reached an end point of 107/g within 2 to 2. 5. enterocolitica. Sodium diacetate was more effective than acetic acid. SA can be used as a boiler additive for food-grade steam (Federal Register.1185) acetates are approved as GRAS substances for miscellaneous and general-purpose usage. monocytogenes were inoculated into the cooked ham products at an initial level of 104 and 102/g of product. 1962). Levels of 0. L. 2.3% sodium diacetate was effective in suppressing total aerobic counts.5% at pH 4. and 28 mM (pH 5.1% or 0.1% to 0. The MIC was determined to be 35 mM (pH 5. Lactobacillus curvatus and L.5% and 3. Enteritidis. monocytogenes. monocytogenes was not significantly different in the presence of the individual inhibitors compared with control samples after 20 days at 10°C. monocytogenes and bactericidal . in products containing buffered sodium citrate. L.0. 2001). 32 mM (pH 5. Aspergillus glaucus. The minimum inhibitory concentration (MIC) of sodium diacetate was determined for L. while having little effect on baker’s yeast. In addition to L.5% or 3.5 weeks in products containing 0.25) at 35°C. S. and when it was incorporated into butter wrappers. the combination 0. The growth of L. and stored for up to 40 days at 4°C. Shewanella putrefaciens. A. such as Bacillus mesentericus (subtilis). 5. SODIUM DIACETATE Antimicrobial Properties Sodium diacetate was effective at a 0. sodium diacetate was also effective against hemorrhagic E. or 1% buffered sodium citrate (Stekelenburg and Kant-Muermans 2001).5% level in malt syrups.0% delayed mold growth in cheese spreads. 1994).4) at 20°C. it prevented mold growth (Chichester and Tanner. coli. fumigatus. However. 6.4% at pH 3. and Penicillium expansum. and B. In ground beef. or 1. The addition of sodium diacetate affected the sensory qualities of the product precluding its use. Sodium diacetate is equally effective in the baking industry where its inhibitory powers prevent the growth of bread mold and rope-forming bacteria. 1972). A.2% SA. Sodium diacetate at concentrations of 0.55) at 5°C. respectively.3% sodium lactate. vacuum-packaged. However. Pseudomonas fluorescens. A concentration of 0. Enterococcus faecalis.5 inhibited these same strains and Mucor pusillus in feeds and silage.3% sodium lactate. niger.29).1721) and calcium (21 CFR 184.5% sodium lactate was bacteriostatic to L.2% sodium diacetate. Saccharomyces species (Glabe and Maryanski. Enteritidis were inoculated into a comminuted beef emulsion formulated with 0.3% and sodium lactate at concentrations of 2% to 3% were equally effective as antilisterial compounds in meat with little effect on pH and sensory characteristics (Mbandi and Shelef. the addition of 0. An interactive effect was noted between temperature and concentration of acid in that growth was inhibited with decreasing concentrations of acid and temperatures. 1994).15% to 0. a concentration of 0. monocytogenes was inhibited by sodium lactate and 0.05% to 0.

sodium lactate (2. 1950). a more complex product. Application of 4 M SA reduced the levels of L.8 logs (Degnan et al. Enteritidis. The growth of L.. stored aerobically at 5 and 10°C. and wrapping material.3% and 0. In bread and cake products. glaucum and A. although it is assumed that this derivative would be assimilated in a manner similar to that for acetic acid. 0. 1994).1754). Sodium lactate did not affect the pH of the meat. producing a 2-log reduction after 7 days at 25°C (Schlyter et al.4% (Glabe and Maryanski. Enterobacteriaceae counts dropped 1000-fold over 6 days at 2°C.2% sodium diacetate alone or in combination in beef bologna.1%.5 logs within 6 days. the combination of acids achieved greater reductions of both organisms at both temperatures compared to individual acids and greatly reduced background spoilage microflora. were pasteurized for 10 minutes at 68°C after which they were inoculated with L. monocytogenes and Salmonella species was demonstrated using 2.104 Antimicrobials in Food to S. 1991). 1962). The powder dissolved in the surface moisture of the carcass to form acetic acid and SA and produced a surface pH of about 4. Although 1 M sodium lactate solution reduced populations within 2 days.59 log reduction). Slurries of uncured turkey breast. Sodium diacetate (0.0 against S.5% concentrations (0. 1989). respectively. Sensory characteristics of roasted chickens were not affected even at levels up to 8 g per chicken (Moye and Chambers. plantarum at levels of 0. monocytogenes in mixtures held at 4°C for 42 days was inhibited at 0. 1981). a fermentation by-product of lactic acid bacteria. .005% to 0. Toxicology No toxicologic data were available for sodium diacetate (Food and Agriculture Organization. Lactate and pediocin enhanced the listericidal properties of diacetate suggesting some type of synergistic activity. and pediocin (5000 U/ml). In subsequent work (Mbandi and Shelef. Sodium dehydroacetate was twice as effective as sodium benzoate at pH 5.25% to 0. Sodium diacetate was determined to be listericidal over the 7-day period at 25°C only at the 0. although it is fungistatic at concentrations of 0.8. cerevisiae and 25 times more effective against P.1%. 1993a). L. butter. populations increased approximately 0. At lower temperatures. DEHYDROACETIC ACID Antimicrobial Properties Dehydroacetic acid (DHA) has one of the highest dissociation constants (Table 4.6 logs in crabmeat stored at 4°C for 6 days when treated with 2 M sodium diacetate.1) of the acidulants and remains effective at higher pH ranges. monocytogenes was reduced by 2. It is used in cheese spread. A surface application of 1 to 3 mg/cm2 (2 to 6 g/chicken) sodium diacetate powder extended the shelf life of chickens about 4 days when held at 2°C. This acid is effective against Enterobacter aerogenes and L. niger (Banwart.5%) was listericidal in combination with ALTA™..5 logs CFU/ml slurry. malt syrups. but sodium diacetate reduced the pH slightly.5% concentration (0. monocytogenes at a level of approximately 4.5% sodium lactate and 0. (1993b) used the multiple-barrier concept to limit growth of L. prepared with sodium diacetate (0. respectively.1% (Wolf. monocytogenes by 0. Application and Regulatory Status Sodium diacetate is approved as a GRAS substance for miscellaneous and general-purpose usage (21 CFR 184.54 log reduction).3%. 0. the reduction of multiple strains of L. use levels range from 0. Mixtures were incubated at 25°C or 4°C and sampled up to 7 and 42 days. 2002). As expected.3% and 0.5%). monocytogenes in turkey mixtures. Schlyter et al. lower concentrations of the antimicrobials were equally as effective.5%).

1953). weight gains were lower and mortality was higher than in the controls (Lang and Bartsch.2). However. sodium p-hydroxybenzoate.130). at the higher dosage levels.1% of the substance showed no changes in growth. and 0. or 100 mg/kg of DHA per day for 34 days.Organic Acids 105 Strawberries dipped in or sprayed with 0. but significant growth depression . thus extending marketability (Watada.25% or 5% sodium dehydroacetate for 60 seconds were considered more marketable after 3 days of storage at 20°C than water-dipped berries. hematology. in addition to acidosis by the former route and intestinal adhesions by the latter route (Horn et al.05%..0% adipic acid showed no effect at the two lowest levels. 0. Toxicology The LD50 for DHA in rats is given in Table 4.02%. Spencer et al. and sodium dehydroacetate acid were investigated for three strains of E. or 5. However. Intravenous and intraperitoneal injection produced hemorrhaging in the lungs. Longer studies using male and female rats given 400 or 800 mg/day did not affect offspring. Toxicology Acute toxicity data for adipic acid in mice and rabbits indicated varied tolerance levels by different routes of administration (Table 4. and sodium dehydroacetate and 16 mg/ml for sodium p-hydroxybenzoate. coli in homogenized beef but not in poultry (Yamamura et al. It can also be used as a fungistat in cheese wrappers. The MIC in broth at pH 6. and caused slight histologic aberrations. The sodium salt of DHA was nonirritating to rabbit’s skin. produced diarrhea. 1.5 was 4 mg/ml for potassium sorbate. Sorbate was bacteriostatic at half the MIC for E. 1971). resulting in the loss of up to 20% of body weight. Adipic acid did not affect female rats fed at levels of up to 40 mg/day or male rats fed up to 800 mg/day in short-term studies (Lang and Bartsch. 30. coli O157:H7 in broth and model meat products. 1950).. Enders (1941) showed similar symptoms in rabbits. which included marked obstruction in the liver and kidneys by the oral route and polyuria by intravenous administration. 1957). benzoic acid. The effects of potassium sorbate. 1953). It is approved for use as a treatment for cut or peeled squash in amounts equal to or less than 65 ppm remaining in or on prepared squash (21 CFR 172. Application and Regulatory Status DHA or its sodium salt is GRAS when used in accordance with good manufacturing practices. 300 mg/kg led to severe weight loss and damage to internal organs.2. Furthermore. although a dose of 200 mg/kg led to growth and organ changes. the 800 mg/day dosage depressed growth.0%.. Oral administration of the acid to mice caused hemorrhaging in the small intestine with distension of the stomach. The 90-day feeding trials conducted on rats fed 0. or histopathology. This acid at the appropriate concentration level would inhibit the growth of any microorganisms susceptible to low pH values. altered behavior. benzoic acid. However. even with prolonged contact (Spencer et al. A 2-year feeding study with male and female rats fed 0. mortality.1%. treatment with sodium dehydroacetate was effective in reducing respiration rate and delaying ripening. appearance. 2000). No toxic effects were noted in monkeys given doses of 50 and 100 mg/kg at a rate of five times per week for 1 year. (1950) did not show any unusual effects in rats receiving 10. ADIPIC ACID Antimicrobial Properties Adipic acid has no antimicrobial properties other than those ascribed to its ability to reduce pH.

0. CAPRYLIC ACID Antimicrobial Properties When tested against a variety of Gram-positive and Gram-negative organisms and yeasts. Inhalation of adipic acid for 6 hours at a rate of 126 µg/l produced no unusual findings (Gage. candies. Application and Regulatory Status Caprylic acid is a colorless. The MIC of caprylic acid was 100 µg/ml in TSB for Vibrio parahaemolyticus (Beuchat.1009). 0.0%. and 5% adipic acid and female rats fed 1. oily liquid that has a slightly unpleasant odor and a burning.8% propionic acid. Caprylic acid is also used as an antimicrobial agent for indirect use in cheese wraps.1%. Teratologic studies using mice.013% for baked goods. Application and Regulatory Status Adipic acid is a nonhygroscopic.8 µmol/ml (Kabara et al.0% level although the food intake level was the same. mildly acid flavor. and hamsters showed no significant difference from controls at levels of 263. meat products. and 0. Toxicology Orö and Wretland (1961) determined the LD50 of caprylic acid for mice by intravenous administration (Table 4.2). The acceptable daily intake for humans is listed in Table 4.5% acetic acid and 1. not to be used in amounts exceeding good manufacturing practice. It is slightly soluble in water.0. Adipic acid is approved as a GRAS substance when used as a buffer or neutralizing agent not to exceed good manufacturing practices (21 CFR 184. 1975b). Caprylic acid is approved as a GRAS substance for miscellaneous and general-purpose usage (21 CFR 184. and 205 mg/kg body weight. however. It is four to five times more soluble than fumaric acid at room temperature and has the lowest acidity of any of the food acids. It is used as a flavoring adjuvant in levels not to exceed 0. Sodium adipate at the 5% level decreased the rate of weight gain (Informatics. It is used in gelatin desserts. 1973b). rats.106 Antimicrobials in Food occurred at the 5. 1957). refrigerated rolls. .04% for cheese. weight gains for rats fed the two highest levels were significantly lower than those for control rats (Horn et al. however.0. At pH 5.. Caprylic acid at a 0.16% for snack foods. 1968). No limit has been set on the acceptable daily intake for humans. respectively (Food and Drug Research Laboratory.. gelatins and puddings.3% concentration inhibited E. caprylic acid inhibited bacteria and fungi at a lower concentration than either acetic or propionic acid. 1972). 1.0%.0% acid did not show any abnormalities. the inhibitory concentrations were not dramatically lower (Woolford. 1980). 3.1025). powdered fruit beverages. 1972). It also serves a function in the canning of vegetables. rancid taste. saturated dicarboxylic acid having low water solubility. and in improving the melting characteristics of processed cheese (Gardner. baking powders. At pH 6. frozen dairy desserts.. and biscuits because it imparts a smooth.005% for fats and oils. and soft candy. Caprylic acid was also more inhibitory to Shigella species than either acetic or propionic acids (Nakamura and Zangar. The 2-year feeding trials using male rats fed 0. caprylic acid was ineffective at concentrations of up to 7. 0. as a sequestrant in oils.3. 1970). coli when grown in a chemically defined medium and was more effective than 1. 1943). 288. Inc.001% or less for other food categories.

then lactic. Canned tomatoes acidified with citric.1% at pH 4. Interaction was demonstrated between citric acid and sodium citrate added to BHI broth adjusted to pH 4. 1997).6 log/ml were seen. aerobic counts at 20°C for pH 3. The activity of the undissociated portion of the acid was hydrochloric followed by lactic propionic. monocytogenes after 4 weeks were pH 4. propionic. lower aerobic plate counts of 3.0 with citric and hydrochloric acids (Conner et al. citric acid was more inhibitory than lactic acid.7 and stored for 7 days at 5°C. Schoenemann et al. citric. Little bacteriostatic activity occurred at pH 5. 6. but inhibition increased with decreasing pH (Murdock. followed by hydrochloric.66 at 30°C. Sodium citrate was also more effective than 2% sodium lactate. monocytogenes after exposure to the acid combinations was dependent on pH and acid concentration whereby pH 5 and 6 appeared to be protective against inactivation. but lower pH levels were toxic. . respectively. monocytogenes survived after 11 to 12 weeks in TSBYE adjusted with acetic. 1994). 2. Fabian and Graham.5% sodium lactate was equally inhibitory as sodium citrate alone. The inhibitory capacity of hydrochloric. A comparison of equimolar concentrations identified citric acid as the most antimicrobial. and the combination of 1. Acidification impaired the color but enhanced the flavor of canned okra. L. who determined that the minimum pH values that allowed survival of L.09.7 juice were 4. Citric acid at a concentration of 0. and 1. The authors suggested the inhibitory activity might be the result of the chelating ability of citric acid. and propionic acids and for 6 weeks in media containing HCl or lactic acid. Based on pH. compared to counts in unacidified juice (6. citric. malic.3. lactic.8 and 2. 1987).7% at pH 6. monocytogenes decreased to undetectable levels within 1 to 3 weeks at 30°C. respectively. lactic. aerobic counts for pH 3.42 log/ml (Bizri and Wahem. acetic.4 and 0.22 log/ml). The effect was temperature dependent in that survival of L. and acetic. Okra. (1974). and degree of dissociation.0 with propionic acid. pH was not considered a reliable indicator of preservation effectiveness (Fabian and Wadsworth.12 log/ml. and 4.5% sodium citrate and 1. The minimum pH that permitted growth within 60 days (>100-fold increase in cell numbers) was the same at 30°C but increased to 4.0.5 with acetic and lactic acids.13. and 4. was processed for 30 minutes in boiling water. enterocolitica based on concentration of acid. 1939. whereas at 10°C. such as texture or appearance. phosphoric. lactic.19 at 5°C. 4. Similar pH-temperature relationships were noted by Cole et al.5) was more effective than lactic acid (pH 3. propionic. Additional interactive effects were also noted in the presence of salt. the highest inhibitory activity was associated with propionic.0 and 3.64 log/ml. 1973. When tomato juice was adjusted with citric acid to pH 4. Yeast and mold counts were the reverse with counts of 2.7 juice incubated at 20°C were 6.. pH. (1990). phosphoric. Citric acid was particularly inhibitory to flat-sour organisms isolated from tomato juice.0.83 at 10°C and 7. fumaric. acetic. and 7 (Buchanan and Golden. (1989) reported that on an equal molar basis.5% (pH 4. Citric acid was more inhibitory to thermophilic bacteria than acetic or lactic acids. Acidification of foods before canning is often used to reduce the thermal process time of foods that are particularly sensitive to changes in sensory qualities. followed by acetic acid. 1994). 1953). indicating that citrate was the more effective compound. canned in a brine containing acetic.0 at 5°C. All acids would be effective antibotulinal agents at that pH level (Nogueira et al. 1999). 1950). citric. or tartaric acid to achieve an equilibrium pH of 4. monocytogenes was achieved in trypticase soy yeast extract broth (TSBYE) adjusted to pH 5. and phosphoric acid was compared in TSB for Y. 1990).36 at 10°C. Rate of inactivation of L.. These experiments used citrate phosphate buffer in which the citric acid content was calculated at 1. citric. 4. or malic acids did not change in physical or chemical attributes during processing compared with unacidified tomatoes (Schoenemann and Lopez. however. Sorrells et al.4) in inhibiting Arcobacter butzleri in a broth system (Phillips. 5. and hydrochloric.Organic Acids 107 CITRIC ACID Antimicrobial Properties Inhibition of L. and acetic (Brackett.

(1989) developed an acid-blanch-chelate (ABC) process designed to reduce mesophilic and thermophilic bacterial spoilage in canned mushrooms. If the amount of lemon juice was increased to 20 to 35 ml per egg yolk. 5.31% CA–2. this process reduced thermophilic spoilage from 68% to 23. Mushroom extract acidified with citric acid to pH 6. 2003). and 4.4% of cans containing mushrooms vacuum hydrated by the ABC process spoiled (Beelman et al. but could grow at 8°C only when the pH was higher (5. and 16°C (Valero et al.22. botulinum type B when held for 70 days at 15°C or 14 days at 20°C.0% TCA-0. Reduction in the thermal process time of canned liver paste was achieved through the addition of 0.3 g/kg) for crawfish (Procambarus clarkii) stored at 4°C were not effective in retarding growth of L.3 when at least 20 ml of lemon juice (citric acid) per fresh egg yolk was used. Only 2. leading the authors to conclude that the extracts have a protective effect on C.. Samples containing the reduced calorie dressing were even lower in microbial numbers than aciddipped vegetables (Eytan et al. 1993). The addition of potassium sorbate did not enhance stability. 1994.05 led to more inactivation of S. 115°C.. The preparation of these products included adjustment of the pH with acetic acid to 3.5 followed by canning in a solution containing 200 ppm ethylenediamine tetraacetic acid (EDTA) controlled the outgrowth of spores of C. Although the heat resistance of C. and 0.9. monocytogenes (Dorsa et al. cereus grew in zucchini broth (pH 6.4.31% CA–2. (1985) dipped potatoes in a solution of 1% citric acid and 2% ascorbic acid for 2 minutes before vacuum packing and cooking. Mayonnaise made with >5% citric acid solution adjusted to pH below 4.. Citric acid dips (0.4 to 4. The authors recommended that the final pH should be <3.. 12°C. Growth curves developed for B.65 and asparagus with added citric acid or glucono-delta-lactone to pH levels of 5.69) and processed with an F0 of 0. Extracts were then heated at 110°C.1.14% CA (pH 5. heat resistance was not affected by higher processing temperatures. cereus could grow at pH 5.0 at temperatures below 16°C. 5. 118°C.5 were inoculated with C. Cabbage and carrots were pretreated with the addition of a reduced calorie dressing or with a dip of 0. 4.... coliforms. 1992). They found that acid blanching in a citric acid (0. indicating a synergistic effect between pH and temperature. Both vegetable broths stimulated germination and growth of spores compared to nutrient broth.2% or 1. B.34. The products were packed with or without modified atmosphere and stored at 4°C for 10 to 21 days.85. Samples pretreated with 1% citric acid displayed significantly lower total numbers.7 (control).2. Microbial stability was achieved in liver paste formulated with 0. sporogenes. Notermans et al. Enteritidis PT4 at 22 than 5°C. Citric acid dips for foods not only retard some spoilage but also act as a chelator of metal ions responsible for enzymatic browning reactions.5). provided temperatures were at 16°C.29% CA. and lactic acid bacteria counts than samples dipped with the lower concentration of citric acid. 6. 2000.3. 1989. Homemade mayonnaise has been a significant health hazard as a result of contamination by Salmonella from raw eggs (Membre et al. 1990a).108 Antimicrobials in Food Beelman et al.5) at 12°C but did not grow in broth acidified to pH 5.14% citric acid (CA). This process improved quality and increased the microbial stability of canned mushrooms (Okereke and Beelman. 0. 8°C.4 and 5.0% trisodium citrate (TCA). 1990b). 0. thereby leading to its survival regardless of the change in pH (Ocio et al.1 and 5.9% and spoilage was further reduced to 16. 1991). or 0.. Liver pastes inoculated with 106 to 107 Bacillus spores per g and 104 C. 1990).1 with holding temperatures between 18°C and 20°C for up to 3 days before consumption. sporogenes PA 3679 spores.8. 0. sporogenes spores per g were heated to an F0 of 0. sporogenes decreased with decreasing pH levels. 5. and 121°C.3.. Okereke et al.0% citric acid for 5 or 30 minutes. the product should .05 M) solution buffered to pH 3.8% with the addition of EDTA.05. Silla Santos et al. By adding citric acid to can brine. 1999). 1997. and 4. but the shelf life was shortened at this higher temperature. Xiong et al. Growth in carrot puree was temperature dependent because B.. 1992). sporogenes PA3679 after a sublethal heating process (Okereke et al.1% potassium sorbate (Houben and Krol. cereus strains grown in model vegetable broths were conducted at typical refrigeration and abuse temperatures of 5°C. This process inhibited C.

Ascospores of heat-resistant molds are difficult to kill during processing of fruit products and an increase in processing temperature can lead to adverse changes in quality (Rajashekhara et al. The addition of garlic or mustard increased the death of S. Concentrations of 12. with Arthrobacter simplex. Sodium citrate can also exhibit bacteriostatic activity..75% concentration of either acid did not affect the growth of P. it did not appear to affect attachment (Appl and Marshall.. lactic. suggesting that citrate was a chelator of ions essential for growth (Rammell. Citrate inhibition of S. A level of 0.6 and incubated at 35°C.016% to 1% malic acid as the sole carbon source because these acids predominate at these concentrations in a ripening mango.08 to 4. 5 hours at 80°C.8% gave a pH of 4.8. During ripening. the time decreased to 48 hours before use. citrate appeared to inhibit glucose utilization (Imai et al.5% lactic acid prevented toxin production. however.1% to 4.2. 80°C. Healthy tissue had a pH of 5. The metabolic effects of citrate on Arthrobacter species were twofold. Thermal resistance studies were conducted in mango juice adjusted to pH 3. 2%. leading to enhanced growth.75% lactic acid inhibited growth of Aspergillus versicolor. No toxin was detected in any of these foods over an 8-week shelf life (Post et al. sodium citrate stimulated growth. aureus showed inhibition both with increasing concentrations of citric acid and decreasing pH (Minor and Marth. The amount of sodium citrate had a dual effect on Lactobacillus casei. 1970). Radford and Board (1993) found that acetic acid was more inhibitory than citric acid and recommended that mayonnaise be prepared with vinegar to a pH or 4. shrimp puree. Branen and Keenan (1970) also suggested that citrate chelation of metal ions inhibited L.0% was not as inhibitory to Streptococcus agalactiae when added to skim milk or fresh milk as citric acid at concentrations of 1%. type E grew and produced toxin at 26°C in media containing citric acid at pH 4. 1985).25% citric acid and 0. hard-cooked eggs allowed to equilibrate in 0. Using the multiple-barrier concept. or 1. 0. A level of 0. botulinum showed no change in numbers when inoculated in tomato puree. Skim milk acidified with citric acid was more inhibitory to S.6.5% sodium citrate were inhibitory to Salmonella anatum and Salmonella oranienburg (Davis and Barnes..5 with citric.25% concentration greatly reduced toxin production. but salt had a protective effect on this organism.0% concentrations of citric acid and 0. aureus was overcome by adding calcium and magnesium ions.3% citric acid lowered the level of Salmonellae on poultry carcasses.. Similar destruction values were seen with potassium sorbate and sodium benzoate.2% sodium benzoate were held for 30 days at 4°C or .0 reduced the numbers of P. When acetic acid was used. A 0. a 0. whereas infected tissue was around 4. sodium citrate inhibited growth. (1985) found no outgrowth or toxin production from spores of types A and B in TPGY medium acidified to pH 4. the level of acidity decreased and sugars and nitrogenous compounds increased. and rinsing with a citric acid solution adjusted to pH 4. As little as 0. fluorescens on beef carcasses (Thomson et al.5% citric or 0.0. Ascospores were able to withstand more than 6 hours of heating at 75°C.. expansum (Reiss. even in media containing up to 1% citrate. Tsang et al. 1984). 85°C. however. 1984). and in concentrations greater than 40 µM/ml. Survival studies for Neosartorya fischeri ascospores were conducted in mango and grape juices and heated at 75°C. However. no growth occurred at pH 5.125% to 4% citric acid or 0. Typhimurium than lactic and hydrochloric acids (Subramanian and Marth.12 (Sinha et al.1 or less. 1968).8. malic. S. Citric acid provided the maximal destruction of ascospores at 85°C.75%. 1952). The MIC of 0. Mangoes are subject to spoilage through infection with Penicillium cyclopium (Palejwala et al. and 3 to 4 hours at 85°C. 1967). and 90°C. With Arthrobacter pascens.. Sabouraud medium was adjusted with 0. Sodium citrate in concentrations of 0. 1962). or tartaric acid. Enteritidis. 1968).Organic Acids 109 be held at 22°C for longer than 72 hours. 1970). when 35 ml lemon juice per egg yolk was added. casei.5%. 1998). In concentrations of 12 to 18 µM/ml.75% for both citric and lactic acids had a slight growth-retarding effect on A. the function of citrate appeared to be one of chelation of metal ions. parasiticus. 1976). or shrimp and tomato sauce acidified with citric or acetic acids to pH 4. and 4%.0% to 12.2 or 4. C. but 0.

slight atrophy occurred in regions of the thymus and spleen (Yokotani et al. fruit preserves.5 depressed growth of L. Application and Regulatory Status Citric acid is a tricarboxylic acid having a pleasant sour taste. plantarum but did not affect P. (1956) fed vitamin D-free diets containing low calcium levels but adequate levels of phosphorus supplemented with 0. Short-term studies on rats fed 0. In another study.. Long-term studies involving rats fed quantities up to 1. salad dressings. Cramer et al. . (1957) evaluated dosage levels in mice given the acid intravenously. It is approved for use in ice cream. Although there was no effect on weight gain. (1982) used a combination of 0. or sodium salt (21 CFR 184. rats.02 mol sodium citrate and citric acid.0 to reduce the growth rate of S. Death by all routes and in all animals resulted from respiratory or cardiac failure and hemorrhaging of the gastric mucosa. Restaino et al. the highest dosage contributed to enamel erosion.110 Antimicrobials in Food in 0.75% glucono-deltalactone incorporated into sausage before fermentation. Studies by Yokotani and coworkers (1971) compared toxicities of commercial citric acid and Takeda citric acid. 1971). Deaths resulted from acute acidosis and lung hemorrhages. and rabbits indicated varied tolerance levels by different routes of administration (Table 4. however. 1985). enterocolitica.1% citric acid and 0. 4. sherbets and ices. a by-product of yeast fermentation.2). The acceptable daily intake for humans is listed in Table 4.7% sodium citrate (5% citric acid) fed to rabbits for 60 days produced no unusual effects (Packman et al. This combination inhibited the growth of S. and it is used as an acidulant in canned vegetables and dairy products. potassium (21 CFR 184.75% citric acid alone for 21 days at 4°C. 1972). Horn et al. Subcutaneous injections of 320 to 1200 mg/kg body weight of sodium citrate into dogs decreased blood calcium levels while increasing calcium levels in urine (Gomori and Gulyas. 1944). (1973) used a combination of 0. and 4.1751).5. and jams and jellies. It is highly water soluble and enhances the flavor of citrus-based foods.2. in the acid form (21 CFR 184. or 5% citric acid (Bonting and Jansen.8% citric acid in a commercial diet showed lowered weight gains as a result of decreased intake of food and minor blood chemistry abnormalities at the 4. or 12 g citric acid per kg of a noncariogenic diet to show the possible effect of citric acid on the development of teeth.. coli. Horn et al. the urine contained calcium citrate. Y. A concentration of 0.4%.3. aureus early in the fermentation and the increased acidity of the meat mixture allowed the fermentation to proceed more quickly. 2. Dalderup (1960) fed rats 1. 1963). aeruginosa (Restaino et al. Citric acid is approved as a GRAS substance for miscellaneous and general-purpose usage when used in accordance with good manufacturing practices. 1956.2%.05% potassium sorbate and citric acid at pH 5. Acute toxicity data of sodium citrate are shown in Table 4. Potassium sorbate at 0. 3%..1625). Gruber and Halbeisen (1948) used comparable levels of acid but suggested that many of the symptoms produced by high levels of citric acid resembled those of calcium deficiency.75% citric acid was sufficient to reduce inoculated populations of S. Citric acid also acts synergistically with antioxidants to prevent rancidity by chelating metal ions (Gardner. 7. 1981). aureus (Fischer et al. Daly et al. It can control the pH for optimum gel formation.. rouxii and Saccharomyces bisporus.2% concentration in combination with citric or lactic acid at pH 5. It is a precursor of diacetyl and therefore indirectly improves the flavor and aroma of a variety of cultured dairy products. 1957) showed no abnormalities other than lowered weight gains and feed consumption in the 5% group. The number of cavities formed did not differ between the control and experimental groups.2%. beverages.1195). The authors concluded that citrate had a rachitogenic effect on the test animals. Toxicology Acute toxicity data for citric acid in mice. E.8% level. Typhimurium. and S.5..1033) or as the calcium (21 CFR 184.

The fumarate esters not only prevented mold growth on bread (Huhtanen et al. However.0) were not lethal to T. 0. and 1.5% level. and di-n-alkyl fumarates were almost inactive (Dymicky et al.125%.0% fumaric acid and 1. Medium chain-length alkyl esters had more activity than aliphatic acids (Dymicky and Trenchard. or 0. citric. 1996a. 0. It was speculated that the antimicrobial properties of fumaric acid were related to a lower pKa than lactic or acetic acids.6 L in the vapor phase were also effective as a mold preventive (Huhtanen and Guy.1% and 1. Human feeding . Fumaric acid was also more effective than lactic or acetic acid when used in meat products (Podolak et al.5% applied for 5 seconds at 55°C to the surface of lean beef inoculated with L.0% acid concentration at 82°C. whereas the presence of fumaric acid esters in the bacon prevented swelling for up to 8 weeks. Fumaric acid by itself was only slightly inhibitory. fumaric acid achieved the greatest reduction of aerobic. 1981. acetic. Toxicology Weanling rats fed 0.0 to 2. Islam. respectively.. 1947). The addition of 0. and coliform bacteria. 1984). 1987). citric.5 to 5... Fumaric acid at concentrations of 1. malic. botulinum swelled in 4 to 6 days at 30°C. and tartaric acids at a concentration of 2.38% sodium fumarate or guinea pigs fed 1.8%. fischeri (Conner and Beuchat..2% showed considerable promise as a substitute for sodium nitrite in bacon. and tartaric acids increased heat inactivation. coli O157:H7 reduced levels by 1 and 1.2% fumaric acid or rabbits fed sodium fumarate at a concentration of 6. respectively. Similar effects were noted with N.15% fumaric acid coupled with 0. and ethyl esters of fumaric acid in concentrations of 0. dimethyl. 0.0% fumaric acid did not show any abnormalities. 1982).1%) controlled fungal growth in tomato juice.3 logs. 1988). In addition to reduction of E. Monomethyl. The use of fumaric acid to effect a 5-log reduction of E. coli O157:H7 inoculated into unpreserved apple cider was investigated by Comes and Beelman (2002). total aerobic counts were reduced to levels below those achieved through pasteurization. 1963).5. These same preservatives provided a similar reduction in less than 5 hours and 2 hours when held at 25°C and 35°C. The n-monoalkyl fumarates and maleates esterified with C13–C18 alcohols also possessed significant antibotulinal activity (Dymicky et al.Organic Acids 111 FUMARIC ACID Antimicrobial Properties Fumaric acid (0. A 0.5% concentration of fumaric acid inactivated ascospores after 20 minutes of heating at 80°C. rats showed an increased mortality rate and more atrophy of the liver (Fitzhugh and Nelson.0% and 1. Monomethyl and monoethyl (0. coli O157:H7.15%. psychrotrophic. and lethality increased as the pH decreased from 5.05%) or sodium propionate (0. When used in vacuum-packaged ground beef patties.5%. malic. Huhtanen (1983) and coworkers (1985) investigated the effect of various esters of fumaric acid on growth of C.b). At the 1. but malic acid did not. monocytogenes and E.5% (pH 2. The 2-year studies using rats fed 0. or tartaric acid based on weight when added to the heating medium.05% sodium benzoate followed by holding at 25°C for 6 hours before refrigeration at 4°C for 24 hours achieved reduction. flavus ascospores (Beuchat. citric.. 1975). but the dimethyl and diethyl esters at a concentration of 20 mg per 2. Fumaric. Cans packed with bacon and inoculated with C.1%.15%) served as an acidulant (Ough and Kunkee.2%) and dimethyl and diethyl fumarates (0. 1974) and inhibitor of malolactic fermentation in wine (Pilone. botulinum types A and B in canned bacon.9% (5% fumaric acid) showed no toxic effects (Packman et al. 1987). Methyl n-alkyl fumarates demonstrated lower activity. Fumaric acid was more fungicidal against Talaromyces flavus ascospores than acetic. 1982). 1987) heated in the presence of 2.

1983. unsaturated.000 mg/kg was proposed (Levey et al. pie fillings. LACTIC ACID Antimicrobial Properties Lactic acid has been used more extensively for its sensory qualities than its antimicrobial properties in the past. 1970). Similar reductions in microbial counts occurred with a 1. Gill and Newton (1982) suggested that the inhibitory effect for psychrotrophic organisms was related to a decrease in pH rather than degree of dissociation. The acceptable daily intake for humans is listed in Table 4.. 1985).3 of veal tongues.25% concentration of acid sprayed on veal carcasses. but a 2% concentration led to discoloration of the carcass surface (Smulders and Woolthuis.25% lactic acid spray of beef carcasses followed by a treatment of hot-boned cuts with 2% acid.. the organism responsible for flat-sour spoilage in tomato juice (Rice and Pederson. 1954). and storage for 10 days.7 log CFU/cm2 (Visser et al. Cold-pack cheese normally prepared with lactic acid was reformulated with acetic acid and inoculated with S. (1984) found that spraying beef and sheep carcasses with a combination of 1% lactic. 1950).1% ascorbic acids selectively inhibited Enterobacteriaceae at 10°C. The inhibitory capacity of this acid lies in its reduction of pH to levels below which bacteria cannot initiate growth. 2% acetic. although more recently this acid has been used as a rinse for beef. In fermented foods. pork. In addition to supplying acidity to a product. Because most other studies chose refrigeration storage temperatures. combined with vacuum packaging and storage at 3°C. A 1. which was reduced to 10% after treatment. an LD50 of 8. Fumaric acid is used in fruit drinks. dicarboxylic acid having low water solubility. it blends with certain flavoring compounds to intensify the aftertaste of a flavor. 0. Typhimurium. and chicken carcasses. Lactic acid was more inhibitory to Mycobacterium tuberculosis as the pH decreased (Dubos. It increases the strength of gelatin gels. propionic.350). and wines. followed by vacuum packaging.3. 1972).. Because of these dosage levels. biscuit doughs. reduced aerobic mesophilic plate counts from 5. . Although this additive has a strong acid taste.. Osthold et al. Fumaric acid and its salts are used for direct addition to food for human consumption (21 CFR 172. fumaric acid does display some antioxidant properties in fat-containing foods (Gardner.0% to 1. 1991).112 Antimicrobials in Food trials using a 500 mg/day oral dose for 1 year were also negative. 1975a). Lactic acid was an excellent inhibitor of spore-forming bacteria at pH 5. lowered microbial counts after storage for 14 days at 2°C. Optimal decontamination was achieved when 1% lactic acid solution at 55°C was sprayed on beef carcasses immediately after dehiding and after evisceration (Prasai et al. gelatin desserts. vacuum packaging. Woolthuis and Smulders. 1946). Lactic acid sprays have been effective in limiting microbial growth on meat carcasses under a variety of storage conditions. 1988). A 2% L-lactic acid treatment at pH 2. Application and Regulatory Status Fumaric acid is a nonhygroscopic. Enterobacteriaceae contaminated 50% of the untreated samples. Lactic acid was about four times more effective than malic. Destruction rates for S. and acetic acid in limiting growth of Bacillus coagulans. Acid treatment when combined with vacuum packaging was more effective in prolonging shelf life than vacuum packaging alone (Smulders and Woolthuis. 1985). and 0. citric. A study using lactic acid sprays of skinned cow heads found that 1% acid was effective in significantly reducing total counts and extending shelf life by 3 days at 4°C and 1 day at 15°C and 20°C (Cudjoe. Typhimurium were similar for both acids (Park et al. 1988).25% citric. this study showed that lactic acid was effective at higher temperatures as well.0 but was ineffective against yeasts and molds (Woolford. lactic acid coupled with other antigrowth factors excreted by lactic acid-producing microorganisms inhibits competing microorganisms.6 to 2.

Acid-treated samples were lighter in color as a result of leaching of the pigment during immersion. Dixon et al. sub-primal meat was injected with one of three treatments: 0. pH 5. coli O157:H7 or S. or hot air at 371°C. All products were stored at 4°C and examined for aerobic. Lactic acid was most effective against S. Aerobic plate counts of meats injected with CAL were significantly higher than with LAC or COM. 0. and 0.5) at a volume equal to 10% of the initial sub-primal weight. The combination of acids performed as well as 1% lactic or acetic acids alone. (1987) compared a combination of 1% lactic. Postchill beef rounds inoculated with E. but control cuts retained more desirable flavor profile (Eilers et al.5 logs (Anderson et al. Typhimurium were subjected to an automated wash water system (Castillo et al. but odor was not affected (Ellebracht et al. 482°C. (1987) confirmed their findings using beef strip loins stored in polyvinyl chloride (PVC) film for 6 days or high-oxygen barrier film for 28 days. There was little difference in bactericidal activity between the combination and either acid alone on sub-primal cuts of beef. 1998. As in beef trim. Acuff et al. Typhimurium levels decreased approximately 1 log.2%.25% citric. 30 psi).. 76°C. 0. and E.2 logs. moisture content.. pH 3. Typhimurium at 70°C. Greater reductions on all microbial loads were seen in BTF compared to BTL. coli counts was detected for beef dipped in acetic or lactic acid solutions compared with a water-dipped control at day 1. b).. and sensory analysis were not affected by acid treatment.3.3 M calcium chloride solution (CAL. Meat color was affected after application and through storage at 4°C. 454°C. E. In addition.0). pH 2. total fecal coliforms. coli.5 logs for E..6 with either 1% lactic acid at pH 2. coli biotype 1 counts. The system alone produced a 3log reduction.3 M lactic acid solution (LAC. 0. COM improved tenderness in muscle cuts. only suggesting that lactic acid can exert a continued antibacterial effect during the shelf life of the product. Kotula and Thelappurate (1994) compared acetic acid and lactic acid solutions applied at two concentrations. but shear values. The inoculated meat was subjected to sprays of tap water (15°C to 17°C.1% ascorbic acids adjusted to pH 2. coli O157:H7 and S.. aerobic bacterial counts and S.b). Furthermore.Organic Acids 113 Hot-boned. and presumptive lactic acid bacteria. <1 log for Enterobacteriaceae and 0. the FPT supported lower microbial numbers than LPT. at 1°C to 2°C as a dip for rib eye steaks. coli (Anderson and Marshall. 2001a. A residual effect was noted for the lactic acid-treated tissue in that microbial counts were significantly decreased compared to the control steaks after 9 days of storage.. combinations of the treatments were examined. Beef trim was fabricated to make predominantly beef trim lean (BTL) and beef trim fat (BTF) products that were inoculated with bovine fecal samples (Kang et al. 65 psi). hot water (30 psi) at 65°C. . Treatments containing lactic acid resulted in the lowest counts without affecting sensory qualities. reducing numbers by 2 logs and Enterobacteriaceae by 1. Similar work was conducted using lean pork trim (LPT) and fat-covered pork trim (FPT) (Castelo et al. Typhimurium-inoculated beef trimmings. 2001). or 82°C. psychrotrophic. Application of 95°C water alone or with 2% L-lactic acid was effective in reducing E. Beef trim meat used to make ground beef is of particular concern because of its higher microbial load. 20 or 120 seconds. 71°C. or a combination (1:1) CAL/LAC (COM.b). but this effect was not seen with acetic acidtreated tissue.1). Lactic acid application as the final step in a multiple-hurdle process led to a residual effect during storage for 7 days at 4°C. 426°C. Microbial counts obtained from ground beef made from the treated beef rounds that were subjected to the multiple sprays were significantly lower than samples receiving a postchill rinse.8 to 7. 1992).9 or 1% acetic acid at pH 3. Sprays containing 4% lactic acid applied at 55°C for 30 seconds or at 65°C for 15 or 30 seconds led to unrecoverable levels of E. When sequential treatments were employed using pre-chill and postchill lactic acid sprays. When temperature changed from 20°C to 70°C or lactic and acetic acid concentrations increased individually to 3%. Significantly higher aerobic plate counts were obtained from hot-boned cuts over 17 days postmortem compared to cold-boned cuts. coli was reduced by 6. 1999).6% or 1. 1990a. Minimal reduction in total plate and E. or 510°C. 2001a. 2% lactic acid (12°C to 15°C. 2% acetic. and at two time intervals. 1994).

Lactic acid treatments alone were only 50% as effective as acid/alginate treatments. After 28 days of storage.. monocytogenes (Ariyapitipun et al. Lactic acid (2%). coli O157:H7 in beef burgers. lactic acid. monocytogenes.. Aeromonas hydrophila. 1993). or hydrochloric acid (Lynch and Potter. The effects of 3% lactic acid at 55°C were studied using pork fat and lean tissue artificially inoculated with 104 to 105 CFU/cm2 of L.6 log/6 cm2. and Lactobacillaceae counts by 2 to 3 logs. coli O157:H7. but the population of microorganisms shifted. A 1% concentration of lactic acid (pH 2. or nisin and lactic acid. 1988). or sodium lactate (4%) alone or in combination with pulse electric field or freezing was ineffective in reducing numbers of E. Numbers of S. or 360 seconds to 2% lactic acid. monocytogenes. Frankfurter emulsions incorporated with acids to adjust pH to 5. lactobacilli and yeasts were the least sensitive. respectively (El-Khateib et al. 1. Salmonella species or Listeria species were not recovered nor were sensory characteristics affected (Prasai et al. Greater inactivation was noted for both organisms at the lower pH level when acetic or lactic acids were used but not with citric. and nisin (400 IU/ml) were used alone or in combination in beef to inhibit L. 1993). 2002). Beef was inoculated with L. nisin. significant reductions were seen with all treatments. 40. After 42 days of storage the initial inoculum of 5 logs was reduced to <1 log after using lactic acid.. monocytogenes before vacuum packaging and storage at 4°C. psychrotrophic Gram-positive bacteria. Van Netten and Hust In’t Veld (1994) developed a pork skin model system to determine the effect of lactic acid decontamination on microbial counts and changes in predominance of the microflora. These compounds reduced the level of L. The use of these compounds was effective in reducing psychrotrophs and Enterobacteriaceae (Ariyapitipun et al. Elimination of these groups shifted the population to Gram-positive bacteria and yeasts. Enterobacteriaceae. monocytogenes and treated with 2% lactic acid. enterocolitica. Beef inoculated with E. and low-molecular-weight polylactic acid. but no differences were seen between polylactic acid and lactic acid.. malic. and 200 IU/ml of nisin (Mustapha et al.5%).000 IU/ml nisin. 2000). 2002).. and polylactic acid. By increasing the concentration of lactic acid to 2%. and 0. these treatments were not deemed usable in the control of E. coli O157:H7 was subjected to treatments of lowmolecular-weight polylactic acid.. 55°C) had little effect on the aerobic plate counts taken from the surface of pork carcasses.6 for Bacillus stearothermophilus and pH 4. monocytogenes on beef tissue.114 Antimicrobials in Food Lactic acid (2%). 1999). and storing for 5 days at 3°C. by immersing porcine livers in 0. and nisin did not enhance the effects of lactic acid against E. 240. . All treatments significantly reduced the number of L. 1993). Therefore. 180. Gram-negative bacteria were the most sensitive to lactic acid.5 and 4. Y. vacuum packaging. The combination of acids and heat treatment led to reduction of spore-forming organisms in frankfurters.2 or 4.8. Lactic acid was effective in decreasing the number of Enterobacteriaceae as well as other Gram-negative mesophilic and psychrotrophic spoilage organisms.7. The use of lactic acid for reduction of microbial numbers or pathogens has been similarly applied to pork products..2 for B. and 3200 units/ml pediocin. A novel approach to shelf life extension was the use of lactic or acetic acid-impregnated calcium alginate gels applied to the surface of beef tissue (Siragusa and Dickson. there was no significant difference between nisin.1. coagulans were heated to 121°C and 105°C or 110°C. noncoated tissue became dehydrated during the 7-day study. Woolthuis et al. followed by mesophilic Enterobacteriaceae. whereas acetic acid was more effective than the acetic acid/gel treatment. low-molecular-weight polylactic acid (2% PLA). Although the gels reduced populations of L. however. Psychrotrophic. monocytogenes by 1. acetic acid (0. lactic acid. numbers of Salmonella species and Campylobacter species were reduced immediately and remained lower 24 hours after slaughter (Epling et al. 1992). (1984) effectively reduced total plate counts. 1992. coli O157:H7 inoculated into beef trimmings (Bolton et al. Typhimurium were reduced more by exposure to acetic acid/gel treatment than acetic acid alone. Not only did the microbial counts decrease as the time of decontamination increased. Five treatments were delivered to the pork skins: water-treated (control) or exposure at 21°C for 120. Beef cubes were inoculated with L.2% lactic acid for 5 minutes.

Consumer evaluation of chicken treated with a 0. Lactic acid dips have also been used successfully in the poultry industry. such as Pseudomonas species. monocytogenes counts were reduced by 0. and a 2% lactic acid dip at pH 2. but no protective effect was observed if the sodium chloride was added to the medium 45 minutes after the inoculation. coli O157:H7 grown in TSB at 37°C (Casey and Condon.. Lactic acid added to scald water alone had minimal effect on reducing the numbers of contaminated birds.2 in media containing lactic.. coli O157:H7 rapidly died at pH 4. . or Brochothrix thermosphacta (Greer and Dilts. None of the pathogens grew on lean tissue. fragi and B.000-fold. E. grew on both tissue types after acid treatment.7 log CFU/g and Enterobacteriaceae from 3.Organic Acids 115 P.9 to 2. In an effort to eliminate carcass discoloration. in sodium chloridefree.5%.3 to 2. Acid treatments of fat tissues led to the reduction of pathogen numbers to nondetectable numbers within 4 days. These treatments inhibited hydrogen sulfide-producing bacteria. monocytogenes compared with monosodium phosphate.7 log CFU/g (van der Marel et al. or formic acids. thermosphacta. 1995.62) were combined with propylene glycol (20%) in chill water.0) for chicken legs increased the shelf life from 6 to 12 days at 6°C.2 decreased from 5. produced similar reductions of approximately 1 log (Zhang and Farber. such as chlorine or chlorine dioxide. pH 2. 1988). Typhimurium to almost nondetectable numbers. Salmonellae were eliminated from broiler carcasses after a 1-hour exposure. Total microbial numbers from skin of birds immersed for 15 seconds at 19°C in 1% or 2% lactic acid at pH 2. hydrophila numbers declined by 5 logs within 11 days of storage.8) and scald water (54°C/2 minutes) reduced the bacterial level of broilers inoculated with S. pH 2. S. coli decreased 10. 1990b). 1996). E. 1995). The interaction of sodium chloride (0. Pretreatment of broiler carcasses with lactic acid buffer increased shelf life by 6 to 7 days at 4°C and 5 to 6 days at 7°C. Cells grown at 46°C were more heat resistant and better able to grow in media adjusted with acetic and lactic acids to pH 5. Hwang.5% to 4%) and pH (4. After inoculation. A 10% lactic acid and sodium lactate-buffered acid spray (pH 3.2) was examined for E. acidified media.1°C..000-fold). although tissue treated with acid affected the growth rate.23 in cells exposed to acid to 5. reduced levels of lactic acid (0.2 logs. pH 2. 2002).88 or 0. however. discoloration still occurred and propylene glycol contributed an objectionable flavor (Izat et al. sodium acid pyrophosphate. Sodium chloride offered a degree of protection against the inhibitory activity of lactic acid when the culture was incubated in the medium containing both ingredients. Although L.79 in acid-free media. 1990a). and A. Lactic acid (1%) added to both chill water (0°C to 1. and Beuchat. fragi.. Chicken skin subjected to washing treatments using 1% lactic acid led to significant reductions of Salmonella species and L. or sodium hexametaphosphate. The treatments did not affect sensory quality.2 to 3. however. The number of Salmonella-positive birds was also reduced as a function of time of the dip (Izat et al.5% sodium benzoate dip concluded that sensory qualities were acceptable (Hathcox et al. E. respectively. that contributed to spoilage..25%. The sodium chloride-sparing effect was partially attributed to a reduction in water activity causing the cell size to shrink as a result of loss of water in the cytoplasm and a concomitant increase of internal pH from 5. The spoilage organisms. 1995).5 and 0. aureus grown at 37°C or 46°C in the presence of 1 M sodium chloride had marked differences in its sensitivity to acetic and lactic acids (El-Banna and Hurst. acidified with lactic acid. shelf life was lengthened to >36 and 35 days at 4°C and 7°C. Levels of psychrotrophs decreased from 3.0 than cells grown at 37°C. Lactic acid was more effective than acetic acid when used as a 10-minute rinse for fresh-cut vegetables. 1983). P. sodium tripolyphosphate. acetic. When treated broilers were packaged using modified atmospheres. 1990). other compounds. coli demonstrated a 10-fold decrease in numbers when grown in media containing sodium chloride. tissue was dipped for 15 seconds in water or lactic acid and held for 15 days at 4°C. Higher concentrations of the acid did not ensure greater decontamination nor did repeated treatments.6 log CFU/g. Lactic acid added to broiler chill water resulted in the development of a brown coloration. respectively (Sawaya et al.5% lactic acid/0. 1995).3 prolonged shelf life to 8 days (Zeitoun and Debevere. This protective effect was even more pronounced with acetic acid (100.

Aflatoxin production also increased during growth of A. Ross et al. Presser et al.. The Food and Agriculture Organization of the United Nations (Food and Agriculture Organization.75% lactic acid adjusted to pH levels of 3. parasiticus in association with L...5 using HCl or NaOH and incubated for 10 days at 28°C produced more aflatoxin B1 than other cultures at 3 days of incubation. Premature infants fed acidified milk containing the isomeric forms of lactic acid.. contributes to water-holding capacity.. Some enamel decalcification was apparent in the experimental groups. Lactococcus.2). jellies.116 Antimicrobials in Food El-Gazzar et al. Toxicology The LD50 of lactic acid for test animals varied according to animal species (Table 4. increases meat yields. parasiticus NRRL 2999 grown in a laboratory medium containing 0. to prevent discoloration in fruit.3. lactis. LACTATES Antimicrobial Properties The salt form of lactic acid has been used successfully as an antimicrobial agent because it provides a slight salty taste that enhances meat flavor. and Staphylococcus ranged from 268 for S. The MIC of sodium lactate was investigated under optimum growth conditions (pH 6. 20°C) for a number of bacteria and yeasts isolated from spoiled and unspoiled meat products (Houtsma et al. aureus and Lactobacillus coryniformis to 804 mM for Brochothrix. Luchese and Harrigan (1990) also noted an increase in aflatoxin production adjusted to pH 4. Calcium lactate is used as a firming agent for apple slices. developed acidosis.. 2003. coli in response to temperature. Application and Regulatory Status Lactic acid is a hygroscopic. Yeasts were very broad in . 2003). (1987) found that A. Ballabriga et al.5% and 0. retains color.2 using HCl or lactic acid stimulated by substituting half of the carbon content by lactate and a reduction in production at pH 6. pH. and in baking powders. MIC values for Salmonella species ranged from 714 to 982 mM and a narrower range for L. The acceptable daily intake for humans is listed in Table 4.5 or 4. 1973) supported this view. although Campylobacter was particularly sensitive with an MIC of 179 mM. With increasing levels of lactic acid. confectionery products. Gram-positive bacteria including lactic acid bacteria.5% lactic acid (Trainer et al. infants suffered stricture of the esophagus following ingestion of milk acidified with 1 teaspoon of 85% (Pitkin. weight loss. Therefore. In two other instances. Hamsters fed a cariogenic diet incorporating lactic acid in the drinking water (40 mg/100 ml) or in the feed (45. 1998. Bacillus. sherbets. 1945). and lactic acid concentration has been developed (Mellefont et al. 1935) or 87. Brochothrix. monocytogenes and L. cultures decreased production of aflatoxin G1. Lactic acid is approved as a GRAS substance for miscellaneous or general-purpose usage (21 CFR 184. D (-) or DL. Infants died after consuming milk acidified with an unknown quantity of lactic acid.5. Gram-negative bacteria including Pseudomonas and Yersinia had a comparable range to Salmonella. appears naturally in meat products. 1949). improves juiciness. Lactic acid is used in the manufacture of jams. 1993).6 mg/100 g) did not differ in growth rate compared with control groups (Granados et al. A predictive model for growth of E. see Shelef (1994) and De Wit and Rombouts (1990). Carnobacterium. water activity. dehydration.1061). For an excellent review of the antimicrobial nature of sodium lactate. and extends the shelf life of products. syrupy liquid having a moderately strong acid taste. and beverages. (1970) recommended that the L (+) form should be used in feeding premature infants. and vomiting. The autopsy revealed hemorrhaging and gangrenous gastritis (Young and Smith.8. It is used to adjust acidity in brines for pickles and olives. 1944). innocua from 804 to 982 mM.

As expected. and 7.5%. 0. spore germination did not lead to growth after 7 days.0 at 4°C.982) did not inhibit toxin production. and the resultant pH. which might explain their prevalence in spoiled meat products. Pork liver sausage was formulated with sodium. There did not appear to be a synergistic effect between a 4% concentration of lactate combined with 140 ppm . toxin production was detected in media containing 4% sodium lactate within 11 days. spore germination. and 4 using 0. 1996). 10°C.. however. monocytogenes was studied in a cooked meat model system containing varying moisture levels and 4% lactate.. At the abuse temperature of 20°C. This level was listeriostatic at a moisture of >55%. innocua was modeled in broth adjusted to pH 5. calcium. 5. Survivor curves were developed using L. The effect of sodium lactate on toxin production. and potassium lactate were equally effective. with Debaryomyces and Candida being the most resistant at 1161 and 1339 mM. no growth or toxin formation was detected after 32 days of incubation for media containing 4. 11 days for 1.5. toxin formation was noted within 5 days for 0%. The pH level was influential in inhibition by sodium lactate but not by sodium chloride. respectively. Sodium chloride at the same water activity as sodium lactate (0. potassium. Lactic acid/sodium lactate and acetic acid/SA were studied in a BHI broth at pH 7. Addition of 2% sodium chloride enhanced the listericidal activity of the lactates (Weaver and Shelef. At 30°C. 20°C. and 30°C (Houtsma et al.Organic Acids 117 their sensitivity. Toxin production occurred within 14 days of incubation at 15°C for cultures grown in 0% and 1% sodium lactate but was delayed in the presence of 2% sodium lactate until day 21. 1993). 1994b). 4% sodium or potassium lactate achieved reductions in L. no toxin formation was detected after 49 days of incubation in media containing 3% sodium lactate. At a higher temperature of 20°C. 3%. comparable to a 3% concentration of calcium lactate.5.0. 1993). monocytogenes per gram of sausage. lactates also contributed to lowering the aw of a food. and 15 days for 2. 1991.. its concentration. with lower amounts of sodium lactate needed as pH and temperature dropped. Calcium lactate (2%) reduced populations below the inoculum level. whereas a 2% level extended the time to 24 days. 1994a). Typhimurium when stored for 4 days at 20°C. Lactic acid was more effective at higher pH levels whereas acetic acid was more effective at lower pH levels and there was a correlation with the concentration of dissociated acid. Calcium lactate was also more effective than sodium lactate in retarding growth of E. Chen and Shelef (1992) suggested that in addition to pH change. 6. Samples were stored up to 50 days at 5°C and up to 10 days at 20°C. L.5%. As expected L.5. Sodium. botulinum strains in peptone yeast extract medium (Houtsma et al. This model was then evaluated for use in a bologna-type sausage system (Houtsma et al.. but the combination of 2% or 3% lactate and 2% sodium chloride at the same moisture level was inhibitory. the rate of inactivation was dependent on the acid. 1995). Salts of lactic acid are equally as effective in muscle food systems. monocytogenes. The effect of sodium lactate and sodium chloride on the growth of L. 6. As expected. 1995) examined the effect of 3% sodium or calcium lactate added to pork liver sausage and various heat treatments. monocytogenes as the target organisms. inhibition by sodium lactate was directly related to pH and temperature. 6. Using 108 CFU/g as a microbial end point.1. Fresh pork sausage formulated with 0% to 3% sodium lactate was vacuum packaged and stored for up to 28 days at 4°C (Brewer et al.. indicating that inhibition was not the result of a lowering of water activity. The calcium form was found to be more inhibitory than the sodium form. Numbers increased to >108 CFU/g after 50 days at 5°C in sausages not containing lactates but were reduced with 2% or 3% sodium lactate and 2% potassium lactate. and 2 M concentrations (Buchanan et al. 1% sodium lactate delayed growth for 10 days. and heat resistance was tested on proteolytic C. Potassium lactate (4%) and sodium chloride (3%) reduced growth somewhat at 5°C. or calcium lactate at concentrations of 2%. or 4% and inoculated with 104 to 105 L.0. monocytogenes was inhibited by combination of 3% lactate and heat sterilization (121°C for 15 minutes) followed by a water bath heat process (70°C internal temperature) and least affected when lactate was added to sausage without heat treatment. 1.0% sodium lactate. coli O157:H7 and S. A subsequent study (Shelef and Potluri.

5 in a meat system prevented anaerobic growth of Serratia liquefaciens. and E. 2002).4%. 2. 1993). monocytogenes and heated to 55°C to simulate a sous vide process (McMahon et al. and A. Papadopoulos et al. Lactate increased cooking yields.05 and 6. S. Sodium lactate was less effective against microorganisms when sausage contained soy concentrate. 1999). 2001) compared the use of 3% or 6% sodium lactate. . uncured bratwurst manufactured with 3..5 reduced surface discoloration in chubs of fresh sausage and extended shelf life from 10 to 20 days. Both concentrations of potassium lactate significantly inhibited or delayed the growth of L. 1997).. Cured meats. Retention of nitrite was linked to higher pH levels and was unaffected by the incorporation of 1.0% or 3. When raised to 210 mM. S. monocytogenes. (1991b) did not find this effect in their studies.. monocytogenes. The microflora changed from predominantly staphylococci and micrococci at 0 days to coryneform. coli O157:H7 and stored at 10°C. 1994). the plate counts of treated roasts increased to 6. The heat resistance of the two organisms was significantly lowered in beef containing either of the two concentrations of sodium lactate. A 3% concentration of sodium lactate at pH 7. or 4.8% sodium lactate was inoculated with Y. Y.0% potassium lactate were inoculated with a five-strain cocktail of L. the amount of L-lactate ordinarily found in muscle was not inhibitory to any of the strains. Typhimurium. The growth of L.1% sodium diacetate (Glass et al. Sodium lactate at concentrations of 3% and 4% were generally effective in limiting growth of L. or 3. such as wieners. or E. Red color was preserved by using 2% to 3% sodium lactate.5°C Lactate influenced the growth of lactic acid bacteria in meat based on the pH level and the atmospheric conditions under which postrigor meat was held (Grau.0% potassium lactate showed differences in spoilage rates. 1980).1. The pH of the product was unaffected by incorporation of sodium lactate.25% sodium diacetate were the most effective treatments throughout the 120-day shelf life conducted at 4°C with minimal change in pH. or 4% sodium lactate were inoculated with L. then cooked and vacuum-packaged the roasts. (1991a. monocytogenes was delayed for 4 and 12 weeks at 7°C and 3°C in unsmoked. Sodium lactate (2%) was also effective in extending the shelf life of lower-fat frankfurters to 6 weeks compared to 3 and 4 weeks for low-fat and high-fat control frankfurters (Bloukas et al. rely on sodium nitrite to inhibit growth of C. perfringens.0 log CFU/cm2 after 84 days of storage.5% SA or sodium diacetate as an antilisterial process for frankfurters. micrococci. 2002).. monocytogenes and stored at 4°C and 10°C for up to 90 and 60 days. monocytogenes during storage at refrigerator and abuse temperatures. Although the aerobic plate counts for control meat increased from 2. 1991).25% or 0. In retail products.5. 2002).7 to 8.2%. coli O157:H7 (Miller and Acuff.c) injected beef top rounds with 0% to 4% sodium lactate. Sodium lactate at a 6% concentration or 0. Although Shelef and Yang (1991) suggested a possible aw effect for sodium lactate. sodium lactate permitted aerobic growth of B. Another study (Bedie et al. A 100-mM concentration of sodium lactate buffered to pH 5.. Cooked beef top rounds containing 1%. nitrite levels are reduced. respectively (Porto et al.. Pork sausages manufactured with 25% fat or 8% fat with and without 3. botulinum.. aureus. thermosphacta down to pH 5..4%. Frankfurters containing 2. enhanced flavor at the 1% level by adding a salty taste.5 log CFU/cm2 in the same period. enterocolitica.6% sodium lactate into the product (Kilic et al. lactobacilli. 1981). hydrophila and aerobic growth of the aeromonads (Grau. 2%. C. but produced a mild throat irritation at a 4% concentration. 3%. and yeasts at 42 days to homofermentative and heterofermentative lactobacilli and streptococci at 84 days. Over the storage life of the product. Papadopoulos et al.b. 2. Lactates inhibited the growth of psychrotrophic organisms and delayed the pH decline (Bradford et al. At pH 6.118 Antimicrobials in Food potassium nitrite.4% sodium lactate/0. 0. Minced beef containing 0%. sodium lactate caused more rapid surface discoloration (Lamkey et al. enterocolitica or L. Typhimurium. S. Enterobacter cloacae. Levels of >3% sodium lactate or 1% each of lactate and diacetate were needed to prevented growth of Listeriae for 60 days at 4.

or 10% lactic acid/sodium lactate buffer rinses (pH 3. Notermans and Dufrenne (1981). botulinum types A. Of all the treatments. B.. (1991) also found that 2% lactate inhibited the growth of C. fragi and S. They concluded that the two salts gave comparable results and could be used interchangeably. 5%. 10°C. found that glyceryl monolaurate at a concentration of 5 g/kg of a meat slurry inhibited C. 7°C. however. which did not provide significant control (Stillmunkes et al. The meat was cooked to an internal temperature of 71. the 2. They also noted that the inhibitory component was the lactate moiety.0% concentration of sodium or potassium lactate delayed the growth of L. 4 to 6.8%. not sodium. monocytogenes in TSB and 4% limited growth of L. (1989) suggested that a possible mechanism for the action of lactate was the inhibition of a major anaerobic energy metabolism pathway necessary for growth.. 7. Typhimurium but did inhibit P. Reduction in production of hydrogen sulfide and other sulfur-containing compounds resulted from the decreased populations of psychrotrophs. . Sodium lactate at the same concentration had no effect. Some treated chicken was packaged without further modification. Lactates have been combined with other compounds in multiple-barrier studies. coli in a chicken model system after 14 days at 22°C but only delayed the growth of Lactobacillus alimentarius by about 2 days (Lemay et al. 5%. or Microgard™ (2%) (Rozbeh et al. calcium lactate up to 0.5% than sodium gluconate. or 5°C. thermosphacta and E. Vacuumpackaged beef was treated with 2% sodium lactate.5%.5% concentration. aureus grown anaerobically (Smith and Palumbo. botulinum in comminuted raw turkey using levels of 2%. 1991). respectively.3%) was very effective in reducing numbers of B.0) alone or combined with modified atmosphere packaging was examined on the survival of L. and E only when lactic acid was used to reduce pH below 5. 3%. or sodium gluconate and inoculated with L. or a combination of the two was inoculated with the two organisms and held at 5°C for 5 days.Organic Acids 119 Shelef and Yang (1991) found that a 5. Maas et al. Product was considered spoiled when microbial numbers reached 7 to 8 log CFU/g. monocytogenes in comminuted chicken or beef when held at 35°C.56 log during 17 days of storage for the legs treated with 10% buffer combined with MAP. (1989) demonstrated that sodium lactate was effective in retarding toxin formation by C. pediocin (1400 U/g). fragi at even at the 4% level. Organoleptically. A 3% concentration of sodium lactate only minimally affected the growth of S. 20°C. Excised skin tissue was analyzed for Listeriae for up to 17 days posttreatment. or glycerol monolaurate enhanced inhibition of S. sodium lactate was the most effective during an 8-week storage period at 3°C. (1991) looked at the interactive effects of sodium alginate and calcium or sodium lactate used in restructured meats on P. nisin (1400 U/g. 10. Nisaplin (500 IU/g). Lactate contributed to water-holding capacity and increased cooking yields. Lactic acid at 25 to 30 mEq in combination with sodium nitrite. suggesting that the lactate moiety was the effective component. vacuum packaged. but other samples were placed into packages that were gas flushed with a 90% carbon dioxide/10% oxygen mixture. monocytogenes and the shelf life of chicken legs (Zeitoun and Debevere. sporogenes were stored at 2°C. and 13 days for the 2%. 1993). None of the treatments affected the growth of either organism. and stored at 27°C. Ground beef containing sodium alginate at concentrations up to 0.5% concentration was acceptable. all were stored at 6°C.1°C. Sodium lactate was more effective at reducing pathogen numbers at levels up to 3.2. sporogenes. Odor concomitant with growth of psychrotrophic spoilage organisms occurred in untreated samples within 8 days and within 9. Toxicity occurred within 3 days in turkey without lactate and within 4 to 5.. respectively. or 3. Unda et al. Modified atmosphere packaging (MAP) extended storage life for products without acid sprays to 17 days and even longer with sprays. 2002). Harmayani et al. Sodium lactate (3. and 10% acid spray. potassium sorbate. glycerol monolaurin. Typhimurium. monocytogenes increased by only 0. The influence of 2%. 2. and 25°C. Beef roasts containing sodium lactate. Levels of L.3%.5%. monocytogenes and C. 1980). whereas off-flavors developed in meat containing the 3. Maas et al. 1993). or 7 to 8 days for each concentration.

primarily for its flavoring and acidification characteristics (Gardner. 1972). jams. A 3% concentration of lactate in combination with 3% sodium chloride or with 125 ppm sodium nitrite and 3% sodium chloride was effective at 10°C (Pelroy et al. 1% kappa carrageenan. dicarboxylic acid with high water solubility. and hydrolyzed vegetable protein and containing 1%. 1971b). aureus at pH 3. a 2% lactate and 3% sodium chloride inhibited L. Nunheimer and Fabian (1940) found that malic acid was inhibitory against S. the acid inhibited yeasts and molds but not to the same extent as bacteria (Woolford. vacuum packaged.8% sodium lactate. and stored at 5°C or 10°C. Application and Regulatory Status Malic acid is a nonhygroscopic.1639). and sodium lactates (21 CFR 184. monocytogenes. sodium lactate was more effective than the other additives in inhibiting growth of L. 1993).1% sodium erythorbate. but the addition of 2% or 3% potassium lactate reduced microbial numbers. monocytogenes and stored aerobically at 4°C. monocytogenes for up to 50 days at 5°C. encapsulated salt.3. 1970). at 50. Malic acid is approved as a GRAS substance for miscellaneous and general-purpose usage (21 CFR 184. and sodium nitrite. jellies. or 3% potassium lactate. anatum. Reproductive experiments using rats fed 1000 and 10. 1994).. The acceptable daily intake for humans is listed in Table 4. In general. subtilis). thus affecting the sensory qualities of the meat (Egbert et al.000 ppm malic acid before mating showed no significant differences from the controls (Hazelton Laboratories. It is used in sherbets and ices. 1975b). As the pH decreased to 5. food consumption decreased and growth rate declined (Hazelton Laboratories.120 Antimicrobials in Food Raw and cooked ground beef were prepared with 1.. Sodium lactate displayed synergistic activity with nitrite and increasing concentrations of sodium chloride. Dogs fed the same three levels of malic acid showed no effects using the same parameters (Hazelton Laboratories. and . The acceptable daily intake for humans is listed in Table 4.. 1971a). especially rope bacteria (B.0. Carrageenan had no effect on bacterial loads. Toxicology Malic acid fed to rats at levels of 500 and 5000 ppm in the basal diet had no effect. potassium (21 CFR 184.1069). however.000 ppm.1768) are also approved. 1992). Chung and Goepfert (1970) compared various organic acids to determine the highest pH level that inhibited growth of S. Some discoloration and lipid oxidation occurred. and an alginate binder. Salmonella Senftenberg. at pH 6. fruit preserves.0 or 4.98. Comminuted salmon was mixed with sodium lactate. It has a strong acid flavor but does not have the same buildup of acid taste as other acids. sodium chloride. 2%. inoculated with L. Application and Regulatory Status Calcium (21 CFR 184. PROPIONIC ACID Antimicrobial Properties Propionic acid inhibited spore-forming bacteria. and beverages. The products were inoculated with L.0. Similar studies used low-fat ground beef patties formulated with carrageenan. MALIC ACID Antimicrobial Properties Malic acid is inhibitory to fungi and bacteria. probably as a direct effect of pH manipulation.1207). monocytogenes and total aerobic plate counts (Harmayani et al. 0.3.

0. pH 4.6 (O’Leary and Kralovec. pyruvate. 2002). When media containing 0.8. Sodium propionate at levels of 0. and cell-wall synthesis decreased. 1941). such as lima beans and peas (Wolford and Anderson. or ZENCP.3. The salts of propionic acid are also effective antimicrobial agents.0 minimized growth at 13°C. toxicity occurred after 7 days for pyruvate. acetate. the levels of L. 4. DNA (deoxyribonucleic acid). 21°C. tartaric. and 0. S. adipic.5 to 0. or 3. 1945).5. At periodic intervals. 1%) and coated onto ready-toeat chicken samples dipped in L. pH 4.000 IU/g) or calcium propionate (CP. Calcium propionate prevented rope formation caused by B. monocytogenes to 8% sodium propionate for 60 minutes caused injury (Buazzi and Marth.3% concentration was also effective at pH 5.156%. This substance not only postponed spoilage in fresh figs. the organism was able to grow at up to a 0. and 35°C. 4 for lactate and acetate. plantarum. (1991) determined that 0.4.6. citrate. 1. mesentericus (subtilis) in bread dough at a level of 0. monocytogenes was the result of incubation temperature. inoculated with C. Eklund (1985) concluded that propionic acid was not as inhibitory as benzoic acid for P. Emphasizing the multiple-barrier concept in retarding growth. At a 5-mM concentration. 1989a). which led to an increase in cell mass without cell division and a decrease in viability (Cherrington et al. Golden et al. and S. 1993). lipid. and stored at 28°C. the lag phase of L.4. aeruginosa. ZPNCP. monocytogenes grown at 13°C was extended by 7.6 with acetic. acetic. ellipsoideus by as much as 5 days. pH levels. botulinum. protein.9% sodium propionate or SA. Sodium propionate at a 0. Turkey breast meat was formulated with sodium salts of lactate. and 35°C and prevented growth at 4°C (El-Shenawy and Marth. adjusted to pH 4.08% EDTA. pH 5. (1995) examined the combination of sodium propionate and SA at a number of concentrations.1. . At pH 5. 1988). or lactic acids in media (El-Shenawy and Marth. respectively. syrup.0 in reducing numbers of L. applesauce. monocytogenes (Janes et al.5 inhibited the growth of Salmonellae at a higher pH level than other acids (pH 5.3% concentration in tryptose broth adjusted to pH 5. the rate of RNA (ribonucleic acid). Sarcina lutea. aureus. succinic. pH 5.6 and incubated at 4°C. 1992).05. tartaric.02% ascorbic acid. pH 5. or at a level of 0. monocytogenes. and lactate. Cherrington et al. 19°C. samples were removed from storage and tested for production of neurotoxin. L. and greater than 18 days for propionate. Proteus vulgaris. coli K12 and Salmonella.. 3 for citrate.0.188%. E. Products were stored for up to 24 days at 4°C or 8°C. and held at 28°C for 0 to 18 days. 21°C. cherries.. pH 4.. and pH. 5.1. little activity was ascribed to EDTA in combination with the acids. concentration of the organic acid. 0. and propionate to a target level of pH 6.3% sodium propionate was adjusted to pH 5. pH 4. acetate. Much higher concentrations of propionic acid were bacteriostatic to B. Exposure of L. Citrate was most effective based on molarity (Miller et al.5.7 M propionic acid at pH 5.0 with hydrochloric acid. Zein (Z) film coatings were dissolved in propylene glycol (ZP) or ethanol (ZE) with and without nisin (N. degree of dissociation of the acid. Incorporation of calcium propionate into coatings for ready-to-eat products was effective in reducing problems with L. 13°C. 18 days for citrate. monocytogenes when used in conjunction with acetic acid in cold-pack cheese food (Ryser and Marth. 1992). citric. Inactivation of L. lactic. and temperatures. Torula species. A three-strain mixture of L. Reduction of pH to 5. The acids contributed to approximately 85% of the antimicrobial activity with 14% contributed by ascorbic acid. 1990). 3.Organic Acids 121 Salmonella Tennessee when all other parameters were at their optimum. Toxin production occurred after 2 days for pyruvate. and Candida albicans. and 5 with a 2% concentration.0 and 13°C. Propionic acid at pH 5. monocytogenes were undetectable. Nondetectable levels of the pathogen were found in products ZEN. aureus. monocytogenes was inoculated into BHI broth containing 0. and 4 days. Propionic acid was more effective than acetic acid as an antimicrobial.0 was more inhibitory in 60 minutes compared to formic acid in 3 hours for E. citric). or 4°C. and berries but also prevented spoilage in vegetables having a more neutral pH. subtilis. coli. At a 6% concentration. pH 4. fumaric and malic. particularly at the higher pH level. monocytogenes. In laboratory studies examining the effect of sodium propionate on L.1% to 5% delayed growth of S. 7.

Although mold spores are destroyed during baking. Studies involving albino rats fed propionic acid at 50 cm3/kg of rice for 110 days showed umbilicate or warty lesions on the stomach (Mori. The inhibitory action could be the result of an interference with β-alanine synthesis (Wright and Skeggs. Concentrations of propionic acid and propionates ranging from 8% to 12% were effective in controlling mold growth on the surface of cheese and butter (Deane and Downs. 0. Mold spoilage is a significant economic problem in bakery products. 0.3% by weight. and Penicillium corylophilum. and 7.2%. 1940). Propionic acid at a concentration of 0. rather than later (Ghosh and Häggblom. postprocess contamination from the atmosphere. sodium benzoate. Not only was the final pH of the substrate critical. rubrum.. but also various organisms displayed different tolerances to the compounds (Olson and Macy.5 with lactic acid was as effective as a 10% unacidified solution in preventing surface mold growth on butter (Olson and Macy. Bread produced with the extracts contained less ethanol and supported less spoilage by molds.8 to 0. or Trichophyton mentagrophytes.0 and a water activity of 0. All preservatives were effective at pH 6. (2001). 1951. Eurotium amstelodami. and potassium sorbate at a level of 0.1% sorbic acid or potassium sorbate. Graham and Grice. 1952a). 1954. Ingle.5 logs.5 and 1. The antimicrobial activity of sodium propionate was most affected at 4°C by fat concentration with a 1. However. monocytogenes by only 1. Adding yeast extracts previously fermented by Propionibacterium freudenreichii provided a source of propionic acid in bread formulation.5. 2002).003%. 0. and wrapping materials reintroduce molds. respectively. pH 4. Additional studies using calcium and sodium propionate fed to mice. Small amounts of β-alanine could overcome bacteriostatic action of sodium propionate for E.. A 5% calcium propionate solution acidified to pH 5. None of the preservatives were effective at neutral pH. Suboptimal doses (0. 1945). herbariorum. and 0. with sodium lactate. Propionic acid at a concentration of 2435 ppm was more effective than acetic acid in limiting growth of Fusarium oxysporum but less effective than sorbic acid and potassium sorbate. and rubrum. and 0.8% sodium lactate.2). Increasing the fat content from 22% to 67% decreased the growth of L. 1.1%. reduced growth and aflatoxin formation of A. no growth occurred. and Hara et al. at 0. however. Molds were inhibited by less calcium propionate by weight than sodium propionate..5. A novel use of propionic acid to retard spoilage of bread was investigated by Gardner et al. herbariorum.03%) led to enhanced growth of Aspergillus and Penicillium isolates.8 log reduction at 67% fat.0. flavus. Toxicology Orö and Wretland (1961) determined the LD50 of propionic acid for mice. Propionic acids and their salts are primarily inhibitory to molds.3%) at pH 4. Pseudomonas species. 1996). 1946).2% sodium propionate.93 in a model agar system on the retardation of growth of E. and humans showed no toxic effects (Graham et al. 1940). B. 1955. 1952b). amstelodami. Subsequent studies (Marin et al. some species of Penicillium grew on media containing 5% propionic acid (Heseltine. 1953). There is evidence that the sodium salt has some local antihistaminic activity (Heseltine. 1985). coli. (1963) determined the LD50 of calcium and sodium propionate for rats (Table 4. Hara. 1965.5 and water activity of 0. This inhibitory effect was more pronounced with the addition of acid at the time of inoculation.5 log reduction of L.03%. Harshbarger.8. monocytogenes was studied in pork liver beaker sausage (Hu and Shelef. A.b) examined several levels of calcium propionate. Spore germination was inhibited by 1402 ppm (Tzatzarakis et al. rats. monocytogenes at 22% fat compared to 2. . and repens were inoculated by needle into cake analogues prepared with calcium propionate. potassium sorbate. and sodium benzoate (0. flavus and niger. 2002a. β-alanine could not reverse the inhibitory effect of propionic acid in Aspergillus clavatus.122 Antimicrobials in Food The effect of fat concentration on the efficacy of antimicrobial agents against L. subtilis.8 log reductions were observed. 2000).85 (Guynot et al. and 0. 6. 1942). cooling surfaces.85. Sausage batter was mixed with 0..

Propionates can be added to bread dough without interfering with leavening because there is little or no effect on yeast. 1980). or eye opening from the control group. Calcium propionate is preferred. A limit of 0.. calcium (21 CFR 184. In addition. are approved as GRAS substances for miscellaneous and generalpurpose usage.38% in whole-wheat products. The acceptable daily intake for humans is listed in Table 4. hair appearance. It can be incorporated into gelatin deserts and cake flavorings (Gardner. Its antimicrobial effect is limited to most yeast and bacteria. for use in bread and rolls because the calcium contributes to the enrichment of the product (Chichester and Tanner. It is normally found in cheese and as a metabolite in the ruminant gastrointestinal tract. Swiss cheese contains up to 1% propionic acid because of the growth and metabolism of propionibacteria. 1974). Typhimurium by 60% on chicken carcasses after spraying with 1. gradually increasing to 2. This naturally formed additive becomes a developed preservative that limits mold growth on Swiss cheese. 0.0 mg/day at 4 weeks and continuing at this level for 100 days.1091). 1972). but surface recontamination can occur during packaging. Thomson et al. 1965). Application and Regulatory Status Succinic acid is a nonhygroscopic. SUCCINIC ACID Antimicrobial Properties Succinic acid successfully lowered microbial loads on poultry carcasses (Mountney and O’Malley.1784).Organic Acids 123 Application and Regulatory Status Propionic acid is a monocarboxylic acid with a slightly pungent.0% succinic acid. Propionic acid (21 CFR 184. The acid form is readily miscible with water. 1972).3% in cheese products (Robach. Toxicology In short-term studies. Sodium propionate is recommended for use in chemically leavened products because the calcium ion interferes with the leavening action. rats received subcutaneous injections of 0. (1967) reduced the population of inoculated S. The test animals did not differ significantly in reproduction rates. Baking destroys most molds.1221) and sodium propionate (21 CFR 184. however a 3% or 5% concentration used at 60°C impaired the appearance of the product (Cox et al. disagreeable odor. Succinic acid is used to modify the plasticity of bread doughs and in the production of edible fats. No limit has been set on the acceptable daily intake for humans. and the sodium salt is more soluble than the calcium form. and growth can be seen under the wrapper during storage. and 0. 1944). No upper limits are prescribed for use of this additive. Propionic acid and its salts are used primarily as mold and rope inhibitors in bread. which are associated with its manufacture and the characteristic Swiss cheese flavor. tooth eruption. however. Succinic acid is approved as a GRAS substance for miscellaneous and general-purpose usage (21 CFR 184.3. . This additive is also used as a mold inhibitor in cheese foods and spreads.1081) and its salts. calcium and sodium propionate are listed as antimycotics when migrating from food-packaging material (21 CFR 181.23).. Salts of the acid have a slight cheeselike flavor.32% can be used in flour and in white bread and rolls. which conform to standards of identity. dicarboxylic acid having low water solubility. Chick embryos developed normally when comparable dosages were injected into the air sac (Dye et al. It has a slightly bitter taste and serves as a flavor enhancer. except bread and rolls.5 mg succinic acid daily.

3. the most soluble of the solid acidulants. Fitzhugh and Nelson (1947) found similar results in long-term studies using 21-day-old weanling rats fed tartaric acid as 0. and phospholipids can be structurally altered by pH changes.5%. and grape-flavored beverages. and rabbits demonstrated normal offspring at levels of 274. such as hydrochloric. Tartaric acid is a GRAS substance for miscellaneous and general-purpose usage in accordance with good manufacturing practices (21 CFR 184. this delicate balance is maintained and alteration of this balance causes destruction of the microbial cell. 1954). Through the interaction of a series of chemical mechanisms. Rose’s (1924) work on subcutaneous administration of 0. 0. proton donation. or gross histology compared with controls (Packman et al. The workers also displayed skin eruptions.1099). and 1. male rabbits consuming a diet containing 7. jellies and preserves.7% sodium tartrate (5% tartaric acid) did not show any changes in food consumption. and cholesterol levels.2% of the diet. growth. rats. has a strong tart taste that enhances grapelike flavors. The microbial cell normally reflects this equilibrium by attempting to maintain an internal pH near neutrality (Baird-Parker. 1980). Biological and chemical systems depend on an interaction between acid and base systems. hamsters. and yeasts. The acceptable daily intake for humans is listed in Table 4. Homeostasis is the tendency of a cell to sustain chemical equilibrium despite a fluctuation in the acid–base environment.. which disappeared after they left the work site (Barsotti et al. sugar. 1963). respectively (Food and Drug Research Laboratory. Toxicology Acute toxicity data for tartaric acid by the intravenous route for mice indicates an LD50 of 485 mg/kg body weight (Table 4.. Proteins. mortality. It is used in fruit jams. sherbets. molds.8%.124 Antimicrobials in Food TARTARIC ACID Antimicrobial Properties Any antimicrobial property of tartaric acid is because of its ability to lower the pH. 225. Inorganic acids. 1978). although some microorganisms such as Acetobacter can exist at and even require extremes of acidity (Langworthy. Tartaric acid acts synergistically with antioxidants to prevent rancidity. . Studies measuring teratogenic activity in pregnant mice. The terms strong or weak are used to describe acids and reflect the degree with which acids readily donate a proton or dissociate in aqueous solutions.25 to 1 g to rabbits produced abnormalities in blood chemistry.1%. including increased nonprotein nitrogen. MODE OF ACTION The mode of action of organic acids in inhibiting microbial growth appears to be related to maintenance of acid–base equilibrium. and 215 mg/kg body weight. 181. These changes in membrane permeability can exert a dual effect by impairing transport of nutrients into the cell or by causing the leakage of internal metabolites to the outside. The availability of metallic ions to the organism is also altered and becomes a function of membrane permeability because membranes are less permeable to charged molecules than to uncharged molecules.2). and the production of energy by the cells. Workers exposed to tartaric acid dust measuring up to 32 mg/m3 in a manufacturing plant that produced the acid experienced tooth erosion. However. 1972). nucleic acids. 1973a). Changes resulting from pH can destroy bacteria. The monopotassium salt (cream of tartar) is commonly found in baking powders. It is thus essential to understand each of these concepts before consideration of the actual mode of action of the organic acids. and it prevents discoloration in cheese (Gardner. 0. Application and Regulatory Status Tartaric acid.

yet easily and without requiring energy penetrate the membrane lipid bilayer.. Further studies by Sheu and coworkers (1972) indicated that acetate uncoupled the amino acid carrier protein from the ETS and inhibited amino acid transport noncompetitively. coli and B. which does not permit H+ or OH. The active transport of a molecule would depend on the proton gradient generated during the oxidation of a substrate. subtilis are equally inhibited by equal concentrations of compounds containing up to six carbons. oxygen consumption would be reduced. 1973). Lipophilic agents. 1973). the bacterial cell was converted to a spheroplast under isotonic conditions and these membrane vesicles were used to study uptake of a substrate against a gradient. inhibition transport would not necessarily be linked with ATP generation. The respiratory chain or ETS transverses the membrane. In a later study. the inhibition by extracellular fatty acids used as antimicrobial agents would increase with decreasing pH.ions to penetrate and ejects accumulated protons to the external environment. but seemed to be a function of the undissociated molecule. However. Sheu et al. confirming that the undissociated molecule was the effective inhibitor.Organic Acids 125 because of their low pKa almost entirely dissociate in solution. Before energy can be produced. Energy produced through chemical reactions is essential for cellular processes. They concluded that this toxicity was not the result of hydrogen ion concentration alone. 1992). Because oxygen consumption was observed in the presence of membrane preparations and an energy source. the problem becomes one of elimination of the excess protons. The energy evolves from the oxidation of the substrates and the respiratory chain. the glycolytic pathways inefficiently generate ATP. materials must be transported into the cell. For this reason. Thus. Early experiments by Levine and Fellers (1940) demonstrated that acetic acid was more lethal at a higher pH than hydrochloric acid or lactic acid. such as the short-chain fatty acids. To understand these processes. twice the amount of a C8 (caprylic) acid is needed to inhibit the growth of E. This process generates a chemical and electric gradient capable of driving metabolic reactions (Dawes and Sutherland. Not all acids are effective against all microorganisms. It was postulated that this interfered with the regeneration of ATP by uncoupling the ETS. One means for substrate entry into the cell is active transport. In aerobic organisms. subtilis when exposed to fatty acids. E. Serine uptake was inhibited in membrane vesicles of B. Synthesis of macromolecules. Sheu and Freese (1972) determined that short-chain fatty acids reversibly reacted with the cell membrane and altered its structure. active transport is coupled with energy-yielding processes. the electron transport system (ETS) principally generates ATP with oxygen as the terminal electron acceptor (Gould et al. and active transport of molecules across the membrane depend on energy generated by the cell in the form of adenosine triphosphate (ATP).. Undissociated acids of short chain length can penetrate the cell more easily because they possess these characteristics. energy must be expended. To maintain this unequal gradient. The . Freese and Levine (1978) further postulated that the most effective antimicrobial agents would be lipophilic enough to attach to microbial membranes yet be soluble in the aqueous phase.. maintenance of osmotic gradients. which also allows greater internal concentration of solute than external concentration. in agreement with pKa values (Freese et al. L-leucine and L-malate were shown to uncouple both substrate transport and oxidative phosphorylation from the ETS (Freese et al. would shuttle protons through the membrane until the proton motive force had been destroyed and transport thus eliminated. coli compared to B. This is because they can approach the membrane from the aqueous medium. 1983). (1975) recognized that if reducing compounds were no longer available to the cell because of transport inhibition by the fatty acids. This ejection process becomes a fundamental issue in how a cell produces energy. In anaerobic microorganisms. Acetic acid and other organic acids only slightly ionize and do not readily give up their proton(s) to water. or the transport of metabolites into the cell was altered. With acetic acid. subtilis. When an acid enters and ionizes within the cell. using the same system. lowering the pH increased the inhibitory activity.

which can be specific to the type of shock conditions. such as intervention strategies that occur during processing of muscle foods (acid sprays. the current data suggest that the mode of action of short-chain lipophilic acids requires the destruction of the proton motive force. 1995). 2001. 1973). Cells strive to maintain a balance between competing stresses that occur through exposure to sublethal conditions. temperature. preservative) to a food. cells can adapt by means of an acid tolerance response. Furthermore. suggesting a static response rather than a killing effect (Freese et al. ACID ADAPTATION AND RESISTANCE Predictable response of cells to stressful situations is the cornerstone of the multiple-hurdle concept. These stresses occur simultaneously. such as the addition of several antimicrobial agents (acid. which possess both lipoidal and aqueous solubilities. there may be more of an opportunity for strains to develop resistances to the antimicrobial compound or treatment provided that time of exposure allows for such development.. Adaptation to sublethal stresses allows organisms to adapt to higher levels of the same stress without being killed. Response to such stresses often takes the form of specific stress proteins induced after sublethal exposure. when placed in fresh medium devoid of the inhibitor.. acidity. organisms survive and function over a wide range of acidity and alkalinity as a result of their ability to maintain homeostasis. Depending on the length of time and exposure to mild acid conditions. 1991). thus acting as a mechanical barrier and preventing passage of the acid into the cell. it does not seem to be the sole cause of the static action. some acids. starvation. among others. or sequentially. however. 1998). namely. Generally. 2002). Kabara et al. lactic and citric. In summary. 2001). coli O157:H7 in salami and apple cider (Leyer et al. 1973). 1973. In sequential treatments. and desiccation. Another theory proposes that Gram-negative organisms can rapidly metabolize the acidulant and therefore not allow it to accumulate within the cell (Freese et al. 1972). During the shock period. can reinitiate transport. 1998. Adaptive responses to sublethal exposure to acids can often offer cross protection to cells when exposed to other stresses. such as E. He states that although inhibition of uptake of nutrients contributes to growth inhibition. and heating (Mazzotta. whereby organisms are subjected to multiple deleterious compounds or processes designed to reduce or eliminate microbial loads. 1999). It is further speculated that acids. coli. Typhimurium in cheeses (Leyer and . The adaptive effect has also translated to survival of microorganisms in acidic foods. It is hypothesized that these proteins protect against subsequent exposure to the same stress or possibly cross protect against other stresses (Davidson and Harrison. bacteriocin. Hunter and Segal (1973) in studies using Penicillium chrysogenum suggest that weak acids at or below their pKa could discharge the proton gradient and ionize within the cell to acidify the interior. are the most effective antimicrobial agents and that some sort of membrane attachment of the acid is involved. Studies have indicated that cells. which can lead to cell death..126 Antimicrobials in Food difference between the two organisms could be because of the lipopolysaccharide layer that surrounds the Gram-negative E. radiation (Buchanan et al. Cross-protective effects have been demonstrated in E. Exposure to greater quantities of acid (lower pH levels) causes acid shock. S.. It was postulated that the rate of proton leakage into the cell versus proton ejection would determine the inhibition of the cell (Freese et al.. exposure to adverse environments frequently induces a stress response in susceptible microorganisms.. coli between initial exposure to organic acids and subsequent exposure to salt (Garren et al. thus potentially allowing them to survive exposure to commonly used antimicrobial intervention strategies. available water. however. there is need for further research on the mode of action of these antimicrobial agents. stress-induced proteins are formed.. For example. Obviously. hot water rinses). Ryu and Beuchat. exposure to low concentrations of organic acids would cause organisms to become more resistant to higher concentrations of acids. reduced the internal pH of the cell more than acetic acid. other changes associated with metabolic or physiologic processes may be similarly affected (Ita and Hutkins. thereby limiting substrate transport. Eklund (1980) has questioned the mode of action of the weak lipophilic acids. Jordan and Davies.

. In addition. pretreatment with acids increased sensitivity of S. and acid-inducible strains of E. J. Preexposure to mild acid conditions led to increased resistance to higher acid environments such as those encountered in the stomach or macrophage phagosome (O’Driscoll et al. 1992). Brul et al. Inducement of stress conditions can result in the shortening of processing systems and can optimize the use of multiple hurdles as a preservation process (Brul and Coote. It was further shown (Uyttendaele et al. coli O157:H7 presented no difference in their survival when inoculated into beef tissue treated with 1% and 2% buffered lactic acid. G. the distillate can be titrated for the particular acid. C. Food Sci. or electrochemical methods. the distillate is separated on a silicic acid column and the component acids can be identified using enzymatic. Qualitative or quantitative methods can be used depending on the degree of sensitivity required. and Hall. Adams. Griffin.90) or low pH (3.. J. 2000) or Handbook of Food Analysis (Nollet. B. monocytogenes in fermented dairy products (Gahan et al. and temperature (Cheng and Kaspar. 1992). 1996).. (2001) postulated that exposure of E. Adams. including oxygen conditions. If only one acid is present. 1991. monocytogenes (Kroll and Patchett. It is interesting that higher survival levels were achieved in acid-injured E. which adds to sample preparation time (Gomis and Alonso. coli when dilutions were made in osmotically stable diluents containing sucrose. M. Vanderzant. Conversely. Measurement of survival or injury of organisms subjected to stresses is dependent on the methods designed to recover organisms. 2001) that acid-resistant. acidulant and temperature on the growth rate of Yersinia enterocolitica. Acid adaptation is also seen in Gram-positive organisms. 1988. thin-layer. Dickson and Kunduru (1995) demonstrated that exposure to organic acid rinses did not lead to more resistant organisms. J. Bacteriol. chromatographic (gas. C. 71:65–71. This was readily apparent if the first stress was low water activity. G. Using acid-adapted strains of salmonellae inoculated into lean beef tissue. Jones. pH rapidly inactivated cells such that a biphasic death phase was not observed... such as L. Often derivatives of the acids are required. or sodium chloride..Organic Acids 127 Johnson. glucose. and Ehlers. J. Shadbolt et al. Methods for determining acid content in foods are found in the Official Methods of Analysis of the Association of Analytical Chemists (Horowitz. 1998). R. Meat Sci.. Effect of acid decontamination of beef subprimal cuts on the microbiological and sensory characteristics of steaks. 23:287–292. growth phase. W. M. and L. Appl. J.. 1999.. 1996). 1999). 2002). Int.. high-performance liquid chromatography). leading to potentially more effective interventions. 1996). coli to stresses such as low water activity (0. Typhimurium to chlorine and iodine (Leyer and Johnson. It is assumed that inhibitory compounds that affect optimal recovery of cells would be avoided. Savell. 1997). R. C. 19:217. The authors hypothesized that disruption of cell homeostasis created a large energy drain. R. acid-sensitive. Little. L. but not glycerol (Jordan et al. Growth inhibition of food-borne pathogens by lactic and acetic acids and their mixtures. REFERENCES Acuff. K. 1987. If the stresses were in reverse order. and Easter. Modelling the effect of pH. Technol. thus sensitizing cells to future environmental stresses.5) could produce a biphasic death phase. D. C. sorbitol. ASSAY The various acids can be assayed by relatively simple procedures. . M. cell survival is affected by its previous growth environment. Volatile acids can be separated from foods using steam distillation. For a mixture of acids. D.. paper.. 1996).

and nisin. Health hazards to workers in a tartaric acid factory. I. J.. M. Food Prot. 1999. J. Freeman.... Food Sci. I. Anderson. 1989. G. Hampshire.. Food Prot. and Reynolds. 13. and Johnston. J. W. J. J. Reducing microbial populations on beef tissues: Concentration and temperature of lactic acid. E. M. Anderson. R. New York. 1980. A. H. Detachment of Pseudomonas fluorescens P26 from beef rinsed in salt and acid solutions. Food Prot.. W. R. 52:178. D. Microbial and sensory quality of fresh camel meat treated with organic acid salts and/or Bifidobacteria. S... Sofos. In Microbial Ecology of Foods. 50:562–566. M. J.. 38:261. D. Food Prot. Int. Carpenter... 1999. Marshall. T. 2nd ed. Al-Dagal. Food Sci. 1980. A. J. K. Marshall. and 35°C in tryptose broth acidified with acetic. J. Belk.. 44:73. D. E. J. Paediatr. J. Interaction of concentration and temperature of acetic acid solution in reduction of various species of microorganisms on beef surfaces. L. H. and Clarke. A. C. Ahamad. 1989. Microbiological and sensory tests of beef treated with acetic and formic acids. C. G. T. Mustapha... L. Food Prot. M. Influence of organic acids on heat resistance characteristics of Talaromyces flavus ascospores. C. N. 1989. Banwart. J. R. Pratt. M. A. 64:336–339. T. J. Citric acid and antimicrobials affect microbiological stability and quality of tomato juice. T. Biemuller.. and Naumann. 49:207. Acid-injury of Listeria monocytogenes. G. 63:131–136. Scanga. Bedie. lactic and two mixed acids in reducing numbers of bacteria on surfaces of lean meat. Helv.. E. Damare. 1995. Lav. N. and Sandine. Gower Publishing Ltd. E. Huff. Anderson. Kilara. 1984.. E. Food Sci. Microbiol. W. lactic acid. Med. 1989. Ballabriga. Ariyapitipun. 53:26. J. Environ. H. J. N.. J. M. Reducing microbial populations on beef tissues: Concentration and temperature of an acid mixture. and Smith. Some characteristics of acid injury and recovery of Salmonella bareilly in a model system. J. W. Anderson. Efficacies of acetic.128 Antimicrobials in Food Ahamad. p. Al-Sheddy... 1988. Survival of Listeria monocytogenes Scott A on vacuumpackaged raw beef treated with polylactic acid. R. M. England. 1986. Food Prot. and Wahem. Ayres. D. Ariyapitipun. Combined and individual effects of washing and sanitizing on bacterial counts of meat — A model system. T. S. M. and Dickson. A. T. San Francisco. M.. 2001. Beelman. A. lactic acid.. J.. P. and Ghetti. H. 47:537. 2000. 1973. J. A. A. . and Marshall. Extension of shelf life of whole and peeled shrimp with organic acid salts and bifidobacteria. J. Microbiology of Foods. Food Microbiol. Handbook of Food Additives. A. E.. J. Doores. E. 52:688. E. T. E. M. 52:312. Eds. 1980. 40:688. E. Evaluation of an automated beef carcass washing and sanitizing system under production conditions. Comparison of anti-Vibrio activities of potassium sorbate. T. Food Prot. W.. Marshall. Beuchat. R. Reduction of bacteria on pork carcasses. M. A. Factors affecting life and death of microorganisms. 1990b. Appl. Sassi. L.. 55:903.. R.. citric. R. and Marth.. J. 1999. J. Metabolic response of prematures to milk formulas with different lactic acid isomers or citric acid. M. Anderson... G. C. A. C. Bell. 6:97. T. and Gallart-Catala. 39:1178.. M. N. and Marshall. 1977. 126. J. Food Prot. and Kuhn. Food Prot. J. 1970. J. 62:913–920. Blankenship. Organic acids. B.. Samelis. E. J. Food Safety 10:181–190. J.. 45:239. Academic Press. sodium benzoate and glycerol and sucrose esters of fatty acids. and Marth. R. J. A. H. E. or lactic acid. Conde.. W. G. and Clarke. R.. M. and Ash. G. International Commission on Microbiological Specifications for Foods. R. A. Food Prot. Behavior of Listeria monocytogenes at 7. M. Acidification process technology to control thermophilic spoilage in canned mushrooms. C. 1987. Anderson. Food Prot. E. 1994. Baird-Parker. 1990a. and Bazaraa. Vol. Acta 25:25. 21. J. Mustapha. I. 59:130–134. and nisin solutions. F. Bizri. Food Prot. Marshall. 1990. Food Sci. Ash. Witowski. A. E. T. Beuchat. M. and Bazaraa. O. Food Prot. Antimicrobials in the formulation to control Listeria monocytogenes postprocessing contamination on frankfurters stored at 4°C in vacuum packages. C. Stringer. New York. and Anderson. 1992. H. Food Safety 12:139. Van Nostrand Reinhold. and Marshall. Naumann. 62:51–56. 1954. C. K.. and Marshall.. Microbial shelf life determination of vacuum-packaged fresh beef treated with polylactic acid. Barsotti. Basic Food Microbiology. R. D. Al-Dagal. 1981. J. 64:1949–1955. M. Mundt. R. Appl. 1..

Brul. J. Egbert. Hellingwerf. and Coote. Nissen. 1992. E. C. E. A.. D. J. A. P. J. 57:567–570. D.. 57:198–203. D. D. Comparison of water wash. G. M. 1956.. P... and Doyle. Food Sci. T... A. J. Physiological actions of preservative agents: Prospective of use of modern microbiological techniques in assessing microbial behaviour in food preservation. T. 76:199–206. and Ayres. Food Prot. Effects of washing and sanitizing on the bacterial flora of vacuum-packaged pork loins. A. J. and Acuff. M. J.. B. B. 64:738–740. I.. G. 41:128. Koohmaraie. A. Brackett. Hagtvedt. Savell.. Mercado.. Nerbrink. and Boyd. Rostogi. Golden... Preservative agents in foods. Lucia..5% lactate and 0.25% acetate controls growth of Listeria monocytogenes in vacuum-packed sensoryacceptable servelat sausage and cooked ham stored at 4°C. 1994. freezing and pulse fields to control Escherichia coli O157:H7 in beef burgers. and Keenan.. R. and Nesbakken. B. and Berry. Buazzi.. Food Microbiol. L. E. R. . 56:474–484. Food Prot. M. R. 62:219–228. A. Growth stimulation of Lactobacillus casei by sodium citrate. Sites of action by propionate on Listeria monocytogenes. L. S. S. trimming.. S. Martin. Sodium lactate and protective culture effects on quality characteristics and shelf-life of low-fat frankfurters produced with olive oil. Brocklehurst. and Klis. Castillo. Sodium chloride decreases the bacteriocidal effect of acid pH on Escherichia coli O157:H7. Food Prot. 2001. C. S. C. Oomes. W. H. 53:593. H. Bloukas. D. McDowell. 1997. Appl. Buchanan. W. L. 50:598–601. J. Sodium lactate/sodium chloride effects on aerobic plate counts and color of aerobically packaged ground pork. 1998. E. 1999. Cacciarelli... D. Ineffectiveness of hot acid sprays to decontaminate Escherichia coli O157:H7 on beef. 64:335–342. E. Food Microbiol. I.. J. S. E. J. Y. Int. and Fournitzis. R. M. and Mikel. I. Addition of 2. R.Organic Acids 129 Blom. M. 2001b. G. The influence of pH. Bradford. McKeith. F. L. J. Mercado. and Acuff.. Dallmier. The ineffectiveness of organic acids. M. M. Brewer. H..-H. and Naumann. Food Prot. E. Effect of sodium acetate on growth and aflatoxin production by Aspergillus parasiticus NRRL 2999. E. J. R... Voeding 17:137.. and Condon. K.. J. R.. Int. B.. Casey. J. J. Brul. Effects of pH and acid resistance on the radiation resistance of Enterohemorrhagic Escherichia coli. L. 2001a. M. R.-Y.. Interaction of citric acid concentration and pH on the kinetics of Listeria monocytogenes inactivation. Hao. Food Prot. M. Int. K. 1970. R. Food Sci.. D. F. K. J. Lactic acid sprays reduce bacterial pathogens on cold beef carcass surfaces and in subsequently produced ground beef.. Food Microbiol. T. Buchanan. H. Paneras. Food Prot. D.. M. J. Sheridan. J. H. Siragusa. J. Bacteriol. 1991. 38:71–76. D. Buchanan. and Blair. 79:55–64. Evaluation of combination treatment processes for the microbial decontamination of pork trim. Mensonides.. 58:1245. 61:823–828. and Whiting... 1993. W.. Int. R. L. F. Castelo. S. Castillo. G. 1983. M. Lett. J.. Food Sci. S. Food Microbiol. J.. S. A. J. Argoudelis. Effects of various acids on growth and survival of Yersinia enterocolitica. Food Prot. K. Food Prot. H.. P. H. G. and combined hot water and lactic acid treatments for reducing bacteria of fecal origin on beef carcasses. 60:58. S. P.. J. C. T. M. temperature and organic acids on the initiation of growth of Yersinia enterocolitica. 1994.. Bonting. M. Edelson.. 34:139–143. M. Int. 56:1176. M. Dairy Sci. and Marth. 2002. and Golden. J. G. L. W. T. and Acuff. 15:109–119. F.. Food Prot. W. G. Sodium lactate effects on shelf-life. J.. 64:58–62. E. J. Brackett. and Jansen. Goodson. J. 1997. Byrne. C. Borch. B. R... Food Sci. L.. 1999. Kang. 45:223–238. Microbiol. R. W. P. L. Castillo.. D.. Stevenson. 1993. C. and Sprouls. Lucia. Huffman. sensory and physical characteristics of fresh pork sausage. Branen. R.. Potassium lactate effects on low-fat fresh pork sausage chubs during simulated retail distribution.. Meat Sci. In-plant evaluation of a lactic acid treatment for reduction of bacteria on chilled beef carcasses. Stringer. 1990. Food Microbiol. 1987. 50:1–17. Catarame. Appl. 1995. Brewer. J. Anderson. Differentiation of the effects of pH and lactic or acetic acid concentration on the kinetics of Listeria monocytogenes inactivation. 2002. S.. J. 46:231. Dainty. G. The effect of a prolonged intake of phosphoric acid and citric acid in rats. Bolton. G. 69:390–397. J. Lucia. Roberson. and Lund. and Meyer. Coote. Food Prot. T. G. M. L. 1976.. 2002. Buchanan. L.

. Cutter. and Downs.. E. Mercuri. S. . Further observations on the relation of pH value to toxicity of preservatives to microorganisms J. Mikel. Furia. S. N. 1980. A. Growth. and Harrison. P. M. R. 1997. H. C. D. In Microbial Ecology of Foods. Cramer. J. Cudjoe. and Granata. E. F. J. W. 68:69. 2002. 35:326. Food Sci. Control of Staphylococcus aureus in sausage by starter cultures and chemical acidulation. L. Arch.. A. A. I. and Beuchat.. 115. 1990. Conner. J. Scott. M. 2001. Bacteriol. I. 61:382–385. Deane. salts of lactic acid. 2nd ed. J. 1960. 1994. D. Cherrington. Parameters affecting the efficacy of spray washes against Escherichia coli O157:H7 and fecal contamination on beef. Growth and processing conditions affecting acid tolerance in Escherichia coli O157:H7.. and Goepfert. and Sutherland. Food Prot. 60:614–618. Growth of Salmonella at low pH. W. Hinton. J. OH. Office of the Federal Register. 1952. 1932. and growth of Listeria monocytogenes in a meat model system. and Irish. edited by T. 1974. Cleveland. J. Dawes. Government Printing Office. W. New York. Corlett.. and Chopra.S. M. and Brown. G. J.. The effect of lactic acid sprays on the keeping qualities of meat during storage. Dent. Food Prot. J. E. Food Sci.. Thomson. 60:58 Cruess. 65:476–483. Food Sci. H. Dalderup.0 kill Escherichia coli and Salmonella spp. W. C. Relationship between water activity. H. B. N. J. 63:33. In Handbook of Food Additives. E. J. K. V. Vol... E. p. 1997. Heat resistance of ascospores of Neosartorya fischeri as affected by sporulation and heating medium. and other process controls. and Shelef. 38:957. and Tamblyn. Bacteriol. Jones. A.. R. Bacteriol. D. N. Hematologic cytochemistry in subjects exposed to occupational risk of intoxication due to acetone and to acetic acid. and Barnes. 34:767. F. Dorsa. A. Antimicrobial food additives. 57:97–103. 38:426. 1970. L. Suppression of growth of Salmonella anatum and Salmonella oranienburg by concentration variation of energy sources in a synthetic basal medium. J. Washington. N. W. 1973. CRC Press. 1990. 23:163. edited by J.. Conner. A. J. Microbial Physiology.. Int. New York.. I.. W. Food Prot. LaChance. Food Sci. 1. T. 60:1560–1563. Efficacy of organic acids against Escherichia coli O157:H7 attached to beef carcass tissue using a pilot scale model carcass washer. J. and Siragusa.. C. R.. and survival of Listeria monocytogenes as affected by acidic conditions. G. and Kaspar. and Chopra. 1951.130 Antimicrobials in Food Cesaro. J. D. V.. Effect of short-chain organic acids on macromolecular synthesis in Escherichia coli. Short-chain organic acids at pH 5. 53:652–655. Daly. Food Prot. Pearson. Microbiol. Academic Press. 7:1. Chen. Appl. M. P. C. Comes. salt concentration and temperature on the survival and growth of Listeria monocytogenes. J. Chichester. Food Microbiol. Davis. and Beelman. Cherrington. G. 1990. N. R. 1988. sanitizers. Biochem. 4:303. D. Dairy Sci. Environ. Cole. J. Cheng. M. P. 1998. E. B. J. J. C. and Chew. V. J. Cox. R. Food Prot. E. C. J.. V. 1992. Bacteriol. Food Microbiol. without causing membrane perturbation. Blackwell Scientific Publications.-M. S. I. A rachitogenic and growth-promoting effect of citrate. 70:161.. A.. R. W. Code of Federal Regulations. Jr. 1987. Growth and survival of Escherichia coli O157:H7 under acidic conditions. Food Microbiol. Folia Med. M. Hinton.. 39:420. A. 55:574–578. Resistance and adaptation to food antimicrobials. Int. Food Technol. 39:985. D. Bacteriol. The effect of pH. 1. B. Evaluation of succinic acid and heat to improve the microbiological quality of poultry meat. 2002. and Bernard. M. A. A. M. K. Silliker. p. F. D. E.. C.. Chung. Juven. Jr. Addition of fumaric acid and sodium benzoate as an alternative method to achieve a 5-log reduction of Escherichia coli O157:H7 populations in apple cider. L. 1955. Sandine. I. E. and Holyoak. 1956. 15:157–166. Flexible wrappers for cheddar cheese. J. inhibition. J. H. C. D. J. J. B.. 92. and Tanner. Inc. and Kotrola. K. N. M. Porrata-Doria. 2nd ed. U. C. Code of Federal Regulations Title 21. J. 56:69–78. Conner.. L. Effects of acetic–lactic acid treatments applied to beef trim on populations of Escherichia coli O157:H7 and Listeria monocytogenes in ground beef. J. A. 1992. 1995. C. Kotrola. Res. Davidson. J. and Siragusa. 69:63. M.. Cutter. and Elliker. W. and Steenbock. The effects of citric and phosphoric acids on the teeth. Vol. 1972. Appl. Appl. Conner. 1991.C. Biophys. D. W. pH and acidity.

M. Buchanan. A. and Kunduru. Int. H. Hale. P. J. M.. alkaline. 2002. Appl. Eds. Eds. Smith. 1950. 57:297–301. Acuff. Sholberg. M. R. C. J. Environ. 1994. Long-term bacterial profile of refrigerated ground beef made from carcass tissue. K. Acuff. Bencivengo. A.. L. Inhibition of growth and uptake processes in bacteria by some chemical food preservatives.. Kaspar.. Salminen. T... W. E. J. G. J. C. and Jones. Z. M. Sci. New York. S. W. Marshall. Food Microbiol. K. J. Acetic acid action on beef tissue surfaces contaminated with Salmonella typhimurium. Food Prot. Oxford. J. M. S. L. and Savell. J.. Dickson. W. Dickson. Marcel Dekker.. U. S. Acidulants. R.. The effect of acetic acid and air injection on appearance. Injections of certain plant growth substances in rats and chick embryos. G.. or organic acid washes.Organic Acids 131 Debevere. 53:110. 1997. 1992.. L. G. D. Food Prot. S. n-alkyl formates. J. Technol.. propionates and butyrates. D. R. Dorsa. Cutter. J. 60:1146–1151. M.. and Anderson. W. 26:480–482.. R. Dubos... Dickens. Inc.. Evaluation of calcium chloride and lactic acid injection on chemical. 1987. Eklund. W. L. Lyon. Savell. 62:953–957. 61:1615–1622. 48:423. J. and Siragusa. experimentally contaminated with pathogens and spoilage bacteria after hot water. S. Microbiol. and Rombouts. Microbiological decontamination of food animal carcasses by washing and sanitizing systems: A review. and Luchansky. Degnan. Int. Meat Sci. 58:973–976. 1990. In Food Additives. Food Microbiol. W. Food Prot. L. Doores. B. and cooked breast meat texture and flavor. Effects of acetic acid. B. The effect of an acetic acid dip on carcass appearance. Exp. acetates. microbiological quality... 1999. Poult. F. Growth 8:1. R.. Listeria innocua. A.. 55:133–140. G. 92:319. J. 7:113–120. 1987. J. and Siragusa. Whittemore. K. P. H. J. 1995. Med. Dickson. and Clostridium sporogenes. J. Poult. 5:181.. Dorsa. Wiss. Eilers. D. G. Otwell. Food Prot. R.. A. S. III. J. Martin. Inhibition of Clostridium botulinum 62A by saturated n-aliphatic acids. 1997. C. Evaluation of lactic acid bacterium fermentation products and food-grade chemicals to control Listeria monocytogenes in blue crab (Callinectes sapidus) meat. D. J. Branen. G. N. D. Dye.. C. Bradford.. T. 73:576–581. D. J. Environ. Overholse. 1988. 1980. L.. C... Food Microbiol. J. M. J. D. R. New and established carcass decontamination procedures commonly used in the beefprocessing industry. lactic acid and trisodium phosphate on the microflora of refrigerated beef carcass surface tissue inoculated with Escherichia coli O157:H7. Delaquis. Dorsa. R. 1994. A. S. 1994. 2003. J. 57:1033–1037.. D. Dixon. W. C. and Semien. and Trenchard. 1985. Disinfection of mung bean seed with gaseous acetic acid. 1994. J. A. D. R. N. W. England. L. C. W. 2nd ed.. Bacteriol. R. Appl. Eklund. and Jones.. Vanderzant. H. Inhibition of Clostridium botulinum 62A by fumarates and maleates and relationship of activity to some physicochemical constants. and Vinson. 38:443–451. 1993. M. de Wit. J.. Dorsa. Microbiol. R. Dymicky. Inhibition of microbial growth at different pH levels by benzoic and propionic acids and esters of p-hydroxy-benzoic acid. . B. 1944. Morgan.. J. and Smith. P. Egbert. Effect of acid treatment of beef strip loin steaks on microbiological and sensory characteristics. W. Tamplin. J. 60:3198–3203. The effect of organic acids on mammalian tubercle bacilli. Appl. and Stanich. 1992. 73:582–586. Food Microbiol. J. J. moisture pick-up. Cutter. Dymicky. J. and Whittemore. Doores. J. S.. 5:135–139. M.. M. M. microbiological quality. Dickens. Miller. Antimicrobial activity of sodium lactate. J. microbiological and descriptive attributes of mature cow beef. W.. L... Food Prot.. and Thorngate. 1982.. 1998. Properties of low-fat ground beef containing potassium lactate during aerobic refrigerated storage. and Salmonella incidence on processed poultry carcasses.. 2:159. and Hong-Shum. Huffman. and Lyon. J.. J. Food Prot. Blackwell Publishing. Davidson. M. 45:1117. A. Lebensm. D. Sci. J. pH control agents and acidulants. 1992. In Food Additives Databook. J. 60:619–624. Food Prot. J. J. Resistance of acid-adapted salmonellae to organic acid rinses on beef. Food Sci. E. S. Food Sci. Effect of buffered acidulant systems on the survival of some food poisoning bacteria in medium acid media. Effect of potassium sorbate and citric acid sprays on growth of Listeria monocytogenes on cooked crawfish (Procambarus clarkii) tail meat at 4°C.

FAO Nutrition Meetings Report Series No.. 1999. Fed. W. E. (Mosk) 47:84. 197:597. D. A. 56:536–537. and Blankenship. Assoc. El-Shenawy. 1973b.. C. and Ramsey.. 56:29–33. Preserving value of acetic acid and lactic acid in the presence of sucrose. E. WHO Technical Report Series No. 52:771. II. 1963. F. L. oxalic and maleic acids. Action mechanisms of preservatives and antiseptics. W. Technol.. Inhibition or inactivation of Listeria monocytogenes by sodium benzoate together with some organic acids. E. Fitzhugh. Am. and Bailey. Food and Agriculture Organization. Inc. 1973. sodium chloride and sugars. Food Prot. M. M. K. and Graham. tartaric. D.. C. Microbiological properties of hard-cooked eggs in a citric acid-based preservative solution. F. Physiological compatibility and excretion of dicarboxylic acids. J. Fabian. 1987. 50:940. B. O. 1973a. J.. J. El-Shenawy. U. Boiler water additives. Arch. and Wadsworth. G. E. I. 48:252. O... L. Castillo. A. Behavior of Listeria monocytogenes in the presence of sodium propionate together with food acids.. 1947. Survival in foods of Staphylococcus aureus grown under optimal and stressed conditions and the effect of some food preservatives. J. W. 1993. H. Fletcher. M. A. on pork carcasses and the reduction effected by spraying with lactic acid. 59:300–305. 1992. WHO Technical Series No. J. Miller. Growth and aflatoxin production by Aspergillus parasiticus NRRL 2999 in the presence of lactic acid and at different initial pH values. E. S. 1992. C. and Marth. and Marth. 53. El-Gazzar. Experimental work on lactic acid in preserving pickles and pickle products. H. Frederick. Epling.. Washington. Toxicological evaluation of certain food additives with a review of general principles and of specifications.. p. R. Food additives. Miller. A. Wiss. Weinert. J. Cox. and Marth. 4:511. G. Food Prot. 207. Behavior of Listeria monocytogenes in the presence of sodium propionate.C. E. T. A. A. Fischer. 35. 8:85. C. J. J. J. edited by L. Food Sci. A. A.. Viability of thermophilic bacteria in the presence of varying concentrations of acids. Contract 71–75. Teratological evaluation of FDA 71–50 (adipic acid) in mice. Effect of salad dressing and citric acid dip on storage quality of shredded cabbage and carrots packed under modified atmospheres. 1985. and Nelson. Federal Register. F. 7th Report of the Joint Food and Agriculture Organization of the United Nations/World Health Organization Expert Committee on Food Additives. The comparative chronic toxicities of fumaric. 31. H. J. J. L. Food and Agriculture Organization. Food Prot. M. Med. H. T. Pathol. 539. K. Specifications for the identity and purity of food additives and their toxicological evaluation: Emulsifiers. L. NAS/NRC Questionnaire on Tartaric Acid. N. Food and Drug Research Laboratory. Fabian. Carpenter. 29:297–302. FAO Nutrition Meetings Report Series No. G. J. 281. J. MD. El-Shenawy.. In Developments in Industrial Microbiology. R.. Food Prot. E. 25:445–450. Rockville. A.132 Antimicrobials in Food El-Banna. 17th Report of the Joint Food and Agriculture Organization of the United Nations/World Health Organization WHO Expert Committee on Food Additives. and Levine. 64:1094–1099. A. J. R. Lebensm. Food and Drug Research Laboratory. Food and Agriculture Organization. Underkofler. Reg. Food Sci. WHO Technical Report Series No. Food Res. 1994. Fin'ko. 7:212. and Ockerman. and McGill. Pharm. 540.. M. 1978.. Inc. A. MD. J. Pharmakol. Freese. Microbiological properties of pork cheek meat as affected by acetic acid and temperature. 1962. L. 1953. A. Can. 1941. Inactivation and attachment of Listeria monocytogenes on beef muscle treated with lactic acid and selected bacteriocins. Food Technol. 1969. A. Prevalence of Campylobacter spp. Food Prot. rats and hamsters. 6th Report of the Joint Food and Agriculture Organization of the United Nations/World Health Organization Expert Committee on Food Additives. Rusul. Food Microbiol. Int. Blood coagulation changes in acetic acid poisoning. and Acuff. El-Khateib. H.. 1962. F. A. 1939. Rockville. Vol. and Salmonella spp. D. and Marth. K. Food and Drug Administration. N. Food and Drug Administration. 36:217. Exp. 55:241–245. H. FAO Nutrition Meetings Report Series No. Enders. 27:6232. Klin. L. E. Eytan. Evaluation of the toxicity of a number of antimicrobials and antioxidants. 1983. Lucia. Food Prot. A. A. Thompson. 228. bleaching and maturing agents. E. A. 1989a. T.. Ellebracht. Reduction of pathogens using hot water and lactic acid on beef trimmings. G. stabilizers. and Hurst. B. . 1993. Food Microbiol. L. 19. 1989b. Yousef. American Institute of Biological Sciences..

.. Food Prot. and Alonso. G. CRC Press. L. R. J. K. E. 54:691–695. Gomori.. Soc. D. Goddard. Graham. and Smith. J. P. Inhibition of Listeria monocytogenes by sodium diacetate and sodium lactate on wieners and cooked bratwurst.. Glavind. Exp. 62:3128–3132. Evaluation of handtrimming. Morgan. Gage. C. H. Effect of parenterally administered citrate on the renal excretion of calcium. J.. Sheu. N.. 1973. J. A. Graham. Pharmacol. Allied Chemical Corporation.. A. H. E.. In Handbook of Food Analysis. and Doyle.. Effect of lactic acid concentration on growth on meat of Gram-negative psychrotrophs from a meatworks. 1966. [A]. W. D. Acid tolerance and acid shock response of Escherichia coli O157:H7 and non-O157:H7 isolates provide cross protection to sodium lactate and sodium chloride. 225. Food Sci. M. Pharm. Food Prot. The subacute inhalation toxicity of 109 industrial chemicals. and Grice. A. Fu. Marcel Dekker. G. Gardner.. Sebranek. J. 6:534. K. 40:433. 1. D. and Grice. Cereal Foods World 26:285. Brown. Role of pH. H. 42:1043.. E. Acidulants in food processing.. Appl.. 2001. 1996. P. Biol. B. B. Environ. 1995. physical. Effect of sodium acetate or sodium propionate with EDTA and ascorbic acid on the inactivation of Listeria monocytogenes. Food Microbiol. F. M. 1998. H. G. Smith. J. M. Teed. Nature 241:321. 67. 61:158–161. E. H.-H. Vol. and Skinner. C. W. In Mechanisms of Food Preservation Procedures. E. F.. H. Changes in the liver in a case of acetic acid poisoning and their significance for the timely measurement of liver regeneration and cirrhotic scar formation. Food Prot. edited by T.. P. 1995. J. Res. J. R. New York. Proc. Gorman. p. O. 67:1855–1858. and Fletcher. C. W. M. Med. Glabe. 65:116–123. Academic Press. C. Inhibition of the anaerobic growth of Brochothrix thermosphacta by lactic acid.. 1985. Edited by L. and Johnson. various sanitizing agents. A. S. F. L. 59:306–309. G. J. New York. J. Virchows Arch. M. Pharmacol. lactate. Ind. E. H.. Harrison. 27:1. Microbiol. K. G. H. Champagne.. 59:849–853. N. D. Environ. 316:456. M. 1996. 2:323. Effect of yeast extracts containing propionic acid on bread dough fermentation and bread properties. and Galliers. Observations on experimental dental caries. R. B. McNamara. C. Microbial and quality characteristics of pork cuts from carcasses treated with sanitizing sprays. W. reducedcalorie mayonnaise. Mechanisms of action of food preservation procedures. edited by Roberts. K. H. 1981. 43:284–288. Acid adaptation of Listeria monocytogenes can enhance survival in acidic foods and during milk fermentation. 1996. J. C. p. Pharm.. The effect of dietary lactic acid. Food Sci. Dent.. Microbiol. Food Prot. Furia.. L. Microbiol. Ghosh. C. Inc. Food Acidulants. B. D. New York. and Jones. OH.. Golden. 1980. and Whiting. J. K. 2nd ed.. W. Environ. A.. A... and Russell. Schmidt. Gardner. and Murano. M. M. Hardin.. J. C. P. Gerhartz. 1944. 58:899–907.. Chronic toxicity of bread additives to rats. Med. 1991. Nollet. Physical Characterization and Nutrient Analysis. M. and Hill. A. M. T. 1955. C. Environ. 1982. H. Microbiol. B. 1954. J. and hot water spray-washing as decontamination interventions for beef brisket adipose tissue. 1981. Food Prot. In Handbook of Food Additives.Organic Acids 133 Freese. G. 1983. Grau. Granados. Gardner. and anaerobiosis in controlling the growth of some fermentative Gramnegative bacteria on beef. Gill. F. J. Glass. and Maryanski.. Appl. 1949. Gahan. G. Sodium diacetate: An effective mold inhibitor.. Br. Food Safety 15:53–65. B. J. Garren. L. A. and Newton. E. and Dam. Glass.. 2002. Ladwig. J. 28:282. H. Buchanan. M. III. Chronic toxicity of bread additives to rats. and Gélinas. D. J. Granberg. R. 1970. A. C. Use of organic acids to improve the chemical.. Cleveland. 7:126. Effect of sublethal concentrations of propionic or butyric acid on growth and aflatoxin production by Aspergillus flavus. J. Appl. 1972.. Grau. J. Sofos. Int. Gould.. and Gulyas. and microbial attributes of beef strip loins stored at –1 degree C for 112 days. W. Mattias. and Häggblom. Part II. Mikel. J. E. 56:226. . 1949. J. W. C. Conner. B.. Analysis for organic acids. Function of lipophilic acids as antimicrobial food additives. Fate of Salmonella and Listeria monocytogenes in commercial. W. Gomis. 1994. Appl. A. O’Driscoll. H. A. J.

Vanderzant. 4:577. Heseltine.. Examination of general pharmacological actions and toxicity of sodium and calcium propionates. Harmayani. 21:1–14. Hathcox. and water activity on growth of Eurotium species on a sponge cake. E. 1969.. 1965. Hazelton Laboratories. E. F. R. Lucia. Effectiveness of acetic acid. amphyll. M. Abstr. P. A note on sodium propionate. Food Microbiol. 1939. M. D. Allied Chemical Corporation. spore germination and heat resistance of proteolytic Clostridium botulinum strains. R. T. and Halbeisen. L. P.. Hardin. P. N. 1979. 44:359 Hazelton Laboratories. Meat Sci. Harshbarger. E. 1995. W. A. J. Holland.. G. G. Takahashi. A. T. Retarding effect of acetic acid on growth of contaminated bacteria during shoyu-koji making process. A study on the comparative toxic effects of citric acid and its sodium salts. College 1–28. 1963. 1971b. 57:327–330. W.. J. 1995. R. (Ed. Oman. Effect of citric acid and citrate on the shelf stability of canned liver paste processed at reduced F0 values. Buffalo. Ramos. S. and Dilts. J. 4:120. Sodium propionate and its derivatives as bacteriostatics and fungistatics... and Beuchat.. Buffalo. and Miller. NY. C. Horn.. J. L. Sanchis. and Savell. B. Hoffman. W. Consumer evaluation of raw and fried chicken after washing in trisodium phosphate or lactic acid/sodium benzoate solutions. Food Sci. Final report submitted to Specialty Chemicals Division. K. W. H. Appl. Acuff. T. Studies on pharmacological and toxic actions of propionates. A. C.. 1942. Agric. Safety of adipic acid as compared with citric and tartaric acid. Hara.. S. Int. Microbiol. Terada. 65:1376–1379. W. X-5120 (malic acid). J. Horowitz. L. J. calcium lactate and sodium alginate on bacterial growth and aminopeptidase activity. NY. Dairy Sci. Food Microbiol. J. J. Allied Chemical Corporation. G. and Dalby. Sofos. Pharmacological and toxic actions of propionates. Int. Lactic acid inhibition of the growth of spoilage bacteria and cold tolerant pathogens on pork. B. 58:368–374.. G. R. 1970. A. 4:539. and Hazelton. G. and Tokizaki.. Gaithersburg. M. M. J. polymyxin B. Resurreccion. K. T.. Sofos... S. Association of Official Analytical Chemists... betadine. Food Prot.. M. R. V. C.. J. W. J. 62:977f. Schweitzer. K. and Schmidt. Food Prot. Comparison of methods for decontamination from beef carcass surfaces. G. 18:223–232. J. Houtsma.. Fungistatic properties of the fatty acids and possible biochemical significance. S. N. Project No. Effect of sodium lactate. T. J. Hazelton Laboratories. Guynot. 2002. C. Report of a study on the toxicity of several food preserving agents. A. W. 18:854. Hamby. Pharmacol. R. and Salverda.. W. Houtsma. J. Mizunuma... 25:141–151. M. J. Food Microbiol. E. J. Meat Sci. Heseltine. Food Chem. C. 165-126 (malic acid). Int. 1991. Kobayashi. and Krol.. and Rombouts. K. Food Microbiol. Chem. MD. A. J. Houben. Buffalo. and Yokotsuka. Dufrenne. and Schmidt. 1994a. J. Food Safety 11:269. H. . Savell. Pharm. D. R. Tokyo Med. de Wit. 104 week dietary administration — Dogs. Takeuchi. D. 1957. M. Fate of Listeria monocytogenes in raw and cooked ground beef with meat processing additives. Acuff. 1971a. and Notermans. 2000.) 2000. Heuvelink. Effect of sodium lactate on toxin production. T. L. and Marín. Food Sci. K.134 Antimicrobials in Food Greer.. G. 25:169. 17th ed. J.. Food Res. Gruber. 1993. J. 1987. Ther. Exp. 30:185–194. W. 1993. Hwang. J. Antimicrobial efficacy of a peroxyacetic/octanoic acid mixture in fresh-cut-vegetable process waters.. Two generation reproduction study — Rats X-5120 (malic acid). Sala. 94: 65. NY. Hilgren. R.. and Cross.. D. J. Official Methods of Analysis of AOAC International. 1991. J. J. Harmayani. J. 1952b. 76:39–46. Int. Pharmacol. Pharm. J.. 5:759. 1995. Shibuya. J. A. Submitted to Specialty Chemicals Division. Hayashi. Hara. C. J.. 1952a. Combined effects of weak acid preservatives. G. V. 20:247–257. C. Food Sci. A. Final report submitted to Specialty Chemicals Division. E... pH.. H. 1948. colistin and gentamicin against Pseudomonas aeruginosa. Allied Chemical Corporation. 60:604–610. K. Pharmacol. Minimum inhibitory concentration (MIC) of sodium lactate for pathogens and spoilage organisms occurring in meat products. 24 month dietary administration — Rats. Yakazu. Hedberg. Spray-chilling and carcass decontamination systems using lactic and acetic acid. S.

Environ. 2001b.. Microbiol. Efficacy of selected chemicals for killing pathogenic and spoilage microorganisms on chicken skin.. R. Hall.. Antimicrob. 1943. Banno. Janes. Food Prot.. Osmotic stress on dilution of acid injured Escherichia coli O157:H7. Food Prot. Antifungal properties of esters of alkenoic and alkynoic acids. P. S. J. K. 2001. Huhtanen. Karapinar. J. 23:509.. N. Some preliminary observations on the effectiveness of propionates as mold inhibitors on dairy products... A. J. Development of a multiple-step process for the microbial decontamination of beef trim.. H. Karapinar. and Zwietering. J. D. M. Dorsa. Huhtanen.. and G... A. C. L. 16:479. Food Microbiol. Kooshesh. and Guy. R. M. Food Prot.. 1983. H. A. Model for the combined effects of temperature.... S.. M. 1995. D.. C. GA. Application of multiple interventions for microbial decontamination of commercial beef trim. Intracellular pH and survival of Listeria monocytogenes Scott A in tryptic soy broth containing acetic. R.Organic Acids 135 Houtsma. Appl.. M. C. and Truant. E. M. Antibotulinal activity of methyl and ethyl fumarates in comminuted nitrate-free bacon. and Johnson.. L. K. J. J. C. N. Izat. M. 64:168–171. and Gönül. D. M. J. Kusters. R. Microbiol. Lett. J. I. and Siragusa. 1982.. L. 1973. vinegar. Johnson. de Wit. N. P. J. Kang. Appl. 2002. Appl. Hierholzer. 113:1184. Microbiol. Islam. Appl. J. Informatics. 1994b.. Food Microbiol. and Beuchat. ready-to-eat chicken coated with edible zein film coatings containing nisin and/or calcium propionate. D. L. W. acetic and citric acids on growth and survival of Yersinia enterocolitica. Guy. and Zwietering. Inc. Seeger. Effect of weak acids on amino acid transport by Penicillium chrysogenum: Evidence for a proton or charge gradient as the driving force.-H. W. L. R. A. J.. Colberg. Environ.. C. A. 1968. Inhibition of mold in bread by dimethyl fumarate. June 7–10. Hu. and Iijima. Atlanta. J. The use of propylene glycol and/or lactic acid in chill water for reducing salmonellae on broilers. A. J. B. Influence of fat content and preservatives on the behavior of Listeria monocytogenes in beaker sausage. 54:15–19. N. E. P. 1984. Effects of lactic acid in processing waters on the incidence of salmonellae on broilers. and Driggers. 1981. J. Gastroenterology 54:8. A. Siragusa.. Izat. Dairy Sci. D. J. Jordan. 1996. Adams.. J.-A. J. 16:343–347. Fatty acids and derivatives as antimicrobial agents. citric. Swieczkowski. 1970. . L. 58:19–23. and Milnes-McCaffrey. 1976. N. M. Gen. J. and Reiber. 1991. J.-H. J. 1996. Appl. M... L.. P. C. N. and Shelef. Thomas. Rombouts. J. K. and Gönül. 28:389–393.. J. Inhibition of Clostridium botulinum in comminuted bacon by short-chain alkynoic and alkenoic acids and esters. and Milnes-McCaffrey. P. 1999. E. lactic. M. W. H. 2001a. Lerke. Ita. E. S. C. 1992b. J.. Rombouts. coli O157:H7. Effect of acid and salt concentration in fresh-pack pickles on the growth of Clostridium botulinum spores. and hydrochloric acids. 62:1616–1622. and sodium lactate on growth rates of Listeria innocua in broth and bologna-type sausages. 14:369–374. Trenchard. Food Sci. and Unverferth.. Int... M. 1990a. Agents Chemother. Presented at the 41st Annual Meeting of the Institute of Food Technologists. Informatics Inc. D. Kabara. Conley.. M. Quoted in Monograph on Adipic Acid — NAS/NRC Questionnaire. M. R. Food Prot. S. J. P. 1990b. 67:2754–2757. 1992a. Source of the histamine released during damage to the gastric mucosa by acetic acid. C. MD. 2:23. Koohmaraie. A. K. Chen. Int. 64:63–71. Kopek. Inhibition of bacterial growth by citrate. Antifungal properties of fumaric acid esters. Modelling growth rates of Listeria innocua as a function of lactate concentration. A. L. McGinnis. J. H. Rockville. G. 49:281. Lett. Hwang. M. K. M. Koohmaraie. 1972. J. M. 13:295–306. 1985. Food Qual. 16:261–264. Huhtanen. J. M. Food Microbiol. Removal of Yersinia enterocolitica from fresh parsley by washing with acetic acid or vinegar. Jordan. L. J. Food Safety 16:175–181. and McClure. 47:1710. Kang. I. K. J. C. Sodium chloride enhances recovery and growth of acid-stressed E. Adams. Hunter. F. Ito. F. A. H. 32:312–315. H. J. P.. Food Sci. G. The toxicity of adipic acid. 48:1574. 24:113–123. Control of Listeria monocytogenes on the surface of refrigerated. and Hutkins. M. T. Houtsma. J. 32:121. Ingle. C. J. and Segel. Pres. J. pH. Food Proc.. 48:570.. Kant-Muermans. Imai. Food Prot. Bacteriol. Microbiol. and Davies. 1940. S. Food Sci.. M. Food Sci. Int. Huhtanen. Microbiol. Effects of sodium bicarbonate. R. A. N.

R. 59:513–516. C. A. E. 47:67. Glass. and Fellers.. Food Microbiol. Food Sci. Growth of. Microbial life in extreme pH values. Boca Raton. 1995a. and Hearnsberger. P. E. J. Food Sci. S. Am. G. Microbiol. J.. and Patchett. G. sodium chloride. C. J.. Levey.. 78:217–226. L. J. Microbiol. J.. Makinen. Kim. Bacteriol. In Microbial Life in Extreme Environments. Kushner. J. 63:461–467. Lett. Adams. S. G. Food Prot.. J. The inhibiting effect of acetic acid upon microorganisms in the presence of sodium chloride and sucrose. M. D. 1991.. Effect of turkey meat. 323:462. H.. J. S. A. Z. acetic acid and essential oil components.. Leyer. P. L. A. A. Microbiol. Microbiol. Smyth. Kurita. Ed.. J. Appl. 1994. K. Atkin. Leak. N. 1992. and Potter. Concerning the metabolism and tolerability of adipic acid. J. Appl. and Johnson. Maas. C. and Thelappurate. J. Effect of acetic acid on the microbiological quality of scalded picked and unpicked broiler carcasses. 1937. 39:499. R. Pharm. Antimicrobial effect of natural preservatives in a cooked and acidified chicken meat model. Appl. 1992. Spermatocyte chromosome aberrations in two species of grasshoppers at two different ionic activities. 1995b. The effect of pH.. R. Lillard.. E. 2002. Nucleus 9:119. H. R. Sci. G. L. C. R. 56:220. C. A. J. and aflatoxin production by.. Microbiol. New York. Microbiol. .. Lemay. Craven. R. Kroll. CRC Press. T. L. Luchese.. and Easter. Academic Press. 58:2075–2080. A. Acid adaptation sensitizes Salmonella typhimurium to hypochlorous acid. M. N. 57:665–670. Appl. edited by D. C. Sodium acetate and bifidobacteria increase shelf life of refrigerated catfish fillets. G. 61:3752–3755... W. A. K. Biochem. A. J. J. C.136 Antimicrobials in Food Kilic. P. P. and Bartsch. CRC Handbook of Chemistry and Physics. Acid adaptation promotes survival of Salmonella spp. Food Sci. and Harrigan. 279. Nucleus 1:131. D.. H. A. W. A. Brimi. G. Levine. Assessment of sodium lactate addition to fresh pork sausage. J. White. 1946. J. Orten. 2002. A. J. Int. F. and Johnson. B. Y. Appl. Lide. Induced acid tolerance in Listeria monocytogenes.. Cereal Chem. and konjac on residual nitrite in cured meats. Food Prot. FL. Levine. C. A. R.. and Marshall. Bacteriol. Lett. 1987. O. and Frey. L. and Smith. Dickens.. Environ. L. J. and Johnson.. Lasichak. and West. and Schackelford. R. The effect of acetic acid and hydrogen peroxide on mitotic frequency in Allium cepa.. C. 55:2226. 1966. and Mukherjee. Vickery. 14:865... L. J. 1992. Lamkey. Langworthy. and Doyle. Lang. N. J. Environ. Kotula. J. Kim. Little. p. J. L. A study to determine the toxicity of fumaric acid. 1995. R. Environ. Choquette. P. D. Hearnsberger. N. G. Gariépy. S... M. B.. Cassens. Extending shelf life of refrigerated catfish fillets using sodium acetate and monopotassium phosphate... 69:512. L. Chem. 2002. Sodium lactate delays toxin production by Clostridium botulinum in cook-in-bag turkey products. Johnson. L. Kirby. R. K. R. Food Prot. acidulant and temperature on the survival of Yersinia enterocolitica. Blankenship.. 1953. J. H. O. 1939. 39:17. and Koike. L. S. C. Kim. 14:148–152. R. C. W. White. H. 58:644–647. 1994. R. Microbiological and sensory attributes of retail cuts of beef treated with acetic and lactic acid solutions. W. W. O. Wang. Effect of organic acids on thermal inactivation of Bacillus stearothermophilus and Bacillus coagulans spores in frankfurter emulsion slurry. Manna. C. Vickery. J. F. sodium lactate. 1958. Food Prot. D. Food Sci. J.. D. M. 35:298. Acid adaptation of Escherichia coli O157:H7 increases survival in acidic foods. S. 83rd ed. Environ.. R. K. and Marshall.. Action of acetic acid on food spoilage microorganisms.. and Saucier. C. 1978. 60:25–27. Biol. Synergistic antimicrobial effect of ethanol. M. K. phosphate. 14:224–227. Agric. 1990. 1988. E. M. Lynch. G. D. 1989. Hearnsberger.. Gram negative bacteria inhibition by lactic acid culture and food preservatives on catfish fillets during refrigerated storage. 50:112. L. Further studies on the growth of bread molds as influenced by acidity. J. N. R. A.. Leyer. Appl. 1983. Rodrigue. Leyer. D. Appl. 51:475–480. J. and Borchert. S. carrageenan. Tuley. Assoc. 1997. Bacteriol. 1940. and Fellers. Delaquis. J. Aspergillus parasiticus when in the presence of either Lactococcus lactis or lactic acid and at different initial pH values. in cheese. 67:29–31.

and Ramos. F... R. 2002a. Thermal inactivation of stationary-phase and acid-adapted Escherichia coli O157:H7. McMahon. R. A. Appl. 2001. M. M. Inhibitory action of citric acid on tomato juice flat-sour organisms.. T. L. H. Physical Characterization and Nutrient Analysis. Effect of glyceryl monolaurate by Clostridium botulinum in meat slurry. 1. sodium acetate. Dufrenne. Influence of organic acids. R. Kraft. J. W. Food Safety 3:83. 56:958–962... Performance evaluation of a model describing the effects of temperature. Microbiol. Mazzotta. Guynot. Cantarelli. 64:640–644. H. W. Arbonés. McDowell. A.. and Walker. I. and physical changes in fresh. G. J. 79:203–211. J. J. and Keybets. phosphates and sodium chloride alone or in combination on shelf life of vacuum-packaged pork chops. E. Food Prot. Gallo. New York. J. Survival of Escherichia coli O157:H7 on vacuumpackaged raw beef treated with polylactic acid. and citric acid on the inactivation of Salmonella typhimurium in reduced calorie mayonnaise. C.. Moye. J. H. Food Prot. McMeekin.Organic Acids 137 Marín.. Comparison of organic acid salts for Clostridium botulinum control in an uncured turkey product. 1968. A.. 28:340–344. L. A. Production of gastric lesions in the rat by the diet containing fatty acids. A. 54:18. Microbiol. K. chemical. D.. Sodium lactate affects pathogens in cooked beef. J. and Tiba. Vol. Mont. Effects of potassium sorbate. M.. 1940. M. J. and Dufrenne. A. pH. J. F. vacuum-packaged pork treated with organic acids and salt. Sanchis. 1965. 1994. H. M. J. Lett. J. J. K. J. 60:1497–1501. and Listeria monocytogenes in fruit juices. 59:15–19. J. S. A. Mustapha. pH and lactic acid concentration on the growth of Escherichia coli. 33:516. 64:315–320. and nisin. S. Int. R.. A. Gann 44:421... Mountney..). Notermans. J. Molins. Effect of fatty acids on Shigella. J. 1950. A. G. R. Bernadó. L.. The production of gastric lesions in the rat by acetic acid feeding. Poultry Sci. Molins. J. Effects of temperature. . and sodium chloride upon strains of food poisoning staphylococci. Aspergillus niger. Food Sci. Food Sci. and Clarke. D. M. L. Appl. Mbandi. J.. T. (editor). lactic acid. 1989b. 43:245. Environ. U. and Ross. C. and Jolly. Am. 56:2120.. Mellefont. J. 1970. 44:582. 48:851. P. M. Archivos Latino Americanos de Nutricion 47:62–65. S. Inc. Call. 1991. Enhanced antimicrobial effects of combination of lactate and diacetate on Listeria monocytogenes and Salmonella spp. 1990. 2002b. Nunheimer.. Salmonella. 15:107. V. L. Doherty. S. 1996. R. sugars.. Food Prot.. Synergistic effect of heat and sodium lactate on the thermal resistance of Yersinia enterocolitica and Listeria monocytogenes in minced beef. in beef bologna. J. L.. 2002. J. Environ.. and Hassan. A. C. 67:2271–2277. 1952. Blair. Food Res. Int. Growth of Staphylococcus aureus in acidified pasteurized milk. Gann 43:443. C. 1993. Influence of pH on inhibitory activity of acetic acid on osmophilic yeasts used in brine fermentation of soy sauce. F. N. Acid tolerance of Leuconostoc mesenteroides and Lactobacillus plantarum.. Food Australia 43:246–249. 67:262–266.. and Whiting. 82:45–58. A. Miller. J. Guynot. Mori.. E.. Microbiological. A. A. Miller. E. Aspergillus flavus. Nollet. Murdock. 1997. Effect of calcium addition and acidification on the quality characteristics of canned okra (Hibiscus esculentus L. Handbook of Food Analysis. T. Sheridan. Moreno. Sanchis.. I. F. Hayashi. V. J. A. and Walker. Proc.. C. Food Microbiol. Acids as poultry meat preservatives. A. A. E. Notermans. 2003. H. K. Enhanced inhibition of Listeria monocytogenes and Salmonella enteritidis in meat by combinations of sodium lactate and diacetate. and Chambers. A. A. 76:191–198. and Ramos. Membre.. T. Mendonca. Marcel Dekker.. J. C.. M. W.. Sci. and Shelef. E. S. Marín. M. 1999. A. Matsuura. 1981. Acad. An innovative technology for salmonella control and shelf life extension. Nakamura. M. P. A. Food Microbiol. A. Neira. M. J. 2001. and Shelef. Mbandi. Mori. 1985.. Appl. McDonald. 1997. Ariyapitipun. J. Milk Food Technol.. M. A. T. and Penicillium corylophilum spoilage prevention of bakery products by means of weak-acid preservatives. A. Food Sci. Food Prot. and Acuff. Use of preservatives to delay toxin formation by Clostridium botulinum (type B strain okra) in vacuum-packed. Risk assessment of the use of sub-optimal levels of weak-acid preservatives in the control of mould growth on bakery products.. I.. Microbiol. A. 1989a. Food Microbiol. E. and Mizunuma. S. D.. and O’Malley. K. 2002. J. and Hegarty. Fleming. H. Food Prot. 54:302. Majchrzak.. Food Sci. 1982. Int. M. and Marth. R. and Fabian. J. D. waster activity. V. J. Noda. Kraft. J. J. I. F. cooked potatoes. Mendonca. Nogueira. Public Health 30:1040. T. 1953. Minor. J. glucose. P. and Zangar. T. Food Sci. 28:51. E.

. R. Papadopoulos. The effect of fumaric acid on malolactic fermentation in wines from warm areas.. and Harrisson. J.. J.. Miller. and Hill. Med. L.. 1986. R. Am. Food Sci. D. W. 1996.. 1990b. Pharmacological effects of fatty acids. Propionic acid and its calcium and sodium salts as inhibitors of mold growth. Cereal Chem. O’Keane.. and Olson. V.. Orö. Olson. H. Lucia. 1961. S. and McPhee. Two fatal cases of poisoning by acetic acid. 1:687 Papadopoulos.. Appl. Okereke. C. O’Driscoll.. R. Okereke. Acuff. Papadopoulos. Vanderzant. Lebensm. pH and organic acids. Environ. H. Bacteriol. Beelman. E. S. D. L. Effect of sodium lactate on microbial and chemical composition of cooked beef during storage. B. and Pfeiffer. and Doores. 28: 701. Ough... and Kunkee.. H. J. J. J. Med. J. 1945. N. S. M. and Wretland. Propionic acid. Toxicol. C.. H. B. and Bock.. Development of B. Cahill.. Owen. S. Salmonella typhimurium and Campylobacter jejuni in poultry scald water. C. C. Fernandez. J. A. L. R. W. 1:255–262. Food Sci. E. J. Food Sci. J. Rodrigo. R. A.138 Antimicrobials in Food Ocio. and Martínez. D. Shin. S... Food Microbiol. V. G. Palmer. Dairy Sci.-K. C. 56:621. G. A. Inhibition of thermally-stressed Bacillus spores by combinations of nisin.. Borton. H.. W. Experientia 27:146. and Leistner. L. 25:188. Dresel. M. J. C. 55:1327. P. Toxicol. R.. Palejwala. A.. 1984. R. D. 1990. M. Adaptive acid tolerance response in Listeria monocytogenes: Isolation of an acid-tolerant mutant which demonstrates increased virulence. M. Control of spoilage of canned mushrooms inoculated with Clostridium sporogenes PA 3679 spores by acid-blanching and EDTA. K. L.. Milk Food Technol. Injury to health caused by acetic acid in the manufacture of acetylcellulose.. B. 1971.. Lav. Pharmacol. J.. Dtsch. Milk Food Technol. J. 56:1141. S. V. J. 1968. Food Sci.. and Goldberg. 56:341. Enol. Paar. mesentericus in bread and control with calcium acid phosphate or calcium propionate. Okabe. Sodium lactate effect on sensory characteristics. Elucidation of the mechanism of the acidblanch and EDTA process inhibition of Clostridium sporogenes PA 36779 spores. Vanderzant. A. 37:203. Fleischwirtschaft 64:828. A. J. L... J. A. 18:730. A. Roth. D. A. and Sassi. A. A.. Food Microbiol. 1954.. Some factors responsible for the spoilage of mangoes by Penicillium cyclopium. Thermal resistance characteristics of PA 3679 in the temperature range of 110–121°C as affected by pH. 1932. Int. C. K. Werner. and Cross. E.. Doores. H. C. Gahan. 1991c. Comparative subacute toxicity for rabbits of citric. G. triolein and cottonseed oil.. M. and Moran. sodium propionate and calcium propionate as inhibitors of mold growth. Ockerman.. and Kralovec. R. A. J.. J. Abbott. Helmsoth. 22:239–247. N. H. C. Acid-blanching and EDTA effects on yield. F. B. R. V. Acuff. Packman. and Beelman. R. and Modi. Consumer and trained sensory comparisons of cooked beef top rounds treated with sodium lactate. Dairy Sci. J. J. K. fumaric and tartaric acids. C. Marth. 1963. R. B. R. R. Observations on the use of propionate-treated parchment in inhibiting mold growth on the surface of butter. O’Leary. Johnston. Ringer. K. Oscroft. T. C. Banks. H. 1984. Haemostatic failure due to consumption of coagulation factors in acute acetic acid poisoning. U. 62:1693–1698. J. E. S. Acta Pharmacol. R. R. 23:538–544. 55:1331. 1974. 18:141. Jr. J.. H. Food Prot. Acetic acid inhibition of gram negative bacilli in culture media. cooked meat color and chemical composition. Technol.. Miller. Osthold. 5:163. Parrett. S. type of acidulant and substrate. Sánchez.. J. R.. 1990. Med. J. W. Park. and Cross. S. 1946. 1991b. S. Smith. D. and Walsh. Beelman. I. 45:319. Okrend. 1990a. Improving the storage life of carcasses by treating their surfaces with an acid spray. Survival of Salmonella typhimurium in cold-pack cheese food during refrigerated storage. 1974. and Macy. G. Parmeggiani.. Olson. 1991a. A. D. 55:1137. V. P. H. L. W.. Vitic.. 33:383. 49:500. Microbiol. L. Miller. 1971... 52:353. and Macy. Appl. L. Effect of acetic acid on the death rates at 52°C of Salmonella newport. A. Differential healing periods of the acetic acid ulcer model in rats and cats. J. R. Monatsschr. 1970. Sattur. 13:421. K. S.. and Hoffman. 1941. J. Aust. quality and microbiological stability of canned mushrooms. and Cross. Food Sci. Lancet 2:795. Wiss. A. Okereke. Food Sci. Gastric erosions caused by home-brewed lager. ... 23:509. 1940. Use of acetic and lactic acid to control the quantity of microorganisms on lamb carcasses. R. J. 1994.

Microbiol. The influence of biopreservatives on the bacterial level of refrigerated vacuum packaged beef. 1935. D. and Board. 2003. Inhibition of Clostridium botulinum type E in model acidified food systems. Appl. M. Food Prot. 59:1037–1040.. J. Microbiological effects of acid decontamination of pork carcasses at various locations in processing. Post. Suresh. F. V. S.. B. W. 1998. Microbiological effects of acid decontamination of beef carcasses at various locations in processing. S. Tartaric. and water activity. p. Rhinol. K. Quartey-Papafio. C. Effects of acids on potassium sorbate inhibition of food-related microorganisms in culture media. M. Call. Field. L. Short-chain fatty acids as sanitizers for beef. E.. Robach. Peterson. S... 34:81.. water activity. W.. D.. Podolak. 1975. B. R. Meat Sci. M. Food Prot. 61:1358–1362. Presser. Acuff. 1978. M... Ross. E. and McMeekin. sodium citrate and sodium lactate. and Solberg. C. E. 19:124. Holland. Y. Carr. 1985. Y. Sant’anna. 1994. D.0 or 3. The nephropathic action of the dicarboxylic acids and their derivatives. Inhibition of Listeria monocytogenes in cold-process (smoked) salmon by sodium lactate. 65:308–315.. A.. Food Safety 13:99–111. Rubin. J. 10:269–278. J. Portner. II. S. R. Microbiol.. and Morgan. K. M.. J. A. Acuff. J.. 64:1773–1779. L. Marshall.. 32:413–423. Porto. C. J. Thermal death rate of ascospores of Neosartorya fischeri ATCC 200957 in the presence of organic acids and preservatives in fruit juices. 1991. The effect of citric acid. J. S. Effect of acids and sorbate combinations on the growth of four osmophilic yeasts. K. J. and Anderson. R. Food Sci. Academic Press. K. Ratkowsky. J. T. Modelling the effects of temperature. Piva. W. Lenovich. Modelling the growth limits (growth/no growth interface) of Escherichia coli as a function of temperature. 29:424–428. J. C. 43:168. Sporicidal effect of peracetic acid vapor. pH and lactic acid concentration on the growth of Escherichia coli.. on the growth of Arcobacter butzleri. Savell. M. Food Sci. and Savell. S. Food Microbiol. A. and Fung. 1993. S. Inhibition by citrate of the growth of coagulase-positive staphylococci. 1999. Microbiol. E. 84:1123. 54:868–872. Lactic acid stricture of esophagus. Podolak. 121. Rajashekhara. 1996a. D. 1996b.. J. J. C. 1993. A. C. 44:842. Pitkin. C. H.. S. L.. 82:33–43. Ther. J. A. K. T. Rozbeh. L. G. Food Prot. 1982. Kastner. 1981. L. Ann. Food Res. R. during extended storage at 4 and 10°C. J. T. D. Prasai. J. Food Prot. I. K. . D. Prasai. R. L. Acidic constituents of tomato juice and specific organic acids. Food Prot. Phillips.. R. 59:370–373. Cutting. 1980. C. C. Environ. R. J.. P. 1968. B. M. Kalchayanand. 47:134. malic and succinic acids. M. G. Rose. and Hoffman. G. K. M. Kastner. G. G. N. Int. 1924. Pharmacol. R. J. Appl. Rice. Zayas. L. 16:1782. London. L. Reiss. G. R.. and Ratkowsky. 1976. J. Inhibition of Listeria monocytogenes and Escherichia coli O157:H7 on beef by application of organic acids.Organic Acids 139 Pelroy.. D. 1980. and Pederson. A. Prevention of the formation of mycotoxins in whole wheat bread by citric and lactic acid. 24:123... and Luchansky. Mellefont. 1962. Food Microbiol. Toxicological model for a two-acid system.. L. T. C. M. G. Bacteriol. G. A. Viability of a five-strain mixture of Listeria monocytogenes in vacuum-sealed packages of frankfurters.. commercially prepared with and without 2. and Ray. 57:108–113. Pilone. R. Food Technol. In Lactic Acid Bacteria in Beverages and Food. J. C. lactic acid concentration. 2002. Rammell. 36:623. lactic acid. K. and Bills.. and Syracuse. Appl. L... Johnson. A. J. C. G. Hale. Appl. Exp.0% added potassium lactate. Fate of pathogens in home-made mayonnaise and related products.. pH. Use of preservatives to control microorganisms in food. Laryngol. Food Prot. Komatsu. E. 45:1138.. A. M.. A. M. E. and G. E.. Factors influencing growth of Bacillus coagulans in canned tomato juice. J. and Ethiraj. 50:966. Food Prot. Environ. and Eklund. and Fung. Restaino. C. 1998.. Whiting. C.. A. W.. E. J. J. B. A. Experientia 32:168. Ross. T. J. J. E.. May. A. J. 1954. Morgan. Microbiol. Food Prot. Lett.. edited by J. Lucia. Radford. Lucia. B. alone and in combination with nisin. M.. 1992. Franco. Zayas. R. Reduction of bacterial populations on vacuum-packaged ground beef patties with fumaric and lactic acids. Amoroso. Otol. Control of malo-lactic fermentation in table wines by addition of fumaric acid. F... S. C. Restaino..

Glass. Inhibition of Listeria monocytogenes. A. The in vitro effect of citric acid and sodium citrate on Streptococcus agalactiae in milk. J. L. and Yang. Smulders. Dis. Antimicrobial effects of lactates: A review. and beef. and Al-Amiri. 1995. C. E. Substitution of vinegar for lactic acid as bactericidal agent in infant milk mixtures. and Woolthuis. Food Sci. N. R. Inhibition of Listeria monocytogenes on beef tissue by application of organic acids immobilized in a calcium alginate gel. 1992. S. 46:1032.. J. J. A. Shelef. J. G. Inhibition of Listeria monocytogenes and other bacteria by sodium diacetate. C. Evaluation of sodium diacetate and ALTA™ 2341 on viability of Listeria monocytogenes in turkey slurries. H. J. Lopez.. Food Microbiol. U. and Palumbo. and Freese. 60:611. C. 51:615. J.. Food Safety 13:147–158. Benedict. Degnan. 45:185–193. and Conner. Al-Otaibi. and Luchansky. S. R. F. pH dependent acetate injury in Staphylococcus aureus: Role of temperature. Shelef. J. Smith. 1993a. J. 1973. Child 88:62. S. C. and Welsh.. 1972. Nuñez Kalasic. K. 1972. A. 1984.. Food Prot. J. R. Lebensm. Ross. W. McMeekin. C. Agents Chemother. D. J. H. Konings. Growth suppression of Listeria monocytogenes by lactates in broth. Taylor. C. Int. A. and Rodrigo Enguidanos. 1954. A. J. J. J. and S. J.. L.. A. Int. W. 12:221–227. T. 1988. 32:99–102. Bacteriol..-H. Am. C. Simmons. A. H. Buchanan. C. J. Influence of acid tolerance responses on survival. G. B. Kalinowski... Food Prot. or pediocin on the viability of Listeria monocytogenes in turkey slurries. 1983. J. 44:149–169. Inhibitory effects of lipophilic acids and related compounds on bacteria and mammalian cells. 1995. Degnan. 45:805.. Salmonella typhimurium and Escherichia coli O157:H7 on beef muscle tissue by lactic or acetic acid contained in calcium alginate gels. 1993b. A. A. Wiss.. J. 111:525. Food Prot. J. and Freese. L. E. Food Sci. A.. and Freese. A. Food Safety 14:103–115. Storage stability of chicken as affected by Map and lactic acid treatment. G. Salomon. J. S. Heat processing effects on physical and chemical characteristics of acidified canned tomatoes. Antimicrob. L. Smulders.. 57:293–296. S. J. and Cooler.. 1992. Food Prot. Food Sci. W. R.. R. K. H. J. Siragusa. J. Food Microbiol. 1994. G. L.. R. Shadbolt.. Q. Food Microbiol. and Dickson. pH and acidic stability during storage of acidified and nonacidified canned tomatoes. D. Seymour. and Woolthuis. Sawaya. Sreevalsan. Sheu. growth. E. 1975. G. The effects of diacetate with nitrite. J. Inhibition of aerobic and anaerobic growth of Staphylococcus aureus in a model sausage system. L.. S. G. J. Effects of acetate and other short-chain fatty acids on sugar and amino acid uptake of Bacillus subtilis. Indian Vet. J. Int.. 1974. Ryu. 1998. Schlyter. Food Microbiol. Lett. Shelef.. Sinha. J.. Differentiation of the effects of lethal pH and water activity: Food safety implications. E. H. M. N. Loeffelholz. R. 17:271–275. Appl. 1985. Schoenemann. W. and Greer.. J. and Beuchat. 54:283–287 Sheu. Survival of Listeria monocytogenes in cold-pack cheese food during refrigerated storage. and Lopez. W. A. 39:257. H. J. Food Microbiol. 1994. H. 2001. 1968. F. Immediate and delayed microbiological effects of lactic acid decontamination of calf carcasses — Influence on conventionally boned versus hot-boned and vacuumpackaged cuts. 38:195. 1998. Smulders. J. chicken.. L. and Potluri. F. The effect of pH on the thermal resistance of Clostridium sporogenes (PA 3679) in asparagus purée acidified with citric acid and glucono-δ-lactone. A. H. P. T. M.. 48:838.. 1980. E. Food Safety 2:221. Glass. Food Prot. D. 111:516. L. and Luchansky. and Addala. J. Integrating microbial decontamination with organic acids in HACCP programmes for muscle foods: Prospects and controversies. Elnawawy.. J. Silla-Santos. Effects of fatty acids on growth and envelope proteins of Bacillus subtilis. Al-Omirah. J. J. J.. 1993. 1991. 19:271–281. Int. R. Microbiol. Bacteriol. T. Palumbo. B. F. L. 16:275–281.. Food Prot. H. Food Sci. Sheu. Behaviour of foodborne pathogens in cooked sausage containing lactates. Influence of two levels of hygiene in the microbiological condition of veal as a product of two slaughtering/processing sequences. Shelef. M. Technol. C. Schlyter. J. A. 57:445–450. T. 56:808–810. L.. 7:349. Drury. A. and Marth. F. J. H. . and thermal cross-protection of Escherichia coli O157:H7 in acidified media and fruit juices. and Dickson. J. Al-Zenki.140 Antimicrobials in Food Ryser. V. Casado Goti. R. lactate. M. Siragusa. Loeffelholz. J. Schoenemann. W. J. J... J. D. A. Smith...

. J. Visser. Dis. W. 1945. 1997. Growth and toxin production by Clostridium botulinum in model acidified systems. A. Effect of common food preservatives on mycelial growth and spore germination of Fusarium oxysporum. Food Sci. J. Food Sci. Exp. J. A. A. Milk Food Technol. and Lagozzino. M. Poult. F. 1971. R. J. 6:31. Influence of pH and temperature on growth of Bacillus cereus in vegetable substrates. C. M. J. acidulant. 64:1661–1666. H... 1989. Effect of pH. N. J. P. K. Hort. 2001. Microbiological safety of cooked beef roasts treated with lactate monolaurin or gluconate. Sci. C.. J. and Mossel. C. Esophageal stenosis due to lactic acid. K.. M. J. S. T.. Tuft. Int. sodium chloride. J. A. D. 1991. H. W. E. Postharvest physiology of strawberry fruits treated with sodium dehydroacetate. van der Marel. and McCollister. J. Studies of chronic intoxication on albino rats. and Hatfield. Ther. and Molins. J.. R. 58:953.. 66:197–203. and Conner. A. Dehydro-acetic acid I. 1985. M..Organic Acids 141 Sollman. 99:57. L. 2003. L. J. 1997. Int. D.. Soc. Tzatzarakis. A. A. W. Sorrells. Am. J. and Johnston. 1969.. Bacteriological quality of broiler carcasses as affected by in-plant lactic acid decontamination. 1994. J. and Huis In’t Veld. E. Liakou. N. E. H. P. Vol. Effect of chlorine. Van Netten.. Acid equilibrium development in mushrooms. 1988. and temperature on the growth and survival of Listeria monocytogenes. H. R. Hygienic standards for acetic acid and acetic anhydride in air. and Walker. and washing on Salmonella typhimurium on eviscerated fryer chickens.. B. Am. 16:463. and Salmerón. Valero. H... Watada. Fernández. E. A. D. 56:198. Philadelphia. 60:625–628. J. 1967. Canker sores from allergy to weak organic acid (citric and acetic). Sci. R. Food Prot. M. and Kant-Muermans. 1988. Acetic and formic acids. J. S. Subramanian.. Environ. W. 1.. Tsang. 1921. K. Tinney. Reduction of microorganisms on beef surfaces with electricity and acetic acid. A. J. 82:71–79. Prabhu. M. Pharmacol. Effect of pH... Valero. Sci. 51:208. J. antibiotics. S. J. Spencer. J. Uyttendaele. Spector. Food Prot. C. Tamblyn. Acute and chronic toxicity. Post. D. C.. Leontidis.. B. C. Pierard. T.. Sebranek. and Vakalounakis. W. C. P. . G. Tutenel. Takhirov. Effect of acid resistance of Escherichia coli O157:H7 on efficacy of buffered lactic acid to decontaminate chilled beef tissue and effect of modified atmosphere packaging on survival of Escherichia coli O157:H7 on red meat. Molins. and Solberg. L. 48:590. Martínez. Food Microbiol.. van Logtestijn.. Food Prot. and Bijker. 2000. Miller. S. M. P. W. W. 31:323. Thomson. F. 50:961. J. A.. H. and Enigl. J. Exp. Tsatsakis. 17:605–612. A. Food Microbiol. S. Multiplication of Salmonella typhimurium in skim milk with and without added hydrochloric. 1985.. 1950. G. J.. Food Safety 14:243–257. D. Jr. acidulant. P. Koolmees. M. 34:122. B. J.. Uradzinski. K. T. 46:146. M. S. Food Safety 11:31... and Salmerón. Child 69:173. Microbiological conditions and keeping quality of veal tongues as affected by lactic acid decontamination and vacuum packaging. D. and temperature on the growth of Listeria monocytogenes. and Ettelson. Food Microbiol. H. Sanders. Ther.. Allergy 27:536. Food Prot. G. Pharmacol. I. M. J. D.. 2000. K. V. Saunders. Food Prot.. Sanit. Stekelenburg. Thompson. and Marth.. G. 2001. Jozwik. M. Health B35:527–537. K. Food Microbiol. J. Enigl. Hyg. Banwart. J.. Growth of Bacillus cereus in natural and acidified substrates over the temperature range 5–30°C. D. Krippaehne. Effects of sodium lactate and other additives in a cooked ham product on sensory quality and development of a strain of Lactobacillus curvatus and Listeria monocytogenes. L.. A. A. Food Sci. and Mercuri. R.. Unda. Ramsey.. J. R. Hunter. Bactericidal activity of organic acids against Salmonella typhimurium attached to broiler chicken skin. M. Stillmunkes. and Carr. 1968. J. A. J.. G. J. The effect of lactic acid decontamination on the microflora on meat. D. W. M. G. C. Food Prot. 96(2):177–179. A. Debevere. Int.. β-propiolactone. time. M.. M. 1993. 52:571. Stroup. J. Clostridium sporogenes and Listeria monocytogenes: Survival and inhibition in microwave-ready beef roasts containing selected antimicrobials. E. L. L. 1956. 1990.. S. M. III. D. Rowe. Handbook of Toxicology. C. pearl onions and cherry peppers. Fernández. D.. De Zutter. 60:629–633. Sorrells. Dickerson. A. Trainer. lactic and citric acids. 1956. J. R. acids.

Allergy 27:50. 1945. 4:294.. and Debevere. P. Assoc. and Smith. and Beuchat. K. R. 30:35. Food Ind. G. R. A. Reversal of sodium propionate inhibition of Escherichia coli with βalanine. Weissinger. survival and growth of Listeria monocytogenes on poultry as influenced by buffered lactic acid treatment and modified atmosphere packaging. Food Agric.... M. Poult. Microbial decontamination of porcine liver with lactic acid and hot water. R. and Skeggs. 1950. Nakaguchi. G. A. J. and Debevere. J. J. 1962. Food Microbiol. 1984. L. Health Sci. Van Logtestijn. A. potassium or calcium lactate in pork liver sausage. and Rogers. Y. Comparison of aqueous chemical treatments to eliminate Salmonella on alfalfa seeds..-Y. T. W. M. J. T. K.-W.. A. 63:1475–1482. Appl.. J. G. and Watabe.. Med. 28:219. L. 1946. and Shelef. J. A. H. Environ. H. H. 2001. Antimicrobial effect of chemical preservatives on enterohemorrhagic Escherichia coli O157:H7. J. Food Prot. H. and Edmondson. A. Allergic reactivity to simple alipathic acids in man. Acetic acid sensitivity as a cause of cold urticaria. Zeitoun. L. Yokotani. Arch. S. A. Mossel. Int.. Zhang. 1996. D. Murai. Weaver. J. Dermatol. J. M. 46(3):204–208. K. The effect of treatment with buffered lactic acid in microbial decontamination and on shelf life of poultry. H. D. R. Sci. J.. Y. Huang. R. E. 1951. R. Antilisterial activity of sodium. 17:227. M. H. feathering. J. The effects of various disinfectants against Listeria monocytogenes on freshcut vegetables. and Farber. 48:832.. F. 54:2179. Food Microbiol. 1990. Woolthuis. 1994. C. C. Lab. Food Agric. 17:622. J. T. Effects of lactic acid bacteria and organic acids on growth and germination of Bacillus cereus. K. J. A. P.. Am. J. Wolf. 1975a. and Anderson. R. Weissinger. 1991. and Smulders. M. Food Sci. Microbial decontamination of calf carcasses by lactic acid sprays. Yamamura. . Microbiol. 1975b. J. A. Woolford. D. Sci. Biochem. S. DeKruiff.. Invest. M. Microbiol. and Aramaki. G. Zeitoun. M. J. Quality changes during refrigerated storage of packaged shrimp and catfish fillets treated with sodium acetate. 2000. Dehydroacetic acid: A new microbiological inhibitor.-C. and Scott. Sci. A. Zhuang. Wederquist. 1985. Int.. Appl. Microbiological screening of the straight chain fatty acids. Wolford. Usui. and Schmidt. Tanda. 1944. Weil. Woolthuis. Sofos. R. J. R. M. 64:442–450. 1971. Food Safety 13:133–146. J. Microbiological screening of food preservatives. and Smulders. J. Young. Xiong. 47:220. Listeria monocytogenes inhibition in refrigerated vacuum packaged turkey bologna by chemical additives. L. H. H. and Beuchat. 11:305. M. A.142 Antimicrobials in Food Waterhouse. Acute and subacute toxicological studies of TAKEDA-citric acid in mice and rats. 14:161–170.. D. Wright. H. 1956. 59:498–500. A.. F. Food Microbiol. Xie. Wiseman. J. and Adler.-L. J. M. Wong. and Chen. (Cl–Cl2) as potential silage additives.. 125:1179. J. J. Propionates control microbial growth in fruits and vegetables. Y. 10:383. Food Prot. 26:229. E.. M. 1988. sodium lactate or propyl gallate. A. 1993. Woolford. and Beuchat. 28:36–40. J. Lactic acid: A corrosive poison. rate of growth and acetates on chick’s need for glycine. 2000. H. H. A. Takamatsu. Food Prot. N. R. R. 61:241. Takeda Res. Food Sci. A. cold sterilants and specific antimicrobial agents as potential silage additives. E. Evaluation of volatile chemical treatments for lethality to Salmonella on alfalfa seeds and sprouts. J. 1996. 1999. The fate of Salmonella enteritidis PT4 in home-made mayonnaise prepared with citric acid. R. R. M. Lett. K. Kanabayashi. McWatters. W. Inhibition. J. N. J.. 13:311–321. Food Prot. 41: 1957. Effect of sex. J. Food Technol. L.

...... According to Schroeter (1966)............................................................. It is fairly soluble in water.................................................155 Toxicology... Sulfur burns in the presence of air to give sulfur dioxide (McAlpine and Soule................................................................. 1933)...5 CONTENTS Sulfur Dioxide and Sulfites Cornelius S..................................................... with some molecules associated with water................................................................................................... occurs in the free state in some regions of the world.155 Amounts in Foods and Regulations.......................143 Form and Solubility ....................................... 2000)......... the monohydrate H2SO3 does not exist. Other uses for sulfites are as an antioxidant...... sulfur dioxide (SO2) has been used as an antiseptic.............................................................................................. on fruit................... Sulfur dioxide gas can be easily liquefied by compression................ SOURCE Sulfur............................................25 atm)........................................151 Molds..................... it is used in fermentations...................................................................................................... Sulfur oxide is especially common to the volcanic areas................ 1996............160 INTRODUCTION It was quoted by Hammond and Carr (1976) that the poet Homer burned sulfur to disinfect his home in Greece around the 8th century B..................................... and in other industries to prevent microbial activity or growth....... dissolved in water...................... or as a gas............................. From that time and perhaps even before................................................ Herraiz and Cabezudo (1989)............145 Antimicrobial Activity ................... existing mainly as sulfur dioxide molecules....................................................................................................................144 Reactivity. and to prevent black spot on crustaceans (Papazian..... which is a colorless gas with an extremely suffocating odor......157 Analytical Methods ... Ough and Lilian Were Introduction .................. sometimes called brimstone........................ In its various forms as salts.................................................................................................................. as a dough conditioner.......................C.................................................................................................143 Source............................... and Bakalinsky (1992).............158 References ................................148 Yeast ......................154 Other Uses...... disinfectant.............................. At 20°C it has a vapor pressure of 329 kPa (3.... Ough and Crowell (1987)..................... It is especially important in the production of wine. 143 .........................148 Bacteria ....... or sanitizer........................................ Sulfites and sulfates are also found in nature............ to inhibit enzymatic browning inhibitor and the Maillard reaction... Gould........... General reviews of the use of sulfites in wine and their effect on yeasts include Schopfer and Aerny (1985)...............................................................

1 Distribution of the ionized forms of sulfurous acid at various pHs. indicating a rather weak dibasic acid (Segal. and solubilities of each in water. heating pyrites. The dry salts are easier to store and are less of a problem to handle than gaseous or liquid sulfur dioxide. The pKa values for sulfur dioxide are 1. The metabisulfite is the anhydride of the acid sulfite: – → 2– 2 HSO3 ← S2O5 + H2O When these salts are exposed to air. 1928). Table 5. Most of these salts are hygroscopic and easily hydrolyzed (Schroeter. Specific gravity for various water solutions of sulfur dioxide is shown in Table 5. It is useful to have the sulfur dioxide in a salt form. FORM AND SOLUBILITY In water solutions. 1928. as well as by other methods.1. and from reducing gypsum. The anhydrous sodium and potassium salts are prepared by precipitation and then dehydrated with the appropriate hydroxide.76 and 7. Phillips. their theoretical yields. sulfur dioxide can be written to show the equilibrium: → SO2 + H2 ← [H2SO3] – → [H2SO3] ← HSO3 + H+ – → HSO3 ← SO2– + H+ 3 The bracketed form indicates the sulfur dioxide associated with water.1. 1968). 1966). .144 Antimicrobials in Food Sulfur dioxide can be prepared commercially from burning sulfur (oxidation). they show increasing stability in the order sulfite > bisulfite > metabisulfite (Mason. A plot of the distribution of the three ionic forms can be calculated and is shown in Figure 5.20. 100 SO2 80 % of Total H2SO3 − HSO3 SO− 3 − 60 40 20 0 0 1 2 3 4 pH 5 6 7 8 FIGURE 5.2 lists the main forms of sulfur dioxide.

If ascorbic acid is being used in combination with sulfur dioxide.0001°F–1 TABLE 5.037 — Corrections are approximately 0.at pH 3.8 25.1 Specific Gravity of Various Sulfur Dioxide Water Solutions at Two Temperatures SO2 (g per 100 g) 1 2 3 6 8 10 a Specific Gravitya 15.0292 1.008 1. 1970).4 H2O Solubility (g/L) 110 (20°C) 250 (20°C) 280 (40°C) 240 (25°C) 1000 (20°C) 3000 (20°C) 250 (0°C) 540 (20°C) REACTIVITY Wedzicha (1984) briefly reviewed the chemical interactions of sulfur dioxide.6 67. the second equation listed previously is a key reaction (Heinmann et al.018 1.4 s-1 for H2O2 with SO32. Holt and Kumar (1986) found an observed k = 41..0091 1.003 1.0040 1.0191 1.028 1.40 and 15°C.6 57.5 61..0493 20°C (68°F) 1. much slower changes occurred in the sulfur dioxide content. After the oxygen disappeared. . The sulfur dioxide stabilizes the dehydroascorbic acid by reacting with the ketone bonds (Wisser et al. The oxidizability of the sulfurous acid salts is indicated by the following equations: 2– 2 SO3 + O2 → 2 SO2– 4 2– 2– SO3 + H2O2 → SO4 + H2O Jacobs (1976) showed that the amount of sulfur dioxide that reacted (oxidized) after 60 days of storage in bottled red wine was proportional to the original dissolved oxygen content of the wine.2 Sulfur Dioxide-Bearing Chemicals Compound Sulfur dioxide Potassium sulfite Sodium sulfite Sodium sulfite heptahydrate Potassium bisulfite Sodium bisulfite Potassium metabisulfite Sodium metabisulfite Formula SO2 K2SO3 Na2SO3 · Na2SO3 7H2O KHSO3 NaHSO3 K2S2O5 Na2S2O5 Theoretical Yield (%) 100 33 50. This reaction is extremely rapid.56°C (60°F) 1.7 ± 3. The sulfur dioxide scavenges the hydrogen peroxide formed and keeps further oxidation of the dehydroascorbic acid and other products to a minimum.4 53. 1970).0393 1.Sulfur Dioxide and Sulfites 145 TABLE 5.

and fructose and sucrose did not react at all.9 6 × 10–5 Note: 1 = Burroughs and Whiting (1960). the pH. and galacturonic acid were significant bisulfite binding forces (Burroughs and Sparks. Glucose is by far the most abundant of the reactive aldehydes and ketones in most fruit juices. 1960. 1944). 1963). pyruvate. 2000). Diethyl ketone reacts slowly and to a limited extent.0 0. and glucose reacted less rapidly.to seven-member carbon ring system will react. mannose. and arabinose reacted rapidly with bisulfite.b). fermented ciders. In moldy apples.6 7. Reactions with the sugars are limited to those with a free aldehyde and are much slower and the products are less stable (Gehman and Osman.3 (Burroughs and Whiting. He also noted that phosphoglyceraldehyde would likely react.1 — 0.1 0. α-ketoglutarate.146 Antimicrobials in Food TABLE 5. Rhem (1964) noted that the rate of formation and the amount of sulfonate formed depended on the concentration of the reactive substances.5 72 44 98 — 66 1 2 — — — — 0. Bisulfite addition products. 1964a. In botrytized grapes made into wine.5-hexodiulose.b). a Aerny (1986 a. the hydroxysulfonic acids (Suter. 1954). natural fruit juices always bind more sulfur dioxide than would be calculated from the glucose present in the juice (Joslyn and Braverman. maltose. Those reacting the most rapidly formed complexes that dissociated the least.7 90 1. 1973. Otherwise. Ingram and Vas (1950) found that galactose. d-threo-2. 1986 a. form rapidly with aldehydes: – – → HSO3 + R-COH ← R-CHOH-SO3 All aldehydes form the hydroxysulfonates. . lactose. 1985). Navara. but not all ketones react. only ketones with a methyl group adjacent to the carbonyl or carbonyls that are part of a four.5 3 5 8 Ka × 10–6 × 10–4 × 10–4 × 10-6 — 2 × 10–4 5 × 10–2 1. 5-oxofructose. 2 = Aerny (1986a. Aerny. Relative percentages of aldehydes reacting with sulfur dioxide and their equilibrium constants are shown in Table 5.5 — 2.b. 2-oxogluconic acid.3 Relative Percentage of Aldehydes that React with Sulfur Dioxide and Their Equilibrium Constants (K) Compound Acetaldehyde Pyruvic acid α-Ketoglutaric acid Glyoxylic acid L-Xylosone Oxaloacetic acid Glucuronic Acid Monogalacturonic Acid Rhamnose Trigalacturonic Acid Arabinose Xylose Fructose Glucose Malvidin-3-glucoside 1 100 66 47 — 27 — — 2. Joslyn and Braverman. Compared with model solutions. 1954). 1954. and wines. 3 = Navara (1985).7 × 102 — — — — 15. acetaldehyde. L-xylosone. Lea et al. raffinose reacted very slowly.12 — 2 99. and the temperature.. as much as 80% of the total sulfur dioxide may be bound by these types of carbonyls (Blouin. 2.5-diketogluconic acid.1 — 1.1 87 3 50 32 18 — — — — — 26 — 45 32 0.

the following formula was used to calculate the equilibrium constant Ks. reacted with bisulfite to form a lemon yellow compound. but these have been reported as the primary reactions. from winery trials. Jurd (1972) suggested the binding site for HSO3. Piracci and Spera (1983) reported similar results. ketones. Dittrich and Barth (1984) did find correlations between the SO2 binding substances and wineries and grape source.. Shapiro and Weisgras (1970) also demonstrated that cytosine was transformed to cytidine by bisulfite.g. 1998). and 15 to 57 and 31. The respective ranges and means for these components were 72 to 287 and 115. but they found no increase in pyruvate or α-ketoglutarate. Undoubtedly other reactions with sulfur dioxide take place that could reduce the amounts of available sulfur dioxide. Lafon-Lafourcade (1985) reviewed the role of yeast and bacteria in the amounts of these components found in wines. The addition product attached at the carbon 4 of the sunset yellow molecule. Farris et al. After approximately 100 days of storage.5 mg/L of thiamine. (1982). sunset yellow. Damant et al. Glories (1984) studied the equilibrium between SO2 and the anthocyanin–bisulfite complex. Traces of the compound could be found in stored commercial soft drinks that had been heated and contained sulfite. (1989) found a food-coloring dye. Sulfur dioxide can loosely bind to anthocyanins.. The reaction affected flavor of the mustard by reducing pungency.. Allyl isothiocyanate in mustard was shown to react with sulfites used as antioxidants to form allylaminothiocarbonyl sulfonate (Cejpek et al. (1983) tested 30 strains of Saccharomyces cerevisiae for production of SO2 binding materials.5-diene (Crowell and Guymon. Farris et al. and other SO2 binding substances limit the effective use of the added sulfite. 1938).Sulfur Dioxide and Sulfites 147 The amounts of aldehydes. a potassium bisulfite solution (148 mg/L) with sorbic acid (200 mg/L) contained 62% less sulfur dioxide compared to a standard solution of potassium bisulfite with no added sorbic acid. This could be significant in wine because the reduction of free sulfur dioxide could result in the malolactic bacteria being available to act on the remaining sorbic acid to form 2-ethoxyhexa-3. 16 to 42 and 27. . the color is reduced by about 25%. and αketoglutarate. They measured acetaldehyde. Bisulfites may also react with nucleotides such as nicotinamide adenine dinucleotide (Meyerhof et al. (1985) found a reduction in the content of the SO2 binding materials by the addition of 0. This contributes to the difficulty in the measurement of free sulfur dioxide in highly colored wines. selected four yeasts that produced low levels of SO2 binding compounds. and at 50 mg/L of SO2 added. Margheri et al. This treatment allows more effective use of SO2 in wine. (1984) also showed that sulfite additions increased acetaldehyde. ˇ Farkas et al.5 mg/L. pyruvate. In model solutions in the range of 30 to 50 mg/L of SO2 added. Heintz (1976) reported the occurrence of an addition product of sorbic acid and bisulfite. malvidin monoglucoside). (1986) and Bach and Hess (1983) could not find any correlation between amino acid levels in the medium and the accumulation of SO2 binding compounds. There is no evidence that these reactions occur in vivo.6 mg/L. – Ks = [AHSO3]/([A+][(S) – (AHSO3 )]) = 10 5M –1 where: A = total anthocyanins + A = anthocyanins in the ionized form (colored) AHSO3 = anthocyanin–bisulfite complex S – = HSO3 added (calculated from Henderson-Hasselbach equation) At 10 mg/L of SO2 added.is on the 4-position rather than the 2-position (e. it is reduced by 80%. Uzuka et al.5 mg/L. 1975).

148 Antimicrobials in Food They analyzed 544 German wines. Hinze et al. Sulfites are used in other foods and pharmaceuticals. Thiamine pyrophosphate. These are as follows: (1) acetic acid–producing and lactic acid-producing bacteria. Anacleto and van Uden (1982) suggested that the antimicrobial effects of SO2 occurred at the surface of the cell. 1964. The other modulated the entropy of activation of the process. Although sulfonates have decreased antimicrobial activity. pyruvate was always found in the smallest amounts. the genus Acetobacter is Gram-negative aerobic rods. Another reaction of significance is that between bisulfite and disulfide bonds: R1-S-S-R2 + HSO3 → R1SH + R2-S-SO3 – – This reaction can cause conformational changes in enzymes. but their major use as an antimicrobial agent is in beverages and fruits. and (3) fruit molds. (1981) also found reduced ATP activity by addition of SO2 to lactic acid bacteria. 1980) and/or its blocking of the cystine disulfide linkages. three main groups of microbes are of interest in the acid beverages and fruits. 1964). 1911). Somers and Wescombe (1987) noted that wines that had undergone malolactic fermentation decreased significantly in the SO2 binding components. nutrient destruction. ANTIMICROBIAL ACTIVITY The growth-inhibiting or lethal effects of sulfurous acid are most intense when the acid is in the un-ionized form (Hailer. aceti.. It has also been noted that bacteria are much more sensitive to sulfur dioxide than are yeasts and molds. the bound forms of sulfurous acid had about 1/30 the antimicrobial effectiveness of the free form. 1935). 1969). Cruess (1912) estimated that.. One type of activity of sulfite against the yeast cell is its reaction with cellular adenosine triphosphate (ATP) (Schimz and Holzer. TPP activity losses were slower than ATP losses. Beech and Thomas (1985) give an excellent review of the many antimicrobial actions possible with SO2. BACTERIA According to Bergey’s Manual of Determinative Bacteriology (Holt et al. These bacteria are able to . Among the activities discussed are blockage of transport. Schroeter. but the overall losses were about the same. 1977. and the sulfites had none. several have been found to inhibit yeast respiration (Rhem. can be destroyed by the action of bisulfite (Williams et al. Excess sulfur dioxide added to grape juice can deplete thiamine and inhibit fermentation (Ournac. 1964). Lenz and Holzer (1985) showed that the depletion of thiamine pyrophosphate (TPP) in Saccharomyces cerevisiae by SO2 at levels used for juice and wine preservation caused the TPPdependent enzymes to decrease in activity. 1979. 1966). 1994). They proposed two receptor sites. a required cofactor for many enzymatic reactions. One site was directly related to the death process. When considering the antimicrobial activity of sulfur dioxide and its salts. and inhibition of general metabolism. (2) fermentation and spoilage yeasts. The bound forms of sulfur generally have reduced antimicrobial activity (Rhem. The order of decreasing antimicrobial activity of the sulfonates was pyruvate > benzaldehyde > arabinose > ketoglutarate > acetone > acetaldehyde > glucose > fructose (Rhem. inhibition of glycolysis. Pyruvate decarboxylase and transketolase lost 42% and 87% of their activity. respectively. in grape juice. with a corresponding increase in free sulfite. Schelhorn (1951) observed that bisulfites had lower activity than sulfur dioxide against yeast. The cytoplasmic membrane has a high affinity for reaction with SO2. The type species is A. Of the three major SO2 binding components. Schimz.

respectively. Lactobacillus brevis. Pediococcus cerevisiae.5 mg/L were bactericidal.0 (Rhem and Wittmann.. 1994). 1962). The juices high in insoluble solids ultimately had lower residual total sulfites. to carbon dioxide and water. 1980). Lactobacillus trichodes (Fornachon et al. The homolactic species found in wines are Lactobacillus acidophilus. Carr and Davies (1971) noted that relatively high amounts of free sulfurous acid were present in ciders that contained viable lactobacilli. hilgardii. after 36 hours. He found that this genus also fixed a certain amount of the sulfur dioxide. and soft drinks were required to control acetic acid bacteria. Cruess (1912) found that 5. Dupuy (1959) postulated that the reversible reaction between sulfur dioxide and cysteine to form thiol esters along with thiamine and NAD+ degradation were the causes for inhibition of Acetobacter. 1949) is described as causing a hairlike growth in fortified wines. O. and Oenococcus oeni (Amerine et al. Pediococcus. Concentrations above 1. Addition of 30 mg/L of free sulfur dioxide killed the bacteria completely in 15 days. Several reports (Karova and Kircheva. Spirov et al. Lactobacillus casei. Leuconostoc mesenteroides subspecies dextranicum can also cause ropiness in beverages through the formation of dextrans. but much larger doses were required for bactericidal action. Liu and Gallander (1983) demonstrated that lower pH wines underwent .Sulfur Dioxide and Sulfites 149 oxidize ethanol in fermented beverages to acetic acid and. Levels above 120 mg/L of total sulfur dioxide (free and bound) decreased the incidence of malolactic fermentation. Leuconostoc. oeni was the most sensitive to SO2. They indicated A. Fornachon (1957) noted that sulfur dioxide and pH were important factors in controlling lactic bacteria in wines. Lactobacillus trichodes.0 in a buffered solution. Lactobacillus delbrueckii. is very heat resistant and alcohol tolerant. Lactobacillus plantarum. Lactobacillus buchneri. Bacteria common in acid fruits and beverages are the lactic acid-producing genera Lactobacillus. They found that up to 1. Manca de Narda and Strosser de Saad (1987) investigated the tolerance of O. and P.3 log CFU/ml. 1983). Rhem and Wittmann (1962) reported that 200 mg/L sulfur dioxide killed Acetobacter at pH 6. red wine. and the heterolactic species are Lactobacillus fermentum. The bacterium. however.. 20 days was required to kill the bacteria when the same amount of aldehyde–bisulfite complex was used. (1984) stated that the amounts of SO2 used in normal wine making are insufficient for acetic acid bacteria control. Dupuy and Maugenet (1963) noted that even small doses of sulfur dioxide inhibited the activity of the cells. They are aerophilic and have pH optimum for growth of around 5. casei.4 (Holt et al.5. Oenococcus oeni grows preferentially at lower pH but seems less tolerant to sulfur dioxide (Mayer. pH.. Amerine et al.5 mg SO2 per liter in the undissociated form was bacteriostatic to L. (1980) reported that 30 mg/L of free sulfur dioxide is sufficient to inhibit malolactic fermentation in wine. pentosaceus to SO2. As low as 75 to 80 mg/L of total sulfur dioxide prevents growth and 100 mg/L kills this species.47 log and 0. 1982. 1982. and Pediococcus pentosaceus.49 log Acetobacter per ml exposed to 100 and 200 mg/L of total sulfur dioxide in grape juice were reduced to 2. Cell growth was completely prevented. Watanabe and Ino (1984) and Juven and Shomen (1985) reported that up to 100 mg/L of total sulfites for grape juice. L. Lactobacillus hilgardii. Lafon-Lafourcade (1975) demonstrated the effectiveness of both free sulfur dioxide and bound sulfur dioxide in inhibiting Leuconostoc gracile in wine at pH 3. further. respectively. LafonLafourcade and Joyeux (1981) and Joyeux et al. In model solutions containing Lactobacillus arabinosus and L. Fermentation of grape juices with larger amounts of insoluble solids present resulted in wines that underwent malolactic fermentation sooner and more rapidly than those juices that contained fewer solids (Liu and Gallander. plantarum. oeni. 1983) indicated that around 50 mg/L of free sulfite could preserve wine vinegar for about half a year. for antimicrobial action at pH 6. and Oenococcus. with lower pH increasing the effectiveness of the sulfur dioxide. and ethanol. 1979). Lactobacillus leichmannii. Additions of 100 mg/L bleached the color of the vinegar. 320 and 651 mg/L of sulfurous acid were required. This organism is particularly intolerant to sulfur dioxide. aceti can grow in red wine with 25 mg/L of unbound sulfur dioxide present. As little as 30 mg/L of added sulfur dioxide may be lethal to the species..

in the Bordeaux area of France. (1983) determined that. E. (1985) reviewed the use of sulfite as an additive to control microbiological changes occurring in meat products. such as Escherichia coli and Pseudomonas. All strains grew at pH 4. This work confirms that wines with high total sulfur dioxide concentrations are more likely to undergo malolactic fermentation with other than O. coli were inhibited to a greater extent by sulfites than other bacteria. 1972). 15–109. The packaging used was a gas-permeable wrapping that allowed oxidative conditions (von Holy et al. Davis et al. a rapid decline in cell numbers occurred. They suggested that these microorganisms could cause spoilage in wines that were considered sterile using conventional counting techniques. although after sulfite addition. O. In addition. oeni was the primary malolactic bacterium associated with wine. (1980) found the addition of 100 mg/kg of SO2 as sodium metabisulfite to canned pork inoculated with Clostridium botulinum spores delayed cell growth.. (1987) found that vacuum packaging and a good oxygen barrier film decreased the . Piracci (1984) found 0. Millet and Lonvaud-Funel (2000) reported that sulfites cause a portion of lactic acid and acetic acid bacterial populations to enter a viable but nonculturable (VBNC) state. The preservation of the color and odor of meats is improved by sulfite treatment and. The delay was proportional to the concentration of the bisulfite addition. oeni in red table wine occurs readily. (1988) tested 146 different strains of wild malolactic bacteria.. The Gram-positive bacteria remaining grew more slowly than the Gram-negative bacteria. 65–136. oeni strains were less tolerant to the sulfur dioxide than Pediococcus paroulus strains or the Lactobacillus species. and Hafnia alvei.150 Antimicrobials in Food malolactic fermentation more slowly and that lower sulfur dioxide levels increased the fermentation rate of the O. the main effect in meat appears to be the antioxidant properties (Roberts and McWeeney. Lafon-Lafourcade et al. 50–195. Serratia marcescens. or in paired combinations. Sensory tests showed no adverse results. Ough et al. Splittstoesser and Stoyla (1989) looked at five different regulatory-approved additives to determine if they could suppress malolactic bacterial growth in grape juice as a replacement for sulfites. the growth of O.0 were as follows: Salmonella.8 days at 7°C storage with no treatment to 12. O. The remaining cells later multiplied to significant numbers. Banks et al. oeni when SO2 was added and quite a variable response between strains. Adams et al. Salmonella and E. Yersinia enterocolitica. The microorganisms tested and the concentration of free sulfite (µg/ml) necessary to inhibit their growth at pH 7. in their review. (1988) demonstrated that without sufficient SO2 and adjusted pH. None of the compounds alone. than in inhibiting Gram-positive bacteria.5 in beef broth and 20% tomato juice serum medium at 64 mg/L of total sulfur dioxide. Tompkin et al. Citrobacter freundii. 1988). The effect was inhibition of growth of the flora. 67–98. were completely effective. coli. (1988) found significant delays in growth of O. 83–142. It survived alcoholic fermentation when others did not.5 mg/L of molecular sulfite was sufficient to control malolactic bacteria growth. Enterobacter agglomerans. oeni PSU-1 used. 200–241. Roberts and McWeeny (1972). There was some additive effect when used with dimethyl dicarbonate but not enough to warrant the use with SO2 for this purpose. Wibowo et al. The cells could not be cultured on nutrient agar plates but demonstrated metabolic activity through hydrolysis of fluorescent esters and were countable using direct epifluorescence microscopy. This is demonstrated in the use of sulfites in meats. It was found to tolerate up to 100 mg/L of SO2. They noted that sulfites shifted the microflora of the meat to Gram-positive bacteria from the normal Gramnegative flora. The shelf life of ground beef was effectively increased from 1. although slowing or prevention of growth of surface bacteria is probably important. They also noted that the interaction of sulfites with nitrites caused a lowering of the nitrites available for nitrosamine formation. 190–241.6 days at 0°C with the addition of 250 mg/kg of sulfur dioxide. state that sulfur dioxide is more effective against the growth of Gram-negative rods. Banks and Board (1982) tested several genera of Enterobacteriaceae isolated from sausage for their metabisulfite sensitivity. Reddy and Mandokhot (1987) found that minced goat meat could be preserved up to 11 to 13 days if held at 7°C with 450 mg/L of sulfur dioxide added. oeni strains with unfavorable sensory results.

spoilage in sulfite-treated sausage.10–20. resistance of yeast to sulfur dioxide ranges from 0.2–8.0 and 3. its use to prevent the growth of yeast in sweet table wines is seldom ever contemplated. bailii at 25°C under aerobic conditions at pH 3. Any rule of thumb addition. In contrast.81).0 mg/L were required. cerevisiae (105 CFU/ml) after 8 hours at 25°C. Rhem and Wittmann (1962) determined the inactivation levels of sulfurous acid for a variety of yeast genera (Table 5. Yeast The use of sulfur dioxide to deplete the wild yeast in grape juice is a standard practice dating back many years (Cruess. which inhibited growth. The amount of molecular SO2 (mg/L) in wine can be calculated using free SO2 mg/L/(1 + 10pH . The concentration of tyramine and putrescine increased in the presence of sulfite.. Goto (1980) determined viable counts of various wild yeasts in grape juice in the presence of sulfur dioxide and found Torulopsis and Saccharomyces were the most tolerant. Once the inhibition was overcome.60 Rhem and Wittmann (1962).8 mM for Z. Dott and Trüper (1978) found “killer yeasts” (those yeasts that when grown in mixed cultures cause the death of other yeasts) were high or medium producers of sulfite and were more resistant to sulfur dioxide.5 in tryptone yeast extract medium. pH is extremely important in the effective use of sulfur dioxide. or tryptamine. A 10-fold increase in molecular sulfur dioxide occurs between pH 4. The yeast also showed increased resistance to the fungicide dimethyl dicarbonate. respectively.0. Haznedari (1979) tested 30 strains of S. was maintained for a longer period.40–0. but the level of cadaverine was reduced (Bover-Cid et al. between 2.” for biological stability can be disastrous. Molecular sulfur dioxide is the most effective form for inhibiting yeast.4) at 25 mg/L was sufficient to kill a culture of S.5 and 1.20 0. Six strains could grow well at 1000 mg/L. cerevisiae that had been characterized as “SO2 resistant” for tolerance to high concentrations of sulfur dioxide. In fact.05 M free sulfur dioxide for Kloeckera apiculata to 2. 2001).4).20 0. Saccharomycodes ludwigii was nearly as tolerant as Z.20 7. with the pressure to reduce sulfur dioxide content.2 mg/L of molecular sulfur dioxide were necessary at the finish of fermentation and during aging. This was because of the lack of oxygen delaying yeast growth and the production of sulfite-binding substances. Sodium sulfite addition in sausage was shown to affect biogenic amines. and several other genera were very susceptible. (1988) found that with yeast acclimatized to sulfur dioxide.Sulfur Dioxide and Sulfites 151 TABLE 5. Delfini (1989) .4 Range of Effective Antimicrobial Concentrations of Sulfurous Acid against Various Genera of Yeast Genus Saccharomyces Zygosaccharomyces Pichia Torulopsis Hansenula Candida a Number of Species 13 2 1 1 1 2 Effective H2SO3 (mg/L)a 0. Ough et al. Pichia. such as “maintain the free SO2 at 20 mg/L. Carr and Davies (1971) found sulfur dioxide incorporated into growth medium (pH 3. 1. 1912).1.0 and 3. phenylethylamine. Rhodotorula. There was no effect on histamine. Thus the free sulfite.60 0. whereas Kloeckera. According to Warth (1985). and five others were able to produce adequate amounts of ethanol. the growth rates and cell yield were similar.7 0. bailii. Sudraud and Chauvet (1985) found that to maintain yeast stability.

(1985) used an initial 1000 mg/L of sulfur dioxide in the last stages of the manufacture . especially wines in barrels. Dott et al. In normal sweet table wines. With modern filtration and sanitation technology.1 but not in the base material. Generally. excess amounts are used. cerevisiae despite a reduction in the bacterial counts in the system at the same concentration.5). minimizing the air around the wine. Growth was found at the highest concentration of free sulfur dioxide used. The treatment was reported to be successful for juice. however. 1982) as an alternative for pretreatment of grape juice before fermentation. 1985). (1977) determined the cause for sulfite production to be sulfate permease inhibition by methionine. if proper sulfur dioxide levels are not maintained.6). Fruit juices and preserves can be protected from microbial spoilage by sulfur dioxide addition. Recommendations include cooling the fruit before crushing. and eliminating oxygen from bottled wine. (1983). because the pH is high.7 (Lloyd. Angelino et al. Patel et al. Chang et al. the sulfate permease was not repressed by methionine. soft drinks. (1983) showed that increasing the dissolved oxygen in the wort from 1 to 2 to 8 to 9 mg/L caused production of 15% to 30% more sulfur dioxide.6 and 3. They found a majority of the wines in the test to contain about 10 mg/L of total sulfur dioxide (Table 5. ludwigii. and Schizosaccharomyces japonicus in the re-fermentation of sweet champagnes. found no correlation with dissolved oxygen levels in wort and no correlation with ATP sulfurase or sulfite reductase. a late-harvest orange juice. 1978). (1997) showed that sulfite up to 400 mg/L had no effect on S. substituting other preservatives at bottling. bailii. Klimovitz and Kindraka (1989) found that endogenous sulfur dioxide varied with starting specific gravity of the brew and the sulfate content of the water used. reducing the amount of sulfur dioxide before or during fermentation. adjusting the pH downward if necessary. Suzzi et al. Spoilage by Z. even at the legal limits. With this genus. This particular species was found to be very resistant to sulfurous acid. 1985. Breweries have an interest in sulfur dioxide as an antioxidant. Galassi and Mancini.. Vernerova et al. Valouyko et al. using yeast that produces minimum amounts of sulfur dioxide-binding components. and wines because of its tolerance to SO2. the main spoilage yeast is Saccharomyces.4). Some yeast strains can form rather large amounts of sulfur dioxide during juice fermentation (Table 5. Asvany. S. 1985. bailii was becoming a problem in juices. Gomes and da Silva Babo. 1985. bailii in comminuted orange drink was controlled by 230 mg/L of SO2 at pH 3. Ranote and Bains (1982) used 350 mg/L of sulfur dioxide to preserve Kinnow. Hydrogen sulfide (H2S) was used (Ubigli et al. This shows that most wines will not be free from sulfite labeling requirements even if no sulfur dioxide is added.152 Antimicrobials in Food reported the existence of very sulfite-resistant S. settling and racking the juices. Sethi and Anand (1984) had to use 692 mg/L in carrot preserves to inhibit Bacillus cereus. stated that the use of sulfide salts failed to protect wine and left negative sensory characteristics in the wine. Z. 1985. 1986a. (1985) tested 1700 Saccharomyces. Strain differences caused variation in the sulfur dioxide produced from 11 to 26 mg/L. There have been numerous reports on techniques to minimize the amount of sulfur dioxide used in wines (Aerny. Schmitt et al. Even at low pH (2. Hernandez. cerevisiae. using sulfide salts before fermentation. Minarik and Navara (1977) reported finding the spoilage yeast S. investigating the sulfur dioxide content of green beer. In a cell recycled ethanol fermentation system. Pearlstein (1988) found that extended aeration decreased sulfite production.. Spoilage yeast in dry wines are fairly rare. which was at pH 3. ludwigii is also a noted spoiler in sweet grape juice containing high amounts of sulfur dioxide (Jakob.b. S. ludwigii in a low-alcohol wine. none seem to be capable of replacing the antioxidant property of SO2. Proper selection of the yeast strain to avoid high levels of sulfite is an obvious choice. Sand (1980) suggested that Z. and low pH. (1989). Yeast can form sulfite from sulfate via sulfate permease. Very little of the sulfur dioxide formed by the yeast remains in the free state. cell numbers are depleted to very low levels at bottling. Brettanomyces can contaminate a winery. Although other agents may act as antimicrobial agents. sulfur dioxide. and there was no correlation with the number of cells in the initial inoculum. does not always inhibit growth and fermentation. In high-sulfite-producing yeast. alcohol. 1975).

160. rosei S. (1976) Poulard and Brelet (1978) Delfini et al. 500 5.5 Formation of Sulfur Dioxide by Various Yeasts during Grape Juice Fermentations Yeast Saccharomyces carlsbergensis Sulfur Dioxide (mg/L) 12 150 80. africana TABLE 5. 35. 3.5. (1976) Delfini et al. 35 20. (1976) Delfini et al. (1976) Delfini et al. italicus S. (1976) Poulard and Brelet (1978) Minarik (1975) Poulard and Brelet (1978) Minarik (1975) Delfini et al.5 12 0 9 Reference Minarik (1975) Eschenbruch and Bonish (1976) Heinzel et al. pastorianus S. 18. 1985) Range of SO2 Found in Synthetic Medium (mg/L) <10 10–20 20–30 30–40 >40 Average Total SO2 in Wine (mg/L) 10 17 22 34 58 Number of Strains 1292 354 50 2 2 . (1976) Minarik (1975) Eschenbruch and Bonish (1976) Heinzel et al. (1976) Minarik (1975) Minarik (1975) Heinzel et al. 21. 20 26 27 0 0. 150 0. 18. 21. acidifacien S.5. 40 0 18.5 0 0 0 1. (1976) Poulard and Brelet (1978) S. magna K. 1300 22. 100.6 Amounts of Sulfur Dioxide Found in Test Wines Fermented by 1700 Saccharomyces Yeasts (Suzzi et al. rouxii Schizosaccharomyces pombe Torulopsis stellata Kloeckera apiculata K. 170. (1976) Delfini et al. chevalieri S. bailii S.Sulfur Dioxide and Sulfites 153 TABLE 5. 56. (1976) Poulard and Brelet (1978) Delfini et al. 3. 5 18–23 85 0. 5. 44. bayanus S. cerevisiae S.5. (1976) Delfini et al. 1. 76 17. 20 1. (1976) Delfini et al. 4. (1976) Delfini et al. 52. 100 5. 500 300. uvarum S..5 14. (1976) Delfini et al.

Sulfur dioxide was tested to preserve apples by Kaul and Mujol (1982). Mohamed et al. for longer stability they recommended redipping every 40 days and storage at 3°C. Botrytis species is probably the most prevalent fungi. The latter used only 150 mg/L of sulfur dioxide to maintain the quality for up to 2 months for mango pulp. Slow-release sulfur dioxide generators placed into storage containers appear to be beneficial for short-term (2 months) shipments (Nelson and Ahmedullah. Fishman and Karalidze (1984) dipped mandarin oranges in concentrated sulfur dioxide solutions and then bagged the fruit in large polyethylene bags. 1976). Botrytis cinerea was inhibited for up to 12 weeks. but severe fruit discoloration occurred. Ballinger et al. Rhizopus. Whole-fruit storage success has depended largely on the use of sulfur dioxide.1% sulfur dioxide gassing weekly had only minor mold growth and only moderate bleaching of the fruit. stored Euvitis hybrid bunch grapes up to 20 weeks. (1986) found that gassing with sulfur dioxide at 200 mg/L three times per week was superior to 2500 mg/L once a week for table grapes in cold storage. humidity.2% and no bagging. Penicillium. 1988. and Uncinula (powdery mildew) also infect fruit. but Aspergillus niger and Rhizopus were only inhibited for 2 to 3 weeks at 20°C. the latter were completely inhibited for up to 4 weeks.2%). Berries have also been successfully preserved with sulfur dioxide. Demetrashvili (1981) stored mulberries for up to 6 months in a solution combination of tartaric acid (1%) and sulfur dioxide (0. Byssochlamys. (1984) found good mold growth inhibition using a sulfur dioxide treatment for drying persimmons and no effect on the composition of the treated versus the controls. (1984) stored mango pulp and tomato pulp in polypropylene or high-density polyethylene pouches using sulfur dioxide as a preservative. They did not recommend the treatment for fresh market fruit. Katsaboxakis et al. Mansour et al. Kalra and Chadha (1984) stored mango pulp and Maini et al. Benkhemar et al. but the Howard mold counts were not significantly different for the control versus the treated berries. Massignan et al. Nelson (1979) reported that grapes held at 2°C with 0. Although this shelf life was superior to other chemical treatments tested. It controlled Trichothecium roseum. using corrugated shipping containers with polyethylene liners and SO2 generators.. Kuwabara et al. (1984) used SO2 generators to store raspberries at several temperatures. Sulfur dioxide is used as a fungicide on grapes because of the physical damage resulting from transit. The fruit maintained quality for longer than a year. Alternaria. At 0°C. sulfur dioxide was effective against G. and transit to market. Spayd et al. Significant bleaching of the anthocyanins occurred. The SO2-treated fruit was preferred in sensory tests. mold inhibition was almost complete. Marois et al. (1984) used SO2 generators to hold Egyptian table grapes for up to 4 weeks at 0°C. (1984) preferred in-package generators. 1988). (1985). . At higher concentrations of sulfur dioxide. Alvarez and Vargas (1983) preferred direct sulfur dioxide fumigation to generators. and temperature. (1981) preserved citron fruit for longer than 560 days in a 2% sulfur dioxide brine solution. fruits similar to litchi nuts. Most other publications favor generator treatments. The former indicated that 350 mg/L of sulfur dioxide was a better treatment than 100 mg/L. The visual mold counts were less using the SO2 generators. Aspergillus.154 Antimicrobials in Food of khoa. Untreated berries molded within 7 days. storage. Longan and rambutans. Stemphyllium. both have shown improved storage by treatments with sulfur dioxide (Wara-Aswopati et al. Yakobashvili and Georgadze (1984) could achieve only 60 days of stability to mold growth in mandarin oranges. Sharma (1986) performed an almost identical experiment using pears. and was partially effective against Rhizopus stolonifer and Monilia taxa. (1989) successfully stored Moroccan table grape varieties using SO2 generators for 3 months. cingulata. Molds A number of molds can infect fruit during processing. but others including Cladosporium.. Again. appreciably controlled Glomerella cingulata. Using only 0. suppressed Penicillium expansum.

OTHER USES Any vegetable or fruit. 1978. The antioxidant effects of ascorbic acid are enhanced by their combined use with sulfites in foods and pharmaceuticals (Schroeter. lung hemorrhage. Spoilage caused by Pseudomonas species and Enterobacteriaceae could be held in the lag phase by 100 mg/L of SO2. the cause of rapid death is pulmonary dysfunction. Chronic symptoms (Reid. Anhydrous sulfur dioxide hitting the eye is readily absorbed. (1984 a. carrots. Vegetables. Giannuzzi and Zaritzky (1990) studied refrigerated prepeeled potatoes held in plastic film bags with vacuum packing at three temperatures. As early as 1896. 1966). The modeling of sulfur dioxide uptake in prepeeled potatoes was studied (Rodriguez and Zaritzky. and velocities of mixing for several different shapes of potatoes. indicated by pulmonary edema. They found sulfur dioxide at 50 mg/L in apple sauce and apple juice was sufficient to kill the mold and was the most efficient. In amounts that were less than lethal doses but greater than tolerable levels (higher than 62 mg SO2 per kg body weight). 1986) with regard to shape of potato pieces. sulfur dioxide resulted in some physiologic changes in rats. dry matter contents. Dried fruits held in an atmosphere of sulfur dioxide maintain a more natural appearance. Coughing. which is highly resistant to thermal processing.. articles appeared in scientific journals suggesting the possible toxicity of sulfur dioxide. and visceral congestion. such as peas. with excess mucous formation and difficulty in clearing the lungs. and tomatoes. and sneezing are outward immediate symptoms. Some people are more sensitive than others. 1975. National Academy of Sciences. Sulfur dioxide injury to the eye is not an uncommon industrial accident. 1980). cabbage. or canned. The level of sulfur dioxide normally used for juice processing would preclude its development. 1963) are hypertrophy of goblet cells and mucous glands. that is subject to nonenzymatic or enzymatic browning can benefit by proper treatment with sulfite compounds. Papazian. and spectacle eyes (Fitzhugh et al. raw. lacrimation. A summary of a number of test studies reported by the Select Committee on GRAS Substances (1976) indicated that no toxic effects were observed for ingested amounts of less than 30 to 100 mg sulfur dioxide per kilogram body weight per day (depending on the experimental conditions and . dried. such as polyneuritis. Equations were derived for holding times at various SO2 concentrations. The mold can exist in the ascospore form. Because of its high solubility in fat. Roland et al. bleached incisors. 1996). visceral organ atrophy. 1947). bone marrow atrophy. Roland and Beuchat (1984) also studied the growth and control of this organism in grape juice. Sulfur dioxide greater than 33 mg/L in air can cause distress or even death when inhaled (Amadur. 1975). have more stable color and less deterioration if so treated. Bolin and Jackson (1985) found that adding an oxygen scavenger to packaged dried apricots or apples greatly increased the effectiveness of the sulfur dioxide. with similar results.. Studies on the median lethal dose (LD50) of sulfiting agents for various animals were compiled by the Select Committee on GRAS (generally recognized as safe) Substances (1976).Sulfur Dioxide and Sulfites 155 Byssochlamys is a mold species that is capable of producing the mycotoxin patulin. potatoes. renal tubular casts. The firmness of whole bananas is increased by dipping them into a 500 mg/L SO2 solution (Levi et al. beans. This organism is not a normal mold found on grapes. it penetrates the cornea and causes deep keratitis and iritis (Grant. In general. frozen. stunting of growth. 1946). TOXICOLOGY The toxicology and safety of sulfur dioxide in its various forms has been the subject of many reviews (Institute of Food Technologists. Patulin is also inactivated by sulfur dioxide rather rapidly.b) studied the best fungicide to use of those legally available.

Early on. including one death. use of SO2 was considered GRAS. Gershwin et al. on volunteer asthmatics.156 Antimicrobials in Food species). They found mixed responses from . sulfite-related. there have been 1132 consumer complaints describing adverse reactions. (1987). In a recent study. There are many more papers and reviews on sulfite-induced reactions in people with asthma. In 1983. (1986) showed that salad fresheners containing sulfites could. The review by Nicklas (1989) particularly stressed that many pharmaceuticals are preserved with sulfur dioxide. Wines with lower concentrations of sulfites (<150 mg/L) did not elicit any response. Howland and Simon (1989).6% of these cases were classified as severe (Warner et al. No significant differences were found between the groups in any lung function parameter.2 mg/kg body weight per day was not a hazard to health. much higher levels would result. studies indicated that certain asthmatic individuals were at risk by consuming relatively small amounts of sulfites. Until the early 1990s. No in vivo carcinogenic or mutagenic effects could be demonstrated with mice or rats. Itami et al. The Select Committee on GRAS Substances (1976) concluded that the average per capita consumption of 0. They found that sulfur dioxide was the more severe causative reagent in all cases. using Wistar rats. Mathison et al. Their report stated that no teratogenic effects had been reported and noted that sulfur dioxide had variable effects on mutagenicity to bacteria. (1985) reported on the effect of wine (white) with and without sulfur dioxide. For humans. the joint FAO/WHO Committee (Joint FAO/WHO. the U. In a reexamination of the findings of the Select Committee on GRAS Substances (1976). The food situation in relation to sulfite reactions is pertinent. Vally and Thompson (2001) exposed 24 patients with asthma and a strong history of wineinduced asthma to a single high-sulfite challenge (300 mg/L). and 48. If the practice is abused. The variation may in part be the result of differing amounts of thiamine administered in these studies. (1985) discussed the possible mechanism for sulfite action on asthmatics and concluded that little is completely understood. Food and Drug Administration (FDA) (1985) agreed with the toxicology findings. Dahl et al. adverse reactions.7 mg SO2 per kilogram body weight per day. The drinker consumed only one glass but was known to have asthma and sulfite hypersensitivity. An extensive review by Gunnison and Jacobsen (1987) discussed in detail the possible mechanisms of sulfite hypersensitivity from which most asthmatics suffer. could not show any teratogenic effects with heavy doses of sulfite but did show evidence of fetal toxicity. leave a 900 mg/kg residue of sulfite on the product. found that normal nonasthmatics were not affected by doses in the airway of up to 0. A joint Food and Agriculture Organization and World Health Organization (Joint FAO/WHO. 1974) established the acceptable daily intake level at 0. Since the monitoring of food reactivity to sulfites started in the 1980s. previously selected as SO2 sensitive by sulfite capsule tests. unless damaging doses are given. sulfite-containing fresheners. to a number of foods treated with sulfites. in controlled amounts. (1988) challenged eight people with asthma. (1989). challenged them with lettuce treated with commercial. 1967) report estimated that 35 mg sulfur dioxide per kilogram body weight per day was the “no observed adverse effect level” (NOEL) in the rat. Taylor et al. Martin et al. when used as recommended. Additionally.. (1987). A death related to a red wine that contained 93 mg/L of total sulfur dioxide was reported by Tsevat et al.6 ppm.S. Only four of the 24 patients had a significant reduction in forced expiratory volume. (1986) tested red wine-sensitive people with asthma for reactions against amines and sulfites in wine. Linn et al. studying patients known to be sulfite sensitive. the FDA (1983) noted that they had received 90 reports of food-caused. However. The researchers suggested that the role of sulfites and/or wine triggering asthmatic responses may be overstated. but the medical aspects are beyond the scope of this chapter. 2000). can recover unaffected. the FDA considered the use of sulfites on fresh fruit and vegetables in restaurant foods a major problem area. Those with asthma had restriction in the airways and in one case a life-threatening reaction. Vally and Thompson (2001) exposed 12 wine-sensitive and 6 control asthmatics to wine with increasing concentrations of sulfites. It appears from all the published reports that normal humans are reasonably tolerant to sulfur dioxide and. but people with asthma responded to this dose level. in a replicated dose-response study. They concluded that some asthmatics could be at risk in consuming wine-containing sulfites.

foods recognized as a sources of vitamin B1. 2000). the Alcohol and Tobacco Tax and Trade Bureau (formerly the Bureau of Alcohol. The amounts of sulfite in foods were limited by the FDA (1988). AMOUNTS IN FOODS AND REGULATIONS Sulfites are considered GRAS substances by the FDA when used in amounts that are in accordance with good manufacturing practices. 1995). They concluded that not all SO2 -sensitive people with asthma would react to all foods. pickled onions and salsa were implicated in adverse reactions in patients with asthma (Gastaminza et al.. The amount of sulfites used in food products is dictated by good manufacturing practice. Banks and Board. 1982). and wine. A number of the foods were over the German limit. An estimate of the concentrations of sulfites used in foods as an antimicrobial was listed by Gould (2000) (Table 5. The total sulfite levels ranged from 10 to 203 mg/L. . directives are set for sulfur dioxide [E220]. Nagy et al. and salmonellae during storage at refrigerated or room temperature (Ingram et al. sodium bisulfite [E222].. 1989). The amounts of sulfite used in foods and beverages vary greatly between countries. In the United States. sodium sulfite [E221].8. then increased levels of thiosulfate can be found in the urine after ingestion of sulfite. Wines with greater than 10 mg/L sulfites must be labeled as containing sulfites. calcium bisulfite [E227]. The FDA (1986a) rescinded the GRAS status of sulfites on raw fruits or vegetables and declared that sulfite could not be used on these items. More recently. 1956. frozen. The FDA (1986b) also made labeling of any product containing 10 mg/L or more of sulfites mandatory and defined the method of analysis.3. Levels of sulfites used in wines in the United States were summarized by Ohkubo and Ough (1987).Sulfur Dioxide and Sulfites 157 the patients to the various foods and drinks. Nordlee et al. In the European Union. Steinman and Weinberg (1986) noted that 5% to 10% of children with asthma were SO2 sensitive and listed foods and beverages to be avoided in South Africa. In the survey. Barnett (1985) reported that levels of sulfites for Australian foods and beverages ranged from 29 mg/L for beer to 3000 mg/Kg for dried fruit. sulfites are not allowed in meats. or dehydrated) was banned but later rescinded in a court action (FDA.7). The maximum level of sulfur dioxide allowed in wine was set at 350 mg/L by the regulating body for the U. In some countries. and many did not declare sulfites on the package. 1974). yeast.1).. The body normally metabolizes sulfite to sulfate by the action of sulfite oxidase (EC 1. Sulfites were permitted in wines at 350 mg/L. 1995.S. 161 white table wines averaged 121 mg/L of total sulfur dioxide. They are allowed in fruit juices and concentrates. or on fruits and vegetables intended to be served or sold raw to consumers or to be presented to consumers as fresh (21CFR 182). He also found that cooking foods frequently lowered the sulfite to below detectable levels. New regulations on sulfites in potatoes are still being developed. Sulfur dioxide restores a bright color but may give a false impression of freshness. sulfites may be used to inhibit the growth of microorganisms on fresh meat and meat products (Kidney. calcium sulfite [E226]. Sulfite use on fresh potatoes (any potato not canned. Tobacco and Firearms) of the Department of the Treasury. (1985) reported on the sulfite levels in 53 different maraschino cherry samples. dehydrated fruits and vegetables. with a mean of 52 mg/L. 1990). Red table wines were generally much lower. sodium metabisulfite [E223]. alcoholic beverage industry. 1987) listed the sulfite composition of a number of foods and beverages. Sulfite or metabisulfite added in sausages is effective in delaying the growth of molds. If this enzyme is less active. and potassium bisulfite [E228] (Gould. A German report (Wever. potassium metabisulfite [E224]. (1989) tested this idea and could demonstrate the effect on people with asthma compared with normal people using white wine as the challenge. Town et al. Table grape sulfite tolerances were set at 10 mg/kg (Environmental Protection Agency. They cautioned that a better test would be direct measurement of sulfite oxidase activity.

and .02 961. Sulfites can be described as free. Reversibly bound sulfites are converted to sulfur dioxide only after heat and acid or alkali treatments. Those sulfites that bind food matrices and are not converted to sulfur dioxide with heating or acidic conditions are irreversibly bound. A sample is placed in a distilling flask.32 980.28a 990. 2000) Method Number 892. The official methods of analysis for agricultural and food products are specific in their recommendations for analysis of sulfur dioxide (Table 5. or irreversibly bound (Beck et al.29 990.30 990.8).. acidified.158 Antimicrobials in Food TABLE 5..8 AOAC International Official Methods of Analysis for Sulfites (Warner et al. reversibly bound. Thiosulfonates are irreversibly bound (Beck et al. and fillings Jams and jellies Nonalcoholic beverages Sausage Sugar confectionary Vinegar Wine a Use Concentration (mg/kg SO2)a 10–30 100 50–1000 10–100 50–100 50–500 50–100 20–200 450 50 50–200 100–300 Sulfites are allowed only in certain foods in different countries. and concentration varies by country. TABLE 5.7 Application of Sulfites in Foods as Antimicrobials and the Concentrations Used (Gould.20 975.31 Year Adopted 1892 1961 1962 1963 1975 1980 1987 1990 1990 1990 1990 Title Sulfurous Acid (Free) in Meats Sulfites in Meats Sulfurous Acid (Total) in Food Sulfurous Acid (Total) in Dried Fruit Sulfurous Acid in Food Preservatives in Ground Beef Sulfites (Total) in Foods Sulfites in Foods Sulfites (Total) in Foods and Beverages Sulfites (Free) in Wine Sulfites in Foods and Beverage Type of Analysis Titrimetry Qualitative Test Modified Monier-Williams Colorimetry Qualitative Test Colorimetry Differential Pulse Polarography Optimized Monier-Williams Flow Injection Analysis Flow Injection Analysis Ion Exclusion Chromatography ANALYTICAL METHODS Ough (1988) and Ough and Amerine (1988) gave very detailed reviews of methods for free and total sulfur dioxide determinations in grapes and wines.04 990. Free sulfites are readily converted to sulfur dioxide upon acidification and can be quantitatively analyzed following distillation. 2000) Food Beer Fresh fruits Fresh vegetables (onion.17 987.16 963. garlic. horseradish) Fruit juices Fruit-based sauces and related products Fruit pulps.09 962.. One official method for measuring total sulfurous acid is the modified Monier-Williams procedure. 2000). purees. 2000).

The direct iodine titration method (Ripper) for measurement of total sulfur dioxide is the method used for routine analysis in the wine industry (Ough and Amerine. Excess iodide is added to the sample. This method is good for most products. Basically the sample is sparged with gas for 12 to 15 minutes. in addition. Burroughs and Sparks (1964b). and the freed sulfurous acid is titrated directly with iodine to a starch end point: I3 + SO2 + H2O → SO3 + 3I– + 2H+ – 2– SO3 + H2O → SO4 + 2H+ This is satisfactory for certain materials. but if other oxidizable substances are present. The reactions are as follows: – 8I– + IO3– + 6H+ → 3I3 + 3H2O I3– + SO2 + H2O → SO3 + 3I– + 2H+ The advantage is a stable standard solution that does not require daily standardization. and it is then titrated with the iodate solution to a starch end point. except dried onions. The optimized Monier-Williams method is used by the FDA to measure sulfites in official samples (Table 5. 2000). Free sulfur dioxide is easily measured in white wines by the Ripper method. Vahl and Converse (1980) made a collaborative study of the Ripper method for the Association of Official Analytical Chemists and concluded that the poor precision and large systematic error precluded it for adoption as an official method. Schwedt (1986) considered the test strips satisfactory but noted that if dyes or natural pigments came in contact with the cellulose. Joslyn and Braverman (1954) reviewed the errors associated with iodine titrations. artificially high results occur. Paul (1958). Rankine and Pocock (1970). This is a direct iodine titration of the substance at acid pH. Kielhofer and Aumann (1957). and cabbage. the sample is made basic to break the bisulfite addition products. Schneyder and Vlcek (1977) reported that iodate was superior to an iodine standard solution. (1979) showed that sodium sulfide and allyl isothiocyanate gave positive results in the MonierWilliams tests. (1988) and Wanderer and Solomons (1987). the sulfate can be precipitated with barium and measured gravimetrically. The sulfurous acid is trapped in hydrogen peroxide solution: 2– SO2 + H2O2 → SO4 + 2H+ The acid produced by the oxidation of sulfur dioxide to sulfate can be titrated with sodium hydroxide. Mitsuhashi et al.Sulfur Dioxide and Sulfites 159 heated. False-negative and false-positive results were found by both groups. 1988). The Ripper method for free sulfur dioxide is given by Ough and Amerine (1988). but determinations on red wines are more accurately done by this variation of the Monier-Williams method. . leeks. and Ough and Amerine (1988) described the method and equipment needed. and the volatile sulfur dioxide is removed by a stream of nitrogen through a reflux condenser.8) (Warner et al. false results were recorded. The reflux condenser must retain all volatile acids except the sulfurous acid. it is then acidified. and the hydrogen ion produced in the peroxide solution is titrated. The Monier-Williams method can be modified to determine free sulfurous acid. The same errors are associated with this procedure as are associated with the Ripper procedure for total sulfur dioxide. Briefly.. Sulfite test strips for protection of people with asthma who are hypersensitive to recognize that the food contained sulfites were questioned by Nordlee et al.

Savini. H. 1998. 1963. In Food Biopreservatives of Microbial Origin. R. 1964b. K. 45:1292. pp. 347. Eds. J. Burroughs. par méthode des sachets générateurs.F. Beech.F. The determination of the free sulfur dioxide content of ciders.. ˘ Cejpek...L. C. OIV 58(652–653):564.. L. Arboric. Sulfite.R. 1989. Agric.G..S. Meat Sci.D. J. 7:161.L. cultivées au Maroc. Sci. 24:207. Aspects pratiques. Bristol. Velísek. J. J. Arboric.. J. E..K. Analyst 89:55. S.. Annual Report. 1985. and Board. Food Agric. Hortic.A. C. Sulfite-inhibition of Enterobacteriaceae including Salmonella in British fresh sausage and in culture systems. Baker. Determination of carbonyl compounds in a wine and calculation of its binding power.. and Nesbitt. F. and Vargas. OIV 58(652–653):621.J. M. M. Sulfites in foods: Their chemistry and analysis. 144–147. Kinetics and activation energetics of death in Saccharomyces cerevisiae induced by sulfur dioxide. Miguélez-Arrizado. L. V.A.. Mocking-Bode. Burroughs. 1980. Technol. Eds. Diminution de la teneur des vins en anhydride sulfureux. Anacleto.R. Biochem. Aerny.H. Bull. 2000. V. Food Process.W.H. L.. and Hrabcová. CT.. Action antimicrobienne de l'anhydride sulfureux. C.P. Westport. Technology of Wine Making. Constituents du vin combinant de l’acide sulfureux. N. Amerine.D... A. 1985. Brauwiss. A. Weinwirtsch. P.A. L. 1975. FL...W. Bolin. Maness. and Vermeire.M.M. Tech. Sultana Agric. and Daeschel..F. 2. en uva almacenada c. H. L. A. A. A.G.. Method. Angelino.A. Tec. Effect of sulphite treatment on allyl isothiocyanate in mustard paste. Metabolites of yeasts as biopreservatives.. 1960. an elective agent in the microbiological and chemical changes occurring in uncooked comminuted meat products. AVI Publishing. Carr. I. 133.v.H. Rev.. Bull. Bull. Bach. 1985. A.P. 2001. A. H. Dalton. and Vidal-Carou. Aust. Efecto de fungicidas aplicados en precosecha y SO2 en postcosecha en la central de Botrytis cinerea Pers. H. Bover-Cid.E. Barnett. and Forrest.160 Antimicrobials in Food REFERENCES Adams. H. B. Eine Möglichkeit zur SO2-Einsparung.C. Blouin. Lactic acid bacteria in juices and fermenting ciders.G. Picomolar quantitation of free sulfite in foods by means of [57Co]hydroxocobalamin and radiometric chromatography of [57Co]sulfitocobalamin. OIV 62(695–696):5. J. and Thomas. J. 59:391. M. The sulfur dioxide combining power of cider. R. Sci. R.G. Food Agric. 62:53. J. Bristol. El Mniai. Chromatography A 881:345–356. Monatsschr. Banks. S. p. R.. Hortic.. Bioeng. and Sparks.G. p. 42:476. and van Uden.H.. Urban. R. Berg. W. K.T.G. Burroughs.B. 1982. 1986a. Ough. 1973. McMillan.F. 1985. Air pollutants. UK. C.. The identification of SO2 binding compounds in apple juices and ciders. Boubekri. H. Aerny. Ray. O..A. 37:503. J..J. Long Ashton Research Station. M..A. H. and Doull. Burroughs. Activity of sulfate-metabolizing enzymes and sulfur dioxide formation during main fermentation. Diminution de la teneur des vins en anhydride sulfureux.J. Beck. Benkhemar. S.G. Les technologies de vinification permettant de diminuer les doses do SO2. Hortic. Bacteriol. L'anhydride sulfureux et ses propriétés utiles en vinification. Bakalinsky. Biogenic amine accumulation in ripened sausages affected by the addition of sodium sulphite. 20:916. Food Chem. J. New York. and Hess. Preserv. J. applications and significance of coexisting sulfides. J.. H. Anes.. J. Banks. 1985. M.F. (7–8):186. .. 1989. 15:176. 1983. and Whiting. 1983. Asvany. H. Suisse Vitic. Appl.C. G. T. CRC Press. 1. Biotechnol.. 1987.B. Nychas. Casarett. R. (Santiago) 43:61. 527–554. Singleton.. J. Rev. 1992. 1982. Annual Report. 1971. Amadur. Lahlou. J. Kunkee. 1985.. Factors affecting sulfur dioxide binding in dried apples and apricots. La conservation frigorifique de six varietiés de raisin de table. Ballinger. 63:227. and Jackson. A note on shelf life extension of British fresh sausage by vacuum packing. p. L. 1986b. and Sparks. and Board. Ann.E. Food Prot. 12:97. J. Alvarez.K. Long Ashton Research Station. Suisse Vitic.. Appl. Boca Raton.M. and Webb. Sci. and Tantaoui-Elakari. 24:2477. D. 18:17.. 1964a. and Sparks. W. 9:25. 18:143. In Toxicology: The Basic Sciences of Poisons. A. M. III. and Davies. Food Technol. and Mateer. Sulfur dioxide for long-term low temperature storage of Euvitis hybrid bunch grapes.. Sulfite binding power of wines and ciders. U..

Heinzel. Davis. B. P. M. Fishman. T. Cardu. and Bonish. Sulfites in foods and drugs. Eschenbruch.K. 26:97. Reg. Reg. Vplyv tiaminu na nacionále sirenie vina. F. 1985. Fatichenti. H. M. and Maugenet. H. Food and Drug Administration. 1988. R. J. H. 61:201.C. 1949. 1990. Agric. C. R. C. and identification of 2-ethoxyhexa-3. 39:184. Vignevini 12(5):13. G.. The occurrence of malo-lactic fermentation in Australian wines. Pharmacol. and Serra. Fleet. Formazione di anidride solforosa en acido solfidrico da parte dei lieviti nel corso della fermentazione alcolica. 31:9. H.H. Enol. 107:299–302. C. Pesticide tolerance for sulfur dioxide. Food Add. F. R. 86:37. Food and Drug Administration.. Environmental Protection Agency. J. 39:137. Microbiol. 55(51):9826.. G. Contam.C. 1976. 1989.H.. Crowell. Dott. 1912. Knudsen. Aspects of intensifying the process of sulfitation of mandarin oranges. Selezione di stipiti di Sacch. H. Hilgardia 19:129. Dittrich. SO2-Gehalte. C. N. Exp. Arch... Sulfiting agents. Vitic. (8):17 [CA 101(17):150141q]. 1989.C. Environ. Kintlerova. Food and Drug Administration. 337. 1946. and Shin. 1986a.. 1982.F. Dahl. P. 1986b. Fed. and Guymon. Lactobacillus trichodes nov.M. Demetrashvili. A. Douglas. revocation of GRAS status of use on fruits and vegetables intended to be served raw to consumers. R. Inst. Vini Ital. D. Wachstumsvorteile yon sulfitbildenden Weinhefen durch damit gepaarter "Killereigenschaft?" Wein Wissensch. Ferment.A. Conditions du développement des bactéries acétiques et mesures préventives dans la conservation des vins. Agric.. and Trüper. Fed.. Reg. Microbiol. G. Dott.G. H. J. declaration of sulfiting agents. L.. 13(2):11. and Mancini. Appl. Enol. 63:1–6. and Harving. Sulfite formation by wine yeasts. Tecnologie per ridurre l'uso di SO2 in enologia... Wein Wissensch. S. 18:251. Farkas. Life Sci. Reg. Appl... J.. P. Riv. Fornachon.D. Am. J.G. and Madau. and KaraIidze. 119:65 [CA99(3):21099y].. Vini Ital. Delfini. P. Exp. A. SO2-dendene Stoffe und Säure abbau in deutschen Weinen. Res. 51(131):25021. 1975. and Barth.. Functional selection of low sulfur dioxide-acceptor producers among 30 Saccharomyces cerevisiae strains. Soc. Production of sulfite and sulfide by low-sulfite and high-sulfite forming wine yeasts. Technol. G. Vinohrad (Bratislavia) ˇ 23(4):86. Sulfiting agents. J. Red wine asthma: A controlled challenge study. cerevisiae bassi produttori di accettori di SO2. Henriksen. Technol.. V.. P. 1989. FDA Drug Bull. Food labeling. Arch. 112:283. Aust. A. W. Bethesda. Deiana. Rifermentazione con lieviti SO2 resistenti di vini dolci imbottigliati. Fed. 78:1126. Technol. Gaia.. 53(243):51065. spec. Konservn. and Trüper.Sulfur Dioxide and Sulfites 161 Chang.. Wine constituents arising from sorbic acid addition. The reexamination of the GRAS status of sulfiting agents.R.5-diene as source of geranium-like off-odor.M. Gruz. The effect of sulfurous acid on fermentation organisms. Ind. MD. BioI. Active uptake of sulfate by "low" and "high" sulfite producing wine yeasts. Delfini. Allergy Clin. G. 1986.A.. and Ková. J. Wibowo. FDA 22383-2020. J. W. Sci.. Galassi. 1957. O.. 1978.D. J. Fed. Damant.H. 1959.. P. and Macrae. P.. Vitic.. Cruess. E. 1977.. Microbiol. 33:143. Vitic.G. Ther. Fed. 1985.. Reynolds.. Sulfiting agents.G. J. . 1997. Tr. affirmation of GRAS status. Ovoshschesush. Fitzhugh. P. Prove di fermentazione in Cantina.A. Dupuy. 1985. Chem. Eng. Deiana. J. S. 1981. Am. 1976. Kim.M. Prom-st. Immuno. 35:376.. Farris. 8:233. and Vaughn.H. Madau. Properties of wine lactic acid bacteria: Their potential enological significance. 6:273. J. Office Fed. Ann.. Ann. S-kh. and Lee.. and Nelson. J. Food and Drug Administration. 1963.. W.. and Bosia. Use of sulfite and hydrogen peroxide to control bacterial contamination in ethanol fermentation. Fornachon.. Ein Untersuchung an 544 Weinen. 1984. Preservation of mulberry berries using preservatives. 8:120. 1983. A. The structural identification of a secondary dye produced from the reaction between sunset yellow and sodium bisulfite. revocation of GRAS status for use on “fresh” potatoes served or sold unpackaged and unlabeled to consumers.S. 54(89):20125. G. 51(131):25012.F. Farris. a bacterium causing spoilage in appetizer and desert wines. The chronic toxicity of sulfites. Food and Drug Administration. Reg. Food and Drug Administration. IV.A. 4:581. Am. P. 12:63. 1983. L'inhibition par I'acide sulfureux de l'oxydation de l'ethanol par Acetobacter rancens. Enol. J. G. I. 1988. Dupuy. 1984.M. Fatichenti.

J. P. B. Adv. Sulfite-treated lettuce challenges in sulfite sensitive subjects.R. 27:42. H. M.. MD. The chemistry of sugar-sulfite reaction and its relationship to food problems.M. 5:1805]. 1987. W. M. L. Rev. Soc. Vini Ital. Dott. Echechipia.. 1:21. Baird-Parker. E. Heinmann. Equilibrium states. 1979. Williams & Wilkins.... Gershwin. Symp.. Changes in the wild yeast flora of sulfited grape musts. 172:389. J. H. J. Howland. Y. K. D. Partie 1.C. 5:369. Herraiz. Arch.. OIV 58(652–653):624. Sci. Ocular injury due to sulfur dioxide. E. Prespectivas del uso del SO2 en vinificación. Gesundh. Food Res. and Trüper. 1981. 1990]. 1989. A.A. Inc. and Decorres. 1976. 1976.. (Yamanashi) 15:29. Bull.. The use of other chemical preservatives: Sulfite and nitrite. M. I. and Kanoh.. C. p. 1911.J. J. In The Microbiology Safety and Quality of Food. Vitic. 1994. 200.H. Obst. Immunol. Combination of sulfur dioxide with concentrated orange juice. Maier.. 1989. of sulfites and a few complex compounds of sulfurous acid in killing germs and retarding their development. S. Forsch. Heinzel.D. Gomes. W. 1947. Vitic. 1976. 2000. Immunol. a scientific status summary. 1995. 1990. 36:297 [CA (1911). N. Enol. M. Technol.V. Food Agric.W. J. 1):139. Bacteriol. 1950.C. Opthalmol. 1954. Grand rounds: Adverse reactions to wine. L. Uber die gegenseitige Beeinflussung von Sorbinsäure un schwefliger Säure. 1975. and Zaritzky. S..F. Exp. a critical review. La couleur des vins rouges. 17(3):185.P.A. R. Lebensm.. Eds. Pickled onion-induced asthma — a model of sulfite-sensitive asthma. D. and Vas. and Carr.. . (London) 42:1154. 31:275.. Vigne Vin 18:195. Lebensm. Adattamento a dosi elevate di SO2 (100 gr/hl) di lieviti (Saccharomyces cerevisiae var. 21:153. Gehman. 1956. H. The antimicrobial activity of sulfur dioxide — with particular reference to fermented and nonfermented fruit juices. OIV 58(652–653):617. J. H. Kais. Giannuzzi...D. Hinze. Krieg. 75:411. The preservative action of acid substances in food. Aspen Publishers. 319:277.S. Toxicol.. Sulfites as food additives. and Cabezudo. and Volter. Evaluation of teratogenic potential of sodium sulfite in rats.. G. Sci.. 38:755. M. 1976..W. 1985. I. J. Z. and Simon. D. Allergy 25:698–703. ellipsoideus) gia caratterizzati da una certa resistenza all'antisellico. and da Silva Babo. Ingram..E. Am. Toxicol.G. and Jacobsen. Munoz. Glories.. 12(2):123.M. Arb. Food Agric.162 Antimicrobials in Food Gastaminza. Hailer. M. Effect of dissolved oxygen on free sulfur dioxide in red wines. 29:117..W. Tabar. N. Störungen im Schwefelstoffwechsel als Ursache der SO2Bildung durch Weinhefen. and Williams.. Flecher.M. Baltimore. Enol. Appl. Food Technol.T... Haznedari. Holt.. 5:53. S. Gunnison. Torres. G. A.. Unters. R. 83:1079 [erratum 85(1 pt. M. MD. Ingram.M.. Holt. H. Ind.. K. Wein Wissensch.G. M. Institute of Food Technologists. Hammond. Ser.F. Heintz.B.. T. Forsch.. Lund. Ser. K. Ottoway. I. 5:89. B. Les technologies de vinification permettant de diminuer les doses de SO2. Inst. S. Itami. 1985. and Holzer. Les equilibres des anthocyanes et des rouges. Allergy Clin. K. Effect of sulfur dioxide on microbial growth in refrigerated pre-peeled potatoes packaged in plastic films. Gaithersburg. Les technologies de vinification permettant de diminuer des doses de SO2. Goto. Oxygen isotopic study of the oxidation of sulfur dioxide by hydrogen peroxide in the atmosphere.. and Coppock.M. 1980. Ough. and Tuft. 1985. 1984. Quirce. T. Experiments on the properties of free sulfurous acid. Rev. M. and Kumar. S. J. D. J.. 1970. 1989. Drug Chem.. S. Gemeueseverwer.H.R. T. G. Sneath.J. Connaiss. Rapid decrease in the adenosine triphosphate content in Lactobacillus malii and Leuconostoc mesentroides after incubation with low concentrations of sulfite. Bull. M. Chem. S. F. J... 1986. ASC Symp.D. W. Staley. Agroquim. Gekoppelte Oxydation von Askorbinsäure und schwefliger Säure. W. Unters. Kawasaki. Hernandez.F. and Gould. Ind. 29:297.N. Bergey’s Manual of Determinative Bacteriology. 61:555. 142:102. Ema. Modellversuche zum Verhalten der schwefligen Säure bei oxydativen Prozessen. Wisser. Z. CRC Crit. 9th ed. Nagy.. Jacobs. S. Clin. Aliment.T. Grant.M. Bock.M. and Osman. Allergy Clin. Gould. Sulfite hypersensitivity. A. J.

A. 1983. Avol. 136:1127. 1954. Unters. J. J.K. 1985.J. Dis.S. M. Food Sci. 1983. 1981. Karova. A. Lafon-Lafourcade. Rome.. and Kindraka. 10:565. Q. Juven.L. Les Bactéries acetiques du vin. 46:874. and Papanecolaou. Intl. Food Technol. Stummschwefelung-Ein Weg zur Konsevierung von Traubemost. Joyeux. C. Levi. Ramirez-Martinez.D. Rep. J.. R. J. D. E. B. Preservation of comminuted orange products. Mitt. Am. 35:105–112. Food Sci. emulsifiers. A. Preservation of citron in brines with some food preservatives. J. and Gallander. 21:317. Nagano-ken Shokuhin Kogyo Shikenjo Kenkyu Hokoku 12:90 [CA 102(15):130646a].G. P. and Chadha. Bull. The use of sulfite in meat processing. Rebe Wein Klosterneuburg 7A:287.C. Codounis. The impact of various antioxidants on flavor stability. 67. C. G. Metabolism of acetic acid bacteria in grape must: Consequences on alcoholic and malolactic fermentation. T.. Food Sci. 1974. 1981. L. Enol. Am. Nakajima. Khranit. and Ribéreau-Gayon. Vitic. Lea.L.. 1985. flour-treatment agents. stabilizers. and Kircheva. I.. Lafon-Lafourcade.-G.M. The chemistry and technology of the pre-treatment and preservation of fruit and vegetable products with sulfur dioxide and sulfites. Studies on the suitability of polypropylene pouches for packing mango pulp. 29:241 [CA 100(23):190455f]. H. Chichester. Food Prot. Technol.C. Food Technol. Lenz. 1972. B.. Occurrence of lactic acid bacteria during different stages of vinification and conservation of wines. 1984. and Joyeux. S. Jurd.. 1984. Analytical techniques for the estimation of sulphite binding components in ciders and wines. Ed. .. Mycol..C. and Munjal. Ford. 1989. Cutting.. M. and Shiozawa.. (London) 1974:717. 48:52. T. Academic Press. Kalra. J. and Anand. Inst. J. 1957. Georgike Ereuna 5:411 [CA 98(3):15685z]. 1985. S. Lloyd. 1982. Assoc.W. Master Brew.R.. 1208. J. p. Effect of pH.. Carr. 1984.D. E. In Lactic Acid Bacteria in Beverages and Foods.. A.J.B. Toxicological evaluations of certain food additives with a review of general principles and of specifications.. Effect of insoluble solids on the sulfur dioxide content and rate of malolactic fermentation in white table wines. 1975. and Fowler. S. 1967. D. H. Lafon-Lafourcade. H. OIV 608:803. J... OIV 58(652–653):590... Meet. A. J. Replicated dose-response studies of sulfur dioxide effects in normal. 4:247. Plant Pathol. 1987. Manca de Narda. Influence of heat and sulfur dioxide treatment on some quality characteristics of intermediate-moisture bananas. Joslyn. Use of high molecular weight high density (HMHD) film pouches for processing and storage of mango and tomato pulps. Vitic. H. and Shomen. 1210. FAO Nutr. J. 1978. sulfur dioxide and ethanol concentrations on the growth of lactic acid bacteria isolated from Cafayate (Argentina) wines.. Technol. Kielhofer. Shamoo.L. 181:417.G. Microbiol. A. London. C. 1982.B. J. Aliment. Lafon-Lafourcade. Klimovitz.. 1984.H.. Technol. acids. Takanami. Eds. Studies on dried persimmons. Cleavage of thiamine pyrophosphate by sulfite in Saccharomyces cerevisiae. 17th Rep. Food and Agriculture Organization of the United Nations. J. antioxidants.. 1975. Plovdiv. Lebensm. K.. S.. M. 5:241.S.R. Oguri. M. New York.. W. Kuwabara. Z. A.. Spoilage of soft drinks caused by bacterial flocculation. 15:557. 1982. Respir. Academic Press... 1980. Aliment. Chem. Joint FAO/WHO Expert Committee on Food Additives. Kaul. Microbiol. atopic and asthmatic volunteers. Yoshida. Vkusova Prom-st.G. Forsch. and Ribéreau-Gayon. B.. and Braverman. Kidney. I. and Whiting.O. Nauchni Tr. Maini. S. 1974.. A. S.A.L. Am. Diwan. A. 2000. J. Linn. Food and Agriculture Organization of the United Nations. C. Liu. J. Indian J. J. E..F. 40A. J. Bull.. Die Bestimmung der gesamten und freien schwefligen Säure im Wein. and Aumann. Lafon-Lafourcade. Food Res. I. Biological stabilization of vinegar by sulfitation. S. Appl. Tech. 5:97. P.W.. Effectiveness of ammonia and sulfur dioxide fumigation on control of certain postharvest diseases of apples. 12:211.. auch Gegenwart von Askorbensäure.F. 33:194. R. and Strosser de Saad. Nutr. Rôle des microorganismes dans la formation de substances combinant le SO2 . Peng. Ind. L. J. Katsaboxakis.. 40. Toxicology evaluations of some antimicrobials. J. Liu... and Holzer. 132–134. Weinwritschaft 114:1206. Sci. S.. Factors of the malo-lactic fermentations of wines. 1987. 26:70. 34:44.L. Ser. and Hackney. Enol. Effect of pH and sulfur dioxide on the rate of malolactic fermentation in red table wines. Environ. and Padau. Rev. K. F. Vissh. p. Am.A.J. pp. E. Carre. and Gallander.Sulfur Dioxide and Sulfites 163 Jakob. and bases.. The Chemistry of Plant Pigments.R. Rome. Adv. Joint FAO/WHO Expert Committee on Food Additives. 21:410.C. S. R.

M. Clustered outbreak of adverse reactions to a salsa containing high-levels of sulfites. Wiley. T. and Ahmedullah. 1987. Adachi. Ough. Stevenson.. Pertanika 11:407.. and Luvisi. 50:256. Unters. sulfites. Analyst 53:142. and Iwaida. D. S. Ough. H. M.M. pp. H.. R. Hortic. Obara.. R.A. S. J. 1976. McAlpine. Sulfite residues in restaurant salads. Othman. Vitic. Am.. D. M.E... 222–235. Teuber.. Vitic. Mayer. Plant Dis. Fujita. Effects of chemical treatments on the shelf life of rambutans (Nephelium lappaceum). G. pp.. Millet..M. E. False positive and false negative reactions encountered in the use of sulfite test strips for the detection of sulfite-treated foods. Lebensm.. 38:171. Sudo. Massignan. 28(3):71. 1979. J.E. Vitic. 70:1050. 30:136. Martin.. Allergy Clin. D. and Amerine. and Taylor. M. Bordeu. 339–358. J. Van Nostrand. W. Nagy. Y. Ogawa. An analytical survey of four California varietal white table wines.G. Chest 87(Suppl. 49:126. 4095. Use of sulfur dioxide in winemaking. Vilas. Am. Nordlee. Allergy Proc. 1988. 1995. and Soule. Vini Ital. 1988. G.. Frücheverwert 27:1.. Qualitative Chemical Analysis. O.... Z. 1987. Fuke. Enol... and Navara. S. Ikuzawa. Vol. . Nordlee. R.A. K. and Ough. pp.. pH and dimethyl dicarbonate on the growth of Saccharomyces cerevisiae Montrachet and Leuconostoc oenos MCW. and Gambacorta. and other drugs.A. Vyznam oxidu siriitého pre kvalitu a stabilitu vin z aspektu jeho koncentrácie vo vinohradnickych vyrobkoch. 130–179. Precipitating factors in asthma.L. A. Martin... Nicklas. 297:113..A. II. No. Ito.. S.S. Toyoda. 393. Biochem. Oblmeyer. G. Springer-Verlag. K. 1979. Y.A. Meyerhof. 1979. and Fahmy. K. M. and Murphy. 1988.. Tanaka. Gubler. F.J. De Leo. M. Reduction du sulfate en sulfite et sa signification taxonomique pour las classification des levures. 1985.. Agric. Z. T. Sulfite residues in maraschino cherries. Immunol. C.F. Pellegrini. J.N.. Sulfur Oxides. R. Nelson. L. Food Prot. 81:537. R. L’azoto assimilabile e la tiamina in fermentazione loro importanza qualifattori di qualita’dei vini. Kunkee. 1985. K. 1988.. J. T. New York. 498–500. 52:386. Am. 1986. R. Lett.):50S.J. J.. University of California. Nordlee. Ind. Mathison.A. T. E. Publ.C. E. Aspirin. Packaging and decay control systems for storage and transit of table grapes for export.. M. Versini. Egypt.E. 1938.A.. Minarik. El-Tobshy. and Lonvaud-Funel. and Taylor. Microbiol. and Tonon. Naidu.. and Crowell.S. L. Note on the oxidation of sulfite by air. Appl. National Academy of Science. A.C.... P. 10:349. 53–54. 1986.. J.. J. and Jackson. 11:11. New Series.. Harvesting and handling California table grapes for market. Über die Koppelung zwischen Oxydoreduktion and Phosphatveresterung bei die anaeroben Kohlen-hydratspaltion.B. Nelson. Ough. E.. Determination of sulfur dioxide in grapes and wines. Loscutoff.. Berlin.. S. J. 1928. Nonogri..F.S. Hamano. D. Matsuiki.D.. S. Committee on Sulfur Oxides. Rech. K.M. J. Die Bedeutung des biologischen Säure-Bedarf der Weine. 1985. J. 7–9. New York. S. C. P.K. 58:95. and Mohle. and Simon. P. Sulfites: A review with emphasis on biochemistry and clinical application. V.L. E. K. 7:279.D.R. 2000.. C.. 1988. The interaction of sulfur dioxide. pp. S.L. National Research Council. K. Food Prot. La Notte.A. 27:74. and Taylor. Modern Methods of Plant Analysis.. Prog. Hasegawa. K. 1933. A. Izumi.M. M.S. J. T. National Academy of Science.A. Conserve 59:130 [CA 101(25):228778z]. and Huang. A..A. 1977. 1978. Effects of controlled atmosphere cold storage on some organoleptic characteristics of table grapes. Washington. Y.. Mitt.164 Antimicrobials in Food Mansour.S. The viable but non-culturable state of wine micro-organisms during storage.. 8th ed. J. Navara.. J. Enol. 1986. Ough. W. S. Minarik.. Sci.. C. Effect of in-package sulfur dioxide generator on postharvest decay and quality of Banati grapes. Z. Klosterneuburg Rebe Wein Obst. 168:299. Viti Vini. Methods for Analysis of Musts and Wines. V. 48–49.. Bledsoe. 1975. Food Sci.B.A. E..S.. 6. Weinwirtschaft 155:223. Marois. 1989. Enol. Comparative determination of free and combined sulphites in foods by the modified Rankine method and flame photometric detection gas chromatography. Nelson. Mitsuhashi. Mason.. Margheri. L. Control of Botrytis cinerea on grape berries during postharvest storage with reduced levels of sulfur dioxide. and Abdullah. Eds. D. Zum vorkommen von Saccharomyces ludwigii Hansen in geschwefelten alkoholarmen Jungweinen. 39:279. pp. Berkeley. B.E. Food Sci.M. Vinohrad (Bratislavia) 23(3):62. In Wine Analysis. Mohamed. Ohkubo. C. Forsch. H. Linskens.. 1984. 1984. A.

1977. pp. 1962. Microbiol. Paul. Sand. Reid. Kohler.E. 1978. U. F. J..222. 125:89. K. 46:108. 1972. Rankine.S. M. R. Exp.J. J. Studies on SO2 treatment of minced goat meat. A review. 1987.J. H. Le rôle de l'anhydride sulfureux en vinification. G. Beitrug zur Kenntnis der antimikrobiellen Wirkung der schwefligen Säure. Cas particuliers de la microflore des mofts dan trois caves du vignoble nantais. A. H.422. Prakash.C.. benzoate. 1970. Unters. Schimz. benzoate and sulfur dioxide and packaged under various oxygen levels.V. L. 1983. 105–115. Keine Einsparung von schwefliger Säure. Food Sci.J. Piracci. 1958.O. Food Sci. Mitt.. dangerous for some. Forsch. Effect on storage life and organoleptic qualities. Roland. VII. M. Lebensm. 118:413. Indian Food Packer 36(5):23. Alkalimetric determination of sulfur dioxide in wine. Diary Res. Kiosterneuburg Rebe Wein Obst. Molin. Biomass and patulin production by Byssochlamys nivea in apple juice as affected by sorbate.. J. Effect of sodium and potassium metabisulfites on the shelflife ofkhoa. Miltenberger. Brew. Zygosaccharomyces baillii Eine zunehmende Gefahr für die Erfrischungsgetränke.L. J. Schimz.. K. Sulfur Dioxide Applications. Arch. B. Fed. 1964. K. Consequence di una corretta acidificazione per la stabilita chimico fisica e biologica dei vini bianchi del Lazio. J.. Rapid decrease of ATP content in intact cells of Saccharomyces cerevisiae after incubation with low concentrations of sulfite. and Wittmann. N. 1980. J. 1979. Food Sci. Rodriguez. Roland. pp. Effects of sorbate. Ed. Roberts. 1980. Beuchat.. Viability of Byssochlamys nivea in applesauce containing sorbate. and Aerny. L. Vignes Vins 275:9. H.. 47:237. 1982. 1963. Sulfites: Safe for most.. N. J. Schneyder. 9:220. H. Technol. E. gebundenen und gesamten schwefligen Säure mittels des Apparates von Lieb and Zacherl. Brauwelt 120:418. R. Pearlstein. Asian J. and Sharma.C. Long Island City. avant et apres sulfitation. An experimental study of hypersecretion of mucus in the bronchial tree. Modeling of sulfur dioxide uptake in pre-peeled potatoes of different geometrical shapes. and Curschmann. and Holzer. in Foods. R. I.. 1966.M. R. K. Beverages. P. Analyst 53:160. H.M. Schroeter.S. Schimz. L. Microbial Inhibition in Foods.L. K. Am. 1984. Rhem. D.L. 1951. J.. sulfur dioxide and temperature. and Heaton. Schmitt. 27:87. Aust.O. Papazian. and McWeeny. 29:40. Influence des constituants de la thiamine. The use of sulfur dioxide in the food industry. and Pharmaceuticals. Biochem. and Hitchcock. Vini Ital.K. and Bains. Fruchteverwert. 49:402. Ranote. Technol. Worthington. Almqvist and Wiksell.S. Ann. and Brelet. Beuchat. H. Roland. Pergamon.F. and Spera. OIV 58(652–653):515. Patel.. Tech. Rhem.V. 1969. Food Technol. J. 1928. Vignevini 11(11):15. Vinificazione in presenza di tiamina di alcuni vini bianchi del Lazio. Rundschau 47:170. 1977. Schelhorn. A. 1984a.. Schopfer. G. Commun. and Holzer. Food Prot. Soc. 1988. Agric. 44:437. Wirksambeit und Wirkungsbereich der schwefligen Säure. A. . sur la vitesse de la fermentation alcoholique. A. 51:618.R. R. 47:685. Spirit Rev. 24:299. Microbiol.. and Zaritzky. Pathol.-F. 1983. 1985.. J. Food Prot. F.S. R. Bull. NY. N. Pilot-scale studies on extended aeration at fermentor fill. 1985. 121:225.L. 18:199. G. Piracci.. Note on the stability of solutions of potassium metabisulfite.E. Phillips. 1984b. Low concentrations of sulfite lead to a rapid decrease in ATP concentration in Saccharomyces cerevisiae X 2180 by activating an ATP-hydrolyzing enzyme located on the cell surface. 11:1977. Goteborg.C. K. 1996. L. and Pocock. 191–227. K. J..J. Lebensm. and Vlcek.J. Mitt. The effect of sulfite on the yeast Saccharomyces cerevisiae. Mass analytische Bestimmung der freien schwefligen Säure in Wein mit Jodsäure. Meet. K. A. Chem. Reddy. Arch. sulfur dioxide and temperature on growth and patulin production by Byssochlamys nivea in grape juice. FDA Consumer December:11–14.. Eur. and Mandokhot.H. Br.. J.O. J. L.S. J. Dtsch.. 7:221.R. Poulard. H. Untersuchunge über Konservierungsmittel. 1986. A. Juice of Kinnow fruit. Die alkalemetrische Bestimmung der Freien... Weinwirtsch.E. 4:89. benzoate. Soc.Sulfur Dioxide and Sulfites 165 Ournac. Wine Brew. and Beuchat... 25(145):167.. B. 1984. Rebe Wein Klosterneuburg 8A:21. Z.

A. Detection characteristics of commercially available sulfite detection test (Sulfitest): Problem of decreasing sensitivity and false negative reactions. Growth of wine yeast and formation of addition compounds.. G. C. Vitic. 1986. Med. and Chauvet. Les technologies de vinification permettant de diminuer les doses de SO2. and Serpentino Opessio. 1985.C.J. .. G. Sulfur dioxide with carbonyl compounds and weakly acidic pH.. Ubigli. and Converse. Washington. 1984. G. An assessment of the role of acetaldehyde.M. 16:73. Enol. T. Vahl. Report SCOGS-15 Bureau of Foods. J. Komatsu. The Organic Chemistry of Sulfur Tetracovalent Sulfur Compounds. 35:458. P.. C. Off. D. Valouyko. Groeneveld. and Solomons.M. 29:121..M. Koepke. P. R. Verernova. Ammonia and sulfur dioxide fumigation of pear fruits in the control of bitter rot of pear caused by Glomerella cingulata.F.. and Thompson.166 Antimicrobials in Food Schwedt. J.O. II. Chem.L. 1982. Mycol. 49:1067. S. J.. 1985.. Food Prot. S. Ann. pp. Somers.. Lebensm. 1983. Clin. 40:839. T. 1985. Gross. A. Response of allergic and normal persons to sulfiting agents in wine: Determination of thiosulfate excretion in urine. 1986. and Weisgras. J. Assoc. Suter. 1976. (Yamanashi Univ. R.H. Wanderer.. packaging and additives on the microbiological shelf-life of ground beef. 1988. S. Lazar. 1989.. Food and Drug Administration. Vally.. Anal. Evolution of red wine. Kvasny Prum. R. T. W. 2001. Vitic. K.M. Bisulfite catalyzed transformation of cytosine and cytidene. S. Kaluim. and Shimada. 1983.T. Suzzi. S. Biophys. Saccharomyces strain selection in minimizing sulfur dioxide requirements during vinification. 35:1792. G. 1980. T. Afr. Christiansen. Indian Food Packer 38(4):64.. M. 344. and Anand. Plant Pathol. Y. A.... Norton.M. J. J. N. Vitis 26:27. Von Holy.. Biological Calculations.P.W. G. A. G. 1989. 1988. Ripper procedure for determining sulfur dioxide in wine: Collaborative study.G. Afr. Enol.A. S. J. J. Andonova. M. and Goranov. Appl. and Shaparis. Vitic. Tsevat. Vinar. Cloette. D. 32(4):23 [CA 99(19):157002u]. Connaiss. Food Sci. Sudraud.E. Prove sull’impiego dell’ H2S in fase prefermentativa in sostituzione della SO2. Holden. Inst. and Yentür. Efficiency of chemical preservatives in controlling fermentations of preserves by Bacillus cereus. OIV 58(652–653):637. N.. Role of sulfite in wine induced asthma: Single dose and cumulative dose studies. Sci. and Zambonelli. Allergy 58:41. pp. 63:194. and Holzapfel.G..W. S.. H. Riv. New York.. 52:240. and Stoyla.) 19:13. di Stefano. and Basarova. Department of Health.C. 136–144. P. and Ogorodnik. J. S. 1987. Activate antilevure de l’anhydride sulfureux moleculaire. Segal. J. 1984. 1968. Education and Welfare. Immunol.. 1987. H... Wiener. Uzuka. W. Med. Taylor... Bayhan. Shapiro. New York.E. Fatal asthma after ingestion of sulfite-containing wine. M. Nitrat/Nitrit und Sulfit in Lebensmitteln...W.preservatives on asthmatic children. Res. Thorax 56:763–769. V. Vigne Vin 19:31. R. J. and Wescombe. H. 84:766. J. Sharma. Effect of sulfur dioxide on the biological stability of wine vinegar.. and Hayrynen. Dtsch. Tvorba oxidu sirtého pivovarskymi kvasinkami. Am. and Dowling..B. Wiley. Enol. Select Committee on GRAS Substances. 81:1159.. Chem. Sethi. 1986. Tanaka. 1970.. Spayd. and Weinberg. 1984. C.... G. 1944.A.D. 36:199.A. J. Commun.K. Indian J. J. 1–25. L. Splittstoesser. 70:404..B.C. K.. L. J. 1980. M. Bush. Nordlee. N. L. The effect of storage temperature. A. Intern. Microbiol.B.. I. Mikyska. Tompkin.E. B... Antibotulinal efficacy of sulfur dioxide in meat.T. Teststäbchen-Reflektometrie für Ascorbinsäure. Ann. 39:1096. Nomura. Romano.C. G. Castino. and Busse.H.. 107:263. Bull. Town. M. M. Selner. p. Wiley. 1987.. J. Biochem.. Effect of various inhibitors on the growth of lactic acid bacteria in model grape juice systems. E. Allergy Clin. Spirov. The effects of soft-drink.G. R. Evaluation of the health aspects of sulfiting agents as food ingredients. J. Environ. Influence of sulfur dioxide generators on red raspberry quality during postharvest storage. Rundschau 82:111. Pavlenko. Steinman.. Sensitivity to sulfited foods among sulfite-sensitive subjects with asthma.

R. Zum Gehalt und Zubereitungsverlust von Sulfit in Lebensmitteln.J.H. ASEAN Food J. Resistance of yeast species to benzoic and sorbic acids and to sulfur dioxide. 1970. 9:155.. Williams. Storage of citrus fruit for processing using antiseptic solution rinsing. A–l during the fermentation and storage of grape must and red wine.. BNF Nutr. and Heimann. 1935. E. Srikok. Bindung von schwefliger Säure an Oxydationprodukte der Askorbinsäure. D. B. P.E. O. S. 1985.L. Yakobashvili. Z. Wisser. Modellveisuche zum Verholten der schwefligen Säure bei oxydativen Prozessen. 1984. Ovoshchesush. and Georgadze. Unters. S. Appl. Fleet. R. 57:536. Waterman. J. A. Volter. 64:421. M. Gomolmanee.. R.Sulfur Dioxide and Sulfites 167 Wara-Aswapati.. C. Sulfur dioxide in foods — chemical interactions. Lebensm.H. Soc. Am. . G. 1984. Food Prot. and Buchman. Growth of the Acetobacter sp. II. Forsch. Wever. G. C. Pranst. Z. Wedzicha.. J. Cleavage of vitamin with sulfite. Lebensm. and Bailey. and Eschenbruch. Part 2. J. 185:457. D. Bull... Lee. Studies on bacteria isolated from Japanese wines. Forsch.. and Ino. 6(4):8–14.. 1984. J. Studies of crystalline vitamin B.E... 2000. 1987. R.D. Unters.A. Yamanashi-ken Shokuhin Kogyo Shidosho Kenkyu Hokoku 16:13. Watanabe.. Food Testing Anal. Wibowo.W. K.. 1988. J. Sulfites: An important food safety issue.. Keresztesy. 48:564. R. 142:180. W.L.. Diachenko. T. 1988. Factors affecting the induction of malolactic fermentation in red wines with Leuconostoc oenos.R. Warner. Konservn. Effect of benomyl and sulfur dioxide on storage life of fresh longan. M. Warth. 7:40. III.C. Bacteriol. Chem. 4(3):73. L. and Boon-Long.R.B.

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......................................................................................206 Fermented Meats and Dry Cured Hams.......................................185 Vacuum-Packaged and Fermented Meats ...........207 Alternatives to the Use of Nitrate or Nitrite.........................193 Perishable Cured Meats ......................................................................................205 Since 1990.....................................179 Summary for 1960–1970 ...............................................................................171 Before 1950....................................................................................................................................................................................................................................................................................................................................................................................................193 1980–1990...................................................................196 Perigo Factor ....................................202 Spoilage of Cured Meats ..........................................................................................................................................171 Summary for before 1950........................................................................................................176 1960–1970..................................................................................174 1950–1960.....................................................................................................................................................188 Bacon............................................................................................................177 Shelf-Stable Cured Meats ............................177 Perishable Cured Meats ............................................................................................................................................................................................................................................. Bruce Tompkin Research History .......................216 169 ........191 Summary for 1970–1980 .........................................................................................................................................................................................................................................................................................................................215 Green Discoloration in Cured Meats.....................................................192 The Perigo Factor in Cured Meats ........................................................................210 Effect of Nitrite on Enteric Pathogens ....................................................................................................199 Mechanism of Nitrite Inhibition ......................................................................................................................................................6 CONTENTS Nitrite R.................................................................................................195 Botulinal Challenge Tests . aureus and Other Bacteria .......................203 Summary for 1980–1990 ....192 The Perigo Factor in Media ..............................................................................................................193 Mechanism of Nitrite Inhibition ...............................................182 Perishable Canned Cured Meat and Slurries of Meat......................................................................200 Miscellaneous Studies in Laboratory Media ................182 Shelf-Stable Cured Meat and the Perigo Factor.....................................................................................212 Effect of Nitrite on Yeasts and Molds ....................................................................174 Summary for 1950–1960 ..........210 Cheese..................................................181 1970–1980......................................................210 Seafood ...........................................................................................................................................................................................................................................................................................................198 Alternatives to Nitrite for Botulinal Protection .........194 Assessment of the Botulinal Risk in Cured Meats .......................................................................189 Tests with S.................................................

............................... Cerveny (1980) described many of the changes in the production and marketing of cured meats in the United States since 1900.......221 This review of the antimicrobial properties of nitrite is one among many........ Ingram............. Reported outbreaks of botulism attributed to meat and poultry products and an assessment of nitrite for botulinal protection have been summarized (Tompkin.. 2002) have been developed.. particularly in the United Kingdom and Europe..... the reader is encouraged to read the historical ............. The use of nitrite and/or nitrate in foods has been influenced by a variety of factors...... Pivnick...... 1980...... and physiologic effects of nitrate and nitrite (Archer............... 1992)... 1963... 1979)... however.................. Skovgaard... 1981............. The status of nitrate and nitrite in food and water until about 1990.................................. in certain respects from other reviews.... It is tempting to delete a significant quantity of information from this third revision of the chapter. Eklund (1982) reviewed the potential risk of botulism in fishery products and the role of nitrite as a preservative....... The review by Gray et al................... Lechowich et al..218 References .. It is a curious thought that if nitrite were disapproved....................... The time and resources expended to resolve the nitrite issue were enormous and worldwide. the reader should consider reactions with heme compounds (Fox.......... At the heart of the issue was whether nitrite would continue to be an approved food additive... the information is being retained for its historical value and to provide a foundation for future research when nitrite and nitrate are next called into question.. 1971... the selection of the experimental procedure.. would disappear from the market.... 1978........216 Regulations Governing the Use of Nitrite and Nitrate .............. Bard and Townsend.. (1981) and Pegg and Shahidi (2000) of the effect of nitrite on the stability of cured meat flavor provides additional possibilities......... Duncan... 1974. Current regulations for the use of nitrite and nitrate and modern..... Extensive updates on the chemistry and impact of nitrite on the color and flavor of cured meats (Pegg and Shahidi..... is discussed in Hill (1991)....... The discovery in 1969 that nitrosamines can be formed in foods containing nitrite led to a series of events................ Pegg and Shahidi........... then cured meats... their use was to fulfill the basic need to preserve food.217 Assay Method ............ It should be remembered that most of the research during that period was driven by the potential risk to consumers from dietary intake of nitrite and the Delaney clause that declared additives found to be carcinogenic in any animal test could not be used in food in the United States.... The many chemical reactions that occur when nitrite is added to meat offer clues to the reactions that may occur with microbial cells. Other comprehensive reviews are available that provide additional information and other perspectives (Anderton. This chapter concentrates on the antimicrobial research. Initially. Binkerd and Kolari (1975) reviewed the history of nitrite and nitrate as curing agents for meats. As questions were raised from about 1970 to 2002 over the toxicologic safety of nitrite.. At the same time a considerable amount of research was directed toward identifying the risks associated with the use of nitrite in food.. 1980).............. insight is provided into why the research was performed. For this reason...................... toxicologic.... this chapter would not exist in this book............. Sofos et al..... considerable effort was directed toward verifying the benefits of nitrite relative to control of certain pathogens and quality attributes.................................. To better understand this complex issue.........217 Toxicology....................... however... 1980.170 Antimicrobials in Food Summary for 1990–2002 ................................ Roberts et al................ 2000) and other components of meat (Cassens et al.. With the new millennium a different perspective has evolved....... 1966..... 1979a.. By generally arranging the information in chronologic order. Roberts et al....... 1991....... Benedict....... 1970. as we know them....... and the information that was available to influence the researchers’ conclusions......... Holley. 2000) and the microbiologic........ If nitrite were not available..... 1991.............. controlled processing conditions provide the benefits of food preservation with no measurable risk to consumers.. It differs...

the value of nitrate. This concept became widely accepted. were not inhibitory to any of the strains. RESEARCH HISTORY BEFORE 1950 Tucker (1930) studied the proteolytic activity of a Clostridium putrificum isolate from sour ham. nitrite was considered to have no value in meat other than for flavor and color development. the value of nitrate as a preservative had been so degraded that it was almost unanimously agreed in the United States that nitrate could be omitted from most cured meats without loss of antimicrobial protection or preservation. of the three. The same results were obtained with Clostridium putrefaciens at 23°C. is responsible for retarding proteolysis by C.000 µg/g cannot be relied on to inhibit putrefactive anaerobes if other conditions are favorable for growth.000 µg/g. Clever as the scientific community has been in more clearly defining the antimicrobial properties of nitrite. and Clostridium sporogenes. research had clearly demonstrated that nitrite has significant antimicrobial properties. and dealing with perishable canned hams that were being temperature abused in the marketplace. By then it was also more firmly believed that nitrous acid derived from nitrite in the acid environment of meat is responsible for microbial inhibition. found that sodium nitrate up to 22. Until the early 1940s. solving a problem of sour hams. Unlike the other antimicrobial agents discussed in this text. Considering that research continues to reveal new interactions among the chemical constituents of cured meat. At 44. or 12. The authors concluded that nitrate at concentrations as low as 22. 44. it remains to be seen whether wisdom has prevailed with the voluntary abandonment of nitrate as an additive to many meat products in the United States.0% sodium chloride (salt).000 µg/g. One of the messages from the 1920s is that nitrate serves as a potential reservoir for conversion to nitrite and thus to nitrous acid. Tanner and Evans (1934) reported that 5900 µg/g of sodium nitrite inhibited 9 of 12 strains of clostridia in nutrient broth and all 12 strains in dextrose broth. using Clostridium botulinum. data appeared that began to prove the opposite. reproducible results in all instances.000 and 44. By the time the nitrosamine controversy arose in 1969 and the early 1970s.000 µg/g of sodium nitrite in a pork infusion broth buffered to pH 7. At levels between 22. By the middle 1950s. irregular inhibition occurred among seven botulinal cultures. The list of recognized factors influencing the antimicrobial efficacy of nitrite in cured meats continues to grow 60 years later. .0.000 µg/g of sodium nitrate. This basic flaw in experimental design led to results that misguided and hardened opinions in the industry for 20 years. 6 of the 7 cultures were inhibited. despite meat having a pH closer to 6. He concluded that.000 µg/g did not inhibit the 12 strains tested in either pork infusion or egg-meat media. Levels of 3100 µg/g in pork infusion and 3900 µg/g in egg-meat. for that matter. Most of the research on sour hams involved test tube experiments with culture media above pH 7.6 and held at 37°C. there has been a long. Proteolysis was inhibited by 4. controversial history over whether nitrate and nitrite have antimicrobial properties. We are still ignorant of all the significant factors that influence the efficacy of nitrite or. and the value of nitrate was relegated to serving merely as a reservoir for the generation of nitrite.Nitrite 171 documentary of Cassens (1990). Research of the 1920s and 1930s focused on using nitrite to cure meat.0. C. Nitrate was believed to provide the antimicrobial effect. During the 1940s and early 1950s. not nitrate. only salt was used in sufficient concentration in commercial hams to prevent proteolytic activity at 37°C. It is still difficult to design experiments that yield predictable. putrificum. the highest levels tested. putrificum. Cassen’s text places the research outlined in this chapter into its proper historical perspective relative to the politics and science of the nitrite issue. Tanner and Evans (1933). has the proper research been conducted and interpreted in an unbiased manner to justify the elimination of nitrate from the majority of cured meats? A primary reason for the controversy over the antimicrobial effect of nitrate and nitrite that prevailed into the 1940s was ignorance of the significance of pH. He suggested that nitrite.

in mixtures containing reducing substances.e. was not pursued. however.. so clearly stated and available to researchers at that time. They further believed that the claim of meat packers that small amounts of nitrate in the pickle produced better preservation of the meat.. Nitrite could not replace nitrate. Perishable (i. the results were quite different. and what factors influence depletion? Would the more rapid chilling process as practiced in North America adversely affect the quality of English-style bacon? Can bacon acceptable to the English trade be produced by the use of rapidly chilled meat and with nitrite in place of nitrate? Not all the questions could be answered. sugar.” Botulinal toxin was produced in the cooked meat and a few samples of raw meat. A marked inhibition was noticed. in 1934 laid the foundation for a new concept. chopped. In acid solutions. and cured meat to detect the gas-producing bacteria in raw materials and the plant environment. insufficiently heated to be stable at room temperature) canned. was borne out by their results. spiced ham and luncheon meats containing nitrate. To obtain gas production in the medium. The authors concluded that reliance cannot be placed on nitrite alone to prevent spoilage by clostridia. It is curious that Tanner and Evans (1933). The conversion . and sugar swelled in 1 to 90 days during temperature abuse at 37°C. 1964)..3).6°C in the meat.4) before autoclaving. Does nitrate serve any function other than as a precursor for nitrite? Does nitrate appreciably retard or inhibit the growth of putrefactive anaerobes? What is the mechanism behind nitrite depletion.49 to 5. citing earlier work by MacNeal and Kerr. The opinions expressed by Jensen et al. MacNeal and Kerr stated that the effect of saltpeter was probably due to the oxidizing action of the nitrate ion in the presence of hydrogen ion. both nitrate and cured meat had to be present. state the following: [P]otassium nitrate in neutral or alkaline solutions exerted no special restrictive effect on bacterial activity. They stated that if nitrate were omitted from the product. and of nitrous acid also. and nitrate in peptic digest broth (pH 7.4). It is surprising that the fundamental importance of pH. Brooks et al. and cooked pork. Satisfactory bacon can be produced with the use of nitrite. Their bold statements that a mixed nitrate–nitrite cure favors most species of aerobes but a nitrite cure “always inhibits fermentation and aids in putrefaction” were to have long-term effects within the meat industry and federal government. After the slaughtering process. The sides were then immersed in a tank and cured using a pickle solution containing potassium nitrate. the sole cause of the spoilage was found to be nitrate-reducing Bacillus species (Jensen et al. From tests with various blends of salt. It seemed that nitrate was especially valuable in preventing high degrees of acidity or souring of meat. the usual fermentative carbon dioxide swells caused by Bacillus species would be prevented and conditions for the growth of clostridia and other anaerobes would prevail. Repression of toxin formation in the raw meat was a result of other bacteria that caused a decline in pH to inhibitory levels (pH 4. nitrite. nitrite. but the research led to the following general conclusions. Perhaps the cured meat provides a source of heme. it was used as a food. as the more recent results suggest (Jacobs et al. Several interesting questions were raised. A medium was developed with nitrate. raw pork. 1934). (1940) described the contemporary method of making bacon in the United Kingdom. Evans and Tanner (1934) concluded that “the most effective component in curing mixtures is sodium chloride” and “the sodium nitrite present apparently produced no effect on the organisms. pork sides were held at ambient temperature overnight and then moved into refrigeration for 24 hours to achieve 5. Potassium nitrate was therefore considered to be particularly effective in restricting acid fermentation of organic substances that are already slightly acid. Examining 1000 cans representing all manufacturers at the time. They said that this effect was incomparably greater than that of salt and was best ascribed to the production of small amounts of nitric acid.172 Antimicrobials in Food although the nitrite was added to the media (pH 7. The characteristic cured flavor of bacon is primarily the result of the action of nitrite. Under these conditions. The reason for this specific combination of ingredients remains unknown.

. Using the spiced ham medium they reported earlier. However. increasing salt and nitrite caused increased inhibition. At pH 5.” They postulated the anticlostridial mechanisms to consist of partial conversion of nitrate to hydroxylamine. In one. Later tests involving C. 1942). Jensen and Hess stated that nitrite reacts with protein during heating and is destroyed. Stumbo et al.. increasing nitrate did not increase the inhibition of C. Subsequent tests (Tarr. Two tests were reported with perishable canned ham.01. Tarr and coworkers published a series of reports on the possible use of nitrite for preservation of fish. little or nothing is known regarding the possible bacteriostatic (or bactericidal) action of nitrites in meat pickles or in cured meats themselves. complete or strong microbial inhibition occurred. 1942). The authors suggested that the combination of heat. the authors concluded that nitrate is beneficial for preventing the growth of putrefactive anaerobes in abused perishable canned ham. which destroys anaerobes. sporogenes yielded similar results (Tarr. 1945b) that nitrite appreciably delayed germination. Even if nitrite had been uniformly accepted as a critical factor in microbial protection. Despite these results. Jensen and Hess found that heat (93°C for 4 hours) combined with 5000 µg/g.” Their tests with fish muscle showed that nitrite delayed spoilage. The effect of pH was more firmly documented in a series of tests in nutrient broth inoculated with a variety of aerobes. Others have shared this concern (Scott. In the other. little or no inhibition was observed. sporogenes 3679. Greatly increased inhibition occurred in tubes of pork heated in the range of 50°C to 65°C for 30 minutes. (1949) also examined the combined effect of heating and curing salts.2°C. Investigations on the mode of action led Tarr to conclude that nitrite is not inhibitory by sole virtue of its toxicity toward aerobic respiratory catalysts (Tarr. prevented growth of C. 1941) demonstrated the importance of pH to the efficacy of nitrite. of sodium nitrate. They also reported (Stumbo et al. although salt was the stronger inhibitor. In both tests some cans with nitrate swelled because of the growth of Bacillus species. longer heating times. Rapid chilling of the meat is not detrimental. Nitrate. which inactivates catalase and allows the accumulation of hydrogen peroxide. While Tarr was demonstrating that nitrite has strong antimicrobial properties. and salt caused destruction of anaerobic spores at much lower temperatures. Yesair and Cameron (1942) pursued this idea but concluded that curing salts do not assist in thermal destruction but inhibit outgrowth. How could reliance be placed on an unstable preservative that disappears during processing and storing? This question is still valid. They stated that the literature shows “unmistakenly that nitrate in the cure exerts a definite inhibitory effect upon bacteria. The purpose of Jensen and Hess’ work in 1941 was to determine the specific value of nitrate as a bacteriostat and as an inhibitor of bacterial spore germination and toxigenicity and its effect on the anaerobic flora of cured meats. Within the levels normally added to canned ham. Jensen et al. At pH 7. alone or in combination with other ingredients. nitrite alone inhibited anaerobic putrefaction during 30 days of abuse at 37. fewer cans became putrid in the product with both nitrate and nitrite than in the product with nitrite only. sporogenes during 4 weeks of incubation at 37.7 and 6. did not appreciably influence spoilage in tubes processed from F0 = 1 to 16 and held for a year at 28°C.2°C. Higher temperatures.0. thus serving as a desirable indicator of temperature abuse. (1945a) reached the same conclusion from tests in meat processed at 116°C. or repetitive heating did not increase this effect. Jensen and Hess (1941) advocated the virtues of nitrate and perpetuated the belief that the sole function of nitrite was for cure color development. 1941) and that the mode of action was still unknown (Tarr.Nitrite 173 of nitrate to nitrite in commercial bacon curing brines is mainly the result of the growth of micrococci. botulinum and C. thus leaving the meat in much the same state as freshly cooked uncured meat. workers at the time would have had difficulty applying the information to commercial practice. but not 2000 µg/g. nitrate. 1955). nitrite. The presence of nitrate or microbial action during the curing process is not essential for bacon flavor. Tarr and Sutherland (1940) concluded that “as far as can be ascertained from the literature.

Salt at the levels used at that time was considered the cornerstone for the preservation of cured meats. but the condition of the meat was a factor. Bulman and Ayres (1952) found that levels of salt in excess of 4. and high heat resistance. Salt was found to be the major factor retarding botulinal outgrowth in temperature-abused products. 1950–1960 Research of the 1950s clarified the relative significance of salt. A mixed cure of salt (3. there was increased inhibition of outgrowth of surviving spores by salt and. however. Halvorson (1955) later discussed the widespread problem of temperature abuse at the retail level. . even in the presence of salt or nitrite.5%) and nitrite (150 to 170 µg/g) was much more effective than either substance alone. especially if the product pH was below 7. SUMMARY FOR BEFORE 1950 A summary of the state of knowledge by 1950 might include the following: 1. In the absence of added salt. The concept of a combined effect of heating in the presence of curing salts was proposed.4% or sodium nitrate in excess of 40.1 to 6.000 µg/g were required to prevent spoilage from anaerobic spore formers in pork.174 Antimicrobials in Food Vinton et al. nitrate. 4. Nitrate was promoted as an inhibitor of anaerobic spoilage and to enhance the swelling of perishable canned cured meat by Bacillus species when temperature abused. intermediate. a combination of both nitrite and nitrate was the most inhibitory. nitrate.” This experience may have led to improvements in meat handling and equipment sanitation. The disappearance of nitrite during processing and storage was assumed to make it an unreliable preservative. and sterilized meat were of low. The basic formulation resulted in a product having a moderately high brine of 5. however. Strong opinions were expressed that nitrite alone was ineffective at the levels used in commercial practice. Steinke and Foster (1951) investigated the effect of packaging liver sausage in an oxygenimpermeable film (Saran™) that was gaining acceptance in the industry. 7. respectively. The question was raised whether the growth of spoilage organisms is influenced by the depletion of nitrite to noninhibitory levels. and nitrite). and nitrite as preservative agents and expands this information to other microorganisms. A mixed cure of all three (salt. 3.05% to 5. (1947) found the heat resistance of spore crops was not altered by adding nitrite and nitrate to the meat on which the spores were grown. yielded the maximum inhibition.37% and a pH range of 6. It is perhaps for this reason that Jensen believed so strongly about the need for nitrate in perishable canned cured meats. increased inhibition of PA 3679 was obtained as sodium nitrite was increased from 400 to 800 µg/g. Nitrite was clearly shown to be an effective antimicrobial agent.0. pasteurized. Adding 200 µg/g of sodium nitrite was considerably more inhibitory to toxin formation than the addition of 1000 µg/g of sodium nitrate. 6. Spores produced in raw. 5.5. to some degree. The relative roles of nitrate and nitrite as preservatives in cured meats were unclear. 2. Although thermal destruction was not shown to be enhanced by the presence of curing salts. An underlying factor in the research before 1940 was the unfavorable experience of 1928 and 1929 with temperature-abused perishable canned hams. During the 1930s some processors incubated perishable canned cured meat at 37°C as an index of “keeping quality. Nitrate at the level permitted in meat (1250 µg/g) was without effect. by nitrite. Nitrate was without effect.

the addition of 1. Department of Agriculture (USDA) issued a regulation to assure the safety of new perishable canned cured meat products (e. The USDA concluded that the high brine would retard the growth of C. The brine requirement of 5. A pH of 5. and 1 ounce of sodium nitrate per 100 pounds of meat.3 or below. a similar debate would include the time elapsed since manufacturing. a very small amount of added nitrite prevented staphylococcal growth when incubated anaerobically. (1954) reported that at pH 7. During the mid-1950s the U. B. aureus growth can occur in any combination of salt. At that time European microbiologists commonly preincubated perishable canned cured meats before microbiological examination.3°C. Jensen concluded that if the brine content were less than 5. In the United Kingdom. Pasteur.55) was autoclaved with glucose. Dack (1949) reported toxin inhibition in meat with brine levels above 6.0%. botulinum. Because the procedures traditionally used for large canned hams had a history of safety. The authors postulated that nitrous acid was responsible for the bacteriostatic action of nitrite. Earlier.S. This effect was reversed by adding sulfhydryl compounds. Additional tests showed that if the broth medium (pH 6. This combination of ingredients was derived from the literature and unpublished results from Jensen. 1955) on semipreserved meats in hermetically sealed containers included a curious debate.5% would have caused consumer . At pH 5.” An international symposium (Ann. (1956) examined whether curing salts plus the anaerobiosis of a vacuum package would inhibit the growth of S. who performed a series of tests with spiced ham inoculated with different strains of C.5%. the growth of Bacillus species would occur. aureus was killed in hams when heated to an internal temperature of 58. if ever. this cautious view of the inhibitory effect of nitrite was expressed by Eddy (1957): “Taken in their totality. The requirements imposed on new products included a brine level of 5. In North America. who concluded that 1 ounce nitrate was optimal for gas formation by Bacillus species in temperature-abused perishable canned luncheon meat. They found S.. Lechowich et al. as suggested by Castellani and Niven (1955).g. and toxin would be produced. 1 1/2 lb canned ham). was unstable. Inst. Nitrite was more inhibitory in the presence of ascorbate. As late as 1957. and a brine level of 3. although effective. salt levels may be expressed as the percentage brine as just described or as the percentage brine or the percentage salt (wt/vol or wt/wt) on the water phase using the equation % brine or % salt = (% salt/% water) × 100. Today. They investigated why Staphylococcus aureus is rarely. nitrite enhanced bacterial growth in curing brine.8 was optimal for antibacterial efficacy. the spores could germinate. S. aureus to die rather rapidly in ham-curing pickle. Halvorson (1955) cited a private communication from L. these observations leave no doubt inhibition by nitrite is at least a possibility.5%. 1/8 ounce sodium nitrite (78 µg/g). Castellani and Niven (1955) stated that nitrite was not known to have any practical preservative value against those organisms not inhibited by the high salt content in cured meats. Jensen. The debated issue was the time and temperature for preenrichment. and in the presence of nitrate. 1/4 ounce sodium nitrate (156 µg/g). growth would occur.5% sugar. the control of salt concentration and resultant water activity was the most reliable bacteriostatic system for cured meats. causing the cans to swell and forewarn consumers of a potential hazard. A typical formula being used for large canned hams at that time included 0.4%. Also.5 or above.05.6 to 5. botulinum. Scott (1955) concluded from the literature that because nitrate exhibited relatively poor antimicrobial inhibition and nitrite.27. At 6. Tests in broth media and raw pork led them to conclude that S. and nitrite that is palatable and permissible. brine values traditionally have been calculated at % brine = (% salt/% salt + % water) × 100. aureus.75% sugar. found in the interior of cured meats. the legal requirement for trichina destruction. unless protected by meat juices. toxin formation was inhibited with a heat treatment as low as F0 = 0. these existing products were not affected. On adding a filter-sterilized solution of nitrite to a broth medium.0% brine or above. nitrite rapidly disappeared and was ineffective.Nitrite 175 Henry et al. nitrate.9 to 5. less nitrite was necessary for inhibition as the pH was decreased from 6. Germination was apparently prevented because viable spores could be recovered.

although there have been several studies under other conditions in which toxin has been detected without obvious organoleptic spoilage. and thermal injury to the low indigenous level of anaerobic spores. toxin was produced without obvious organoleptic spoilage. 5. There have been no reported attempts to duplicate this effect of brine at levels approaching total inhibition. the following became increasingly clear: 1. It was also believed within the industry that the higher nitrate level would have enhanced can corrosion and product discoloration owing to reduction of nitrate to nitrite by iron at imperfections in the lacquer coating of the cans. Silliker et al. The system providing stability consisted of the combined effect of nitrite. . as postulated by test tube experiments. Nitrate. 6. 3. At 5. 7. Verhoeven. Because viable spores could be recovered from stable commercial product. 1950. They concluded that the stability of shelf-stable canned cured meat given less than a botulinal cook was the result of the combined effect of nitrite.25%. S. toxin was produced without concomitant organoleptic spoilage. other than its possible influence on water activity. and 7. Brine level (0%. Nitrate could serve as an electron acceptor.0%) alone was not responsible for stability. (1958) used this class of product to show that nitrate played no role in retarding putrid spoilage. salt. At brine levels approaching the inhibitory level. toxin was not produced. Brine content was further shown to influence botulinal outgrowth and toxin formation. thermal injury to the spores. permitting the growth of aerobes. A significant observation was that at brine levels of 6. As expected. 4. Although nitrite was recognized as an effective antimicrobial agent. aureus was killed during the heating process given to hams.. Although shelf-stable canned cured luncheon meat had been produced for years. was not confirmed. the primary effect of nitrite was to prevent germination and/or outgrowth of heat-injured spores.176 Antimicrobials in Food complaints of excessive saltiness in the new products. This research and a subsequent review by Silliker (1959) established the future direction for research on nitrite. 2. the product was organoleptically unacceptable — as the cans swelled — and toxin assays were performed.125% and abused at 29. Anaerobic intolerance to nitrite within the interior of ham.8°C for up to 90 days. The suspected role of nitrite was to prevent germination or outgrowth of surviving heat-injured spores.12%. Nitrite was shown to play a significant role in the stability of shelf-stable canned meat. 1950. 1956).0% to 7. the reasons for stability had not been defined.4°C to 37. salt. its value as a preservative in perishable cured meats was still in doubt. Several new reports of unstable commercially canned cured meats containing nitrate appeared (Hankins et al. At 8.5%. Sodium nitrite levels were 22 µg/g or less at the time the meat was inoculated. nitrate actively stimulated spoilage by aerobic spore formers. had no antimicrobial effect. such as micrococci and bacilli.95% brine.14% brine or less. or 5. and a low indigenous spore level. Eddy and Ingram. The authors considered the key to stability to be the addition of sodium nitrite (78 µg/g) and heat injury to the small number of indigenous spores. Greenberg et al. per se. This brought nitrate under renewed attack.09%. (1959) subsequently demonstrated botulinal spore outgrowth in perishable canned cured meat having brine levels from 3. 3. SUMMARY FOR 1950–1960 As 1960 approached.

with a median of 0. The thermal process used during the early 1960s by seven U. He concluded that most of the spores are killed during the thermal processing of shelf-stable canned cured meat. (1962) explored whether nitrite or the oxides of nitrogen were involved in the inhibition of Gram-negative bacteria in cured meats.Nitrite 177 1960–1970 After 1960 increased emphasis was directed toward the role of nitrite in the total inhibitory system in cured meats and its effect on thermally injured spores. inhibition. Immediately after germination. development ceased before lysis or rupture of the spore walls. They concluded that nitrite and nitrate have either neutral or stimulatory effects on bacteria and that nitric oxide has virtually no effect. The impaired synthesis of heme prevents the formation of the required cytochrome systems. Hemin had no effect on nitrate reduction or growth of Escherichia coli or Bacillus polymyxa. however. The toxicity of nitrite was 3 to 5 times greater at pH 6 than at pH 7.to 5-log reduction occurred. canned chopped ham was inoculated with botulinal spores and given 2 thermal processes. even if nitrate is present as an alternative electron acceptor. heme synthesis is impaired. The surviving spores are subsequently inhibited by the salt and nitrite. Another factor in spore destruction is that a thermal process of F0 = 0.203) were compared. In the course of this research. Nitrous acid was considered primarily responsible for the quantitative as well as qualitative changes that occur in the bacterial flora when meat is cured. the larger number of spores that survive the heat process increases the probability that at least one will be able to grow despite the preservative system.. One reason given was that spores indigenous to meat emulsions have lower heat resistance than spores produced in laboratory media. The authors concluded that when oxygen is absent during the growth of some bacteria. but not in others. It was postulated that nitrous acid reacts either with the cell itself or with various constituents of the medium.2. Nitrate and oxygen were considered interchangeable as electron acceptors required for heme synthesis in some bacteria. and outgrowth of spores with unpublished data from the Danish Meat Research Institute. If the surviving spore counts between the enzyme-inactivating (74°C internal temperature) and the shelf-stable process (F0 = 0. Shank et al. making them unavailable for subsequent metabolism. Shelf-Stable Cured Meats Riemann (1963) supplemented existing information on the incidence.0.5. . and nitrate is not reduced anaerobically. The inhibited spores did not appear to become fully phase dark.S. Gould (1964) observed germination. 1965). Product inoculated with the lowest inoculum level (173 spores/g) had 0. the initial spore load in the product is too high.7°C for 6 months.6 at the center of the can would yield 10-fold higher values near the surface of the can. A requirement of hemin for nitrate reduction and growth stimulation under anaerobic conditions for six strains of staphylococci and one strain of Bacillus subtilis was shown by Jacobs et al. Research was initiated on irradiation for shelf-stable canned cured meats. (1967) opened new possibilities. He estimated the thermal process caused a 4-log reduction of the indigenous spores. the observations of Perigo et al. producers of shelf-stable canned cured meat was reported to range from F0 = 0. In a study comparing irradiation with conventional thermal processing. but at a reduced rate. of spores of Bacillus species in the presence of less than 300 µg/g of sodium nitrite at pH 6.2 (Greenberg et al. spoilage. heat resistance. (1964).1 to 0. a 4. Outbreaks of botulism from temperature-abused vacuum-packed smoked fish during the early 1960s added impetus to this research.017 viable cells/g remaining after processing to F0 = 0. If. The presence of 25 µg/g of residual nitrite after processing and a relatively low brine level of 3. and toxin formation from this low level of survivors during storage at 30°C to 37.36% did not prevent outgrowth. Germination was completely prevented by sodium nitrite levels of 750 µg/g or higher at pH 6. The increased use of vacuum packaging raised new questions about the microbiological safety of cured meats.4 to 0.

The thermal process required to achieve shelf stability of canned hams larger than 1 1/2 pounds caused excessive purge (e.0015 to 1. nitrite. 1 psig). This process caused the surface of the meat in the can to reach 96°C to 107°C. First.0. When heated and cultured in the presence of the agents.. The effect of nitrite was pH dependent and led the authors to support the position that undissociated nitrous acid is the inhibitory agent. Curing salts interfered with some stage of germination and development of the surviving heated spores at concentrations that were ineffective with unheated spores. A 3-pound meat portion was filled into each can. was then added and mixed into the meat while under a positive pressure (e. but the process was not dependable and was abandoned.5% to 4. 30%) and poor meat texture. The refrigerated storage period was necessary to assure stability.g. This process was tested extensively in at least one commercial plant. The product was then heated for a short time at a high temperature (e. but nitrite enhanced heat injury. (2) partial destruction of spores by the initial long thermal process but at the same time retaining a high level of residual nitrite. A nitric oxide-generating material. heat resistance was decreased. Duncan and Foster (1968a) also reported that heated putrefactive anaerobe spores were less tolerant of salt. . Roberts and Ingram (1966) heated spores in water from F0 = 0. The two Bacillus species were rendered more sensitive to salt by lower heat treatments than clostridia. sealed. 15 minutes at 113°C to 118°C). The process generally consisted of injecting a curing solution and then immersion in a cover pickle for 1 to 3 days as was typical for that time. and appeared to be the chief preservative against putrefactive anaerobic spoilage. The success of the process was believed to be the result of a combination of factors. The meat mixture was then filled into cans. The process was considered to greatly increase the amount of nitric oxide available for use as an antibacterial agent. The process involved a unique sequence of adding the curing ingredients. Their results confirmed the concept of spore injury and increased sensitivity to salt and nitrite proposed by Silliker et al.3°C) before release as a shelf-stable product.g.5% to 1% (5000 to 10. and a low incidence of clostridial spores (less than 1/g) were necessary for the safety and stability of shelf-stable canned cured meats. such as sodium nitrite.g. and nitrate than unheated spores.000 µg/g) increased the apparent heat resistance of the spores. The product was then cooled in tap water and placed into refrigerated storage (e. Nitrite was strongly inhibitory. They demonstrated that increased stability could be achieved by omitting sodium nitrate and sucrose from the formulation.. These data reemphasized the importance of spore contamination level for the stability of shelf-stable canned cured meats. The process had to be properly controlled to be successful (Kueck.4. (1965) developed a new process for the production of 3-pound shelf-stable canned hams with a purge of about 17% and acceptable texture and flavor. salt and sodium nitrate at 0. Schack and Taylor (1966) described a procedure to produce shelf-stable canned cured meat with improved quality as a result of reduced thermal process. nitrite. When heated in the presence of these agents. (3) destruction of spores on the surface of the meat by the short but high thermal process. 2 to 2 1/2 hours in 74°C water) to achieve an internal temperature of at least 71°C. personal communication). spores tended to be protected by salt and nitrate. especially at pH 6.. the normal pH value of canned luncheon meat. and the product was given a long thermal process at a low temperature (e.1 to 0. and nitrate.. 30 days at 6°C to 8. and then thermally processed. Kueck et al. At 2% to 4%. 75 to 150 µg/g of added sodium nitrite to provide about 20 µg/g after processing to F0 = 0.5 before placing them onto culture media containing salt.. Ascorbic acid was then uniformly distributed throughout the meat.178 Antimicrobials in Food Spencer (1966) concluded from the literature that 3.g. This process was used commercially with the approval of the USDA.0% brine. and (4) the germination and autolysis of surviving spores during refrigerated storage of the meat because of the adverse conditions for outgrowth (low temperature and high residual nitrite). Nitrate had no effect beyond that on water activity. (1958). including (1) the destruction of vegetative cells by heating to the initial internal temperature of 71°C. Increased stability also resulted from improving the canning process to reduce or prevent voids.g. the meat was ground or chopped and then deaerated and backflushed with nitrogen to displace free oxygen.

. The significance of spore level on the thermal process required for preventing botulinal outgrowth was again demonstrated.6 became toxic in the absence of salt and nitrite.000 µg/g. perfringens spores. (1967) offered a new possibility for the stability of shelf-stable canned cured meat. Even high nitrite levels (i. The bacon exposed to air or carbon dioxide in oxygen was obviously spoiled. Streptococcus faecalis var. Meat inoculated with 106 spores/g remained nontoxic after the same process if sufficient salt and nitrite were present. their results supported only one: the inhibition of outgrowth of heat-injured spores. Thus.. The authors postulated that the unknown substance may be formed from the nitrite that disappears during heating. Perigo et al. zymogenes. Perigo and Roberts (1968) subsequently expanded the observation of Perigo et al. sporogenes. Four possible mechanisms by which nitrite may inhibit heat-injured spores in shelf-stable canned cured meats were investigated by Labbe and Duncan (1970). Because formation of the inhibitor required substantial heating. and enterotoxin was reportedly produced under all four packaging conditions. but not Streptococcus faecium.e. an unknown substance was formed that was extremely inhibitory to the growth of vegetative cells of C. aureus were similar in back bacon packaged under nitrogen. or intermediate levels of brine and nitrite. 1969). Despite the research and experience since the commercialization of these products. Because the bacon incubated under . levels within the range of brine in shelf-stable canned cured meat (i. Perishable Cured Meats Thatcher et al. The interrelation of thermal process. The stability of perishable cured meats was assigned to undissociated nitrous acid.000 µg/g) did not prevent germination or swelling of the spores. and spore level in shelf-stable canned cured meat was further substantiated by Pivnick et al. The first attempt to demonstrate the presence of a Perigo-type factor (PTF) in cured meat was unsuccessful (Johnston et al.1% alone. air. The newly emerged cells then lysed. When testing the PTF formed with nitrite levels up to 10 times greater than those commercially accepted. the inhibitor did not prevent germination of most of the intact or heat-injured C. (1967) to 30 different clostridial strains. Growth levels of S. and 5% carbon dioxide in oxygen. they selected a medium that enhanced the disappearance of nitrite during autoclaving. Sodium nitrate affected neither germination nor outgrowth at levels up to 20. 40. and the thermal process required for the safety of shelf-stable cured meats given less than a botulinal cooking. salt. Streptococcus faecalis.. When nitrite was heated in a laboratory medium. between 3% and 6%) allowed germination and vegetative cell formation.Nitrite 179 Duncan and Foster (1968b) also reported that nitrite levels up to 600 µg/g at pH 6. in the case of shelf-stable canned cured meat. vacuum. Roberts and Garcia (1973) later reported 9 of 14 strains of Bacillus species and Streptococcus durans to be sensitive to the inhibitor. they reasoned the inhibitor might play a complementary role in the stability of shelf-stable but not perishable canned cured meats. For this reason.0 allowed emergence and elongation of vegetative putrefactive anaerobe cells but blocked cell division. (1962) questioned the safety of vacuum packaging. to date there has been no agreement on the lower limits of brine. The questions were raised as to what constitutes a safe process and at what point are the products underprocessed.e. nitrite. one of 4. Of the four. Viable botulinal cells were recovered in stable inoculated product 18 months after processing. Salt levels above 6% prevented germination.6% with 300 µg/g of sodium nitrite. nitrite. This required either a brine level of 6. the level of residual nitrite remaining after processing may be an inappropriate measurement of the inhibitory capacity of the product. (1969). or Salmonella Typhimurium. Ground pork inoculated with 1 botulinal spore/g and processed to F0 = 0. The authors concluded that the safety of some shelf-stable cured meats is the result of the scarcity of botulinal spores in the raw materials. concluded that the inhibitor produced in media is of little or no consequence for explaining the inhibitory effect of nitrite in commercially produced canned cured meat. Johnston et al.

The significance of brine level and storage temperature on botulinal toxin formation (types A and B) in vacuum-packaged meats was studied by Pivnick and Barnett (1965). Their findings that the type of cured meat influenced toxin production were particularly significant. which was thought to be the result of product differences in brine and residual nitrite levels.8% brine). toxin was produced at 20°C to 30°C but not at 15°C. The rate of toxin formation decreased as the brine level increased from 3.0 and below. Similar results were obtained with jellied corned beef stored at 15°C and 25°C. The four strains failed to produce toxin during 5 weeks at 10°C in sliced bologna (2.0. In jellied ox tongue.35% and 3. At the highest brine level (4.0 to 4. Toxin was not produced at 10°C or lower. and temperature on a Microbacterium species in all-purpose Tween (APT) broth.1. Pivnick and Bird (1965) found the oxygen permeability of packaging films to influence spoilage but not toxigenesis of sliced cured meats inoculated with C. A total of 90% of the samples held at 25°C remained above pH 6. 34 µg/g of residual nitrite). and nitrite. The effect of brine on type E toxin production was more .180 Antimicrobials in Food nitrogen or vacuum was organoleptically acceptable. In sliced ham. the authors recommended that vacuum-packaged nonsterile processed foods be stored in the frozen state.5% brine and in 4 days at 4. Nitrite was more inhibitory at 0°C than at the other temperatures tested (10°C and 25°C). The time required for toxin production decreased as the abuse temperature was increased (22°C. botulinum. Toxin was produced within 7 days at 30°C in bologna (3. At pH 6.5 in the presence of 200 µg/g of sodium nitrite.7% brine. From studies in thioglycollate medium containing salt. Cooked sliced ham from five producers inoculated with type E spores yielded variable results at 30°C. Failure to obtain toxin production at 5°C may have been influenced by pH because 13 of 15 samples had a pH of 5.7 or less after 4 weeks. The combination of brine (3. the authors concluded that the levels used in ham would permit growth. 21 were able to grow under the same conditions. toxin was produced only at 30°C. but not at 25°C if the brine was 5. A similar effect occurred with increasing salt from 0% to 4% during incubation at 7. type E toxin was not produced. pH 6. This would help to explain the dominance of the lactobacilli as spoilage organisms in many perishable cured meats.8%). Within 1 week the product pH had decreased from 6. aureus was inoculated onto sliced chopped ham. but no attempt was made to determine whether they could multiply if the hams were subsequently temperature abused. Schmidt and Segner (1964) observed increased delay in the growth of four type E strains in culture media (neutral pH) as the nitrite level increased from 0 to 200 µg/g. Bologna was relatively resistant to type A toxin production but not ham. Schmidt and Segner (1964) concluded that factors or a combination of factors provided a much longer shelf life than might be expected from salt content alone.4% brine at 30°C. toxin was detected in 2 days at 2.8%.4°C. In jellied pork tongue. When S.8°C or 10°C. and 37°C). Christiansen and Foster (1965) found type A botulinal toxin production to occur at the same rate on bologna whether packaged with vacuum or without. This suggested that salt becomes more inhibitory as storage temperatures are decreased.75%). Gough and Alford (1965) showed that spores of C. At pH 7. No mention was made of pH and none of the agents were tested in combination. perfringens inoculated into raw hams could survive the thermal process.6. even at 30°C. botulinum in vacuum-packaged perishable cured meats were reported during this period. Another 25 strains of microbacteria failed to grow at pH 5. a fermentable carbohydrate. and decreasing pH during temperature abuse were the suggested inhibitory factors.1% to 4. Several studies on the growth of C. A decrease in pH likely occurred during storage and prevented toxin formation in the other variables. lower maximum population levels occurred in vacuum packages than in nonvacuum packages at 15°C and 22°C. residual nitrite. but not at 29.0. type E toxin was produced at 10°C to 25°C but not at 5°C. 30°C.1% or higher. Brownlie (1966) demonstrated the combined inhibitory effect of sodium nitrite concentration. Of 25 lactobacilli from cured meat. nitrate. pH. On sliced bologna. the presence of nitrite caused very little or no inhibition. increasing the amount of nitrite from 25 to 200 µg/g caused progressively greater inhibition.

2°C. during which time naturally occurring lactics drop the pH to 5.3.8.5% brine at pH 6. C. 25°C. botulinum. botulinum type A seemed to be completely inhibited by 4. All of them reached the same general conclusions as those set forth by Silliker et al. . The system responsible for the shelf stability of canned cured meats given less than a botulinal cooking was studied by several research groups.5. A new explanation for the shelf stability of canned cured meat was proposed. a brine level of 4. Toxin was also detected in bacon held at 12.5% brine at 20°C.9. increasing the nitrite level increased the time for toxin production. and 8. 1968) emphasized maintaining adequate salt levels to assure the microbiological safety of perishable vacuum-packaged cured meats. Riemann (Anon. and an input nitrite level of 500 µg/g or more. The concept of thermal injury to spores that survive the processing of shelf-stable canned cured meats was confirmed. and more than 10% brine at pH 6. which disappeared during the thermal process. considering the bacon had a brine level of 5. The growth of S. more importantly.. ham. An additional factor influencing the apparent significance of nitrite is the growth of competitive flora.2°C for 3 to 18 days.5% or above. and jellied pork tongue) were formulated with commercial levels of nitrate and nitrite. 4.3.1% brine) and jellied pork tongue (3. 5. Although not mentioned.4% brine. The process of outgrowth was more sensitive than spore germination to nitrite. aureus is inhibited by more than 4% brine at pH 5. He concluded that C.35% and after 8 days with 3.6% brine at pH 6. At each temperature (20°C. perfringens.2 and 3% brine at all pH values. 1967). Blanche-Koelensmid and van Rhee (1968) described a smoke ring sausage produced by stuffing the sausage mixture into casings and smoking at 25°C to 35°C. sporogenes. Evidence was presented that the antimicrobial effect of nitrite is related to the formation of nitrous acid. (1958). Perigo et al. 9% brine at pH 5. Kafel and Ayres (1969) demonstrated that the growth of enterococci in culture media and. they determined that type B botulinal outgrowth was inhibited during 4 weeks at 30°C if the juice had a combined pH of 5. Type E botulinal growth is inhibited by 2% brine at pH 5. 2.8% to 4. and Lactobacillus viridescens. The authors concluded that heat injury of spores is not necessary for nitrite to prevent toxin production in cured meats. In jellied pork tongue. and 30°C). (1967) suggested an inhibitory substance was formed from nitrite. In both products a marked delay in toxin production occurred at 20°C. SUMMARY FOR 1960–1970 Progress made during the 1960s toward defining the role of nitrite in cured meats included the following: 1. The product had been changed to a shelf-stable fermented product precooked to 80°C in an oxygen-impermeable casing.8°C and 22. The effect of nitrite levels on types A and B botulinal toxin production in vacuum-packaged sliced ham (4. 5. as the brine level increased in the bologna and ham. Toxin was not produced in either bologna or country cured ham.1. in perishable canned hams can inhibit the growth of C. The raw product was then sold at ambient temperature for cooking at home. Toxin was not produced in product with 4.5% brine at pH 5.1.Nitrite 181 dramatic. but this is highly questionable. (1967) reported type E botulinal toxin production in ground beef held at 22.8 or less. 3. All three products (bologna.. C. an increase in the level of residual nitrite also occurred. Warnecke et al.5 to 5. toxin was detected after 4 days with 2.3% brine) was studied next (Pivnick et al.28%. Using juices expressed from the newer sausage product.

Lücke et al. but their presence in the culture medium markedly reduced the recovery of the heated spores. 7. The significance of decreasing pH on botulinal inhibition in cooked cured meats with sufficient fermentable carbohydrate was reported for the first time.01% . (4) meat products produced and packaged in pilot plants. The complex. The chief value of nitrite appeared to be its ability to prevent growth from spores that survive and germinate following processing. (1980) would later report growth at 8°C in liver sausage. Limited data indicated the lowest temperature reported for growth of type E on cured meats to be 10°C. residual nitrite. Shelf-Stable Cured Meat and the Perigo Factor Pivnick et al. Of the four possible roles. The data were discussed in terms of applying the D concept to shelf-stable canned cured meat. An early test on the effect of vacuum packaging on the safety of perishable cured meat led to the recommendation that they be frozen to prevent the development of microbial health hazards.182 Antimicrobials in Food 6. Each of the approaches had its favorable and unfavorable features. (3) nitrite increases the germination rate of spores that survive heating. and inoculum level. pH. Neither sodium chloride (3%) nor sodium nitrate (0. and (5) commercially produced meats. Research during this period can be grouped into five categories by the approaches used. and 120°C) and curing salts on the destruction of Clostridium subterminale spores. (2) nitrite potentiates direct killing by heat without the need for prior germination. storage temperature. and 0. nitrite at the level used commercially (200 µg/g) did not enhance destruction during heating or germination during subsequent storage. (1970) used an aqueous meat mixture without salt or other additives to investigate four possible roles of nitrite in shelf-stable canned cured meats: (1) nitrite induces germination during the heating process. 9. with the goals of greater understanding and its possible use as a preservative to replace nitrite. As in the past. They include tests in (1) test tubes or Petri plates using controlled media and conditions. in 1967 opened the possibility of a breakthrough on the mechanism of nitrite inhibition. and the germinated spores are killed by heat. Salt and nitrite had little effect on the degree of thermal destruction. Sodium nitrite alone caused increased destruction. 0. Considerable effort was applied toward isolating and characterizing the Perigo factor. (2) water slurries of meat processed in bottles. The timely report of Perigo et al.1% sodium nitrate. dynamic system in which nitrite plays a role in cured meats has been virtually impossible to duplicate in microbiological media. 1970–1980 From the outset of the 1970s. A combination of 3. the formation of nitrosamines in cured meats was an issue of immediate concern. but not all the results were directly applicable to commercially cured products. Matsuda and Sekiguchi (1971) studied the combined effect of heat (105°C. (3) meat products processed in tubes or cans. Ingram and Roberts (1971) heated botulinal spores in water and in water with salt (4%) and sodium nitrite (200 µg/ml) before subculturing into media with and without salt (2%) and sodium nitrite (50 µg/ml). Factors influencing growth included brine level. 110°C. 8. and (4) these rapidly germinating spores are then inhibited by the nitrite remaining after processing and subsequently die before the nitrite has decreased appreciably. Certain cured meats are more prone to supporting botulinal growth. the test tube approach was productive. The growth of enterococci in perishable canned hams was reported to inhibit several clostridial species.1%) affected thermal destruction when tested alone. Throughout the decade pressure increased to reduce or eliminate nitrate and/or nitrite from cured meat.0% salt.

Omitting any one or all three of the additives did not affect thermal destruction. and 0. cooked. Reducing agents (e. and pH 6. nitrite and salt levels had lesser effects. The extract had 2. salt. however.. but not at levels above 0.35 were as stable as sausages in brine without nitrite and processed to F0 = 2. the liquid placed into tubes. cysteine. normal. It was reported that less nitrite was required for inhibition when it was added before autoclaving. (1967) medium and reinforced clostridial medium but not in liver veal or Wynne fluid media. These observations led to the conclusion that a Perigo-type effect was detected in the meat system.3.6% salt. (1973) studied the effect of adding nitrite to the brine solution for canned Vienna sausages. The sausages were prepared with nitrite-containing curing salt. sporogenes spores to provide a level of 104 spores per can. sodium ascorbate.4.” The effect of all three factors (pH. Outgrowth was determined by blackening of the tubes as a result of ferric citrate having been added to the meat. The major factor influencing outgrowth was pH. Aqueous meat suspensions prepared from raw. A significant observation was that residual nitrite did not undergo normal depletion in the extract. and then inoculated with vegetative clostridial cells. One of the sausages in each can was inoculated with C. The effect of heating spores of PA 3679 at 115. The magnitude of the effect was much less than was previously reported in culture media. Preliminary data. Ashworth and Spencer (1972) reported the equivalent levels to be . Reducing agents (e. and heated to varying degrees.g.8% salt. Considerable differences were noted in their redox potentials. Between 120 and 200 µg/g sodium nitrite provided stability through 5 weeks of storage. inoculated (105/ml). and salt) appeared to be additive.g. nitrite.5°C on the outgrowth of surviving spores was also examined by placing the heated tubes at 23°C for up to 1 year.966. In ground pork.5°C in ground pork containing 2. botulinum. and overheated (115°C for 90 minutes) canned luncheon meat required 500. Ashworth and Spencer (1972) described a model system of ground pork (25 g) in 1-ounce bottles. aw 0. (1975).01% strongly influenced the results.01%. Because it would later be found that iron concentration can influence the antibotulinal efficacy of nitrite in perishable canned cured meat given a milder heating. although they were “clear and undisputable subsidiary effects.. to some degree. A similar response with strain 3679 would not be unexpected. Farkas et al. Perigo et al.4). Sausages packed in brine with 400 µg/g of sodium nitrite and processed to F0 = 0. respectively. held overnight for nitrite equilibration. exist (Christiansen et al. This emphasized the importance of medium selection when studying the antimicrobial activity of nitrite and relating the results to commercially produced meats.Nitrite 183 sodium nitrite caused the greatest destruction when heated. After a loss of 15% to 25% during processing and an initial decline. 200 µg/g sodium nitrite. and. (1967) reported equivalent inhibition with 8 µg/g of nitrite added before heating the medium and 52 µg/g added after heating the medium. The pork was autoclaved before or after adding nitrite. thereby confirming earlier research.. thioglycollate) enhanced the effect of nitrite.. as opposed to perishable canned cured meats heated in hot water to a minimum internal temperature of 66°C. A 1:1 extract was next prepared with 3% saline and ground. showing a similar iron–nitrite interaction with C. The cans were then filled with brine (3% salt). Johnston and Loynes (1971) obtained Perigo factor formation in the Perigo et al. Small differences in the amount of sodium nitrite below 0. The residual nitrite values of the four media and three meat suspensions were about the same after preparation. the solids removed. The inhibitory level of nitrite was not influenced by pH within the range 5. personal communication). and 50 µg/g of sodium nitrite. and then packed into cans. The effect of nitrite. cooked sausages. 150. did the addition of ferric citrate influence the results? The interaction of iron and nitrite has not been investigated in shelf-stable canned cured meats heated in steam under pressure (e. and heating at 115. pH. ascorbate and cysteine) enhanced the antibotulinal effect of the meat suspensions but did not induce Perigo factor formation.g.5% sodium tripolyphosphate was studied by Nordin et al. 115°C).6. residual nitrite levels remained above 125 µg/g throughout 80 days at 30°C. for inhibition. as already discussed. overlaid with paraffin oil. sealed. and heated (F0 = 0.7 to 6. sporogenes strain PA 6982 in perishable canned cured meat as occurs with C.

the relative inhibitory effect was quantitatively small. which indicates that residual nitrite influenced the results. such as perishable canned ham. autoclaving. Residual nitrite. It was originally suggested (Perigo et al. (1967) based their conclusions on the added level of nitrite. The depletion rate of the nitrite added to the suspensions was not influenced by the amount of nitrite originally added to the meat. The authors found that as the original input nitrite level increased. results similar to those just mentioned were obtained during the early stages of temperature abuse. This was considered evidence for an inhibitory factor other than residual nitrite. The additional nitrite provided comparable residual levels after the various heat treatments. Shelf-stable canned cured luncheon meat was prepared with 0 to 300 µg/g of sodium nitrite and processed to F0 = 0. Ashworth et al.g. (1973) extended this research to the milder heating given to large perishable canned hams and obtained similar results. Actually.184 Antimicrobials in Food 60 to 70 and 90 to 100 µg/g. however. Perigo et al. another inhibitory factor may be formed when nitrite is heated with meat. more added nitrite was required in the pork if the nitrite was added before (300 µg/g).. In this case alone. In summary. The meat was then inoculated with botulinal spores. The amount of PTF formed was related to the amount of original nitrite added to the meat before heating. 100. (1969) found the inhibitor of Perigo et al. per se. Similar residual nitrite levels were required to inhibit vegetative cells (160.4. With excessive heating. 1967) that the nitrite that disappears during heating may be involved in the formation of the unknown inhibitor. Chang and Akhtar (1974) prepared buffered saline suspensions of canned luncheon meat made with 0. and 200 µg/g of sodium nitrite and processed to F0 = 0. and inoculated. on the basis of added nitrite.5 or 22. However. The term Perigo-type factor (or PTF) was selected to describe the effect observed in their test. more was required when added before heating the meat. Ashworth et al. The nitrite level of the suspensions was adjusted after heating to 53. deaerated in boiling water. the conclusion that a Perigo effect was demonstrated must be tempered with the observation that.5% salt and 150 µg/g of sodium nitrite.1 µg/g) were the residual nitrite levels after 0 and 2 weeks in product made with 200 µg/g of nitrite. When spores were used. (1967) to be inactivated when added to meat. The levels selected (53. Inhibition by nitrite diminished gradually over a period of 8 weeks at 37°C. higher initial residual nitrite levels were required for inhibition. less residual nitrite was required for inhibition when nitrite was added before heating. higher levels of added nitrite were required for inhibition. outgrowth and toxin production decreased.4 in raw meat juice with 4..4 The product was held until the highest level of nitrite declined to less than 2 µg/g. thermal destruction of spores and inhibition of the survivors by the salt). the Perigo effect was of low magnitude. Adding salt (3.000/g) added after heating and spores (20/g) added before or after heating. They concluded that although a PTF was formed. 150.5 and 22. that although the Perigo inhibitor may not be present in meat. Ashworth and Spencer based their conclusions on the residual level of nitrite. the levels of nitrite required to obtain inhibition were about the same or lower if added before heating than if added after heating. strongly influenced clostridial inhibition. The equation of Pivnick and Petrasovits (1973) was used to estimate the inhibitory effect of the PTF relative to the protection provided by other factors (e.5% brine) to the meat had no effect on the inhibitory levels of residual nitrite but appeared to reduce the level of added nitrite required for inhibition. Pivnick and Chang (1974) considered. concluded that a PTF was produced. Johnston et al. If residual nitrite is a valid means for comparison. The inhibitory levels of residual nitrite for two heating processes — heating from 80°C for 4 hours and heating from 20°C to 70°C over a 4-hour period — did not differ.1 µg/g. Ashworth and Spencer (1972) found no relation between the amount of nitrite lost during heating and the inhibition obtained. with prolonged storage (42 days at 37°C). and it may also play a role in the safety and stability of pasteurized cured meats. Botulinal . In all cases. which had survived heating to F0 = 0. The data obtained with the spore inoculum should be more relevant to shelf-stable canned cured meats. rather than after (150 µg/g).

and 4 weeks before preparing the suspensions. At 37°C. 1975b) that inhibition was apparently at the cellular level.. The authors concluded that it was not possible to demonstrate any preservative action of nitrite at the levels (i.1 µg/g of sodium nitrite yielded similar results. Perishable Canned Cured Meat and Slurries of Meat In addition to research on shelf-stable canned cured meats and the composition and role of the Perigo factor in commercial products. 1974. and fewer treatments proved stable. Incze et al. Asan and Solberg. At 4°C. Hansen and Levin.Nitrite 185 inhibition was related to the original amount of nitrite. 200 µg/g) usually used in meat products. toxin was not detected. Van Roon (1979a. age of product must be considered when investigating the presence or activity of PTF in commercial product or model meat systems. A second test using canned product held for 4 weeks at 35°C before preparing suspensions with 22. Decreasing inhibition occurred as the product was held for 0. . causes inhibition by reacting with enzymes containing functional sulfhydryl groups. van Roon. Additional studies were reported by 1980 that dealt with the Perigo factor (Mirna and Hofmann. heat injured. Huhtanen and Wasserman. considerable effort was devoted to perishable cured meats. Inoculation of the medium after heating with nitrite and aging for 3 months showed that it was less inhibitory. Roberts and Smart (1974) found that the inhibitor in the medium of Perigo et al. Wasserman and Huhtanen. common agreement that nitrite. Moran et al. with sulfhydryl-containing constituents of the bacterial cell. indicating the Perigo factor is not stable.. This correlation agreed with results obtained when canned product was held at 35°C to allow the depletion of residual nitrite and then challenged directly (Chang et al. 1974). Debatable as the formation of a Perigo-type in meat may be. Nitrate had a slight but statistically significant inhibitory effect that may have been caused by nitrite generated from nitrate. in some form.. 1972. There was no evidence of germination (i. swelling and toxin production ranged from 3 to 17 days. although the spores remained viable during prolonged incubation. (1973). 1975. Neither swelling nor toxin occurred in some treatments having 240 or 480 µg/g of nitrite. and 37°C. the rate of swelling ranged from 11 to 48 days across all variables. b) concluded that Perigo-type inhibitors do not contribute to clostridial inhibition in heated cured meats. They hypothesized that nitrite. 1974. At 22°C. 1979a. van Roon. perfringens may involve an interaction of nitrite. Additional data by O’Leary and Solberg (1976) suggested that inhibition of C. Ashworth et al. Nontoxic spoilage occurred at 7°C in uninoculated product made without nitrite. shelf-stable canned meat. It was stated that botulinal inhibition was related to the level of nitrite added during formulation rather than residual nitrite. 1975. Tjaberg and Kvale (1972) studied the antibotulinal effect of nitrite (0 to 480 µg/g) added to canned raw meat. and sulfhydryl compounds are involved in the formation of the Perigo factor in broth media may be significant. 1974. 1974.. 22°C. who concluded that residual nitrite is a significant factor influencing botulinal inhibition. iron. Research on perishable canned cured meat or meat slurries is discussed first. or irradiation injured. perfringens in a chemically defined medium. Botulinal outgrowth in product abused at 27°C was influenced by the spore inoculum level and the level of nitrite. Perishable canned cured pork was studied by Christiansen et al. vegetative cells) in the media with inhibitory levels of nitrite. 1978.e. respectively.. regardless of the inoculum level or whether the spores were untreated. and perishable canned meat and held at 4°C. 1969. (1967) had a comparable effect on the inhibition of clostridial spores. Riha and Solberg (1975a) studied the formation and effect of a PTF on seven strains of C. 2. They subsequently concluded (Riha and Solberg.. 1976. as nitrous acid.e. predictably. It was concluded that a PTF is formed during the heating of canned luncheon meat and that the inhibitor is not stable and loses its activity during storage. 1975. Lee et al. This was later reassessed by Christiansen (1980). Mirna and Coretti..b). The degree of inhibition was correlated with the amount of nitrite originally added to the product. Thus.

a race occurs between death of germinated botulinal cells and depletion of residual nitrite. Isoascorbate had enhanced the antibotulinal efficacy of nitrite in the canned meat. pro or con.5). the more the residual nitrite levels declined and the less inhibitory the product became when placed at 27°C. Sodium nitrite and L-ascorbic acid were added as filter-sterilized solutions. 5. or 95°C decreased the number that could grow in a recovery medium containing 2. At 17. The longer the product was held at refrigeration temperatures. 22. Although less effective. 1978a). and inoculum level on botulinal growth and toxin production at pH 6. The spores heated at 70°C and 95°C were equally sensitive to the autoclaved nitrite. 20 µg/g of nitrite in the Perigo medium and 210 µg/g in the slurry were equally inhibitory. Botulinal outgrowth was dependent on the relative levels of residual nitrite and surviving botulinal cells.5% or above. At the lower temperatures. or 25°C.5% salt. (1977).5%.0. On investigation (Tompkin et al. Spores heated at lower temperatures (70°C or 80°C) were inhibited by 4. The emulsions were filled into cans and heated. 6.1%) also increased the efficacy of nitrite in broth but had no effect. At 20°C. 150. Temperature of abuse was the most significant factor influencing the rate of botulinal growth. and whether the products were treated by a perishable or shelf-stable thermal process. (1976) found that heating botulinal spores in an aqueous extract of lean pork at 85°C. In another study. recommended that large-scale tests be used to quantify the many interacting factors influencing the safety of perishable cured meats. 15°C). Adding L-ascorbic acid (1. A statistically significant salt–nitrite interaction was observed after heating (70°C to 95°C) only if the recovery medium contained salt levels of 4. It was suggested that Bacillus species are less sensitive to nitrite than clostridia.5°C. nitrite. The rate at which inoculated cans swelled at 27°C was inversely related to the level of nitrite added to the product. (1976). level of clostridial spores.5°C. thermal processing within the range of 63°C to 74°C (internal temperature) influenced neither residual nitrite levels nor botulinal inhibition in perishable canned cured meat . 6. 103. 101).5% salt. 90°C.4°C or 10°C for up to half a year before placing at 27°C. This led to the pork slurry system of Rhodes and Jarvis (1976) and used by Roberts et al. 100. Grever (1974) examined the stability of emulsions normally used for preparing cooked sausage and liver sausage. decreasing pH (7.0%. a preliminary test in bacon led to the conclusion that L-ascorbic acid does not alter the antibotulinal properties of nitrite in bacon. (1978) concluded that when inoculated. Furthermore. decreasing temperature (25°C. 3.5%. and decreasing inoculum level (106..0. 6.8% or 3. The antibotulinal effect of nitrite was considerably greater than that reported for the meat slurries of Roberts et al. one reason was found to be the addition of sodium isoascorbate to the canned meat but not to the meat slurries. the impact of increasing the nitrite level from 40 to 300 µg/g was minor and became less so as the temperature of abuse increased to 22. Christiansen et al. a lower concentration of L-ascorbic acid (0. less inhibition occurred. neither salt level provided much inhibition. 200 µg/g). The number of strains showing growth in broth was found to decrease with increasing nitrite (50.186 Antimicrobials in Food Baird-Parker and Baillie (1974) studied the growth of a large.5.0.5% but not 3. Jarvis et al. perishable canned cured meat is placed at 27°C. 4.0% w/v). Stability was influenced by nitrite content. diverse collection of botulinal strains in a broth medium and a cooked pork medium. salt. product pH..5% salt (on water phase). At 15°C. a pronounced salt–nitrite interaction occurred. the greatest inhibition occurred with 1. 1978b) involving product held at 4. (1976) to study the interactions of temperature of abuse. even with 300 µg/g of sodium nitrite. 20°C. This agreed with an earlier observation (Tompkin et al. Jarvis et al.5°C and 25°C. Spores heated at either 70°C or 95°C were more sensitive to nitrite that was autoclaved in a meat slurry or the Perigo medium than to nitrite added after autoclaving.0%) markedly increased the effectiveness of nitrite with none of the 38 strains tested showing growth in broth containing 50 µg/g sodium nitrite. increasing salt (1. in the cooked pork medium. Autoclaving nitrite in the Perigo medium resulted in significantly more inhibition than autoclaving the nitrite in the meat slurry. The effect of nitrite in perishable canned cured meat was also reported by Tompkin et al. At 20°C.

and enzyme activity. sporogenes.Nitrite 187 (Tompkin et al. ferredoxin) within the germinated cell. The product was formulated with 2. (1979b–e).. single-electron transfer sites consisting of four iron atoms. A third possibility is the germination of dormant spores after residual nitrite has declined to noninhibitory levels.g. Substituting turkey thigh meat. . When testing a frankfurter emulsion prepared with mechanically deboned chicken meat. energy production. Bacillus species. 1979b). ascorbate. One possibility involved the reaction of nitrite with an essential iron-containing compound (e. 100. This made it possible to explain the differences in the relative efficacy of nitrite in various meats on the basis of their iron content. (1978) studied the effect of nitrite on the stability of naturally contaminated perishable canned pork when temperature abused at 37°C. perfringens.. Spoiled cans yielded C.5% salt and sodium nitrite (0. peroxidases. 1978d). This was based on the rationale that in clostridia. cysteine. nonheated) heme compounds. with one exception: beef liver. or 200 µg/g) and then processed to an internal temperature of 69°C. Suggested mechanisms were proposed for the relation of iron to the antibotulinal efficacy of nitrite (Tompkin.g. and myoglobin).g. ferredoxin) are essential for electron transport. Although isoascorbate enhances the antibotulinal effect of nitrite in freshly prepared perishable cured meat that is temperature abused. low levels of nitrite (20 and 40 µg/g) did not influence botulinal growth or toxin production (Sofos et al. however. decreased botulinal inhibition.. beef liver did not cause a substantial loss in the antibotulinal efficacy of nitrite. which later was demonstrated to be the sequestering of iron (Tompkin et al. It was assumed that some form of nitrogen oxide–iron reaction could occur with the nonheme iron–sulfur proteins as occurs with nitric oxide and the iron in heme compounds (e. nitric oxide)–iron reaction as in ferredoxin. It was also shown that the type of meat could influence the degree of botulinal inhibition. 1978c)..5°C. 1978. Clostridium bifermentans. Isoascorbate. as can occur with nondenatured (i. which resulted in a lower level of residual nitrite after processing. beef round. nonheme iron–sulfur proteins (e. no botulinal inhibition was observed. The efficacy of nitrite in frankfurters was explored by Sofos et al. a background flora of thermodurics existed from the ingredients. Subsequent outgrowth could occur when (1) residual nitrite depletes to noninhibitory levels. 1979b). a nitrogen oxide (e. the degree of inhibition was less than that reported by others. Adding 156 µg/g of sodium nitrite delayed botulinal toxin production.. 1970). cytochromes.. beef hearts.e. The reason isoascorbate enhanced the antibotulinal effect of nitrite was pursued (Tompkin et al. This avoided the problem of recontamination inherent in some studies after heating. isoascorbate also reduces the efficacy of nitrite by causing more rapid depletion of residual nitrite (Tompkin et al. a simplistic explanation for the inhibition of clostridia consisted of the uptake of undissociated nitrous acid. catalase. C. heating to 68. cooling. Tompkin et al. however. On the basis of the data existing in 1978. allowing the dissociation of the nitrogen oxide from the iron.. and interference with energy metabolism in the vegetative cell to prevent outgrowth. cytochrome oxidase. overlaying with vaspar. As the level of nitrite decreased. In a later unpublished test with pork liver that has a much higher iron content than beef liver (225 to 243 versus 49 µg/g). 1979a)... the rate at which the product spoiled increased. Clostridial ferredoxin is believed to contain two independent. and/or (2) repair through the formation of new unreacted ferredoxin by the vegetative cell. Despite its relatively high iron content (49 µg/g). 1978e). 50. or pork hearts for fresh pork ham reduced the efficacy of nitrite. Their test system mainly consisted of extruding a frankfurter emulsion into test tubes. Each active site is comprised of iron bonded to both cysteine and sulfur (Buchanan and Arnon.. and enterococci. but as in other model systems receiving a mild heat treatment. 1978e). hemoglobin..g. and then storing the tubes at 27°C. and ethylenediaminetetraacetic acid (EDTA) were found to share a common function in meat.. Subsequent research more clearly confirmed the relation of available iron to the antibotulinal efficacy of nitrite (Tompkin et al. Wojton et al. Adding hemoglobin.

Under these circumstances. the levels of nitrite (0 to 150 µg/g) and ascorbate (9. Toxic product occurred with 50 µg/g of sodium nitrite at all levels of ascorbate. reported by Bowen and Deibel (1974) and Bowen et al. In the first. 40.6. 1973). and then only 2 of the 55 franks became toxic. and 655 µg/g). beef.188 Antimicrobials in Food Sofos et al. the two studies indicate that the combination of 4. Toxin was produced in all variables formulated without dextrose and more quickly in product having 0 or 50 µg/g of nitrite. Adding 50 µg/g of sodium nitrite delayed botulinal toxin formation for more than 4 weeks during abuse at 27°C. The rate of botulinal toxin production in formulations with soy protein was equal to or slower than in an all-meat formulation. a fairly rapid decrease in product pH during abuse. and fewer toxic samples occurred in product with 100 or 150 µg/g of nitrite. One toxic sample occurred at 100 µg/g and none at 150 µg/g of sodium nitrite. The second test. (1979c) evaluated various soy proteins in their system. The pH of the dextrosefree product remained above 5. The depletion of residual nitrite was faster as the amount of beef was increased in a formulation containing soy. more importantly. about 2.08 or lower within 1 week at 27°C.0 or less within 21 days. in both the presence and absence of nitrite. Although product made with 150 µg/g of sodium nitrite did not become toxic. the addition of 156 µg/g of sodium nitrite was very effective in delaying botulinal toxin production. and omission of nitrate. The franks were produced in the same facilities and formulated as before (Hustad et al. It was concluded that ascorbate did not affect the antibotulinal efficacy of nitrite. 105. The rate of botulinal toxin production was slower in all soy protein formulations than in all meat formulations. The strong inhibitory effect observed was likely the result of the combined effects of nitrite. Nitrite was ineffective in a formulation of soy isolate and pork fat. 1975). In a meat–soy mixture. A significant delay in botulinal toxin production occurred in pork. (1973) formulated franks with various levels of nitrite and nitrate. and. In mildly heated (58..52% brine. They concluded that the effect of nitrite was on delaying the development of the cells after germination and before toxin was produced. botulinum spores in a chicken frank formulation to be unaffected by the addition of sodium nitrite (0. (1979d) compared the efficacy of nitrite (0 and 80 µg/g) in chicken. A later review of the pH data showed that all variables had a pH of 5. Sofos et al. The rate of nitrite depletion was influenced by the type of soy. 450 µg/g of nitrate gave a variable response. 400 µg/g of sodium ascorbate. Sofos et al. . The pH of product made with 50.8% fermentable carbohydrate. Assuming a similar pH response in the first test (Hustad et al. the relatively high brine. and the effect of ascorbate in franks should be restated or reexamined.5% to 4. or 150 µg/g of nitrite declined to 5. In that variable the pH was 5. the exception being one variable with 150 µg/g of nitrite. The finished franks had a brine of 4. The addition of 80 µg/g of sodium nitrite was relatively ineffective in chicken and beef. The type of soy protein influenced the results. The decline in pH caused by a lactic fermentation was also the dominant factor preventing botulinal outgrowth in fermented sausage (Christiansen et al. only those variables without nitrite became toxic at 27°C. and 156 µg/g). input versus residual nitrite. Toxin production was slower.. and 4. 20.83%.. Hustad et al. (1979e) found the rate of germination of C. and pork in their system. 100. Vacuum-Packaged and Fermented Meats The effect of nitrite in vacuum-packaged franks was studied on two occasions. the conclusions in both studies concerning the inhibitory levels of nitrite.8% brine and nitrite levels of 50 to 100 µg/g delayed botulinal outgrowth until the lactic flora decreased the pH to an inhibitory level.3°C) sausages containing dextrose. (1974). examined whether an increase in ascorbate over normal levels would alter the antibotulinal effect of nitrite.0 or less within 14 days. except for changes in the ratio of pork to beef. 1973). and the products remained nontoxic.

Nitrite 189 A request was made by the poultry industry to the U..9% became toxic. 20°C. Raevouri encouraged more widespread use of erythorbate for sausage production in Finland. respectively.9%. 67. If all samples with no mixed cure. Food and Drug Administration (FDA) for approval of sodium nitrite as an additive in poultry products. If all samples containing mixed cure with and without ascorbate were compared.80 and 0.7% and 18.98. It also was reported that S. growth was faster at 25°C than at 15°C. respectively. higher populations of S. 42. cereus tested. 19. Bacon that was sliced and vacuum packaged developed a flora mainly of micrococci and lactics. The predominant flora of the bacon after curing consisted of micrococci. 500. ascorbate (0.4%. 1000. Omitting nitrite from commercially produced liver sausage in Finland led to rapid spoilage by gas-producing anaerobes and suggested a potential risk of botulism (Ala-Huikku et al.3%. After cooking in Saran™ casings. and 2000 µg/g). The naturally occurring B. Including nitrate in the bacon enhanced the growth of micrococci. the interaction of nitrite and ascorbate is difficult to interpret. A combination of 200 µg/g of sodium nitrite and 500 µg/g of sodium erythorbate prevented the growth of the two strains of B. These values raise a question concerning the conclusions that (1) protection was greater if the level of nitrite was increased to 200 µg/g and (2) sodium ascorbate at a level up to 2000 µg/g did not reduce the protection afforded by nitrite against C. respectively. Inoculation studies examined the effect of nitrite (0 and 100 µg/g) and storage temperature (15°C. The request included results by Christiansen et al. . and 34. Product without nitrite became toxic within 3 days at 25°C and within 6 days at 20°C and remained nontoxic through 14 days at 15°C. When all samples having the mild and medium brine were compared.. (1976) in the United Kingdom studied the effects of a mixture of nitrite (100 or 200 µg/g) and nitrate (250 µg/g). In opened packages. 100. Product with 100 µg/g of sodium nitrite remained nontoxic through 14 days at all three temperatures.9% to 4. The addition of ascorbate enhanced the antibotulinal effect of 100 µg/g but not 200 µg/g of nitrite. As expected. Bacon Crowther et al. mixed cure with 100 µg/g of nitrite. botulinum. B. Shaw and Harding (1978) studied the effect of nitrate and nitrite on the microbial flora of Wiltshire bacon. and 25°C) on the rate of botulinal toxin formation. and 200 µg/g) and sodium erythorbate (0. regardless of the level of nitrite or ascorbate. cereus was evidently more resistant to nitrite than C. Erythorbate caused reductions in the residual nitrite and the redox potential of the product. 35. A higher percentage of samples were toxic with the addition of 200 µg/g of nitrite than with 100 µg/g of nitrite. lower populations resulted and enterotoxin was not detected. aureus developed and enterotoxin A was produced. and Moraxella-like bacteria. The data included botulinal challenge tests in frankfurters made with chicken meat and ham made with turkey meat.6%) on botulinal toxin production in vacuum-packaged back bacon. The nitrate likely served as an electron acceptor for the growth of micrococci in the vacuum package. Omitting nitrate led to higher numbers of Moraxella species in the cured bacon. Erythorbate alone was not inhibitory. and mixed cure with 200 µg/g of nitrite were compared. There was a general trend toward reduced residual nitrite levels at the tune of inoculation when ascorbate was added. 1977). the sausages had relatively high pH and water activity values of 6. Because brine levels could not be held constant. botulinum. A summary of the data showing the antibotulinal effect of nitrite in the two poultry products appears in Hauschild (1982). In vacuum packages. Noninoculated product containing nitrite developed a slight unusual taste at 20°C owing to the growth of spore-forming spoilage organisms (e. Moraxella species. cereus). medium 4. 2. and brine level (mild. (1977) that demonstrated the efficacy of sodium nitrite in poultry meat.9% became toxic.7% to 6. Erythorbate was found to enhance the inhibitory effect of nitrite. and 1000 µg/g) on the growth of Bacillus cereus in cooked sausage placed at 20°C for 48 hours.S.8% and 21. Raevuori (1975) studied the combined effect of sodium nitrite (0.g.7% became toxic.65 to 6. aureus grew well in the medium-salted bacon.

The results demonstrated that the combination of nitrite–sorbate was equal to or better than nitrite alone. Temperatures below 27°C or 30°C increase the delay in outgrowth and the inhibitory effects of the foregoing factors. There was considerable variation in toxin production in the bacon from the four plants. Brine levels below 4.26% potassium sorbate.. it is possible to glean the relative effect of nitrite by examining the nitrite-containing control variables from each test. 7. At least on the basis of the botulinal results. As the brine level exceeds 4. 1979). the combination of relatively higher brine and decreasing pH can prevent botulinal outgrowth. fermentable carbohydrate. A total of 30 botulinal studies were performed with vacuum-packaged bacon during the 1970s in the United States and Canada. outgrowth is increasingly delayed. Sofos et al.0% are not inhibitory to botulinal outgrowth. The initial period of delay depends on the inoculum level. Too high a level of botulinal cells when the bacon is abused can overwhelm the inhibitory system. phosphate. Within the levels and types used. 1974. 4. and fermentable carbohydrate (e. The level of nitrite added to the product is not important. 10 appear in the technical literature (Christiansen et al. 1974. sucrose) were left to the discretion of the producing plant. Of the 30 tests. 1980). Although the studies were performed for a variety of reasons. The addition of ascorbate or isoascorbate can act in concert with residual nitrite to retard botulinal outgrowth in freshly produced bacon. ascorbate and isoascorbate can also have a negative effect by causing more rapid loss of residual nitrite during processing and storage.. The levels of sodium chloride. . This was likely the result of the interaction of the other components of the product (brine level.7% sugar (sucrose) or more provides sufficient fermentable carbohydrate that naturally occurring lactics cause a decline in pH to inhibitory levels. The pH of the bacon during abuse significantly influences the potential for botulinal outgrowth. bacon with 120 µg/g of sodium nitrite. and vacuum packaged (100 g per package). The data indicate the following: 1. aside from the fact that the amount of added nitrite partially determines the level of residual nitrite. The USDA conducted a test to verify the antibotulinal effectiveness of a combined nitrite–sorbate mixture in bacon (Johnston. Because phosphates were present in almost all the studies. and growth of a spoilage flora). Botulinal outgrowth is influenced by the temperature of abuse. They can be more readily compared because they were similar in processing and final product. and bacon with 40 µg/g of sodium nitrite plus 0. All three bacons contained 550 µg/g of sodium ascorbate or sodium isoascorbate. The bacon was then incubated at 27°C for up to 56 days. The level of residual nitrite at the time the bacon is abused influences the extent of the delay in botulinal outgrowth. Ivey et al. 1980b.190 Antimicrobials in Food Because higher numbers of lactics were present in bacon with the lowest initial nitrite concentration. Collins-Thompson et al. 6. Bowen and Deibel.. The bacon was shipped to a laboratory. Vacuum-packaged bacon prepared with 0. 3. Tanaka et al.g. 5. These factors interact to influence botulinal inhibition. phosphates do not appear to influence botulinal outgrowth in bacon. 1978.. 8. Three types of bacon were produced in four commercial plants: bacon with no preservative. If a lactic fermentation develops in the interim.. 1974. However. 2. inoculated with 1000 spores/g. it was suggested that nitrite could be important in delaying the sour spoilage caused by the growth of lactics.0%. the data supported the potential approved use of a nitrite–sorbate blend to produce bacon.. it is difficult to assess their effect.

Of significant importance was a report that the growth of S. and Pediococcus cerevisiae (1 strain) were examined for their tolerance to nitrite in the presence and absence of 4. This suggested either that nitrite was not being used as an electron acceptor or the nitrite reductase system was blocked anaerobically. Heterofermentative lactobacilli were more tolerant of salt than the homofermentative strains.3 in two samples with negligible nitrite levels. aureus is less acid tolerant under anaerobic conditions. 500. At higher temperatures (22°C and 30°C). and 0% oxygen yielded lower S. aureus under aerobic but not anaerobic conditions. nitrite. 1972). to a slight extent. The authors questioned the hypothesis that nitrite inhibition is dependent on the conversion of nitrite to nitrous acid. At 40 µg/g growth was comparable to that in the control without nitrite. because high nitrous acid values (>0. and 2000 µg/g).34. Understanding why this occurs would be very instructive. brine. low oxygen level. They also concluded that vacuum packaging should improve the safety of cured meats relative to S.01% salt. Very high levels of nitrite (1000 and 5000 µg/g) either prevented or severely retarded the growth of all strains. reduced pH. and product pH) contributes to this effect. 5%. or both prevented anaerobic toxin production. oxygen pressure. and an abnormally low pH of 5. and initial pH (6. Collectively. Extensive tests were also reported for laboratory-cured ham. At 200 µg/g growth was delayed or slower. Buchanan and Solberg (1972) studied the effect of nitrite (0. 8 µg/g enhanced the growth of the lactobacilli and. but it is unclear whether nitrite level influenced anaerobic enterotoxin production.6 µg/g) were possible only at reduced pH levels (pH 5. growth of competitive flora.3.6%.3) on the growth of S.0). (1976) indicate that the growth of S. aureus populations as . Nitrite was metabolized by S. but it remains debatable how much each of the possible factors (e.9% salt and 100 µg/g of sodium nitrite) incubated at 37°C in environments of 20%.58. no residual nitrite. the data along with the aforementioned results of Christiansen and Foster (1965) and Crowther et al. 15%.159 µg/g of nitrous acid also became toxic. A third sample with a pH of 5. it is uncertain whether high nitrous acid content. aureus and Other Bacteria A cluster of reports appeared during the early 1970s that dealt with the effect of nitrite on S. 22 µg/g of nitrite. aureus by blocking the sulfhydryl sites of either coenzyme A or α-lipoic acid. the micrococci.3. Reasons for the reduced growth and enterotoxin production are evident from the reports. Nitrous acid levels were calculated from the residual nitrite and pH values as described by Castellani and Niven (1955). aureus. nitrite was without effect in an aerobic environment and 500 µg/g or less was without effect anaerobically. Lactobacilli (78 strains).3.6 or less). Genigeorgis et al. aureus in brain heart infusion broth (BHI). and 0. micrococci and staphylococci (24 strains). At pH 7. the increase in the adjustment phase was 10 hours or less aerobically and about 30 hours anaerobically. However.Nitrite 191 Tests with S. (1969) found the production of type B staphylococcal enterotoxin to occur earlier on ham at 30°C than at 22°C to 24°C and earlier under aerobic than anaerobic conditions. The commercially canned ham used for the test had a brine level of 3. After considering the effects of changing pH during the growth of S. anaerobic toxin production occurred at pH 5. The probability of toxin production at 10°C reportedly decreased as the level of nitrous acid increased. At pH 6.. thus preventing the normal metabolism of pyruvate. Toxin was produced in certain variables having residual nitrite levels as high as 145 µg/g. Inoculated raw sausage (2. Nurmi and Turunen (1970) studied the effect of adding nitrite to a previously autoclaved broth medium (pH 6. 1000.g. Anaerobic toxin production was prevented with 0. aureus was found to be restricted to the periphery of commercially fermented sausage (Barber and Deibel. 10%. future research might demonstrate the involvement of additional factors. aureus. 7.6 µg/g or more nitrous acid and pH values below 5. Buchanan and Solberg suggested that nitrite inhibits the growth of S.3 and 200 µg/g. aureus is reduced and enterotoxin production can be prevented by the environmental conditions existing within vacuum-packaged cured meats. aureus and a review of the literature. 200. One reason given was that S. however.

residual nitrite. and decreasing pH. increasing the level of nitrite had the general effect of reducing the recovery of both heat-damaged and undamaged cells.g. Subjecting the cells to anaerobic shock (sparging with 95% N2 and 5% CO2) to inhibit pyruvate decarboxylation caused an increase in the rate and quantity of toxin produced. The formation of the Perigo factor in commercially cured meat is highly debatable. and pH). the sum of which influences stability. There was a general pattern of increased heat resistance as salt level (0. 6. Nitrite did not affect glucose transport by S. The inhibitory effect of nitrite in the recovery medium increased with increasing salt content. cytochrome oxidase) to the ferric state. If manipulations of thermally processed meat were attempted to extract such an inhibitor. Markus and Silverman (1970) reported enterotoxin A production in broth media (pH 6.6 to 6.0 but not at pH 5. both of which lack cytochromes. Contrasting results were subsequently reported that showed the production of enterotoxin A to decrease as pH decreased.8) to be unaffected by sodium nitrite (200 µg/ml) or sodium nitrate (1000 µg/ml). Adding nitrite as an electron acceptor at the time of anaerobic shock prevented increased toxin production. Bean and Roberts (1974. enterotoxin was not detected through 5 days of storage in the absence of oxygen.0 but to a lesser degree than at pH 7. and. 4.5 or 5. faecalis or Streptococcus lactis. 2. 1975) demonstrated the combined effect of pH.192 Antimicrobials in Food the oxygen level decreased. .. and nitrate on either growth or enterotoxin production. Rowe et al. Glucose repression of enterotoxin B production was also reported to occur at pH 6. Morse and Mah (1973) studied the effect of glucose on enterotoxin B synthesis in a broth medium buffered to an alkaline pH (7. Adding glucose caused decreased toxin production as a result of the rapid oxidative decarboxylation of pyruvate. and oxidative phosphorylation in Pseudomonas aeruginosa. and 8%) increased at pH 6. salt.g. (1979) reported that nitrite inhibits active transport. presumably. and nitrate was converted to nitrite. They instead rely on glycolysis for generating adenosine triphosphate (ATP) when grown on glucose and transport glucose via phosphoenolpyruvate-phosphotransferase rather than by active transport. and 7. aureus. Perhaps because of the atypically high pH. no synergistic effect occurred between salt. and nitrite on the heat resistance and recovery of S. The effect of nitrite on heat resistance was inconsistent. Although adequate growth occurred within 48 hours.0. 1973). The evidence strongly suggested that nitrite exerted its inhibitory effect by oxidizing ferrous ion of an electron carrier(s) (e. more available energy. It has been difficult to design tests that prove the presence or absence of an unknown inhibitor that is likely unstable and is apparently formed in low quantity and functions within the midst of a complex system (e. oxygen uptake. when added to the recovery medium. 1973). salt level. the percentage of lactic acid as an end product. the question could be raised whether the inhibitor would be altered or destroyed. However. salt increased.5.7). In another report on non-spore-forming bacteria. SUMMARY FOR 1970–1980 The Perigo Factor in Cured Meats 1. Formation of some similar inhibitor(s) (PTF) during the thermal processing of shelfstable canned cured meat appears possible. decreasing incubation temperature. The data of Pivnick and Chang (1974) are convincing evidence that a small percentage of the total inhibition can occur that is not the result of residual nitrite per se.7 (Morse and Baldwin... and nitrite increased (Tompkin et al. nitrite.

Mechanism of Nitrite Inhibition 1. . The low level of residual nitrite that continues to be detected after prolonged storage does not appear to be inhibitory. as used in certain experiments with bacon and large-diameter sausages. aureus was suggested to involve the blocking of sulfhydryl sites within the bacterial cells. sequestering iron) remained unclear.g. All the factors are interrelated. Destruction of residual nitrite could occur directly by utilization of nitrite by certain microbes and perhaps indirectly by reaction with degradation products produced during the growth of the flora. 9. heating times. Perishable Cured Meats Tests on a variety of cured meats showed the efficacy of nitrite to be highly variable among the laboratories and the products tested. aeruginosa.. 8. It was suggested that nitrite inhibition of C. 2. certain competitive flora are inhibitory to botulinal outgrowth (e. pH of the product during abuse Brine level Residual nitrite upon abuse and the rate of depletion during abuse Level of viable botulinal spores and vegetative cells at the time of abuse Temperature of abuse Level of ascorbate or isoascorbate Level of “available” iron in the product Type of meat and other formulation ingredients The thermal process applied to the product The growth of competitive flora The level and type of phosphate might play a role through their influence on product pH. Inhibition seems to be influenced only by the amount of residual nitrite in excess of the asymptotic level on the depletion curve. but this effect was not clearly defined in meats. of the germinated cells as the temperature is increased. botulinum may be the result of a reaction of nitric oxide with an essential iron-containing compound (e. 5. iron. In addition.g.. Whether phosphates can contribute to inhibition by some other mechanism (e. can provide the opportunity for germination followed by destruction.Nitrite 193 The Perigo Factor in Media The data appear conclusive that nitrite. and sulfhydryl groups are involved in the formation of the inhibitor. perfringens and S. Glucose transport was not affected in two bacterial species that lack cytochromes. long. Nitrite was reported to inhibit active transport. Although the thermal process given to perishable cured meats does not appear to cause thermal injury to the spores. 4. 3. 11.g. 2. 10. and oxidative phosphorylation in P. slow.. 6. 7. The effect appeared to be at the electron carrier level. Inhibition of C. The disappearance of residual nitrite in cured meat is faster as product pH is decreased and isoascorbate level and storage temperature are increased. enterococci). and perhaps injury. 3. ferredoxin) within the germinated cells. oxygen uptake. The list of recognized factors influencing the antibotulinal efficacy of nitrite continued to grow and included the following: 1. PTFs do not appear to play any role in perishable cured meats.

7. Most N-nitroso compounds (NOCs) are carcinogenic in laboratory animals and mutagenic in microbial and mammalian test systems. the regulatory agencies would have had to disapprove the use of nitrite as a food additive. The momentum established in the previous decade led to new information on the possible mechanisms involved in nitrite inhibition. 8. and methods to reduce exposure should be developed. and its effect against spoilage microorganisms and pathogens other than C.g. for public health safety it is prudent to consider that certain preserved foods may contain C. In view of the possible but unquantified risk resulting from the use of nitrite as a curing agent. Evidence does not indicate that nitrite acts directly as a carcinogen in animals. 2. there was also interest in other microorganisms. 4.. and some are teratogenic in laboratory animals. Ascorbate is now added to bacon to inhibit the formation of nitrosamines. 9. Results of limited experiments suggest that nitrate is neither carcinogenic nor mutagenic. botulinum. The investigation was conducted at the request of the FDA and the USDA. and other dietary components deserve attention. Except for its use in dry-cured and fermented products. Further research is needed to determine the amount of nitrite that is destroyed in the human stomach and the extent to which nitrosation reactions are modified by various inhibitors. botulinum spores. This includes the role of bacteria in the reduction of nitrate to nitrite and NOC formation. Concern . Exposure to nitrite should be reduced to the extent that protection against botulism is not compromised. Further studies are required to determine the mechanisms whereby nitrite controls the outgrowth of C. Although it is not possible to estimate the potential morbidity or mortality from C. 5.. pesticides and drugs) should be modified to reduce exposure. 3. Improved methods are needed to distinguish between free and bound nitrite and whether residual nitrite is a true measure of nitrosating capacity. The NAS conclusions were then used to determine regulatory policy for the use of nitrite and nitrate in foods. Interactions among inhibitors. catalysts. Attention should be given to reducing the nitrate content of vegetables and drinking water. the use of nitrate should be discontinued in meat and poultry products. Research on alternative agents and/or processing methods to replace nitrite as a preservative was actively pursued. 10. 1981. its antioxidant activity. botulinum if nitrite were removed as a curing agent from certain products. botulinum and may be abused. Additional research is needed on the metabolism and pharmacokinetics of nitrate in humans. 1982). Amino compounds that can be nitrosated in vivo should be identified and investigated. 11. Sources of human exposure to amines and nitrosamines (e. If the NAS committee had concluded that nitrite is carcinogenic.194 Antimicrobials in Food 1980–1990 The National Academy of Sciences (NAS) examined the health effects of dietary nitrate and nitrite and evaluated possible alternatives to the use of nitrite as a preservative in food (Assembly of Life Sciences. Such action would have eliminated all cured meat and poultry products from the market. its action in cured meats. Environmental exposure to NOCs should be determined. especially in certain clinical conditions. the search for alternatives to the use of nitrite should be continued. Although there was continued emphasis on the anticlostridial effect of nitrite. The exposure of humans to nitrite and nitrate should be reduced. The recommendations are also important because they influenced the intensity and direction of research. 6. Following is a summary of the 11 points in the recommendations: 1. No new agent or combination of agents should be used to replace nitrite until the safety of the new agent(s) has been ensured.

Nitrite 195 for sodium intake in the diet brought new pressure to reduce the salt content of cured meats. Their review was to assist in the development of a document for these products. where P is the probability of outgrowth and toxigenesis from one spore. The level of risk could also differ among products of the same type as a result of differences in their brine concentrations. Throughout the decade. They recommended that the bacterial spore levels in the raw product be rigidly controlled and that 150 µg/g of sodium nitrite be used to produce products with the following brine and thermoprocess values. The data provided estimates of the relative degree of botulinal risk for each product. Bacon was considered a low-risk product. Assessment of the Botulinal Risk in Cured Meats Hauschild (1982) summarized the existing data from botulinal challenge tests in a wide variety of meat products. This information was reiterated by Hauschild and Simonsen (1986). 1990).5–1. In 1983. the FDA acknowledged that prior sanction existed for the use of nitrite as an additive to poultry meat (Cassens. and erythorbate are controlled. whereas turkey ham was a high-risk product.2–0. Reductions in the recommended levels should be based on extensive plant experience and/or experimental work and controls to assure minimal spore levels. They summarized research. Hauschild and Simonsen (1985) conducted an extensive review of the technology for producing shelf-stable canned cured meats (Table 6.3–0. existing government regulations.5 1. The level of risk differed among the various products.5 3.5 Thermoprocess (F0 ) 1. He concluded that significant reductions in nitrite should not be made for all cured meats.1 Shelf Stable Canned Cured Meats: Minimal Brine and Thermoprocess with 150 mg/g Sodium Nitrite Product Luncheon meat % Brine 3–4 4. Reductions in the level of nitrite can be compensated for by moderate increases in the brine level and/or thermoprocess. Since that time.0–5. Considering the recent reductions in the amount of nitrite used for curing meats and the voluntary elimination of nitrate from most cured meats in the United States.3 3. there has been a very rapid growth in the volume and variety of cured poultry products in the United States.5 0.1). . and commercial practices to arrive at guidelines to assure the microbiological safety of these products. TABLE 6. carbohydrate. the per capita consumption of red meats decreased while the consumption of poultry products increased. further accelerating the trend of the past 40 years. Another major trend developed during this time.1–0.5–5. selective reductions in nitrite could safely be made provided the levels of brine.3 0. He proposed expressing botulinal inhibition as log I/P.5 0.0–1.0 5. However.5 Ham and shoulder Sausages Source: Hauschild and Simonsen (1985).2 1.5 4. the impact of further reductions in salt deserved close examination.0–4. This was used to quantitatively compare the data from the various challenge tests.0 0.7 4 2.0 0.

Storage at 8°C until a nearly complete loss of nitrite occurred did not diminish the effect of the nitrite. Toxin production by a mixture of type E and nonproteolytic type B botulinal strains in liver sausage. Another possibility could be offered for this observation. Product with low levels of toxin often appeared acceptable. Hauschild et al. pH 6. cooled.2% brine. The number of botulinal spores was estimated to be about 0. but the recovery of viable botulinal cells became increasingly difficult.3%. (1987) demonstrated that adding sodium nitrite to obtain an initial level of 156 µg/g could reduce the amount of salt needed for botulinal protection.2. nitrite was more effective and an appreciable delay in toxicity was obtained with 50 and 100 µg/g of nitrite. and nitrate at 8°C. The tests involved vacuum-packaged smoked whitefish fillets prepared with about 2. heated. (1980). The total C.2. botulinum was estimated to be about 0. 4.0 being required. The pH of the fillets decreased during 27°C incubation from about pH 6. (1980a) studied the significance of pH on the efficacy of nitrite and sorbic acid mixtures in a chicken frank emulsion.2 without loss of the benefit of sorbic acid. As temperature was increased to 8°C. overlaid with vaspar. 10°C. They concluded that nonproteolytic.4%). By increasing the processing temperature and/or prolonging the time of processing. Cuppett et al. After curing.24/kg. In fermented sausage. and 6.15 spores/kg in the product. 1982). The hams had a slight off-odor but no apparent putrefaction. competitive flora could have been destroyed. and aw 0. with levels of at least below pH 6. Hauschild and Hilsheimer (1983) examined 138 samples of liver sausage from retail outlets in Canada. and potassium sorbate (0% and .8% to 4. Some consumers find this amount of salt unacceptable. Toxin was not produced at 5°C. (1982). Methods and media were recommended to facilitate the recovery of both the proteolytic and the psychrotrophic nonproteolytic strains of C. (1982) assessed the relative risk of botulism from temperature-abused liver sausage. toxin production was increasingly more rapid. nitrite in the range of 0 to 100 µg/g had little or no effect on toxigenesis. botulinum. psychrotrophic strains can grow and produce toxin during the early stages of curing before the salt and nitrite levels penetrate and reach inhibitory levels. the inhibitory effect of the lactic fermentation and decreasing water activity values during fermentation and subsequent drying prevented botulinal toxicity.4 to as low as 5. Liver sausage was prepared with 83 µg/g of added sodium nitrite. sodium acid pyrophosphate (0% and 0. toxin levels remained high. a high level of salt (5% brine) has been required. If a low level of nitrite (40 µg/g) was added. Tests in model systems.2%.4%. The nitrite (40 µg/g) also significantly increased the inhibitory effect of the sorbic acid. Greater protection was obtained with 150 µg/g. The test system consisted of a frank emulsion prepared from mechanically deboned chicken meat that was extruded into test tubes. To prevent the growth of C. nitrite. Additional data on the ham experiments were presented by Lücke et al. thereby permitting the more heat resistant C. The use of nitrite in smoked fish in the United States has been restricted. botulinum to dominate and develop toxin. By increasing the thermal process. and hams was investigated by Liicke et al. and incubated at 27°C. The antibotulinal efficacy of sorbic acid was pH dependent. Similar results were obtained during the dry curing of hams at 8°C or 10°C. the pH of the formulation could be increased to pH 6. Wagner and Busta (1983) compared the antibotulinal effect of combinations of sodium nitrite (40 and 120 µg/g). and 15°C. The presence of sorbic acid slowed the rate of nitrite depletion. Sofos et al. At brine levels of 3.98. As the hams continued through the completion of curing. The latter were suspected to be of particular importance as a cause of botulism from raw cured meat products in Europe (Lücke. botulinum. fermented sausage.196 Antimicrobials in Food Botulinal Challenge Tests Tests in meat and fish products. Large bone-in hams were inoculated with nonproteolytic botulinal spores before curing in a solution of salt. toxin was detectable 10 cm from the injection site. The rate of pH decrease was slower as the brine level increased. Perhaps the milder thermal process permitted the survival of a competitive flora that prevented toxigenesis. Toxin was detected after 9 to 11 days. At a higher brine level of 5. the inhibitory effect of the nitrite was reduced.44%.

Statistically significant reductions in toxin production occurred with increasing salt level. Nelson et al. The frankfurters were made from chicken instead of beef and pork. The pH of the pork used for the higher pH slurries ranged from 6. sodium acid pyrophosphate (0. The combination of 40 µg/g of sodium nitrite. It is unclear whether the target or analytical values for salt were used for statistical analysis. (1982) examined the effect of source of the meat on the antibotulinal effect of 100 µg/g of sodium nitrite in a pork slurry system. polyphosphate. No systematic difference in inhibition was observed with the meat from three breeds of pig.03.6 to 6. or decreasing the abuse temperature.78 to 6. The combination of nitrite (40 µg/g). Of the polyphosphates tested.19.5%.4 units in the lower pH slurries and about 0. The pH of the pork used for the lower pH slurries ranged from 5. 1983). A separate formula was developed for slurries prepared with low-pH pork (pH 5.26% potassium sorbate.56. (1982) examined the interaction of potassium sorbate in their cured pork slurry system. potassium sorbate (0.4 to 6. and 4. and 0. nitrite. The increase in pH could explain the decreased inhibition in the lower pH slurries. Jarvis et al. although the conclusions were stated on the basis of the target values of 2. The pH was important in all treatments tested. This increased inhibition was not observed with nitrite levels of 75 to 125 µg/g. Tubes showing gas or other spoilage were preferentially selected for toxin assay. The effect of sorbate was greater as salt levels . There appeared to be more botulinal inhibition with the addition of sodium acid pyrophosphate.3) and high-pH pork (pH 6.72). although the trends in the data may not change. and thermal process on botulinal outgrowth in pork slurries of two different pH ranges (pH 5. 1980a). Gibson et al.b) evaluated the interactions of salt. but the reason for the increased inhibition when adding polyphosphate to the higher pH meat slurries is less evident. They found the nitrite to be less effective in slurries prepared with meat from the shoulder than meat from the leg.4%). They found that increasing the heating from P80°C = 0. They also found that the antibotulinal efficacy of nitrite could be increased by the addition of phosphate. The polyphosphates did not influence botulinal inhibition in the absence of nitrite. nitrite level. and pH control of the emulsion was the most effective to achieve reduced nitrite levels and provide enhanced botulinal inhibition.26%). The addition of polyphosphate caused an increase of about 0.4 and 5. They used the same test system as in Sofos et al. The test system avoided the problem of recontamination that occurs when peeling and packaging franks.5% salt (wt/vol water).3 to 6. The initial pH for the various products was 5. 0. more like that of a reformed ham after heating. isoascorbate.54. except for two outliers of 5.1 to 0. with certain phosphates being more effective than others.2 units in the higher pH slurries. nitrate. (1981c) analyzed results from large multifactorial pork slurry tests to arrive at formulas for estimating the probability of toxin formation in their pork slurry system. sodium acid pyrophosphate was more effective than sodium hexametaphosphate or sodium tripolyphosphate.Nitrite 197 0. 3.27 to 6. (1979) modified their pork slurry to a thicker consistency.65. At a given spore inoculum.7 and 6. potassium sorbate significantly decreased toxin production. adding isoascorbate or nitrate. (1981a. (1983) assessed the antibotulinal effect of three phosphates in the test system just described (Wagner and Busta.5%. (1979b–e.4% sodium acid pyrophosphate caused the greatest delay in toxin production. The pH of the four product variables was between 5. One nitrite level (40 µg/g) was used throughout. This may alter the quantitative assessment of the individual and combined effects of the additives.27 to 6. The levels of salt found in the slurries were often as much as 0. Polyphosphate increased botulinal inhibition when added to combinations of sorbic acid or potassium sorbate and a low level of nitrite (40 µg/g). Roberts et al.0.7).5 to 6.36 and 6. Roberts et al. nitrite in the range of 175 to 300 µg/g was even more inhibitory to botulinal outgrowth as measured by the percentage of slurries that became toxic. Considerable variation occurred between the meat from different animals within each breed. Roberts et al.26%) in frankfurters.65 to P80°C = 6.5% (wt/vol water) below the targeted values. or thermal process.

A rather high level of isoascorbate (1000 µg/g) caused nitrite to disappear rapidly and delayed the outgrowth of C. No suggestion was offered for the increased botulinal inhibition when nitrate was added. The probability of botulinal outgrowth decreased as the level of added nitrite increased above 50 µg/g. Their approach was to conduct large multifactorial experiments using pork slurries and then to analyze the data statistically and develop equations that could be used for predictive modeling. which resulted in rapid botulinal outgrowth. However. thioglycollate. After higher doses of irradiation (10. They concluded that measuring the residual nitrite of a product cannot be used to assess compliance with the nitrite regulations unless the analysis is performed soon after manufacturing the product. forms an inhibitor when autoclaved in the presence of nitrite. as pH levels decreased to below 6. Transferrin. 50. Residual nitrite levels after irradiation were inversely related to the irradiation dose. The slurries were irradiated. They found that the rate of depletion in their pork slurry system was influenced by many intrinsic and extrinsic factors.K. and as the storage temperature decreased. the addition of nitrate. The authors suggested that Bactin formation may be responsible for the inhibition observed in Perigo-type media. there was a marked decrease in the residual nitrite. 30. They suggested that the bacteriostatic effect is the result of facilitated conversion of cysteine to Bactin and a concomitant removal of thio compounds.5%). sealed with vaspar. the rate of nitrite depletion was influenced by storage temperature. an iron-binding protein in casein. The significance of microbial growth on nitrite depletion in the slurries during storage was not addressed. 100. Higher or lower levels prevented the inhibitory effect. For example. the . and stored at 30°C for 28 days. The bacteriostatic action was reversed by the addition of cysteine. then inoculated with botulinal spores. The amount of iron required for inhibitor formation was reportedly similar to the level found in muscle tissue. Perigo Factor Jonsson and Raa (1980) heated cysteine at pH 6 to produce bis-(2-amino-2-carboxyethyl) trisulfide (Bactin) and found it to be bacteriostatic to Lactobacillus plantarum. At lower dose levels of 0 to 9 kGy. and 50 kGy). This led Gibson et al. and the addition of isoascorbate. 40. and iron in culture media. The addition of nitrate led to higher residual nitrite levels for a longer period. legislation was proposed to control the use of nitrite by monitoring the residual nitrite in cured meat products.198 Antimicrobials in Food increased (2. 20. U. Pretreatment of the lactoferrin and transferrin with a protease was necessary for inhibitor formation. The results are intriguing and leave relatively unexplored the underlying mechanisms influencing the results. which otherwise prevent bacteriostatic activity. 156. unless ferric chloride was present and the pH was 6. (1984) to examine the effect of various factors on the rate of nitrite depletion and botulinal outgrowth. Custer and Hansen (1980) reported that lactoferrin.5% to 3. As expected. and 200 µg/g of sodium nitrite. responded similarly. (1989) prepared ground pork slurries with 0. there was a slight reduction of residual nitrite and the probability of botulinal outgrowth increased in only one variable (50 µg/g of sodium nitrite and the lower spore inoculum level). a similar protein in blood serum. Bactin was not produced when heating cysteine. The primary objective of the Bristol research group was to arrive at a model system to quantitate the effect of the various factors influencing botulinal inhibition in cured meats. why was a high heat treatment (80°C for 7 minutes followed by additional heating at 70°C for 1 hour) more effective for increasing botulinal inhibition? Why should the initial pH of the meat influence the degree of botulinal inhibition? Szczawinski et al. In another study of Perigo factor formation. They concluded that the level of residual nitrite did not influence botulinal outgrowth in their experiments. They concluded that lactoferrin and transferrin are precursors of the Perigo factor and that it is likely that nitrite-modified transferrin accounts for some of the antimicrobial effects of nitrite in cured meats. botulinum.0.

two additional options were added to the USDA regulations (Code of Federal Regulations. the high temperature can result in the formation of NOCs.. a listing of some of the proposed chemical alternatives along with references was later provided by Roberts and Gibson (1986). 1985). A PTF was produced when tryptone was heated with nitrite at 121°C for 20 minutes (Miwa and Matsuura. coli or S. oxidative stability. 550 µg/g of sodium ascorbate or isoascorbate. Nitrite is unique in being able to produce cured meats with the proper attributes of color. There was evidence that the factor may be unstable toward oxygen.e. The research and subsequent regulation ignore the significance of brine level as an important factor that should also be controlled. The process also does not acknowledge the potential value of the natural contaminating flora associated with most bacon processing environments.2% thioglycollate with more than 50 µg/g of sodium nitrite.Nitrite 199 requirements of pretreatment with protease and autoclaving reduce the likelihood that this would occur under natural conditions in meats. Hansen and coworkers published a series of papers devoted to the Perigo factor with the intent of learning the mechanism of nitrite inhibition. cereus was used throughout the research. The relevance of this information to the effect of nitrite on C. or cured color and flavor development). botulinum but not against E. special requirements were established by the USDA for the production of bacon and verifying compliance with a nitrosamine limit.e. a minimum of 0.. in the bacon section for 1970–1980. and sufficient sodium nitrite is added to produce an acceptable cured product. flavor. The factor was inhibitory against S. The system consists of using nitric oxide to cure the heme portion of beef blood. antimicrobial agent. and C. This led the USDA to reduce the input level of sodium nitrite to a maximum of 120 µg/g and to require the addition of 550 µg/g of either ascorbate or isoascorbate (Code of Federal Regulations. If the brine level is controlled. The second option is to manufacture the bacon using a combination of 40 to 80 µg/g of sodium nitrite. antioxidant. B. Subsequently.g. 1982) is the most complete assessment of the possible replacements and the issues involved in finding a replacement.. Thus. The system was developed to avoid the addition of nitrite. Alternatives to Nitrite for Botulinal Protection A considerable amount of research has been devoted to finding an acceptable replacement for nitrite. The second option in the USDA regulation is based on a three-plant study that verified the efficacy of this concept (Tanaka et al. It was learned that the addition of ascorbate or isoascorbate (erythorbate) can block nitrosamine formation during the frying process. The committee considered the different functions of nitrite as a food additive (e. When bacon is fried. 2002a). They concluded that nitrite inhibits the germination of spores of B. subtilis. 1983). and a lactic acid-producing bacterial culture (i. cereus by inactivating membrane sulfhydryl groups (Buchman and Hansen. Bacon was identified as the one cured meat in which unacceptable levels of nitrosamine could be produced. Typhimurium. and microbial stability. A new approach to replace nitrite was developed in Canada. B.7% sucrose). The maximum inhibitory activity was achieved by heating the medium containing 4% tryptone and 0. botulinum and the stability of cured meats that are mildly heated remains to be established. . A key to this approach is to not eliminate the lactics from the environment through an overly aggressive sanitation program. In addition. and a USDA-approved partial quality-control program to assure compliance. The rationale for this approach is apparent from the summary outlined earlier in this chapter. an adequate quantity of fermentable carbohydrate is present. None of the chemicals investigated have shown promise to date. Inhibition was not observed when casein or an acid hydrolysate of tryptone was used. a readily fermentable carbohydrate (i. The NAS report (Assembly of Life Sciences.. One option is to use a combination of 100 µg/g of sodium nitrite. 1987). This cured pigment is then further processed. Pediococcus acidilactici). 2002a). then the naturally occurring lactic flora would reduce the pH of the product to inhibitory levels in a high percentage of the packages if the bacon were temperature abused. aureus.

(1980) reported nitrite inhibition of oxygen uptake and proton-dependent proline transport in E. Fang et al. phosphate.6. The accumulation of glucose decreased in relation to increasing the nitrite level and the time of exposure to nitrite before adding sugar. This effect was explainable by their modes of action. and ammonia were not end products of the sodium nitrite disappearance. along with other ingredients (e. They found that RSNO was less effective than nitrite for preventing the recovery of heat-shocked C. toward increasing inhibition with an increasing level of origanum oil in three of the four sets of nitrite levels. very little synergistic effect was observed with RSNO and salt. which is formed during the curing of meats. Because streptococcal aldolase was sensitive to nitrite but glucose accumulation was unaffected. Third. The anticlostridial effect of RSNO increased as the . but a high level of synergism was observed with nitrite and salt. Because glucose transport was not affected. alone or in combination with sodium nitrite. The origanum oil affects germination and vegetative growth. (1985) investigated the effect of nitrite on S. This was thought to be the result of the highly soluble oil components being absorbed into the lipid fraction of the meat. lactis and S.200 Antimicrobials in Food dried. and then microencapsulated to provide stability against degradation by oxygen and light. Perhaps the results would be more favorable if challenged with a lower botulinal spore level. or absorption to the cells. (1990) and Shahidi and Pegg (1992) have summarized the system and provided the status on the various aspects of the research. could prevent botulinal outgrowth in a broth medium. oxidative phosphorylation. 400. interactions with metabolic products. The proposed system also requires the addition of an antimicrobial agent (e. and tertiary butylhydroquinone). At levels of 200..g. Recovery of the heat-injured cells was comparable in media with no nitrite and media containing 100 µg/g of sodium nitrite.. sodium hypophosphite). Second. The two additives acted synergistically. to provide oxidative stability. Unfortunately. origanum oil was not as effective when tested in pork. Kanner and Juven (1980) compared the anticlostridial effect of nitrite and S-nitrosocysteine (RSNO). S. At the lowest spore levels tested. Under less rigid conditions of anaerobiosis. 1981). coli. sporogenes spores. aureus hastens the loss of nitrite was not attributable to nitrite reductase activity. Palumbo and Smith (1982) found heat-injured S. although statistically insignificant. and 800 µg/g. Nitrite was found to inhibit aldolase. nitrite acts as an uncoupler. The increased efficacy of nitrite under strict anaerobic conditions could be a factor affecting the growth of other microbes in cured meat and poultry products.g. aldolase) are inhibited. causing a collapse of the proton gradient. First. ascorbate.g. nitric oxide. the recovery was progressively lower. The inhibitory effect of nitrite was enhanced by applying stricter anaerobic conditions. this suggested that nitrite was inhibiting glucose catabolism. The cured meat pigment would then be added. faecalis is the result of the impermeability to nitrite in these bacteria. The authors concluded that nitrite inhibits bacteria by several mechanisms.. certain metabolic enzymes (e. the authors suggested the high resistance of S. aureus have been reported to be inhibited by nitrite under anaerobic conditions (Simone and Solberg. The mechanism by which S. Mechanism of Nitrite Inhibition Yarbrough et al. aureus. Nitrite. Glucose and 2-deoxyglucose transport and accumulation by S. and protondependent active transport. aureus was more resistant to the nitrite and also caused a more rapid depletion of nitrite in the medium. This suggested that RSNO is not the major anticlostridial factor in cured meat. aureus. dismutation resulting from the reduced pH during growth. nitrite interferes with energy conservation by inhibiting oxygen uptake. When salt was also present in the recovery medium. nitrous oxide. sodium nitrite affects outgrowth and vegetative growth. but hexokinase was not affected. aureus to undergo repair and grow in a nitritecontaining agar medium at pH 5. The disappearance of nitrite was faster with S. Ismaiel and Pierson (1990) demonstrated that origanum oil. O’Boyle et al. there was a consistent trend.

. Reddy et al. sporogenes in a glucose medium caused a rapid decrease in intracellular ATP concentration and an accumulation of pyruvate in the medium. one of which is an iron protein containing nonheme iron and sulfide as a cubic Fe4S4 cluster. RSNO (540 µg/g) was considerably less inhibitory than sodium nitrite (200 µg/g) when tested in inoculated perishable canned turkey at 27°C. Adding EDTA or denatured nitrosylated myoglobin increased the efficacy of nitrite.. Nitric oxide was inhibitory. Woods and Wood (1982) next reported that adding sodium nitrite to cell suspensions of C. Nitrite did not react directly with ferredoxin. The enzyme system consists of two proteins. but this could be attributed to the formation of analytically detectable nitrite in the meat. thereby verifying observations that ascorbate increases the antibotulinal efficacy of nitrite in meat products. The results suggested that nitrite is converted to nitric oxide. Adding nitrosylated myoglobin to the meat system resulted in a rather inhibitory system compared with adding untreated myoglobin. Recovery of heat-injured spores in a basal medium containing 3% salt or 1% salt with 200 µg/g of nitrite yielded comparable recovery rates. Chumney and Adams (1980) found C. Adding ferric chloride or myoglobin decreased the antibotulinal efficacy of nitrite. (1981) reported that ferric chloride or hemoglobin stimulates the growth of C. They concluded that botulinal cells contain iron–sulfur proteins that react with nitrite to form iron–nitric oxide complexes. The decreased inhibition by nitrite when iron compounds were added appeared to be the result of a reduction in residual nitrite levels in the product rather than to the iron serving as nutrient to stimulate botulinal growth. This enzyme consists of a single protein molecule containing thiamine pyrophosphate and a single nonheme chromophore. (1983) used electron spin resonance to examine the spectra of botulinal cells treated with nitrite and ascorbate. Woods et al. They suggested the increased sensitivity to each agent was caused by the same cellular damage. The authors concluded that. With addition of sodium ascorbate the intensity of the reaction was stronger. botulinum led to an accumulation of pyruvic acid. botulinum (as opposed to reducing the antibotulinal efficacy of nitrite) in canned cured meat. although nitric oxide can react with ferredoxin in vitro. a nutritional requirement for growth. Their results supported the theory that nitrite is inhibitory to C. suggesting that nitrite inhibits the phosphoroclastic system. ferredoxin) that are essential for growth. with resultant destruction of the iron–sulfur cluster. perfringens spores to be sensitive to inhibition by salt. the nitrite was relatively ineffective as an inhibitor. there was no noticeable relation between botulinal growth rate and residual nitrite level in the meat to which nitrosylmyoglobin had been added. but there was decreased activity in the ferredoxin from cells preincubated with nitrite.. Collinge et al. Meyer (1981) investigated the effect of nitrite and nitric oxide as inhibitors of the nitrogenase enzyme system of Clostridium pasteurianum. This may have been the result of sulfite in the basal medium (tryptone sulfite neomycin agar) because sulfite destroys nitrite (Tompkin et al. Also. The accumulation of pyruvate was the result of inhibition of the phosphoroclastic oxidoreductase.6. Compared with earlier research. botulinum by inactivating iron–sulfur enzymes (e. They concluded that the antibotulinal effect of nitrite and related compounds is probably the result of inhibition of the iron-containing enzymes within the botulinal cell rather than to the complexing of iron. 1980). Vahabzadeh et al. Nitric oxide was found to rapidly and irreversibly inactivate the iron protein.4 to 5. (1981) found that the addition of nitrite to a suspension of C. They considered that nitrite inhibits C. botulinum by making the iron of the heme groups in meat unavailable for growth.Nitrite 201 pH of the culture medium was lowered from 6. nitrite. which then disrupts the iron–sulfur cluster of the protein and thereby inhibits nitrogenase activity. and a mixture of polymyxin and neomycin after heat injury at 70°C to 100°C. On an equimolar basis. the main site of inhibition within intact cells is the reaction of nitric oxide with the enzyme pyruvate-ferredoxin oxidoreductase. botulinum in pork.g. (1983) found carbon monoxide to be ineffective as a replacement of nitrite for inhibition of C.

it is fairly certain that the mechanism of nitrite involves the inactivation of iron–sulfur proteins. incubation temperature. sodium chloride. 1978). and 5. The inhibitory effect of nitrite against C. The fecal streptococci proved to be quite resistant to nitrite. Miscellaneous Studies in Laboratory Media Botha and Holzapfel (1987) tested the effect of nitrite on Sporolactobacillus isolates from a variety of sources. In addition.0. The cell walls showed a 90% decrease in thiol content when compared with untreated cells. and 200 µg/g). a decrease in temperature of incubation. A high salt concentration and low incubation temperatures were the more important factors influencing the inhibition of these bacteria.5. Buchanan et al. comparable to the inhibitory properties of the anion of Roussin’s black salt. 120.5%) and sodium nitrite (50. leading them to doubt that the inhibitory effect of nitrite is on preformed iron–sulfur proteins of the bacterial cell. perfringens to 3% to 4% salt could explain why there have been few outbreaks in C.0) found in cured meats. decreasing pH and aw) influenced the rate of death. 28°C. At pH 6. botulinum was again addressed by Carpenter et al. Research by Payne et al. Sodium nitrite was added to the broth before autoclaving. Juntilla et al. At the pH values (6. sporogenes. (1980). In the research of Duncan and Foster (1968b) such blister formation was not reported following exposure to sodium nitrite. the data more strongly demonstrate that L. which is more typical of cured meat and .5. some reduction in growth was observed with the addition of 200 µg/g of sodium nitrite. (1991) and Roberts and Dainty (1991). perfringens was enhanced with a decrease in pH. more generally.202 Antimicrobials in Food The debate over whether clostridial ferredoxin or pyruvate-ferredoxin oxidoreductase is the site of nitrite inhibition for C. such as ferredoxin and pyruvate oxidoreductase. (1990a.g. 30% to 50% less growth was observed after 5 days at 35°C with 100 or 200 µg/ml of sodium nitrite compared with no added nitrite. although the nitrite had been autoclaved with the broth.5. Consistent with previous reports over the past 60 years. 19°C. On the basis of the research conducted by these researchers and others. The incubation temperature (5°C. After continued research on the mechanism of compounds associated with the Perigo factor.. Sodium nitroprusside was found to be a very effective inhibitor of C.. The final pH of the products was about pH 4. blisters at the surface of the cells were observed when viewed under the electron microscope.5 and 6. and sodium nitrite on the inhibition of C. (1989) found the rate of death of L. (1989) tested the effect of nitrite on L. The effect of nitrite on the growth of Listeria monocytogenes was examined in trypticase soy broth by Shahamat et al. Most cured meats have traditionally had brine levels of 3% or greater. In a broth medium at pH 6. perfringens and fecal streptococci in broth media. It appears that the hypothesis proposed earlier is fairly close (Tompkin et al. sporogenes cells and isolated proteins. 5. and/or an increase in the salt concentration. monocytogenes to be similar in fermented sausage manufactured with different levels of salt (3. nitrite was not an effective inhibitor at pH 7. perfringens foodborne illness involving cured meats. One aspect that the more recent data suggests is that the interaction of nitric oxide with iron–sulfur complexes is irreversible. It can be concluded that factors (e. Nitroprusside was used as a model to investigate the inhibitory mechanism of sodium nitroprusside and. Their results led to the conclusion that both are inactivated.0.6. They were unable to demonstrate a direct action of the compounds on the iron–sulfur proteins. Additional information and views on the mechanism of nitrite are available in Roberts et al. monocytogenes in a broth medium at pH 7. Gibson and Roberts (1986b) demonstrated the interactions of pH. the efficacy of sodium nitroprusside was explored (Joannou et al.b) examined the effect of nitrite and iron–sulfur–nitrosyl complexes on iron–sulfur clusters in C. (1987). monocytogenes is quite resistant to the inhibitory effect of nitrite. the toxicity of nitrosyl compounds to bacteria. and 37°C) had the strongest effect on growth rate and lag-phase duration..0% and 3. However.0. 1998). The relative sensitivity of C. The cell-wall surface was the primary point of attack for the nitrosyl complex associated with nitroprusside.

monocytogenes will grow in processed meat and poultry products at refrigeration temperature. buchneri (Wolf and Hammes. Inhibitory species caused the pH of the bologna to decrease to 5. Under certain circumstances the level of analytically detectable residual nitrite can influence microbial inhibition. CollinsThompson and Lopez (1982) and Dodds and Collins-Thompson (1984) further demonstrated that lactic acid bacteria. viridescens. CollinsThompson and Lopez (1982) reported the inhibition to be species dependent. The growth of Brochothrix thermosphacta was restricted when grown on sliced vacuum-packaged bologna with Lactobacillus brevis or L. viridescens or Leuconostoc mesenteroides. plantarum. 1985). and 200 µg/g of sodium nitrite. The data of Grau (1980). Fresh pork was divided into two groups: low-pH meat (5. vacuum packaged. The depletion of nitrite was attributed to enzymatic activity (i. the canned hams were opened. can accelerate nitrite depletion. lactis. nitrite reductase) rather than lowering the pH of the medium.5% and 4. lactis was able to reduce nitrite to N2O (Dodds and CollinsThompson.5).5%).Nitrite 203 poultry products. and placed at 5°C. the lactic acid bacteria . The higher pH meat resulted in a ham with a pH of 6. During the 7 to 8 weeks of storage at 5°C.e. The authors questioned the role of nitrite during cured meat spoilage because there is evidence demonstrating that nitrite can either limit or enhance the population of lactics. L. 1988). the degree of inhibition was most strongly enhanced by reducing the incubation temperature. Both products had a brine level of approximately 4. manganese). both strains accumulated manganese in their cells. L. viridescens was among the most active species. By 1988 it was established that nitrite reduction can occur in some strains of L. Competitive inhibition can be a factor affecting the spoilage flora of cured meats.3 to 6. When comparing 0. Spoilage of Cured Meats Zeuthen (1980) studied the effect of meat pH on the rate of microbial growth on sliced ham..5 to 5. sliced. that were resistant to the addition of 50 µg/g of sodium nitrite if grown aerobically in APT broth. commonly involved in the spoilage of cured meats. 50. The significance of these observations was discussed. the rate of microbial growth was considerably slower in the sliced ham prepared from the lower pH meat. The loss of residual nitrite in bologna as a result of the action of the lactic acid bacteria was estimated as 30%. Collins-Thompson and Lopez (1981) demonstrated that nitrite depletion rate can be increased by certain lactic acid bacteria that are typically responsible for the spoilage of many cured meats. nitrite may stimulate the uptake and transport of ions (e. For example.5 within 4 weeks. however.0 and a residual nitrite level of 38 µg/g after processing. however. particularly homofermentative lactobacilli.. plantarum but not with L. demonstrate that inhibition can be dependent on the concentration of undissociated lactic acid and the degree of anaerobiosis existing at the site of growth. to a lesser extent by anaerobiosis. 100. After cooking and chilling.0%. leichmannii. The results of Glass and Doyle (1989) and Schmidt and Kaya (1990) indicate that the presence of nitrite is of questionable value in determining whether L. plantarum. When grown anaerobically. L. the rate of nitrite depletion in cured meats can be important. Collins-Thompson and Thomson (1986) found two strains of L. The hams were boned and pumped with a pickle solution containing sodium tripolyphosphate. Subsequent tests demonstrated that a strain of L. Noninhibitory strains decreased the pH of the bologna to only 5. and even less by increasing the salt level (0. Thus.3 within 2 weeks at 5°C.g. it was suggested that in trace metal metabolism. The nitrite reductases generally are heme-containing enzymes. L. acidophilus. The authors suggested that inhibitory strains produced specific antibiotic substances other than hydrogen peroxide or lactic acid. and some interesting questions were posed. The lower pH meat resulted in a ham with a pH of about 6. Both cultures were sensitive to the inhibitory effect of nitrite under anaerobic but not under aerobic conditions. nitrite was inhibitory.35 and a residual nitrite level of 90 µg/g. The meat was then used for processing as canned hams.7) and high-pH meat (6. and L. L.

hemoglobin. Kafel and Jozwik (1987) investigated the spoilage rates of commercially produced products from 6 meat processing plants in Poland. 100. cereus can grow in the product even with the normal curing ingredients of salt. and 200 µg/g) on the spoilage flora of cooked pork loin. did not activate nitrite reductase or catalase activity. myoglobin. (1980) found the most important factors influencing the rate of microbial spoilage of liver sausage to be the degree of cooking and the level of nitrite. Increasing the level of sodium nitrite (0.8. This led to the lactic acid bacteria becoming the dominant spoilage flora in the vacuum-packed product. and Moraxella and Moraxella-like organisms were increasingly inhibited by increasing the nitrite concentration and/or decreasing the temperature of storage (2°C. A correlation was found between the nitrite concentration at the time of slicing and packaging and the time for spoilage to occur.e. all the strains of L. sake tested were able to produce catalase when provided with hematin. plantarum tested were able to reduce nitrite when hematin was provided to cell suspensions.. and 74°C). Following the clue provided by Whittenbury (1964) that some strains of lactobacilli produce heme-containing catalase when grown in the presence of blood or hematin. cereus in liver sausage has been a problem in Finland. sliced. and lactic acid bacteria were inhibited to a limited extent. cooked. thermosphacta and Enterobacteriaceae at all temperatures of storage (2°C. 63°C to 68°C) led to the growth of enterococci during storage at 7°C. chilled. and sodium nitrite (120 µg/g). and processing to 68°C resulted in a shelf life of more than 16 weeks at 5°C. 10°C. an iron-free compound. 5°C. The level of surviving enterococci decreased as the processing temperature increased (63°C. 5°C. Grampositive cocci. Also. and then vacuum packed. and 20°C).e. The spoilage flora of the liver sausage prepared without nitrite also included Pediococcus pentosaceus. thermosphacta. A marginal thermal process (i. hematin) did activate the enzymes. cereus was delayed or prevented by including a combination of erythorbate. Nitrite inhibited the growth of B.. plantarum and L. dominated the spoilage flora in product with nitrite. 1981). Product with GDL had a pH value of 5. Protoporphyrin. (1988) reported that the growth of B. It was concluded that nitrite has a significant effect in extending the keeping quality of vacuum-packed cured meat when used in combination with low storage temperatures and/or the use of packaging films with a low oxygen permeability. whereas other iron-containing porphyrins (i. 50. being insensitive to the presence of nitrite. 68°C. and 150 µg/g) retarded microbial growth. Chyr et al.2 to 0. Adding GDL was considered the most important. 100.7 to 5. and 10°C). The growth of B. Nielsen (1983b) subsequently studied the effect of sodium nitrite (0. The Pediococcus isolate was not as heat resistant as the enterococcus. Using good quality raw materials. Wolf and Hammes (1988) found all the strains of L. thermosphacta may also have significantly influenced spoilage of two of the products. phosphate. The enterococcus survived 65°C for 5 minutes or 60°C for 60 minutes with only 103 cells/ml broth. about 0. particularly in the summer. Polish regulations require the heating of perishable canned . Silla and Simonsen (1985) studied the rate and type of spoilage of four cured meat products packaged under vacuum and in modified atmospheres. Gramnegative bacteria often constituted the major flora of the product without nitrite.. If the product is temperature abused. and citric acid in the product. The formulated raw meat was stuffed into a casing. Omitting nitrite resulted in a perfume-like odor that was associated with the growth of enterococci.3 units lower than with the other variables tested. the Gram-negative flora proliferated in product with nitrite. An industry survey found that liver sausage was commonly cooked to an internal temperature of 65°C. The spoilage of liver sausage was shown to be the result of S. faecalis (Chyr et al. yeasts. and 200 µg/g) on the spoilage flora of a sliced vacuum-packed bologna-type sausage. B. Again. Additional additives were tested to reduce this risk. as the storage temperature increased. Asplund et al. GDL). In addition. Nielsen (1983a) studied the effect of sodium nitrite (0. gluconolactone (glucono delta lactone. the lactic acid bacteria. Lactobacilli were the dominant flora in all four products at the time of spoilage. B. 100. 156 µg/g of sodium nitrite.204 Antimicrobials in Food were considered incapable of synthesizing heme compounds. High levels of B. Enterobacteriaceae.

A test was not conducted in which the pH of the two meats was adjusted and iron was added along with the nitrite. The NAS concluded that the evidence available about nitrite does not indicate that nitrite can act directly as a carcinogen in animals. The effect of nitrite on the aerobic spoilage flora of raw chicken patties was studied by Bushway et al. This could be influenced by the fact that white meat is lower in pH and iron content than dark meat. 3. The authors concluded that the differences in iron content between the dark and white meat may contribute in a small way to the differences observed. isoascorbate. Adding EDTA alone had no significant impact on the rate of aerobic microbial growth. Tests demonstrated that nonproteolytic. Extensive studies in meat and meat slurries demonstrated the interactions of nitrite. The authors also investigated the effect of the inherent pH of the meat on the efficacy of nitrite. Bushway and Serreze (1984) also studied the effect of adding EDTA to raw dark chicken meat. psychrotrophic strains of C. respectively. The spoilage rate of this second group of cans was 4%. pH. the efficacy of nitrite increased when the pH of dark meat was adjusted to 5. the effect of iron on aerobic microbial growth depended on the amount of iron added. This led to the development of a model system for predicting botulinal outgrowth in food. Adding EDTA enhanced the efficacy of nitrite and caused an increase in the lag phase for aerobic microbial growth. even a low level of nitrite (40 µg/g) caused some inhibition in white meat but not in dark meat. 2. phosphate. With the addition of sodium chloride (2. a concept . SUMMARY FOR 1980–1990 1. This provided a basis for assessing the risk associated with different cured meats. The time elapsed was normally 7 to 10 days after production. The ground meat was aerobically packaged and stored at 4°C to 5°C. Low levels of iron (5 to 15 µg/g) reduced the efficacy of nitrite. additional cans (8290) were removed from storage at around 8°C and incubated at 37°C for 3 days. Nitrite was considerably more effective in white meat than in dark meat. and thermal process on botulinal outgrowth. high levels of iron (40 to 160 µg/g) increased the efficacy of nitrite.6. From among 4322 cans of 8 different classes of perishable canned cured meats. These data reflect the influence of a mixture. This led to the conclusion that the inherent pH of the meat is the major factor determining the effectiveness of nitrite on the rate of aerobic spoilage in chicken meat. Perhaps some adjustment occurred in the microbial population.5%). The regulations also require that product from each “lot” be incubated at 37°C for 3 days to assess the relative stability and quality of the product.4. The pH of the white and dark meat was 5. When spoilage first occurred in each lot.Nitrite 205 meats to a minimum internal temperature of 68. If nitrite (130 µg/g) was also added. 1980). botulinum can multiply and produce toxin in dry cured hams during the early stages before the salt and nitrite levels penetrate and reach inhibitory levels. or 23%. the NAS was not able to conclude that nitrate can act as either a carcinogen or mutagen. 4.6 and 6. A means to quantitatively compare the data from various challenge tests based on the probability of outgrowth and toxigenesis from one spore was proposed. The authors assumed that the lower spoilage rate was the result of the death of spores that had been heat injured and then subsequently exposed to the effect of salt and nitrite. whether cooked or raw. Furthermore. (1982). However.8°C. of naturally occurring spores in fairly large cans of commercial product. The antimicrobial effect of nitrite was blocked when the pH of the white meat was adjusted to 6. The reason for this effect on the aerobic spoilage flora was not known. 980 cans. Likewise. These conclusions meant that neither nitrite nor nitrate would be banned as food additives. Bushway and Serreze (1984) found that adding iron to raw white meat caused an increase in the rate of aerobic microbial growth. Bushway and Jen (1984) found lower residual nitrite levels in chicken white meat than in dark meat. This could explain the high number of cases that have occurred during certain periods in Europe (Tompkin.4. swelled.

1991). The potential impact of various blood fractions as ingredients in sausage was investigated (Miller and Menichello. SINCE 1990 C. A beef sausage formulated with 156 µg/g sodium nitrite and an average iron level of 20 ± 5 µg/g was used as the control.. The debate over the risk of C. (1991). McClure et al. Adding hemoglobin. Alternatives to nitrite continued to be investigated. 1993). or whole blood reduced the time for botulinal outgrowth and toxin formation compared with the control with nitrite. cured products that receive no heat treatment or are cooked to final internal temperatures in the range of 60°C to 75°C. delayed the time for toxin to be detected. The USDA adopted a regulation for bacon that requires a maximum of 120 µg/g sodium nitrite and the addition of 550 µg/g sodium ascorbate or isoascorbate. and the addition of plasma delayed the time for toxigenesis compared to the control sausage (Miller et al. that has been expanded to include other pathogens and has gained acceptance throughout the world. At pH values of 6. Buchanan and Phillips (1990) studied the growth of L. “What we need at the present time. in my opinion. using an automated tubidimetric system with multiwelled plates containing trypticase soy broth. Again.0 and 5°C no growth was observed with any of the levels of sodium nitrite evaluated (50. monocytogenes in tryptose phosphate buffer under a wide variety of conditions (salt. Other available options have not received commercial acceptance.0 sodium nitrite had little effect in delaying the time to detect visible growth except at the highest level tested (200 ppm) and a temperature of 15°C or below. Some research continued on the Perigo factor. Adding dried plasma. the USDA in 1988 initiated a series of increasingly restrictive policies on the rate of chilling for perishable cured meat and poultry products manufactured under USDA inspection. 7. temperature. found the efficacy of sodium nitrite was temperature and pH dependent. pH. At 20°C and below no visible growth occurred even with 50 µg/g sodium nitrite and a pH of 5. This situation is a reminder of Morris Ingram’s frustration with the increase in research on nitrite’s role in botulinal inhibition in the 1970s. 400 µg/g).206 Antimicrobials in Food 5. aerobic/anaerobic conditions) and found sodium nitrite to interact with combinations of the factors tested. however. monocytogenes to the degree that . 6. The mechanism of nitrite inhibition differs in different bacterial species. In the absence of nitrite. 8. red blood cells. In subsequent tests the effect of various decolorized blood fractions was evaluated. but none have been adopted commercially. At pH 6. Sausage formulated without sodium nitrite became toxic within 1 week compared with 3 weeks when nitrite was present. 200. but no evidence was generated to link the Perigo factor to the antimicrobial effect that has been demonstrated in perishable. monocytogenes in ready-to-eat foods.3. Since 1990 there has been increased interest in control of L. 1974). Autoclaving the sodium nitrite in the medium did not increase the degree of inhibition against L. perfringens in commercially manufactured cured meat and poultry products has continued into the new millennium and serves as an example for the need of a risk assessment to clarify appropriate risk management policies. Despite this evidence. The database was incorporated into the USDA Pathogen Modeling Program. very slow growth did occur. This is a case where the epidemiologic data indicate a negligible public health concern for cured meats but the evidence from challenge studies and predictive modeling suggests otherwise. 100. At the time he stated. perfringens has not been a concern in cured meat and poultry products as evidenced by the relative lack of research on this pathogen during the past 40 years. the rate of toxin formation was inversely related to the level of iron. is not more inoculated pack experiments but a rationale for interpreting them” (Ingram. This probably reflects the effect of the higher salt levels and nitrite found in cured products compared with noncured products.

. and temperature of storage (4°C. 10°C) on the rate of growth of L. Since about 1990. In regions of Northern Europe. sporogenes (9 × 105/ml) in reinforced clostridial medium. 1997). Adding 200 µg/g sodium nitrite to the sausage increased the time required to multiply from 10 cfu/g to 105 cfu/g by 9. and sodium nitrite in laboratory medium and a predictive model for the growth of L. Perhaps the high iron content of liver pâté negated the effect of sodium nitrite and sodium erythorbate in this product. 1992b). the effectiveness of sodium nitrite was significantly increased by the addition of sodium ascorbate.7. Sameshima et al. and the United States. mesenteroides. salt (1%. Three µM of residual undissociated nitrite doubled the time taken for a 3 log10 increase in numbers. monocytogenes in the sliced vacuumpackaged product when stored at 4°C or 8°C. Duffy et al. irradiation. although sodium nitrite concentration was not varied. 1995. (1997) examined the effect of nitrite on three lactics in vacuum-packed cooked sausage made with and without nitrite. smoked flavor can be found. 4. viridescens. and additives to achieve shelf stability of canned luncheon meat (Farkas and Andrássy. Farber et al. inoculum level. An extensive series of experiments led to the conclusion that nonthermal inactivation of L. If the broth had been adjusted to pH 6.0 and a sour. Farkas and Andrássy (1992a) described a test system to evaluate the combined effects of formulation.Nitrite 207 had been observed with clostridia (see Perigo Factor). (1994) inoculated a variety of vacuum-packaged cooked sliced meats with L. monocytogenes in liver pâté. L. 3%). Using C. Qvist and Bernbom (2000) manufactured bologna-style sausage with 60 and 150 µg/g sodium nitrite and found neither level retarded the growth of L. they estimated the various formulations and thermal processes provided the equivalent of 4 to 7 log10 destruction of C. as had been reported earlier for C. and Enterococcus faecalis. Liver paté has been implicated in outbreaks of listeriosis in the United Kingdom. 550 µg/g). some . botulinum. (1992). but levels above 3% were inhibitory. monocytogenes and found the lag time increased and the rate of growth decreased at 0°C and 5°C with the addition of sodium nitrite (0 to 315 µg/g). pH. sodium erythorbate (0. As in the case of earlier research with C. sausages made with nitrite and having pH 5.8 and containing 3. These sausages are produced at temperatures below those generally used in the United States but higher than the temperatures used to manufacture traditional raw fermented dry sausage. botulinum. and 15 days for L. monocytogenes by sodium nitrite is pH dependent and related to the concentration of undissociated nitrous acid (Buchanan and Golden. heat. The procedures described show promise for estimating and comparing the combined effects of multiple treatments on pathogen survival and growth. respectively. Low levels of salt (1% to 2%) enhanced growth. (1995) examined the effect of sodium nitrite (0 and 200 ppm). Buchanan et al. sporogenes. F0 = 0. monocytogenes.5% sodium chloride and 50 or 200 µg/g sodium nitrite inhibited growth during 125 days storage at 37°C. McClure et al. Applying the procedures described by Hauschild (1982). Australia. nitrate continued to be used in the manufacture of fermented meats and dry cured hams throughout much of Europe and to a lesser degree in the United States. Only temperature proved to be a significant factor. The effect of salt and nitrite on the growth of 24 lactics isolated from vacuum-packed cooked sausage was evaluated by Korkeala et al. Leuconostoc isolates seemed more sensitive to the effects of sodium nitrite and salt in comparison to the homofermentative lactobacilli.7 with 50 µg/g). and other factors for inactivating spores and preventing the outgrowth of survivors. temperature. The model test system was then used to evaluate various combinations of heat. they found that broth adjusted to pH 5. a combination of heat and sodium nitrite was necessary to prevent survival and growth (F0 = 0. (1996) subsequently investigated the effects of salt. Fermented Meats and Dry Cured Hams Although removed from most cured meat products in the United States during the 1970s.2 with 200 µg/g.

8) and flavors that are more balanced. rich.3°C –0. Tests by Mares et al. rubbery or spreadable texture Low-Acid Sausage <12°C 14°C –16°C ~ 6. Flores and Bermell. perhaps. Lücke. plantarum but not P. loss of the traditional flavor and increased acidity has led to a decrease in consumer acceptance and declining sales. In pursuit of more effective starter cultures for use in manufacturing dry sausage. was hematin dependent for nitrite reduction and yielded ammonia as the sole product. Flores. Hungary. nonacidic flavor. The basic parameters of raw fermented sausage processing in Europe are summarized from Incze (1992) in Table 6. nitrate Yes Salty. Fernandez et al. Italy. In Europe. 1991). It is curious that very little information has been published during that period from researchers in North America.g. and nonacidic. the rapid production of acid renders the cocci inactive and. 2001). 1994. (1994) found that sodium nitrite (80 µg/g) reduced the catalase activity of a strain of L. incapable of reducing nitrate or influencing flavor development. Scannell et al. In Spain.0 None Nitrate Not necessary Salty. complex. 1989. Wolf et al. softer. The other group was able to reduce nitrite in the absence of hematin and yielded nitric oxide and N2O.2 Basics of Raw Fermented Sausage Processing in Europe Processing Characteristics: Fermentation Drying/aging Final pH Added carbohydrate Added nitrate/nitrite Added starter culture(s) Product characteristics: Source: Incze (1992). pentosus. In studies involving faster fermentation. Several reviews. 1989. consisting of L. >5.208 Antimicrobials in Food TABLE 6.. more than 50% of the initial nitrite disappearance was attributed to conversion to . low-temperature processing that involves long holding times and reduction of nitrate by micrococci or staphylococci to yield nitrite and other nitrogen oxides that are necessary for cure color development. these traditional raw fermented sausages have had higher pH values (e. In more modern processes. The cocci also play an important role in flavor and aroma development. 1996. L. and the Balkan countries. pentosus. Specifically. and Pediococcus pentosaceus. Tests on 70 strains of lactic acid bacteria using cell suspensions incubated anaerobically led to two groups. High-Acid Sausage 22°C –25°C 15°C –18°C <5. describe the procedures used and changes occurring in Europe (Incze. Spain. plantarum. 1997.. 1987.7% Nitrite and. Nagy et al.. firm texture manufacturers in the Mediterranean countries have been shifting away from the more traditional. 50. Subsequent research found the lactic acid bacteria could be separated into heme-dependent and nonhemedependent catalase producers (Wolf et al. the rate of fermentation is faster and micrococci become inactive as the result of an accumulation of acid. (1990) conducted further investigations on nitrite reductase activity among the lactic acid bacteria.. nitrate has traditionally been used for dry sausages and hams requiring long ripening times. One group. published since 1987. In France. (2001) found the growth of two strains of Lactococcus lactis and production of the bacteriocin lacticin 3147 was inversely related to the added level of sodium nitrite (20. and 1992. acidilactici or L. which is responsible for the reddening and other effects. Nychas and Arkoudelos. and 100 µg/g) in salami. A few of the studies pertaining to the manufacture of fermented meats will be summarized. round. strongly acidic flavor. thus.3 0. 1990. The trend toward using starter cultures and slightly higher fermentation temperatures to reduce processing times is accompanied by certain negative features.2. In these processes micrococci convert the nitrate to nitrite.

Nitrite 209 nitrate. (1996) demonstrated that the red pigment found in Parma ham was much more stable and different from nitrosomyoglobin. Certain bacteria. which is then either excreted or further reduced to ammonia. Color stability was mainly affected by the presence of residual added ascorbate (Alley et al. Of the ten strains selected for further study. A study comparing sausages made with only nitrate or nitrite and a 26-day process at temperatures that did not exceed 15°C found the aroma and flavor to be more acceptable when the product was made with nitrate. 1986). Typhimurium. Morita et al. Two main forms have been described for respiration and. and S. carnosus and S. warneri. are able to perform assimilatory nitrate reduction but cannot use nitrate as an electron acceptor.(1994) found that S. Adding nitrate to the growth medium favored the synthesis of catalase. the red pigment that is associated with products made with nitrate or nitrite. In assimilation. nitrate is taken up.. warneri had previously been reported to have no nitrate reductase activity and a low catalase activity and when inoculated into sausage caused the product to become rancid (Berdagué et al.. The mechanism for the formation of the pigment in the absence of nitrate and nitrite was not determined. in both. 1999). which is used commercially as starter cultures for fermented sausages and dry cured ham. In respiration. Parma and San Daniele hams made in Italy. but nitrate alone is more common (Sanz et al. epidermidis. Tests with two strains of S. nitrate is used as an alternative electron acceptor when oxygen is not present. S. Obligately respiring bacteria (e. Karst ham made in Yugoslavia. respectively. Neubauer and Götz (1996) summarized nitrate utilization as being either assimilation or respiration. First. lentus in a sausage mix without nitrite or nitrate found both to produce a pigment in the meat that had the same spectral pattern as the pigment found in Parma ham. 1993). reduced to nitrite. A new class of nonfermented raw dried sausages of small diameter that has gained popularity in countries such as Spain and Italy is made with nitrate and/or nitrite. pseudomonads) reduce nitrate to gaseous oxides of nitric oxide and nitrous oxide. particularly in S. Spectral analysis and electron spin resonance studies determined the pigment was nitrosomyoglobin. Second. carnosus reduces nitrate to ammonia in two steps. Subsequently. lentus). certain other bacteria use both assimilatory and respiratory nitrate reduction...g. 1998). xylosus and found it to produce a red myoglobin derivative in the same meat system without any added nitrate or nitrite. (1998) then evaluated a strain of S. Morita and coworkers investigated the red pigment found in Parma ham and its formation.. Microbiological examination of 471 isolates from Parma ham found the majority were able to convert metmyoglobin to a red myoglobin derivative. such as S. carnosus. and excreted. Synthesis of the nitrate and nitrite reductases is inhibited by oxygen and induced to greater or lesser degrees by nitrate or nitrite. Neubauer and Götz (1996) investigated the nitrate reducing system of Staphylococcus carnosus. which may occur aerobically or anaerobically. . nitrate is reduced to ammonia that is then incorporated into the cell and used as a source of nitrogen. the pigment found in hams made with nitrate and/or nitrite. after nitrate is depleted the accumulated nitrite is taken up and reduced to ammonia. Other bacteria. and Savoie ham made in France involve long ripening times and are made without any added nitrate or nitrite (Leistner. do not assimilate nitrogen through nitrate reduction during aerobic growth but grow anaerobically with nitrate as the terminal acceptor. all were staphylococci (S. warneri) was able to synthesize nitrate reductase (Talon et al. S. Morita et al. nitrate reduction is coupled to the generation of a proton motive force that is directly used as a source of energy or transformed into ATP. They found S. Members of Enterobacteriaceae reduce nitrate to nitrite. A study of cell suspensions from seven strains of staphylococci isolated from dry sausage (six) and cheese (one) found all but one (S. caseolyticus isolated from fresh pork were able to convert metmyoglobin to red myoglobin derivatives. such as strict aerobes. 1992). In contrast. Morita et al. which may be further reduced to nitrogen.

4% salt in the water phase. pork. and 6 kg sucrose (Knøchel and Huss. (1997) found the addition of sodium nitrite delayed or prevented toxin production by C. Cuppett et al. botulinum type E in vacuum-packed. Assembly of Life Sciences (1981). butyricum). Cheese Nitrate has been added during the manufacture of certain European cheeses for more than 150 years (Pivnick. 1991). (1987) found that toxin production by C.. botulinum type E (103 spores/g) at 27°C was inhibited for 35 days in smoked whitefish containing 4. The 600 barrels were topped off with brine solution. 1991. Seafood The comprehensive review by Jarvis (1950) provides very few examples for the use of nitrate (i. 2000)..g. respectively. Although not permitted in the United States. 1984a. 2001a. fish. In Denmark. They conducted an extensive study in which 0. 8. saltpeter) in the curing of fishery products prior to 1950 throughout the world. 1995). or putrid off-odors and offflavors. 1988.. Gilles and Fryer.. 1983. and keeping quality (Leistner et al. late fermentation and gassiness resulting from Clostridium tyrobutyricum or C. Monascus purpureus (Leistner et al. 1984. Park et al. sweet/sour.b). 1983. rice is fermented with molds of the genus Monascus to form a pleasant red color. and 50 g of sodium nitrate. This chapter will not review the extensive literature on the use of nitrate to prevent cheese defects (e. When sodium nitrite (156 µg/g) and sodium ascorbate (550 µg/g) were added. 1999. cold-smoked trout having 3. Galesloot and Hassing. 1980). but no differences were noted for flavor or texture. In one study with 166 µg/g sodium nitrite.. tofu. Klijn et al. Knøchel and Huss (1984b) state that nitrate traditionally has been added to the salt used in producing sugar salted fish in Scandinavia. fewer barrels were judged to be spoiled with sour. particularly in China. nitrous acid) because a high percentage of the isolates from all three variables were able to reduce nitrate. or 50 g of potassium nitrate was added to each of 200 barrels containing 90 to 100 kg fish. and others (Mayenobe et al. As the amount of sodium nitrate increased.. The additives provided no additional protection when the salt level in the fish was reduced to about 2.. toxin production was inhibited for 56 days. 1981). toxin was detected at 4 weeks at 4°C with a high inoculum level (20. Hyytiä et al.. Fink-Gremmels et al. Tseng et al. botulinum and L. poultry. Among the options considered was the use of a red pigment producing mold used to ferment rice. An opening to the literature on this subject can be obtained through references cited in Langsrud and Reinbold (1974). 1991. ground. flavor. monocytogenes (Gram. rice wine) to improve color. 1984. the allowable level has been 500 mg potassium nitrate per kg fish to provide about 300 µg/g nitrate. and evaluated over 18 months storage at 4°C to 6°C. and virtually no mention is made of its value as a preservative. Two microbial hazards of concern in ready-to-eat fish products are C. The reader may recall that during the 1950s nitrate was found to be an electron acceptor in certain cured meat products. added to a variety of foods (e.g.210 Antimicrobials in Food Alternatives to the Use of Nitrate or Nitrite Concern for the potential toxicologic effects of nitrite and nitrate led to continued research to find an alternative. for example. 25 g. with 0. Gouda and El-Zayat. Adding nitrate caused the herring to be redder in color. 25 g.. and 2 barrels. sealed. nitric oxide.000 cfu/kg) and after 4 weeks with a low inoculum (800 cfu/kg). Nitrate was found to delay the depletion of trimethylamineoxide (TMAO). Ariga et al. and. Nitrate and TMAO serve as alternative electron acceptors in barrel-cured herring and when in sufficient concentration delay growth of the strict anaerobes that are responsible for the most common type of spoilage. Canadian regulations permit this practice (Assembly of Life Sciences. 16 kg salt. in this form (called Angkak). In a second .e.4% salt in the water phase. The incidence of spoilage was 13. For more than 1500 years the rice has been dried.. fruity. In the Far East. The reduction in spoilage was not considered to be the result of microbial inhibition by nitrate or its reaction products (nitrite.b)..3%.

will prevent toxin production in cold smoked fish for several weeks beyond its sensory shelf life when packaged under reduced oxygen conditions. Two options are available in the event commercially manufactured product is implicated in botulism. if present. Following a review of the literature. however. Controlling the salt in the water phase and temperature should be adequate.. NOCs were not detected in two studies in which nitrite had been added to smoked fish (Pelroy et al. shad.. 1982. It is possible. botulinum (Huss. with a production of 12.. In many countries there is no legal requirement for the minimum amount of salt. however. but the panel did not consider the more recent risk assessments demonstrating the safety of dietary nitrite (see section on toxicology). Considering the lack of regulations and apparent variability in salt concentration in large quantities of cold smoked salmon distributed domestically and internationally. questions still remain about “aerobic packaging” and its value for controlling botulinal outgrowth. Following its review of the literature the panel of experts concluded that it is not possible to consistently produce cold smoked salmon that is free of L. depending on the conditions of processing.5% salt in the water phase). In response to outbreaks of botulism in the United States in the early 1960s when less information was available about commercial practices and from research. Cuppett et al. Analysis demonstrated that nitrite was produced during storage. Surveys conducted before the year 2000 indicate L. 1999). Toxin production can be controlled with a combination of a moderate level of salt (3. . 1997). gravad fish.380 tons of cold smoked salmon in 1997. federal regulations were adopted requiring vacuumpacked. sablefish. that combination of salt and temperature. and with refrigeration this combination is effective in inhibiting growth of C. botulinum can be expected to occur in smoked fish. The panel expressed concern for the potential carcinogenic effects of nitrosamines. Furthermore. it is highly unlikely that sodium nitrite will be adopted as an additive in smoked fish in Europe. The addition of sodium nitrite is limited to smoked salmon. and chub. but. lightly preserved fish products (cold smoked fish. although the numbers. are low. 2001a).e. whereas with salt alone. monocytogenes is common in smoked fish. 2001b).6% (Duffes.5% in the water phase) and cold storage (<4. brined shrimp).. 1997). 1987). 2001a): • • Psychrotrophic C. 1982. if necessary.. The addition of nitrite or nitrate increased the time the fish was considered acceptable by 3 or 4 weeks at 4°C. If packaged aerobically. botulinum would be further inhibited. Cuppett et al. a lower salt content is permitted (i.. the salt in the water phase ranged from 2% to 4. A level of 3% salt in the water phase will yield perfectly acceptable. smoked fish to contain at least 3. particularly in combination with controlled levels of salt (Pelroy et al. sodium nitrite could be used as an additional control measure. This may reflect survival or contamination.5% salt in the water phase or 3. a minimum concentration for salt in the water phase can be established. 2. The panel was reluctant to recommend expanded use of sodium nitrite despite evidence showing that psychrotrophic C. With the availability of lower cost farm-raised salmon the international market for cold smoked salmon has continued to increase. toxin was detected in 3 to 4 weeks. 1987. Hyytiä et al. In France. 1995). a panel of experts reached the following consensus for its report to the FDA (Gram.0% salt if combined with 200 µg/g sodium nitrite (Gram.000 tons shipped internationally from 28 exporting countries to 52 importing countries (Duffes. monocytogenes (Gram. although a minimum of 3% salt in the water phase is strongly recommended (Huss et al. allowing for short periods of elevated temperatures up to 10°C. it would appear the risk of botulism is lower than the research has suggested. First. In 1992 more than 32. Adding potassium nitrate at 347 or 686 µg/g was equal to or more effective than sodium nitrite. 1999).4°C) for at least 4 weeks.Nitrite 211 study with 109 µg/g sodium nitrite and lower inoculum levels (4000 and 140 cfu/kg) toxin was not detected through 6 weeks at 4°C or 8°C.. Until there is evidence of a need.

S. nitrite accelerated the death of all four strains. Sodium lactate (2%. because S. The authors implied that. Dehydration prevented growth if the packages were opened and left exposed at room temperature. Viable S.26% brine. Peterson et al. A lower maximum population (106 versus 108/g) and slower die-off occurred in the imported product. Previous studies had found levels of 0. Similar results were obtained on ham. In subsequent research. cans inoculated with C. The growth of S. Typhimurium grew in both vacuumpackaged products when held at room temperature. 1993. there was a gradual decline. Typhi strains. monocytogenes in salmon containing 3% salt in the water phase. The domestic product contained 4. At the end of storage. Typhi remained after 20 months at 27°C to 38°C. Meers and Goode (1965) reported the rapid growth of S. Goepfert and Chung (1970) inoculated the surface of sliced bologna and liver cheese with S. 15 µg/g of sodium nitrite. Chemical data were not provided for either luncheon meat. Growth occurred in both products. At 5°C. Pelroy et al. it uses the nitrate in the product in place of oxygen as an alternative hydrogen acceptor. Kittaka and Greenberg (personal communication) obtained similar results in corned beef inoculated with three different S.13% brine. and 45 µg/g of sodium nitrate. 1991. perfringens swelled within 2 days at 37°C. Cans inoculated with E. 14 µg/g of sodium nitrite.7 cfu/g in positive samples of cold smoked fish from five plants. After reaching 107/g. often less than 1 cfu/g and at a low prevalence. one domestic and one imported. but a steady increase in numbers occurred at 24°C. It is reasonable to assume the products were recently produced. Very erratic growth occurred on bologna at 18°C. Anatum grew in bologna but not in liver cheese. Because of the relatively high tolerance of L. to produce cold smoked salmon with low levels. Peterson et al. Typhimurium declined during the first 2 weeks at 5°C and then remained stable through 6 weeks. coli remained flat. Typhimurium occurred more rapidly and to higher levels when the packages were opened. (1994b) found the addition of sodium nitrite (190 to 200 µg/g) to be very effective in slowing the growth of L.5 to 11. and 720 µg/g of sodium nitrate at the time of opening and inoculation. Davidson and Webb (1973) also studied the growth of S. Mock chicken supported the best growth at both temperatures tested (24°C and 37°C). nor were analytical data presented for the product tested. Pelroy et al. including an isolate from the 1964 Aberdeen outbreak involving canned corned beef. increasing the salt concentration above the normal range for smoked fish is an unacceptable option for control. the imported product had 4. Such levels would ensure the number does not increase above 100 cfu/g when the product is held at 5°C and eaten within 3 to 4 weeks. 1994. Typhimurium in vacuum-packaged luncheon meats. and held at room temperature. Survival of the inoculum in product held at 7°C varied with the product and inoculum level. however. 1994a). Product from two sources. rewrapped in Saran™. Effect of Nitrite on Enteric Pathogens Blanche Koelensmid and van Rhee (1974) observed the growth of three of four Salmonella strains at 20°C in ham jelly containing 500 µg/g of sodium nitrite and 5.6% salt. although not before some initial growth at 24°C. tests were not performed to show whether the presence of nitrate influenced growth.. were used for testing. 2000). (1993) found the rate of growth to be similar at 5°C for salt concentrations of 3% to 5% in the water phase. viable cells remained even after 6 months of storage.212 Antimicrobials in Food with strict adherence to good manufacturing practices. the pH of mock chicken . Typhi in inoculated canned corned beef stored at room temperature and at 37°C. In both products.. S. w/w) was much more effective and prevented growth through 50 days at 5°C when used in combination with 3% salt. Rørvik. Typhimurium or Salmonella anatum. however. This combination was more effective when a low inoculum level (10 cfu/g) was used as a challenge. Typhi has a nitrate reductase. The inocula died in wieners at both 18°C and 24°C. The initial level of contamination is an important factor influencing growth (Guyer and Jemmi. adding 125 µg/g sodium nitrite resulted in a slower rate of growth in vacuum-packed salmon having 3% salt in the water phase (Pelroy et al. monocytogenes to salt and the adverse effect on flavor.. S.

Nitrite 213 and bologna was about 6. that of the ham was near 7. No other chemical data were provided. whereas the other frequently decreased to below pH 5. Stiles and Ng (1979) studied ham and chopped ham obtained fresh from two processors. Salmonella Enteritidis was able to grow at 17°C but not at 10°C. Death occurred after 3 days in the sausage with the lower inoculum and 130 µg/g of nitrite. Two strains of S. Again. This probably reflects the quantity of fermentable carbohydrate added to the product and/or the nature of the competitive flora. Typhimurium to be resistant to nitrite (2000 to 4000 µg/g) when added as a filter-sterilized solution to a broth medium having a higher pH of 6. and 130 µg/g) and two Salmonella inoculum levels (100 and 100. no data were provided for brine or nitrite levels.47 and 5. whether the nitrite was added before or after autoclaving the medium of Perigo et al. The authors did not state whether the nitrite was added before or after autoclaving the medium. cereus) and then vacuum packaged. commercially produced sliced ham and chopped ham did support the growth of the pathogens studied. 50. At a low pH of 5.0 by 160 µg/g of sodium nitrite. The products were inoculated with five “enteropathogens” (C. The mean pH for the ham during storage decreased to no lower than 5. Fermented sausage (teewurst) was prepared with four sodium nitrite levels (0.78. growth was reduced by only about 30%. 98. nitrite. A low-brine ham was prepared with 1. perfringens. Leistner et al. E. nitrite was without effect. coli. Klebsiella. (1979) tested the same bacteria on sliced bologna from two processors. and 100 µg/g) and the level of inoculum (100 or 100.8. It was concluded that cooked ham is a more likely source of salmonellosis if prepared with a low salt content. the primary factor influencing growth was product pH. and ammonia as the sole nitrogen source in a chemically defined minimal medium.7 to 4. and B. 75. and Hafnia strains readily multiplied at all nitrite levels. Enteritidis were then used to study the effect of nitrite and salt on growth in nutrient broth at 22°C. pH values were followed through storage. The pH of the chopped ham with 70% and 59% moisture decreased to 5. In the high-brine ham held at 22°C.7. A high-brine ham had a brine level of about 6%.000). The product from one supplier remained above pH 6. The moisture content of the hams was 70%. (1973) inoculated vacuum-packaged luncheon meat with a mixture of Salmonella strains and held the product at 8°C for 22 days. Castellani and Niven (1955) reported S. Page and Solberg (1980) found S.6 to 6. Both genera were prevalent in commercially produced luncheon meats and fermented sausages. the fastest generation times occurred in . and the pH for the wieners was 4. 65.0. The resistance of Salmonella to nitrite in culture media was demonstrated by Roberts and Garcia (1973). that of the chopped hams was 70% and 59%.2% salt and 60 µg/g of potassium nitrite and a finished brine level of about 2.0 and 5% salt. coli. Typhimurium to utilize nitrate. At a high pH of 7. Typhimurium.0. (1974).58. Neither of two strains of S. The trend in the United Kingdom toward using less salt for processing ham was studied by Akman and Park (1974). All the enteropathogens grew in one or more of the products within 24 hours at 30°C.8%. but a mixture of Enterobacter. respectively. Stiles et al.5. Although salt and nitrite values were not reported. perfringens did not occur under any condition. 4 of 5 strains of Salmonella grew readily. The growth of Klebsiella and Enterobacter species on vacuum-packaged luncheon meats at 3°C was reported earlier by Hechelmann et al. Aside from temperature of storage. (1967). A similar response occurred with enteropathogenic E. Nitrite and ammonia were assimilated at comparable rates. It is not possible to assess the influence of nitrite from the data.000/g). and 100 µg/g of sodium nitrite. Growth of C. Typhimurium was inhibited at pH 6. given the proper circumstances. Growth occurred in the sausage containing 0 and 65 µg/g of nitrite. In the low-brine ham at 22°C. nitrite values were not given. viable counts of the 5 strains remained near the inoculum level.0. The primary factors influencing growth were the temperature of storage and growth of competitive flora.0. The other four species were able to grow under certain conditions. S.8% to 2. Under anaerobic conditions. 5% salt. including Salmonella. other than to say that fresh. S. The rate of growth or death was influenced by the sodium nitrite content (0. aureus. but pH changes were noted.

Hughes and McDermott (1989) used a procedure similar to that of Gibson and Roberts (1986a. The death rate was slower with potassium nitrate (150 or 300 µg/g) or a combination of nitrate and a low level of nitrite (75 µg/g of potassium nitrate and 50 µg/g of sodium nitrite). The presence of nitrite had no effect on aerobically grown cultures. (1984) prepared fermented sausages with sodium nitrite (0. Growth did not occur during fermentation or drying. and 6. coli was also apparent in tests involving two-dimensional gradient plates incubated at 20°C. (1985) identified a nitrite reductase in the cytoplasmic fraction of S. 50. S. 27°C. 50. Page et al. vacuum packaged. Collins-Thompson et al.0 to 6. complete inhibition occurred. At pH 5. except when the cultures were incubated at the lowest temperature tested (10°C). Typhimurium numbers decreased in BHI broth adjusted to pH 4. The reductase required NADH and was most active at pH 8. The nitrite resistance of E. (1982) were able to isolate Gram-negative bacteria from commercial packages of vacuum-packaged bologna only after storage for 7 to 8 weeks at 5°C. 1991). using a gradient gel plate system. and the presence of lactic acid-producing bacteria. This research was primarily concerned with preventing the growth of Salmonella. 1977). and 37°C (McClure and Roberts. The franks were inoculated on the surface. sporogenes. Isolates of Enterobacter and Serratia species were tolerant to nitrite. S. numbers increased during 72 hours in the presence of 0.2). the addition of sugar. When grown anaerobically in a glucose-limited minimal medium with peptone and nitrite. When examined after 10 weeks of incubation. coli.8 and held at 24°C independent of sodium nitrite concentration. Schillinger and Lücke (1989) measured Salmonella growth and/or survival in mettwurst.6 to 5. the effect of added phosphate was relatively minor.214 Antimicrobials in Food media containing both nitrate and ammonia. Typhimurium when grown anaerobically with nitrite as the sole nitrogen source. Typhimurium had shorter generation times and increased cell yields compared with nitrite-free cultures. The results demonstrate that these bacteria are quite resistant to the effect of salt and nitrite at the levels most commonly present in such foods as perishable cured meats and smoked fish. (1991). coli. Similar results were observed with S. Typhimurium to be relatively resistant to sodium nitrite across the ranges of salt and pH values found in nonfermented and acidulated foods.b) to determine the additional effect of eight different phosphates of E. The presence of nitrite did not influence the survival of salmonellae.8 versus 6. The rate of death of Salmonella Senftenberg in fermented sausage was fastest with 200 µg/g of sodium nitrite and next fastest with 100 µg/g of sodium nitrite (Puolanne. S. 6. At concentrations of 500 µg/g and higher. being able to use nitrite in broth culture under anaerobic conditions at 5°C. Rice and Pierson (1982) tested the effect of sodium nitrite (0. Nitrite was only inhibitory with the highest level of nitrite and when the franks were incubated at 15°C. and 100 µg/g sodium nitrite. None of the variables tested would likely assure the destruction of Salmonella in this product category.0. Collins-Thompson et al.8. a cured raw spreadable sausage. 100. pH (5. aureus and C. coli. the inherent pH of the meat (normal pH 5.8). 50. and 200 µg/g) and three strains of Salmonella. Factors influencing Salmonella growth in mettwurst that was cured and stored at 20°C include the amount of nitrite and salt. Typhimurium used nitrite as the sole nitrogen source at concentrations as high as 400 µg/g. 150. and 156 µg/g) in frankfurters on a mixture of two strains of Salmonella. and incubated at 15°C or 27°C for 21 days. found S. Gibson and Roberts (1986a) examined the interaction of salt (1% to 10% wt/vol). The phosphates generally delayed the outgrowth of E. but growth was delayed with 200 µg/g (Turanta and Ünlütürk. and sodium nitrite (0 to 400 µg/g) in BHI and incubation at 10°C to 35°C on the growth of Salmonella and E.6. In a traditional Turkish dry sausage that is fermented and dried at temperatures . Thomas et al. 1987). He recommended that a starter culture and/or gluconolactone be added to provide optimum safety.2.

The effect of nitrite in Chinese-style sausage during drying at 50ºC to 60ºC for different time periods (2. coli O157:H7 when lower temperature and/or shorter time combinations (e. and. salt level. storage temperature. That cured meats have been implicated in outbreaks of salmonellosis supports the available research that Salmonella can survive. 1993). Inhibition of Shigella flexneri in broth incubated aerobically was significantly influenced by the combined effect of nitrite concentration. This sausage is not fermented or cooked and relies on relatively rapid dehydration to achieve microbial stability. nitrite. the lowest pH tested and with increasing salt levels.5. 1994). fermented to pH 4. enterocolitica 0:3 to grow and survive during the manufacture of raw dry fermented sausage was examined by Asplund et al. The data indicate that the nitrite content of cured meats can influence the growth and survival of Salmonella and E. Raccach and Henningsen (1997) reported sodium nitrite and salt to act synergistically in the reduction of Y. These factors include water activity. and 120 µg/g) on Y..5% salt and 100 µg/g sodium nitrite. (1993). 55ºC for 4 hours) were used. This is not true for the clostridia. S. the death rate of S. 78. coli O157:H7 (Chikthimmah et al. and then dried for 7 days. pH 5.. but even 1 g/L sodium nitrite was ineffective at higher pH values (Tsai and Chou.5) and enhanced by lowering the incubation temperature (Buchanan and Bagi. and incubation temperature (Chou and Tsai.g. The inhibitory effect of nitrite was much greater at pH 5. The data for Salmonella can be summarized as follows. 1994 and 1995). 1994). salt level.Nitrite 215 not exceeding 24°C. coli.. the addition of sodium nitrite (0. and temperature. In tryptic soy broth 4 strains of E. They found some increased death of E.7 × 105/g to nondetectable levels by 35 days in the sausages formulated with 80 µg/g nitrite. 1997). coli O157:H7 at 37ºC were inhibited by 200 µg/g sodium nitrite at pH 5. 1996). 50. coli O157:H7 in a broth system containing sodium nitrite was strongly pH dependent (i. In ground pork held at 5ºC or 25ºC.8. The inoculum decreased from 1. enterocolitica when tested in a meat system. Inhibition was much greater at 28ºC than at 37ºC (Zaika et al. The effect of sodium nitrite (0. pH. Pin et al. Another study involving anaerobic conditions yielded similar trends (Zaika et al. the inhibitory effect of smoke. coli O157:H7 numbers decreased less than 1 log10 in pepperoni formulated with 2.5 to 6 hours) was next studied (Yu and Chou. Inhibition of Aeromonas in broth has been found to be highly dependent on nitrite concentration. 1996).0. in a variety of commercially cured products. the availability of oxygen.5. a fermented noncooked beef sausage. In Lebanon bologna. The highest survival involved a starter culture that produced the least amount of acid and yielded a relatively high final pH of 5.. 1996. Adding sodium nitrite did not increase death of E. (1998) found E. pH. sodium nitrite at 70 to 150 µg/g did not affect the growth or survival of E. coli O157:H7 (Yu and Chou... Inhibition of Yersinia enterocolitica in broth incubated aerobically was significantly influenced by the combined effect of nitrite concentration. Preventing salmonellosis from cured meats is best accomplished by means other than relying on the level of nitrite in the product. 1996). 2001). 100. The lack of information on the significance of nitrite and nitrate stems from the general acceptance that other factors largely determine the potential for yeast and mold spoilage of cured meats. pH. if not multiply. and temperature (Bhaduri et al. Typhimurium was not affected by the addition of 200 µg/g sodium nitrite (Turanta and Ünlütürk. and 156 µg/g) had no effect on the death rate of E. Riordan et al. coli O157:H7 with the addition of curing agents and drying at time–temperature combinations that appear to cause stress of the inoculum. Effect of Nitrite on Yeasts and Molds A wide variety of yeasts and molds have been isolated from commercially cured meats. where . Inhibition of E. Typhimurium has a nitrate reductase to reduce nitrate to nitrite and can assimilate inorganic nitrogen from nitrate. 80. or ammonia under anaerobic conditions.. 1991). but there is no published information that nitrite or nitrate inhibits their growth. particularly because salmonellae are destroyed during the manufacturing process for most cured meats.e.

but not all. the remaining nitrite enhanced growth and aflatoxin production. (1983). The heat resistance of these bacteria has been reported (Niven et al. 1971). Despite this observation. The role of nitrate and nitrite in the manufacture of traditional and more rapidly produced fermented meats was clarified. Control of these bacteria lies in proper handling of cooked product to be reworked into succeeding lots of meat. viridescens was given to these bacteria (Niven and Evans. The mechanism of green discoloration was further elucidated by Shank and Lundquist (1963).. A system was developed to evaluate the combined effects of formulation. 1957).5 to 15 minutes. Vrchlabsky and Leistner. All were salt tolerant and capable of growth at low temperature (5°C). 1954. At 26°C and 37°C. circumstances. Nitrite burn is another form of green discoloration that is caused by a combination of excessive levels of nitrite and reduced pH (Deibel and Evans. Unpublished research by Shaparis and Christiansen has shown D values in minced ham to be D63 = 30. Additional information on factors affecting green discoloration by L. Niven et al. The more rapidly produced products closely resemble those that have been produced in the United States over the past 30 to 40 years. monocytogenes under certain. viridescens and other lactics has been developed by Grant and McCurdy (1986) and Grant et al.5 to 6. . The effect of nitrite on aflatoxin production by Aspergillus parasiticus in fresh pork sausage was examined by Obioha et al. 3. no aflatoxin production or mold growth occurred. 5. 1972). and D71 = 1 to 2 minutes (Tompkin. Except for a limited number of products to which nitrite and nitrate might be added. D68 = 5. The most significant development during this period was the growing body of data that dietary nitrate and nitrite are not carcinogenic (see Toxicology). As residual nitrite decreased to less inhibitory levels. they are not major causes of spoilage. 1965. the results in pork sausage plus a test with media led to the conclusion that high levels of nitrite (e. and other factors for controlling mesophilic clostridia. Nitrite can contribute to the inhibition of L. During storage at 5°C for 28 days. Iwata et al. (1949) described the characteristics of Lactobacillus and Leuconostoc strains isolated from commercially cured meats with greenish discoloration. Endogenously formed nitric oxide and oxygen radicals can play an important role in human health. In addition. the product spoiled before aflatoxin reached detectable levels. (1988). The name L... 4. These bacteria must not be given the opportunity to develop a population of increased heat resistance whereby the process will no longer be adequate to assure their destruction. the use of preservatives (e.216 Antimicrobials in Food permitted. sorbate). Increased heat resistance could be acquired by repetitive exposure to thermal processing. Evidence was presented indicating that the discoloration results from a reaction of hydrogen peroxide with the cured meat pigment. 1986). 6.5 minutes. heat.g. (1954) demonstrated that the heterofermentative lactobacilli responsible for greening on the surface of cured meats were more sensitive to heat than those developing in the center of sausages. inoculum level. 156 to 200 µg/g) initially restricted the profuse growth of mold.5 = 12. D65. Conversion of nitrate in the saliva is the major source of nitrite in humans.g. Naturally occurring nitrate in vegetables accounts for more than 85% of the dietary intake of nitrate.5 minutes. 1957). Green Discoloration in Cured Meats Niven et al. Gardner. 2.. High levels of nitrite can result from the conversion of nitrate to nitrite by micrococci near the periphery of sausage or by Staphylococcus species within the sausage (Bacus and Deibel. 1967. yeasts and molds are of little or no public health importance. SUMMARY FOR 1990–2002 1.

160. 1995). 1974. and Food.S. Other countries limit the amount of nitrite and nitrate on the basis of the total weight of the product formulation. the method of assay applicable to each country should be used. REGULATIONS GOVERNING THE USE OF NITRITE AND NITRATE The regulations limiting the use of nitrite and nitrate in cured meats vary widely among countries and have continued to change. Gerhardt. leads to lower residual nitrite levels after processing. 8.S. regulations do not specify minimum nitrite levels for cured products. meat and poultry regulations is that nitrite and nitrate are added on the basis of the uncured meat in the product formulation. The status of nitrite and nitrate in cured meats and cheese in the United Kingdom was reviewed in 1978 (Ministry of Agriculture. As the percentage of extenders and other nonmeat ingredients is increased in the products. An example of rework is the round ends of sausages that are trimmed off before slicing the product. 1981. The regulations were summarized in two comprehensive surveys (Meester.170. The net effect of the U. The use of nitrite and nitrate in the processing of fish products is specified in 21 CFR 172. 172. The most recent summary of the regulations in different countries appears in Cassens (1990). as proposed by the Expert Panel on Nitrites and Nitrosamines (Food Safety and Quality Service. The current USDA regulations controlling the use of nitrite and nitrate in meat and poultry products are specified in 9 CFR 317.21-23 of the federal regulations (Code of Federal Regulations. Within the United States the methods of assay appear in the Official Methods of Analysis of the Association of Official Analytical Chemists . 2002a). Examples of the effect of this regulation on the maximum allowable level of nitrite in several sausage and loaf products are listed in Table 6. It is also common to generate a certain amount of rework during the manufacture of sausage products. the weight of this meat is subtracted from the fresh meat portion of the formulation and the amount of nitrite added is reduced accordingly. 1978).Nitrite 217 7. Nitrite and nitrate levels for curing meats in the United States were recommended by a USDA Expert Panel on Nitrites and Nitrosamines (Food Safety and Quality Service. With the exception of bacon and smoked chubs. A unique feature of the U. The use of nitrate or nitrite as additives in cheese and seafood products was clarified. Braathen. Procedures for calculating the amount of nitrite and nitrate that can be added to curing solutions and product were specified in the USDA calculation handbook (Food Safety and Inspection Service. 1986). Germany reduced the permitted level of nitrite in nitrite-containing curing salt by 20% (Leistner. depending on the circumstances. This material is normally used as an ingredient at a level of about 5% in the manufacture of succeeding lots of the same product. 2002b). Also listed is the actual amount of nitrite that could be added to the total formulation of the products if the permitted level of nitrite was reduced from 156 to 100 µg/g. Fisheries. 1978). regulations is that less nitrite is added to many products than might be assumed with a regulation that permits 156 µg/g of sodium nitrite. the level of added nitrite is decreased. 1979). and 172. Nitrite is of relatively little value for controlling Gram-negative enteric pathogens in commercially prepared foods.175.S.3. This further reduces the nitrite level in the product formulation and. 9.177 (Code of Federal Regulations. ASSAY METHOD Because the levels of nitrite and nitrate permitted in foods are established by regulation. U. Because it has already been cured. 172. 1978). The cured ham appearance of traditional European dry cured hams manufactured without the addition of nitrate or nitrite was found to be the result of the formation of stable pigments by naturally occurring staphylococci.17 and 424.

whereas 30 to 60 g of sodium nitrate have been given for 2 months as an acidifying diuretic without manifestation of adverse effects. the uppermost level being found in infants and pregnant women. (AOAC international.7 and 0 to 0. 1994). estimates for the lethal dose have ranged from 2 to 9 g (about 33 to 250 mg/kg body weight). respectively (JECFA. occurs at methemoglobin levels in excess of 10%. TOXICOLOGY The acceptable daily intake for potassium nitrate and sodium nitrite have been reported to be 0 to 3. respectively.218 Antimicrobials in Food TABLE 6. For sodium nitrite. as evidence of toxicity. A realistic estimate of a lethal dose for adults is about 20 g nitrate ion or 330 mg nitrate ion/kg body weight (Gangolli et al. 1995).5% to 2.3 Examples of Sodium Nitrite Levels Added to Cured Meats in the United States Actual NaNO2 (µg/g) in Total Formulation if Input NaNO2 Is Reduced to 100 µg/g in the Meat 78 75 67 74 69 73 97 90 92 83 58 60 50 47 Product Bologna Bologna with NFDMa Bologna (imitation) Franks (meat)a Franks (meat)a Franks with NFDM Liver sausagea Liver sausagea Cured beef loaf Blood pudding Pickle and pimento loaf Olive loaf Family loaf Veal loaf a NaNo2 (µg/g) Added on Basis of Meat Content 156 156 156 156 156 156 156 156 156 156 156 156 78 78 Actual NaNO2 (µg/g) in Total Formulation 121 117 105 115 107 114 152 140 143 130 91 94 50 47 NFDM. Data on reproductive toxicity for nitrate were limited. Concern for the formation of NOCs in foods began with the discovery that adding sodium nitrite to preserve raw herring for fish meal production resulted in malignant liver disease in cattle .. and apply to all sources of intake but do not apply to infants younger than age 3 months. Cyanosis.3 mg sodium nitrite/kg body weight (Gangolli et al. International agreement has been reached on the specifications and analytical methods for potassium nitrate and sodium nitrite (JECFA. 2002). Two years of rat studies on nitrate showed no evidence of carcinogenicity. estimates of the lethal dose have ranged from 4 to 30 g (about 70 to 500 mg/kg body weight). 1994). The normal range of methemoglobin levels is 0. Source: Based on formulations in Pearson and Tauber (1974). Using this criterion.. For potassium nitrate. and it appears that nitrate per se is not genotoxic. The values are expressed as nitrate and nitrite ions. the criterion most commonly used for nitrite toxicity. 1995). The no-adverseeffect dose level was estimated to be 2500 mg sodium nitrate/kg body weight/day (Gangolli et al.0%. estimates for the toxic dose range from 1 to 8.. with the lower doses applying to infants who are more sensitive to methemoglobinemia. 1994). nonfat dry milk.06 mg/kg body weight.

1996). Controls adopted by the food industry over the past 20 years have led to reductions in the risk of exposure to nitrosamines (Lijinsky. and NOCs have concluded that dietary nitrite and NOCs represent a relatively small contribution to the total body burden of these compounds (Cassens. 1997). particularly among young children. nitrite. An extensive review was conducted during the development of two reports by the NAS (Assembly of Life Sciences. per se. not to list sodium nitrite as a developmental toxicant under the state’s Proposition 65 law. 1998). Endogenous synthesis of nitrate is an additional important source of nitrate. Gangolli et al. Assessments conducted during the past decade have led to the conclusion that nitrite. 1982). and beer from exposing the malt to nitrogen oxides. and epidemiologic surveys led to the conclusion that there is no firm scientific evidence to recommend drastic reductions beyond the average levels of nitrate encountered in vegetables grown following good agricultural practice. In 1963 it was proposed that nitrosodimethylamine formed during the herring meal process was the toxic agent. (1994) conducted a comprehensive risk assessment on nitrate. (1994) concluded that dietary sources of nitrite and NOCs contribute relatively small amounts to the body burden. 2001).Nitrite 219 and sheep. In addition. The major source of these compounds in the body is derived . 1999). especially bacon when prepared and cooked under certain conditions. 1500. an extensive review of the literature and testimony from experts led the California Developmental and Reproductive Toxicant Identification Committee to vote on June 2. including a review of epidemiologic studies seeking to establish a link between intake of nitrate and nitrite from food and water and cancer. Gangolli et al. is not carcinogenic. A review of the pros and cons of nitrate and nitrite as food additives in light of the information available by 2001 has been provided by Archer (2002). Cassens (1997) reported that residual nitrite levels in cured meats at the retail level in the United States had decreased over the past 2 decades by approximately 80%. higher residual ascorbate levels (mean value of 209 µg/g) in the cured meat products would reduce the risk of nitrosation reaction products being formed. 1981. For humans the greatest exposure to NDMA is from tobacco smoke (Koppang. An assessment of NOCs by Hotchkiss et al. (1992) concluded that there was only indirect evidence that some portion of human cancer risk is related to NOC exposure and that tobacco was the major source. 2000. 1997). Sodium nitrite is now viewed in an entirely different light than during 1970 to 1990 (CAST. 1995). Investigations for NDMA in foods led to cured meats. Endogenous formation of NOCs is influenced by dietary intake and a variety of other factors. Murphy et al. or 3000 µg/ml. nitrite. For example. (1998) concluded that the increase in the incidence of brain and nervous system cancer observed among both children and adults between 1970 and 1990 was not consistent with the decrease in the consumption of cured meats and marked reductions in residual nitrite content during that period. Those reports provide the most complete assessment of the safety of nitrite as a food additive for that period. Several countries that monitor their foods for nitrate. Information presented at a Ceres Forum led to the overall conclusion that available information either does not or is inadequate to support a link between dietary intake of nitrite and NOCs and brain and nervous system cancers in children and adults (Ceres Forum. human effects. summarizing studies conducted over a 2-year period on the carcinogenesis and genetic toxicology of sodium nitrite in drinking water. A review of animal toxicologic studies. There was equivocal evidence of carcinogenic activity in the forestomach of the female mice tested (National Toxicology Program. Even the American Cancer Society has concluded that “nitrites in foods are not a significant cause of cancer among Americans” (American Cancer Society. More recently. and NOC and determined that vegetables provide more than 85% of the average daily human dietary intake of nitrate. The report concluded that there was no evidence of carcinogenic activity for sodium nitrite in rats and mice exposed to 750. This was followed by the final report from the National Toxicology Program in May 2001. Cassens (1995) has provided an update of the nitrite controversy.

2002). factors considered of importance in asthma (Sanders. Expelled stomach air contains a high concentration of the antimicrobial gaseous nitric oxide that is enhanced by dietary nitrate intake. and perhaps antimicrobial defense. and antioxidative capacity (Gewaltig and Kojda. 1997). superoxide) are generated in response to various infectious diseases (Akaika and Maeda. 2001. blood clotting. Nitric oxide is produced by three different nitric oxide synthases (Bogdan. which can cause oxidative tissue injury through oxidation and nitration reactions of various biomolecules. Cherayil and Antos. Generation of nitric oxide in response to viral infections such as influenza virus and certain other neurotropic viruses. autoimmunity. Kurowska. 1999). Excessive amounts of nitric oxide produced for long periods allow generation of peroxynitrite. Inhaled nitric oxide therapy shows promise for improving oxygenation and reducing mortality in preterm infants with pulmonary illness (Srisuparp et al. 2002. in vitro tests demonstrated the bactericidal effect of acidified nitrite against several enteric pathogens (Dykhuizen et al.. may have an overall detrimental effect on the host. leukocyte adhesion. and cigarette smoking (Gewaltig and Kojda. In humans. diabetes. hypercholesterolemia. 2000). Salivary nitrate is converted to nitrite by nitrate-reducing microorganisms in the mouth. and blood pressure control (CAST. This has been found in atherosclerosis. We have also begun to learn about the possible role of NO in thymic education” (Bogdan.220 Antimicrobials in Food through metabolism of ingested nitrate. Evidence suggests that nitric oxide and oxygen radicals (e. Bogdan. particularly through action of an inducible nitric oxide synthase. Some of the most important effects that nitric oxide exerts in the vascular wall are potentially vasoprotective because these effects maintain important physiologic functions such as vasodilation.g. (1996) reported that ingested nitrate is absorbed from the gastrointestinal tract into the bloodstream and concentrated in the saliva.. hypertension. 2002). cell adhesion. Shiloh and Nathan. 2001. Impairment of endothelium-dependent nitric oxide activity is believed to be the result of increased production of reactive oxygen species. increasing in concentration up to ten times the level found in plasma. 2002).. 1995). Nitric oxide also appears to adversely affect the host’s immune response (Akaika and Maeda. There is a growing body of evidence showing that endogenously produced nitric oxide is an important component of the natural defense system to infectious disease in humans (Karupiah et al. It is now clear that inducible nitric oxide synthase (iNOS) “is detrimental in some infectious disease processes” and that it helps to counteract excessive immune reactions. It has been speculated that nitric oxide plays an important antiinflammatory and antiviral role in rhinovirus infections and that nitric oxide donors may . Nitric oxide biosynthesis. 2002). protects to some degree against autoimmunity and functions as an intra. In addition.. recent studies demonstrate that nitric oxide is a potent inhibitor of both rhinovirus-induced cytokine production and viral replication. smooth-muscle proliferation. Patients with infective gastroenteritis have increased plasma nitrate levels compared with healthy controls (Dykhuizen et al. 1996).. The enzyme nitric oxide synthase catalyzes the oxidation of L-arginine and L-citrulline to release nitric oxide that can be converted to nitrite and nitrate and subsequently excreted (CAST. Alam et al. 1997). As might be expected from other information in this chapter. is beneficial in the case of bacterial and protozoal infections. 2000). 2001). tumors. 2001). anticoagulation. 2000. On the positive side. Endogenously formed nitric oxide also plays a role in neurotransmission. Although this source of nitrite would increase the risk of NOC formation when introduced into the acid conditions of the stomach.and intercellular signaling molecule shaping the immune response. particularly superoxide. 2002). More recent research indicates that nitric oxide plays many diverse roles in infection and immunity. Chen et al.. however. the acidified nitrite also has antimicrobial activity that coincides with the formation of nitric oxide. the saliva is the major site for formation of nitrite. nNOS and eNOS (neuronal and endothelial NOS) are now known to participate in important immunological processes such as apoptosis.. 2000. Dykhuizen et al. 2001. and chronic degenerative diseases (Bogdan. autoimmune processes. heart failure. Nitric oxide is involved in the pathogenesis and control of infectious diseases.

Appl. J. R. 65:872. Reduction of nitrate and nitrite in cheese and nitrate reducibility of Penicillium candidum as fungal starter. K. E. Appl.. 1977. and Him. and Krol. 1999). nitrite. Wageningen. and Maeda. 1984.aoac.. The inhibition of the growth of Bacillus cereus in liver sausage. DC. T. Microbiol. Microbiol... 1963. P. L. A.. Baird-Parker. Akaika. and Jarvis. Microbiol. and Spencer. sulfur. 2000. Asplund. Eur. R. J. H. and Demeyers.. and Maeda. and Baillie. I. Food Technol.. DC. Alley. A. Nippon Shokuhin Kogyo Gakkaishi 31:710. Okamoto. G.. H. 24:405. This has impaired the development of new drugs that would take advantage of the beneficial properties of nitric oxide (Cirino et al. British Food Manufacturing Industries Research Association.. J. and Solberg. E. Immunol. L. Didcock. 1981. Food Prot. 40. H. 32:279. and cancer prevention: Reducing the risk of cancer with healthy food choices and physical activity. and Raevuori. Hargreaves. Nitric oxide and virus infection.” Meat Sci. J. 1974. 2002. Pasteur (Lille) 7:265. Food Microbiol. Alam. Food Technol. 70:3130. Part 2 of a 2-part study by the committee on nitrite and alternative curing agents in food. American Cancer Society. and Park. Pudoc. perfringens by heated combinations of nitrite. J. Ashworth. L. nutrition. M. Eds. Available at: http://www. Yoshitake. nitrite and ascorbate on colour and colour stability of dry. Ashworth. C. Ohtsuka. 84:403. Environ. 1988. B. Hyg. Inhibition of C. Ann.. and N-nitroso compounds. 1982.. 101:300. The Perigo effect in pork. An approach to the standardization of the bacteriological examination of semipreserved canned meats. Ann. 31:49.. T. F. A. L. K. M. REFERENCES Akaika.. Y. Inst. 1972. T.. Effect of nitrite. The health effects of nitrate. and Larkworthy. Pajulahti. 4:145. J.. M. J. J. Surrey. 8:477. 2002. J. Part 1 of 2-part study by the committee on nitrite and alternative curing agents in food. 1974. Official Methods of Analysis of AOAC International.. botulinum type A toxin formation in liver sausage. The growth of Salmonella on cooked cured pork. M.. L. Guidelines on diet. Washington. T. Microbiol. R.. T. Gen. 72:369.. 1992. . Nurmi. W.. Atlanta. Nitrite burn in fermented sausage. Pasteur. F. Association of Analytical Communities International. T. National Research Council. Asan. The American Cancer Society 1996 Dietary Guidelines Advisory Committee. The Netherlands. S. Miyamoto. 1976. D. Ala-Huikku. 1996. p. D. Infect. National Academy Press. 1972. Archer. and Deibel. Scientific and Technical Surveys No. J. MD. Walters. Assembly of Life Sciences.. National Research Council. Int. Leatherhead. Ohnishi. 77. S. Pathogenic organisms in relation to pasteurized cured meats. Immun. Assembly of Life Sciences. N. Hattori. National Academy Press. L. K. American Cancer Society.. L.. Sawa. 7:349.. J. Role of nitric oxide in host defense in murine salmonellosis as a function of its antibacterial and antiapoptotic activities. B. A. Nurmi. Effect of nitrate. and Sukegawa. Kubota.Nitrite 221 represent a novel therapeutic approach for the treatment of colds and their related complications (Sanders. Evidence that ingested nitrite and nitrate are beneficial to health. J. B. Inst. H. H. Cours. In Proceedings of the International Symposium on Nitrite in Meat Products. D. Research over the past 20 years has clarified many aspects of the role of nitric oxide in physiology and pathology. J. Hargreaves. storage temperature and time on C. Ashworth..htm Bacus. The production of an antimicrobial effect in pork heated with sodium nitrite under simulated commercial pasteurization conditions.. Washington.. 1974. S. H. Chemical and microbiological comparisons of inhibitors derived thermally from nitrite with iron thionitrosyl (Roussin black salt). Jarvis. 1973. Alternatives to the current use of nitrite in foods. Tinbergen. 2002). C. Anderton. B. 1955. J. 2002. The inhibition of Clostridium botulinum by nitrite and sodium chloride.org/index. fermented sausage prepared using “back slopping. T. M. Appl. Ariga. 7:111. Gaithersburg. Tamura. Association of Analytical Communities International.. and ferrous or ferric ions.. Hill. Akman. The research has demonstrated that nitric oxide is a double-edged sword mediator in that it can exert beneficial or detrimental effect depending on the physiopathologic context. J..

A. Bard. Buchanan. Brownlie. Bhaduri. H. Buchanan. Bowen. 35:275. Botha. Bhaduri. 43:877. and Holzapfel. J.. The function of nitrate. G. 82:567. Montel. J. and Arnon. M. 17:605. G. T. and Roberts. B. Immunol. E. Intl. Brooks. and Roberts. Food Prot. Effects of nitrite and ascorbate on botulinal toxin formation in wieners and bacon. Monteil. Braathen. H. L. G. J. London.. and Townsend. L. 1970. Expanded models for the non-thermal inactivation of Listeria monocytogenes. Int. Effect of pH and oxygen tension on staphylococcal growth and enterotoxin formation in fermented sausage. sodium nitrite concentration and atmosphere on the growth of Listeria monocytogenes. 23:317. Intrinsic factors in meat products counteracting botulinogenic conditions.452.. 1995. Food Technol. Ann. 49. H. R. 10:327. J. 143. Response surface model of the effect of pH. 1990. Food Sci. Effect of sorbate and sodium nitrite on toxin formation of Clostridium botulinum in wieners. and Kolari. and van Rhee. and Solberg. 1994. Salmonella in meat products. sodium chloride content. 33:119. J. P. J. Bean.. M. G. 1974. R. J. and Phillips. R. G. J. C. 2:907. J.. and van Rhee. and Bagi. Chicago. Enzymol. M. sodium chloride. R. 24:891. 1966. F. G. Update: Nitrite free meat products. P.. Haines. Cong. W. Food Microbiol.. V. Buchanan. Biochemical basis for nitrite-inhibition of Clostridium botulinum in cured meat. 1972. p. R. L. and Phillips. K. W.. sodium chloride and sodium nitrite on growth of Yersinia enterocolitica at low temperatures. Effect of some environmental factors on psychrophilic microbacteria. J. Meat Sci. p. D. oxygen and pH on growth of Staphylococcus aureus. Department of Scientific and Industrial Research. and Golden. R. . E. Turner-Jones. 1987. Technol. E. Int. Bowen. Bogdan. C. and Phillips. 23:333. Blanche Koelensmid. B. sodium chloride content and sodium nitrite concentration on the growth rate of Yersinia enterocolitica. R. O. p. R. 5:331. S. Expansion of response surface models for the growth of Escherichia coli O157:H7 to include sodium nitrite as a variable. and Pace. 53:370.. Buchanan. Eds.. The history and use of nitrate and nitrite in the curing of meat. W. J. Pasteur (Lille) 15:85. Intl. and Deibel. Bacteriol.. and Deibel. R. J. Antonie Van Leeuwenhoek 34:287. 3:93. Resistance of Sporolactobacillus to potassium sorbate and sodium nitrite. In The Science of Meat and Meat Products. C. H. J. J. M. Food Microbiol. R. Moran. Benedict. 1971. San Francisco. Food Investigation Special Report No. 2001. R. Interaction of sodium nitrite. 1993. 1975. S. Appl. 1975.. 12:203. and fermentative metabolism. 1974. 29:447. pH. Food Sci. J. and Phillips. and sodium nitrite on heat resistance of Staphylococcus aureus and growth of damaged cells in laboratory media. Inst. L. The effect of pH. L. A. 63. J. W. S. A. E. Golden. R. Effects of starter cultures on the formation of flavour compounds in dry sausage. 1994. O. Berdagué. 1979. G. 13:655. E. R.. L. Microbiol. Response surface model for predicting the effects of temperature. nitrogen fixation. T. A. Appl. Food Microbiol. Buchanan. Cerveny. G. 1968. V. Meat curing. Ferredoxins: Chemistry and function in photosynthesis. L. 1997. J.. J. L. His Majesty’s Stationery Office. Appl. Brookings. In Proceedings of the Meat Industry Research Conference.. 1940. H. 1972. Expanded response surface model for predicting the effects of temperature. J. 1980. nitrite and bacteria in the curing of bacon and hams. Blanche Koelensmid. G. J. T. F. Food Microbiol. H. J.. R. and Freeman. B. 1974. Schweigert. South Dakota. pH. and Deibel. Binkerd. R. A. American Meat Institute. W. O. Nitric oxide and the immune response. Buchanan. Toxicol. Microbiol. C. A. P. I. Bacteriol. 1974. In Proceedings of the 32nd Annual Reciprocal Meat Conference of the American Meat Science Association. Nat. 1995.222 Antimicrobials in Food Barber. Proc. Price. Food Cosmet. L. Appl. Buchanan. Food Prot. Model for the non-thermal inactivation of Listeria monocytogenes in a reduced oxygen environment. Appl. 37:81. Effect of sodium chloride and sodium nitrite on the heat resistance of Staphylococcus aureus NCTC 10652 in buffer and meat macerate. Bean.. National Livestock and Meat Board. S. 79:163.. L. Buchanan. B. Chicago. and Talon. H. Adv. Microbiol.. L.

J. 27:733. atmosphere.. and Ayres. J. J. 53:549. Nitrite-Cured Meat. Akhtar. Tompkin. 1974. T. 33:46. R. G. 25:357 (erratum 26:653). 1974. Buchman. R. R. R. L. 1955. Howard. The Perigo effect in luncheon meat... Antimicrobial activity of sodium nitrite against Aeromonas hydrophila at various pH and temperatures. Food Technol. 7:209. G. Food Sci. A. W. 1987. R. Microbiol.. Appl. Stahl. Food Sci. J. Pharmacol. K. Influence of sodium chloride on growth of lactic acid bacteria and subsequent destruction of Escherichia coli O157:H7 during processing of Lebanon bologna. Food Sci. 6:255. E.. CAST (Council for Agricultural Science and Technology).. 47:119.. Ames. 7:117. Christiansen. Inst.. Effects and interactions of temperature. Kautter. Microbiol. M. Cassens. Anantheswaran. Johnston. M. 53:79. A. A Food Safety Issue in Perspective. Nitric oxide synthase gene therapy for cardiovascular disease. C.. and Kautter. A. 1974. N. C. and Serreze. Residual nitrite concentration and total plate counts in white and dark chicken patties. A. D. L. pH.. E. Effect of sodium nitrite on toxin production by Clostridium botulinum in bacon. B. T. Technol. Appl. 1987. A. S. L. Cherayil. Food Technol. N. Food Technol. E.. Microbiol. 1997. smoked. J. R. Appl. 1996. Factors affecting the bacteriostatic action of sodium nitrite. C. A. R. M. Ren. Bulman. 34:237. and Jen.. 1965. W. J. G. J. S. J. 1984. N.. A. 52:844. W. 17:214.. CT. N. Microbiol.-W. H-J. G. Bushway. Burke. C. W. D. 47:858.. D. 34:240. A. C. C. B. J... S. Appl. Taiwan 23:159. L. Shaparis. Roberts. 2001. Food Prot. Appl. Christiansen. W. A. 1973. Tompkin. F. 49:72. Food Sci. Microbiol. Chou. 3:15. Factors influencing botulinal inhibition by nitrite. Castellani. 3:771. and Niven. Johnston. A. Food Technol. C. R.. G. and Antos. R. Difference in the PH and iron content of chicken white and dark meat that affect the action of nitrite against aerobes. and Lee. B. C. W.. . J. Chang. Can. 1980. Appl. Cassens. 13:1023.. R. Food Sci. L. Picker. and Whiting. and Tsai. Christiansen. Chang. M. A Ceres Forum organized by Georgetown University’s Center for Food and Nutrition Policy. Issue Paper number 8. W. 1989. N. 1952. and Jen. Inst. G. C. 40:488. Residual nitrite in cured meat.. Shaparis.. J. L. N. and Cornforth. A. R.. Modification of membrane sulfhydryl groups in bacteriostatic action of nitrite. Cassens. Jpn. F. 1982. Environ. N. Kautter. P. 1997. Christiansen. C.. Effect of sodium nitrite on Clostridium botulinum in canned luncheon meat: Evidence for a Perigo-type factor in the absence of nitrite. Food Prot. Trumbull. Cassens. 2001. Can. W. R. L.. Effect of vacuum packaging on growth of Clostridium botulinum and Staphylococcus aureus in cured meats. S. Effect of sodium nitrite and nitrate on Clostridium botulinum growth and toxin production in a summer style sausage. and sodium nitrite on the growth of Listeria monocytogenes. Effect of nitrite and nitrate on toxin production by Clostridium botulinum and on nitrosamine formation in perishable canned comminuted cured meat. J. Food Prot. and Miao. J..A. 89:327. 1990. H. Use of sodium nitrite in cured meats today. and Hansen. 51:53. December 10–11. C. C. Inst.. Y. Food Technol. Inducible nitric oxide synthase and Salmonella infection. J. and nitrite-preserved foods. D. 1979. Chen. D. G. Washington. Mills. Ito. H. November. O. F. Reddy.. Bushway. and Knabel. and Pivnick. Christiansen.. J. and Foster. G. Examination of dietary recommendations for salt-cured. J. V. 1975. Nitrite as a food additive: State of the science. sodium chloride. C. Greaser. A. Cerveny. Food Sci. and Kolari. Preservative effect of various concentrations of curing salts in comminuted pork. Microbes Infect. DC.Nitrite 223 Buchanan. Effects of changes in the production and marketing of cured meats on the risk of botulism. Can. Technol. R. Inactivation of clostridial ferredoxin and pyruvateferredoxin oxidoreductase by sodium nitrite. N. Council for Agricultural Science and Technology. T. and Akhtar. Kueper. Food & Nutrition Press. J. Food Technol. Microbiol. 1984. 1980. P. 2002. 1997. V. Bushway. 64:1145. A. Ceres Forum. B. Johnston. J. B. Reactions of nitrite in meat. 1995.. Chikthimmah.. Effect of nitrite and sorbate on total number of aerobic microorganisms in chicken white and dark meat patties. and Aunan. Environ. IA. D. Technol. P. Carpenter. D. M.

and Shaparis. Presented at the 80th Annual Meeting of the American Society for Microbiology. L. M. D.gov/nara/cfr/index. and Thomson.. Special Poultry Research Committee.html. L. S. Food Sci. J. E. Eds. 39:607. Government Printing Office. B. B. 1982. 1977. and Krol. Walker. Atlanta. and 172. . Influence of raw ingredients. Chry. 2002b. H. 1986. 1987. Manuel. G. Effect of nitrite and storage temperature on the organoleptic quality and toxinogenesis by Clostridium botulinum in vacuum-packaged side bacon. Price. L. S.. N. Collins-Thompson.gov/nara/cfr/index. K. L. Cornforth. 1973. J. Relationship between the increased sensitivity of heat injured Clostridium perfringens spores to surface active antibiotics and to sodium chloride and sodium nitrite. H. Collins-Thompson. C. J. Food Sci. Holbrook.. and Webb. Effect of nitrosylation of myoglobin on gas production by Clostridium botulinum in pork sausage product. G. p. 32. W. D. W. 41:354. Food Prot. 42:44376. R. Walker. J. 15:307. Food and Drugs.. Washington. A. and Post. D. 46:468.. Bacteria associated with spoilage of braunschweiger. I. Crowther. Can. Food Sci. and Hansen. Reaction of nitrite with lactoferrin or transferrin forms an inhibitor of bacterial spore outgrowth. Animals and animal products. DC. Technol. Technol. C. Krusky. 6:41. P. J.S. W. R. Usborne. Mahoney. L.175.. L. Inst. Baird-Parker. 172. 13. G. 2. Tompkin. K. 2002a. Effect of salt level and nitrite on toxin production by Clostridium botulinum type E spores in smoked great lakes whitefish.S. May 11–16. J. D. A.. Washington. C. H. C. C. K. Chicago. R.170.17 and 424. Factors controlling the growth of gram-negative bacteria in vacuum-packed bologna. “Nitrite burn” in cured meat products — particularly in fermented sausages. B. Botulism. and Hauschild. G. 17:102. Technol. Collins-Thompson. B. 1980. Can. D. Food Prot. Bacteriol. R. J. Role of nitrite and ascorbate in the microbiological safety of vacuum-packed sliced bacon. Response to FDA Notice Sept. M. F. Deibel. and Lopez. University of Chicago Press. H.. Reg. D. 8:233.. Docket No. J. Inst. Food Sci.. and Collins-Thompson. L.. J.-Y. L. Booren. Christiansen.. Incidence of nitrite depleting lactic acid bacteria in cured meats and in meat starter cultures. 1981. 1981. and Sebranek. C. The effect of nitrite on the growth of pathogens during manufacture of dry and semi-dry sausage.-Y. 49:55. G. Dodds. 1981. 1982. 1949.. Cirino. Presented at the 41st Annual Meeting of the Institute of Food Technology. C. B. J.. U. W. Appl. M. 172. and Sebranek. Collins-Thompson. The Netherlands. Larmond. D. Available at: http://www. 1980. B. 1957. C.html. 50:1550... and Caliendo. p.. Collins-Thompson. Food Prot. J. Dodds.. Inst. J. 50:212. R. Depletion of sodium nitrite by lactic acid bacteria isolated from vacuum-packed bologna. Miami Beach. S. L. D. J. J. N. Code of Federal Regulations.. Food Prot. nitrite levels.. 44:593. Code of Federal Regulations. M. and cooking temperatures on the microbiological quality of braunschweiger. American Meat Institute Foundation. The behavior of Salmonella in vacuum-packaged cooked cured meat products.. L. and Divilbiss. J. Chicago. A. W.177. J. L.. The nitric oxide related therapeutic phenomenon: A challenging task. Production of N2O and CO2 during the reduction of NO2– by Lactobacillus lactis TS4.. H. J. and Lopez. and Austin. V. G.. U. 1978. 1984.. 49:602. Available at: http://www. Fed. J. L. 45:1732. 21CFR 172. Custer. and Evans. 1977. K. In Food Poisoning. Food Prot. M. Dack. and Collins-Thompson. L. J.. Curr. L.. and Adams. Wageningen. C. J.. Bulletin No. Tinbergen. In Proceedings of the 2nd International Symposium on Nitrite in Meat Products.. 1974.160. J. 1980. J. Can. 1985. Changes in manganese content in Lactobacillus plantarum during inhibition with sodium nitrite. Fate of Clostridium botulinum in perishable canned cured meat at abuse temperature. L. 77N-0222. Georgia. J. D. Q. G. DC. Chang. Control of Brochothrix thermosphacta by Lactobacillus species in vacuum-packed bologna. J. Santagada. A. 2002.gpo. 45:305.224 Antimicrobials in Food Christiansen. Pudoc. L. 1976. Wood. June 7–10. Pharm.21-23. Gray. Food Prot. 1984. Effect of sodium nitrite on cured poultry products. J.. R. 47:7. Pestka. M. Chry. B.. D. and Pivnick. Collins-Thompson.. Florida. and Jones. Collinge. G. A. Des. 65. D. S. F. Food Prot. 9CFR 317. Government Printing Office. P. N.gpo. Davidson. and Kuth. M. J.. R. Food Sci. Davidson. I.. Chumney. Food Sci. Cuppett.

Antimicrobial effect of acidified nitrite on gut pathogens: Importance of dietary nitrate in host defense. Environ. Gardner.. Combined effects of heat treatment. aw. H. 1995. L. Alimentaria 21:49. Brandt. Her Majesty’s Stationery Office. J. 71:1184. J. F. 36(12):307. M. L. and Hoz. 2:361. and Hassing. J. Smith. J. W. S. A salt-tolerant denitrifying Bacillus strain which “blows” canned bacon. II. 16:401.. Technol. Washington. G. É. J. J. Eddy. E. 1992a. and Andrássy.. nitrite and N-nitrosos compounds.. R. Farkas. 1996. 1968b. DC. and Solberg. Trends Food Sci. Processing technologies and main differences. nitrite. Dresel. Combined effects of physical treatments and sporostatic factors on Clostridium sporogenes spores. 31:73. H. Mediterranean vs Northern European meat products.. 50:1412. and sodium nitrate on germination and outgrowth of anaerobic spores. F. J. Feron.. R. Abt. Effect of nitrate and mixtures of these salts on the growth of coliform bacteria. Incze. Proc. Hung. 1967. and Bermell. Results of model experiments related to gas defects in cheese. and Foster. 14:207. W. 1957. 40:1422. Duncan.S. F. 33:60.. Food Microbiol. J. J. nitrite and ascorbate.. Fraser.. I. W. F. Walker. B. Final report on nitrites and nitrosamines. Copland. A. G. C. S. 16:406. 76:163. Appl. Report to the secretary of agriculture by the expert panel on nitrites and nitrosamines. B. A. D. Acta. Assessment: Nitrate. Dykhuizen. TX.S. M.. C.. Acta Aliment. C. Microbiol. Fleischwirt. Department of Agriculture. S. Food Safety and Inspection Service Program Training Division. B. Vanderlinde. Biochem. 19:62. Factors influencing souring and their consequences. M. Food Safety and Inspection Service. The inhibitory action of nitrite... Farber. C. Bakteriol. The effect of mixed curing solutions. Post. and Ross.. 1968a. Combined effects of physical treatments and sporostatic factors on Clostridium sporogenes spores. 1970. C. Zentralbl.. Food Chem. Agric. L. In Proceedings of the 2nd International Symposium on Food Microbiology. C. McKellar. Gangolli.. L. C. Effect of sodium nitrite. Milk Dairy J. Food Sci. E. 1992b. Alimentaria 21:39. Benjamin... Ordóñez. Douglas. Dykhuizen. N. Eklund. Appl. Eur. Antimicrob. C.. 1966. B. 2:49. London. sodium chloride. 1982. Growth of Listeria monocytogenes on vacuum-packed cooked meats: Effects of pH. Food Chem. E. 1997. R.. Antimicrobial effect and disappearance of sodium nitrite in Staphylococcus aureus cultures. S. 1983. Fernández. Role of curing agents in the preservation of shelf-stable canned meat products. A. 23:377. Fleischwirt. J. N. B. L. L. 2000. 1991. 1996. 1994. L.. Farkas. Spiegelhalder. P. Arrest of growth from spores in semipreserved foods. 1995. Effect of nitrite on the microbiological stability of canned Vienna sausages preserved by mild heat treatment or combinations of heat and irradiation. Koeman. R. Food Safety and Quality Service. 1956. Eddy. M. Discoloration in cooked ham. Toxicol. T. 1994. M. 1934. 1999. reduced aw and reduced pH in an anaerobic nutrient medium. Accelerated ripening of dry fermented sausages. C. and Zukal. Duncan.Nitrite 225 Duffes. Farkas. S. Duncan. Bacteriol.. Processing Inspectors’ Calculations Handbook. R. J. J. Duffy. Pharmacol. 59:505. É. K. M. 2 91:135. Plasma nitrate concentration and urinary nitrate excretion in patients with gastroenteritis. 10:211. M. Bruna. Acad. Sci. Janzowsky. Infektionskr. Herranz. Appl. Smith. 37:1. Significance of Clostridium botulinum in fishery products preserved short of sterilization. J. Denton. F. The effect of meat curing solutions on anaerobic bacteria. 1978. Fox. Flores. Fang. L. U. B. 292:1. Agents Chemother. The chemistry of meat pigments.. S.. Duncan.. Trends Food Sci. Galesloot. Bacteriol. Food Technol. M. 1973. Speijers. and Tanner.-S. .. U. Hyg. Modelling the effects of various parameters on the growth of Listeria monocytogenes on liver pate. Technol. and Leifert. and Grau. and Andrássy. C. P... Flores. 11:201. J. P. J. Infect. M. and Foster. Department of Agriculture. Fink-Gremmels. reduced aw and reduced pH in canned luncheon meat. and Ingram. M. Evans. L.. IV. 1986. Neth. P. Appl. J. and Benjamin. Food Microbiol. Improving the control of Listeria monocytogenes in cold smoked salmon. C. G. Acta. heat treatment. Intl. Use of monascus extracts as an alternative to nitrite in meat products. A.. and Wishnok. J. and Leistner. J. H. 1985.. Microbiol. J. Parasitenkd. E. Golden.. Combined effects of gamma radiation. J. C. J. Dry-cured sausages. V. 12:447. Pharmacol.

Gram. R. The effect of nitrite and starter culture on microbiological quality of “chorizo” — a Spanish dry cured sausage. and Fryer. E. R. Bladel. A. Meat Sci. A. Res. Guyer. and Roberts. 55:1565. 1991. 1981. Food Sci. sodium nitrite and storage temperature on the growth of enteropathogenic Escherichia coli and Salmonella in a laboratory medium. 1986. R. G. V. McCurdy.. Stockholm. sodium nitrite. B. Effect of food preservatives on the growth of bacteria from spores. 2001a. 13:743. and Jemmi. J. 2001b. Bacterial greening in cured meats: A review. M. N. F. H. Microbiol. Factors determining survival and development of Clostridium botulinum in semipreserved meats. Gough. Inst. Environ. Factors controlling the growth of Clostridium botulinum types A and B in pasteurized cured meats. Int. W. Hankins. 17. G. water activity. 57:1523.. IV. W. Environ. Behavior of salmonellae in sliced luncheon meats. A. Cardiovasc. Food Sci. 16:135.. sodium chloride. 1965. and McCurdy. 55:250. M. T. and storage temperature on the growth of Clostridium perfringens and faecal streptococci in laboratory media. H. J. Food Sci. J. Production of enterotoxin-B in cured meats.. The effect of pH. and Robinson. and Diez. 1984. Gray. Gonzales.. S. Appl. Pudoc. W.) 66:S1082. Food Sci. A. O. Eds.. 1988. J. and Krol. Goepfert. J.. Tinbergen. cut and batch of pork. Egypt. C. Appl. 19:78. 1950. Pasteur (Lille) 7:53. 44:302. 1988. and Doyle. T. Effect of curing agents on the growth and survival of food-poisoning strains of Clostridium perfringens. 40:433. A. 1980. Grau. A. 30:1025. 19:83. J. 1959. and Chung. B. L. 1986a. 4:33. J. 3:195. Roberts. 1955. Inst. Molin. T. Riemann. A. L. B. C. Can. Greenberg. 3:183. J. Gibson. A. W. Food. F. J. Grant. Microbiol. Food Sci. J. J. A. D. B. D. and Kojda. A. Roberts. Minimum nitrite concentrations for inhibition of clostridia in cooked meat products. Eds. I. The influence of sodium chloride on toxin production and organoleptic breakdown in perishable cured meat inoculated with Clostridium botulinum. Pearson. T. 1989. Silliker. Can.. 17:471. Microbiol. 19:190. M. B. Gould. M. Gilles. Grant. A. Technol. The Netherlands. In Microbial Inhibitors in Food. P. A. Technol.. M. Role of nitrite in cured meat flavor: A review. H. J. Behavior of Listeria monocytogenes during fabrication and storage of experimentally contaminated smoked salmon. 1995. Environ. 1984. Preserving agents in meat products — a real necessity? Fleischerei 10:VI–IX. Grever.. and Alford. and El-Zayat. . 2002. 60:295. Gibson. (Suppl. Food Sci. D. Fate of Listeria monocytogenes in processed meat products during refrigerated storage. Effect of curing agents on the growth of Lactobacillus viridescens in model meat systems. Food Technol. G. W. W. F. Microbiol. T. Food Microbiol. A. and Robinson. G. 1974. 1986b.) 66:S1072. Ann. Gram. M. Sci. 1970. Wageningen. F. J... Microbiol. K. Gerhardt. A. A.. Potential hazards in cold smoked fish: Listeria monocytogenes. G. 19:29. L. J. A. 34:62. Gibson. Kauffman.. and Fatta. Inhibition of the anaerobic growth of Brochothrix thermosphacta by lactic acid. A. MacDonald. Greenberg. VI. Inst. New Zealand J. J. 1965. and Roberts. M..226 Antimicrobials in Food Genigeorgis. and Osborne. Potential hazards in cold smoked fish: Clostridium botulinum. M. Radiation injury of Clostridium botulinum spores in cured meat. In Proceedings of the International Symposium on Nitrite in Meat Products. The effect of storage temperature on “late blowing” in Gouda cheese. I. R. Vasoprotection by nitric oxide: mechanisms and therapeutic potential. 1964. Int. 1982. Sulzbacher. Nitrite monitoring during storage of pasteurized pork slurries. Food Prot. J. Technol. K. Factors affecting the keeping quality of bacon. T. R. I. 13:509. Gibson. M.. U. Glass. L. Food Technol. H. and Zingelmann. and Mayo. The effect of pig breed.. (Suppl.. A. Food Sci. J. Appl.. p. A. Effect of some preservatives on the quality of processed cheese spreads. 2002.. Halvorson. B. p. Factors controlling the growth of Clostridium botulinum types A and B in pasteurized cured meats. and Sadler. Food Technol. The effect of pH. Technol. J. Gouda. Almqvist and Wiksell. A.. 103. T. A. Food Microbiol. O. 21:50. Appl. Gewaltig. O. Appl. G. J. J. and Morton.. B. 1969.

and Weng. Food Prot. Hughes. Fleischwirtschaft 54:1515. 1989. Effect of sodium nitrite and origanum oil on growth and toxin production of Clostridium botulinum in TYG broth and ground pork. M. Incidence of Clostridium botulinum in commercial liver sausage. 148:819. 26:22. and Pierson. 1990. M.. 36(12):95. M.. A. J. W. The microbiological effects of nitrite. D. and Armstrong. Food Control 6:335. K. DC.. 30:768. 6:21. Food Prot. and Leistner. P. 1987. V. 45:500. Reg. and Tompkin.Nitrite 227 Hansen. Hauschild. C. H. H. A. K.. Wageningen. Effect of some inhibitors derived from nitrite on macromolecular synthesis in Bacillus cereus. 1982. Robinson.. J. Christiansen. A. Food Microbiol. Washington. G. H. Appl. P. and Krol. A. W. Food Prot. K. N. S. H. K. Huss. Ingram. and Simonsen.. J. B.. 1989. sodium chloride. Ivey. H. Nitrates and nitrites in food and water. and Kolari. Inst. Deibel. 40(4):155. H. Huss. Holley. Fazio. L. R. 1975. Both evaluations are available at: http:// jecfa. The Netherlands. Fleischwirt. The effect of phosphate. Ingram.ilsi. Effect of added sodium nitrite on the formation of clostridial inhibitors. Trenk. Ripening and storage of Hungarian salami. 1995. 72:58. H. Biol. Microbiol...... Ben Embarek. Nitrates and nitrites. 1973. B. Farkas... J. 400–418. Appl. K. J. S.. Technol. Food Prot. B. and Hilsheimer. L. M. B. Food Prot. H. A. J. Effect of sodium nitrite and sodium nitrate on botulinal growth production and nitrosamine formation in wieners.. and Levin. J. and Korkeala. The technology and microbiology of Hungarian salami. Eds. Appl. L. . In ACS Symposium Series: Food Safety Assessment. Cerveny.W. Hauschild. Pudoc. D. Incze. J. Hechelmann. p. Contribution of nitrite to the control of Clostridium botulinum in liver sausage. Food Technol. Antibacterial effect of cysteine-nitrosothiol and possible precursors thereof. 53:958. R. Mecanisme biochimique de Faction du nitrite dans la conservation des viandes. 1991.. W. C.. Food Sci. 37:63. R. Hauschild. Can. A. Soc. Int.... Microbiol. Microbiol.. Ellis Horwood. G. Finley. O. International Life Sciences Institute. p. Tinbergen. 48:997.. H. A. Food Technol. storage temperature and pH on the growth of enteropathogenic Escherichia coli in a laboratory medium. Z. J. J. Control of indigenous bacteria in seafood.. R. C. J. Intl. 46:242. N. A. Tradition and current status. New York. 69:587. Nitrate.. 14:183. W. K. J. D. Joubert.. and Goret. Washington. In Proceedings of the International Symposium on Nitrite in Meat Products. Effect of potassium sorbate on toxinogenesis by Clostridium botulinum in bacon. and Jeppesen. 1985. American Chemical Society. H. H.. and McDermott. Raw fermented and dried meat products. and Zukal. A. T. 1997. Kautter.. K. 1992. J. Eds. R. 1992. Control of biological hazards in cold smoked salmon production. and Simonsen. R. Sodium nitrite and potassium nitrate in control of nonproteolytic Clostridium botulinum outgrowth and toxigenesis in vacuum packed cold smoked rainbow trout. nitrite and N-nitroso compounds: Food safety and biological implications. Safety assessment for shelf-stable canned cured meats — an unconventional approach. Hauschild. D. 63. E. 1981. 9:215. J. 1971. D. M. J. F. Maragos. E. A. Incze. 27:202. and Roberts. 1997. H. Appl. L. 44:48959. 30:862. Safety of shelf-stable canned cured meats. N. 1954. Hotchkiss. and Raymond. 1975. 67:445. J. Microbiol. J.. A.org/index. Incze. 1979. J.. Hilsheimer. Food Technol.htm. Hyytiä. A. E. Review of the potential hazard from botulism in cured meats. Fleischwirt. A. Food Microbiol. Food Control 8:91–98. H. Hill. 1983. M. O. Huhtanen. Helser. F. Henry. T. G. 41:621. P. W. A. Y. S. 1978. Houston. E. R.. Mihalyi. DC. J. Assessment of botulism hazards from cured meat products. 1974. Chemical and organoleptic changes. J. Application of the “D-Concept” to heat treatments involving curing salts. Fleischwirt. 1974. Uchida. Fed.. Incze. Johnston. H. 1982. Jarvis. Shaver.. R.. H. 1986. C. W. Eerola. M. M. V. and Wasserman. B. sodium nitrite. Hielm. F. 1974. Ismaiel. Conditions physicochimiques favorables a son action bacteriostatique. Bern. Hauschild. Hustad. Vorkommen des tribus Klebsielleae bei kuhlgelagertem fleisch und fleischwaren.

Immunogenet. Jpn. King. Conversion of cystine or cysteine to the antibacterial compound bis-(2-amino2-carboxyethyl) trisulfide (bactin) enhanced by ferric chloride and sodium nitrite. Food Prot. Food Prot. Bacteriol. H. Jarvis. 50:56.. J. 1998.. and Jansen. ILSI Press. 32:217. N. X-Y. J..org/es/ESN/Jecfa/database/cover. and Hess. 1980.. Knøchel. Microbiol. Inst. Food Sci. B. Sci. J.. 1969. J. R. Hill. Wood. 4:179. Jarvis. S.. 1979. M.. Jensen. A. Jonsson. B. Juntilla. N. Curing of fishery products. Kog-ningens haemmende betydning overfor bombagefremkaldende bakterier.htm. sodium nitrite.. R.. Fish. J. H. and Sakamoto. King. May 10... Kogning af dasekinker. S. 2000.r. Ripening and spoilage of sugar salted herring with and without nitrate. Ripening and spoilage of sugar salted herring with and without nitrate. 2:52. and Samson. Nieuwenhof. E. J. M. Konserves 7:105. and Jozwik. Jacobs. 1971. Eng. L. D. G. 2:387. Taxonomic characteristics of lactic acid bacteria which cause the greening.. B.. JECFA (Joint FAO/WHO Expert Committee on Food Additives). Food Technol. Vugman. Tomishima. Rhodes. 1979. J. Microbiological studies on the greening of cured meat products. R. Environ. and Chaudri. sodium nitrite. 18... L.. and Huss. J. Hoolwerf. Hughes. Washington. 1989. I. 1987. Environ. B. DC. Soc. Madsen. M. J. and Deibel. L. Effect of a short cold storage on frequency of spoilage in pasteurized (perishable) canned meat products subjected to the incubation test. Characterization of the bactericidal effects of sodium nitroprusside and other pentacyanonitrosyl complexes on the food spoilage bacterium Clostridium sporogenes. Parma. R. Appl. I. Can. 1950. Rogers. C. A. J. and Nurmi. Nramp1 and nitric oxide synthase in the host antimicrobial response. Administrator. H. Martinez. Jarvis. J. M. C. 1949. F. W. N. Hirn. In Food Microbiology and Technology. H. Karupiah. J. J. Ind. Chem. Johnston. Johnston. E. P. Identification of Clostridium tyrobutyricum as the causative agent of late blowing in cheese by species-specific PCR amplification. type B to sodium chloride and sodium nitrite. R. Food Sci. 1995. 1964. Kafel. 251. I... Report submitted to D. B.. 19:203. A. S-nitrosocysteine as an antioxidant. C.. p. J. 31:527.. E. S. S. Washington. 1984a. Food Technol. J. Swelling in canned chopped hams.. Potassium nitrate. Inhibition of Clostridium botulinum by sodium nitrite in a bacteriological medium and in meat. H. NADPH oxidase.l. Vielotte. 11:41. Pivnick. 45:1105. van der Waals. A. Eds. 1941. and Huss. H. and Loynes.. H. J. JECFA (Joint FAO/WHO Expert Committee on Food Additives). J. Cui. C. A.. J. Can. 1965. 1980. Summary of Evaluations Performed by the Joint FAO/WHO Expert Committee on Food Additives (JECFA 1956-2001). Jensen. J. Chan. B. Food Technol. Microbiological and related chemical changes. and Michener. Food Technol. M. 1976. Food Technol.. Internet edition. USDA. Medicina Viva Servizio Congressi S.. 1984b. N. 26:1118. V. N. Fish and Wildlife Service. J. H. F. Italy. N. 61:2919. Appl. I. G. nitrite/sorbate and control variables. M. L. Heady. Both specifications are available in the online edition of JECFA Compendium of food additive specifications at: http://www. Research Report No. and Ronnenberg. J. T. Sensitization of heat-damaged spores of Clostridium botulinum. Inst. 64:3195. Klijn. and Ayres. J... Inhibition of Clostridium botulinum by sodium nitrite as affected by bacteriological media and meat suspensions. color-developing and anticlostridial agent in comminuted turkey meat. C. 1934.. B. 1969. 1995.228 Antimicrobials in Food Iwata. 19:215. J. Joannou. Appl. M. Houston.. Jacobs. 52:158. N. J.. Effect of different levels of nitrite and nitrate on the survival of Listeria monocytogenes during the manufacture of fermented sausage. H. and Patel.. J. Rhodes. C. T.. and Raa. and Patel. Kanner. K. P. Effect of hemin and oxygen tension on growth and nitrate reduction by bacteria. Potassium nitrate. Rev. and Juven. R.fao. United States Department of the Interior. 1979. and Cammack. N.. Johnston. 1995.. Knøchel. and Weerkamp. B. Bacteriol. Jensen. Christian. A study of the effects of sodium nitrate on bacteria in meat. Convention Canner 92:82. D. S.. II. Effect of nitrate.. The antagonism of enterococci on other bacteria in canned hams. L. Microbiol.. DC. Microbiological safety of pasteurized cured meats: Inhibition of Clostridium botulinum by curing salts and other additives. Jarvis. H. H. 45:1641. C. FSQS. H. S. A. Kafel.. E. W. 87:1406. D. Microbiological results from the four plant commercial bacon study with nitrite. Hunt. N. M. M. J. .

A. Characterization of the late blowing in St Nectaire cheese and some pressed cheese. salt and nitrite on the heme-dependent catalase activity of lactic acid bacteria. J. 1541. Effect of curing ingredients and procedures on the survival and growth of staphylococci in and on cured meats. Vet. p. Food Microbiol. Comparison of carbon monoxide. Jpn. Lechowich. 1991. Christian. einschliesslich salmonellen. J. 1994. Biophys. J. 1982. L. 1991. T. B. Roberts. 1956. 265.. Factors affecting inhibition of Clostridium botulinum in canned meats. Didienne. J. . Effect of curing agents on the heat tolerance of putrefactive anaerobic bacterial spores. and Duncan. 12:384. Des. 508. Appl. W. 491. and Pradel.. and Leistner. Z. Fleischwirtschaft 62:203. T. D. J. Leistner. Bacteriol. Appl. Fermented meat products. and Krol. The effects of temperature. Patent 3. 1978. W. Academic Press. Fleischwirt. Congress Meat Sci. 1971. 37th Intl. 1974. L. 37:26.. The role of nitrite in the production of canned cured meat products. In Proceedings of the 19th of the European Meeting Meat Research Workers. Evans. Deibel. Le Lait 63:15. Kelly. H. 1981. J. and Debevere. J. Patented June 29. and Tiusanen. L. Leistner. 1970. R. 1982. pH.. H. M. Salmonella typhi in corned beef. Pharm. B. France. Alanko.-K. Sutherland. Flavor development and microbiology of Swiss cheese — a review. Lebensmittelhyg. Cassens. 3:1252-1256.. Influence of pH. Kurowska. and Skovgaard. In Psychrotrophic Microorganisms in Spoilage and Pathogenicity.. The enrichment and isolation of Clostridium botulinum from raw ham. 1965. 34:221. F. Acta. 63:401. nitric oxide. 1996. 19:353. F. 1983. 443:129. T. Beaumont. Factors affecting the secretion of staphylococcal enterotoxin A. G. Meyer.. 33:1. C.. Monascus extract — a possible alternative to nitrite in meats. 43:1371. Langsrud. and nitrite as inhibitors of nitrogenase from Clostridium pasteurianum. and Niven. Scand. Nitrosodimethylamine as a toxic and carcinogenic substance in herring meal. Hobbs.. Lancet 1:426. Lücke. Leistner. and Sekiguchi. Mutation Res. T. S. Intl. Proc. T. L. H. R. Matsuda. Korkeala... 4:360. 1994. and Uchida.. p. Arch.S. 1998. pH. 1981. I. L. R. DC. N. Labbe.. 1999. and Leistner... Defects. Wageningen.. L. Food Technol. L. 24:191. Eds. Meers. Neyts. Lücke.. Arch. Meester.. The relevance to meat products of psychrotrophic strains of Clostridium botulinum. J. and Goode. J. The effects on growth of NaCl. A. V. Washington. F. 1965.-K. Intl. Patent Office. Microbiol. New nitrite regulation of West Germany. P. Food Sci. H. New York. 498. p. 1978. F. Hemmung von Enterobacteriaceae. 504.. Biochem. R. Hechelmann. McClure.-K. D. G. 567. Growth from spores of Clostridium perfringens in the presence of sodium nitrite. Mares. T. Brown.. Food Microbiol. K. 20:492.053. Milk Food Technol. 2002. N. McClure. L. G. 503.. 8:155. 506. H. T. Lechowich. Lücke.Nitrite 229 Koppang. Fink-Gremmels. 14:77. Technol. P. 33:27. Adams. Lücke. Hechelmann. Process for canned meat. and Somers. 1986. T. 210:246. K. Appl. R. H. U. Food Res. J.. G. Paris. 510. J.192. Kulmbach. J. H. A. V.. A. B. Intl. Soc. Markus. J. H. 1970. C. J. and Sugiyama. and Roberts. and Reinbold.. H. 1973. G. 32:45.. 1974. M. D. D. L.. Kueck.. sodium chloride and sodium nitrite on the growth of Listeria monocytogenes. I.. R. Microbiol. and Froning.. IV. J. Pudoc. Effect of sodium nitrite and sodium chloride on growth of lactic acid bacteria. 27:299. P. N. storage temperature and NaNO2. Appl.. W. T. Mayenobe. Hechelmann. J. J. Food Microbiol. 1992. A. P. 1987. A. The Netherlands. 1980.-K.S. U.. 1965. Curr. Predictive modelling of growth of Listeria monocytogenes. M. Microbiol. A. Botulism after consumption of raw hams. Norsk Vetrinaertidsskrift 110:493. McClure.. J. E. Eds. The effect of incubation time and temperature on growth of Escherchia coli on gradient plates containing sodium chloride and sodium nitrite. September 1–6. Nitrate and nitrite allowances in meat products. J. N-nitroso compounds in the diet. and Roberts. Lee. Nitric oxide therapies in vascular diseases. G. Leistner. In Proceedings of the International Symposium on Nitrite in Meat Products. 66:496.. Food Hyg. in fleischerzeugnissen durch nitrit. Tinbergen B. Germany. and Roberts.. Lijinsky. and Dresel. An overall look at raw hams. L. R. F. L. J. Fleischwirtschaft 61:252.. P. Intl. and Silverman.

H.. Sonoki. Appl.. and Götz. 2:26. S. 1973. and Hofmann. F. Y. 8:58. S. Konieczny..-J. 2001. Bacteriol. Murphey. Agric. Fleischwirtsch. and Archer. 73:758. Effect of pH. Nagy. R. Untersuchungen iiber chemische und bakteriostatische eigenschaften verschiedener reaktionsprodukte des nitrite. and van Binnendyke. Fisheries. 01-3954. Nychas. R. . V. Effect of polyphosphates in combination with nitrite-sorbate or sorbate on Clostridium botulinum growth and toxin production in chicken frankfurter emulsions. Formation of antibacterial factor by heating system containing nitrite and tryptone.. R. Sadler. A. Niven. 1991. Uber den verbleib von nitrit in fleischwaren. R. G.. 1975.. Neubauer. Microbiol.. J. J. E.. Nelson. J. S. Composition of bacterial flora in sliced vacuum packed bologna-type sausage as influenced by nitrite. Sakata. 25:553. Food Prot.. Food Control 9:299. 63:352. J. 7:839. L. Ministry of Agriculture. Bacteriol. spec. Mirna. salt and nitrite in heat processed meat on destruction and out-growth of PA3679. P. Bacteriol. 1989. J. 56:1158. S. 1996. R. Appl. and Blot. Thermal tolerance studies on the heterofermentative lactobacilli that cause greening of cured meat products. Inst. C. Nordin. C. Food Technol.. G. Morse. E and Arkoudelos. S. Morita. W. 1996. 1983.. 1975. and Nagata. Miller. A. C. K. 46:846. Appl. Influence of nitrite addition and gas permeability of packaging film on the microflora in a sliced vacuum-packed whole meat product under refrigerated storage. 65:1026. Sakata. (Jpn). Miwa. 69:587. S. J. Food. Food Sci. 1983a. N. NIH Publication No. 1983b. Niven. Nitric oxide complex of iron (II) myoglobin converted from metmyoglobin by Staphylococcus xylosus. K. I. 1969. Biol. and Evans. and Nagata. H. 18:371. A. L. S. 30:838.O. H. Immun. Technol. Report on the review of nitrites and nitrates in cured meats and cheese. Y. Ripening and storage of Hungarian salami: chemical and organoleptic changes. 1957. J. J. J. F. and Incze. A. and Nagata. J. R. A. H. T. Mihályi. B. and Allanson. Niven. Box 12233. Mitteilung. W. Mitteilung. J.. K. Anim. Burke. 1998. 1978. M. C. Mirna. A. and Food. and Wagner. Nielsen. Microbiol. Regulation of staphylococcal enterotoxin B: Effect of anaerobic shock. J. a heterofermentative species that produces a green discoloration of cured meat pigments.. 1983. 61:1021. A.. Chem. C. Blood fraction effects on the antibotulinal efficacy of nitrite in model beef sausages.230 Antimicrobials in Food Miller.-J. A. D. and Evans. Food Sci. Infect. Red pigment of Parma ham and bacterial influence on its formation. K. 1998. 1954. Buettner. Webb. Sci. Technol. Niu. Appl. Lactobacillus viridescens nov.. C. F. Y.. Letters Appl. Microbiol. Staphylococci: Their role in fermented sausages. J. Castellani. and Coretti. and Menichello. Call.. J. Sofos. M. Food additives and contaminants committee. 1973. 1993. Umsatz von nitrit mit sulfhydrylverbindungen. B. Trends in cured meat consumption in relation to childhood and adult brain cancer in the United States. 18:573.. H. A. Morita. 1974. M. A. N. Can.. Busta. National Toxicology Program.. Fleischwirtschaft 54:507. 7632-00-0) in F344/N Rats and B6C3F1 Mice (Drinking Water Studies). Technol. A. NTP Report on the Toxicology and Carcinogenesis Studies of sodium nitrite (CAS No. J. National Toxicology Program.. 178:2005. F. M.. G. D. K. J. Morse. Factors affecting the regulation of staphylococcal enterotoxin B. 1949. 1994. Uber den verbleib von nitrit in fleischwaren. J. J.. S. 167S–188S. J.. J. Tannenbaum. NC. Moran. J. Microbiol. 58:633.. 47:687. 16:80. S. A. and Mah. J. Murphy. Morita. R. Inhibitor of Clostridium perfringens formed by heating sodium nitrite in a chemically denned medium.. Research Triangle Park.. G. and Matsuura. Physiology and interaction of nitrate and nitrite reduction in Staphylococcus carnosus. London. Food Sci. II. Enhanced botulinal toxin development in beef sausages containing decolourized red blood cell fractions. Bacteriol. Nielsen. H. J. F. F. H. Sakata. and Baldwin. Rubin.. NTP TR 495. Fleischwirtschaft 49:1361. J. P. Food Sci.. Metmyoglobin conversion to red myoglobin derivatives and citrate utilization by bacteria obtained from meat products and pickles for curing. A study of the lactic acid bacteria that cause surface discolorations of sausages. D.. 1990. and Uchman. V.

J. and Chang. R. and Barnett. A. Gen.. 2:377. 3:91. Optimization of red pigmentation and effect of the metabolites produced by Monascus strains on microbial inhibition and colorization in processed ham. Food Prot. H. B. Benito. N. H. Solberg. 111. Food Prot. G. Almond. R. Food Prot. E. M. P. Pelroy. Inhibition of clostridia by nitrite. F. Payne. H. Pudoc. Bristol. J. Peterson.. Food Prot. J. Stahr. 31:208. Food Technol.. Food Soc.. 1970. 1990b. The Netherlands. 45:833. Food Safety 7:1. A. Pegg. W. Microbiol. 136:2077. Agric. 136. V. 1967.. B. 27:172. M. and Bashford. T. and Brightwell.. 1974. M.. New York. O’Boyle. R.. M. S. Woods. M. F. L. V. P. J. Processed meats. M. J. A.. and Eklund. 1980. Aladin-Kassam... J. Suzuki. J. R. Poysky. E. Curing salts and related materials. 1994b. Nitrogen assimilation by Salmonella typhimurium in a chemically defined minimal medium containing nitrate. and Shahidi. L. 31:1039.. Toxinogenesis by Clostridium botulinum types A and E m perishable cooked meats vacuum packed in plastic pouches. Pelroy. Comer. A.. J. R. In Proceedings of the International Symposium on Nitrite in Meat Products. and Bird. Choi. Food Prot... S. N. R. and Casas. R. J. G. Pivnick. Page. and Hwang. Peterson. Arch. W. Observations on the inhibition of vegetative cells of Clostridium sporogenes by nitrite which has been autoclaved in a laboratory medium. Holland. p. A. Eklund. T. J. 1983. 1980. Nitrite reductase in Salmonella typhimurium. Vol. E. and Peterson. and Eklund. 1. A. Wageningen. L. Inhibition of Clostridium botulinum types A and E toxin formation by sodium nitrite and sodium chloride in hotprocess (smoked) salmon. E. J. 1999. A nitrite-free curing system and its application to the production of wieners. G.. O’Leary. AVI Publishing. Westport. D. Pelroy. I. M. Academic Press. discussed in the context of sublethally processed cured meats.Nitrite 231 Nurmi. J. England. and Kraft. J. strains. Pivnick. A. Food Technol. H. and Cammack. CT. CT. E. 1976. J. Y. M. Payne. J. 2000. E. C. F. 1984. Carcia. p. Obioha. Pearson... H. Effect of salt and temperature on toxinogenesis by Clostridium botulinum in perishable cooked meats vacuum-packed air-impermeable plastic pouches. and Roberts. Gen. Paranjpye. 1985. E. M. A. 19:1164. A. V. Microbiol. 1965. Food and Nutrition Press. J. O. G. Tormo. J. W. Whiting. 136:2067. W. Nitrite curing of meat. Electron paramagnetic resonance spectroscopic investigation of the inhibition of the phosphoroclastic system of Clostridium sporogenes by nitrite. Y. S. p.. Effect of sodium nitrite on aflatoxin production in pork sausage at different temperatures and the effect of nitrite on growth of Aspergillus parasiticus in culture. A.. Palumbo. S.. and Solberg. 44(5):88. H. J... Glidewell. and Turunen. Biotechnol. nitrite or ammonia. and Carman. H. Appl. Gibbs. Rubin.. Kim. G. W. I. Perigo effect in pork. B. Peterson. 45:458... 1993. 1994a. M.. Environ.. Almond. and Eklund. L.. Paranjpye. Selgas. Food Chem. M.... C... Y. Inhibition of Listeria monocytogenes in cold process (smoked) salmon by sodium nitrite and packaging method. 19:1156. G. J. H. 56:938. In Proceedings of the 16th European Meeting of Meat Research Workers.. Trumbull. Microbiol. F. 1996. Food Technol. J... Park. Pivnick. p. Appl. 211. H. 1990a. Eds. A. and Smith.. Interactions of iron-thiol-nitrosyl compounds with the phosphoroclastic system of Clostridium sporogenes. Page. J. Paranjpye. 413. M. The N-nitrosamine problem and nitrite alternatives. Effect of sodium nitrite inhibition on intracellular thiol groups and on the activity of certain glycolytic enzymes in Clostridium perfringens. 1982. and Krol. 1968. pH.. V. P. and Cammack. Perigo. . Food Technol. M. L. Mah.. and Solberg. and Tauber. W.. M. M.. 1982. 1965. G. Pin. F. sodium chloride and sodium nitrite on the growth of clinical and food motile Aeromonas spp. L. Influence of temperature. J. D. Pivnick. Parameters for control of Listeria monocytogenes in smoked fishery products: Sodium chloride and packaging method. T. Diosady. Microbiol. H. A. M. 1990. 45:75. In Microbial Ecology of Foods. J. Korean J. Tinbergen. Pelroy. Food Technol. Perigo.-C. M. 57:114. C. Inhibition of Listeria monocytogenes in cold process (smoked) salmon by sodium lactate. M. 57:108. The effect of nitrite on the growth curve of lactobacilli and micrococci. Repair of heat-injured Staphylococcus aureus 196E on food substrates and additives and at different temperatures.. J. Lebensmittelhygiene 47:50. A. J.

. Hyg. Effect of nitrite on destruction and germination of Clostridium botulinum and putrefactive anaerobes 3679 and 3679h in meat and in buffer. 37:261. W. Conf.. Rice. H. Lancaster. and Gibson. 3:103. A. J. J. C. cured shelf-stable luncheon meat inoculated with Clostridium botulinum. E. T. I. 1982.. A. T. Ed. B. Roberts. J. L. A. H.. Food Technol. S. Ferguson. Food Prot. A. E. faecal streptococci and Salmonella typhimurium to an inhibitor of Clostridium spp. Can. 16:239. 17:39. and Pierson. T. Factors affecting the safety of canned. D. Effect of nitrite and erythorbate on growth of Bacillus cereus in cooked sausage and in laboratory media. and Loynes. J. Clostridium perfringens inhibition by sodium nitrite as a function of pH. pH and temperature on growth of Listeria monocytogenes. Reddy. Johnston. and Solberg. Sheridan. Bakteriol. 2:141. and Rhodes. B. D. J. Inst. Eblen. Zentralbl. B. Effect of sodium nitrite and temperature on toxinogenesis by Clostridium botulinum in perishable cooked meats vacuum-packed in air-impermeable plastic pouches. . Roberts.. Raccach. N. Food Technol. Clostridium perfringens growth in a nitrite containing defined medium sterilized by heat or filtration.. A. 1997. P. M. 11:25. A. and Smart. R. A. 47:1615. B. J. C.. 40(4):164. 1975a.. Savannah. GA. Presented at the Second NSF Intl. M. 1976.. H.. Nitrite inhibition of Clostridium botulinum: Electron spin resonance detection of iron-nitric oxide complexes. Roberts. Riha. 1981c. Abt. L. Roberts. 1998. Effect of sodium lactate.. potassium nitrate and sodium nitrite on the recovery of heated bacterial spores. Food Technol. H. A. L. H.. Roberts. Riha. 1970.8). A. M. Food Technol. R. and Robinson. Sci. 1974. and Jarvis. Food Technol. J. Hill. Bacteriol. C. Roberts. Rhodes. J. R. A. 1991. 1975. W. M. M. Can. C. II. K. C. 1966. S. G. C. A. Food Sci. Whiting. M. A. 1976. D. 8:463. J. M. The effect of chloride salts on Yersinia enterocolitica in meat. 1973.. Ellis Horwood. and Solberg. C. sodium nitrite. A.. Gibson. and Bernbom. Food Technol.. and Dainty. 2000.. Inhibition of spores of Clostridium spp. Barnett. 40:443. Food Technol. and Perrin.. Effect of sodium chloride. and McDowell. Chemical methods for controlling Clostridium botulinum in processed meats. 40:439. 1975b. by sodium nitrite.. Riemann. and Henningsen. A rationale for the safety of canned shelf stable cured meat: Protection = destruction + inhibition. 16:337.. 16:267. Inhibition of Clostridium botulinum by curing salts in pasteurized pork slurry. Safe heat processing of canned cured meats with regard to bacterial spores. M. Survival of Escherichia coli O157:H7 during the manufacture of pepperoni. J. A. E. T. C. J. T. R. cured meats. Food Technol. E. 161:280. 11:13. Jarvis. Agric. I. Food Technol. J.. J. M. D. J. A. W. Duffy.3–6. R.. A. Rubin. Inhibition of Salmonella by sodium nitrite and potassium sorbate in frankfurters. M. M. Thacker. Food Sci. R. cured meats. The effect of lowered addition of nitrite and nitrate on the properties of dry sausage. New York. and Petrosovits. Gibson. October 11–13. Qvist. 1977. A. formed by heating sodium nitrite.232 Antimicrobials in Food Pivnick. Pivnick. and Garcia. J. p. J. and Robinson. Barnett. France. Food Sci. Puolanne.. Factors controlling the growth of Clostridium botulinum types A and B in pasteurized. Riordan. H.. Science 221:769. Factors controlling the growth of Clostridium botulinum types A and B in pasteurized. A. J. Blair. Raevuori. A pork slurry system for studying inhibition of Clostridium botulinum by curing salts. Roberts. W. 21:100. S. Nordin.. A. 1983. Gibson... and Rubin. Soc. Pivnick. J. and Ingram. E. H.. 1973. 1981b. inoculum size and heat. In Proceedings of the 19th European Meeting of Meat Research Workers... In Nitrates and nitrites in food and water. Prediction of toxin production by Clostridium botulinum in pasteurized pork slurry. J. Orig. Roberts. Nordin. A. T. H. 1981a. I. 1:147. H. A. Growth in pork slurries prepared from “low” pH meat (pH ranges 5. and Cornforth.. Food Microbiol. A note on the resistance of Bacillus spp. T. M. D. 1963. 1969. Food Technol. 61:146. J. Roberts. H. Finland 49:1. Growth in pork slurries prepared from “high” pH meat (range 6. Paris. and Robinson. M. On Food Safety. Food Technol. T. 14:431. 1967. T. J. 1086. Pivnick.3). 1986. Food Technol. H. Nitrite and nitrate as food additives: Rationale and mode of action. Inst. P.5–6. A. Appl.

F. . Eds. 1999. Extending shelf life of cured meats. Microbiological aspects and technological need: Technological needs for nitrates and nitrites. Pasteur (Lille) 7:68. J. N. 10:185. Influence of sodium chloride. 1979b. H. W. 1979. Russell N. Fleischwirt. and Flores. Food Chem. Georgia. K. and Corry. and Lundquist. Food Sci. M. Effect of individual curing ingredients on the shelf stability of canned comminuted meats.. Glasgow. S. Effect of nitrite on anaerobic glucose transport. p. R. J. R.. B. Schmidt. J. J. U.. 1989. P. botulinum and Staph. Blackie and Son. Vila. Inst. Sodium nitrite and sorbic acid effects on Clostridium botulinum toxin formation in chicken frankfurter-type emulsions. F. 1991. 1978. W. Schmidt. G. Scott. Silliker. and Kaya.-K. 1955. June 7–10.. and Harding. Ross.. and Nathan. W. Effect of nitrate and nitrite curing salts on microbial changes and sensory quality of non-fermented sausages.S. W. Bacteriol. R.. Rørvik. Jap. Shahidi. and Robinson. Bhothipaska. J. cooked and sliced meat products. Yarbrough. and Allen. U. J.. and Simonsen. and nitric oxide. M. 2000. 1997. and Schack. C. 44:668. J. J. 2000. G. F. R. 1966. J. Sofos. 213:48. A. B. R. W. Chicago. C. Roberts. J. R. Effect of sodium nitrite and sodium lactate on the growth of lactic acid spoilage bacteria isolated from cured meat products. color. 1982. Shiloh. Inhibiting salmonellae growth in fresh. 1966... DC. American Meat Institute Foundation. Rowe. Influence of composition. W. The effect of potassium sorbate. C.. T. E. Gibson.. Technol. C. J. A. In Proceedings of the Meat Industry Research Conference. N. 45:39. Med.Nitrite 233 Roberts. J. and Lücke. In Proceedings of the Meat Industry Research Conference. Presented at the 41st Annual Meeting of the Institute of Food Technology. G. Sofos. C. Toldrá. Patent 3. In Microbial Growth and Survival in Extremes of Environment. 1992. E. 44:1267. and Harper. Shelf life of cured. A. Sofos. The effect of packaging conditions on the bacteriology. Skovgaard. M. Eds. R. U. and Segner. 43:185. Food Res. III. P. Intl. J. Y. and Taylor. A. The effect of nitric oxide on bacteria. Arendt. Food Sci. Fleischwirt. F. viruses. 1990. Itoh.. Effect of nitrite on a bacteriocinogenic Lactococcus lactis transconjugant in fermented sausage. and Allen. N. Food Addit. M. M. 1979a. T. L. Silla. Fleischwirt. H. Shank. Food Prot.. F. Arihara. In Food preservatives. L. Busta. J. and Allen. and flavor of table-ready meats. 227.258. J. and Schwarz. and Solberg. Asthma. R. 69:879. p. Curr. J. 1980. spreadable Mettwurst (dry sausage eaten relatively fresh) made with sugar. J.. J. E.. 13:159. 12:551. The bacteriology of type E Clostridium botulinum. Busta. Silliker. F. Nitrite inhibition of aerobic bacteria. and Woodbine. American Meat Institute Foundation. Shahamat. Scannell. A. Chicago. Opin. J.. Takeshita. 17:307. Miki. 42:213. F. 51. T. 1958. Shank. Food Microbiol. Food Technol. R. Hill. F. 2:51. Woods. M. R. L. Silliker. 65:66. T. D.. Factors in canned ham controlling Cl. F. Food Technol. J. Seaman. 1981. I. E. Listeria monocytogenes in the smoked fish industry.. Nitrite-free meat curing systems: Update and review. L. 17:83.. Microbiol. 2001. C. Curr.. J. Y. Schillinger.345. J. U. 220:123. Sanz. Schack. Effects of sodium nitrite on Clostridium botulinum toxin production in frankfurter emulsions formulated with meat and soy proteins. The effect of nitrate and nitrite on the microbial flora of Wiltshire bacon after maturation and vacuum-packed storage. E. M. and Kondo. M. Food Microbiol. Botulism control by nitrite and sorbate in cured meats: A review. P.. Greenberg. 70:236. New York. 13. K. Ltd. Ann. Appl. pH and temperature on the inhibitory activity of sodium nitrite on Listeria monocytogenes. The effect of curing salts on bacterial spores. p. Eur. and Eagan. F. Nitrite.. Experimental Biol. M.. Food Microbiol. H. Gould G. 1985.. Behaviour of Listeria monocytogenes in vacuum-packed sliced frankfurtertype sausage. Simone. Payne. K. A. Washington. N.. Microbiol. M. vacuum packaging and modified atmospheres. 9:391. 1962. 3:35. Factors controlling the growth of Clostridium botulinum types A and B in pasteurized.. and Cammack. H.. 1979c. G. E. Shaw. J. B. C.. Contamin.. cured meats. 1963. and Gould. aureus. Intl. Reactive nitrogen intermediates and the pathogenesis of Salmonella and mycobacteria. 42:739. A. J. Academic Press. and Pegg. B. U.. M. J... 1998. L. Patent Office. G. Appl. B. H.S.. R. J... 1964. Patented June 28. 1959. R. 62:183–190. Microbiol. K. Sameshima.. F. F.. J. J. Food Technol. Sanders. Atlanta. Rake. Busta. 1992..

. The effect of sodium nitrite on germination and growth of spores from Clostridium botulinum type B in a meat product. W. C.. The action of nitrites on bacteria: Further experiments. The action of nitrites on bacteria. C. L. H. p. and Ng. J. F. and Foster. W. 45:1285. 1991. Board Can. Parasitenkd. C. Sci. 1980a. and Erdman. Cassens. M. Can. V. 54:1313. sucrose and lowered sodium nitrite. 5:265. Srisuparp. and Kvale. W. N. Storrs. Tanaka. E. Allen. and Allen. M. HI. A. B. A. L. Busta. Technol.. Szczawinska.-K. F. Food Prot. and Peters. Lee. E. E. 10:293. R. M. J. J. 91(2):1. Bakteriol. J. The role and mechanism of the inhibition of C. R. M. Food Microbiol. F. Board Can. L. L. Processing factors affecting stability and safety of non-sterile canned cured meats. E. Intl. Stiles. Infektionskr. J. Effect of meat curing solutions on anaerobic bacteria. 1962. Inst. E.. 41:39. Food Microbiol. 37:1103. M.. 1934. Thayer.. Botulism from meat and poultry products — a historical perspective. 14:261. Meske. J. J. K. F. Food Sci. II. Robinson. 1972. 1979e. and Evans. and Sutherland. Thai. 16:477. P. H. Survival of enteropathogenic bacteria on artificially contaminated bologna. 1979d. and Evans. and Johnston. Szczawinski. F. Influence of a putrefactive anaerobic bacterium in meat. 44:1662. W. W. R. S... and Foster. Board Can. Med. P. J. E. Microbiol... 12:128. Influence of pH on Clostridium botulinum control by sodium nitrite and sorbic acid in chicken emulsions. and Schreiber. Tarr. A. E. C. M. Fish Res. J. and Paradis. F. Ng.. Food Sci. Steinke. Meat Conference of American Meat Science Association. Inhaled nitric oxide therapy in premature infants with mild to moderate respiratory distress syndrome. K. 52:47–56. Environ. P. 1999. 25:120. Food Prot.. The comparative value of preservatives for fresh fillets. 1985. Busta. A. Tompkin. Appl. An investigation of the effects of four variables on the growth of Salmonella typhimurium using two types of gradient gel plates. III. 1989. 6:74. Doyle. F. B. C. D. 1980. M.-K. P. Sofos. 42:464. I. Stiles. 1980. Food Sci. The “vacuum pack” method of packaging foods in relation to the formation of the botulinum and staphylococcal toxins. Sodium nitrite and sorbic acid effects on Clostridium botulinum spore germination and total microbial growth in chicken frankfurter emulsions during temperature abuse. 1951. Infektionskr. Sodium nitrite. Tarr. R. J. 1940. 119. Assoc. 48:679. R. M. E. and Vinton. 1945a. D. CT. and Montel. Food Res. Tanner.. W. H. Traisman. Gross. Heitschmidt. 1945b... L. B. Tjaberg. Gross. C. C. Food Res. A... C. F. Inhibition of botulinum toxin formation in bacon by acid development.. 1979. Tarr. T. 1942. N. Food Sci. M.. L.. J. Effects of various concentrations of sodium nitrite and potassium sor-bate on Clostridium botulinum toxin production in commercially prepared bacon.. Baketeriol. Food Prot. 1980b. Effect of nitrate and incubation conditions on the production of catalase and nitrate reductase by staphylococci. Stumbo. Food Technol. 1933. Bacteriological studies relating to thermal processing of canned meats. Food Manuf. Geulph. 1979. N.. Bacteriol. E. C.. 88(2):48. Wimpenny. M. Parasitenkd.234 Antimicrobials in Food Sofos. J. Bhothipaksa. Hyg. C. 45:7. S. Food Res. M.. J. J. and Allen. L. Thomas. Effect of meat curing solutions on anaerobic bacteria. Influence of meat-curing agents upon growth of a putrefactive anaerobic bacterium in meat. W. R. In Proceedings of the 18th Meeting of Meat Research Workers. Effect of irradiation on antibotulinal efficacy of nitrite. D. C. Traisman. Zentrabl. F. Intl J. Thatcher... Sofos.. P.. M. T. E. Hyg. Plant trials of bacon made with lactic acid bacteria. p.. C. Barrière. botulinum by nitrite — is a replacement available? In Proceedings of the 31st Annual Reciprocal. Sofos. and Vinton. F. R. 5:148.. N. and Szulc. 10:283. Sodium nitrate. Spencer. Tompkin. Tanner. E. O. Appl. 43:450. Walter. J. 1941. M. Fish Res. 1966. 34:229. Fate of pathogens inoculated onto vacuum-packaged sliced hams to simulate contamination during packaging. Fish Res. J. Busta.. Clostridium botulinum control by sodium nitrite and sorbic acid in various meat and soy protein formulations.. and Paquette. 85:S469. Tanaka.. F. C. Talon. Botulinum toxin formation in liver sausage. Zentralbl. E. N. .. Robach. L. J. N. J... J. L. J. R.. G. F. J. and Allen. Food. C. 2002. Stumbo. Bacteriological studies relating to thermal processing of canned meats. H. 1978. II.. Chartier. C. Busta. D. Ontario. 135.

Environ.. B. Van Roon. Antibotulinal role of isoascorbate in cured meat. Christiansen. Utrecht. and Shaparis. Eds. L. B. S. Evaluation of ironbinding compounds as inhibitors of gas and toxin production by Clostridium botulinum in ground pork. F. Fleischwirtschaft 59:542. Food Sci. J. R. M. Environ. 35:59... Mahoney. 13:521. R. Christiansen. A. B. 1978d. and Chou. A. A. and Shaparis. N. Meat and Poultry Microbiology. p. 1971.. B. S. Christiansen. W. F. 2000. and Krol. Antonie Van Leeuwenhoek 16:269. Sci. sodium nitrite. R.. 1986. P. 48:990. C. B. AVI Publishing. In Proceedings of the International Symposium on Nitrite in Meat Products. Sci. Food Sci. B. and Lin. S. Bacteriological studies relating to thermal processing of canned meats. The effect of nitrite. M. W. 43:1368. 42:1046. R. and Shaparis. Drukkerij Elinkwij BV. Mikrobiol. 37:351.Nitrite 235 Tompkin. Turanta. Growth. R. Environ. Iron and the antibotulinal efficacy of nitrite. Isoascorbate level and botulinal inhibition in perishable canned cured meat. 1950. R. T. M. J. P. Effect of prior refrigeration on botulinal outgrowth in perishable canned cured meat when temperature abused. 1978a. B. Enhancing nitrite inhibition of Clostridium botulinum with isoascorbate in perishable canned cured meat. Food Agric. Wagner. B. Microbiol. L. B. pigment production and protease activity of Monascus purpureus as affected by salt.. Martin. A. Appl. C. A.. Microbiol. L. and Dutson.. Variation in inhibition of C. Y. Chicago. B. T. J. Appl.. 1978b. Causes of variation in botulinal inhibition in perishable canned cured meat. botulinum by nitrite in perishable canned comminuted cured meat. J. Verhoeven. Microbiol. sodium chloride. B. Christiansen. L. Ambrosino. C. 48:1445–1451.. Christiansen. Eds. In Advances in Meat Research. B. and Shaparis. Appl. Injury. and Leistner. K.. Vinton. Westport. 35:863.. 35:886. Chem. Tompkin. J. and Ünlütürk. Volume 2. 1979a. N. 26:833. B. Tsai. Y. 1978c. Effect of reaction products from nitrite on Clostridium sporogenes. 1974.. 1979b. J. H. L. B. Technol. and sodium nitrite on enterotoxin A production. Wageningen. Microbiol. B. 12:173. Institute of American Meat Packers. 71:10.. Inhibitors in cooked meat products. and Busta.. J. 1947. Environ. and Post. 1993. 1977. B. and Shaparis. E. D. The effect of sodium nitrite and pH on the growth of Salmonella typhimurium and Staphylococcus aureus. P. N. Tseng. 1996. VII. H. 1930. B. and Gross. Fleischwirtschaft 54:1368. . N. Effect of pH. R. J. L. S. Tompkin. 44:1147. R. F. Tompkin. L. Tompkin. p. Christiansen. Van Roon. 117. 1983. L. 1979b... Appl. Microbiology of ready-to-eat meat products. Effect of substrate upon thermal resistance of spores. Lebensmittel 13:167. The Netherlands.. J. Turanta. F. J. N. and Shaparis.. Tucker. Food Technol. 61:95. R. Appl. Microbiol. Vahabzadeh. Christiansen.. 89. A. L. Tompkin. and Shaparis. Food Res... Antibotulinal efficacy of sulfur dioxide in meat. Tinbergen.. Appl. 39:1096. N. A. Environ. polyphosphate and various sugars. F. 1978e. Microbiol. F. J. C. M. Effect of reaction products from nitrite on Clostridium sporogenes in heated cured meats... W. Christiansen. 88:31. Appl. K. F. A. Effect of sodium acid pyrophosphate in combination with sodium nitrite or sodium nitrite/potassium sorbate on Clostridium botulinum growth and toxin production in beef/pork frankfurter emulsions. N.. J. 1979a.. and Shaparis. CT. On a spore-forming bacterium causing swelling of cans containing cured hams. B.. The heat resistance of lactobacilli at different aw values. inhibition and inactivation of Escherichia coli O157:H7 by potassium sorbate and sodium nitrite as affected by pH and temperature. B. Tompkin.. L.. R. 1980. Studies on Clostridium putrificum and Clostridium putrefaciens.. Vrchlabsky. A. B. S.. 1973. Food Sci. Microbiol. J. P. B. Tompkin. S. 1983. and Shaparis. Cornforth. N. K. Pearson. R. C. Van Roon. and Stozek. Christiansen. Tompkin. Chen. 1991. A. Tompkin. A. garlic and starter culture on the survival of Salmonella typhimurium in Turkish soudjuk. N. Food Agric. Food Sci. S. Collinge. Pudoc. R. B. Food Sci. and Ünlütürk. The effect of iron on botulinal inhibition in perishable canned cured meat. Tompkin..

Meat Ind. C. P. Woods. p. 1988. E.. J. J. M. R. Yesair. Environ. Wood. Chicago. 1967. Z... West. 125:399.. Chem. J. G. Appl. G.. and Chou. J. Appl. O. Vet. Microbiol. F. L. Zaika. H. Effect of sodium nitrite on the stability of pasteurized canned meat. Yarbrough. 35:13–26. 1978. L. In Toxicological Evaluation of Certain Food Additives. 1976.. Woods. 12:133. Inst. J. P. Carpenter. Wolf. 1968. Strahl. 92.. 74:551. Yu. Kim. Intl.. and Eagon. The involvement of nitric oxide in the inhibition of the phosphoroclastic system in Clostridium sporogenes by sodium nitrite. Convention Canner 94:89. Wasserman.236 Antimicrobials in Food Warnecke. initial pH. E. and Gibbs. Bacteriol. 34:165. B. J. P.. G.. Rake. C. G. Bull. J. Heme-dependent catalase activity of lactobacilli. J. Soc. Geneva. in vacuum packaged meat.. Fate of Escherichia coli O157:H7 in Chinese-style sausage during the drying step of the manufacturing process as affected by the drying condition and curing agent. 1964. Microbiol. 10:323. Pfähler. A study of Clostridium botulinum type E. M. R. Food Prot. P. 14:15. p. H. L. Model for the combined effects of temperature. J. vacuum-packed ham in relation to pH of the product. 1991. A note on the effect of nitrite inhibition on the metabolism of Clostridium botulinum. and of intracellular enzymes. C. 1942. Kossakowska. Hydrogen peroxide formation and catalase activity in the lactic acid bacteria. Yu. Geneva. . A. Moulden. L. Food Sci. W. C. Emulsifiers and Thickening Agents. Anon. L. 37:785. sodium chloride and sodium nitrite concentrations on anaerobic growth of Shigella flexneri. 1981. C. and Huhtanen. K. 1990. Food Microbiol. 1994. M. American Meat Science Association National Live Stock and Meat Board. 149:220. 265. 1991.. Proceedings. R. Microbiol. Sci. Zaika. Food Agric. 1980. 39:831. Arendt. J. Gen. C. Effect of sodium nitrite on growth of Shigella flexneri. Antimicrobials. potassium and sodium salts. W. 1982. H. Volume 2. P.. 1974. Heme-dependent and heme-independendent nitrite reduction by lactic acid bacteria results in different N-containing products. A. J. and Buchanan. and Cameron. Phillips. Intl. 52:109. and Ford. 86. Effect of curing salts on the growth of hemorrhagic Escherichia coli O157:H7 in ground pork at different temperatures. Inhibitive effect of curing agents on anaerobic spores.. World Health Organization. and Saffle. In 26th European Meeting of Meat Research Workers. J. J. L.. A. Antioxidants. J. G. U. F. Chinese Agric. J. 1980. potassium and sodium salts. Weimer. World Health Organization. Gen Microbiol. Nitrate. Food Technol. Wojton. Effect of hematin on the activities of nitrite reductase and catalase in lactobacilli. Food Microbiol.. N. and Wood. J. A. Whittenbury. and Hammes. and Moczybroda. In Toxicological Evaluation of Some Food Additives Including Anticaking Agents. Nitrosamines and the inhibition of clostridia in medium heated with sodium nitrite. and Chou. A. Food Microbiol.. L. and Hammes. Intl J. 1997. M. B. p. 1972. J. Arch. J. Some observations on the bacterial growth in sliced.. E.. J. but not of group translocation. R. 23:345. Bacterial inhibitory effects of nitrite: Inhibition of active transport.. 21:433. W. J. Ryglewicz. Wolf. Meat Ind. Meisel. Cured meats need built-in safety factor. Nitrate. Pulawy 22:38. Wolf. L. and Hammes. Zeuthen. 1996. L. L. C. E. A. J.. 54:424.

.....................................................................................................................................................................................................................................................241 Mode of Action...................................................249 Natural Cheese ................................257 Fresh Pasteurized Chilled Soups..............................................7 CONTENTS Nisin Linda V....................................................................................................................................248 Dairy Applications ........................................................................................................................................................................................................................................................................................................240 Antimicrobial Spectrum.....................................................................................................................................................257 Cooked Potato Products ....................................................................................................................................................................255 Fruit Juice................250 Pasteurized Milk and Other Dairy Products........................................................................................................................................................................................................................................240 Units of Measurement and Assay...............................................................249 Pasteurized Processed Cheese.........................................................................................................257 Genetics ...........................................................................................................................................................................................................................................................................................259 References ...............262 237 ..............256 Salad Dressings ...................................................................................................................................258 Toxicity.....................................................................................................................................................................................................................................239 Antimicrobial Activity ................................................................................... Thomas and Joss Delves-Broughton Chemical and Physical Properties ............................................................................................................255 Beverages ..................253 Raw Meat ............................................................259 Legal Status..................................................................256 Crumpets.................257 Fresh Pizza .......................................................................246 Factors Affecting Antimicrobial Activity .........................................................................246 Applications .......................................................252 Meat Applications .....................................................................................................................................................................253 Cooked Meat Products ............................255 Alcoholic Beverages.................................................................................................................................................257 Soy and Vegetarian Products....................................256 Miscellaneous Applications .....................................................................................................................244 Vegetative Cells ............................................................................................................................................244 Spores .......................254 Fish and Shellfish Applications .....................................................................................................................256 Pasteurized Liquid Egg .......................251 Canned Foods.................................................

. several reviews have been published. We recommend that nisin (and other bacteriocins) should be considered as antimicrobial peptides and not referred to as antibiotics. passaging of bacteria in media containing sublethal concentrations of nisin did not alter the sensitivity of the test organisms to therapeutic antibiotics and other chemotherapeutic drugs (Hossack et al. the first commercial extract of nisin was developed by Aplin & Barrett Ltd. Nisin was also shown to be present in farmhouse cheese (Chevalier et al. 1992). the bacteriocin nisin has now been in use as a food preservative for almost 50 years (McLintock et al. 1962). (2000). Food safety is a major concern internationally. Delves-Broughton (1990). When its potential as a food preservative was realized (Hirsch et al. Nisin has been studied by many research groups. Although there is no doubt that therapeutic drugs associated with medical usage should not be used in food applications. being active against a wide range of Grampositive bacteria. Thomas et al. 1933). (1996). Delves-Broughton et al. Nisin is not used therapeutically in human or veterinary medicine nor is it used as an animal feed additive or for growth promotion. 1983). It remains the only bacteriocin allowed in food as an added preservative. 1957). Therefore. de Vuyst and Vandamme (1994).238 Antimicrobials in Food Following its discovery in the late 1920s. The characterization of the molecule was first conducted by Mattick and Hirsch (1947). Hurst and Hoover (1993). and Thomas and Delves-Broughton (2001). It has a much broader spectrum than most other bacteriocins. excellent safety record — and its designation as a natural food additive. (now part of Danisco) in 1957. Hurst. Ray (1992). Toxicity testing results demonstrating its safety for human consumption were published in 1962 (Frazer et al. Generally antibiotics are secondary metabolites. Since this group of organisms includes heat-resistant bacteria. but these attempts failed because of nisin’s lack of activity against Gram-negative bacteria and its rapid digestion and breakdown within the body. Nisin was approved for use in food in 1969 by the Joint Food and Agriculture Organization/World Health Organization (FAO/WHO) Committee on Food Additives and was awarded generally recognized as safe (GRAS) status in the United States in 1988 (FDA. Consumers do not want high levels of chemical food preservatives in their food and yet they increasingly look for convenient.1983). Nisin represents a safe. the producer organism was classified as Lancefield serologic group N Streptococcus. 1993. most notably those by Hurst (1981. Various studies have shown that nisin will not contribute to antibiotic drug resistance. long-life food that is of high quality but that is not severely processed (Gould. 1951).” At that time. both academic and industrial.. Nisin is a primary metabolite produced by a process involving ribosomal transcription and translation.. who coined its name from the term “N inhibitory substance. One of the earliest reports about “inhibitory streptococci” concerned milk in which cheese starter organisms failed to develop (Whitehead and Riddet. Nisin is a low-molecular-weight polypeptide produced by the bacterial dairy starter culture Lactococcus lactis subspecies lactis.. Delves-Broughton and Gasson (1994). Nisaplin (which was nisin A) is the first commercial extract of nisin.. There has been misplaced concern expressed in some quarters resulting from a confusion arising from references to nisin as an antibiotic (Hansen. Nisin-producing lactococci occur naturally in milk. 1981). long history of successful usage as a food preservative. Hara et al. 1962. There are clear differences between this bacteriocin and pharmaceutical antibiotics (Cleveland and Tchikindas. developed between 1962 and 1965. 2001). because of its many positive attributes — broad-spectrum antimicrobial activity.. natural method of preservation that can be used as part of a combined preservative system to increase the safety and shelf life of food. For instance. there were attempts to use nisin as a medical drug. Before its development as a food preservative. Increasing mass production and the longer distribution chains operating in many countries have led to an increase in foodborne illnesses with associated fatalities. nisin has become a widely used food preservative because it can maintain or even extend the shelf life of heat-treated foods and contribute to their safety. 1952). nisin is not one of these agents. 1988). consequently nisin can be present naturally at low levels in both soured milk and cheese. Apart from numerous research papers on nisin. it is likely that nisin or substances similar to it have been consumed for a significant length of time without apparent ill effects.

1964). 1994). Nisaplin® produced by Danisco). Nisin will be less effective in a processed food that has a high spore count. Bailey and Hurst. 1973. is shown in Figure 7. molecules characterized by their unusual amino acids. Gross and Morell (1971) first elucidated the unusual structure of nisin. ALA-S-ALA.. 1976. β-methyllanthionine. 1971. with its distinctive five internal ring structures formed by the disulphide bridges.. but this activity was highly dependent on pH and nisin concentration (Bani-Jaber et al. ABU.1. 1994. It can form dimers or even oligomers. 1985). The natural variant nisin Z differs by a substitution of histidine for asparagine at position 27 (Mulders et al. . and extended shelf life can be achieved with a combination of nisin and a reduced heat treatment. Thermally injured spores are more sensitive to nisin. Methods for its concentration and purification have been described (Cheeseman and Berridge. as well as amino butyric acid (Abu). The effectiveness of nisin is also dependent on the bacterial load (Scott and Taylor. Nisin contains the thioether amino acids lanthionine (Ala-S-Ala) and β-methyllanthionine (Abu-S-Ala). Nisin is available commercially (e. 1991).1 The structure of nisin. It is classified as a Class I bacteriocin. It does not contain aspartate or glutamate. Kupke and Gotz. and molds. It is a cationic molecule because of its three lysines and either one histidine in nisin Z or two in nisin A (de Vuyst and Vandamme. or microorganisms that should be killed by normal pasteurization treatments and that are only present in such foods if the heat treatment is inadequate or if postprocessing contamination has occurred. ABU-S-ALA. The structure of nisin A. Nisin has a flexible. the N-terminal contains several hydrophobic residues. which is a 34-amino acid polypeptide with a molecular mass of 3510 Daltons. and its synthetic production has been accomplished (Fukase et al. Wilimowska-Pelc et al.Nisin 239 Regulatory authorities and food producers acknowledge and appreciate that nisin does not hide poor manufacturing practice. nisin offers the possibility (within safety limits) of energy saving and consequent cost cutting. aminobutyric acid. It is amphipathic in character.. 1957. DHB. 2000). The net charge of nisin is pH-dependent. this would usually be the result of poor-quality ingredients (Gibbs and Hurst. also known as β-methyldehydroalanine). 1996). 1981b)... whereas the C-terminal is more hydrophilic. yeasts. Lipinska et al. which is determined by its internal thioether rings.. dehydroalanine. Lee and Kim. Nisin has been shown to have emulsifying activity in a comparative study with Tween80 (a common nonionic food emulsifier) and β-casein (a food emulsion stabilizer). Nisin is not effective against Gram-negative bacteria. lanthionine. and dehydrobutyrine (Dhb. 1988). The structure and synthesis of nisin have been reviewed (de Vuyst and Vandamme. DHA. Apart from the advantages already mentioned. CHEMICAL AND PHYSICAL PROPERTIES The polypeptide can be prepared from culture fluids or the cells of the producer organism.g. dehydroalanine (Dha). three-dimensional structure. dehydrobutyrine (β-methyldehydroalanine). DH ILE H2N ILE DHB ALA S S LEU ALA ABU ALA GLY LEU MET GLY ALA S S LYS AS MET ALA ALA ABU SER ALA HIS ABU ILE S HIS VAL DH LYS ALA LYS ABU PRO GLY COOH FIGURE 7.

In triangle taste trials conducted by Danisco. The nisin preparation Nisaplin® was developed by Aplin & Barrett (now Danisco) between 1962 and 1965. An international nisin reference preparation was later established by the WHO Committee on Biological Standardisation (Anonymous. 1970). is the same value as the Reading Unit. Delves-Broughton et al. The current term. The horizontal agar diffusion test. suggesting that the molecule is protected to some extent by food components (Delves-Broughton. 1990. ANTIMICROBIAL ACTIVITY UNITS OF MEASUREMENT AND ASSAY The activity unit for nisin was first defined as a Reading Unit: the minimum amount of nisin required to inhibit the growth of one cell of Streptococcus agalactiae in 1 ml of broth (Tramer and Fowler.25% biologically active nisin. Optimum heat stability occurs at pH 3 to 3.240 Antimicrobials in Food Nisin as a powder is extremely stable if stored at temperatures not exceeding 25°C away from direct sunlight in a sealed container allowing no moisture ingress. 1 µg/g pure nisin is equivalent to 40 IU/g or 40 mg Nisaplin®/kg. Nisin is. It is recommended that such solutions should not exceed a maximum concentration of 250 µg/ml.025 mg/ml (equivalent to 1000 mg/kg Nisaplin®). 1969).3 to 4. heating for 3 minutes at 250°F (121°C) destroyed 25% to 50% of the added nisin.25 mg/ml at pH 8.5 (Davies et al. A similar degree of destruction was reported for highly acid foods having pH values of 3. The specifications for the purity and identity of nisin were set in 1969 by the FAO/WHO Joint Expert Committee on Food Additives (Anonymous. and contains 2. (1965). Their results show that in low-acid foods at pH 6. International Unit. research in Danisco laboratories has shown that care must be taken when preparing high-concentration nisin solutions from commercial preparations such as Nisaplin®. is the most widely used nisin assay. successfully used in high-pH/heat-treated food products such as canned vegetables and pasteurized liquid egg. the levels of pure nisin should be multiplied by 40. Pure nisin was deemed too potent for use in food.5. IU/g). The Gram-positive bacterium Micrococcus luteus is used as an indicator organism because it is very nisin-sensitive and grows well overnight at 30°C. For example. At high pH. To convert these units to equivalent Nisaplin® levels (mg/L. 1992). producing clearly distinguished and measurable zones of inhibition in the presence of nisin. dropping to 1. volunteers could not detect 200 mg/L Nisaplin® in mineral water (equivalent to 5 µg pure nisin/g). however. and nisin activity was then defined in International Units.5. otherwise a loss of activity may occur as a result of salt precipitation or filtering. 1964). Solubility in foods is never a problem because nisin levels are always very low. and this bioassay method contains . 1966). Nisin has no significant taste and imparts no negative taste to foods in which it is used. The effect of processing temperature and pH on the stability of nisin in different foods was studied by Heinemann et al.5 mg/ml at pH 6 and 0. with levels of usage in food rarely above 0. and a specification was set for commercial nisin concentrates containing at least 2. Liu and Hansen (1990) reported nisin solubility at pH 2.. However. Both the solubility and stability of nisin in solution are affected by pH. This has a standard potency of 1 million International Units per g. This was contrary to previous work by Tramer (1964. equivalent to 1 µg of this preparation. Liu and Hansen (1990) suggested that the Dha and Dhb residues become more susceptible to modification as a result of the presence of nucleophiles. Nisin is extracted from the test food by an acid-heat treatment. mg/kg) or to International (Reading) Units (IU/ml.5% nisin A with the remainder made up of added salts and milk solids derived from the fermentation of a modified milk medium by L lactis subspecies lactis.9.1 to 6.2 as approximately 56 mg/ml. 1998). These may be intended as stock solutions for the preparation of test dilutions in smallscale laboratory experiments. first developed by Tramer and Fowler (1964) and Fowler et al. In this chapter nisin is expressed as levels of pure nisin (µg/ml or µg/g). (1975).

Rose et al. capable of transducing the signal into detectable bioluminescence (Wahlstrom and Saris. unpublished results). or alcoholic beverages (e. 1987).1. 2001). The meat spoilage organism Brochothrix thermosphacta. nisin-sensitive organisms relevant to food are shown in Table 7. Nisin has a stronger bactericidal effect against energized cells. ANTIMICROBIAL SPECTRUM Nisin has a broad spectrum of activity against Gram-positive bacteria.5 µg/ml prevented the growth of 103 cells/ml (Thomas and Delves-Broughton. A relatively new species.Nisin 241 numerous control procedures to ensure nisin activity is not confused with other antimicrobial agents that may be present in the food. Nisin is also effective against another food-associated pathogen. detectable by bioluminescence (Waites and Ogden. Bacillus sporothermodurans. Another important group of nisin-sensitive bacteria is lactic acid bacteria. Such heat treatments usually eliminate all vegetative cells so that such foods are vulnerable to spoilage only by these surviving spore-forming bacteria. is extremely nisin sensitive. Joosten and Nunez. This species is extremely nisin sensitive. An incorrect measurement of activity would be obtained by assaying nisin Z samples against the nisin A FAO/WHO standard used for calibration in the horizontal agar diffusion assay. again most probably because of detection of inactive degraded nisin or possibly biologically active nisin that was bound within the food (Bouksaim et al. 1996). Important pathogens in this group include Bacillus cereus and Clostridium botulinum. Wolf and Gibbons. For example. as its name suggests. 1999). was first described in 1985 and. Figure 7. 1989a). the Gram-positive endospore-forming genera that include Bacillus and Clostridium. Hurst (1981) reported that lower nisin levels prevent spore outgrowth compared with the levels required for inhibition of vegetative cells.. first described in 1951. wine and beer) that are often not heat processed. The growth of 104 cfu/ml of four different strains of this species was controlled for 7 days at 37°C by 0.2 shows the activity of nisin against three different strains of Bacillus. with considerable variation being observed even between strains of the same species (Gupta and Prasad. because nisin Z diffuses more rapidly than nisin A. enabling it to be used as a preservative in pasteurized or heat-treated foods that are not fully sterilized. 1995. molds. 1991. An enzyme-linked immunosorbent assay (ELISA) has been developed using polyclonal antiserum raised in sheep to nisin A and conjugated to horseradish peroxidase (Falahee et al. The outcome of nisin activity can also vary from achieving total kill of the cells to a growth inhibitory effect (DelvesBroughton et al. lactis construct strain sensitive to nisin has been reported. In normal circumstances nisin does not significantly inhibit yeasts. which was thought to result from the test measuring a partially degraded nisin molecule that had lost biological activity. ensuring that nondegraded nisin only would be measured.. Listeria monocytogenes.g. It is active against both bacterial cells and their heat-resistant spores. or Gram-negative bacteria. Falahee and Adams. A rapid nisin assay method has been described that measures nisin activity by adenosine triphosphate (ATP) release from Lactobacillus casei as a result of exposure to nisin. (1999) described detection of nisin by matrix-assisted laser/desorption time-offlight spectrometry. Comparative studies of processed cheese assayed by ELISA and the horizontal agar diffusion bioassay showed a lack of correlation (Aplin & Barrett Ltd. nisin at 0. sauces... An ELISA system using nisin Z polyclonal antibodies in rabbits also suffered from similar problems.. An L. this bacterium produces extremely heat-resistant spores capable of surviving ultra-high temperature (UHT) treatments. 1999). Its activity range encompasses. 1996).625 to 2.125 µg nisin/ml (Thomas and DelvesBroughton. whereas . 1990. There have been several reported improvements to this test (Rogers and Montville. This latter test does not measure biological activity. 2001). most importantly for its use as a food preservative. This could be corrected by developing an antibody that detected distal parts of the molecule. After 7 days at 25°C. Wide variation in nisin sensitivities has been reported. organisms that may cause spoilage of low pH food such as salad dressings. 1992).

licheniformis. Nisin activity against spores is predominantly bacteriostatic. Can grow at low pH. butyricum. Includes psychrotrophs. Aerobe/anaerobe. Heat-resistant spore-forming obligate anaerobes. Varied nisin sensitivity. luteus is indicator strain for the bioassay. Growth at low pH. Growth between 0°C–30°C. brevis. putrificum. stearothermophilus Brochothrix thermosphacta Clostridium Desulfotomaculum bifermentans. tertium. 2000.1 Gram-Positive Species Sensitive to Nisin Genus Alicyclobacillus acidoterrestris Species Description Heat-resistant spore former. acidresistant. pumilis. 1992). Spore forming. low water activity. Heat-sensitive spoilage organisms of meat. bulgaricus. sporogenes. pentosaceus inulinus aureus Source: From Thomas et al.. Cause spoilage. Aerobes/anaerobes. Gram-negative bacteria are resistant to nisin because the antimicrobial is unable to penetrate their complex cell wall to