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

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

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

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

other bacteria 172.1207. bacteria Yeasts. cooked meat. diacetates. Generally. wines 184. 184.1733 172. (1998). fermented foods Meats Cheese.3795 Various For meat products. dairy products Most foods.3640. wines Fruits. syrups. Title 9. molds Bakery products. Section 424.1081. lactates Lactoferrin Lysozyme Microbial Target Yeasts. cooked meat. 182. 184.1550.175. 184. a combination of chemical preservatives and other preservation methods is needed (Leistner. molds. dry sausage CFR Designation 184.1538. 182. beverages. propionates Sorbic acid.145 Molds Yeasts. Extensive reviews on natural antimicrobials may be found in Dillon and Board (1994).S. methyl.170. condiments. GRN 000065 Nitrite.1221.1490. The target pathogen or spoilage microorganisms must be identified first. dairy products.130 184.1021. casings for frankfurters. Sofos et al. 184. margarine Beverages Meats. Naidu (2000). 2002 to foods (Tables 1.3225. confections. potato products. 172. casings for frankfurters. 184. Source: Davidson and Harrison. 184.177 184. food antimicrobials permitted by USDA Food Safety and Inspection Service are listed in the Code of Federal Regulations. bacteria (Gram positive) 172.1768 GRAS Notice No. other bacteria Natamycin Nisin Molds Clostridium botulinum. baked goods.1061.22.160.Food Antimicrobials — An Introduction 3 TABLE 1.21 and 424.2). Food and Drug Administration) Food Antimicrobialsa (Title 21 of the Code of Federal Regulations) Compound(s) Acetic acid. 184. dehydroacetic acid Benzoic acid. molds Yeasts Bacteria Bacteria Clostridium botulinum. benzoates Dimethyl dicarbonate Lactic acid. SELECTION OF ANTIMICROBIALS It is not an easy process to select the appropriate preservation system for a particular food product. heptyl) of p-hydroxybenzoic acid) Propionic acid. GRN 000067 184. sorbates Sulfites a Clostridium botulinum Yeasts. and then the possible preservation systems must be evaluated via model studies and studies in the food product in question. fruit products. 182. 172. sauces Beverages. 172. 172. nitrate Parabens (alkyl esters (propyl.6197.1721. 182. GRAS Notice No. 184.1784 182. GRAS Notice No. 2000). 172.1754.3089.1 and 1. and Davidson and Zivanovic (2003). GRN 000064 Yeasts. and poultry products Cured meats Beverages. fruit products.1005. meats.1670.1185. and poultry products Cheese Cheese. acetates. 184. bacteria Primary Food Applications Baked goods.1 Traditional or Regulatory-Approved (U.133 184.1639. .155 184. fats/oils. molds. 184.

and the type of preservation or processing and storage systems used. or strains of microorganism. Few chemicals have the ability to inhibit several different types. the chemical properties of the antimicrobial. the physicochemical properties and composition of the food product in question. The antimicrobial spectrum should involve an evaluation of the compound against various types of microorganisms (e. which have the ability to screen the antimicrobials because of the outer membrane lipopolysaccharide layer.. molds) and forms of those microorganisms (vegetative cells vs. bacteria. does growth in a synthetic . negative) can have dramatic influences on apparent activity. Source: Verbruggen. Selection of the proper antimicrobial depends on several primary factors. species. ANTIMICROBIAL SPECTRUM The initial selection of the antimicrobial is normally based on an assessment of the overall microbial spectrum of the chemical in question. the activity of very hydrophobic antimicrobials may be limited against Gram-negative bacteria.g. yeasts. The antimicrobial spectrum of a compound is generally determined by following the growth of organisms in the presence of various concentrations of the antimicrobial. Even species. Each of these factors is discussed in detail. Appropriate methods for in vitro evaluation of the activity of food antimicrobials are described in Chapter 21.2 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. For example. including the spectrum of antimicrobial activity. a broad spectrum of activity. spores).” Seldom. however. is not easy to achieve. Quite often. and Gram reaction (positive vs. 2002. strain. “Methods for Activity of Assay and Evaluation of Results. although desired.4 Antimicrobials in Food TABLE 1.

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

Once inside the cell. there are very few compounds that are effective at low concentrations. Because protons generated by the organic acid inside the cell must be extruded using energy in the form of adenosine triphosphate (ATP).5 or greater.79 4. some Gram-positive bacteria are more susceptible to certain antimicrobials in the presence of chelators including EDTA. A lowering of the water activity can select for those organisms that have the ability to survive and/or grow at lower water activity. molds survive and yeasts can grow at a lower water activity than bacteria. thus indicating the need for a different antimicrobial. thus requiring an antimicrobial capable of limiting the growth and activity of these organisms.6). nisin. Certain preservation processes may result in the need to control sporeformers that have the ability to survive the heating process.6 Antimicrobials in Food TABLE 1. Protons generated from intracellular dissociation of the organic acid acidify the cytoplasm and must be extruded to the exterior. organic acids can penetrate the cell membrane lipid bilayer more easily.0 (Table 1. organic acids function at low concentrations only in high-acid foods (generally less than pH 4. For food products with a pH of 5. where microbial growth occurs (Branen et al. pH of the food can result in ionization of an antimicrobial and a change in activity. In the undissociated form.19 3. 1995.3 pKa of Regulatory-Approved Organic Acids Compound or Group of Compounds Acetic acid Benzoic acid Lactic acid Propionic acid Sorbic acid pKa 4.75 into the lipid areas of the food and away from the water phase.. They expand the activity of certain antimicrobials (e. which exists in the majority only at a pH below the pKa of the compound.g. the constant influx of these protons will eventually deplete cellular energy. Vacuum or modified atmosphere packaging results in . It is theorized that chelators may destabilize the LPS layer of the outer cell membrane of the Gram-negative bacteria. Packaging can directly alter the environment of the food and thus influence the overall growth pattern and type of organisms in the food. allowing the antimicrobials access to the inner cell membrane. For example. Branen and Davidson. Bacteria maintain internal pH near neutrality to prevent conformational changes to the cell structural proteins. the acid dissociates because the cell interior has a higher pH than the exterior. Eklund (1983) demonstrated that the anion does contribute slightly to antimicrobial activity.75 4. Rico-Munoz Davidson. Refrigeration generally selects for psychrotrophic Gram-negative microorganisms. which means their maximum activity will be in high-acid foods. lysozyme) to include Gram-negative bacteria. In addition. Generally. Although the undissociated form of a weak acid has most of the antimicrobial activity. PROCESS FACTORS The type of preservation process used in conjunction with antimicrobials has a significant influence on the type and level of antimicrobial needed. 1983). Chelating compounds such as ethylenediamine tetraacetic acid (EDTA) or its salts have a potentiating effect on some antimicrobials. 2004).87 4.5 to 4. and phospholipids. This is because the most effective antimicrobial form is the undissociated acid. All regulatory-approved organic acids used as antimicrobials have pKa values less than 5.. nucleic acids. which are not normally inhibited by the compounds alone (Cutter and Siragusa. 1980.3). enzymes.

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

This would involve very expensive and time-consuming toxicologic testing. or texture. SENSORY EFFECTS Another major factor that needs to be addressed when applying antimicrobials is their potential impact on the sensory characteristics of a food.S. 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. LABELING One of the alleged attractions of naturally occurring antimicrobials is their reduced negative impact on the labeling of foods. . If antimicrobials are to be used exclusively as inhibitors of pathogens in food products. In many cases. 21CFR 184. just as thermal processes need validation. For that reason. In addition. Obviously. This is because. The reason for these assays is that various conditions of process or storage could reduce the effectiveness of the compound. Code of Federal Regulations. In addition to adverse effects on flavor. For example. 21CFR 184. of course. 2003). they should be nonallergenic or bind or destroy important nutrients in a food product (Harlander. they may need to be approved as food additives. Code of Federal Regulations. assays need to be developed that evaluate the activity of these compounds against the pathogen they are designed to kill. odor. If a product is simply an “extract of” a commonly consumed plant or animal food product.8 Antimicrobials in Food common potential natural antimicrobials such as spice extracts. there are methods for the determination of the activity of lysozyme (U. the compound would probably have to be listed using a chemical name on a food label. 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. it is known that peptides. less purification may be better.1550) and nisin (U.S. 1993). an additional 2 or 3 days of shelf life can significantly help to offset the cost of using an antimicrobial. are susceptible to inactivation by enzymes in foods. Finally. such as nisin. there are no other activity assays specified or required for food antimicrobials. so should there be validation for the activity of food antimicrobials. Many antimicrobials must be used at high concentrations to achieve activity against target microorganisms. Therefore.1538). it is much less likely to require complex regulatory approval for use (Davidson and Zivanovic. although spices have been consumed for centuries. In the United States. there are few standardized methods for validation of the activity of regulatory-approved food antimicrobials. compounds that negatively affect flavor and odor or contribute inappropriate flavors and odors would be unacceptable. would defeat the purpose of using a natural compound. ACTIVITY VALIDATION METHODS Currently. Extensive studies at a pilot-plant level are necessary on any food additive to prove its overall usefulness. The efficiency of these compounds can be extremely important in determining the overall economics of their use. This is only possible if the product from which the extract is taken is known to be nontoxic. 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. they are not normally consumed in the concentrations necessary to achieve antimicrobial activity. This. Consumers are reportedly concerned about the presence of synthetic chemicals in their foods and would prefer natural compounds.

and allyl isothiocyanate (AIT). Buchanan and Edelson.Food Antimicrobials — An Introduction 9 RESISTANCE DEVELOPMENT Because the activity spectra are often different for each antimicrobial. The information should also serve as a basis for selection of any new antimicrobial developed in the future. however. preservatives will be needed. carvacrol. heat. regulators. phenolic compounds may inhibit certain Gram-positive food-poisoning bacteria. In addition. One should be cautious. starvation. it has been demonstrated that some bacterial pathogens may develop a tolerance or adaptation to organic acids following prior exposure to low pH. favorable conditions can be created for growth and spoilage by these latter organisms. natural antimicrobials will be increasingly looked on as adjuncts in hurdle technology and used with milder nonsterilizing. Although this increased resistance may be a problem in application of organic acids for controlling pathogens. 1999). It is hoped through the information presented in the following chapters that food scientists. 2002). The global economy in which we live results in foods being transported throughout the world. 1995. certain compounds. the microflora contaminating a food product significantly influences the choice of the antimicrobial needed.. not to select an antimicrobial solely according to its ability to control the predominant microorganism present. The future of research in the area of food antimicrobials will likely be on two fronts. For example. For example. Microorganisms exposed to a stress may become more resistant and have enhanced survival to subsequent stresses (Foster. First is the expansion of information on the antimicrobial spectrum of natural antimicrobials. but because of the reduced activity against Gram-negative bacteria. Attaining synergistic activity with antimicrobial combinations requires that the components have different mechanisms. FUTURE OF ANTIMICROBIALS Antimicrobials will undoubtedly continue to be needed to provide the food supply that will be demanded in the future. If foods are to arrive in the condition expected.g. are not compatible with certain foods. selecting antimicrobials that control some genera but not others may result in selecting for and creating favorable conditions for growth of other organisms. 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. 1999). cold. low pH/organic acids) on developed resistance of microorganisms to subsequent stressors. such as thymol. To more effectively apply antimicrobials so that synergistic activity is possible will require knowledge of the mechanisms of action of the compounds. Because of their specificity. . A second major area of research involves use of antimicrobials in combinations with each other and with traditional or novel processing methods. There has been much interest in the effect of environmental stress factors (e. 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. This research will be more focused on the appropriate use of natural antimicrobials or utilization of compounds in situations in which they are compatible. it has not been shown to occur in an actual food processing system (Davidson and Harrison. This will also require the development of uniform worldwide regulations regarding the use of these chemicals in food products. For example. 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. nonthermal processing methods such as high hydrostatic pressure or pulsed electric fields (Smid and Gorris.

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

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

1). 1992). oxidation of benzaldehyde. 18°C. Potassium benzoate and calcium benzoate have also been approved for use. and 74.2). Its presence appears to occur as a by-product of the microbial degradation of either hippuric acid or phenylalanine in these products (Figure 2. 1991).2 g dissolves in 100 ml water at 0°C. O OH Benzoic Acid O O− Na+ Sodium Benzoate FIGURE 2.6 mg/l. although their solubility in water is less than that of the sodium salt. pineapple. was found to have antimicrobial properties.1% and in neutral media at 0.1. It is soluble to a limited extent in water (0.1 Structures of benzoic acid and sodium benzoate... 1999). For this reason.. respectively). 1995).27. and 100°C. 66. and 2.18.1). 0. 1984. Teuber. 20°C.1) is a white granular or crystalline powder. Benzoic acid has also been identified as a major constituent in extracts of blackberries (Humpf and Schreier. Similar results were reported for fungi and yeasts (Cruess and Richert. Gabel (1921) was one of the first to demonstrate that benzoic acid was effective against bacteria in acid media at a level of 0. occurs in pure form as colorless or white needles or leaflets. 1994). In an extensive review. It is much more soluble in water than benzoic acid (62.8. MECHANISM OF ACTION It is the undissociated molecule of benzoic acid that is responsible for antimicrobial activity (Table 2. In four of the seven juices. Benzoic acid has also been identified as a natural by-product in culture filtrates of Lactobacillus plantarum (Niku-Paavola et al.3). Sodium benzoate (molecular weight 144. Orange. Sieber et al. Benzoic acid occurs naturally in several foods and commodities (Table 2. apricot. pear.0. p-hydroxybenzoic acid was detected at 0. . Several new types of low-molecular-mass compounds were also identified. it is preferred for use in many cases.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.. Benzoic acid (molecular weight 122. and a mixture of these. Naturally occurring nonflavonoid phenolic compounds. respectively).2 to 2. It accounted for approximately 16% of the growth inhibition of Saccharomyces bayanus and Pseudomonas fluorescens from ethanolic extracts of cranberries (Marwan and Nagel. peach. may also contribute to benzoic acid generation.2). apple. 1929). and of fresh tomatoes (Marlatt et al. including derivatives of benzoic acid. along with benzoic acid. This may be of significance in countries like Switzerland. 1986a). were used to characterize commercial fruit juices (Fernandez de Simon et al. depending on the variety (Abdullah et al. where benzoic acid has not been approved as a food additive (Sieber et al. and 75°C.2% but inactive in alkaline media. A third pathway. 1990).2 g dissolves in 100 ml water at 4°C. 1992). and grape juices were analyzed to establish phenolic profiles unique to each type of juice. of mushrooms. also called phenylformic acid or benzenecarboxylic acid.. (1995) surveyed and analyzed many types of cultured dairy products and cheeses for the natural occurrence of benzoic acid (Table 2. Yogurts have been found to contain natural levels of benzoic acid (Stijve and Hischenhuber. 1989.

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

Source: Adapted from Sieber et al.44 0. Macris (1975) observed a rapid uptake of benzoic acid in Saccharomyces cerevisiae.144 4. sucrose. Zygosaccharomyces bailii and Yarrowia lipolytica were the most resistant to the two preservatives at pH 5. and that the toxicity of sodium benzoate in solution was a result of the undissociated benzoic acid molecule. TABLE 2. The combined effects of pH and benzoate on the growth of these strains are presented in Table 2.5 59.14 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. As expected. Saturation was reached in about 2 minutes and remained constant thereafter. and sorbic and benzoic acids on the growth of 30 strains of food spoilage yeasts have been reported (Praphailong and Fleet.3 Effect of pH on the Dissociation of Benzoic Acid pH 3 4 5 6 7 pK Undissociated Acid (%) 93. NaCl.8 1.3 12. . Proteinaceous material was apparently involved in the uptake of this preservative.0. 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. that only the undissociated acid was antimicrobial.2 Biosynthesis of benzoic acid by lactic acid bacteria in fermented dairy products. Sorbate was more inhibitory than benzoate on the strains that were tested.4.19 Source: From Baird-Parker (1980). not to the pH itself. The strong dependence of uptake on pH was because of the relative distribution of undissociated and dissociated forms in solution. The undissociated form was the only one taken up by cells. 1997). increasing the pH of the culture medium decreased the effectiveness of this preservative. The effects of pH. The effect of temperature on the uptake was similar to that observed in enzymatic reactions. (1995).

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

1977. 1982). Chipley and Uraih. B. 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.5. 1978). such as benzoic acid. 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. 1960. 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.. Synthesis of messenger ribonucleic acid (mRNA) was critical for restoration of salt tolerance. They also stated that although both the undissociated and dissociated forms of these acids cause the intracellular pH to fall. enzymes involved in acetic acid metabolism and oxidative phosphorylation can be inhibited (Bosund. 1986). 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. growth inhibition is the result predominantly of the undissociated acid... coli have been reported (Salmond et al.0 as well as pH 6. yielding protons that acidify the alkaline interior of the cell. In Bacillus subtilis (Freese. 1962). The authors suggested that the potency of weak acids. More than 99% of the viable cells of Listeria monocytogenes were injured after exposure to a solution of 8. Acid-susceptible bacteria (Bacillus and Alicyclobacillus) and acid-resistant bacteria (Lactobacillus and Escherichia coli) were grown in media containing organic acids..0) for 1 hour (Buazzi and Marth. membrane transport of amino acids is inhibited. In yeast cells. One hypothesis is that they inhibit or kill microorganisms by interfering with the permeability of the microbial cell membrane. 1973). MICs were determined for each acid and used with principal components to produce models with good correlations. Gould. A pH-related increase in protein production was observed when cultures of E. 50% of the trimethylamineN-oxide reductase activity was inhibited by 1. 1989). 1992). coli. in many bacteria and yeasts. In resting cells of E. Reduction . as food preservatives is related to their capacity specifically to reduce the intracellular pH. benzoic acid or sodium benzoate can also inhibit specific enzyme systems within cells (Webb. 1980. benzoate addition resulted in an increased production of 33 proteins.16 Antimicrobials in Food 3.. The production of lipase by P. 1956. The inhibitory potency of this acid can be determined by its lipid/water partition coefficient. 1973).2 mM benzoic acid (Kruk and Lee. Uraih et al. Increased permeability of cell membranes during the course of benzoate injury was not observed. 1983. 2001). Eklund. Corlett and Brown. In addition to these mechanisms. 1980). Aflatoxin production by a toxigenic strain of Aspergillus flavus was greatly reduced by the presence of these compounds in synthetic media (Uraih and Chipley. Sheu et al. causing uncoupling of both substrate transport and oxidative phosphorylation from the electron transport system (Freese et al.. At pH 6. α-ketoglutarate and succinate dehydrogenases appear to be quite sensitive (Bosund.. 1989. 1980).. 1966). Benzoate also inhibits amino acid uptake in Penicillium chrysogenum (Hunter and Segel. Theoretical ATP consumption for proton pumping can be directly correlated with reduction in cell yield. 1997).3). Davidson. coli.5. such as benzoic. 1975. subtilis. its pK value (Table 2. Production of 12 of these was induced at pH 8. and the effectiveness of undissociated acids as preservatives has been noted for many years (Ingram et al. E. fluorescens was completely inhibited by sodium benzoate (Andersson et al. coli were grown in liquid media with or without 20 mM benzoate (Lambert et al. Eklund. resulting in nutritional starvation of cells (Gould et al.. For example. and Pseudomonas aeruginosa (Freese et al. Only those organic acids that are lipophilic. 1962). phosphofructokinase activity was inhibited (Francois et al. Duration of the lag phase can be predicted from the model. 1984). 1976. The effects of organic acid preservatives on the growth and intracellular pH of E.5% sodium benzoate (pH 7. In the citric acid cycle. 4. Similar effects have been found using a variety of microorganisms. 1980). show antimicrobial activity. 1973.. 1997).

1980).6. . with the greatest increase occurring when the medium contained 0. prior exposure to the preservative. and environment from which the microorganism was originally isolated. 4-dinitrophenol and cell envelope-associated enzymes of E. as an inhibitor of Damino acid oxidases (Quay and Massay. the use of benzoic acid or sodium benzoate as a food preservative has been limited to those products that are acid in nature. The effects of several preservatives and antimicrobial agents on aflatoxin B1 production by A. 1983).. Several factors interact to determine the MIC. Benzoate may also serve as a scavenger for free radicals (Harvath. however. Sodium benzoate was found to control both growth and aflatoxin production by Aspergillus parasiticus in liquid media (El-Gazzar and Marth. In benzoic acid-supplemented cultures. potassium sorbate. 1977. Currently. 1980). 1972.3). The scattering of MIC values was lower with potassium sorbate and benzoic acid and higher with sorbic acid and sodium benzoate. The results suggested that these preservatives blocked an enzymatic step late in the biosynthetic pathway of aflatoxin B1. as an inhibitor of passive anion transport (Lucas-Heron and Fontenaille.. Increasing the concentration of sodium benzoate increased the percentage of inhibition at the end of incubation (10 days). This might result in inhibition of membrane-bound enzymes as a secondary effect. and as an inhibitor of nitrosamine formation (Sung et al. 1987). 1980). ANTIMICROBIAL ACTIVITY SPECTRUM OF ACTION Because the quantity of undissociated acid decreases with increasing pH (Table 2. The sensitivity of 42 yeast cultures to sorbic and benzoic acids. 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.02% undissociated acid.5 and 2. 1974). composition of the growth medium. 1979). Yagi et al. The authors concluded that subinhibitory concentrations of these compounds may stimulate toxin production in some cases. temperature. 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. cerevisiae decreased the biomass and increased the specific oxygen uptake rate of cells (Verduyn et al.1% of the undissociated acid.. 1981). Addition of benzoate to chemostat cultures of S. Fusarium species were more sensitive to benzoic acid (210 to 420 µg/ml) than were Penicillium species (250 to 3000 µg/ml). Therefore. In general. 1992). including pH. With the previously mentioned results in mind. benzoic acid cannot be relied on to effectively preserve foods capable of supporting bacterial growth (Chichester and Tanner.3% sodium benzoate. Baird-Parker.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. genus and species of the microorganism in question. 1997). and fungi involved in food poisoning and food spoilage are given in Tables 2. and most yeasts and fungi are inhibited by 0. aflatoxin B1 production was higher than in controls. flavus have been reported (Bauer et al..1979). The average accumulation of mycelial dry weight was greater in the presence of benzoate than in its absence. and sodium benzoate was determined (Manganelli and Casolari. yeasts. these compounds are used primarily as antimycotic agents. as a weak inhibitor of poly(ADP ribose) polymerase (Oikawa et al. In liquid media. coli and Salmonella Enteritidis (Chipley. 1988). all treated cultures produced measurable levels of toxin 3 to 7 days later than controls. MICs for some of the bacteria. 1979). Food-poisoning and spore-forming bacteria are generally inhibited by 0. but many spoilage bacteria are much more resistant. Conversion could be prevented by addition of benzoic acid or sodium benzoate. Similar results were observed with 2. It has been reported to have a flower-inducing effect in plants (Watanabe and Takimoto.01% to 0..

8 Fungi 5.0 5.2–5.6 6.0 4.5 Antimicrobial Spectrum of Benzoic Acid against Selected Bacteria.5–5.0 4.6 (4°C) 5.0 4. cerevisiae Torulopsis sp. 1993). Russell (1991).6–4. Hansenula subpelliculosa Oospora lactis Pichia membranefaciens P.1 5.3–6. pastori Rhodotorula sp. (1999).5 5.0 5. Aspergillus parasiticus Aspergillus niger Byssochlamys nivea Chaetomonium globosum Cladosporium herbarum Mucor racemosus Penicillium sp.8 4.0 5.0 5. Source: Adapted from Chipley (1983.0 6. Listeria monocytogenes Micrococcus sp.0 4.8 4.0 5.0 5. and Steels et al.0 5. lentus Z.2–5. Pseudomonas sp.0–5. rouxii 4.3 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.0 3. Saccharomyces bayanus S.6 Yeasts 2.6–5.0 4.6 (21°C) 5. 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. 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).0–5. .0 6.0 2.0 4.5 4. Davidson and Juneja (1990). Pseudomonas aeruginosa Staphylococcus aureus Streptococcus sp.3 5. and Fungi Microorganisms pH Bacteria 6.6 6.0 3. Yeasts. Zygosaccharomyces bailii Z.0 MICa Bacillus cereus Escherichia coli Klebsiella pneumoniae Lactobacillus sp.0 6.18 Antimicrobials in Food TABLE 2.

1979). selected organic acids promoted growth and toxin production when added to the media.. 1989).5. monocytogenes (El-Shenawy and Marth. bacteriostatic and bactericidal properties of benzoic acid can also be demonstrated. including benzoic (Rusul and Marth. Most were isolated from spoiled foods that had contained preservative. Source: Adapted from Warth (1989c). Yousef et al. including benzoic acid..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. Eight of these had a lethal effect on both morphotypes of this bacterium. 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. Growth and aflatoxin production by toxigenic strains of Aspergillus were partially or completely inhibited by the undissociated form of six organic acid preservatives. This organism was isolated from milk. but relatively modest bactericidal. according to the results of a study involving several strains of Trichophyton and Microsporum (Pelayo. 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. such as sodium and potassium chlorides and sodium nitrate. it was found that benzoic acid at concentrations of approximately 1000 to 3000 ppm had strong bacteriostatic. Both fungistatic and fungicidal properties have been attributed to benzoic acid. Incubation of cells in minimal media caused injury that depended on the temperature of incubation but not on the presence of benzoic acid. 1988. activities against cells in a liquid minimal medium. were tested against both the opaque and translucent morphotypes of Vibrio vulnificus (Sun and Oliver. Fungal growth was reduced by lowering the pH of laboratory media from 7. Salts.5) without addition of benzoic acid. 1977). enhanced aflatoxin production when present at low levels but became inhibitory at higher levels. 1995). 1989). Growth of this fungus was accompanied by production of fumitremorgin mycotoxins. The authors questioned the suitability of benzoic acid alone to control this pathogen in foods. Sodium benzoate has been suggested as an inhibitor of cellulose-decomposing bacteria and fungi (Sauer. Small amounts (75 mg/L) of potassium sorbate or sodium benzoate completely inhibited germination of ascospores and subsequent outgrowth. Isolates were grown at 25°C in yeast extract medium containing 5% glucose (pH 3. In a series of studies involving L. Ten generally regarded as safe (GRAS) substances.0 to 2.Sodium Benzoate and Benzoic Acid 19 TABLE 2. and its survival . Under appropriate conditions.

However. 1963). . The growth patterns of S. Chichester and Tanner. sorbate was more effective than sodium benzoate against S. bayanus and Hansenula species were 330 and 180 ppm benzoic acid. 1980). benzoic acid. sodium chloride. The x-axis intercept was the concentration of the inhibitor that gave infinite microbial inhibition. 1988). when these organisms were grown in Trypticase® soy broth. At 35°C. respectively.20 Antimicrobials in Food and growth were determined in media supplemented with organic acids and sodium benzoate (ElShenawy and Marth. bailii were determined and expressed in polynomial equations (Cole and Keenan. like benzoic acid. carbon dioxide. Synergistic effects have also been reported using combinations of benzoate and sulfur dioxide. pH. stearothermophilus spores (Lopez et al. It should then be possible to achieve (1) a broader spectrum of action or (2) an increased antimicrobial action by using this combination (Lueck. combinations of sorbic acid and benzoic acid inhibit several bacterial strains better than either antimicrobial alone (Rushing and Senn. 1986b). bayanus in the presence of different concentrations of benzoic acid are shown in Figure 2. Increasing the acidity and/or NaCl generally improved the effect of all the preservatives. and antagonistic effects reported in several early studies have been extensively reviewed by Lueck (1980) for benzoic acid as well as for other preservatives. Additive. A plot of the inhibitor concentration versus the reciprocal of relative effectiveness was linear (Figure 2. INFLUENCE OF OTHER CHEMICALS AND PHYSICAL ENVIRONMENT No antimicrobial is completely effective against all microorganisms present in a given foodstuff. aureus. synergistic.0) were used and required lower levels of sodium benzoate. coli at all temperature/pH/NaCl combinations. and sorbic acid on the growth rate of the yeast Z. or sucrose (Rehm. monocytogenes than when each preservative was tested alone (Oh and Marshall. especially when used at pH <6. cereus. The influence of combinations of chemical antimicrobials on the growth of food-poisoning and spoilage microorganisms has been reported (Banks et al. 1989). For example. 1996).3). Infinite inhibition concentrations could be affected by the growth medium.0. 1994). An interesting method to determine concentrations of antimicrobial agents that provide infinite microbial stability has been developed (Marwan and Nagel. 1998). at various concentrations. Sodium nitrite was the least effective preservative tested. At <25°C. The effects of temperature. The infinite inhibition concentrations for S. The anionic form of benzoic acid was shown to have a distinct effect on doubling time.. aureus when used with higher concentrations of NaCl.. 1986). bayanus and Hansenula species.. Potassium sorbate was completely effective against Vero cytotoxigenic E. This method was based on finding the relative effectiveness of an inhibitor. A typical dose-response effect may be observed. and three preservatives on the growth of three foodborne bacterial pathogens were studied using gradient gel plates (Thomas et al. 1960. Synergism between benzoic and sorbic acids was pH dependent. It was also the most effective against B. one should be able to combine various antimicrobials having different modes of action to compensate for this deficiency. sodium chloride. when these preservatives were added to recovery media. boric acid. The relative effectiveness values of benzoic acid were established for S. benzoate was the most effective preservative against S. Effectiveness increased as the pH of the recovery media decreased to 5.4. 1993). Potassium sorbate and sodium benzoate did not have any significant effects on the heat resistance of four strains of B. In general. Combining glycerol monolaurate with benzoic acid gave greater inhibition of L. 1972).. Individual and synergistic effects of pH. they were very effective inhibitory agents for heatdamaged spores (Lopez et al. inactivation or inhibition of growth occurred in inoculated media when a lower incubation temperature (13°C) and pH (5. In theory.

3 Effect of the medium on the inhibition of Saccharomyces bayanus by benzoic acid at 30°C and pH 4. depending on the chemical in question.Sodium Benzoate and Benzoic Acid 21 1. However.8 0. Sulfur dioxide had the most significant effect on the rate of biomass and patulin production by this organism. in hours. 150 ppm potassium sorbate. RE.0. 1961).6 1/RE 0. Antagonistic as well as synergistic effects were observed when a wine yeast strain of S.2 0. 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. and 37°C over a 25-day incubation period was significantly retarded by 75 ppm sulfur dioxide. In addition to combining several preservatives together.. 1988). 1990). followed by potassium sorbate and sodium benzoate. 30°C. relative effectiveness (the time. Likewise.0 50 100 150 ppm 200 250 300 350 FIGURE 2.0 0. and 500 ppm sodium benzoate. 1984). Open circles: Trypticase® soy broth.5 volumes of CO2 have been reported (Kelly. Sodium benzoate prevented the growth of Staphylococcus aureus in an intermediate-moisture food (Boylan et al. it may also be advantageous to combine preservatives with physical methods of food preservation. 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).. Sulfur dioxide was apparently antagonistic when used in combination with any of the other antimicrobial agents (including benzoate) tested in this study. 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. 1956). respectively. Closed circles: yeast nitrogen base. cerevisiae was inoculated in a laboratory medium supplemented with several combinations of preservatives (Parish and Carroll. refrigeration. Growth at 21°C.4 0. . 1976). 1975). The effects of pH and benzoate on the growth of yeasts in an experimental grape drink containing 1. A combination of benzoic acid and low temperatures was used effectively to reduce the numbers of yeasts in grape juice (Pederson et al.b). 1958). 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. or drying. The presence of other chemicals may be beneficial or detrimental to the action of the preservative.9) (Okoli and Ezenweke. For example. such as heating. Source: Marwan and Nagel (1986b). benzoic acid can be solubilized with a surfactant to hold it in solution in its undissociated form (Lewis and Payne. the antimicrobial activity of benzoic acid is reduced by nonionic surfactants (de Navarre and Bailey. irradiation.

Carbonated and noncarbonated citrus drinks and fruit juices were involved. The concentrations of benzoic acid tested (in parts per million) are indicated.025% sodium benzoate acted synergistically against three food spoilage yeasts suspended in saline solutions at pH 6. but mold growth was accelerated in the presence of quinic acid. this might enhance the antimicrobial effects of benzoate. cited by Lueck (1980).2 and 4. Adding 1% quinic acid to media almost completely eliminated the inhibitory effect of 0. Source: Marwan and Nagel (1986b).5 (Sagoo et al.01% to 0. They had been supplemented with the maximum levels of benzoic acid permitted for use in these products.02% sodium benzoate.0. This acid was antagonistic to the antifungal effects of both potassium sorbate and sodium benzoate. 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. At these pH values. However. Growth of yeasts was unaltered.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. In a German study of 1959. . A combination of the polysaccharide chitosan (0.005%) and 0. recent studies imply that this may no longer be the case. the authors proposed that chitosan is highly polycationic and could react with the anionic components of the microbial surface. The effects of quinic acid on yeasts and molds were investigated (Kallio et al.4 Inhibition of Saccharomyces bayanus by benzoic acid in Trypticase® soy broth at 30°C and pH 4. coli after 14 passages in media containing benzoic acid.. no resistance could be observed in cells of E. 1985).. 2002).

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. Resistance resulted primarily from an inducible energy-requiring system that transported benzoate from the cell. respectively (Ethiraj and Suresh. Warth (1977) reported that cells of Z. Because this system required energy. Heat resistance increased with age. However. resulting in reduced growth yields and rates of cell production by yeasts. In a later study (Warth.. the energy source was unavailable for growth. In an inoculated peach-based system. sorbic. . were tested for tolerance to several organic acids during and after heating (Beuchat. The heat-resistant yeast Talaromyces flavus was isolated from fruit juice concentrates involved in spoilage of packaged reconstituted fruit juice (King and Halbrook.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. heat tolerance appeared to be related to the morphology of the ascospores. this isolate was less resistant to potassium sorbate. Fumaric. 1990). flavus. TABLE 2. Source: Adapted from Warth (1988). with the required concentrations lower when the pH of the growth media was reduced from 5. 1987). Three strains of T. and in some cases.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. 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. Thus. and lethality was enhanced as the pH of the heating medium was reduced. such as glucose.7).5. cells were resistant only when a sufficient energy source.4 to 3. and benzoic acids were the most effective. 1988) it was determined that energy was required for the reduction in cytoplasmic benzoate concentration and for the maintenance of internal pH. The authors observed that preservative resistance may be strain specific. 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.25 mM benzoic acid (first five species) or 2 mM benzoic acid (last four species). 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. cerevisiae was found to have multiple resistances to both physical treatment and chemical preservatives (Guerzoni et al. 1988). isolated from spoiled fruit products.19). A strain of S. Potassium sorbate and sodium benzoate prevented outgrowth. was present.0 and 4. 1988).

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

0 (Goodson . For example. Henriques et al. 1985). This appears unlikely because uptake occurs rapidly by passive diffusion.Sodium Benzoate and Benzoic Acid 25 2. 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. were isolated and found to be resistant to benzoic acid. Weak organic acid preservatives have maximum inhibitory activity at low pH. Acquired resistance to benzoic acid has been reported for other microorganisms. b. Cells of E. and adaptive mechanisms have been suggested. In yeasts. coli initially grown in media at pH 5. resulting in the release of charged anions and protons. which is freely permeable across the plasma membrane and is thus able to enter the cell. undissociated state of the molecule. Prevention of uptake. 1998)..8. cerevisiae were able to extrude benzoic acid by an energy-dependent mechanism. They included the acid tolerance responses of E. 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. 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. Holyoak et al.0 survived exposures to inorganic acid or to acid pH plus organic acid that prevented subsequent growth of cells grown at pH 7. However. This favors the uncharged. 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. Reducing the pH of the media to 3. The net result is that the preservative diffuses into the cell until equilibrium is reached in accordance with the pH gradient across the membrane. When G. 3. inhibition of microbial cell growth occurs because various key metabolic reactions are inhibited. Anions and protons accumulate inside the cell. (1997) found that cells of S. 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. 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. Therefore. monocytogenes and the role of membrane ATPase in the resistance of food spoilage yeasts. Benzoic acid occurs naturally as a phytoalexin of immature apples (Seng et al. When this happens. The inhibitory action is classically believed to occur when the molecule encounters the higher pH inside the cell and then dissociates. Adaptation to or repair of damage. MICs of sorbic and benzoic acids for Gluconobacter oxydans were 1000 and 900 mg/L.. 1996. as cited previously (see “Mechanism of Action”). which can infect apple tissues. Several microbial-resistance mechanisms induced by weak organic acid preservatives were also reviewed by Brul and Coote (1999). in media at pH 3. However. The pathogenicity of this fungus could be increased by growth on a medium supplemented with benzoic acid before inoculation into fruit. adaptation could involve enhanced removal of protons using H+ATPase. earlier studies by Henriques et al. respectively. coli O157:H7 and L. In an extensive review. oxydans was grown in the presence of sublethal concentrations of either compound before the MIC was determined. 1989). however.3 reduced the MIC of both antimicrobials by about 300 mg/L (Eyles and Warth. Piper et al.. Bracey et al. (1998) demonstrated the existence of a multidrug-resistance pump that actively extruded preservative anions from cells of this yeast. 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. which cannot cross the plasma membrane. Whether efflux pumps are involved is open to debate. 1997. The authors proposed that in yeasts.. Mutants of the fungus Nectria galligena. the MIC of both antimicrobials increased substantially within 1 hour.

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

This isolate was able to oxidize benzoate completely to carbon dioxide. whereas salicylate induced catechol 1. Similar results have been reported with Bacillus stearothermophilus (Adams and Ribbons.2-dioxygenase. Kinetic models have been derived for the metabolism of [14C]-benzoate by Pseudomonas species (Simkins and Alexander.. 1992). (4) ring opening. 1984). cis. including Pseudomonas. fumarylpyruvate. 1982). which are central intermediates in the anaerobic metabolism of many aromatic compounds (Elder and Kelly. A mutant incapable of growth on benzoate was isolated and characterized. A permease-like protein involved in benzoate transport and degradation has now been isolated from cells of Acinetobacter (Collier et al.. and with Micrococcus (Haribabu et al. benzoate and salicylate are metabolized via the protocatechuate and β-ketoadipate pathways. Hugo (1991) described the ability of nine species of Pseudomonas and Vibrio to degrade 18 aromatic compounds used as preservatives in medicines. Niemetz et al. Harwood and Gibson (1986) stated that aerobic degradation of benzoate by various Gramnegative bacteria. 1990). Benzoate was converted to catechol by the strains able to grow on this substrate. (3) introduction of a carbonyl group. Outlines of the new aerobic pathway were deduced. 1995). 3-hydroxybenzoyl-CoA. respectively (Shailubhai et al.. 1988). The enzymes of these pathways appeared to be under coordinate control and were repressed in the presence of a primary substrate. In Alcaligenes eutrophus. and fumarate + pyruvate. with Rhodococcus (Janke et al. 1995).. 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. Active transport of benzoate may occur in this organism (Thayer and Wheelis. 1997). 1982).. 1997). cis-muconate. . and β-ketoadipate were identified as intermediates of benzoate metabolism (Figure 2. The proposed intermediates included benzoyl-CoA. 1996). Compared to relatively well-characterized aerobic mechanisms for metabolism of benzoate and other aromatic compounds. gentisate. (2) ring reduction. a commonly occurring soil bacterium. Further metabolism proceeded via the catechol branch of the β-ketoadipate pathway with the occurrence of high specific activities of catechol 1.. 1994).5). The enzymes responsible for ring cleavage were induced depending on the substrate used for growth. In Aspergillus niger. Nitrate.. One pathway proposed for the anaerobic microbial metabolism of benzoate is shown in Figure 2.. He reported that benzoic acid was easily metabolized by all nine bacterial species tested. In the presence of benzoate. such as glucose. 1997). 1988). 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. Benzoate was metabolized by 16 of these strains when added as the sole carbon source to liquid mineral media. both aerobically and anaerobically.Sodium Benzoate and Benzoic Acid 27 β-ketoadipate pathway. Pseudomonas solanacearum. This is followed by conversion of the remainder of the molecule to acetyl-CoA. protocatechuate 3. maleylpyruvate. iron. putida (Jeffrey et al. Seventeen strains of aerobic bacteria were tested for their ability to degrade 15 aromatic substrates (Lang. foods. the rate of benzoate metabolism may be regulated by the presence of succinate. one of the causal agents of wilt in banana plants. and (5) a β-oxidation sequence..2-dioxygenase for ring fission. Catechol. Harwood and Gibson (1997) stated that this pathway could be divided into several phases: (1) formation of CoA thioesters. Both chromosomal and plasmid-encoded pathways have been described for the catabolism of benzoate in P. 1984). 4-dioxygenase was induced. was also capable of metabolizing benzoate (Kumari and Mahadevan. 1993. anaerobic pathways have been far less studied and reported.6. end product of the β-ketoadipate pathway (Figure 2.5) (Ampe et al. 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. These results were confirmed by assaying key enzymes from benzoate-grown cells. Anaerobic metabolism can also occur but involves reductive saturation of the nucleus. and cosmetics. and sulfate can be used as electron acceptors during metabolism (Colberg and Young. 1984).

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

ease of incorporation into products. Inc. Calcium benzoate has also been approved for use.8 and 2. However. 184. 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). 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 authors recommended that the combination of ascorbic acid and sodium benzoate in foods and beverages be evaluated more carefully. 1990). under certain conditions. .). their main advantages include low price. 1990). although its solubility in water is much less than the sodium salt (Pollard.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.5 or that can be brought into this range by acidification.1%.1%. A Food Technology special report (1986) reviewed the characteristics. (1993) when they analyzed several foods and fruit-flavored soft drinks for benzene. Subsequent studies indicated that soft drinks containing both ascorbic acid and sodium benzoate could produce very low levels of detectable benzene. the off-flavor they may impart to the foods they are to preserve. benzoic acid and sodium benzoate are GRAS preservatives (Code of Federal Regulations. Applications of these preservatives to foods have been reviewed by Chichester and Tanner (1972). An ADI (average daily intake) value of 0 to 5 mg/kg of body weight has been specified for benzoic acid (Pollard. The potassium salt is less water soluble (42.4 g per 100 ml solution) than the sodium salt. Kimble (1977). the maximum permissible quantities generally range between 0.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. They may also be used in certain standardized foods. Approximately 1. Title 21. Product information for both salts is available (Miles Laboratories. 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. REGULATORY STATUS In the United States.9). the narrow pH range in which they are effective.25%.1733) up to a maximum permitted level of 0. and Chipley (1983. generate detectable levels of benzene. and limitations of preservatives and antimicrobial agents. Similar results were reported by McNeal et al. Lueck (1980). Although the detectable yield was extremely low (<1 ppb). Potassium benzoate is now commercially available and apparently evolved in response to consumers’ interest in reduced sodium intake. APPLICATIONS Benzoic acid and sodium benzoate are most suitable for foods and beverages that naturally are in a pH range below 4. bakery products. 1993). Benzoate does not control the growth of high levels of microorganisms and therefore cannot be used to compensate for poor ingredients or processing. They may also include foods from which benzoate is excluded in the United States. As food preservatives. and lack of color. In addition. Secs. fruit products.15% and 0. In most other countries. 1977/1988. USE IN VARIOUS FOOD SYSTEMS Benzoic acid and sodium benzoate have been widely used to preserve beverages. the use of benzoic acid and other chemicals in the processing and freezing of shrimp has been reviewed (Chandrasekaran. In one case (a fruit juice–mineral water combination). benzene levels higher than the permissible levels for mineral waters prompted a recall in one state. applications. including benzoic acid. and other foods in several countries (Tables 2. 1994).1021 and 184.

Halotolerant fungi were isolated from salted dried fish (Chakrabarti and Varma. Fresh catfish were dipped in various concentrations of sodium benzoate or potassium sorbate and smoked. U. A. 4-hexylresorcinol was the most effective inhibitor of both melanosis and microbial spoilage in prawns. was evaluated against the growth of Z. was most sensitive to propionic acid and sodium benzoate. Penicillium sp.. separately or combined. then evaluated during shelf life (Efiuvwevwere and Ajiboye. A combination of sodium benzoate and kojic acid was effective in inhibiting melanosis.04%) inhibited growth of all three fungi.S. A maximum level of 2000 ppm may be used in orange juice for manufacturing. yolk or whole Margarine. separately or combined. 1996). 2001).06%) or potassium sorbate (0.8 Maximum Permitted Levels (ppm) for Benzoic Acid and Its Salts in Foods in Selected Countries Food Fish products Fish. bailii in an inoculated salsa mayonnaise stored at room temperature (Wind and Restaino.a 1000 200 500 500 5000 1500 1500 1500 5000 2000 1000 1000 1000 10. The shelf life of sardines stored in an acid brine containing 0.. salad dressings Sauces. ketchup Soft drinks containing fruit juice Soft drinks. Source: Adapted from Chipley (1993). Several additives and preservatives were evaluated as inhibitors of melanosis (black spot) and microbial spoilage in prawns in chilled storage (Montero et al. However. Potassium sorbate was more effective than sodium benzoate. b Soft drinks for consumption after dilution. 1994). prevented spoilage of the product by Z.30 Antimicrobials in Food TABLE 2. Propionic acid (0. Sorbate treatment was more effective than benzoate and increased the shelf life by 8 days.3% sodium benzoate was three times longer than that of the controls (Ponce de Leon et al. were dominant. relishes Salads. bailii. carbonated a Canada 1000 1000 1000 1000 1000 Denmark Norway Sweden U. flavus was most sensitive to potassium sorbate. Aspergillus flavus. Recent reports indicate that these preservatives continue to be of benefit in a variety of products. neither preservative. niger.K. The effectiveness of sodium benzoate and potassium sorbate. a maximum level of 1000 ppm benzoic acid or its salts may be used for all products listed.02%) or sodium benzoate (0. Orange juice not for manufacturing may not contain a preservative (federal standards of identity). However.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. 1995). d Good manufacturing practice. but A. c Soft drinks for consumption without dilution. salted Mayonnaise Mustard Pickles. semipreserved Fruit juice Fruit pulp Jam. and Penicillium sp. . jellies Liquid egg white. 2000).

were observed in any samples containing preservatives. benzoic acid and its sodium. 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 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. Growth and control of two strains of E.. and potassium sorbate (Uboldi Eiroa et al. yolk.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. bailii. coli O157:H7 were also evaluated in this cheese (Kasrazadeh and Genigeorgis.. In both studies.. dried fish Shrimp. Tomato concentrate could be kept for up to 12 months at room temperature with the addition of salt. 1994). (1995). potassium sorbate was more effective than sodium benzoate or their combination. Growth and control of four Salmonella serotypes in a soft Hispanic type cheese were evaluated (Kasrazadeh and Genigeorgis. In the EU. jellies. 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. In grape juice. and other fruit-based spreads Candied fruits and vegetables Vegetables in vinegar. sodium benzoate..Sodium Benzoate and Benzoic Acid 31 TABLE 2. 1995). Fleming et al. Source: Adapted from Anon. 1998). No viable microorganisms. The minimum temperature that allowed growth of the E. coli strains was 10°C. The effects of sodium benzoate and potassium sorbate on the thermal death rates of ascospores from the heat-resistant mold N. 1995). brine. models were developed relating lag time and specific growth rate to . including Z. cooked Low-sugar jams. 1996. and calcium salts are all permitted for use (E210–E 213) (Pollard. condiments. fischeri were evaluated in fruit juices (Rajashekhara et al. The minimum temperature that allowed growth was 8°C. acetic acid. Comparable rates were noted when each preservative or the combination of both was used in mango juice heated to 85°C. Montano et al. 1990). marmalades. 1994. mustard Liquid egg white. potassium. 1997). The levels of some nutrients were reduced when tomato juice was treated with dimethyl dicarbonate. unfermented Liquid tea concentrates Semi-preserved fish products (including fish roe) Salted.

low pH. However. 1989).. Both preservatives reduced the heat resistance of E. 2001). Ethanol-enhanced killing correlated with damage to the bacterial cytoplasmic membrane. However. The treatment that was successful included addition of 0. and luncheon meats. jejuni. 1995). 1999). USE AS A POSTHARVEST FUNGICIDE Because of the long and successful use of benzoic acid in the processed food industry. several factors may influence the effectiveness of benzoic acid in the treatment of fresh . or 5% potassium benzoate for 120 days (Samelis et al. No significant increase in the numbers of this pathogen occurred on bologna slices treated with 2. milk. the combination of sorbate and benzoate was more effective than either preservative alone (Zhao et al. Contamination of many of these foods frequently occurs after processing. 2001).5% lactic acid combined with 0. or S. Dock et al. Combinations of organic acids.5% or 5% acetic acid. C. Application of antimicrobials as sprays or mists to meats following processing may be more effective than their addition in the meat formulation.. Large outbreaks of infection have been linked to consumption of contaminated coleslaw.3% or 0. Listeria monocytogenes is recognized as a foodborne pathogen of major concern to humans. Benzoate was one of the most effective compounds tested in the presence or absence of ethanol. monocytogenes on sliced. 1999... Washing the skin with solutions of either 0. Survival of E. L. monocytogenes (Barker and Park. A study was conducted to evaluate aqueous dipping solutions of organic acids or salts to control L. this antimicrobial was evaluated for control of postharvest diseases of various fruits and vegetables.05% sodium benzoate followed by holding at 25°C for 6 hours and at 4°C for 24 hours. Sorbates and benzoates are currently approved in various countries for use as dipping solutions to prevent fungal growth in dry sausages (Sofos. At 8°C. growth was inhibited by benzoate or sorbate (Koodie and Dhople. respectively. monocytogenes usually attach following cooking and during slicing and packaging (Farber and Peterkin. These solutions could be used for dips to sanitize chickens intended for frying before presentation to consumers (Hathcox et al..32 Antimicrobials in Food temperature. monocytogenes. coli O157:H7 in cider because of the severity of illness this pathogen causes.3%) to cheese (pH 6. the growth of some O157:H7 strains in apple cider was not affected by the presence of either preservative (Miller and Kaspar. but benzoate was about two to eight times more effective than sorbate (Splittstoesser et al. coli O157:H7. The antimicrobial can be applied directly onto the product surface where cells of L. Most research efforts have been directed toward E. cheeses. Growth was either prevented or delayed by adding sodium benzoate (0.0) made from milk acidified to pH 5. and ethanol were very effective bactericidal treatments for L. 1994). A preservative treatment was developed that was capable of achieving the FDA mandate for a 5-log reduction of E. coli O157:H7 in apple cider (Comes and Beelman.15% fumaric acid and 0. Chicken skin was inoculated with Salmonella spp.3%) to cheese (pH 6. coli failed to grow in either type of apple juice. 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.6) or adding potassium sorbate (0. These strains grew well in unpasteurized and pasteurized apple juice. 2000). 1996. hot dogs.. Resistance to these preservatives was greater at 4°C than at 25°C (Fisher and Golden. 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. 2001). Control strains of E. 5% sodium diacetate. 1993). 1995).. coli O157:H7 in apple juice and apple cider with or without potassium sorbate or sodium benzoate has been investigated. 2002). and wash solutions were evaluated for effectiveness in decontaminating the skin (Hwang and Beuchat.9 with propionic acid.05% sodium benzoate reduced the numbers of these pathogens compared to washing with water. Unpasteurized apple cider has been implicated in several foodborne disease outbreaks caused by pathogenic microorganisms. vacuum-packaged bologna stored at 4°C for up to 120 days. 1998). Tompkin et al. aureus. especially in younger children and the elderly.

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. and as a preservative in certain brands of cough syrup (Chen et al. 1976). The minimum inhibitory concentrations of sodium benzoate and dichlorobenzyl alcohol for 115 strains of dental plaque microorganisms were determined (Ostergaard. 1996). following official methods of analysis (Zani et al. A mixture of fruit-coating polymers and potassium sorbate or sodium benzoate completely inhibited postharvest fungal growth on bananas (Al Zaemey et al. sealing gaskets for food containers. as a disinfectant for artificial kidneys (Kolmos. used in cosmetics has been published (Bach et al. Patents have also been issued for the use of benzoic acid or sodium benzoate as an industrial fungicide in several products. Although its use in pharmaceuticals has largely been displaced by other compounds (Grundy. insoles for shoes. and treatment for microbial infections in trees.. in peanuts (Uraih and Offonry. In the pharmaceutical industry. modified starch adhesives. Methylcellulose was mixed with chitosan and 4% sodium benzoate or potassium sorbate to produce a food-grade film (Chen et al. flavus. rubber manufacturing. Sodium benzoate did not inhibit growth of any Gram-positive cocci. Generally. concentrations of 0.1% and 0. 1967). lubricants. For example. 1990).. possible areas for specialized applications still exist — for example. 1994). Significant antifungal properties were demonstrated against food spoilage fungi. 1986). 1988. It is currently used in animal feeds and in some tobacco products as a fungicide at levels of up to 0.. Sodium benzoate has also been used to inhibit postharvest changes in fruits and vegetables. and leather.1% to 0. However. liquid coolants.. 1994). as a control for dentureborne Candida albicans (Lambert and Kolstad. Currently.1%. this preservative system was ineffective against an adaptiveresistant strain. A single oral rinse with a mouth rinse containing these antimicrobials did not affect plaque removal (Danielsen et al. 1968). A benzoic acid-based polymer coating for apples has been developed (Ivanov et al. Hewala. 1989). saliva samples from volunteers who used a dentifrice containing these antimicrobials indicated that for 5 to 10 minutes after toothbrushing. their concentrations were high enough to inhibit growth of periodontal pathogens. The chilling-induced production of ethylene in cucumbers was inhibited by the addition of sodium benzoate (Wang and Adams. incorporated as sodium benzoate. adhesives for paper.. 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. . 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. 1994). 1996). benzoic acid was used for oral preparations in concentrations of 0. varnishes.Sodium Benzoate and Benzoic Acid 33 fruits and vegetables. have been used to preserve cosmetic formulations. OTHER APPLICATIONS Benzoic acid is one of the oldest preservatives in the cosmetic and pharmaceutical industries. However. These include animal feedstuffs. 1996). A summary of the antimicrobial activities of 30 preservatives. especially against toxigenic strains of A.. 1997). with perhaps the most pertinent consideration the pH of the superficial tissues (Eckert. Benzoic acid and its derivatives have been proposed for use as fungicides.5%. including benzoic acid. cepacia grown under different conditions. 1981). A p-hydroxybenzoate-based system was effective in protecting the product against a variety of strains of P. 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.05% to 0. 1968).025%. wood preservatives. respectively...

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 1.10.10 Toxicity of Benzoic Acid and Sodium Benzoate by Exposure Categorya Category Acute Species Tested Rat Guinea pig. the second is catalyzed by an acyltransferase enzyme. However. Containers should be kept closed as much as possible.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 0.34 Antimicrobials in Food STORAGE AND HANDLING Sodium benzoate should be stored in a cool. dry place. (1909) and Dakin (1909) to conclude that sodium benzoate was not deleterious to human health. 1964).7 (White et al. Route of administration was per os. Sax and Lewis (1989) reported data for several animal species based on the route of administration (Table 2. In addition. Excretion of benzoic TABLE 2. TOXICOLOGY Several toxicologic studies involving different animal species have been conducted using both benzoic acid and sodium benzoate. Hippuric acid. both benzoic acid and sodium benzoate are moderately toxic by ingestive. 1964). The overall reaction sequence proceeds as shown in Figure 2. Benzoate does not appear to be accumulated in the body. This product is not corrosive.11). These have been summarized by exposure category in Table 2. Extensive human feeding trials conducted early in the twentieth century led Chittenden et al.. intramuscular. The first reaction is catalyzed by a synthetase enzyme. cat.4–2..0 g/kg body weight 1.5% 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. and intraperitoneal routes (Sax and Lewis. Source: Adapted from Lueck (1980). is then excreted in the urine (White et al.7–4.b rabbit. 1989).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) 1.3–4. There is little danger of its being a toxicant or fire hazard under normal conditions of use. . Its formation can be increased greatly by administration of benzoic acid. 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. synthesized in the liver.

intraperitoneal. lowest published toxic dose. 35 . e Total dosage for 3 days to pregnant rats. Ipr. severe irritation effects. subcutaneous. LD50. mild irritation effects. SEV.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. MLD. b Source: Adapted from Sax and Lewis (1989). c Total dosage. d Total dosage for 22 days to pregnant rats. Ims. TDLo. intramuscular. lowest published lethal dose. lethal dose 50 percent kill. LDLo.

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. Source: White et al. 1990). 1999). 80. 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. 1993). Akira et al. acid by this particular pathway was first reported by Dakin (1909). or 160 mg/kg) were given at least 1 week apart to 6 healthy male volunteers. 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. When applied topically. cerevisiae was used as a model system for testing antioxidant or prooxidant properties of benzoic acid and other weak organic acid food preservatives (Piper. only a small portion (6. 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. In guinea pigs. (1994) used deuterated benzoic acid and nuclear magnetic resonance spectroscopy to monitor metabolism in a healthy male volunteer. 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. 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. the route of administration significantly affected the efficiency of benzoic acid metabolism (Nathan et al.. (1964). Different animal species differ in their ability to metabolize benzoic acid. 1991).. The food preservatives were shown to have a strong prooxidant effect on aerobically grown yeast cells. . 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. They were also mutagenic to the yeast mitochondrial genome but only in the presence of oxygen.. it was excreted almost completely as hippuric acid after systemic administration. 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.7 Metabolism of benzoic acid by mammals. 1991). However. S. Cell sensitivity to these acids was enhanced by aerobic rather than anaerobic growth conditions. A single oral dose of 250 mg of benzoic acid was quantitatively metabolized to hippuric acid and excreted in the urine within 4 hours. These results have been confirmed in several animal studies. These authors analyzed plasma concentrations of benzoic and hippuric acids and urinary levels of hippuric acid after oral doses of sodium benzoate (40. 1991). Inhibition was caused by accumulation of benzoyl CoA with a resultant depletion of free CoA and acetyl CoA.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. This may be related to differences in the ability of liver and kidney cells to carry out glycine and glucuronic acid conjugation (for review.9%) of the absorbed benzoic acid was conjugated with glycine to form hippuric acid. see Chipley. Since the late 1970s.

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

GLC/TLC. highperformance thin-layer chromatography. Biochem. fruit juices. Adams. (1994) Chen and Fu (1995) de Luca et al. HPTLC. 2002). (1990) HPLC. I dedicate this review to my sons. James and John. high-performance liquid chromatography. gas chromatography-mass spectrometry. Supercritical fluid extraction of carboxylic and fatty acids from Agaricus spp.. (1996) Waldron and Li (1996) Fazio (1999) Mahalanobis et al. Three isolates of the heat-resistant fungus Neosartorya fischeri were tested for their resistance to sodium benzoate. C. and to Dr. selected ion monitoring. D. 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. and temperature. 1999) Serrano-Olea et al. (1995) Pant and Trenerry (1995) Mannino and Cosio (1996) Vogels et al. Agric. ultraviolet. . and it was recommended for screening purposes. UV. NMR. W. 1988. GC-MS. (1995) Lee (1995) Montano et al. E. SIM. Young. REFERENCES Abdullah. and Ribbons. J. 1994. Inhibitory effects were strongly affected by pH. ACKNOWLEDGMENTS Sincere appreciation is given to Miriam Chipley for compiling the reference citations. 42:718.12. M..38 Antimicrobials in Food TABLE 2. Food Chem.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. gas–liquid chromatography/thin-layer chromatography. and Games. The use of impedance microbiology to study effects of environmental factors on fungi has been reported by Nielsen (1991). nuclear magnetic resonance. 1996). Biotechnol. to Barbara Borrelli for library searches. 17:231. The metabolism of aromatic compounds by thermophilic bacilli. D. D. citrus juices. mushrooms.. I. In addition. a biosensor prepared from tyrosinase-containing mushroom tissues has been developed and used to assay for the presence of benzoic acid (Wang et al. Advantages of the method were noted. was performed. Appl. to Sharon McGinness for her patience in typing the manuscript. (1991) Kakemoto (1992) Khan et al. Tod Miller for figure preparation. 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. inoculum amount. Recent developments in detection and quantitation methodologies for several food products are outlined in Table 2. J.

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

The carboxyl group of sorbic acid is highly reactive and results in formation of various salts and esters. The salts (e. Under certain conditions.. however. depending on differences in microbial types. especially potassium sorbate. The molecular weight of potassium sorbate is 150. Commercially available salts include calcium. stability. 1989. Robach and Sofos. During the 1970s extensive research was performed on the potential for using sorbic acid and its salts as antibotulinal agents in meat products. with a molecular weight of 112.2% w/v at 20°C). the pH of the solution. including yeasts.2). 1982). 1989). Currently.g. substrate properties. 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.4-hexadienoic acid. especially in combination with reduced nitrite levels for a reduction in nitrosamine formation (Sofos et al. 1979d) by sorbate is believed to occur at the connecting reactions of the germination process. has an antimicrobial potency of 74%. while the health effects of sorbate were reexamined (Sofos.. 1981). CHEMISTRY Sorbic acid is a straight-chain. Inhibition of bacterial spore germination (Sofos et al. 1986).50 Antimicrobials in Food activity against microbial growth and the potential application of the compound in additional food products (Sofos and Busta. however. The solubility of sorbic acid (Table 3. The solubility of sorbic acid is also higher in alcohols. 1986. CH3CH=CH-CH=CH-COOH). and potassium sorbates. In general.13 (Table 3. and inhibition of enzymes or transport functions (Sofos. some microbial strains are resistant to inhibition by sorbate or even metabolize the compound. 1992). and it has a weak but characteristic acrid odor and acid taste. The volatility of the compound in steam is useful in its isolation for quantitative detection in foods or other materials. but it increases with temperature. Sorbic acid forms colorless flakes or needles when crystallized. 1981. 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. The calcium salt is formed as a white odorless and tasteless powder. changes in the genetic material. Robach and Sofos. or both. Sofos et al. trans-trans unsaturated fatty acid (2. in inhibition of microorganisms by sorbates. on an equivalent weight basis with the acid.2) in water at room temperature is only 0. The mechanisms of antimicrobial activity of sorbates are not fully defined (Sofos and Busta. 1989).. and bacteria.22. alterations in cell membranes. Inhibition of microbial metabolic function is probably the result of morphologic alterations in the structure of the cells. and in other industrial applications (Sofos.. sorbic acid and its more water-soluble salts. Sofos. especially ethanol. and in glacial acetic acid. There is variation. 1981. are collectively known as sorbates and are used widely throughout the world as preservatives for various foods. and it constitutes the most soluble form of sorbate (Table 3. species and strains. Potassium sorbate may be manufactured as a powder or granules and. Sofos et al. 1982). 1989.2% and is insoluble in fats. 1989. and . 1980). The sodium salt has a water solubility of 32% (wt/vol). Sofos. 1989). 1985. Calcium sorbate has a water solubility of 1. and environmental factors. it is available as a white freeflowing powder or as granules. as animal feeds. molds. probably through action on spore membranes or protease enzymes involved in germination (Blocher and Busta.. Good solubility (58. as pharmaceuticals and cosmetics. which makes it valuable as a delayed-release form of sorbic acid (Sofos.1). The extensive use of sorbates as preservatives is based on their ability to inhibit or delay growth of numerous microorganisms. sorbates are considered effective food preservatives when used under sanitary conditions and in products processed using good manufacturing practices. sodium. potassium sorbate) of sorbic acid may find more frequent applications in foods because of their greater solubility in water. Sofos. 1979a.15 g per 100 ml. In food systems the solubility of the compound is estimated as higher than 50%. 1989). 1981.

°C Heat of combustion at 25°C.1 Physical and Chemical Properties of Sorbic Acid and Potassium Sorbate Property Chemical name Molecular formula Molecular weight Flash point. maximum% Ash. Oxidation of sorbic acid yields various carbonyl compounds.. the type and amount of fat. and other ingredients present (Sofos. whereas in the pure dry form they are stable. and anaerobic conditions (Sofos.Sorbic Acid and Sorbates 51 TABLE 3. 1981). such as sugars and salts (Sofos and Busta. °C (COC ASTM D-92) Ionization constant at 25°C Density at 20°C g/ml Melting range.36 Decomposes higher than 270°C — ml 0.001 10 43 Decomposes 143 119 >98 0.73 ∞ 10-5 — 132–137 11. The partition quotient is increased from 3. °C 760 mm Hg 50 mm Hg 10 mm Hg Purity.0 with increased levels (50%) of soluble food components.927 — Potassium Sorbate 2. such as crotonaldehyde. 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).13 126 1.0 to 7.0 10 3 — <0. Sorbic Acid 2. Oxidation and losses of sorbic acid are inhibited by antioxidants. and β-carboxylactolein (Arya and Thakur.. acrolein.1 g — — — — — — >98 1. appropriate packaging materials. maximum% Heavy metal content. increasing the production of carbonyls. at 20°C 120°C 140°C Boiling point. Aqueous solutions of sorbates are unstable and degrade through oxidation. 1995). The rate of oxidation in aqueous solutions is increased at lower pH values by the presence of light and acids or increased temperatures. 1989.1 N HCL per 1. malonaldehyde. maximum% Source: From Sofos (1989). mm Hg. 1989). 1996). 1988.4-Hexadienoic acid CH3-CH=CH-CH=CH-COOH 112. Thus. 1992). Sofos. acetaldehyde.2 ease of manufacture make potassium sorbate the most widely used form in food systems (Sofos. In general.1 N NaOH to 0. It is proposed that EDTA-Fe2+ complexes catalyze sorbic acid autoxidation. Btu/Lb Alkalinity/acidity Vapor pressure.8 ml 0. Ethylenediamine tetraacetic acid (EDTA) enhances sorbate degradation probably as a result of iron scavenging from the packaging material (glass or polypropylene) through EDTA complexation.5 10 3 0.4-Hexadienoic acid potassium salt CH3-CH=CH-CH=CH=COOK 150. malonic acid. 1992). formic acid. % Water content. maximum ppm Arsenic content.22 — — 1. which take part in nonenzymatic browning. the partitioning of sorbic acid between the water and fat phases of foods depends on the pH of the food. . 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.

5% and 8.10 0. and storage temperature and time (Bolin et al. 100% Propylene glycol.80 2. 20°C Acetone.00 0. 40% solution Sucrose. 20% Ethanol.0% enhanced sorbic acid destruction.08 0.20 — — 61. 20°C (pH 5.14 24. 1983.50 2. 50°C Water.29 — 12.00–15. 20°C Cyclohexane. 1986a.2 Solubility (%) of Sorbic Acid and Potassium Sorbate Solvent Water. oxidative degradation of .03 — 0. 20°C Glycerol. processing conditions. Sorbic Acid 0.02 0. 20°C (pH 4.60 45. 1989. 25°C Pentane. 75°C Ethyl ether.28 1.15 0. 1992.00 64.07 0.01 — — 0.00 45. 20°C Carbon tetrachloride.01 Losses of sorbic acid during storage of foods depend on sorbate levels. 20°C Propylene glycol.12 0. 10% solution Sucrose. 15% solution Acetic acid.30 6.00 5.55 4.25 0. 20°C Source: From Sofos (1989). 100°C Pentane. but at 13. Sofos. 85% Ethanol.90–14.16 0..9) Water.00 55. Vidyasagar and Arya.20 0.0% it had a protective effect (Guerrero et al.10 0.00 0. 1994). 95% Ethanol. 75°C Benzene.00 — — — <0. 5% solution Sodium chloride. packaging material.00 — — — — 57. 20°C (pH 3. 1987. other additives present. 20°C Corn oil.5% Citric acid.00 34. 20°C Cottonseed oil. 50% Phosphoric acid.00 47.00 28.05 5.15 0. amino acids.00 0.60–14. 50°C Pentane.40 54. metal ions. 60% solution Sodium chloride.. 1980.. glacial Lactic acid. 50°C Benzene. 100°C Corn oil.90 0. antioxidants. Gerschenson et al.01 58. In general. 20°C Methanol.30 2.80 0.4) Water.52 1.00–5.10 16. 20°C Sucrose.08 2. 1984. 5% Ethanol.01 0.30 Potassium Sorbate 58.34 8.1) Water. Sodium chloride at 3.22 1.60 1. 85.30 0. 1990).00 — <0.02 0.50 0. types of food materials present.15 0.00 12. 50°C Soybean oil.50 12. 50°C Propylene glycol.00 48.b.50–12.11 0. the nature and pH of the food.26 0. 50% Ethanol.00 0.31 9. 25°C Benzene.20 12. 10% solution Sodium chloride.04 11. moisture content.52 Antimicrobials in Food TABLE 3.00 20. light.

and a further loss is apparent after the dough is cooked to give the traditional West African food. suspensions. which are available as powders.3%). such as composition. and ethanol-soluble components of fura from pearl millet. 1989).6 to 9.5±0.. and dark storage areas. Charvalos et al. Dialysis of the ethanol-. salt-. Sofos and Busta. New controlled-release. the water-soluble (6. sorbamide. moisture. In general. is hindered by disadvantages. such as low solubility in water. structure. the ethanol-soluble (4. Although the salts of sorbic acid have been commercially developed.. was an effective mold inhibitor over a wide pH range (3.7%). 1995). and sorbic anhydride. dry. Sofos. 1963).8. and storage conditions (Sofos.9±0. salt-. 1990). but commercial production of sorbic acid has mostly involved the methods of oxidation of . Fractionation of the lipids and proteins from fura showed the 14C to be associated with the petroleum spirit crude fat extract (28±5%). moisture. 1994). a significant amount of sorbic acid is not recovered by extraction with methanol. and the acetic acid-soluble components (0. 1981). sorbohydroxamic acid.6%).5±0. Sorbamide was 1000 times more inhibitory than sorbic acid against yeast alcohol dehydrogenase (Martoadiprawito and Whitaker. aldehydes. the salt-soluble (3. Because the compounds are sensitive to heat. handling. These cartons should be kept closed in cool. Commercial application of such derivatives. or solutions. 1963. other derivatives of sorbic acid have also been examined. broadly tested. granules. 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. 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. A proportion of the binding is with the low-molecular-weight components of these fractions (Jideani and Wedzicha. Several procedures have been patented for the manufacture of sorbates (Sofos. and widely used as antimicrobial agents. strong offflavors and odors. some of these compounds are sorboyl palmitate. and light. Vojdani and Torres. When wheat flour doughs are treated with sorbic acid and heated. 2001). A mixed anhydride of sorbic and palmitic acids called sorboyl palmitate has found some application in yeast-leavened bakery products. 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.. These include esters. 1992). amine salts. 1989). fura. 1976). 1989. 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.b. and sorbic aldehydes were also effective antimicrobials (Troller and Olsen. 1986a.Sorbic Acid and Sorbates 53 sorbic acid in foods is less extensive than in aqueous solutions. methyl sorbate. In addition. ethyl sorbate. alcohols. and amide derivatives. sorbohydroxamic acid. and it is influenced by the nature of the food and its components and by processing. physical state. Commercial applications of sorbates include use of the free acid or its potassium and. Troller and Olsen. to a lesser extent. calcium or sodium (salts). The results show that sorbic acid binds with the lipid components. the water-. with a dissociation constant (pKa value) of 8. 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. Specifically. This suggests that sorbic acid is indeed reacting with thiol groups in the flour protein. 1992).7%). 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. 1980. they are shipped and stored in fiber drums with a moisture barrier in the carton wall. 1976).2 ± 0. and water activity (Giannakopoulos and Guilbert. 2000. and potential health problems (Lück.2) (Dudman. however. Uptake and diffusion of sorbate into foods should be influenced by properties of the substrate. The lipid and protein fractions that appear to bind sorbic acid or its degradation products were identified with the help of 14C-sorbic acid. and acetic acid-soluble fractions suggested that 14C activity tends to be associated with the low-molecular-weight components of these fractions..

wines. and others.02% to 0. as well as against many bacteria (Sofos. 1954a. Liewen and Marth.54 Antimicrobials in Food 2. Aly. 1970. 1998). tomato products. Geminder. Sorbates inhibit the formation of mycotoxins by various molds in culture media and in foods (Bullerman. Sporotrichum. Ascochyta. Ascosphaera. Curvularia. Penicillium. Gliocladium. Piper et al. Aspergillus. In addition to their effectiveness in fermented vegetables. after 21 days.. 1954a.80 to 0.. salad dressings. 1998. Torulopsis. and Zygosaccharomyces (Sofos. cakes. 1999).. 1984. dried fruits. 1972). ANTIMICROBIAL ACTIVITY INHIBITION Sorbic acid is active against yeasts and molds. Kloeckera. Debaryomyces. jellies. Pederson et al. Colletotrichum. Geotrichum. 1984. Numerous studies have also documented the effectiveness of sorbates against molds (Emard and Vaughn. Cunninghamella. whereas no growth was observed when the preservative concentration was increased to 220 ppm (El Halouat and Debevere.. Bhattacharya and Majumdar. Melnick and Luckmann. Alkaline salts of sorbic acid are formed by its neutralization with alkali metal carbonates. Bracey et al. CO2 atmospheres. Truncatella.. Cephalosporium. and chocolate syrup (Restaino et al. 1985a. Endomycopsis. jams. Effective antimicrobial concentrations of sorbates in most foods are in the range of 0.4-hexadienal and the condensation reaction of ketene and crotonaldehyde. Gourama and Bullerman. Ferguson and Powrie.b. 1988). Rhodotorula. Fusarium. Sofos. hydroxides. 1989). and sorbic acid. 1983. such as carbonated beverages. 1984. 1986. Fan and Chen. 1993. 1998. 1983. 1952. Huang and Armstrong. Garza et al.. 1985. Baldock et al. Sorbates also inhibit molds in butter.. 1959. Rhizoctonia. Kivanç.. 1954a. Humicola. Under an 80% CO2 atmosphere. 1984. 1984. were able to depress mycelial weight and aflatoxin production by Aspergillus flavus or Aspergillus parasiticus cultured in yeast extract-sucrose (YES) broth (Fan . Tong and Draughon. Although. which is most widely used.90). 1979. Huang and Armstrong. Monilia. Numerous patents describing catalysts. candy. Heterosporium. 1989). lower than the inoculum level (103 CFU/g). Pichia. Helminthosporium. cottage cheese. 1985. the inhibitory amounts of sorbic acid were reduced by 40% to 50%.. Candida. 1996). Botrytis.b. grains. A major application of sorbates in food products is their use for inhibition of molds in cheeses (Chichester and Tanner. 1996). Pullularia. neither sorbate nor propionate. 1961. Hurdles used in combination included aw. Kaul et al. Roland and Beuchat. sorbates inhibit yeasts in fruit juices. Liewen and Marth. bread. Sporobolomyces. El Halouat et al. reaction conditions.. 1970.. and other suitable salts in the presence of necessary additives. and meat and fish products. applied at 10 mg/ml. Under this atmosphere and at aw values in the studied range (0. 1957. 2000). Torulaspora. Lennox and McElory.b. Mucor. 1952. 1996. syrups. use of a hurdle approach resulted in a synergistic effect (El Halouat and Debevere. 1979. Skirdal and Eklund. potassium sorbate used at 150 ppm resulted in final counts. Tsai et al. Ulocladium. 1999. However. Hansenula. Rhizopus. The inhibitory action of sorbate against yeasts was first documented in the 1950s in fermented vegetable products. Mold species inhibited by sorbates belong to the genera Alternaria. Pestalotiopsis. Cladosporium. Yeasts inhibited by sorbates include species of the genera Brettanomyces. Pepularia. Phoma. fruits and juices. 1982. Saccharomyces.b. Marshall and Bullerman. Chaetomium. sausages. Smith and Rollin. 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. 1993. and smoked fish (Liewen and Marth.30%. 1992. and purification procedures follow the second method. Cryptococccus. Matamoras-León. Zygosaccharomyces rouxii is considered resistant to sorbate treatments. Melnick et al. The effectiveness of sorbates against yeasts has been documented by numerous studies (Emard and Vaughn. 1985a). 1954a. Use of sorbates for inhibition of yeasts is especially important in low-pH and/or intermediate water activity (aw) products. Deuel et al. Rosellinia. 1984. Trichoderma.

Alteromonas. Tsay and Chou.b. Escherichia. Overall. Zhao et al. Chung and Lee. aw. and some may even metabolize the compound (Sofos. The combination of 0. Palou et al. Lund et al. sorbates can inhibit Gram-positive and Gramnegative. 2001)..5% (Kumar et al. the addition of sorbic acid (0. 1987).b.b. Rusul and Marth.04% to 0. however. 1984. Sofos. increasing its survival. monocytogenes in broth. Klebsiella. Aeromonas. Salmonella. and other factors (Sofos.2% to 0. Yersinia. and mesophilic and psychrotrophic microorganisms. type . pH. strains.05% sorbic acid was found to inhibit growth of E. Clostridium. subinhibitory levels of sorbate may simulate the production of mycotoxins (Bullerman and Olivigni. In fact. Proteus. 1994). Sofos. aerobic and anaerobic. Greer. 1993). with a lesser influence on rate and extent of growth (Larocco and Martin. Campylobacter. 1981... Kouassi and Shelef. Potassium sorbate sensitized cells of L. The effect of sorbate on spore-forming bacteria may be exerted on spore germination. Lactobacillus.. coli O157:H7 during storage of apple cider (Miller and Kaspar. 1988). 1981. storage temperature. food-processing treatments. However. Gareis et al. 1985. Bacillus. Smoot and Pierson.. gas atmosphere. Stimulation of mycotoxin formation by low levels of sorbate depends on species and strains of molds. however. composition of substrate. monocytogenes presence in two commercial cheese brines and thus could be used as an antilisterial agent in commercial brines. 1993. Pseudomonas.. 1979a–d. sorbate inhibited or inactivated Listeria monocytogenes in a broth substrate (El-Shenawy and Marth. Achromobacter..1% sodium benzoate substantially decreased Escherichia coli O157:H7 contamination and suppressed the growth of yeasts and molds. 1988) and in a coldpack cheese food (Ryser and Marth. Seward et al. Enterobacter. 1989). outgrowth.. In other studies (Kouassi and Shelef. 1989).. 1995a. Vibrio. It was concluded that combinations of sorbate with propionate or lactate. Alcaligenes. 1989). however. 1995. A hydroxypropyl methylcellulose (HPMC) coating significantly reduced the number of viable Salmonella Montevideo cells on the surface of tomatoes.. 2000. which inhibited growth. 1995a. monocytogenes and Zygosaccharomyces bailii to inactivation by high hydrostatic pressure (Mackey et al. temperature of storage. and result in spoilage of the product (Zhao et al. could extend shelf life and increase safety (Kouassi and Shelef. potassium sorbate slightly decreased L. 2001). although sorbate did not affect growth of L. Inhibition of bacteria by sorbate appears to cause an extension of the lag phase. 1982.. 1999). coli O157:H7. which could have a protective effect on E. Pediococcus.4%) to HPMC did not substantially enhance bactericidal activity (Zhuang et al. 1981. 2000). 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. 1982. however. 2000). and others (El-Shenawy and Marth. Depending on pH and concentration. as well as spoilage and pathogenic bacteria. At the relatively high level of 1%. but the cost effectiveness of adding high levels (1%) of substrate is questionable. additives present. 1993.. 1987a.. 1980a. under certain conditions. catalase-positive and catalase-negative. 1985a. 1974. 1989). Koodie and Dhople. coli O157:H7 in apple cider (Koodie and Dhople. 1996). 1988.b. 1997). Bacterial species inhibited by sorbate belong to the genera Acetobacter. Certain bacterial strains are not inhibited by sorbate. 1988). Monnet et al. Blocher and Busta. 1987. Acinetobacter.b). Overall. it has also been shown that potassium sorbate or sodium benzoate did not affect survival of E. SELECTIVE ACTION Microbial inhibition by sorbate is variable and depends on species.1% potassium sorbate and 0. 1995a. Sofos. 1982. Zamora and Zaritzky. and their long-term stability in the high-salt and low pH environment of brines is not well documented (Larson et al. and/or vegetative cell division (Sofos et al. Sanchis et al. it suppressed cysteine activation of listeriolysin. Moraxella. 1999). Arthrobacter. sorbic acid is considered as a more effective inhibitor of yeasts and molds than bacteria (Skirdal and Eklund. 1988. Addition of 0. 1986. Micrococcus.Sorbic Acid and Sorbates 55 and Chen. Serratia. Liewen and Marth. Mycobacterium. Staphylococcus.

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

temperature. coli O157:H7 from 36 to 5.3-pentadiene. that there is no apparent relationship between sorbate resistance and the toxigenic properties of molds (Tsai et al. 1985a–c).20%) sorbate levels (Marth et al. sorbate is completely oxidized to carbon dioxide and water. inoculum size. Degradation of sorbate by molds depends on species and strains. Mold strains of the genus Penicillium isolated from cheese treated with sorbate were able to grow and metabolize high (0. 1988).b. pH. amount of sorbate present. Fusarium. These factors can act synergistically or be antagonistic and either enhance or negate the antimicrobial activity of sorbate (Sofos. 1989). under normal conditions of alimentation. 2000). 1954b. aw. 1981. It appears that selection may occur in sorbate-treated cheeses for certain molds tolerant to the compound (Schroeder and Bullerman. Finol et al. 1989. Sofos. under certain conditions. although many molds are sensitive to inhibition by sorbate. using it as a carbon source. This metabolism is mostly associated with lactic acid-producing bacterial strains present as high inocula in sublethal concentrations of sorbate (Crowell and Guymon.. certain strains are resistant and can metabolize the compound. and other additives (Sofos and Busta. 1966.. caused by ethyl sorbate. large inocula at high cell concentrations therefore require considerably higher concentrations of the inhibitor to prevent growth than do dilute cell suspensions. In general. a geranium-like odor is usually associated with wines treated with sorbate and contaminated with high microbial loads. however.5-diene (Edinger and Splittstoesser.. A study found a pronounced positive inoculum effect of Z. of which 50% is biologically usable.2 min. which was not an artifact caused by insufficient growth time. 1-ethoxyhexa-2. or hydrocarbon-like odor (Marth et al. which is a volatile compound formed through a decarboxylation reaction and has a kerosene-like. other ingredients.4-diene. 1954b. In addition to certain molds. 1989). Horwood et al. substantial metabolism of sorbic acid.. Products of sorbate metabolism by molds include 1. some bacterial strains may also degrade sorbate under appropriate conditions. 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.. such as concentration.. or binding of sorbic acid to dead cells. The study showed that the resistance of Z. as a fatty acid through β-oxidation. coli O157:H7 during storage .. 1986a. Degradation of sorbate by lactic acid bacteria has been associated with geranium-type off-odors in wines and fermented vegetables. 1985). and Geotrichum (Sofos. 1989). dehydration of cultures. Because it is metabolized like other fatty acids. 1995).. INTERACTIONS The antimicrobial activity of sorbates is influenced by compositional.1%) reduced the D50°C of E. Mucor. Liewen and Marth. Like caproic and butyric acids. 2000).18% to 1. 1980). 1966. and environmental factors. and 2-ethoxyhexa-3. It appears. 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.. 1992). about a 7-fold increase in lethality (Splittstoesser et al. microbial flora. 4hexenoic acid. Lück. 1982). gas atmosphere. level of inoculum.1% sorbate is usually sufficient to inhibit sensitive molds (Liewen and Marth. Sorbic acid (0. The MIC of sorbic acid increases with the size of the inoculum. there is also evidence of some ω-oxidation (Deuel et al. sorbate yields 6.Sorbic Acid and Sorbates 57 DEGRADATION Animals and certain microorganisms can metabolize sorbate. Liewen and Marth.. 1989. In general. 1985a. 2000).b). prior exposure to subinhibitory levels of sorbate. 1975. It should be noted that 0. Other strains of molds that may degrade sorbate belong to the genera Aspergillus. Bullerman. packaging. 1977. and type of substrate (Sofos. 1989). 1981. plastic paint. Sofos. processing. Increasing the concentration of sorbic acid from 200 to 1000 mg/L in apple cider decreased the D50°C value of E. When sorbate levels are high. Some mold strains can grow and metabolize sorbate under certain conditions as detected in cheeses and fruit products (Melnick et al.6 kcal/g. 1985a). Steels et al. bailii resistance to sorbic acid activity. Sofos..

0 to 4. 1956. solutes should increase the inhibitory activity of sorbate by reducing the aw of the substrate (Sofos. might not be effective owing to their increased solubility in fat. 1993). 1982. have reduced the synergistic effect of sorbate and heat on thermal inactivation of microorganisms (Beuchat. 1981a–c. Sofos and Busta. 1959.. such as parabens. 1981).76) (Cowles.2 minutes. 1987.. Acott et al. 1944. 1981. Hoffman et al. 1981.1% sorbate (Bills et al. 1976. Sofos and Busta. 1985a. Gooding et al.5 (Bell et al.0 to 5. Sofos et al. Sorbate may enhance heat activation and destruction of spores. 1993). The increased antimicrobial activity of sorbate at lower pH values has been attributed to the increased amount of undissociated acid present. whereas benzoic acid reduced it to 0. Chung and Lee. Although activity is greater at low pH values. Lund et al.b).5 and 4. Increased amounts of fat in a product reduce the concentration of sorbate in the water phase. 1981.. In general. 1990). 1980. 1989). 1988).. 1988). Statham and McMeekin. Lück.. 1989). 1954a. it is believed that both undissociated and dissociated sorbic acids have antimicrobial activity (Skirdal and Eklund. 1988). Use of artificial saltwater prevented dissociation of the 1% sorbic acid. as well as dormancy and recovery of heated microorganisms (Sofos. Studies have indicated that the dissociated sorbic acid also had antimicrobial activity. 1960). 1990). Lowering the aw enhanced the resistance of the same organism to increasing concentrations of sorbate (Restaino et al. 1955. 1941. more than 50% of the inhibition was the result of dissociated sorbic acid (Eklund. act synergistically to enhance the antimicrobial activity of sorbate (Costilow et al. Cerruti et al.. 1980.. 1996). Lück. Beuchat. 1985a). 1989.5 for propionate and benzoate. 1982). 1976.. however. 1955. Sodium chloride (1.. the maximum pH for antimicrobial activity by most other common food preservatives is lower — for example. Sorbic acid (0. Interactions of sorbate with heat may affect the rate and extent of microbial destruction during heating. and sodium chloride. 1944.64 min (Splittstoesser et al.g. which exhibited favorable antimicrobial properties against Vibrio vulnificus. The increased activity of sorbates at pH values higher than 5.5%) reduced the inhibition of Clostridium botulinum by sorbate in a nutrient broth (Wagner and Busta. This popular theory has been questioned.. 1981). 5. 1983). however.. 1983). Sofos and Busta. 1983.4 but not at pH 6..5 is advantageous because it allows for their use in foods of higher pH values in which preservatives. Cerruti et al. In environments of pH higher than 6. Sheneman and Costilow.0 (Raevuori. Sucrose. sorbates have the advantage of being effective at pH values as high as 6. which is believed to be the effective antimicrobial form (Lück. 1997). salt and sugars) also reduce the concentration of sorbate in the aqueous phase (Gooding et al. 1979a. respectively (Sofos and Busta.25 and 2.1% sorbate rendered the cells more sensitive to inhibition by sorbate than preconditioning in 0% sucrose + 0. Skirdal and Eklund.. Sofos and Busta. 1993)... Sugar and salt. supporting previous evidence that sorbic acid is effective in its undissociated form (Sun and Oliver. where it is needed for microbial control (Oka. 1984. Rahn and Conn. 1976. Thus.0. Other food ingredients (e. sorbates should be used to preserve foods processed using good manufacturing practices. Cerruti et al. but increased levels of microbial contamination reduce antimicrobial activity. Preconditioning of Saccharomyces rouxii cells in 60% sucrose + 0. however (Sofos.58 Antimicrobials in Food of apple juice from 18 to 5. 1980. 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. Not only are certain strains of microorganisms resistant to inhibition by sorbate. Liewen and Marth. In contrast. 1982. however. 1989. 1981. 1955. Statham and McMeekin. 1994). 1981c). Sofos. 1980. 1957.4 (Liu et al.. In certain instances.1%) enhanced destruction of E. certain studies have indicated antimicrobial activity by sorbate at pH values as high as 7. Nevertheless. Robach and Stateler. coli O157:H7 cells at pH 3. not as a substitute for appropriate sanitation and hygienic practices. 1955. glucose. The antimicrobial action of sorbate is pH dependent and increases as the pH of the substrate decreases. and it may inhibit the repair and growth of . approaching its dissociation constant (pKa = 4. 1976. however. but it was 10 to 600 times less inhibitory than the dissociated acid (Eklund..

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

Guerrrero et al.. 1990). 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. bailii is highly resistant to individual inhibitory factors. In addition. 1987. Robach et al.. in a medium containing 0. coli at all temperature/pH/NaCl combinations and was the most effective preservative tested against B. As indicated. 100 ppm potassium sorbate.. Kadam et al.5. At <25°C. However. 1993).. 1989.98. 1990). 1993). At temperatures of 12°C and below. 1984. Z. 1994).60 Antimicrobials in Food et al. Numerous publications and patents describe interactions of sorbate with many other factors. the log probability of growth of a single vegetative cell is approximately –4. Morad et al. Interactions and increased antimicrobial effects have also been observed in combinations of sorbates with propionate. antioxidants. 1981. 1968. These combinations offer the advantage of simultaneous inhibition of microbial growth and development of rancidity. Mendonca et al.. After 14 days at 30°C in the presence of 280 mg undissociated sorbic acid/L. Increase in the acidity and/or the NaCl concentration improved the effect of all the preservatives. 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. pH. Sorbate was completely effective against E. certain amino acids.. 1981. Variations existed. Wagner and Busta... 1981. and potassium sorbate was evaluated against growth of three foodborne bacterial pathogens (Bacillus cereus. sodium chloride concentration. Gourama and Bullerman.. Nelson et al. 1995). 1987.. however.8. at aw of 0. 1983. The ability of C. verocytotoxigenic Escherichia coli.06%) combined with aw values lower than 0. and mild heat (Guerrero et al. The microbial stability of shelf-stable banana puree was enhanced through inhibition of inoculated osmophilic and nonosmophilic yeasts. sucrose fatty acid esters. Potassium sorbate was found more inhibitory against yeasts than hydroxycinnamic acid (Stead. 1994). Lahellec et al. 1989). aureus when used with higher concentrations of NaCl (Thomas et al. aureus. Ough and Ingraham. 1986. and substrates... TellezGiron et al. Sofos. 1983. 1960.06% potassium sorbate. or at 10°C (Deak and Beuchat. ascorbate. and other compounds (Ferguson and Powrie. 1982. including sorbate. equivalent to a total sorbic acid concentration of 1300 mg/L. 1988). 1979b. Deak and Beuchat.93. and aw 0. Thomas et al. aureus) using gradient gel plates (Thomas et al. 1994).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. glucose oxidase.95 (Deak and Beuchat. Bell and De Lacy. Seward et al. Kabara. 1999). fatty acids. and S.. Marshall and Bullerman. 1994. Several studies have also indicated increased antimicrobial effects when sorbate was combined with various phosphates (Ivey and Robach. 1982. 1999). cereus. 1957. 1984. these concentrations of chemicals represented a greater than 50% reduction of the amounts needed to suppress growth when used individually (Matamoras-León et al. 1994). as well as multifactorial interactions (Sofos. 400 ppm sodium bisulfite. Combinations of sorbate with benzoate or propionate may be used to expand the range of microorganisms inhibited with reduced concentrations of each preservative. 1978. Thomas and Wagner. . The effect of temperature. A study found that growth was not inhibited at pH 3. Bacillus coagulans.. and Clostridium butyricum by adjustment of aw to 0. 1960. Amano et al. Miller and Brown.97 and pH to 3.4 and addition of 250 ppm ascorbic acid. 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. pH 3... molds. 1993. Clostridium pasteurianum. sorbate was more effective than benzoate against S. 1988). the probability of growth was approximately –6 (Lund et al.. mixtures of sorbate with various antibiotics have demonstrated increased antimicrobial activity (Schmidt. sulfur dioxide. with types of microorganisms. the log probability of growth of a single bacterium at 12°C after 60 d at pH 5. Poerschke and Cunningham.. propylene glycol. 1985.. except nitrite when used against S. 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. 1986a. 1985). sorbic acid resulted in marked inhibition. Robach.

02–0. Selection of the most appropriate method depends on processing procedures.03–0. federal standards of identity permit the use of sorbates as preservatives in more than 40 types of cheese. cheese spreads. 1981.30 In general. animal feeds.02% to 0. Part 133 (2001)]. In the United States.3). sour cream. and convenience (Sofos. higher levels may cause undesirable changes in the taste of most foods.1% may be detectable in some foods.25 0. syrups. product processing. amounts of 0. dry sausages Miscellaneous: dry sausage casings.3%. 1989) (Table 3. cheese dips. In the United States. cheese spreads.R.1% to 0.20 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. 1989.3% are tolerated. pharmaceuticals. packaging materials. objectives to be accomplished. purees. Amounts of sorbate used in foods are in the range of 0. pickles. fresh salads Fruit products: dried fruit.02–0. salad dressings Meat and fish products: smoked and salted fish. cake mixes. jellies.05–0. fudges. These factors include product pH and composition (fat and moisture). toppings. The compounds find application in human foods of all types. margarine. 1989). edible fat emulsion products.05–0.F. including dairy products. Levels (%) 0. fruit drinks. sorbates have found wide application in various foods. 1992). and storage temperature (Sofos and Busta. presence of other ingredients and preservatives.30 0. However. gas atmosphere. equipment available. confectionary Source: From Sofos (1989). and cheese food [21 C. low-calorie drinks Food emulsions: mayonnaise. packaging.10 0. jams. They should be considered as adjuncts to proper sanitation and hygiene. .02–0.4). the extent of the antimicrobial activity of sorbate under commercial conditions is difficult to predict because it is influenced by a variety of factors. type of food. carbonated and noncarbonated beverages. cosmetic products. As yeast and mold inhibitors. juices. yogurt Bakery products: cakes.10 0.05–0. fillings.30 0. spraying or immersing the food material in a solution. DAIRY PRODUCTS Cheese preservation is one of the most common applications of sorbates. mixes. In general. APPLICATIONS Sorbates are widely used food preservatives throughout the world. fruit salads. or addition in a coating or packaging material (Table 3. and sugar and confectionery items (Sofos. olives. but levels as low as 0. dusting with a powder. cottage cheese. concentrates Beverages: still wines.03 0.3 Common Applications of Sorbates as Antimicrobial Agents Products Dairy products: aged cheeses. as is the case with all food preservatives.05–0. relishes. Application methods for sorbates include direct addition in the formulation. The most commonly used forms include sorbic acid and the potassium salt. certain meat and fish products. Although these concentrations have no major impact on food quality. pies. fruit and vegetable products. bakery items. 1989). processed cheeses. doughnuts Vegetable products: fermented. icings.Sorbic Acid and Sorbates 61 TABLE 3. semimoist pet foods. Sofos. 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. sanitation and contamination. and technical preparations that come in contact with foods or the human body.

1976.3 0. depending on the technique used to treat the cheese (i. however. fudges Vegetables: fresh salads (potato.02–0. toppings. which may vary among countries (Lück. is limited to the surface of cheese.1–0. In these applications sorbates not only prevent cheese spoilage by mold. fillings. and domestic and imported cheeses. For application on .1 0. Caciocarallo.3 Spraying Immersion or spraying Dry mixing Direct addition Dry mixing or direct addition Source: From Sofos (1989). preservation objectives. available facilities and equipment.05–0.04 0. Swiss cheese.15 0. however (e. sorbic acid may be incorporated into the cheese curd.05–0.1–0. there may develop a slight objectionable flavor (Aly.5–10 20–40 20–40 2. Kivanç (1992) found that Penicillium was the major genus found on cheddar cheese.03–0. and the improved action of sorbates at high pH values compared to other common preservatives (Lück.06 0.4 Methods of Application of Sorbates Methods Immersion Products Dairy: provolone.05–0.05–0.g. pasta filata.62 Antimicrobials in Food TABLE 3. pickles. slaw.25 0. in which they can interfere with the desirable molds involved in the manufacture of these cheeses.1–0. 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. relishes. Siciliano.1 0. Emmentaler Dairy: cheddar. vegetable.3 0. sauerkraut) Fruits: syrups.01–0.3 0.05–0.4 0. however.3 0. and regulations. but they also protect human health by preventing the formation of toxic mold metabolites (mycotoxins). especially surface mold growth during storage. aging.075 0.05–0. 1981). blue. cottage cheese). beverages. It is obvious. in general. such as Roquefort or blue cheese and Camembert cheese.05–0. 1980. Colby.075–0.05–0. Gruyere.1 0.06 0. In certain products.1 0.1–0. Sorbate application.. drinks Still wines Other: Margarine Semimoist pet food Bakery: pound cake Devil’s food cake Chocolate cake Solution (%) 20–40 2.05–0.15 0.. Addition of sorbate does not significantly affect the organoleptic properties of mozzarella cheese.5–5 5 2. and distribution. Edam. The method of application varies with the type of cheese. dipping or in brine salting).02–0. olives. Sofos and Busta. 1976. Gouda Dry sausage or casings Dairy: Swiss. that sorbates should not be used in mold-ripened cheeses. 1996).5–5 5 Application Level (%) 0.3 0.1 0.3 0.1–0. Sorbates are widely used in preserving cheese owing to the susceptibility of all types of cheese to microbial deterioration. where mold development usually occurs.1–0.05–0. spreads Cottage cheese Bakery: icings. the high activity of sorbates against molds. Monterey Jack.075 0.05–0.1–0.15 0.1 0.3 0. 1980).025–0. Muenster.e.2 0.1 0.

1994). acetic. 1994). including fresh. 1998. 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. or calcium sorbate impregnated into the packaging material or other coatings (e.30% (Lück. initial numbers of yeasts were low and relatively low concentrations of potassium sorbate and sodium benzoate (<150 and <250 mg/kg. when applied to the surface of hard. thermophilus and Lactobacillus delbrueckii var. one of the following procedures may be followed: (1) potassium sorbate in solution or in a brine. 1997). Labaneh with higher initial yeast counts needed higher concentrations of preservative (>300 and >400 mg/kg for K-sorbate and Na-benzoate. potassium sorbate inhibited L. 1976). and the cheese was stored at ≤30°C (Kasrazadeh and Genigeorgis.07% sorbic acid are generally used in direct addition to cheese. Inclusion of sorbic acid in cottage cheese was effective as a preservative but not against L. which can be used to dip.3 g/dm2.0) at levels of ≥0. Therefore. 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. In addition to reducing cheese spoilage and the possibility of aflatoxin formation. potassium sorbate (0. 1999). ripened cheeses. and was especially effective against mold and yeast contamination by Penicillium roqueforti and Mucor miehi. however. potassium. Sofos. 1989. at the relatively high level of 1%. In labaneh (a yogurt product) produced under strict hygienic processing measures and effectively chilled. monocytogenes (Piccinin and Shelef. 1980). bulgaricus. 1996). especially at lower (≤6. Sofos and Busta.1 to 0. wax) used in cheese storage and marketing (Han et al. spray. 1996). Growth of Salmonella in soft cheese was inhibited if the cheese was prepared from milk that was acidified with propionic acid to pH 5. These concentrations are in agreement with use of these preservatives in other dairy products. 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.3%) showed a significant effect in delaying growth of E.g. or wash. monocytogenes survival in two commercial cheese brines (Larson et al.. amounts of 2 to 4 g/m2 are used (Lück. Treatment of mozzarella cheese with potassium sorbate in the kneading water or brine may be more effective than dipping (Aly. and it was initiated through extensive research performed during the early stages of sorbate testing. or propionic acid significantly increased the effectiveness of potassium sorbate. Calcium sorbate may be preferred in cheese preservation over other forms of sorbate owing to its solubility properties. This research demonstrated the effectiveness of sorbates against undesirable organisms without interfering with the desirable . such as cottage cheese and yogurt (Mihyar et al. and pickled vegetables (Sofos. respectively) extended the shelf life by 14 days. soft cheese (Kasrazadeh and Genigeorgis. fermented.. 1995). respectively (Aly.9. 1989). respectively) to maintain stability when kept at 5°C for 7 or 14 days. Levels of 0. coli O157:H7 in queso fresco. Weng and Chen. 1996). and 25°C after 30 days (Kivanç.6°C) temperatures (Kasrazadeh and Genigeorgis. VEGETABLE PRODUCTS The water-soluble salts of sorbic acid are used widely in the preservation of a variety of plant products. potassium sorbate was added to the cheese (pH 6. and (3) sorbic acid. Amounts of sorbic acid permitted and applied in cheese preservation range from 0. 1980. use of sorbates in aged cheeses also reduces labor and other costs through reduction of the need for cheese washing and trimming. 1981)..Sorbic Acid and Sorbates 63 the surface of the cheese.0 with hydrochloric. The appearance of Penicillium verrucosum var. Milk acidification to pH 6. Concentrations applied to cheese surfaces range from 0. Furthermore. addition of potassium sorbate may increase the meltability and improve the fat leakage of certain cheeses (Aly.3%. Addition of potassium sorbate (≤6%) to mozzarella cheese inhibited growth of Streptococcus salivarius var. 1997). The compound is insoluble in fat and only slightly soluble in water.. 15°C.05% to 0.3%) or sodium benzoate (0. whereas when applied to packaging films. 1992). (2) a sorbic acid powder for dusting. 1995). In another study.05% to 0. The use of sorbates in the processing of vegetables was one of the first applications.

sorbate is more effective than benzoate owing to the pH of these products. 0. In sweet cucumber packs. cucumbers and olives). and putrefactive bacteria. Addition of 0. In wine processing. In fruit juice and drink processing. 1980.. sorbate is either added directly into the product or applied to the surface of the product or packaging material. Other vegetable products preserved with sorbates include tomato products and fermented Asian sauces (Chichester and Tanner. and wines..05% and 0.g. 1980).g. Combinations of sorbic acid and sulfur dioxide are also used in the preservation of high-pulp fruit juices.02% to 0. molds..02% sorbic acid in combination with . in high-sugar products (e. Potassium sorbate is preferred because of its solubility. or microbiological deterioration (e. 1988a. coli O157:H7 growth was inhibited in both tryptic soy broth and in apple cider by sorbic acid applied at 0. In these products sorbic acid acts as the microbial inhibitor and sulfur dioxide prevents oxidation and enzymatic spoilage (Lück. preserves.64 Antimicrobials in Food fermentations. In these products. Use of sorbates at levels between 0.. The higher moisture content in these products is preferred by the consumers. together with sulfur dioxide and pasteurization.02% to 0. At these concentrations the selective action of sorbic acid allows growth of the desirable lactic acidproducing organisms. however. Lück. The lower the moisture content of the product (e. Sorbate levels in the range of 0. Other fruit products that may be preserved with sorbates include maraschino cherries and strawberry puree.. prunes. Costilow et al. 1972. salt concentration).10% are adequate in improving the preservation of soft drinks.1% sorbate and 0. 1993). jams and jellies). jellies. 1994). Potassium sorbate and sodium benzoate were highly effective in reducing yeast and mold populations in tomato juice (Bizri and Wahem. Good wine preservation is achieved with concentrations of 0. 2001). 1994). sorbate acts as an inhibitor of yeasts. Sorbate concentrations as low as 0.02% to 0. Because potassium may result in potassium bitartrate precipitation in some wines.02% to 0. enzymatic. In sweet relishes. 1980). 1980).b). coli O157:H7 and result in spoilage of the product (Zhao et al. depends on products and specific fermentation conditions (e. or bacteria. smaller quantities of sorbic acid are adequate for preservation because of the synergistic action of sorbate with sugar (Lück.05% sorbates in the fermenting and holding tanks. Depending on the particular product. jams. such as yeasts.05% to 0.. Also.g. but it makes them susceptible to mold and yeast spoilage. fermentation). figs). 1955.5% acetic acid completely inhibit microbial spoilage at sucrose concentrations ranging from 2% to 40%. the pH values of fermented green olives favor the use of 0.10% may be used to preserve refrigerated fresh salads. which may influence the survival of E.1% potassium sorbate substantially inhibited E. sodium sorbate may be used to avoid the problem. 1989).1% sodium benzoate and 0. Concentrations as low as 0. Levels of 0. dried fruits). Mold and yeast spoilage is also prevented by sorbates in pickled vegetable products (e.05% and as such may be an option that processors have to reduce contamination in their products (Koodie and Dhople. fruit cocktails.20% inhibits growth of organisms not desired in vegetable fermentations. the lower is the sorbate level needed for preservation. Selection of precise levels of sorbate.. Also. Lück. Sofos..g.05% sorbate to a 15% to 20% brine retards yeast scum formation. sorbates are used to prevent refermentation (Parish and Carroll. 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. fruit juices and syrups.05% are adequate for preserving high-moisture dried fruits (e. to inhibit chemical.g. coli O157:H7 in apple cider. Another study showed that E. Potassium sorbate was found more effective than chitosan in inhibiting growth of Aspergillus niger on lowsugar candied “kumquat” (Fang et al..g. FRUIT PRODUCTS Fruit products preserved with sorbates throughout the world include dried fruits. Use of the combination of 0. 1952. raisins.02% to 0. molds. sorbates are used mostly at the preprocessing stage. beverages.10% sorbate reduce the number of bloaters and poor seed cavities. however. while additionally suppressing the growth of yeasts and molds.

brown-and-serve products). such as Japan and Korea (Sofos. especially when used in combination with calcium propionate (Tellez-Giron et al. BAKERY PRODUCTS Although propionic acid-based preservatives are the most widely used compounds in preserving bakery goods. 1989). In addition to undesirable molds.Sorbic Acid and Sorbates 65 0.03% are not recommended in wine preservation because they may impart an off-flavor to the delicate taste of wine. In fruit preserves (e.. In addition to preventing mold spoilage. and sulfur dioxide protects against enzymatic and oxidative changes and bacterial fermentation. sorbates are also valuable because they are active at higher pH values and effective against molds (Chichester and Tanner.. Tompkin et al. examined its activity against various pathogenic bacteria in an uncured. in cream pies (Schmidt et al. the dipping method of application is more effective (Sofos. figs).30% sorbic acid are adequate for preserving bakery products. 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. however. unless the amount of yeast and the leavening time are increased. sorbates may also be useful in preventing aflatoxin formation (Lück. including cakes and confectionery.. Sofos. Tompkin et al. toppings.. mold inhibition may be achieved by sprinkling sorbate on the surface of the product. 1972. Often.. may be more suitable for use in fruit products than other common preservatives because of their milder organoleptic properties and their neutral taste. Sorbates. 2000). Sorbates are also used in the preservation of pie crusts and fillings. jams. which is a mixed anhydride of sorbic and palmitic acids that has no direct antimicrobial activity. 1989. sorbate also inhibits rope-forming bacteria and pathogens. 1989). In 1974. York and Vaughn. 1980). and other beverages. the derivative sorboyl palmitate. 1999). 1989). jellies. It has also been used in champagne preservation (Sofos. 1980).004% sulfur dioxide. 1969. Sorbate can also be added before bottling the wine and is very valuable in the preservation of sweet wines. aureus.002% to 0. soft drinks. 1988). In such applications sorbate may be used in the coating oil or the pan grease (e. (1974) .10% sorbate inhibit the spoilage of cake icings.g. However. 1954). and doughnut mixes (Sofos. Sorbate levels above 0. Yeast-leavened products may be protected with sorbate spraying after baking.g. Use of sorbates must be avoided in products raised by yeasts.05% to 0. Use of this compound has the advantage of preserving yeast-leavened bread without interfering with the fermentation process (Lück. which is especially important in preservation of juices. upon heating in the baking process. may also be used. Lück. cooked sausage product.03% to 0. 1952. 1980). 1989). in general. refrigerated dough products. sorbates are used in combination with benzoate in preserving fruit juices and drinks. sorbates are more common meat preservatives in other countries. A few sporadic studies in the 1950s and 1960s also indicated its potential as a preservative for meat products (Sofos. To avoid the effect of sorbates on desirable yeasts in yeast-leavened products. 1976. muffins. and fillings. 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. Concentrations of 0. the compound is chemically degraded and releases sorbic acid after the yeast-leavened dough is raised. pizza shells and toppings. 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. such as S. Concentrations of 0. and marmalades). Sorbate acts as the inhibitor of yeast refermentation during storage. For this purpose a solution of up to 10% potassium sorbate may be used to protect unrefrigerated dry sausages. In dried products with an irregular surface conformation (e. Sorbates should be valuable in baking powder-raised products. 1989). Potassium sorbate increased the shelf life of acidified tortillas.g...

1989) and cooked pork chops (Prabhu et al. U. Vareltzis and Buck. Department of Agriculture.. Clostridium sporogenes. Ivey et al. Yersinia enterocolitica. National Academy of Sciences 1982). Robach. Wagner and Busta. Nelson et al. Escherichia coli. 1983. Amundson et al. botulinum.. 1981. It should be noted. 1982. These studies have been thoroughly reviewed by Sofos and Busta (1980). The antimicrobial activity of sorbate in these products has been enhanced when combined with nitrite. Greer. 1980. and it can react with amines to form carcinogenic nitrosamines during meat product processing and cooking (Sofos et al. 1983. In addition to C. 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. 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. 1980. phosphates. low pH.b. 1979a. Robach et al. 1982). 1981a. aureus. National Academy of Sciences. 1982).. Salmonella. 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 . Draughon et al.b.. botulinum.S. Robach and Sofos. Serratia liquefaciens. 1983. 1989). (1979a).. Bauermann.. Pierson et al. aureus and delayed growth and toxin production by C. Pseudomonas. Sofos et.. 1979b). and the National Academy of Sciences (1981. 1979. Brochothrix thermosphacta. that no other studies reported undesirable flavors or allergic reactions (Sofos. Gray et al. 1978. 1989. al. Bacillus licheniformis. Nitrite was reported to be a potential carcinogen. 1979a. 1982. antioxidants. In addition.... In addition.. Sofos (1981. 1978. 1981). botulinum in cured meat products. Morad et al. uncured cooked sausage (Tompkin et al. 1981. 1981. 2000). 1974) was followed by extensive testing that examined the action of sorbates against clostridia and other pathogens in a variety of fresh... Lactobacillus. 1977. 1983). 1979. 1980c. eating quality. Cunningham. sodium chloride. Zamora and Zaritzky. such as color and flavor (Frank. 1989). Wagner and Gehrke. Based on the results of the various studies. pork slurries (Roberts et al.3%) had no major adverse effects on sensory qualities. McMeekin et al. sorbate was proposed as a means of reducing nitrite and nitrosamine levels in bacon while maintaining antimicrobial activity and inhibition of C. Hallerbach and Potter. Tompkin et al. 1982. and safety were examined. Paquette et al. Rice and Pierson. The antimicrobial activity of sorbate was demonstrated in bacon (Ivey et al.c. other pathogenic and spoilage bacteria inhibited by sorbate in various meat products include S.. 1981. the effect of sorbates on overall product shelf life. 1979b. Perry et al. Sorbate in meat products (<0.. 1982. and hamburger beef (Yamamura et al. Vareltzis et al. 1979.. and lipolytic organisms (Kaloyereas et al.S. 1974). Robach and Sofos (1982).b.. Elliot et al. 1984). 1979. 1983).66 Antimicrobials in Food showed that sorbate retarded growth of Salmonella species and S.. 1979a. 1974. Huhtanen et al. 1982. 1983... did not take effect following a report that consumption of experimental bacon resulted in allergic-type reactions in certain individuals (Berry and Blumer. Bacillus cereus. nitrosamine formation. raw (Mendonca et al.. uncured. Wagner et al. 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. 1981).. 1979. To and Robach. poultry products (Robach et al. 1980. psychrotrophs.. 1983). 1980b). 1982.. 1981. 1984. 1987a.. Berry et al.. 1978. 1961. 1984. 1988). 1984. 1985. and increased carbon dioxide atmospheres (Sofos. 1990). Petaja et al. Sofos.. Kemp et al. 1978. Huhtanen and Feinberg. 1981. comminuted pork products (Ivey and Robach. and processed cured meat products (Sofos. 1979b.. beef steaks (Unda et al. however. raw beef and pork. 1978. U. Robach and Ivey. low storage temperature.. sliced bologna (Chang et al. Sofos et al. 1982.. beef and pork frankfurters (Sofos et al. low oxygen. 1982.. Chambers et al. acids.. Clostridium perfringens. This proposal. Hall and Maurer. 1980a. Chang et al. 1980b. 1978. Sofos et al.. botulinum spores during temperature abuse of the product. Sofos et al. Myers et al. 1986b.. and uncured poultry meat and carcasses... however.. Robach et al..b).. Products in which these microorganisms are inhibited include bacon and other cured meats.. This work (Tompkin et al. 1980a.. Berry and Blumer. mesophiles. cured poultry products. 1964. Robach. 1980b). 1979b. Bushway et al. Department of Agriculture.

5% did not adversely affect the germination energies and germination capacities of sorghum grain and did not significantly affect malting loss.. Application of potassium sorbate with bifidobacteria extended the shelf life of whole and peeled shrimp by 3 days (Al-Dagal and Bazaraa. Bremner and Statham. 1978. in which levels of up to 0. 1995). however. Sofos and Busta. 1978. 1982. 1983. Mexican-type hot sauces. 1982. tangerine sherbet base. 1972. In Asian countries sorbates are commonly used in combination with other preservatives to preserve fish sausages and similarly processed fish and meat products. 1989). This optimal concentration of sorbic acid coincided with the lethal dosage for most of the associated fungal microorganisms during malting. 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). delicatessen items. the esters of sorbic acid (e. Lynch and Potter. Bremner et al. are also effective in preventing mold growth on animal feeds during storage.03% to 0. Lück. tomato juice. Sorbates may be particularly important in intermediate-moisture (25% moisture content) pet foods. One study. 1972.Sorbic Acid and Sorbates 67 of bacon treated only with nitrite. Debevere and Voets. and pancake batter (Sofos. inhibiting their growth... 1983.20%. 1988). Fey and Regenstein. 1989).. Shaw et al. diastatic powers. 1976.. Other food-related applications of sorbates include sugar-based and confectionery products at concentrations of 0.g. Robach and Hickey. Several studies have reported improved preservation of various types of fish with sorbate (Geminder. 1972. Sorbic acid levels in the range of 0. and cold and hot water extracts of the malt samples. Chung and Lee. 1989). shredded cabbage. it may be desirable to apply sorbic acid during malting (Ogundiwin et al. Samples treated with sorbic acid lost biological activity within a few days of aerobic storage (confirmed by aerobic .1%) was used (Fletcher et al. potato chips. and animal feeds (Sofos. 1980. strawberry puree. 1989).10%.. in which the emulsifiers may be inactivated by nonionic compounds (Chichester and Tanner. 1972. 1991). The literature contains reports on several other food products that may be preserved with sorbates (Sofos. Sharp et al. Lück. thus. In certain instances and for better preservation. which is longer than the preservation (31 days) achieved by the most effective bacteriocin (nisin) tested (Einarsson and Lauzon. Sorbic acid at a concentration of 0. at which they prevent growth of molds and osmophilic yeasts in items such as fillings for chocolate and pralines (Chichester and Tanner. 1959. pineapples. and similar products.1%) solution preserved brined shrimp for 59 days. cut squash. Regenstein. 1981. Lück. 1981). 1986). Sofos. botulinum in shucked scallops developed slightly more rapidly at 27°C in vacuum packages when sorbate (0. cucumbers. as well as fermented plant foods (Chichester and Tanner. such as benzoates. 1980. indicated that toxin production by C. orange peels. sour cream. 1976. such as margarine. A sorbate (0. dried cod). garlic oil. They include high-moisture marshmallow candy. MISCELLANEOUS FOOD PRODUCTS Sorbates are commonly used in the preservation of oil-in-water or water-in-oil emulsion-based products.1%)–benzoate (0. prepeeled carrots. In addition. Statham and Bremner. depending on moisture content. 1983.g. mayonnaise.30% can be effective mold inhibitors.05% to 0. This should assure mycotoxin-free animal feeds and thus eliminate any hazards associated with mycotoxin-contaminated feed. 1982. the flavors of both types of bacon were equally desirable (National Academy of Sciences. 1999). OTHER APPLICATIONS Other items that may be preserved by sorbic acid and its salts include pharmaceuticals. Sorbates are also used to prevent mold growth on the surface of dried and smoked fish (e. cosmetic products. sorbic acid may be mixed with its potassium salt or with other preservatives. 1983. 1982). 1976). especially those of the emulsion type.. salad dressings. ethyl and propyl) may find applications in cosmetics and pharmaceuticals. Robach and Sofos.

Death of microorganisms exposed to high concentrations of preservatives. 1985.. and function of cell membranes and inhibition of transport functions and metabolic activity (Sofos. whereas higher amounts may result in death. Several mechanisms of inhibition of metabolic function by sorbate have been proposed. when the sorbate hurdle is reduced or removed. Sorbate inhibits cell growth and multiplication as well as germination and outgrowth of spore-forming bacteria. Seward et al.. 1997). It was also postulated that sorbate probably inhibits a postgerminant binding step in the process of germination. which could be overcome to some extent by addition of magnesium ions. 1996). adhesives. increased numbers and variable sizes of mitochondria. and glues (Lück. Sofos. 2001). and it may be possible that several of them may be functional under various conditions. 1986. the outer cell wall was absent in many areas (Ronning and Frank. Treatment of Alteromonas putrefaciens with sorbate at pH 7. strains. Sofos et al. MECHANISMS OF ACTION Sorbate concentrations used in foods (<0. 1989). 1989). 1978. molds. 1985). 1988. Cells of C. Other proposed mechanisms of inhibition of microbial growth by sorbate include alterations in the morphology. Sofos et al. and not triggering of germination. Freese and Levin. 1986. enterobacteria. 1985). 1989. and it was thus concluded that it inhibits spore commitment to germination. 1989). irregular nuclei. 1989). did not influence the susceptibility of the bees to the fungus Ascosphaera aggregate (Goettel and Duke. 1985a. environmental conditions. 1979d. therefore. Under certain conditions sorbates have changed the morphology and appearance of microbial cells (Statham and McMeekin. 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. type of substrate. resulted in minicells. sporogenes were usually filamentous and nonseptate but with distorted shapes characterized by numerous bends and bulges. Such changes have been observed in yeast cells as dense phosphoprotein granules. with bulbous formation and defective division (Seward et al. has been attributed to generation of holes in the cell membrane . The preservatives sorbic acid and methyl paraben. 1989). botulinum were long. There was also evidence of other membrane damage in sorbate-treated cells (Statham and McMeekin. Blanching (80°C) and the application of 0. Ronning and Frank. Sorbate-treated cells of C. Sofos. Smoot and Pierson. 1986). 1989). and vacuoles. Sofos. 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.3%) usually inhibit microorganisms. the surviving microorganisms may resume growth and spoil the food. but the exact mechanisms of antimicrobial activity are not well defined (Freese et al. reduced ensiling losses and increased the feed value characteristics of citrus pulp silages (Filya et al.. 1982. Another study reported that sorbate inhibited spores triggered to germinate or after germinant binding (Blocher and Busta. 1986.0 increased cell hydrophobicity and cell wall lysis on exposure to lysozyme.68 Antimicrobials in Food degradation of starch and triglycerides). such as sorbate. 1981). and the type of food processing. Inhibition has involved various species of bacteria in laboratory culture media and in foods and has been influenced by species. pH. Consequently... Several studies have indicated that sorbate inhibits bacterial spore germination (Sofos et al. Other reported uses of sorbates include preservation of tobacco products. Wagner and Busta. Blocher and Busta. Septation..1% sorbic acid to citrus pulp were effective in inhibiting yeasts. including types and species of microorganisms. 1982.b). Although the presence of enzyme activities was shown. and sorbate concentration (Sofos. and clostridia common in the spoilage of silage and.. 1973. Although the significance of such alterations is unknown. Published data have suggested that sorbate acts as a competitive and reversible inhibitor of amino acid-induced germination (Smoot and Pierson. there was no detection of pectinolytic activities (Mayer and Hillebrandt. and the inner cell wall appeared to be thickened. when present. 1988). 1976. integrity. Sofos.. 1989). 1981. when used individually or in combination as preservatives of an artificial diet for leafcutting bees.

. 1978). such as sorbate. Freese et al. aureus metabolism were significantly affected by sorbic acid in a concentration-dependent manner (Sayeed and Sankaran. succinic dehydrogenase. a membrane-active compound. Sofos and Busta. enolase. 1961. inhibition of the electron transport system. 1978). 1964. 1992). α-ketoglutarate. 1981).Sorbic Acid and Sorbates 69 (Freese and Levin. Harada et al. 1989). Sofos. Troller. 1961). 1991). nutrient uptake. 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. Some reports. Sorbate has decreased the assimilation of carbon from several substrates. Inhibition of nutrient uptake may be the result of neutralization of the proton-motive force (PMF) needed for substrate uptake. oxaloacetate. 1959) that sorbate inhibits the sulfhydryl enzymes through the formation of a thiohexenoic acid derivative (CH3-CH=CH=RSCH-CH2CO2H). may interfere with substrate and electron transport mechanisms. 1995a. Binding and inhibition of CoA should result in inhibition of oxygen uptake and microbial growth (Sofos. however. 1964. lactate. growth. 1954b. including glucose. 1968. 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. 1973. This effect may be occurring through incorporation of sorbic acid. Sheu and Freese. Sorbic acid has inhibited the uptake of glucose (Yousef and Marth. microcalorimetric measurement of the effects of sorbic acid on S. 1992). 1991). 1963).b). Azukas et al. fumarase. 1991).2%) for use in foods. into the cell membrane. and ficin (Melnick et al. Whitaker. 1972. and inhibition of metabolic energy utilization by the amino acid transport systems (Sofos. succinate. Lipophilic acid food preservatives. ethanol. and acetaldehyde (York and Vaughn... . inhibition of transport enzymes. Inhibition of catalase by sorbate was attributed to the formation of sorbyl peroxides through the autoxidation of sorbic acid. 1992). 1965). The highly reproducible and characteristic thermograms of S. Thus. where it may cause steric disorganization of active membrane transport proteins (Sofos. 1972. Sorbate is known to inhibit the in vitro activity of many enzymes. Harada et al.. inhibition of synthesis or depletion of ATP. Sorbate inhibits the activity of several enzyme systems. have indicated no inhibition of enzymatic activity by sorbate (Sofos. 1970. acetate. 1963. 1989). 1989. 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. 1983) and amino acids (Hunter and Segel. catalase. 1965). Inhibition of cell metabolism by sorbate in these studies may have been the result of inhibition of enzymes. 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. 1973). pyruvate. Sheu et al.. 1960. aspartase.and/or β-carbon of sorbate (Martoadiprawito and Whitaker. Another postulation has indicated that sorbate may act competitively with acetate at the site of acetyl coenzyme A (CoA) formation (Wakil and Hubscher. or a specific inhibitor of metabolism (Azukas et al. It was also proposed (Whitaker. Both the peak heat and total heat dissipation profiles were affected by 50% of the common concentration (0. 1985). cell metabolism. 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. These peroxides then would inactivate catalase (Troller. which may lead to disruption of vital processes involved in transport functions. and replication. malate dehydrogenase.. Additionally.0 in Alteromonas putrefaciens (Sayeed and Sankaran. Inhibition of substrate transport into the cell by uncoupling it from the electron transport system results in cell starvation (Deak and Novak. especially sulfhydrylcontaining enzymes (Kouassi and Shelef.. α-ketoglutarate dehydrogenase. Tuncan and Martin. 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. 1973. as well as the electron transport system (Anderson and Costilow. Sorbic acid could conceivably inhibit microorganisms as a weak-acid preservative. 1975. Martoadiprawito and Whitaker. 1959. Enzymes inhibited by sorbate include alcohol dehydrogenase. Freese and Levin. or various transport systems. York and Vaughn. 1968. 1963. or unsaturated fatty acid.

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. 2000). 1996). Similar degrees of inhibition by sorbic alcohol and sorbic aldehyde suggest that sorbic acid does not act as a weak-acid preservative. Indeed. Conversely. as indicated. 1988. 1997). Thus. In another study using a novel method to measure pH in growing cells.. Sofos. Preincubation of S. 1980. which is one of the components of the PMF (Sofos. 2000). 1998)..... 1992. cerevisiae (Piper et al.8 elicited a set of metabolic effects on sugar metabolism. Salmond et al. According to this theory. Hunter and Segal. In lower pH environments. 1998). Inhibition of uptake of such components is believed to induce a stringent-type regulatory response in the cells. In fact. Sorbic acid in its MIC releases far fewer protons than other weak-acid preservatives. found no decreases in ATP in the presence of sorbic acid (Sofos.70 Antimicrobials in Food which act as molecular chaperones (de Nobel et al. Because inhibition of microbial growth by sorbate has been associated with decreased levels of ATP (Harada et al. 2000). A stringent response involves readjustments occurring in bacteria when amino acids become limiting or their specific ratios are disturbed (Sofos. Previous studies with bacteria. 2000). 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 became . which decreases the intracellular pH and dissipates the PMF of the membrane that energizes transport of compounds such as amino and keto acids.. 1980). 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. growth inhibition correlated with an increase in the intracellular adenosine diphosphate (ADP)/ATP ratio as a result of increased ATP consumption by the cells. such as increased proton pumping by the membrane H+-ATPase. 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. Therefore. 1989. Cells of Z. 1989). 2000). 1998). Specific inhibition of metabolism is also unlikely because inhibitory activity is shared by several small carbon compounds. weak-acid preservatives such as sorbic acid and lead to starvation of cells from compounds that are transported actively by the PMF (Eklund. Inhibition of microbial growth by sorbate may be based on neutralization of the PMF that exists across the cell membranes by lipophilic. undissociated sorbic acid acts as a protonophore. 1983. resulting in inhibition of growth but in maintenance of cell viability (Sofos. 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.. a shift in charge may potentially take place and lead to a decrease in the net negative intercellular charge. the difference across the cell membrane is large. This could then discharge the pH gradient required for ATP formation according to the chemostatic theory of oxidative phosphorylation (Sofos. 1968. however. 1973). little correlation was found between reduced growth on exposure to sorbic acid and reduction of intracellular pH (pHi) (Bracey et al. cerevisiae yeast cells in the presence of benzoate or sorbate at an extracellular pH value of 6. it has been shown that sorbic acid acts at high pH where weak-acid preservatives are not expected to be active (Stratford and Anslow. 1987. 1998).. 1984. As the hydrogen influx exceeds the pumped efflux. 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. Ronning and Frank. 1997). 1985. and the amount of acid entering and dissociating in the cytoplasm is higher. 1994). a pump recently identified as the Pdr12 protein is induced by sorbic acid in S. 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. This was partly attributed to the activation of protective mechanisms. which ensured that pHi did not decline when cells were exposed to sorbic acid (Henriques et al. 2001).

the growth rate and liver weight of the animals increased (Demaree et al. sorbate is considered a GRAS compound. These effects can be summarized as follows: (1) reduced glucose consumption. its effect on the other component of the PMF. the LD50 for common salt (NaCl) is 5 g/kg body weight. this pattern resulted in prevention of glucose-induced switch of metabolism from a gluconeogenic to a glycolytic state. On the whole. 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. 1976). Sofos. Thus. Although dissociation of the acid inside the cell may eliminate the pH difference across the membrane. however. and (5) block of catabolite inactivation of fructose-1. 1954a. even at pH values near neutrality. 1973. Acute toxicity studies with rats have determined LD50 (mean lethal dose) values for sorbates in the range of 4. is smaller. although less efficiently. uptake of the compound based on pH difference across the cell membrane would not be adequate to explain the observed antimicrobial activity. depending on the type of microorganism. but not of cytoplasmic malate dehydrogenase (Burlini et al. . Demaree et al.2 to 10. 1980. (3) suppression of glucose-triggered peak of hexose monophosphates. in such situations.and fructose-phosphorylating activities. substrates. Sofos. the World Health Organization has set the acceptable daily intake for sorbate at 25 mg/kg body weight per day. 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. sorbate is considered one of the least harmful preservatives in use. Lück. unlike former assumptions.. as well as influence on metabolism. Sorbic acid has been shown to inhibit microbial growth. 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. 1955). and environmental conditions (Sofos. Food and Drug Research Laboratories. 1948.6-bisphosphatase and phosphoenolpyruvate carboxy-kinase. 1992). On extending the feeding period to 120 days. 1955. The compounds were fed to various animal species for determination of acute toxicity.6-bisphosphate. Overall. (2) inhibition of glucose. Lück. these studies demonstrated the relative harmlessness of sorbates and their relative superiority in safety compared to other chemical additives. 1998.5 g/kg body weight (Smyth and Carpenter. which is the difference in electrical potential.Sorbic Acid and Sorbates 71 apparent after the subsequent addition of glucose. For comparison. Rat feeding studies have indicated that a dosage of 10% sorbic acid in the diet could be tolerated for 40 days (Lück. and because it is a metabolizable fatty acid. 1998.. Overall. However. 1954a). it is believed to be adequate to energize the uptake of substances needed for cell maintenance and growth. 1993). Reasons for this conclusion include the following: 1. 1993). 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... TOXICOLOGY AND SAFETY In the United States. based on acute toxicity. 1980). The relative nontoxicity of sorbates was established early in their testing as food preservatives. but 8% sorbic acid resulted in an increase in liver weight (Deuel et al. 1989). 2000).. carcinogenicity.. 1989. and teratogenicity after short. Thus. 3. Bracey et al. (4) substantial reduction of glucose-triggered peak of fructose 2. 2.or longterm exposure. the mechanisms involved in inhibition of microbial metabolism and proliferation by sorbic and other similar lipophilic acid preservatives appear to be different. 1954a. Additional studies with rats and dogs have shown no damage with feeding 5% sorbic acid for 90 days. Thus. Deuel et al.. 2000)..

which is the average pH of most meat products. It has been regarded that amino-compounds. This reaction was demonstrated in model systems but. not at 6. Department of Agriculture. Sorbic acid has a conjugated system of double bonds. Considering the average use levels of 0.. sometimes giving mutagenic products (Ferrand et al. Osawa and Namiki. 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. 1975.. may undergo nonenzymatic browning reactions when stored with sorbic acid at a favorable range of aw and temperature (Quattrucci and Masci. Some sensitive individuals. 1989). leading to decreased amino acid availability (Quattrucci and Masci. Under conditions typical of . no direct relation could be proved between symptoms and specific ingredients used in formulating the bacon (U. and vegetable juices and is inactivated by heat (Kada. Berry and Blumer. National Academy of Sciences. No abnormalities have been observed. 2000a. Kito et al. does not rule out the possibility that high concentrations of sorbate may act as irritants to certain susceptible individuals (Sofos. however.b). 1982. Namiki. Because there is evidence of reactions between nitrite and fatty acids (Benedict. Dejobert et al. but it indicates that most people are not affected by sorbic acid applied to the skin. This. Department of Agriculture. 1981. sorbates have been extensively examined for skin tolerance (Lück. Some taste panelists reported certain “allergic-type” symptoms after tasting uninoculated experimental bacon (U. botulinum. 1976). The literature is contradictory. 1982. As preservatives for cosmetics and pharmaceutical products.S. of course. Average sorbic acid concentrations for skin irritations are in the range of 1%. show skin irritations when exposed to sorbic acid (National Academy of Sciences. Hendy et al. 1992). 1980. 1979. Khoudokormoff. and (3) mutagen formation is inhibited by such ingredients as ascorbic acid. Sorbates were found to react easily with proteins.. does not appear to create toxicologic problems. 1989). Shtenberg and Ignat’ev. 1979. no such symptoms were observed by other individuals who tasted sorbate–nitrate experimental bacon from other studies. various studies have indicated the following: (1) nitrite reacts with sorbic acid to form mutagens optimally at pH 3. Osawa et al. however. 1981. 1982). Patrizi et al. Sofos and Busta. the potential for such irritations in commercial products is minor.S. to some extent. 1979b.. In general. forming high-molecular-weight products that do not seem to result in a nutritional loss. Walker (1990) concluded that sorbate use. Lück. it may react with nitrite in products such as cured meats. 1994).5. 2001). There has been some concern about possible sorbate–nitrite reactions when added to the same substrate (Sofos. 1993. Although it was implied that sorbate might have been involved in producing those symptoms. 1983. 1989).. Thus. 1977. which makes it susceptible to nucleophilic attack. apparently because of an increased caloric intake resulting from the metabolizable sorbic acid. 1974.10% to 0. (2) formation of the c-nitroso mutagens requires the presence of excess nitrite. 1992). 1968. 1981).. 1981.. and in some instances the growth rate of sorbate-fed animals increased significantly. 1999). No carcinogenic or mutagenic effects have been observed with sorbate alone (Dickens et al. 1980) and because sorbic acid is an unsaturated fatty acid. The products of such reactions have the potential of being mutagenic (Sofos. 1975. 1981. Sofos. 1992).0. 1980. and some very sensitive individuals may show irritations even at lower levels (Lück. probably because the acid environment of the stomach leads to the breakdown of the sorbic-protein adducts (Quattrucci and Masci.. Gaunt et al. 1979b). cysteine. 1974.. 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. 1980a.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. it may apply also to more complex systems such as foods. 1976. and Robach and Adam (1980) were able to induce such symptoms from panelists consuming commercial bacon manufactured without sorbate. 1982). 1976. Food and Drug Research Laboratories. In addition. 1980. Litton Biometrics. in general. Robach et al. 1970. 1978. including lysine and glutamate. 1986.30% in food processing. 1981.

and propionate (Sofos. spectrophotometry. 1989). Massey et al. Lathia and Schellhoeh. to cyclic derivatives resulting from double addition (Ferrand et al. wine. selective gas diffusion. .10) by the color reaction with αthiobarbituric acid (Roy et al. The spectrophotometric (ultraviolet/UV. 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. method 975. 1981a.. whereas combinations of various treatments have also been used to improve extraction and reduce interference (Sofos. it involves measurement of absorbance at 260 nm (250 to 290 nm). The most widely used methods. 1997). 1976. bromometry.. No synergism in acute toxicity has been detected for combinations of sorbic acid with various other additives. including parabens. Detection methods require quantitative extraction and separation of sorbic acid from the food material without food ingredient interference (DeLuca et al. 1972. 1982. Potential injury in cell hepatocytes induced by potassium sorbate was prevented by antioxidants (Sugihara et al. and chromatography. 1973.. 2000). In some foods.. 1980). Extraction by steam distillation has been used extensively. enzymatic. or direct analysis has been used (Sofos. 1981. 1989). cheese. colorimetry. method 975. however. 1978. 1982.. 1995. 1955.. 1962. including fruit products. Overall. 1983. 1976. cheese (AOAC Int. 1962) and spectrophotometric (Melnick and Luckman.. 1988). 1989). at 20°C.. After development by Melnick and Luckman (1954a). successive extractions with ether. 1989). 2000a. that certain studies have reported inhibition by sorbate of carcinogenic nitrosamine formation in model systems (Tanaka et al. Mandrou et al. AOAC Int. Larsson. Stafford.b). involving the Ames test and genotoxicity studies with HeLa cells and on plasmid DNA. 2000). 1995. Rao et al. Several modifications have been proposed as useful in avoiding interference and in improving the accuracy of the steam distillation procedure (Luckmann and Melnick. Sofos. benzoate. 1972. and methylene chloride.b). filtration. showed that none of the products studied presented mutagenic or genotoxic activities (Ferrand et al.. Sofos. 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. at 50°C. Wilamowski. 1983. Sofos. bakery items. to linear monoadducts and. Puttermans et al. Sofos. 1981. It should be noted. dialysis. Holley and Millard. The method is simple and usually very specific (Lück.b.22).15 and 975. 1989). and wine (AOAC Int. and isooctane. sorbates appear to be one of the safest food preservatives available. 1999. methods 971.. however.. 1980). Noda et al.. absorption) procedure has also been used in many foods. 1989). the method was modified by Alderton and Lewis (1958). and double distillation and ether extraction (Tjan and Konter. in cyclic interaction products. Special extraction and purification steps have been proposed in the literature to reduce interference problems (Sofos.31). had no effect on nitrosamine formation from aminopyrine and sodium nitrite in animal stomachs (Kawanishi et al. The derivatives of sorbic acid are also relatively nontoxic. sodium hydroxide. Extraction methods include acid-steam distillation.. have been colorimetric (Schmidt. however. did not protect against the toxic effects of other substances (Daoud and Griffin. Sorbic acid. although chromatographic methods have gained acceptance in recent years. 1989). Such combinations have involved extraction under acid conditions from the steam distillate and reextraction with sodium hydroxide.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. and sausage products (Maxstadt and Karasz. however.. polarography. Chung. Sofos. Montano et al. 2000a. 1960.. dialysis and solvent extraction. Sorbate. Mutagenesis studies. DETECTION AND ANALYSIS Analytic methods used or tested for qualitative and quantitative detection of sorbates in foods include acidimetry. dichloromethane. The formation of new products by addition reactions using ethyl sorbate and various amines led.. and solvent extension using ethyl or petroleum ether. 1974. Yamamoto et al. 1980). 1954a).

In addition.. caffeine. 2001). It is obvious. Solid extraction has been used as a cleanup procedure for the determination of sorbic acid by liquid chromatography in fruit products (Mandrou et al. method 974. The method is an official first action procedure for ground beef (AOAC Int. and this status has been reaffirmed (U. theobromine.16) for sorbic and benzoic acids in foods (AOAC Int. 2002). sorbates in the United States . Dobiasova et al. Thus. 2000). 2000). Moreover. sorbic acid and potassium sorbate are GRAS. In the United States. 2000).. and theophylline and as such may be a beneficial alternative to conventional HPLC. Castineira et al. 1999).. In addition. A gas chromatographic method is a first action procedure (AOAC Int. preservatives. The literature contains numerous reports of chromatographic methods used or evaluated for determination of sorbate in food products (Sofos. Inhibition of microorganisms has also been evaluated as a procedure for the qualitative detection of sorbate (Ellerman. HPLC was found to be adequate in obtaining accurate stability data for sorbic acid in creams (de Villiers and Bergh.74 Antimicrobials in Food 1989). 1978) by a select committee of the U. 1999. They include gas chromatography. 1989). such inhibition may be caused by a number of other inhibitors. The use of reversed-phase highperformance liquid chromatography (RP-HPLC) was found to be a simple. method 980. and other substrates (Cancalon. Food and Drug Administration. 1995). 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. have reported that cleanup procedures did not improve determination by liquid chromatography (Benassi and Cecchi.. Capillary electrophoresis was also applied for detection in citrus juices. however. The method was found to be fast. and micellar electrokinetic capillary chromatography (MECC) and ion chromatography. ion chromatography was effective in simultaneously determining artificial sweeteners. where the absorbance is measured at 300 nm and is specific for sorbyl CoA. 2000. considering their extensive testing. 2000). 1996). Microdialysis was used to extract sorbic acid and benzoic acid from food to be separated and detected by HPLC (Mannino and Cosio.. low toxicity. 1977). This major use may expand in the future. and advantages over other preservatives. 1998). 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. 1990).08) and dairy products (AOAC Int. Department of Agriculture. precise. method 983.10) (AOAC Int. REGULATORY STATUS Several forms of sorbate are allowed for use in a variety of foods throughout the world (Sofos. wine. Others. 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. 2000). In addition.S. especially for high sample throughputs (Hofer and Jenewein. which is proportional to sorbic acid levels in the sample.17) and final action procedure for wines (AOAC Int. The reaction is quantified by irreversibly hydrolyzing pyrophosphate to phosphate in the presence of inorganic pyrophosphatase. thin-layer chromatography. and reliable and is thus well suited for routine determinations. 1998.S. 2001). 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. that in addition to sorbate.. 1989). 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. method 974. Mercier et al.. 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. 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. it can be used for the analysis of intensely colored juice samples. rapid method with high reproducibility for determining sorbic acid and other additives in fruit juices (Zou et al. highperformance liquid chromatography (HPLC). however. paper chromatography.

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

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

They can control pectin gel formation.39 times. and protect color.08 3.98 pK2 pK3 5.03 3. pK1 4. Therefore. it would be advantageous to use the acids near these values. organic acids are usually more effective as antimycotic agents.11 5. The pKa of most organic acids lies between pH 3 and 5 (Table 4. Effective use of an acidulant depends on the dissociation constant (pKa) or the pH at which 50% of the total acid is dissociated.43 4.16 2. water activity. In addition to any antimicrobial effects an acid possesses. Hoffman et al.89 3. 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. The inhibitory effect of acids has been compared based on pH.0.1).14 3. 2003). 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. Given that this value lies at the lower limits of growth for many bacteria. type.77 4. but also the decreased processing time would aid in preserving the palatability of the product (Corlett and Brown. concentration. and degree of branching to inhibit or kill a wide variety of microorganisms. Not only would this interaction ensure commercial sterility of the food.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).40 4. physical parameters. the choice of an acidulant may depend on secondary effects. normality. This primary concern limits the use of acidulants to foods with pH values of less than 5.75 4. They can create a synergistic relationship with antioxidants by chelating metal ions.87 4. and other inhibitory compounds. Comprehensive texts describing applications.27 4. 1980). adjustments in pH levels can be coupled with changes in temperature. prevent browning. Some can be used .Organic Acids 93 TABLE 4. aid in the inversion of sucrose.75 5. chain length. Because some experiments express concentration in percent. EFFECTIVE USE Various approaches have been used to study the efficiency of antimicrobial effects on cells.61 4. gaseous atmosphere. 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. (1939) observed that acids with fewer than 7 carbons were more effective at lower pH.34 6. Acids contribute to the taste and tartness of a product. The multiple-barrier concept (“hurdle” concept) has gained popularity for use in a variety of foods in recent years.41 4. and chemical analyses can be found in the references by Ash and Ash (1995) and Doores (2002. and acids with 8 to 12 carbons were more effective at neutrality and above. or molarity to achieve final pH.

Numerous studies included in this chapter compare several organic acids for their antimicrobial effects. ACETIC ACID Antimicrobial Properties Acetobacter and heterofermentative lactic acid bacteria (heterolactics) produce acetic acid as a byproduct of their metabolism and. as the name implies. are more tolerant to this acid than homofermentative lactic acid bacteria (homolactics). would further indicate that hazards are unlikely. 1966) The toxicology and safety of the acids must be considered when selecting an acid for use.8.7. sauerkraut. McDonald et al.6 to 4. although they could also serve other functions in foods. many of these studies used high levels of the concentrated acids at low pH values. At the levels and pH values in which these acids are normally used in foods. plantarum at pH 4. are relatively nonpolar compounds. The succession typically begins with growth of coliforms and natural microflora followed by Leuconostoc mesenteroides (heterolactic). The lipophilic acids include acetic acid and acetic acid derivatives. a more acid-tolerant organism. which do not.4 to 5. Microorganisms vary in their susceptibility to acetic acid. In addition. however. application. (1990) found that growth of L. L. Several animal studies have indicated possible safety hazards. Salmonella Aertrycke (pH 4. THE ACIDS Information on the toxicology. These organisms are found naturally associated with fermented products. Concentrations of acid lower than those needed to inhibit Saccharomyces cerevisiae (pH 3. the information appears separately for each acid. The results of these studies are usually presented in the section describing the acid that gave the most antimicrobial effects.94 Antimicrobials in Food as chemical leavening agents.9) and Aspergillus niger (pH 4. as the pH decreased to 4. 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. all these acids can be metabolized by the body primarily through lipid oxidation and via the tricarboxylic acid cycle.0). and vinegar. In some instances. propionic acid.9). all of the groups were similarly affected (Woolford. These acids.1) (Levine and Fellers.0 than lactic acid bacteria. such as sauerkraut. 1975b). Gram-positive bacteria. Sauerkraut manufacture is a fermentation process brought about by a natural succession of microbes that inhabit the surface of cabbage leaves. whereas growth stopped in L. They are usually added to foods for their antimicrobial properties. and molds. 1939) inhibited Bacillus cereus (pH 4. Adams and Hall (1988) showed a weakly synergistic inhibitory effect between acetic and lactic acids with Salmonella enteritidis and Escherichia coli.0. this apparent synergy has not been demonstrated in other foods. and caprylic acid.9). however. such as pickles. The salts of the acids are important in regulating the acidity of the foods (Gardner.0. as such. which exist naturally in many foods. Most of the acids appear safe for use in food products. Although these acid mixtures might be expected to occur naturally during fermentations by heterolactic bacteria. and regulatory status is presented here for each acid. antimicrobial properties. This association could explain the apparent stability that occurs in natural fermentations. then by Lactobacillus plantarum (homolactic). 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 long-term consumption of these acids. the same hazards would not be expected. Owen (1946) suggested that acetic acid inhibited Gram-positive organisms. yeasts. and Staphylococcus aureus (pH 5. mesenteroides ceased when the internal pH of the cells reached 5. Bacillus and Clostridium species and Gram-negative bacteria were more inhibited at pH 6. .

7% acetic acid. Salmonella were inactivated within 48 hours in both RCM and CFM formulated with 0. which has recognized antimicrobial properties. It is unlikely that these products would contain such high initial levels of either of these organisms. coli O157:H7 grew in all media at pH 5.0. tartaric. Reduced-calorie mayonnaise (RCM) manufactured with 0.5 for all acidified media at 10°C except for acetic acid.4% to 0. 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. respectively. aureus preconditioned in acetate buffer (pH 4. cereus was inhibited at pH 5.. 0. and caused 50% inhibition of spore germination at pH 4. malic. 1991).0 with scant growth in TSB containing acetic acid.5% and 0. respectively. and E.5 (Noda et al. malic. pH 4. and HCl.2 and 4.6) at 20°C shifted to pH 7.Organic Acids 95 S. 1984). E. some strains of S. 1970).3% acetic acid formulations. Both studies concluded that the antimicrobial effect was the result of the undissociated molecule. followed by lactic. A 0. citric. Gram-negative aerobic bacteria.7% acetic acid in the aqueous phase and adjusted to pH 4. monocytogenes and Yersinia enterocolitica. such as mayonnaise (Debevere.3% and 0. therefore the standard use of 0.8% reduced growth of Micrococcus and Bacillus species. most likely because of the absence of egg yolks in CFM and the presence of egg white. 1940). monocytogenes than lactic and citric acids and increased inhibition as the temperature of incubation . monocytogenes.15 M acetic acid and with 0. The effect of temperature-acid interactions is noteworthy for the psychrotrophic pathogens. coli than unbuffered systems. E. aureus. malic.7 and 4. although more resistant than Salmonella. aureus was most sensitive to acetic. 0. At 25°C and 37°C. 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 23.7% acetic acid (Glass and Doyle.1-M concentration of acetate and lactate completely inhibited multiplication at pH 6.8. and Enterobacteriaceae in Japanese fermented soy sauce (Hayashi et al. 1979). pH 4. tartaric. coli. L. In addition. 22°C) containing 1.. A concentration of 0. 1988)..0. 1982). and tartaric acid permitted growth at pH 4.6. pH 4. pH 4. in the aqueous phase of CFM and RCM would still produce a microbiologically sound product.6 and 4.9°C. or 0. monocytogenes was also reduced to undetectable levels in RCM within 10 days and in CFM within 14 days compared to 2 days for Salmonella. and exposed to 40°C became injured.0 (Conner and Kotrola.0 buffer.3%. Similar results were noted when various acids were compared at the pH level required for a 90% and 99% reduction in numbers of S. 1997). followed by citric. citric. aureus. especially with the use of pasteurized egg products. When E.7% acetic acid. 1988). or citric acid were far more effective in reducing numbers of S.3. Acetic acid caused greater inactivation of L. and tartaric acids. This finding is important in the manufacture of medium acid foods containing more than one organic acid. Conner et al. and hydrochloric acids (Nunheimer and Fabian.0% acetic acid and 1. Acetic acid exerted the most inhibition but had the lowest dissociation constant.0 with hydrochloric acid was compared to cholesterol-free (egg yolk-free) reduced-calorie mayonnaise (CFM) manufactured with 0. aureus in 12 hours (Minor and Marth. other ingredients can interact with acid to influence the degree of inhibitory action. whereas tartaric acid had one of the highest dissociation constants yet exerted the weakest inhibitory action. coli O157:H7 was inoculated into tryptic soy broth (TSB) containing acetic.7% acetic acid with reduction to undetectable levels in 1 and 2 weeks with 0.0. respectively. respectively.4 to 4. but no injury occurred in cells not exposed to acidic conditions before heating (Smith et al. B.33 M lactic acid (Wong and Chen. L.. was reduced by 4 logs in both mayonnaises within 72 hours in formulations containing 0.0 with 0.5. 1995.0% lactic.2 and 5. Acetic acid was effective at pH 5. Buffered acidulant systems (brain heart infusion [BHI] broth. L. Malic acid allowed growth at pH 4. S. Traditionally acidic foods contain a single or multiple acids.5%. Destruction of Salmonella occurred more quickly in CFM than RCM.9 and 4. lactic.1%.5. Streptococcus faecalis. lactic. growth occurred at pH 5.5 to 3. Salmonella Blockley. pH 4.1 and 5.6. added or developed through fermentation. coli O157:H7 is considered more acid tolerant than strains of E.

This treatment reduced S. enterocolitica. Dorsa.5. such as Y. injury and death rates were not affected by refrigeration temperatures for Salmonella Bareilly. 1990).2 N for 4 min. Mung bean seeds inoculated with 3 to 5 logs of S. 0. Conversely.6 for lactic acid. but acetic acid was less effective. 1992a. A variety of organic acid treatments has been used to control microbial loads associated with meat carcasses and products (Dickson and Anderson. 1981). Karapinar and Gönül. When comparisons were based on equal molar concentrations. but resulted in changes in sensory characteristics. monocytogenes were exposed to acetic acid at a concentration of 242 µL/L of air for 12 hours at 45°C. there can be adverse sensory effects including discoloration of meat.. 1997.. Sorrells and Enigl (1990) also demonstrated interactive effects between sodium chloride. Acetic acid displayed the greatest antimicrobial action against L. The greatest antimicrobial effect occurred at 35°C with the greatest survival at 10°C.0 have been used to rinse pork.5 or 2. In addition. and 0.96 Antimicrobials in Food decreased from 35°C to 13°C. seed germination was effected (Weissinger and Beuchat.. Parsley.5.9 log reduction for the nontreated seeds (Weissinger et al. 40%. Typhimurium and E.5 min. acidulants.10 N for 5 min. citric acid caused greater injury than either lactic or acetic acids and injured organisms survived longer at low temperatures (Ahamad and Marth. 1 log unit at pH 2. and temperature. inoculated with approximately 107 CFU/g Y. or 5%) or vinegar (30%. 2001).0 for 30 or 60 seconds (Biemuller et al. coli O157:H7 to nondetectable levels by enrichment methods. monocytogenes when compared to citric. malic. monocytogenes irrespective of temperature. Smulders and Greer.7 log without affecting germination of seeds. 1999). 1998. 0. A 90% reduction of the population required exposure to 0. 1992. However. a mesophilic organism. Dorsa et al. monocytogenes was isolated from 2 of 10. 1991. 2%. Acid injury did not appear to involve damage to ribosomes or other nucleic acid material. and pH 4. and beef carcasses or meat tissue. was dipped in acetic acid solutions (1%. respectively. but recovery required the presence of amino acids or peptones (Blankenship..4 for malic. 1989). 2000). Total plate counts were reduced by 4 logs at pH 1. Immersion of alfalfa seeds in mixtures containing 5% lactic or citric acid for 10 minutes resulted in several log reductions. off-odors. concentration ≥0. a twofold reduction at pH 2. Because produce is typically refrigerated with only minimal processing.72 logs. Acetic acid was more effective than lactic or citric acids in producing this effect (Adams et al. and hydrochloric acids. Treatment at low temperature (4 days at 10°C) with 200 and 500 mg/L of air was effective in reducing Salmonella by 2. pH 4. and no reduction in microbial . Little et al. Depending on the treatments. 1992b). 1990.8 to 5. or 50%) for 15 or 30 minutes. and L. enterocolitica at lower pH levels as the temperature increased. 1998).0 for acetic acid.. lamb. E. but malic acid was more bacteriostatic than other acids at 10°C. lactic. but L. In addition. 25-g samples. these foods can be the carriers of psychrotrophic pathogens. Enteritidis were sprayed with acetic acid at pH 1. citric. The minimum inhibitory pH level was pH 4. Numerous organic acid combinations. enterocolitica was not detectable after exposure to 2% and 5% acetic acid or 40% and 50% vinegar for 30 minutes (Karapinar and Gönül.6% to 3. and hydrochloric acids at equal pH values and at all incubation times and temperatures (Sorrells et al. 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. containing from 0. Y.. Typhimurium.0. 1992). Similar growth inhibition was demonstrated for Y. Treatments spanned varying amounts of time with concomitant decrease in microbial loads up to 3 logs. and off-flavors. vapor treatment also reduced levels of resident microflora on seed but did not affect seed germination rates (Delaquis et al.01 N for 75 min.5 to 3. 1989.0% acetic acid at pH levels from 1.33 and 5.3% inhibited L. 1973). 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.4 to 4.05 N for 8. however. coli O157:H7. Exposure at 50°C for 12 hours with lower levels (100 and 300 mg/L of air) reduced the population >1. 1997. citric acid was more bactericidal than other acids at 25°C and 35°C. Pork carcasses inoculated with 106 CFU/ml S. enterocolitica.. Brocklehurst and Lund.

respectively. such as rumen fluid.5. anaerobic. Department of Agriculture Food Safety Inspection Service (FSIS). (1977) reduced bacterial counts by 99. 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. S.6% acetic and 0. some discoloration occurred (Cacciarelli et al. dirt. Typhimurium was reduced by 0.. resulted in less effective reduction of S. They found that acetic acid was a superior sanitizer compared to hypochlorite and had a greater residual effect. Dickson (1992) contaminated lean and beef tissue surfaces with S. Hardin et al.. Although washing of pork carcasses with 1. Meat was sprayed. Unda et al.S. 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.. Typhimurium followed by treatment with 2% acetic acid.8 log CFU/cm2. however. but beef treated with the mixture of acids did not differ in flavor from untreated beef (Bell et al.4°C (Anderson et al. or manure slurry. coli O157:H7. 1995).. Beef cubes treated with 1.3 log/cm2). Hamby et al. 1997.5% acetic. 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. 1983).8 to 4. Quartey-Papafio et al. Cutter et al.5% acetic acid were reduced more when treatments were conducted at 52°C than at 14. Temperature played a greater role in reducing numbers than did the acid concentration.6% on meat using a 3% concentration of acetic acid before washing. 1989b)... 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. Anderson et al. coli. At this concentration. suggesting that acids had limited effectiveness (Fu et al. vacuum packaged. Typhimurium reductions were greater when the bacteria were attached to fatty tissue.. 1994). however.. 1994. An increase in organic material.046% formic acids for 10 seconds caused discoloration from both treatments. (1980). this was not significantly different from the controls. 1997.. The treatment effectively reduced recovery of S. Dorsa et al. Lactobacillus species predominated as storage progressed. Reductions in microbial counts depend on the water temperature. and application rate and sequence in the process (Gorman et al. 1974).1 and 1. 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. 1994. 1997). however. Incorporation of pulsed power electricity and a 2% acetic acid spray led to improved reduction of inoculated E. (1991) inoculated beef roasts by injection and on the . L. which resulted in significant reductions in aerobic plate count (1. type of chemicals. Typhimurium. 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. 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. a final application of acid washes could provide some residual effect during storage (Brackett et al...0.5 to 0.. Enteritidis to 1 of 6 samples at pH 1. Cutter and Siragusa. 1995).5 and 11 of 72 for pH 2. Typhimurium.. and lactic acid bacteria counts. possibly because this tissue retained less moisture (20%) than lean tissue (75%) and the reduced water activity could have enhanced the antimicrobial effects. Typhimurium on beefsteaks (Tinney et al. S.Organic Acids 97 numbers at pH 3. the pH level decreased to 2. 1994).2% acetic acid or 0.. coli O157:H7 and S. It was noted that the use of acetic acid as a rinse for beef tissue led to sublethal injury of bacterial cells. or manure. and stored for 28 days at 2°C in a high-oxygen barrier film. 1996). 1987).0. citric. Microbial loads on beef carcasses subjected to machine washing and sanitization with 1. An 18% acetic acid or 12% lactic acid spray significantly reduced bacterial counts on lamb carcasses. As part of recently established guidelines by the U. 1986). (1987) used intermittent sprays of 1% acetic acid or lactic acids. monocytogenes was detected in 69% of loins and 33% of chops. organic acids can be used in preevisceration rinse systems consisting of a water rinse followed by an organic acid rinse. and bleaching of the carcasses occurred (Ockerman et al. S. 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.

5 with acetic acid was the most effective antimicrobial treatment followed by other acids in descending order of effectiveness: adipic. Expanding on their work. lactic.6% acid solutions. monocytogenes. 1994).5 mM. 1 mM perillaldehyde—or 0. monocytogenes. although the skin of the 0. but Enterobacteriaceae counts were significantly reduced by 0. Typhimurium using a model system. As the incubation temperature to recover spores was lowered. Acetic and tartaric acids were more inhibitory than lactic and citric acids.5 logs (Okrend et al. greater than a 4% concentration of acid was needed to reduce levels by 2 logs (Tamblyn and Conner.5% concentrations of acetic.0 on inhibition of L. There was no significant difference in texture or sensory characteristics between the treatments. Addition of 1. Because of the possible attachment of Salmonella to broiler chicken skin. Rubin (1978) found that acetic and lactic acid acted synergistically to retard growth of S.. with and without the use of air injection to agitate the chill water..6 or 5. citric. 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.98 Antimicrobials in Food surface with approximately 103 CFU of Clostridium sporogenes and L. fumaric. 1994). Typhimurium and Campylobacter jejuni by 0. 1987). 0.5%.7% citric acid. and tartaric acid adjusted to pH 5. aerobic plate counts were unaffected by the treatments. Acidified media showed greater inhibitory activity at 35°C than at 13°C.2% to 0. El-Shenawy and Marth (1989b) investigated the effect of acetic.6% acetic acid under the same conditions.. acetic acid caused the skin of the poultry to be hard and leathery. Poultry scald water containing 0.05% and 0. lactic. and lactic (Mountney and O’Malley. The acetic acid in the brine inhibited anaerobic and aerobic bacteria. Levels of 0. germination and outgrowth of Bacillus . of the same components—with 2% salt to inhibit growth of contaminating microorganisms for more than 20 days at 27°C. Other studies continued to explore the use of acetic acid in chill water.1% acetic acid at 52°C decreased levels of S. Intervention steps incorporated into poultry processing have been effective in reducing microbial contamination.71 log most probable number (MPN)/ml (Dickens et al. succinic. The acid level could be reduced when used in combination with brines and sugar. Products are now being preserved by a number of antimicrobial compounds using a multiplebarrier approach to preservation. Dickens and Whittemore (1994) exposed broilers to 0. see Chapter 7) coupled with 0.3% acid and 2. 20°C.3% and 0. further poultry processing can reintroduce microorganisms onto carcasses. but changes in pH and concentration of acid were more successful.6% acetic acid-treated carcasses was darkened or yellowed (Dickens et al.5 to 1. acetic acid was the most destructive acid at all combinations of parameters. (1985) found that mushrooms reached an equilibrium pH of 4. a 0.0% acetic acid caused instantaneous death. 0.. Air injection did not affect reduction of these counts.0% acetic acid compared to 2 days for 0. and 0. citric.24 log (Lillard et al. and 30°C (Oscroft et al.6 depending on the concentration of acid in can brine within 1 day with 1.5% acetic acid reduced Enterobacteriaceae counts by 2. Whole pickles preserved with brine at 12° Brix and 0. For pearl onions.7% acetic and 4 days for the same concentration of citric acid.86 log MPN/ml for the 0.35 log MPN/ml for the 0.35% solution required 7 days for equilibration for acetic and citric acid but 1 day for 0. Again.6% acetic solution acid (pH 3.05%. Adjustment of the chill water to pH 2. Although each individual acid influenced the heat resistance of Bacillus spores differently. respectively.05% acetic acid.. 1997). 1994). Nisin (an approved bacteriocin for some foods. 1976). 1990). Water pockets occurred under the skin of chicken carcasses with air-agitated samples (Dickens and Whittemore. 1965).0) did not result in a significant reduction of aerobic plate counts but did significantly reduce levels of Enterobacteriaceae by 0. Despite these treatments. Kurita and Koike (1983) combined 3% ethanol. 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. 1986). Despite the reduction in microbial numbers at this pH.. Immersion of broilers for 10 minutes in 0. Stroup et al.9% acetic acid did not support growth or toxin formation of Clostridium botulinum (Ito et al.

60 mg/m3. 1976). Aspergillus fumigatus required 0. niger. and citric acid when spores were heated at 95°C for 15 minutes and acetic. On contact with organic substrates. Peracetic acid is formed by an oxygen molecule bound to the carboxyl carbon atom of acetic acid. particularly precooked.. and Rhizopus nigrificans at pH 3. Rats exposed to vapor levels of acetic acid showed muscle imbalance at 0.0. however.2 g/kg body weight) did not show any adverse effects. Johnson (1968) noted that rat gastric mucosa responded to a 100-mM concentration of acetic acid by releasing histamine into the interstitial fluid. pneumonia (Gerhartz.0% concentration of acetic acid. 1971).Organic Acids 99 spores were more restricted.. A. Mori (1952) fed rats acetic acid ranging from 10 to 50 cm3 acid/kg of rice. however. 1969).8% to 1.2 or 5 mg/m3 but not at 0. Toxicology Acute toxicity data for acetic acid in mice and rats indicate varied tolerance levels by different routes of administration (Table 4. renal failure (Paar et al. cabbage.5 inhibited Saccharomyces ellipsoideus and Penicillium glaucum. suggesting its use as a decontamination agent for equipment (Hedberg and Miller. 1921). 1968). chilled. Nisin. it was not possible to determine the actual quantity of acid consumed.0001 and 40% relative humidity within 10 minutes (Portner and Hoffman. 1971). Allium cepa (Makinen.2).04 M acetic acid at pH 3 to 4 showed chromosomal and chromatid breaks. in combination with organic acids.48 mg/m3 altered sensitivity to light.999% after exposure to a 1. Lower concentrations of 0. It was found that acetic acid was the most effective acid. followed by glucono-delta lactone. gastric erosion (O’Keane et al. displayed synergistic effects in increasing the destruction of the organism. lactic. 2000). interference with blood coagulation (Fin'ko. Ingestion of acetic acid by humans has caused burned lips (Palmer.0 and below to inhibit growth (Kirby et al.5.8 and 0. rats decreased their food intake level and lost 2.. aerobic bacteria. Damage from acetic acid resembled that from ionizing radiation or radiometric chemical damage (Manna and Mukherjee. it decomposes to yield oxygen and acetic acid.0001 and 0. Other forms of acetic acid have antimicrobial uses.001 M accelerated spindle activity and retarded anaphase in the mitotic cycle of the root tips of the onion. Studies on nonmammalian species showed that acetic acid at a concentration of 0.2% (0. glucono-delta lactone.4% acetic acid at pH 5. and coliforms. 1958). Spore-forming bacteria are killed on surfaces at a concentration of 0. Grasshoppers treated with 0. 1932). and 0. Although acetic acid is generally used as an inhibitor of bacteria and yeasts. lactic.. respectively (Buchanan and Ayres. Foods.01% to 0. This treatment could also be used in recycling of water to reduce levels of yeasts and molds. Levels of 0. Parmeggiani and Sassi (1954) reported that workers .06 mg/m3. 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.8% partially inhibited growth and decreased toxin formation by 70% and 90%.2% acid at pH 5.6% or 0. Rats consuming acetic acid in drinking water in concentrations of 0. but a concentration greater than 4% was needed when the pH was at 7. Takhirov (1969) suggested limits of 0. 1966). are especially susceptible to spoilage because of indigenous microbial populations. 1937). A combination of these parameters could be used to extend the shelf life of these products. 1968). 1969). Cruess and Irish (1932) showed that apple juice containing 0. At the 0. and death.01 mg/m3.5% level. Rats fed 10% acetic acid and cats fed 20% acid developed ulcers in the gastric mucosa (Okabe et al. a 1% concentration of acetic acid at pH 4. and potatoes than peroxyacetic (80 ppm) acid alone (Hilgren and Salverda. it is effective against the black bread molds.5 completely inhibited growth and aflatoxin production by Aspergillus parasiticus. 1949). All rats showed mucosa damage owing to the feeding method. and citric acids when temperatures were reduced to 65°C for 60 minutes.0% acetic acid adjusted to pH 3. Populations of Pseudomonas aeruginosa were reduced by 99. When used as a surface application. ready-to-eat products. Human olfactory thresholds to acetic acid vapor were 0.29 mg/m3 caused changes in electroencephalograms.6% of their body weight (Sollman.

380–3. as well as citric.900 680 275 2.500 330 44 1. sodium Dehydroacetic Adipic Rabbit Caprylic Citric Mouse Mouse Enders (1941) Orö and Wretland (1961) Yokotani et al.040–5. (1950) Horn et al. sodium Tartaric Spector (1956) Orö and Wretland (1961) Hara et al. cold sensitivities (Wiseman and Adler.100 Antimicrobials in Food TABLE 4. and epidermal reactions (Weil and Rogers.860 2.700 725 884 5. (1957) Acetate.730 1. No toxicologic data were available for peracetic acid.810 625 3. (1957) exposed for 7 to 12 years to acetic acid vapors in the production of acetyl cellulose displayed corrosion of the hands.020 5. pharynx. (1971) Gruber and Halbeisen (1948) Gruber and Halbeisen (1948) Lactic Propionic Propionate. (1957) Gruber and Halbeisen (1948) Yokotani et al.340 580–1. eyes. and lungs with an air concentration of 0.460 1. In rare instances acetic acid.65 mg/L. (1963) Horn et al. (1963) Hara et al.2 to 0. although it is assumed that this derivative would be assimilated in a manner similar to that for acetic acid.700 11.000 1.530 380 1. .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. lactic. Men having 7 to 25 years of exposure displayed blood abnormalities (Cesaro and Granata.430 600 5. 1956). and tartaric acids. (1971) Rat Citrate. sodium Rabbit Mouse Rat Rabbit Rat Guinea pig Mouse Rat Rat Mouse Gruber and Halbeisen (1948) Yokotani et al.960 525 3. teeth.200 485 Reference Spector (1956) Orö and Wretland (1961) Spector (1956) Spector (1956) Spencer et al.430–4. 1951). 1955). (1971) Gruber and Halbeisen (1948) Horn et al.100 1. caused allergic reactions by producing canker sores (Tuft and Ettelson.210 338 3.790 940–960 42 203 961 2.310–3. calcium Propionate. 1956).

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

respectively (Buchanan and Ayres.7% SA and 0. 1996). 1994). and trisodium citrate extended shelf life to 6 days. Histologic findings showed hypertrophy of the kidneys and ureters.3 or 1..5%) was also effective in reducing populations of L. SA alone or SA-MKP-treated fillets were not significantly different in odor from fresh controls up to 9 days. or trisodium citrate were used alone or in combination with Bifidobacterium breve (5%) as a dip for fresh camel meat (Al-Sheddy et al. Waterhouse and Scott (1962) attributed the toxic effect to the sodium ion rather than the acid moiety. B. 1995a). Treatment with SA alone or in combination with bifidobacteria extended shelf life at 4°C by 3 days (Kim et al. At lower acid concentrations of 0. 1. the indicator of spoilage. breve and SA exhibited an additive effect that extended shelf life still further. SA in combination with potassium sorbate and Lactococcus lactis ssp. Psychrotrophic counts from untreated whole shrimp increased to >107/g.5% potassium sorbate. Toxicology The LD50 (mean lethal dose) for SA in mice is presented in Table 4.2. sodium lactate (3%).5% lactic cultures or 0.5% SA. potassium sorbate (1. SA (0. breve. but bifidobacteria did not grow within 9 days. whereas potassium sorbate increased shelf life to 9 days. 5% sodium lactate.26% potassium sorbate or 2% sodium citrate in its efficacy (Wederquist et al.6% or 0. vacuum packaged. 1999). although a mild acetic acid odor was detected. SA extended shelf life to 12 days at 4°C.5%). A combination of 0. but they did appear brownish and watery (Kim et al. sodium lactate.0% SA alone inhibited growth for more than 6 days at 4°C and counts increased only by 1.5% lactate was effective as an antimicrobial treatment in sliced servelat sausage over a 4. untreated samples reached the spoilage mark within 10 days. cremoris culture added to catfish fillets was an effective treatment against growth of Gram-negative spoilage bacteria at 4°C (Kim and Hearnsberger. The combination of 0. and 1.44% SA showed decreased appetite. 1994). Chickens fed diets supplemented with 5.0% concentration of SA at pH 4. No toxicologic data were available for calcium acetate (Food and Agricultural Organization. and increased mortality. although it is assumed that this derivative would be assimilated in a manner similar to that for acetic acid. depressed growth rate. breve culture and stored at 4°C for 9 days (Al-Dagal and Bazaraa.5 to 1. monocytogenes in vacuum-packaged turkey bologna during storage at 4°C and was similar to 0.4% monopotassium phosphate (MKP) more effectively prolonged shelf life to 12 days compared with SA alone.7 logs when stored at 4°C compared with the nontreated fillets and increased shelf life by 6 days...75% and 1% significantly reduced the initial aerobic plate counts of catfish fillets by 0. 0. and 2. 1997).102 Antimicrobials in Food SA (10%). SA at concentrations of 0. A combination of B.5% trisodium citrate with or without B. but SA provided better sensory characteristics and more controlled growth of psychrotrophs than potassium sorbate with bifidobacteria. 1963). The combination of 0. and held at 4°C for 12 days contained lower population levels of psychrotrophic and anaerobes than untreated samples (Zhuang et al.5. although MKP alone had no effect. 1995b).3% to 0. parasiticus was inhibited by a 1.25% potassium sorbate. Color remained acceptable.2 logs by day 12. 1999). . For peeled shrimp. 1951).8%.to 6-week storage life (Blom et al. 1976).. over 6 days. Whole and peeled shrimp were immersed for 2 minutes in solutions containing 10% SA. Growth and aflatoxin production of A. A 22-year-old woman with sensitivity to acetic anhydride was not allergic to SA (Weil and Rogers. 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...6 to 0. Catfish fillets treated with 2% SA.25% acetate and 2.

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. A. The growth of L.25) at 35°C. coli.1% and 0. SODIUM DIACETATE Antimicrobial Properties Sodium diacetate was effective at a 0.5 inhibited Aspergillus flavus. L.3 and incubated at 5°C. A concentration of 0. the addition of 0. S.8% and 2.4. it prevented mold growth (Chichester and Tanner.3% sodium lactate. such as Bacillus mesentericus (subtilis).2% sodium diacetate or 1% buffered sodium citrate and within 3 and 5 weeks for products containing 2.4% at pH 3. and 28 mM (pH 5. Sodium diacetate was more effective than acetic acid.2% sodium diacetate. Lactobacillus fermentans.5 weeks in products containing 0. vacuum-packaged.2% SA. 6. or 1% buffered sodium citrate (Stekelenburg and Kant-Muermans 2001). 1981).1% or 0. Sodium diacetate at concentrations of 0. 1994). Enteritidis. 1972). 32 mM (pH 5.5% and 3. The minimum inhibitory concentration (MIC) of sodium diacetate was determined for L. Enteritidis were inoculated into a comminuted beef emulsion formulated with 0. monocytogenes and bactericidal .5% or 3. Saccharomyces species (Glabe and Maryanski. 1994).2% sodium diacetate and 2. monocytogenes increased to approximately 106/g within 3 weeks.5% at pH 4. Enterococcus faecalis. fumigatus. monocytogenes was not significantly different in the presence of the individual inhibitors compared with control samples after 20 days at 10°C.1% to 0. In addition to L. However. 2001). monocytogenes were inoculated into the cooked ham products at an initial level of 104 and 102/g of product. monocytogenes.1% and 0. The addition of sodium diacetate affected the sensory qualities of the product precluding its use. However. L. 2.5% sodium lactate was bacteriostatic to L. and B.55) at 5°C.Organic Acids 103 Application and Regulatory Status Sodium (21 CFR 184. Sodium diacetate is equally effective in the baking industry where its inhibitory powers prevent the growth of bread mold and rope-forming bacteria. monocytogenes was inhibited by sodium lactate and 0.0% delayed mold growth in cheese spreads. sodium diacetate was also effective against hemorrhagic E. The MIC was determined to be 35 mM (pH 5. 20°C. In ground beef. 5. Calcium acetate is classified as a stabilizer when migrating from food packaging materials (21 CFR 181. Shewanella putrefaciens. or S.55. and when it was incorporated into butter wrappers. monocytogenes grown in BHI broth adjusted to pH 5. L. cereus with no effect against Pseudomonas fragi. and the inhibitory effect was attributed to the diacetate moiety rather than pH alone. while having little effect on baker’s yeast. and 6.15% to 0. the combination 0. and 35°C (Shelef and Addala.0.1721) and calcium (21 CFR 184.3% sodium diacetate was effective in suppressing total aerobic counts. enterocolitica. Cooked-in-bag ham was formulated with additions of 0. 5. curvatus reached an end point of 107/g within 2 to 2. and Penicillium expansum. in products containing buffered sodium citrate. or 1. 1962). Levels of 0. a concentration of 0.25. and stored for up to 40 days at 4°C.5% sodium lactate or combinations of the acids and held at 5°C or 10°C.4) at 20°C. Y. monocytogenes and S. aureus (Shelef and Addala. An interactive effect was noted between temperature and concentration of acid in that growth was inhibited with decreasing concentrations of acid and temperatures. niger.1185) acetates are approved as GRAS substances for miscellaneous and general-purpose usage.2% sodium diacetate.05% to 0.5% level in malt syrups. Lactobacillus curvatus and L.2% sodium diacetate. A.3% sodium lactate. Aspergillus glaucus. SA can be used as a boiler additive for food-grade steam (Federal Register. respectively. L. 0.1% to 2.5 inhibited these same strains and Mucor pusillus in feeds and silage. Pseudomonas fluorescens.29).

1962). use levels range from 0. .6 logs in crabmeat stored at 4°C for 6 days when treated with 2 M sodium diacetate. L.5% sodium lactate and 0.3% and 0. were pasteurized for 10 minutes at 68°C after which they were inoculated with L. 0.8 logs (Degnan et al. monocytogenes at a level of approximately 4. malt syrups.1) of the acidulants and remains effective at higher pH ranges. Although 1 M sodium lactate solution reduced populations within 2 days. Schlyter et al.1%. populations increased approximately 0. the reduction of multiple strains of L. This acid is effective against Enterobacter aerogenes and L.5% concentration (0. lower concentrations of the antimicrobials were equally as effective. Sodium diacetate was determined to be listericidal over the 7-day period at 25°C only at the 0. 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. producing a 2-log reduction after 7 days at 25°C (Schlyter et al.5 logs CFU/ml slurry. glaucum and A.5% concentrations (0.0 against S. 1993a).2% sodium diacetate alone or in combination in beef bologna. In bread and cake products. 1981). niger (Banwart. Enterobacteriaceae counts dropped 1000-fold over 6 days at 2°C. Lactate and pediocin enhanced the listericidal properties of diacetate suggesting some type of synergistic activity. Slurries of uncured turkey breast. 0.1% (Wolf. Enteritidis.54 log reduction). sodium lactate (2. Toxicology No toxicologic data were available for sodium diacetate (Food and Agriculture Organization. In subsequent work (Mbandi and Shelef. monocytogenes in turkey mixtures. monocytogenes was reduced by 2. Sensory characteristics of roasted chickens were not affected even at levels up to 8 g per chicken (Moye and Chambers..5 logs within 6 days. respectively. As expected. 1991). Mixtures were incubated at 25°C or 4°C and sampled up to 7 and 42 days.. (1993b) used the multiple-barrier concept to limit growth of L. DEHYDROACETIC ACID Antimicrobial Properties Dehydroacetic acid (DHA) has one of the highest dissociation constants (Table 4.005% to 0. The growth of L. It is used in cheese spread. a more complex product. Sodium diacetate (0.3% and 0. and wrapping material. The powder dissolved in the surface moisture of the carcass to form acetic acid and SA and produced a surface pH of about 4. but sodium diacetate reduced the pH slightly.25% to 0.1%.104 Antimicrobials in Food to S. a fermentation by-product of lactic acid bacteria. and pediocin (5000 U/ml). Sodium dehydroacetate was twice as effective as sodium benzoate at pH 5.3%. although it is fungistatic at concentrations of 0.5%) was listericidal in combination with ALTA™.5%). Application and Regulatory Status Sodium diacetate is approved as a GRAS substance for miscellaneous and general-purpose usage (21 CFR 184. stored aerobically at 5 and 10°C.8. monocytogenes in mixtures held at 4°C for 42 days was inhibited at 0. 1950). butter. although it is assumed that this derivative would be assimilated in a manner similar to that for acetic acid. 1994).59 log reduction). Application of 4 M SA reduced the levels of L.1754).4% (Glabe and Maryanski. respectively. 1989). 2002). cerevisiae and 25 times more effective against P. plantarum at levels of 0. monocytogenes by 0. monocytogenes and Salmonella species was demonstrated using 2.5%). prepared with sodium diacetate (0. At lower temperatures. the combination of acids achieved greater reductions of both organisms at both temperatures compared to individual acids and greatly reduced background spoilage microflora. Sodium lactate did not affect the pH of the meat.

benzoic acid. Toxicology Acute toxicity data for adipic acid in mice and rabbits indicated varied tolerance levels by different routes of administration (Table 4. 1957).130).. coli in homogenized beef but not in poultry (Yamamura et al.0% adipic acid showed no effect at the two lowest levels. Intravenous and intraperitoneal injection produced hemorrhaging in the lungs. The sodium salt of DHA was nonirritating to rabbit’s skin.5 was 4 mg/ml for potassium sorbate. ADIPIC ACID Antimicrobial Properties Adipic acid has no antimicrobial properties other than those ascribed to its ability to reduce pH. The 90-day feeding trials conducted on rats fed 0. 1971). produced diarrhea. treatment with sodium dehydroacetate was effective in reducing respiration rate and delaying ripening. It can also be used as a fungistat in cheese wrappers. or 5.0%. 0. although a dose of 200 mg/kg led to growth and organ changes. The MIC in broth at pH 6.1% of the substance showed no changes in growth.2). at the higher dosage levels. appearance. which included marked obstruction in the liver and kidneys by the oral route and polyuria by intravenous administration. or 100 mg/kg of DHA per day for 34 days. However.. hematology. resulting in the loss of up to 20% of body weight. the 800 mg/day dosage depressed growth. and 0. The effects of potassium sorbate. Oral administration of the acid to mice caused hemorrhaging in the small intestine with distension of the stomach. Toxicology The LD50 for DHA in rats is given in Table 4. 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. Furthermore.25% or 5% sodium dehydroacetate for 60 seconds were considered more marketable after 3 days of storage at 20°C than water-dipped berries. coli O157:H7 in broth and model meat products. thus extending marketability (Watada. even with prolonged contact (Spencer et al. or histopathology. Application and Regulatory Status DHA or its sodium salt is GRAS when used in accordance with good manufacturing practices. 1. 30. 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.Organic Acids 105 Strawberries dipped in or sprayed with 0. This acid at the appropriate concentration level would inhibit the growth of any microorganisms susceptible to low pH values. benzoic acid. 1953). and sodium dehydroacetate and 16 mg/ml for sodium p-hydroxybenzoate. However. 2000).02%. weight gains were lower and mortality was higher than in the controls (Lang and Bartsch. mortality. 1953). Longer studies using male and female rats given 400 or 800 mg/day did not affect offspring. However. Enders (1941) showed similar symptoms in rabbits. Sorbate was bacteriostatic at half the MIC for E.05%.2. 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. A 2-year feeding study with male and female rats fed 0. 300 mg/kg led to severe weight loss and damage to internal organs. 1950). sodium p-hydroxybenzoate.1%. but significant growth depression . and caused slight histologic aberrations. Spencer et al. (1950) did not show any unusual effects in rats receiving 10. in addition to acidosis by the former route and intestinal adhesions by the latter route (Horn et al.. altered behavior. and sodium dehydroacetate acid were investigated for three strains of E.

caprylic acid was ineffective at concentrations of up to 7.013% for baked goods. mildly acid flavor. and biscuits because it imparts a smooth.04% for cheese. 288. not to be used in amounts exceeding good manufacturing practice.8% propionic acid. 0. frozen dairy desserts. refrigerated rolls. 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. rats. 0. and hamsters showed no significant difference from controls at levels of 263. Sodium adipate at the 5% level decreased the rate of weight gain (Informatics.0%. respectively (Food and Drug Research Laboratory.0% level although the food intake level was the same.8 µmol/ml (Kabara et al. . 1980).005% for fats and oils. 3. and 0. CAPRYLIC ACID Antimicrobial Properties When tested against a variety of Gram-positive and Gram-negative organisms and yeasts.0.3. 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. Application and Regulatory Status Adipic acid is a nonhygroscopic. gelatins and puddings. however. saturated dicarboxylic acid having low water solubility.. It is used as a flavoring adjuvant in levels not to exceed 0. caprylic acid inhibited bacteria and fungi at a lower concentration than either acetic or propionic acid. and 5% adipic acid and female rats fed 1. Inc. powdered fruit beverages. At pH 6. 1972). Inhalation of adipic acid for 6 hours at a rate of 126 µg/l produced no unusual findings (Gage.3% concentration inhibited E. 1970).001% or less for other food categories. baking powders. 1968). oily liquid that has a slightly unpleasant odor and a burning.1025). The 2-year feeding trials using male rats fed 0. candies. meat products.0% acid did not show any abnormalities. 1957). 1. No limit has been set on the acceptable daily intake for humans. 0.106 Antimicrobials in Food occurred at the 5. Caprylic acid at a 0.. At pH 5. Toxicology Orö and Wretland (1961) determined the LD50 of caprylic acid for mice by intravenous administration (Table 4. 1943). It also serves a function in the canning of vegetables.16% for snack foods. The MIC of caprylic acid was 100 µg/ml in TSB for Vibrio parahaemolyticus (Beuchat. 1972).0.0%..1%. Caprylic acid was also more inhibitory to Shigella species than either acetic or propionic acids (Nakamura and Zangar. however. and in improving the melting characteristics of processed cheese (Gardner. coli when grown in a chemically defined medium and was more effective than 1. and 205 mg/kg body weight. Teratologic studies using mice.1009). and soft candy.2). Caprylic acid is also used as an antimicrobial agent for indirect use in cheese wraps. weight gains for rats fed the two highest levels were significantly lower than those for control rats (Horn et al. It is used in gelatin desserts. It is slightly soluble in water. 1975b). rancid taste. the inhibitory concentrations were not dramatically lower (Woolford. as a sequestrant in oils.5% acetic acid and 1. Caprylic acid is approved as a GRAS substance for miscellaneous and general-purpose usage (21 CFR 184. 1973b). Application and Regulatory Status Caprylic acid is a colorless. The acceptable daily intake for humans is listed in Table 4.

propionic. Little bacteriostatic activity occurred at pH 5. and acetic. but lower pH levels were toxic. was processed for 30 minutes in boiling water. Additional interactive effects were also noted in the presence of salt. citric. Fabian and Graham. 1950).83 at 10°C and 7. Interaction was demonstrated between citric acid and sodium citrate added to BHI broth adjusted to pH 4.0. 1994). fumaric.4) in inhibiting Arcobacter butzleri in a broth system (Phillips. 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. pH. 1939.66 at 30°C. respectively. then lactic. lactic.7 juice incubated at 20°C were 6. L. followed by acetic acid. citric. followed by hydrochloric.7% at pH 6. Citric acid was more inhibitory to thermophilic bacteria than acetic or lactic acids. and 4. Schoenemann et al. aerobic counts for pH 3. the highest inhibitory activity was associated with propionic. canned in a brine containing acetic. Acidification impaired the color but enhanced the flavor of canned okra. Sodium citrate was also more effective than 2% sodium lactate. 1987).Organic Acids 107 CITRIC ACID Antimicrobial Properties Inhibition of L. lactic. enterocolitica based on concentration of acid.12 log/ml. 1990). aerobic counts at 20°C for pH 3. such as texture or appearance. The effect was temperature dependent in that survival of L.64 log/ml. and 7 (Buchanan and Golden.6 log/ml were seen.5 with acetic and lactic acids. lower aerobic plate counts of 3.0 with citric and hydrochloric acids (Conner et al. 1999).4 and 0. lactic. and the combination of 1. or tartaric acid to achieve an equilibrium pH of 4. The authors suggested the inhibitory activity might be the result of the chelating ability of citric acid. 4. malic. and acetic (Brackett.1% at pH 4. citric acid was more inhibitory than lactic acid. The activity of the undissociated portion of the acid was hydrochloric followed by lactic propionic. (1989) reported that on an equal molar basis.5% sodium lactate was equally inhibitory as sodium citrate alone. phosphoric. 5. monocytogenes after 4 weeks were pH 4. whereas at 10°C.19 at 5°C. When tomato juice was adjusted with citric acid to pH 4. Citric acid was particularly inhibitory to flat-sour organisms isolated from tomato juice.0 with propionic acid. and phosphoric acid was compared in TSB for Y.22 log/ml). Rate of inactivation of L. citric. acetic. Canned tomatoes acidified with citric. 1997). (1974). .09. and propionic acids and for 6 weeks in media containing HCl or lactic acid. Okra.7 juice were 4. These experiments used citrate phosphate buffer in which the citric acid content was calculated at 1. 1953). and 1. citric.3. Similar pH-temperature relationships were noted by Cole et al. Sorrells et al.7 and stored for 7 days at 5°C.0.5% sodium citrate and 1.13. but inhibition increased with decreasing pH (Murdock. monocytogenes survived after 11 to 12 weeks in TSBYE adjusted with acetic. compared to counts in unacidified juice (6. 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. propionic. 6. 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. who determined that the minimum pH values that allowed survival of L. (1990). Citric acid at a concentration of 0. and degree of dissociation. 1994). however.0 at 5°C. 4. phosphoric. A comparison of equimolar concentrations identified citric acid as the most antimicrobial. All acids would be effective antibotulinal agents at that pH level (Nogueira et al. and hydrochloric. Yeast and mold counts were the reverse with counts of 2..8 and 2. 1973. Based on pH. respectively. pH was not considered a reliable indicator of preservation effectiveness (Fabian and Wadsworth.0 and 3. acetic. indicating that citrate was the more effective compound.5% (pH 4. or malic acids did not change in physical or chemical attributes during processing compared with unacidified tomatoes (Schoenemann and Lopez. 2. monocytogenes was achieved in trypticase soy yeast extract broth (TSBYE) adjusted to pH 5. monocytogenes decreased to undetectable levels within 1 to 3 weeks at 30°C. and 4.5) was more effective than lactic acid (pH 3. The inhibitory capacity of hydrochloric.36 at 10°C..42 log/ml (Bizri and Wahem.

cereus strains grown in model vegetable broths were conducted at typical refrigeration and abuse temperatures of 5°C. 118°C.85.4 to 4. and 16°C (Valero et al. indicating a synergistic effect between pH and temperature. 1990a). Although the heat resistance of C.4. If the amount of lemon juice was increased to 20 to 35 ml per egg yolk. B. 1990b). 8°C. coliforms. This process inhibited C.14% CA (pH 5.0 at temperatures below 16°C. 1989. This process improved quality and increased the microbial stability of canned mushrooms (Okereke and Beelman. sporogenes PA 3679 spores. (1985) dipped potatoes in a solution of 1% citric acid and 2% ascorbic acid for 2 minutes before vacuum packing and cooking. and 121°C.7 (control).. The addition of potassium sorbate did not enhance stability. They found that acid blanching in a citric acid (0.4% of cans containing mushrooms vacuum hydrated by the ABC process spoiled (Beelman et al. and 4.5 were inoculated with C. provided temperatures were at 16°C. 5.34. sporogenes PA3679 after a sublethal heating process (Okereke et al. 115°C. 0.4 and 5. Silla Santos et al. but the shelf life was shortened at this higher temperature. 1992).0% TCA-0. and 0. 1994.2. monocytogenes (Dorsa et al... 12°C. 5.5) at 12°C but did not grow in broth acidified to pH 5. Citric acid dips (0.65 and asparagus with added citric acid or glucono-delta-lactone to pH levels of 5.9% and spoilage was further reduced to 16.5). leading the authors to conclude that the extracts have a protective effect on C.05 M) solution buffered to pH 3.3. thereby leading to its survival regardless of the change in pH (Ocio et al. Microbial stability was achieved in liver paste formulated with 0.. Extracts were then heated at 110°C. 0.1 with holding temperatures between 18°C and 20°C for up to 3 days before consumption. Notermans et al. and 4. Reduction in the thermal process time of canned liver paste was achieved through the addition of 0.29% CA.3 g/kg) for crawfish (Procambarus clarkii) stored at 4°C were not effective in retarding growth of L. or 0. 0. 5.14% citric acid (CA).69) and processed with an F0 of 0. Liver pastes inoculated with 106 to 107 Bacillus spores per g and 104 C. 1991).1 and 5.. The authors recommended that the final pH should be <3. and lactic acid bacteria counts than samples dipped with the lower concentration of citric acid. cereus grew in zucchini broth (pH 6. but could grow at 8°C only when the pH was higher (5. Samples pretreated with 1% citric acid displayed significantly lower total numbers. 1993).0% citric acid for 5 or 30 minutes. 4. 2003).. heat resistance was not affected by higher processing temperatures.8. 1997. Okereke et al. the product should . Xiong et al. Citric acid dips for foods not only retard some spoilage but also act as a chelator of metal ions responsible for enzymatic browning reactions.3 when at least 20 ml of lemon juice (citric acid) per fresh egg yolk was used. sporogenes decreased with decreasing pH levels. (1989) developed an acid-blanch-chelate (ABC) process designed to reduce mesophilic and thermophilic bacterial spoilage in canned mushrooms.05 led to more inactivation of S.1% potassium sorbate (Houben and Krol.31% CA–2.5 followed by canning in a solution containing 200 ppm ethylenediamine tetraacetic acid (EDTA) controlled the outgrowth of spores of C.. this process reduced thermophilic spoilage from 68% to 23. Homemade mayonnaise has been a significant health hazard as a result of contamination by Salmonella from raw eggs (Membre et al.31% CA–2.05. Both vegetable broths stimulated germination and growth of spores compared to nutrient broth. sporogenes spores per g were heated to an F0 of 0.2% or 1. Samples containing the reduced calorie dressing were even lower in microbial numbers than aciddipped vegetables (Eytan et al.. 6. 1990).8% with the addition of EDTA. Growth in carrot puree was temperature dependent because B. 1999). Mayonnaise made with >5% citric acid solution adjusted to pH below 4. 1992). Enteritidis PT4 at 22 than 5°C. Growth curves developed for B.9.1.0% trisodium citrate (TCA). Mushroom extract acidified with citric acid to pH 6.3.108 Antimicrobials in Food Beelman et al.22. The preparation of these products included adjustment of the pH with acetic acid to 3. 2000. Only 2. cereus could grow at pH 5.. sporogenes. Cabbage and carrots were pretreated with the addition of a reduced calorie dressing or with a dip of 0. By adding citric acid to can brine.. The products were packed with or without modified atmosphere and stored at 4°C for 10 to 21 days. botulinum type B when held for 70 days at 15°C or 14 days at 20°C.

Ascospores were able to withstand more than 6 hours of heating at 75°C. Sodium citrate can also exhibit bacteriostatic activity. 1952). 5 hours at 80°C. 1976).0.5% sodium citrate were inhibitory to Salmonella anatum and Salmonella oranienburg (Davis and Barnes. however. but salt had a protective effect on this organism. sodium citrate inhibited growth.75% lactic acid inhibited growth of Aspergillus versicolor.. malic. casei. No toxin was detected in any of these foods over an 8-week shelf life (Post et al. Citrate inhibition of S.8% gave a pH of 4. and 3 to 4 hours at 85°C.5% lactic acid prevented toxin production. Mangoes are subject to spoilage through infection with Penicillium cyclopium (Palejwala et al. suggesting that citrate was a chelator of ions essential for growth (Rammell.75%.0 reduced the numbers of P. A level of 0. A 0. 1968). Concentrations of 12. Similar destruction values were seen with potassium sorbate and sodium benzoate.2% sodium benzoate were held for 30 days at 4°C or . Sabouraud medium was adjusted with 0..8. the function of citrate appeared to be one of chelation of metal ions.6 and incubated at 35°C. Branen and Keenan (1970) also suggested that citrate chelation of metal ions inhibited L.08 to 4. Using the multiple-barrier concept. it did not appear to affect attachment (Appl and Marshall. 1970). Tsang et al. leading to enhanced growth. and 90°C. In concentrations of 12 to 18 µM/ml.2. even in media containing up to 1% citrate. 1984). whereas infected tissue was around 4.0% was not as inhibitory to Streptococcus agalactiae when added to skim milk or fresh milk as citric acid at concentrations of 1%.. expansum (Reiss.0% to 12. When acetic acid was used. Typhimurium than lactic and hydrochloric acids (Subramanian and Marth.25% concentration greatly reduced toxin production. and 4%. Healthy tissue had a pH of 5. citrate appeared to inhibit glucose utilization (Imai et al. aureus showed inhibition both with increasing concentrations of citric acid and decreasing pH (Minor and Marth.8. hard-cooked eggs allowed to equilibrate in 0.5% citric or 0. when 35 ml lemon juice per egg yolk was added. However. Thermal resistance studies were conducted in mango juice adjusted to pH 3. During ripening..12 (Sinha et al. type E grew and produced toxin at 26°C in media containing citric acid at pH 4. sodium citrate stimulated growth. botulinum showed no change in numbers when inoculated in tomato puree. 1984). 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. or 1. parasiticus. 2%. but 0. no growth occurred at pH 5. C. The addition of garlic or mustard increased the death of S.5 with citric.1% to 4. 1985). A level of 0. 80°C. however.125% to 4% citric acid or 0. the level of acidity decreased and sugars and nitrogenous compounds increased.5%. lactic. (1985) found no outgrowth or toxin production from spores of types A and B in TPGY medium acidified to pH 4. With Arthrobacter pascens. 1967). The MIC of 0. fluorescens on beef carcasses (Thomson et al. 0. shrimp puree. the time decreased to 48 hours before use. Enteritidis. 1968).3% citric acid lowered the level of Salmonellae on poultry carcasses.016% to 1% malic acid as the sole carbon source because these acids predominate at these concentrations in a ripening mango. and rinsing with a citric acid solution adjusted to pH 4. Skim milk acidified with citric acid was more inhibitory to S. 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. 1970). Citric acid provided the maximal destruction of ascospores at 85°C.25% citric acid and 0.. 1998).. As little as 0. aureus was overcome by adding calcium and magnesium ions.Organic Acids 109 be held at 22°C for longer than 72 hours.0% concentrations of citric acid and 0. Survival studies for Neosartorya fischeri ascospores were conducted in mango and grape juices and heated at 75°C. or shrimp and tomato sauce acidified with citric or acetic acids to pH 4.2 or 4. The metabolic effects of citrate on Arthrobacter species were twofold. 85°C. a 0. and in concentrations greater than 40 µM/ml. S.1 or less.75% concentration of either acid did not affect the growth of P. The amount of sodium citrate had a dual effect on Lactobacillus casei. with Arthrobacter simplex.75% for both citric and lactic acids had a slight growth-retarding effect on A. Sodium citrate in concentrations of 0. 1962).6. or tartaric acid.

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. (1982) used a combination of 0.. coli. in the acid form (21 CFR 184. Short-term studies on rats fed 0.3. enterocolitica. and it is used as an acidulant in canned vegetables and dairy products.2). Daly et al.1% citric acid and 0.5. 1972).75% citric acid alone for 21 days at 4°C. the urine contained calcium citrate. however. 4. A concentration of 0.2%. aeruginosa (Restaino et al. beverages. It is highly water soluble and enhances the flavor of citrus-based foods.1033) or as the calcium (21 CFR 184.05% potassium sorbate and citric acid at pH 5. In another study. E.8% level. potassium (21 CFR 184. or 5% citric acid (Bonting and Jansen.. 1963). 1957) showed no abnormalities other than lowered weight gains and feed consumption in the 5% group.75% glucono-deltalactone incorporated into sausage before fermentation. It can control the pH for optimum gel formation. 1956. Application and Regulatory Status Citric acid is a tricarboxylic acid having a pleasant sour taste. Citric acid also acts synergistically with antioxidants to prevent rancidity by chelating metal ions (Gardner. the highest dosage contributed to enamel erosion. a by-product of yeast fermentation. sherbets and ices. It is a precursor of diacetyl and therefore indirectly improves the flavor and aroma of a variety of cultured dairy products. 1985). salad dressings. Citric acid is approved as a GRAS substance for miscellaneous and general-purpose usage when used in accordance with good manufacturing practices. Cramer et al. Dalderup (1960) fed rats 1. Toxicology Acute toxicity data for citric acid in mice.0 to reduce the growth rate of S. and S. Long-term studies involving rats fed quantities up to 1.1751). 3%. This combination inhibited the growth of S. and jams and jellies. or 12 g citric acid per kg of a noncariogenic diet to show the possible effect of citric acid on the development of teeth.75% citric acid was sufficient to reduce inoculated populations of S. 2. Horn et al. slight atrophy occurred in regions of the thymus and spleen (Yokotani et al. Although there was no effect on weight gain. Y.1625). (1956) fed vitamin D-free diets containing low calcium levels but adequate levels of phosphorus supplemented with 0. plantarum but did not affect P. 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.1195). 1944). 7.02 mol sodium citrate and citric acid. Studies by Yokotani and coworkers (1971) compared toxicities of commercial citric acid and Takeda citric acid. Restaino et al. fruit preserves. rouxii and Saccharomyces bisporus. Potassium sorbate at 0. Deaths resulted from acute acidosis and lung hemorrhages.7% sodium citrate (5% citric acid) fed to rabbits for 60 days produced no unusual effects (Packman et al. rats. 1981).5. The number of cavities formed did not differ between the control and experimental groups.. Typhimurium. 1971).110 Antimicrobials in Food in 0.2%. It is approved for use in ice cream. or sodium salt (21 CFR 184.8% 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 4.4%. aureus (Fischer et al.. The authors concluded that citrate had a rachitogenic effect on the test animals. aureus early in the fermentation and the increased acidity of the meat mixture allowed the fermentation to proceed more quickly. Horn et al.2% concentration in combination with citric or lactic acid at pH 5.. Death by all routes and in all animals resulted from respiratory or cardiac failure and hemorrhaging of the gastric mucosa. Acute toxicity data of sodium citrate are shown in Table 4. .5 depressed growth of L. (1973) used a combination of 0. and 4.2. The acceptable daily intake for humans is listed in Table 4. (1957) evaluated dosage levels in mice given the acid intravenously. and rabbits indicated varied tolerance levels by different routes of administration (Table 4.

botulinum types A and B in canned bacon. 1996a. citric. When used in vacuum-packaged ground beef patties.0 to 2. Huhtanen (1983) and coworkers (1985) investigated the effect of various esters of fumaric acid on growth of C.1%. 1974) and inhibitor of malolactic fermentation in wine (Pilone. However.5% applied for 5 seconds at 55°C to the surface of lean beef inoculated with L. 1987).0% and 1. 0.0% acid concentration at 82°C. and 1. 0.05%) or sodium propionate (0. 1947). and tartaric acids increased heat inactivation.1% and 1.. flavus ascospores (Beuchat.Organic Acids 111 FUMARIC ACID Antimicrobial Properties Fumaric acid (0. The addition of 0. Similar effects were noted with N.3 logs. 1987). whereas the presence of fumaric acid esters in the bacon prevented swelling for up to 8 weeks.2%) and dimethyl and diethyl fumarates (0. coli O157:H7 reduced levels by 1 and 1.1%) controlled fungal growth in tomato juice..5%. Islam.15% fumaric acid coupled with 0. psychrotrophic. It was speculated that the antimicrobial properties of fumaric acid were related to a lower pKa than lactic or acetic acids. 0. monocytogenes and E.b).5% concentration of fumaric acid inactivated ascospores after 20 minutes of heating at 80°C. malic.. fumaric acid achieved the greatest reduction of aerobic.38% sodium fumarate or guinea pigs fed 1. respectively.5. and di-n-alkyl fumarates were almost inactive (Dymicky et al. and coliform bacteria. citric. rats showed an increased mortality rate and more atrophy of the liver (Fitzhugh and Nelson. A 0. The fumarate esters not only prevented mold growth on bread (Huhtanen et al.5% level.9% (5% fumaric acid) showed no toxic effects (Packman et al. and tartaric acids at a concentration of 2. Monomethyl. At the 1.15%. dimethyl. In addition to reduction of E. 1963). Fumaric acid was also more effective than lactic or acetic acid when used in meat products (Podolak et al. Human feeding . fischeri (Conner and Beuchat. coli O157:H7 inoculated into unpreserved apple cider was investigated by Comes and Beelman (2002). Fumaric acid by itself was only slightly inhibitory.2% showed considerable promise as a substitute for sodium nitrite in bacon. 1988). total aerobic counts were reduced to levels below those achieved through pasteurization. and ethyl esters of fumaric acid in concentrations of 0. citric. and lethality increased as the pH decreased from 5. 1975).. Fumaric acid was more fungicidal against Talaromyces flavus ascospores than acetic. coli O157:H7. These same preservatives provided a similar reduction in less than 5 hours and 2 hours when held at 25°C and 35°C. The 2-year studies using rats fed 0.2% fumaric acid or rabbits fed sodium fumarate at a concentration of 6. Fumaric.5 to 5. Cans packed with bacon and inoculated with C. but the dimethyl and diethyl esters at a concentration of 20 mg per 2. 1987) heated in the presence of 2. or tartaric acid based on weight when added to the heating medium. respectively. malic. but malic acid did not. or 0. 1982).6 L in the vapor phase were also effective as a mold preventive (Huhtanen and Guy.05% sodium benzoate followed by holding at 25°C for 6 hours before refrigeration at 4°C for 24 hours achieved reduction. The use of fumaric acid to effect a 5-log reduction of E.8%. Toxicology Weanling rats fed 0. The n-monoalkyl fumarates and maleates esterified with C13–C18 alcohols also possessed significant antibotulinal activity (Dymicky et al. Medium chain-length alkyl esters had more activity than aliphatic acids (Dymicky and Trenchard. 1982). Monomethyl and monoethyl (0.5% (pH 2. 1984). 1981.0) were not lethal to T. Methyl n-alkyl fumarates demonstrated lower activity.15%) served as an acidulant (Ough and Kunkee.. Fumaric acid at concentrations of 1.0% fumaric acid and 1.125%. acetic.0% fumaric acid did not show any abnormalities. botulinum swelled in 4 to 6 days at 30°C.

1972). . 1983. Typhimurium were similar for both acids (Park et al. Similar reductions in microbial counts occurred with a 1.0 but was ineffective against yeasts and molds (Woolford. which was reduced to 10% after treatment.25% concentration of acid sprayed on veal carcasses.1% ascorbic acids selectively inhibited Enterobacteriaceae at 10°C. 1946).0% to 1. followed by vacuum packaging. 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. pork. 1970). 1988). but a 2% concentration led to discoloration of the carcass surface (Smulders and Woolthuis. fumaric acid does display some antioxidant properties in fat-containing foods (Gardner. and 0.3 of veal tongues. 1985). Application and Regulatory Status Fumaric acid is a nonhygroscopic.. vacuum packaging. Lactic acid sprays have been effective in limiting microbial growth on meat carcasses under a variety of storage conditions. Because of these dosage levels. Osthold et al. 1975a). citric. Although this additive has a strong acid taste.6 to 2. Fumaric acid is used in fruit drinks. The inhibitory capacity of this acid lies in its reduction of pH to levels below which bacteria cannot initiate growth. Fumaric acid and its salts are used for direct addition to food for human consumption (21 CFR 172. 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. pie fillings. Destruction rates for S. Gill and Newton (1982) suggested that the inhibitory effect for psychrotrophic organisms was related to a decrease in pH rather than degree of dissociation. 1985). Lactic acid was about four times more effective than malic. 1991).. LACTIC ACID Antimicrobial Properties Lactic acid has been used more extensively for its sensory qualities than its antimicrobial properties in the past. Acid treatment when combined with vacuum packaging was more effective in prolonging shelf life than vacuum packaging alone (Smulders and Woolthuis. (1984) found that spraying beef and sheep carcasses with a combination of 1% lactic. combined with vacuum packaging and storage at 3°C. Lactic acid was more inhibitory to Mycobacterium tuberculosis as the pH decreased (Dubos.. 1954). A 1. 1988). although more recently this acid has been used as a rinse for beef. and chicken carcasses. reduced aerobic mesophilic plate counts from 5. and storage for 10 days. The acceptable daily intake for humans is listed in Table 4. an LD50 of 8. propionic. 2% acetic.3. In addition to supplying acidity to a product. 0. It increases the strength of gelatin gels. In fermented foods. dicarboxylic acid having low water solubility.7 log CFU/cm2 (Visser et al. gelatin desserts. the organism responsible for flat-sour spoilage in tomato juice (Rice and Pederson.112 Antimicrobials in Food trials using a 500 mg/day oral dose for 1 year were also negative.. and acetic acid in limiting growth of Bacillus coagulans. and wines. 1950).25% lactic acid spray of beef carcasses followed by a treatment of hot-boned cuts with 2% acid.25% citric. this study showed that lactic acid was effective at higher temperatures as well. it blends with certain flavoring compounds to intensify the aftertaste of a flavor. A 2% L-lactic acid treatment at pH 2. Woolthuis and Smulders.350). Typhimurium. Cold-pack cheese normally prepared with lactic acid was reformulated with acetic acid and inoculated with S. Lactic acid was an excellent inhibitor of spore-forming bacteria at pH 5. lactic acid coupled with other antigrowth factors excreted by lactic acid-producing microorganisms inhibits competing microorganisms. unsaturated.000 mg/kg was proposed (Levey et al. biscuit doughs. Because most other studies chose refrigeration storage temperatures. lowered microbial counts after storage for 14 days at 2°C. Enterobacteriaceae contaminated 50% of the untreated samples.

76°C.25% citric. and 0. . 426°C. All products were stored at 4°C and examined for aerobic.3 M calcium chloride solution (CAL. Lactic acid was most effective against S. 1992). Furthermore. or a combination (1:1) CAL/LAC (COM. 2% acetic. psychrotrophic. 65 psi). 0. combinations of the treatments were examined. coli counts was detected for beef dipped in acetic or lactic acid solutions compared with a water-dipped control at day 1. Kotula and Thelappurate (1994) compared acetic acid and lactic acid solutions applied at two concentrations. Acid-treated samples were lighter in color as a result of leaching of the pigment during immersion. but this effect was not seen with acetic acidtreated tissue. coli O157:H7 or S. In addition. and at two time intervals. coli (Anderson and Marshall. reducing numbers by 2 logs and Enterobacteriaceae by 1. (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. 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. E.3 M lactic acid solution (LAC. and E. There was little difference in bactericidal activity between the combination and either acid alone on sub-primal cuts of beef. Greater reductions on all microbial loads were seen in BTF compared to BTL. When sequential treatments were employed using pre-chill and postchill lactic acid sprays. Beef trim meat used to make ground beef is of particular concern because of its higher microbial load. Application of 95°C water alone or with 2% L-lactic acid was effective in reducing E. 2% lactic acid (12°C to 15°C..1).. 2001a. Postchill beef rounds inoculated with E. 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. at 1°C to 2°C as a dip for rib eye steaks. 2001). Typhimurium at 70°C. Treatments containing lactic acid resulted in the lowest counts without affecting sensory qualities. 1990a.5) at a volume equal to 10% of the initial sub-primal weight.5 logs (Anderson et al..Organic Acids 113 Hot-boned. and sensory analysis were not affected by acid treatment. but shear values. The inoculated meat was subjected to sprays of tap water (15°C to 17°C. but control cuts retained more desirable flavor profile (Eilers et al. coli. pH 2. Typhimurium-inoculated beef trimmings. When temperature changed from 20°C to 70°C or lactic and acetic acid concentrations increased individually to 3%. Dixon et al. 482°C. 20 or 120 seconds. Similar work was conducted using lean pork trim (LPT) and fat-covered pork trim (FPT) (Castelo et al. coli biotype 1 counts. or hot air at 371°C. pH 3.b).9 or 1% acetic acid at pH 3. coli O157:H7 and S. moisture content. 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. 2001a. Acuff et al. sub-primal meat was injected with one of three treatments: 0. aerobic bacterial counts and S. 0. and presumptive lactic acid bacteria.b). The system alone produced a 3log reduction.. Meat color was affected after application and through storage at 4°C.8 to 7.2%. Significantly higher aerobic plate counts were obtained from hot-boned cuts over 17 days postmortem compared to cold-boned cuts. or 82°C. hot water (30 psi) at 65°C. <1 log for Enterobacteriaceae and 0. COM improved tenderness in muscle cuts. 1999).5 logs for E. Typhimurium levels decreased approximately 1 log.2 logs.6% or 1. the FPT supported lower microbial numbers than LPT. but odor was not affected (Ellebracht et al. or 510°C. 454°C.. pH 5. b).6 with either 1% lactic acid at pH 2. total fecal coliforms. Minimal reduction in total plate and E.. Typhimurium were subjected to an automated wash water system (Castillo et al. only suggesting that lactic acid can exert a continued antibacterial effect during the shelf life of the product.1% ascorbic acids adjusted to pH 2. (1987) compared a combination of 1% lactic. 1994). 30 psi).3. Aerobic plate counts of meats injected with CAL were significantly higher than with LAC or COM. coli was reduced by 6. 71°C. 1998. 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. 0. The combination of acids performed as well as 1% lactic or acetic acids alone. 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.0). As in beef trim.

coagulans were heated to 121°C and 105°C or 110°C. malic. however. 1993). Not only did the microbial counts decrease as the time of decontamination increased.000 IU/ml nisin.. these treatments were not deemed usable in the control of E.7. Salmonella species or Listeria species were not recovered nor were sensory characteristics affected (Prasai et al. 180. vacuum packaging. 1992). and Lactobacillaceae counts by 2 to 3 logs. psychrotrophic Gram-positive bacteria. After 28 days of storage. monocytogenes and treated with 2% lactic acid. These compounds reduced the level of L.6 log/6 cm2. 2002). Psychrotrophic. respectively (El-Khateib et al. Y.. All treatments significantly reduced the number of L... Numbers of S. but the population of microorganisms shifted. Woolthuis et al. coli O157:H7 was subjected to treatments of lowmolecular-weight polylactic acid. Lactic acid was effective in decreasing the number of Enterobacteriaceae as well as other Gram-negative mesophilic and psychrotrophic spoilage organisms. lactobacilli and yeasts were the least sensitive. coli O157:H7 inoculated into beef trimmings (Bolton et al. coli O157:H7 in beef burgers. or hydrochloric acid (Lynch and Potter. .2 or 4.1. and 200 IU/ml of nisin (Mustapha et al. The use of lactic acid for reduction of microbial numbers or pathogens has been similarly applied to pork products. and 3200 units/ml pediocin. Five treatments were delivered to the pork skins: water-treated (control) or exposure at 21°C for 120. The use of these compounds was effective in reducing psychrotrophs and Enterobacteriaceae (Ariyapitipun et al. significant reductions were seen with all treatments. 2000). Lactic acid (2%). Typhimurium were reduced more by exposure to acetic acid/gel treatment than acetic acid alone. whereas acetic acid was more effective than the acetic acid/gel treatment. Frankfurter emulsions incorporated with acids to adjust pH to 5. monocytogenes by 1. monocytogenes before vacuum packaging and storage at 4°C.5%).8. and polylactic acid. by immersing porcine livers in 0.5 and 4. Beef cubes were inoculated with L. and nisin did not enhance the effects of lactic acid against E. 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. 55°C) had little effect on the aerobic plate counts taken from the surface of pork carcasses. 240. noncoated tissue became dehydrated during the 7-day study. 1993). 1988).2% lactic acid for 5 minutes. monocytogenes..6 for Bacillus stearothermophilus and pH 4. 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. and low-molecular-weight polylactic acid.. 1999). (1984) effectively reduced total plate counts. After 42 days of storage the initial inoculum of 5 logs was reduced to <1 log after using lactic acid. Elimination of these groups shifted the population to Gram-positive bacteria and yeasts. lactic acid. or sodium lactate (4%) alone or in combination with pulse electric field or freezing was ineffective in reducing numbers of E. Beef inoculated with E. nisin. Therefore. Lactic acid treatments alone were only 50% as effective as acid/alginate treatments. and nisin (400 IU/ml) were used alone or in combination in beef to inhibit L. A 1% concentration of lactic acid (pH 2. monocytogenes on beef tissue. but no differences were seen between polylactic acid and lactic acid. Greater inactivation was noted for both organisms at the lower pH level when acetic or lactic acids were used but not with citric.2 for B. 1993). By increasing the concentration of lactic acid to 2%. monocytogenes. coli O157:H7. Gram-negative bacteria were the most sensitive to lactic acid. Enterobacteriaceae. and 0. The combination of acids and heat treatment led to reduction of spore-forming organisms in frankfurters. numbers of Salmonella species and Campylobacter species were reduced immediately and remained lower 24 hours after slaughter (Epling et al. enterocolitica. Beef was inoculated with L. there was no significant difference between nisin. Aeromonas hydrophila. followed by mesophilic Enterobacteriaceae. lactic acid. acetic acid (0. 40. and storing for 5 days at 3°C. or nisin and lactic acid. 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. Although the gels reduced populations of L. monocytogenes (Ariyapitipun et al. low-molecular-weight polylactic acid (2% PLA). 1.114 Antimicrobials in Food Lactic acid (2%).. 2002). 1992. or 360 seconds to 2% lactic acid.

000-fold.5%. 1983). 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. coli decreased 10.Organic Acids 115 P. or Brochothrix thermosphacta (Greer and Dilts.1°C. After inoculation. 1995). and a 2% lactic acid dip at pH 2. The number of Salmonella-positive birds was also reduced as a function of time of the dip (Izat et al. pH 2. Chicken skin subjected to washing treatments using 1% lactic acid led to significant reductions of Salmonella species and L. 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.. Lactic acid added to broiler chill water resulted in the development of a brown coloration. 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.. Salmonellae were eliminated from broiler carcasses after a 1-hour exposure.3 prolonged shelf life to 8 days (Zeitoun and Debevere. monocytogenes counts were reduced by 0.0) for chicken legs increased the shelf life from 6 to 12 days at 6°C. P. coli demonstrated a 10-fold decrease in numbers when grown in media containing sodium chloride.7 log CFU/g and Enterobacteriaceae from 3. tissue was dipped for 15 seconds in water or lactic acid and held for 15 days at 4°C. In an effort to eliminate carcass discoloration. None of the pathogens grew on lean tissue.7 log CFU/g (van der Marel et al. however. but no protective effect was observed if the sodium chloride was added to the medium 45 minutes after the inoculation. respectively.8) and scald water (54°C/2 minutes) reduced the bacterial level of broilers inoculated with S. Lactic acid added to scald water alone had minimal effect on reducing the numbers of contaminated birds. 1995).5% sodium benzoate dip concluded that sensory qualities were acceptable (Hathcox et al.. such as chlorine or chlorine dioxide. These treatments inhibited hydrogen sulfide-producing bacteria. 1995).23 in cells exposed to acid to 5. acidified media.62) were combined with propylene glycol (20%) in chill water. sodium tripolyphosphate.. pH 2. 2002). and Beuchat.2 logs. coli O157:H7 grown in TSB at 37°C (Casey and Condon. 1995. discoloration still occurred and propylene glycol contributed an objectionable flavor (Izat et al. Although L. Typhimurium to almost nondetectable numbers. produced similar reductions of approximately 1 log (Zhang and Farber.000-fold). that contributed to spoilage. other compounds. or sodium hexametaphosphate.6 log CFU/g. E. pH 2. such as Pseudomonas species. fragi. in sodium chloridefree. Levels of psychrotrophs decreased from 3. 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. E. fragi and B. or formic acids. . grew on both tissue types after acid treatment. 1988). shelf life was lengthened to >36 and 35 days at 4°C and 7°C.. 1990a). Acid treatments of fat tissues led to the reduction of pathogen numbers to nondetectable numbers within 4 days. 1990b).2 decreased from 5. A 10% lactic acid and sodium lactate-buffered acid spray (pH 3.9 to 2. 1990). Lactic acid (1%) added to both chill water (0°C to 1.79 in acid-free media. Consumer evaluation of chicken treated with a 0. however. The spoilage organisms. sodium acid pyrophosphate.88 or 0. S. although tissue treated with acid affected the growth rate. The treatments did not affect sensory quality.2 in media containing lactic. and A. 1996).5 and 0. Lactic acid dips have also been used successfully in the poultry industry. When treated broilers were packaged using modified atmospheres. E. This protective effect was even more pronounced with acetic acid (100. monocytogenes compared with monosodium phosphate. coli O157:H7 rapidly died at pH 4. reduced levels of lactic acid (0. hydrophila numbers declined by 5 logs within 11 days of storage. Hwang. The interaction of sodium chloride (0. respectively (Sawaya et al. 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. Lactic acid was more effective than acetic acid when used as a 10-minute rinse for fresh-cut vegetables.5% to 4%) and pH (4.3 to 2.5% lactic acid/0.0 than cells grown at 37°C.2) was examined for E. Higher concentrations of the acid did not ensure greater decontamination nor did repeated treatments. thermosphacta. acetic.25%.2 to 3. acidified with lactic acid. Total microbial numbers from skin of birds immersed for 15 seconds at 19°C in 1% or 2% lactic acid at pH 2.

see Shelef (1994) and De Wit and Rombouts (1990)..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. MIC values for Salmonella species ranged from 714 to 982 mM and a narrower range for L.. Gram-negative bacteria including Pseudomonas and Yersinia had a comparable range to Salmonella. 2003. syrupy liquid having a moderately strong acid taste. although Campylobacter was particularly sensitive with an MIC of 179 mM. and lactic acid concentration has been developed (Mellefont et al. and extends the shelf life of products. Yeasts were very broad in . 1993). In two other instances. 1935) or 87.116 Antimicrobials in Food El-Gazzar et al. Toxicology The LD50 of lactic acid for test animals varied according to animal species (Table 4. parasiticus NRRL 2999 grown in a laboratory medium containing 0.. The autopsy revealed hemorrhaging and gangrenous gastritis (Young and Smith. improves juiciness.. Ross et al. Presser et al. dehydration. retains color. 1944). coli in response to temperature. D (-) or DL. Application and Regulatory Status Lactic acid is a hygroscopic. 2003). Infants died after consuming milk acidified with an unknown quantity of lactic acid. confectionery products. Gram-positive bacteria including lactic acid bacteria. lactis. Lactococcus. developed acidosis. Bacillus. Some enamel decalcification was apparent in the experimental groups.2). The acceptable daily intake for humans is listed in Table 4.2 using HCl or lactic acid stimulated by substituting half of the carbon content by lactate and a reduction in production at pH 6. Hamsters fed a cariogenic diet incorporating lactic acid in the drinking water (40 mg/100 ml) or in the feed (45. 20°C) for a number of bacteria and yeasts isolated from spoiled and unspoiled meat products (Houtsma et al. 1949). and in baking powders. Premature infants fed acidified milk containing the isomeric forms of lactic acid. It is used to adjust acidity in brines for pickles and olives. The Food and Agriculture Organization of the United Nations (Food and Agriculture Organization. and Staphylococcus ranged from 268 for S. A predictive model for growth of E. With increasing levels of lactic acid. 1945)..5% lactic acid (Trainer et al. Lactic acid is used in the manufacture of jams. 1973) supported this view.5. aureus and Lactobacillus coryniformis to 804 mM for Brochothrix.75% lactic acid adjusted to pH levels of 3. and beverages. The MIC of sodium lactate was investigated under optimum growth conditions (pH 6.. contributes to water-holding capacity.5% and 0. 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. 1998.8. infants suffered stricture of the esophagus following ingestion of milk acidified with 1 teaspoon of 85% (Pitkin. parasiticus in association with L. (1987) found that A. and vomiting. Luchese and Harrigan (1990) also noted an increase in aflatoxin production adjusted to pH 4.3. water activity. weight loss. pH. increases meat yields. (1970) recommended that the L (+) form should be used in feeding premature infants.1061).6 mg/100 g) did not differ in growth rate compared with control groups (Granados et al. monocytogenes and L. Carnobacterium. jellies. Brochothrix. to prevent discoloration in fruit. Calcium lactate is used as a firming agent for apple slices. Ballabriga et al. cultures decreased production of aflatoxin G1. innocua from 804 to 982 mM.5 or 4. appears naturally in meat products. For an excellent review of the antimicrobial nature of sodium lactate. sherbets. Lactic acid is approved as a GRAS substance for miscellaneous or general-purpose usage (21 CFR 184. Aflatoxin production also increased during growth of A. Therefore.

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. 6. Typhimurium when stored for 4 days at 20°C. botulinum strains in peptone yeast extract medium (Houtsma et al. Addition of 2% sodium chloride enhanced the listericidal activity of the lactates (Weaver and Shelef. however. Samples were stored up to 50 days at 5°C and up to 10 days at 20°C. There did not appear to be a synergistic effect between a 4% concentration of lactate combined with 140 ppm . This model was then evaluated for use in a bologna-type sausage system (Houtsma et al. 1995). Salts of lactic acid are equally as effective in muscle food systems. spore germination did not lead to growth after 7 days. respectively. with lower amounts of sodium lactate needed as pH and temperature dropped. The effect of sodium lactate on toxin production. spore germination. Sodium. which might explain their prevalence in spoiled meat products.5. At the abuse temperature of 20°C. Survivor curves were developed using L. This level was listeriostatic at a moisture of >55%. 1993). At a higher temperature of 20°C. monocytogenes. Lactic acid/sodium lactate and acetic acid/SA were studied in a BHI broth at pH 7. potassium. innocua was modeled in broth adjusted to pH 5. but the combination of 2% or 3% lactate and 2% sodium chloride at the same moisture level was inhibitory. 1994b).5%. As expected L. As expected. calcium. and 30°C (Houtsma et al. 1991. no toxin formation was detected after 49 days of incubation in media containing 3% sodium lactate. Pork liver sausage was formulated with sodium. or calcium lactate at concentrations of 2%. monocytogenes per gram of sausage. 0. toxin formation was noted within 5 days for 0%. and 7. whereas a 2% level extended the time to 24 days..982) did not inhibit toxin production. Calcium lactate (2%) reduced populations below the inoculum level. A subsequent study (Shelef and Potluri.. 6. toxin production was detected in media containing 4% sodium lactate within 11 days. Using 108 CFU/g as a microbial end point. comparable to a 3% concentration of calcium lactate. or 4% and inoculated with 104 to 105 L. indicating that inhibition was not the result of a lowering of water activity. 4% sodium or potassium lactate achieved reductions in L. 1996). and the resultant pH. The effect of sodium lactate and sodium chloride on the growth of L. Chen and Shelef (1992) suggested that in addition to pH change. coli O157:H7 and S.. Sodium chloride at the same water activity as sodium lactate (0.Organic Acids 117 their sensitivity.0 at 4°C. the rate of inactivation was dependent on the acid. 1993). 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. 20°C. At 30°C. 1995) examined the effect of 3% sodium or calcium lactate added to pork liver sausage and various heat treatments. monocytogenes as the target organisms. 6. lactates also contributed to lowering the aw of a food. and potassium lactate were equally effective. 3%.. 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. Potassium lactate (4%) and sodium chloride (3%) reduced growth somewhat at 5°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. and 15 days for 2.1. inhibition by sodium lactate was directly related to pH and temperature. and heat resistance was tested on proteolytic C.5.0% sodium lactate. and 2 M concentrations (Buchanan et al. 1994a).5%. 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. monocytogenes was studied in a cooked meat model system containing varying moisture levels and 4% lactate. The calcium form was found to be more inhibitory than the sodium form. and 4 using 0. its concentration. 5.0. As expected. 1% sodium lactate delayed growth for 10 days. no growth or toxin formation was detected after 32 days of incubation for media containing 4.. L. 1. with Debaryomyces and Candida being the most resistant at 1161 and 1339 mM. 10°C.0. The pH level was influential in inhibition by sodium lactate but not by sodium chloride. 11 days for 1. Calcium lactate was also more effective than sodium lactate in retarding growth of E.5.

C.b. The pH of the product was unaffected by incorporation of sodium lactate. Cured meats. In retail products. Papadopoulos et al. and E. the amount of L-lactate ordinarily found in muscle was not inhibitory to any of the strains. 1997). monocytogenes. Cooked beef top rounds containing 1%. monocytogenes during storage at refrigerator and abuse temperatures. but produced a mild throat irritation at a 4% concentration. 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.4%. and A. botulinum..5.2%. monocytogenes was delayed for 4 and 12 weeks at 7°C and 3°C in unsmoked. or 4% sodium lactate were inoculated with L...c) injected beef top rounds with 0% to 4% sodium lactate.4% sodium lactate/0. A 100-mM concentration of sodium lactate buffered to pH 5.7 to 8. monocytogenes. Lactate increased cooking yields. The heat resistance of the two organisms was significantly lowered in beef containing either of the two concentrations of sodium lactate.0% or 3.118 Antimicrobials in Food potassium nitrite. 1981). Sodium lactate at concentrations of 3% and 4% were generally effective in limiting growth of L. 1991). sodium lactate caused more rapid surface discoloration (Lamkey et al. 0.. 2002). Although Shelef and Yang (1991) suggested a possible aw effect for sodium lactate. and yeasts at 42 days to homofermentative and heterofermentative lactobacilli and streptococci at 84 days. Another study (Bedie et al. Both concentrations of potassium lactate significantly inhibited or delayed the growth of L. 1999). enterocolitica or L.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. or 4. or 3. When raised to 210 mM.6% sodium lactate into the product (Kilic et al. 1993). then cooked and vacuum-packaged the roasts.1% sodium diacetate (Glass et al. uncured bratwurst manufactured with 3. Over the storage life of the product. such as wieners. Enterobacter cloacae. 1980). nitrite levels are reduced. aureus. Papadopoulos et al. Sodium lactate was less effective against microorganisms when sausage contained soy concentrate. Typhimurium. The growth of L. Retention of nitrite was linked to higher pH levels and was unaffected by the incorporation of 1. Red color was preserved by using 2% to 3% sodium lactate. rely on sodium nitrite to inhibit growth of C.5% SA or sodium diacetate as an antilisterial process for frankfurters. enhanced flavor at the 1% level by adding a salty taste.8% sodium lactate was inoculated with Y. or E. 2002). Typhimurium.5 in a meat system prevented anaerobic growth of Serratia liquefaciens. Although the aerobic plate counts for control meat increased from 2.4%. A 3% concentration of sodium lactate at pH 7. 2.0% potassium lactate showed differences in spoilage rates. 2002). Levels of >3% sodium lactate or 1% each of lactate and diacetate were needed to prevented growth of Listeriae for 60 days at 4. Sodium lactate at a 6% concentration or 0.25% sodium diacetate were the most effective treatments throughout the 120-day shelf life conducted at 4°C with minimal change in pH. enterocolitica. S. (1991a. 3%. perfringens.1. hydrophila and aerobic growth of the aeromonads (Grau. Frankfurters containing 2. coli O157:H7 and stored at 10°C. respectively (Porto et al. thermosphacta down to pH 5. (1991b) did not find this effect in their studies. Y. 2.5 log CFU/cm2 in the same period. the plate counts of treated roasts increased to 6. lactobacilli.. coli O157:H7 (Miller and Acuff. At pH 6. The microflora changed from predominantly staphylococci and micrococci at 0 days to coryneform. .. monocytogenes and stored at 4°C and 10°C for up to 90 and 60 days. 1994). 2001) compared the use of 3% or 6% sodium lactate.0% potassium lactate were inoculated with a five-strain cocktail of L. S. monocytogenes and heated to 55°C to simulate a sous vide process (McMahon et al. sodium lactate permitted aerobic growth of B. micrococci.05 and 6. 2%.. Minced beef containing 0%. Lactates inhibited the growth of psychrotrophic organisms and delayed the pH decline (Bradford et al. S.25% or 0..5 reduced surface discoloration in chubs of fresh sausage and extended shelf life from 10 to 20 days. Pork sausages manufactured with 25% fat or 8% fat with and without 3.0 log CFU/cm2 after 84 days of storage.

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

3.000 ppm. The acceptable daily intake for humans is listed in Table 4. fruit preserves. a 2% lactate and 3% sodium chloride inhibited L. monocytogenes and total aerobic plate counts (Harmayani et al. In general. and sodium nitrite. 1% kappa carrageenan. monocytogenes for up to 50 days at 5°C. 1994). Some discoloration and lipid oxidation occurred. and hydrolyzed vegetable protein and containing 1%. Similar studies used low-fat ground beef patties formulated with carrageenan. 0. MALIC ACID Antimicrobial Properties Malic acid is inhibitory to fungi and bacteria. Nunheimer and Fabian (1940) found that malic acid was inhibitory against S. or 3% potassium lactate. Sodium lactate displayed synergistic activity with nitrite and increasing concentrations of sodium chloride. primarily for its flavoring and acidification characteristics (Gardner. sodium lactate was more effective than the other additives in inhibiting growth of L.1207). potassium (21 CFR 184. 1970).. It has a strong acid flavor but does not have the same buildup of acid taste as other acids.1% sodium erythorbate. subtilis). and sodium lactates (21 CFR 184. sodium chloride. especially rope bacteria (B. dicarboxylic acid with high water solubility.1069).000 ppm malic acid before mating showed no significant differences from the controls (Hazelton Laboratories. Carrageenan had no effect on bacterial loads. probably as a direct effect of pH manipulation. As the pH decreased to 5. monocytogenes. monocytogenes and stored aerobically at 4°C. at 50. and beverages. but the addition of 2% or 3% potassium lactate reduced microbial numbers.. encapsulated salt. Application and Regulatory Status Malic acid is a nonhygroscopic. 2%. The acceptable daily intake for humans is listed in Table 4. the acid inhibited yeasts and molds but not to the same extent as bacteria (Woolford. jams. and stored at 5°C or 10°C. and .0. 1992). 1993). Application and Regulatory Status Calcium (21 CFR 184.1768) are also approved.8% sodium lactate. PROPIONIC ACID Antimicrobial Properties Propionic acid inhibited spore-forming bacteria. Reproductive experiments using rats fed 1000 and 10. vacuum packaged. thus affecting the sensory qualities of the meat (Egbert et al.98. Chung and Goepfert (1970) compared various organic acids to determine the highest pH level that inhibited growth of S. aureus at pH 3. food consumption decreased and growth rate declined (Hazelton Laboratories. at pH 6. 1972).0.3. 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. anatum. Malic acid is approved as a GRAS substance for miscellaneous and general-purpose usage (21 CFR 184. jellies. however. Dogs fed the same three levels of malic acid showed no effects using the same parameters (Hazelton Laboratories. The products were inoculated with L..0 or 4. and an alginate binder. Toxicology Malic acid fed to rats at levels of 500 and 5000 ppm in the basal diet had no effect. Comminuted salmon was mixed with sodium lactate. Salmonella Senftenberg. 1971a). It is used in sherbets and ices. 1971b).120 Antimicrobials in Food Raw and cooked ground beef were prepared with 1.1639). inoculated with L. 1975b).

such as lima beans and peas (Wolford and Anderson.9% sodium propionate or SA.0 and 13°C. 1992). Nondetectable levels of the pathogen were found in products ZEN. pH levels. inoculated with C. 1989a). and temperatures. Proteus vulgaris. and 4 days. acetate. the organism was able to grow at up to a 0. protein. aureus. or at a level of 0. Toxin production occurred after 2 days for pyruvate. the lag phase of L. tartaric. monocytogenes (Janes et al. The salts of propionic acid are also effective antimicrobial agents. adipic. succinic. concentration of the organic acid. acetate. ZPNCP. DNA (deoxyribonucleic acid). 18 days for citrate. monocytogenes was inoculated into BHI broth containing 0. 7. and 35°C and prevented growth at 4°C (El-Shenawy and Marth. aureus. monocytogenes when used in conjunction with acetic acid in cold-pack cheese food (Ryser and Marth. 2002). pH 4. Golden et al. Calcium propionate prevented rope formation caused by B. monocytogenes.02% ascorbic acid. and held at 28°C for 0 to 18 days. and propionate to a target level of pH 6.8.0 in reducing numbers of L. or 4°C. 1%) and coated onto ready-toeat chicken samples dipped in L. and Candida albicans.000 IU/g) or calcium propionate (CP. which led to an increase in cell mass without cell division and a decrease in viability (Cherrington et al.6 (O’Leary and Kralovec. and berries but also prevented spoilage in vegetables having a more neutral pH. Reduction of pH to 5. pH 5. respectively. At a 5-mM concentration.1. and 35°C.6. 19°C.5 to 0. At a 6% concentration. particularly at the higher pH level. the rate of RNA (ribonucleic acid). or lactic acids in media (El-Shenawy and Marth. 5. or ZENCP. and 5 with a 2% concentration. mesentericus (subtilis) in bread dough at a level of 0. pH 5. Products were stored for up to 24 days at 4°C or 8°C. 1990). monocytogenes to 8% sodium propionate for 60 minutes caused injury (Buazzi and Marth. monocytogenes were undetectable. pH 4. toxicity occurred after 7 days for pyruvate. the levels of L...6 with acetic. 4 for lactate and acetate. pH 4.3% sodium propionate was adjusted to pH 5.1% to 5% delayed growth of S. L. Sodium propionate at levels of 0. applesauce. Propionic acid at pH 5. 13°C. 1992). The acids contributed to approximately 85% of the antimicrobial activity with 14% contributed by ascorbic acid. and pH. coli K12 and Salmonella. ellipsoideus by as much as 5 days. Exposure of L.0 minimized growth at 13°C. and cell-wall synthesis decreased. In laboratory studies examining the effect of sodium propionate on L. 1993). Much higher concentrations of propionic acid were bacteriostatic to B. tartaric. adjusted to pH 4.6 and incubated at 4°C.188%. pH 5. lactic.0 was more inhibitory in 60 minutes compared to formic acid in 3 hours for E. (1991) determined that 0. At pH 5. When media containing 0. S.0 with hydrochloric acid.7 M propionic acid at pH 5. Cherrington et al.0. syrup. lipid. Inactivation of L. 3. coli. Citrate was most effective based on molarity (Miller et al. 1945). Sodium propionate at a 0. Eklund (1985) concluded that propionic acid was not as inhibitory as benzoic acid for P. Sarcina lutea. monocytogenes was the result of incubation temperature. little activity was ascribed to EDTA in combination with the acids.3% concentration in tryptose broth adjusted to pH 5. monocytogenes grown at 13°C was extended by 7. E. acetic. 1. and 0. citric). pH 4. cherries.5.3. aeruginosa. . samples were removed from storage and tested for production of neurotoxin. 0. Zein (Z) film coatings were dissolved in propylene glycol (ZP) or ethanol (ZE) with and without nisin (N.05. pyruvate. Turkey breast meat was formulated with sodium salts of lactate. Propionic acid was more effective than acetic acid as an antimicrobial. botulinum. 21°C.08% EDTA. Incorporation of calcium propionate into coatings for ready-to-eat products was effective in reducing problems with L. 21°C. 4. 1941).5 inhibited the growth of Salmonellae at a higher pH level than other acids (pH 5. degree of dissociation of the acid. Torula species.4.4.Organic Acids 121 Salmonella Tennessee when all other parameters were at their optimum.0. A three-strain mixture of L.1.3% concentration was also effective at pH 5. and greater than 18 days for propionate. and stored at 28°C. monocytogenes. Emphasizing the multiple-barrier concept in retarding growth. At periodic intervals. plantarum. This substance not only postponed spoilage in fresh figs. citrate. or 3.. citric.156%. (1995) examined the combination of sodium propionate and SA at a number of concentrations. pH 4. subtilis. and lactate. 1988). 3 for citrate. fumaric and malic. and S.5.

0. and Penicillium corylophilum. and Hara et al. Additional studies using calcium and sodium propionate fed to mice. 2000). B. 1965.0 and a water activity of 0.2). 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. .8 to 0. All preservatives were effective at pH 6. A 5% calcium propionate solution acidified to pH 5. There is evidence that the sodium salt has some local antihistaminic activity (Heseltine. Eurotium amstelodami. A novel use of propionic acid to retard spoilage of bread was investigated by Gardner et al. 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. no growth occurred. however. pH 4. and rubrum. subtilis. 1942). or Trichophyton mentagrophytes. Mold spoilage is a significant economic problem in bakery products. and humans showed no toxic effects (Graham et al. monocytogenes was studied in pork liver beaker sausage (Hu and Shelef.b) examined several levels of calcium propionate. Bread produced with the extracts contained less ethanol and supported less spoilage by molds. reduced growth and aflatoxin formation of A. Ingle.3%) at pH 4. flavus. Propionic acids and their salts are primarily inhibitory to molds. 1. 1940). None of the preservatives were effective at neutral pH. herbariorum. 1953).5 logs.003%. some species of Penicillium grew on media containing 5% propionic acid (Heseltine. rats.1% sorbic acid or potassium sorbate. However. 1985).5 and water activity of 0.8 log reductions were observed. (1963) determined the LD50 of calcium and sodium propionate for rats (Table 4. 2002). monocytogenes at 22% fat compared to 2..85 (Guynot et al. Propionic acid at a concentration of 0. and 0.5. Molds were inhibited by less calcium propionate by weight than sodium propionate. flavus and niger. Small amounts of β-alanine could overcome bacteriostatic action of sodium propionate for E..5 and 1. The antimicrobial activity of sodium propionate was most affected at 4°C by fat concentration with a 1. cooling surfaces. The inhibitory action could be the result of an interference with β-alanine synthesis (Wright and Skeggs. Adding yeast extracts previously fermented by Propionibacterium freudenreichii provided a source of propionic acid in bread formulation. and 7. 1952a). This inhibitory effect was more pronounced with the addition of acid at the time of inoculation.. 1945). and 0. Toxicology Orö and Wretland (1961) determined the LD50 of propionic acid for mice. Increasing the fat content from 22% to 67% decreased the growth of L. respectively. Spore germination was inhibited by 1402 ppm (Tzatzarakis et al. but also various organisms displayed different tolerances to the compounds (Olson and Macy. potassium sorbate. Subsequent studies (Marin et al. β-alanine could not reverse the inhibitory effect of propionic acid in Aspergillus clavatus. 1946). 1940). (2001). Pseudomonas species.8% sodium lactate. and sodium benzoate (0. coli. 2002a. and wrapping materials reintroduce molds.5 log reduction of L.03%) led to enhanced growth of Aspergillus and Penicillium isolates. at 0.2%. sodium benzoate. postprocess contamination from the atmosphere. 1954. with sodium lactate. rubrum.0. and 0. Sausage batter was mixed with 0. rather than later (Ghosh and Häggblom.122 Antimicrobials in Food The effect of fat concentration on the efficacy of antimicrobial agents against L. Harshbarger.85. 0. Hara.3% by weight. herbariorum. and potassium sorbate at a level of 0. Not only was the final pH of the substrate critical. 1996). amstelodami. 1951.8. 1955. Graham and Grice.2% sodium propionate.03%. monocytogenes by only 1. Although mold spores are destroyed during baking. 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. A.1%. 1952b).5 with lactic acid was as effective as a 10% unacidified solution in preventing surface mold growth on butter (Olson and Macy. 0..5.8 log reduction at 67% fat. 6. and repens were inoculated by needle into cake analogues prepared with calcium propionate. Suboptimal doses (0.93 in a model agar system on the retardation of growth of E.

0% succinic acid. (1967) reduced the population of inoculated S. 1944). It can be incorporated into gelatin deserts and cake flavorings (Gardner. and the sodium salt is more soluble than the calcium form. except bread and rolls. 1972). Swiss cheese contains up to 1% propionic acid because of the growth and metabolism of propionibacteria.. 1974).1091). The acid form is readily miscible with water. 0. calcium (21 CFR 184. however.3% in cheese products (Robach. which are associated with its manufacture and the characteristic Swiss cheese flavor..38% in whole-wheat products. A limit of 0. hair appearance. No limit has been set on the acceptable daily intake for humans. Propionic acid (21 CFR 184. but surface recontamination can occur during packaging. Sodium propionate is recommended for use in chemically leavened products because the calcium ion interferes with the leavening action. 1972). It is normally found in cheese and as a metabolite in the ruminant gastrointestinal tract.32% can be used in flour and in white bread and rolls. The test animals did not differ significantly in reproduction rates. Thomson et al.23).1221) and sodium propionate (21 CFR 184. 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. Chick embryos developed normally when comparable dosages were injected into the air sac (Dye et al. tooth eruption. and growth can be seen under the wrapper during storage.Organic Acids 123 Application and Regulatory Status Propionic acid is a monocarboxylic acid with a slightly pungent. Baking destroys most molds.1081) and its salts. Propionic acid and its salts are used primarily as mold and rope inhibitors in bread. 1965). dicarboxylic acid having low water solubility. calcium and sodium propionate are listed as antimycotics when migrating from food-packaging material (21 CFR 181. or eye opening from the control group. which conform to standards of identity. Propionates can be added to bread dough without interfering with leavening because there is little or no effect on yeast. It has a slightly bitter taste and serves as a flavor enhancer.0 mg/day at 4 weeks and continuing at this level for 100 days.1784). The acceptable daily intake for humans is listed in Table 4. SUCCINIC ACID Antimicrobial Properties Succinic acid successfully lowered microbial loads on poultry carcasses (Mountney and O’Malley. Calcium propionate is preferred. rats received subcutaneous injections of 0.3. and 0. This naturally formed additive becomes a developed preservative that limits mold growth on Swiss cheese.5 mg succinic acid daily. however a 3% or 5% concentration used at 60°C impaired the appearance of the product (Cox et al. are approved as GRAS substances for miscellaneous and generalpurpose usage. No upper limits are prescribed for use of this additive. gradually increasing to 2. Succinic acid is used to modify the plasticity of bread doughs and in the production of edible fats. 1980). Succinic acid is approved as a GRAS substance for miscellaneous and general-purpose usage (21 CFR 184. . Salts of the acid have a slight cheeselike flavor. Application and Regulatory Status Succinic acid is a nonhygroscopic. In addition. This additive is also used as a mold inhibitor in cheese foods and spreads. disagreeable odor. Toxicology In short-term studies. Typhimurium by 60% on chicken carcasses after spraying with 1.

25 to 1 g to rabbits produced abnormalities in blood chemistry. sherbets.1%.5%. 225. Tartaric acid acts synergistically with antioxidants to prevent rancidity. Application and Regulatory Status Tartaric acid. 1973a). Homeostasis is the tendency of a cell to sustain chemical equilibrium despite a fluctuation in the acid–base environment. and phospholipids can be structurally altered by pH changes. Proteins. Toxicology Acute toxicity data for tartaric acid by the intravenous route for mice indicates an LD50 of 485 mg/kg body weight (Table 4. nucleic acids. 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. growth. and yeasts.7% sodium tartrate (5% tartaric acid) did not show any changes in food consumption. sugar. However. 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. 1978). or gross histology compared with controls (Packman et al. The microbial cell normally reflects this equilibrium by attempting to maintain an internal pH near neutrality (Baird-Parker. and 215 mg/kg body weight. It is used in fruit jams. and it prevents discoloration in cheese (Gardner. mortality. 1954). although some microorganisms such as Acetobacter can exist at and even require extremes of acidity (Langworthy. Tartaric acid is a GRAS substance for miscellaneous and general-purpose usage in accordance with good manufacturing practices (21 CFR 184. molds.1099). The monopotassium salt (cream of tartar) is commonly found in baking powders. and the production of energy by the cells.. The acceptable daily intake for humans is listed in Table 4. Inorganic acids. which disappeared after they left the work site (Barsotti et al. and cholesterol levels.3. The workers also displayed skin eruptions. the most soluble of the solid acidulants. Workers exposed to tartaric acid dust measuring up to 32 mg/m3 in a manufacturing plant that produced the acid experienced tooth erosion. Fitzhugh and Nelson (1947) found similar results in long-term studies using 21-day-old weanling rats fed tartaric acid as 0. 1963).124 Antimicrobials in Food TARTARIC ACID Antimicrobial Properties Any antimicrobial property of tartaric acid is because of its ability to lower the pH.8%. this delicate balance is maintained and alteration of this balance causes destruction of the microbial cell. and 1. MODE OF ACTION The mode of action of organic acids in inhibiting microbial growth appears to be related to maintenance of acid–base equilibrium. Biological and chemical systems depend on an interaction between acid and base systems.. proton donation. and grape-flavored beverages. jellies and preserves. including increased nonprotein nitrogen. Rose’s (1924) work on subcutaneous administration of 0. hamsters. male rabbits consuming a diet containing 7. has a strong tart taste that enhances grapelike flavors. Through the interaction of a series of chemical mechanisms. 0. respectively (Food and Drug Research Laboratory. 0. . Studies measuring teratogenic activity in pregnant mice. Changes resulting from pH can destroy bacteria.2% of the diet. It is thus essential to understand each of these concepts before consideration of the actual mode of action of the organic acids. and rabbits demonstrated normal offspring at levels of 274.2). such as hydrochloric. 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. 1972). rats. 181. 1980).

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

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

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

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

The metabisulfite is the anhydride of the acid sulfite: – → 2– 2 HSO3 ← S2O5 + H2O When these salts are exposed to air. 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. . as well as by other methods.1.76 and 7. indicating a rather weak dibasic acid (Segal. 1928). and from reducing gypsum. The anhydrous sodium and potassium salts are prepared by precipitation and then dehydrated with the appropriate hydroxide. Most of these salts are hygroscopic and easily hydrolyzed (Schroeter.2 lists the main forms of sulfur dioxide. 1966). they show increasing stability in the order sulfite > bisulfite > metabisulfite (Mason. 1968).1. FORM AND SOLUBILITY In water solutions. The dry salts are easier to store and are less of a problem to handle than gaseous or liquid sulfur dioxide. Specific gravity for various water solutions of sulfur dioxide is shown in Table 5. 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. A plot of the distribution of the three ionic forms can be calculated and is shown in Figure 5.20.144 Antimicrobials in Food Sulfur dioxide can be prepared commercially from burning sulfur (oxidation). Table 5. their theoretical yields. 1928. Phillips. and solubilities of each in water. heating pyrites.1 Distribution of the ionized forms of sulfurous acid at various pHs. It is useful to have the sulfur dioxide in a salt form. The pKa values for sulfur dioxide are 1.

This reaction is extremely rapid.4 53.6 57.8 25.0091 1.0393 1. The sulfur dioxide scavenges the hydrogen peroxide formed and keeps further oxidation of the dehydroascorbic acid and other products to a minimum.0191 1. The sulfur dioxide stabilizes the dehydroascorbic acid by reacting with the ketone bonds (Wisser et al.0292 1. Holt and Kumar (1986) found an observed k = 41.008 1. After the oxygen disappeared.40 and 15°C.4 s-1 for H2O2 with SO32.at pH 3.Sulfur Dioxide and Sulfites 145 TABLE 5.56°C (60°F) 1.0040 1. 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.003 1.7 ± 3.6 67.. .028 1. If ascorbic acid is being used in combination with sulfur dioxide.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. much slower changes occurred in the sulfur dioxide content.0001°F–1 TABLE 5.4 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.. 1970).0493 20°C (68°F) 1. the second equation listed previously is a key reaction (Heinmann et al.037 — Corrections are approximately 0.2 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 50.5 61.018 1. 1970).

1954). Navara. Those reacting the most rapidly formed complexes that dissociated the least. Otherwise. form rapidly with aldehydes: – – → HSO3 + R-COH ← R-CHOH-SO3 All aldehydes form the hydroxysulfonates. He also noted that phosphoglyceraldehyde would likely react. Relative percentages of aldehydes reacting with sulfur dioxide and their equilibrium constants are shown in Table 5.0 0. Diethyl ketone reacts slowly and to a limited extent.146 Antimicrobials in Food TABLE 5. In moldy apples.to seven-member carbon ring system will react. Bisulfite addition products. fermented ciders..5-hexodiulose. 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. 1985). d-threo-2. a Aerny (1986 a.5 — 2.3 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. Glucose is by far the most abundant of the reactive aldehydes and ketones in most fruit juices. 1954. α-ketoglutarate. and galacturonic acid were significant bisulfite binding forces (Burroughs and Sparks. Rhem (1964) noted that the rate of formation and the amount of sulfonate formed depended on the concentration of the reactive substances.6 7. In botrytized grapes made into wine. 1986 a. natural fruit juices always bind more sulfur dioxide than would be calculated from the glucose present in the juice (Joslyn and Braverman. and glucose reacted less rapidly.b). Compared with model solutions. 1960. raffinose reacted very slowly.9 6 × 10–5 Note: 1 = Burroughs and Whiting (1960).5-diketogluconic acid. 1963).7 × 102 — — — — 15.1 — 1. 1944). as much as 80% of the total sulfur dioxide may be bound by these types of carbonyls (Blouin. the hydroxysulfonic acids (Suter.3 (Burroughs and Whiting. mannose. and the temperature.12 — 2 99. 1964a.1 — 0.1 87 3 50 32 18 — — — — — 26 — 45 32 0. 5-oxofructose. Lea et al. 3 = Navara (1985). the pH. and arabinose reacted rapidly with bisulfite.b). Aerny.b.5 72 44 98 — 66 1 2 — — — — 0. 2000). but not all ketones react.1 0. . 2-oxogluconic acid. Ingram and Vas (1950) found that galactose.7 90 1. L-xylosone. pyruvate. 2 = Aerny (1986a. 1973. and fructose and sucrose did not react at all. only ketones with a methyl group adjacent to the carbonyl or carbonyls that are part of a four. lactose. 2. and wines. acetaldehyde. Joslyn and Braverman. 1954).5 3 5 8 Ka × 10–6 × 10–4 × 10–4 × 10-6 — 2 × 10–4 5 × 10–2 1. maltose.

5 mg/L. and αketoglutarate. The respective ranges and means for these components were 72 to 287 and 115. Heintz (1976) reported the occurrence of an addition product of sorbic acid and bisulfite. ˇ Farkas et al. (1982). 1975). Bisulfites may also react with nucleotides such as nicotinamide adenine dinucleotide (Meyerhof et al. The addition product attached at the carbon 4 of the sunset yellow molecule. . Margheri et al. (1989) found a food-coloring dye. malvidin monoglucoside). 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. 1938). In model solutions in the range of 30 to 50 mg/L of SO2 added. (1983) tested 30 strains of Saccharomyces cerevisiae for production of SO2 binding materials. ketones. – 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. from winery trials. Glories (1984) studied the equilibrium between SO2 and the anthocyanin–bisulfite complex. Farris et al. 1998). (1984) also showed that sulfite additions increased acetaldehyde. selected four yeasts that produced low levels of SO2 binding compounds. (1985) found a reduction in the content of the SO2 binding materials by the addition of 0. There is no evidence that these reactions occur in vivo.Sulfur Dioxide and Sulfites 147 The amounts of aldehydes. (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. Lafon-Lafourcade (1985) reviewed the role of yeast and bacteria in the amounts of these components found in wines. but they found no increase in pyruvate or α-ketoglutarate. pyruvate. reacted with bisulfite to form a lemon yellow compound. Jurd (1972) suggested the binding site for HSO3. Farris et al.6 mg/L.. Undoubtedly other reactions with sulfur dioxide take place that could reduce the amounts of available sulfur dioxide. This contributes to the difficulty in the measurement of free sulfur dioxide in highly colored wines. Damant et al. Allyl isothiocyanate in mustard was shown to react with sulfites used as antioxidants to form allylaminothiocarbonyl sulfonate (Cejpek et al. They measured acetaldehyde. Uzuka et al. the color is reduced by about 25%.5-diene (Crowell and Guymon.5 mg/L of thiamine. and other SO2 binding substances limit the effective use of the added sulfite.. 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. and at 50 mg/L of SO2 added. Dittrich and Barth (1984) did find correlations between the SO2 binding substances and wineries and grape source.. and 15 to 57 and 31. but these have been reported as the primary reactions. it is reduced by 80%. Shapiro and Weisgras (1970) also demonstrated that cytosine was transformed to cytidine by bisulfite. Piracci and Spera (1983) reported similar results. the following formula was used to calculate the equilibrium constant Ks. sunset yellow.g. After approximately 100 days of storage.5 mg/L. This treatment allows more effective use of SO2 in wine. The reaction affected flavor of the mustard by reducing pungency. Sulfur dioxide can loosely bind to anthocyanins. 16 to 42 and 27.is on the 4-position rather than the 2-position (e. Traces of the compound could be found in stored commercial soft drinks that had been heated and contained sulfite.

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

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

The delay was proportional to the concentration of the bisulfite addition. (1985) reviewed the use of sulfite as an additive to control microbiological changes occurring in meat products. a rapid decline in cell numbers occurred. 83–142. 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. 50–195. 67–98. Tompkin et al. O. The packaging used was a gas-permeable wrapping that allowed oxidative conditions (von Holy et al. Piracci (1984) found 0. in their review. or in paired combinations. coli were inhibited to a greater extent by sulfites than other bacteria. 200–241. than in inhibiting Gram-positive bacteria. 1988). It survived alcoholic fermentation when others did not. and Hafnia alvei. oeni was the primary malolactic bacterium associated with wine.. They noted that sulfites shifted the microflora of the meat to Gram-positive bacteria from the normal Gramnegative flora. Salmonella and E. 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. O. The Gram-positive bacteria remaining grew more slowly than the Gram-negative bacteria. 1972). The shelf life of ground beef was effectively increased from 1. Wibowo et al. This is demonstrated in the use of sulfites in meats. (1987) found that vacuum packaging and a good oxygen barrier film decreased the . oeni when SO2 was added and quite a variable response between strains. Banks and Board (1982) tested several genera of Enterobacteriaceae isolated from sausage for their metabisulfite sensitivity. Ough et al. E. (1988) tested 146 different strains of wild malolactic bacteria. the growth of O. They also noted that the interaction of sulfites with nitrites caused a lowering of the nitrites available for nitrosamine formation. The remaining cells later multiplied to significant numbers. Serratia marcescens. There was some additive effect when used with dimethyl dicarbonate but not enough to warrant the use with SO2 for this purpose.. coli. Banks et al. although slowing or prevention of growth of surface bacteria is probably important. None of the compounds alone. Adams et al. Sensory tests showed no adverse results. This work confirms that wines with high total sulfur dioxide concentrations are more likely to undergo malolactic fermentation with other than O.0 were as follows: Salmonella. Citrobacter freundii. in the Bordeaux area of France. 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. Yersinia enterocolitica. 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. (1980) found the addition of 100 mg/kg of SO2 as sodium metabisulfite to canned pork inoculated with Clostridium botulinum spores delayed cell growth.150 Antimicrobials in Food malolactic fermentation more slowly and that lower sulfur dioxide levels increased the fermentation rate of the O. All strains grew at pH 4. Davis et al. The effect was inhibition of growth of the flora. Roberts and McWeeny (1972). oeni strains were less tolerant to the sulfur dioxide than Pediococcus paroulus strains or the Lactobacillus species. In addition. 15–109. (1983) determined that.5 mg/L of molecular sulfite was sufficient to control malolactic bacteria growth. oeni PSU-1 used. state that sulfur dioxide is more effective against the growth of Gram-negative rods. (1988) found significant delays in growth of O. were completely effective. oeni strains with unfavorable sensory results. (1988) demonstrated that without sufficient SO2 and adjusted pH. 65–136. Lafon-Lafourcade et al.5 in beef broth and 20% tomato juice serum medium at 64 mg/L of total sulfur dioxide. such as Escherichia coli and Pseudomonas. The microorganisms tested and the concentration of free sulfite (µg/ml) necessary to inhibit their growth at pH 7.8 days at 7°C storage with no treatment to 12. oeni in red table wine occurs readily. 190–241. It was found to tolerate up to 100 mg/L of SO2. They suggested that these microorganisms could cause spoilage in wines that were considered sterile using conventional counting techniques.6 days at 0°C with the addition of 250 mg/kg of sulfur dioxide. the main effect in meat appears to be the antioxidant properties (Roberts and McWeeney. The preservation of the color and odor of meats is improved by sulfite treatment and. although after sulfite addition. Enterobacter agglomerans.

with the pressure to reduce sulfur dioxide content.05 M free sulfur dioxide for Kloeckera apiculata to 2. The concentration of tyramine and putrescine increased in the presence of sulfite. which inhibited growth. Sodium sulfite addition in sausage was shown to affect biogenic amines. A 10-fold increase in molecular sulfur dioxide occurs between pH 4. and five others were able to produce adequate amounts of ethanol. Sudraud and Chauvet (1985) found that to maintain yeast stability. Rhem and Wittmann (1962) determined the inactivation levels of sulfurous acid for a variety of yeast genera (Table 5.20 7. spoilage in sulfite-treated sausage.8 mM for Z. (1988) found that with yeast acclimatized to sulfur dioxide.60 0. Yeast The use of sulfur dioxide to deplete the wild yeast in grape juice is a standard practice dating back many years (Cruess.81). Rhodotorula. Once the inhibition was overcome. 1. whereas Kloeckera.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. The amount of molecular SO2 (mg/L) in wine can be calculated using free SO2 mg/L/(1 + 10pH . Delfini (1989) . its use to prevent the growth of yeast in sweet table wines is seldom ever contemplated.4). bailii at 25°C under aerobic conditions at pH 3. 2001). such as “maintain the free SO2 at 20 mg/L. This was because of the lack of oxygen delaying yeast growth and the production of sulfite-binding substances. phenylethylamine. Haznedari (1979) tested 30 strains of S.20 0. and several other genera were very susceptible.60 Rhem and Wittmann (1962).Sulfur Dioxide and Sulfites 151 TABLE 5. bailii. resistance of yeast to sulfur dioxide ranges from 0.5 and 1. Six strains could grow well at 1000 mg/L. cerevisiae (105 CFU/ml) after 8 hours at 25°C.2 mg/L of molecular sulfur dioxide were necessary at the finish of fermentation and during aging.2–8. Carr and Davies (1971) found sulfur dioxide incorporated into growth medium (pH 3. Any rule of thumb addition.5 in tryptone yeast extract medium. The yeast also showed increased resistance to the fungicide dimethyl dicarbonate. In contrast.7 0. cerevisiae that had been characterized as “SO2 resistant” for tolerance to high concentrations of sulfur dioxide. respectively.20 0. pH is extremely important in the effective use of sulfur dioxide. Ough et al.” for biological stability can be disastrous. the growth rates and cell yield were similar. According to Warth (1985).0 and 3. but the level of cadaverine was reduced (Bover-Cid et al.0 and 3.10–20. Thus the free sulfite. 1912). 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. was maintained for a longer period.1. In fact..40–0. 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. Saccharomycodes ludwigii was nearly as tolerant as Z.0 mg/L were required.0.4) at 25 mg/L was sufficient to kill a culture of S. Molecular sulfur dioxide is the most effective form for inhibiting yeast. There was no effect on histamine. between 2. or tryptamine. Pichia.

substituting other preservatives at bottling. Very little of the sulfur dioxide formed by the yeast remains in the free state. Z. cell numbers are depleted to very low levels at bottling. (1985) used an initial 1000 mg/L of sulfur dioxide in the last stages of the manufacture . stated that the use of sulfide salts failed to protect wine and left negative sensory characteristics in the wine. (1977) determined the cause for sulfite production to be sulfate permease inhibition by methionine. adjusting the pH downward if necessary. Fruit juices and preserves can be protected from microbial spoilage by sulfur dioxide addition. In a cell recycled ethanol fermentation system. Even at low pH (2.. using yeast that produces minimum amounts of sulfur dioxide-binding components. Dott et al. Brettanomyces can contaminate a winery. soft drinks. found no correlation with dissolved oxygen levels in wort and no correlation with ATP sulfurase or sulfite reductase. Hernandez. ludwigii. sulfur dioxide. none seem to be capable of replacing the antioxidant property of SO2. Strain differences caused variation in the sulfur dioxide produced from 11 to 26 mg/L. reducing the amount of sulfur dioxide before or during fermentation. excess amounts are used. Yeast can form sulfite from sulfate via sulfate permease. and Schizosaccharomyces japonicus in the re-fermentation of sweet champagnes. cerevisiae despite a reduction in the bacterial counts in the system at the same concentration. Pearlstein (1988) found that extended aeration decreased sulfite production. alcohol. Ranote and Bains (1982) used 350 mg/L of sulfur dioxide to preserve Kinnow. 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. They found a majority of the wines in the test to contain about 10 mg/L of total sulfur dioxide (Table 5. Gomes and da Silva Babo. In high-sulfite-producing yeast. minimizing the air around the wine. Angelino et al. With this genus.b. 1985. This particular species was found to be very resistant to sulfurous acid. 1982) as an alternative for pretreatment of grape juice before fermentation. With modern filtration and sanitation technology. if proper sulfur dioxide levels are not maintained. The treatment was reported to be successful for juice. settling and racking the juices. (1983). Sand (1980) suggested that Z. 1985. Growth was found at the highest concentration of free sulfur dioxide used. Hydrogen sulfide (H2S) was used (Ubigli et al. cerevisiae.. because the pH is high. Minarik and Navara (1977) reported finding the spoilage yeast S.6 and 3.4). however. the sulfate permease was not repressed by methionine. Generally.5). using sulfide salts before fermentation. bailii. 1986a. Galassi and Mancini. does not always inhibit growth and fermentation.152 Antimicrobials in Food reported the existence of very sulfite-resistant S. Spoilage yeast in dry wines are fairly rare. 1985. even at the legal limits. (1997) showed that sulfite up to 400 mg/L had no effect on S. Proper selection of the yeast strain to avoid high levels of sulfite is an obvious choice. Asvany. Valouyko et al. Patel et al. a late-harvest orange juice. which was at pH 3. (1985) tested 1700 Saccharomyces. 1985. (1989). and low pH. especially wines in barrels. S. (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.1 but not in the base material. Recommendations include cooling the fruit before crushing. This shows that most wines will not be free from sulfite labeling requirements even if no sulfur dioxide is added. ludwigii is also a noted spoiler in sweet grape juice containing high amounts of sulfur dioxide (Jakob. S. Vernerova et al. ludwigii in a low-alcohol wine. There have been numerous reports on techniques to minimize the amount of sulfur dioxide used in wines (Aerny. In normal sweet table wines. 1985). Breweries have an interest in sulfur dioxide as an antioxidant.6). and there was no correlation with the number of cells in the initial inoculum. and eliminating oxygen from bottled wine. Suzzi et al. bailii was becoming a problem in juices. 1978). bailii in comminuted orange drink was controlled by 230 mg/L of SO2 at pH 3. Spoilage by Z.7 (Lloyd. the main spoilage yeast is Saccharomyces. and wines because of its tolerance to SO2. Schmitt et al. Chang et al. investigating the sulfur dioxide content of green beer. Although other agents may act as antimicrobial agents. Sethi and Anand (1984) had to use 692 mg/L in carrot preserves to inhibit Bacillus cereus. 1975). Some yeast strains can form rather large amounts of sulfur dioxide during juice fermentation (Table 5.

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 .6 Amounts of Sulfur Dioxide Found in Test Wines Fermented by 1700 Saccharomyces Yeasts (Suzzi et al. (1976) Delfini et al. (1976) Minarik (1975) Minarik (1975) Heinzel et al. bayanus S. (1976) Poulard and Brelet (1978) Minarik (1975) Poulard and Brelet (1978) Minarik (1975) Delfini et al. 35. pastorianus S. cerevisiae S. 44.5 0 0 0 1. 21. 1. 4. (1976) Poulard and Brelet (1978) Delfini et al. rouxii Schizosaccharomyces pombe Torulopsis stellata Kloeckera apiculata K. 18. 150 0.. 5.5 12 0 9 Reference Minarik (1975) Eschenbruch and Bonish (1976) Heinzel et al. acidifacien S. rosei S. (1976) Minarik (1975) Eschenbruch and Bonish (1976) Heinzel et al. 18. (1976) Delfini et al. 21. uvarum S.Sulfur Dioxide and Sulfites 153 TABLE 5.5. (1976) Delfini et al. (1976) Poulard and Brelet (1978) S.5 Formation of Sulfur Dioxide by Various Yeasts during Grape Juice Fermentations Yeast Saccharomyces carlsbergensis Sulfur Dioxide (mg/L) 12 150 80. 100. chevalieri S. 56. italicus S. 35 20. 100 5. 76 17. (1976) Delfini et al. 40 0 18. 3.5. 52. 5 18–23 85 0.5. 1300 22. 3. 500 300. 160. (1976) Delfini et al. magna K. bailii S. (1976) Delfini et al. 170. 20 1. (1976) Poulard and Brelet (1978) Delfini et al. (1976) Delfini et al.5 14. 500 5. africana TABLE 5. 20 26 27 0 0. (1976) Delfini et al.

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

Chronic symptoms (Reid. Dried fruits held in an atmosphere of sulfur dioxide maintain a more natural appearance. stunting of growth. In amounts that were less than lethal doses but greater than tolerable levels (higher than 62 mg SO2 per kg body weight).. dry matter contents. 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 . The mold can exist in the ascospore form. (1984 a. 1963) are hypertrophy of goblet cells and mucous glands. potatoes. carrots.b) studied the best fungicide to use of those legally available. indicated by pulmonary edema.Sulfur Dioxide and Sulfites 155 Byssochlamys is a mold species that is capable of producing the mycotoxin patulin. and velocities of mixing for several different shapes of potatoes. Some people are more sensitive than others. Anhydrous sulfur dioxide hitting the eye is readily absorbed. such as peas. Vegetables. with similar results. Because of its high solubility in fat. which is highly resistant to thermal processing. National Academy of Sciences. 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. Papazian. and tomatoes. 1996). that is subject to nonenzymatic or enzymatic browning can benefit by proper treatment with sulfite compounds. This organism is not a normal mold found on grapes. Bolin and Jackson (1985) found that adding an oxygen scavenger to packaged dried apricots or apples greatly increased the effectiveness of the sulfur dioxide. In general. The level of sulfur dioxide normally used for juice processing would preclude its development. with excess mucous formation and difficulty in clearing the lungs. beans. articles appeared in scientific journals suggesting the possible toxicity of sulfur dioxide. 1978. 1966). lacrimation. Sulfur dioxide injury to the eye is not an uncommon industrial accident. The firmness of whole bananas is increased by dipping them into a 500 mg/L SO2 solution (Levi et al. frozen. OTHER USES Any vegetable or fruit. As early as 1896. Coughing. 1947).. renal tubular casts. lung hemorrhage. raw. cabbage. Spoilage caused by Pseudomonas species and Enterobacteriaceae could be held in the lag phase by 100 mg/L of SO2. and spectacle eyes (Fitzhugh et al. and visceral congestion. Giannuzzi and Zaritzky (1990) studied refrigerated prepeeled potatoes held in plastic film bags with vacuum packing at three temperatures. 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). 1980). TOXICOLOGY The toxicology and safety of sulfur dioxide in its various forms has been the subject of many reviews (Institute of Food Technologists. Equations were derived for holding times at various SO2 concentrations. Roland and Beuchat (1984) also studied the growth and control of this organism in grape juice. bone marrow atrophy. it penetrates the cornea and causes deep keratitis and iritis (Grant. 1946). The modeling of sulfur dioxide uptake in prepeeled potatoes was studied (Rodriguez and Zaritzky. Patulin is also inactivated by sulfur dioxide rather rapidly. The antioxidant effects of ascorbic acid are enhanced by their combined use with sulfites in foods and pharmaceuticals (Schroeter. the cause of rapid death is pulmonary dysfunction. bleached incisors. such as polyneuritis. Roland et al. Sulfur dioxide greater than 33 mg/L in air can cause distress or even death when inhaled (Amadur. 1975. or canned. and sneezing are outward immediate symptoms. visceral organ atrophy. 1975). sulfur dioxide resulted in some physiologic changes in rats. have more stable color and less deterioration if so treated. 1986) with regard to shape of potato pieces. dried.

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

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

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. Those sulfites that bind food matrices and are not converted to sulfur dioxide with heating or acidic conditions are irreversibly bound.28a 990. horseradish) Fruit juices Fruit-based sauces and related products Fruit pulps.. Thiosulfonates are irreversibly bound (Beck et al. Free sulfites are readily converted to sulfur dioxide upon acidification and can be quantitatively analyzed following distillation. acidified.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. and concentration varies by country..16 963.32 980.30 990.158 Antimicrobials in Food TABLE 5. 2000). purees. reversibly bound. 2000). Sulfites can be described as free.8).29 990.7 Application of Sulfites in Foods as Antimicrobials and the Concentrations Used (Gould. Reversibly bound sulfites are converted to sulfur dioxide only after heat and acid or alkali treatments. or irreversibly bound (Beck et al.04 990. and . garlic..09 962. The official methods of analysis for agricultural and food products are specific in their recommendations for analysis of sulfur dioxide (Table 5. TABLE 5. 2000) Method Number 892. 2000) Food Beer Fresh fruits Fresh vegetables (onion.20 975.02 961. One official method for measuring total sulfurous acid is the modified Monier-Williams procedure.8 AOAC International Official Methods of Analysis for Sulfites (Warner et al. A sample is placed in a distilling flask.17 987.

Kielhofer and Aumann (1957). in addition. The optimized Monier-Williams method is used by the FDA to measure sulfites in official samples (Table 5. . Briefly. and Ough and Amerine (1988) described the method and equipment needed. but determinations on red wines are more accurately done by this variation of the Monier-Williams method.Sulfur Dioxide and Sulfites 159 heated.. and the volatile sulfur dioxide is removed by a stream of nitrogen through a reflux condenser. Burroughs and Sparks (1964b). 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. it is then acidified. 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 cabbage. 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. Schwedt (1986) considered the test strips satisfactory but noted that if dyes or natural pigments came in contact with the cellulose. False-negative and false-positive results were found by both groups. false results were recorded. This is a direct iodine titration of the substance at acid pH. Excess iodide is added to the sample. and the hydrogen ion produced in the peroxide solution is titrated.8) (Warner et al. The same errors are associated with this procedure as are associated with the Ripper procedure for total sulfur dioxide. This method is good for most products. artificially high results occur. Basically the sample is sparged with gas for 12 to 15 minutes. 1988). 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. Free sulfur dioxide is easily measured in white wines by the Ripper method. but if other oxidizable substances are present. and it is then titrated with the iodate solution to a starch end point. Schneyder and Vlcek (1977) reported that iodate was superior to an iodine standard solution. 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. the sample is made basic to break the bisulfite addition products. leeks. Mitsuhashi et al. Paul (1958). Rankine and Pocock (1970). the sulfate can be precipitated with barium and measured gravimetrically. The Ripper method for free sulfur dioxide is given by Ough and Amerine (1988). 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. 2000). except dried onions. Joslyn and Braverman (1954) reviewed the errors associated with iodine titrations. The Monier-Williams method can be modified to determine free sulfurous acid. (1988) and Wanderer and Solomons (1987). The reflux condenser must retain all volatile acids except the sulfurous acid. (1979) showed that sodium sulfide and allyl isothiocyanate gave positive results in the MonierWilliams tests.

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

...........221 This review of the antimicrobial properties of nitrite is one among many.......... The discovery in 1969 that nitrosamines can be formed in foods containing nitrite led to a series of events.... It is tempting to delete a significant quantity of information from this third revision of the chapter.................. Holley.. Bard and Townsend. the reader is encouraged to read the historical . however... toxicologic............. If nitrite were not available. 2002) have been developed...... To better understand this complex issue. Binkerd and Kolari (1975) reviewed the history of nitrite and nitrate as curing agents for meats...... then cured meats........ 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.............. Duncan...217 Assay Method .... 1979)....... is discussed in Hill (1991)..... Skovgaard......... however............. Cerveny (1980) described many of the changes in the production and marketing of cured meats in the United States since 1900............... Initially......... Reported outbreaks of botulism attributed to meat and poultry products and an assessment of nitrite for botulinal protection have been summarized (Tompkin.. Current regulations for the use of nitrite and nitrate and modern. It differs.. Pegg and Shahidi.. 1970...... Eklund (1982) reviewed the potential risk of botulism in fishery products and the role of nitrite as a preservative.......... The many chemical reactions that occur when nitrite is added to meat offer clues to the reactions that may occur with microbial cells........................... 1980.216 Regulations Governing the Use of Nitrite and Nitrate .. The use of nitrite and/or nitrate in foods has been influenced by a variety of factors.............. Sofos et al........ particularly in the United Kingdom and Europe............. 1966.......................... The review by Gray et al............... and the information that was available to influence the researchers’ conclusions................. 1971......... Ingram.... this chapter would not exist in this book........ 1974.. considerable effort was directed toward verifying the benefits of nitrite relative to control of certain pathogens and quality attributes........ 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. controlled processing conditions provide the benefits of food preservation with no measurable risk to consumers.................. and physiologic effects of nitrate and nitrite (Archer.... in certain respects from other reviews........ 1978..170 Antimicrobials in Food Summary for 1990–2002 ......218 References .. The status of nitrate and nitrite in food and water until about 1990...... For this reason............. Roberts et al.. as we know them......... 2000) and the microbiologic...... 1991. insight is provided into why the research was performed..... The time and resources expended to resolve the nitrite issue were enormous and worldwide......... their use was to fulfill the basic need to preserve food....................... By generally arranging the information in chronologic order. would disappear from the market....... Pivnick.. As questions were raised from about 1970 to 2002 over the toxicologic safety of nitrite... 1981............................. 1963...... Lechowich et al...................... With the new millennium a different perspective has evolved. At the same time a considerable amount of research was directed toward identifying the risks associated with the use of nitrite in food. 1992)......................... Roberts et al. Benedict...... At the heart of the issue was whether nitrite would continue to be an approved food additive............. the reader should consider reactions with heme compounds (Fox...... This chapter concentrates on the antimicrobial research.. Other comprehensive reviews are available that provide additional information and other perspectives (Anderton.... the selection of the experimental procedure.217 Toxicology..... 1980)........... Extensive updates on the chemistry and impact of nitrite on the color and flavor of cured meats (Pegg and Shahidi..... 1991....... 1979a. 1980....... It is a curious thought that if nitrite were disapproved................. (1981) and Pegg and Shahidi (2000) of the effect of nitrite on the stability of cured meat flavor provides additional possibilities... 2000) and other components of meat (Cassens et al.......

Most of the research on sour hams involved test tube experiments with culture media above pH 7. and dealing with perishable canned hams that were being temperature abused in the marketplace.000 µg/g cannot be relied on to inhibit putrefactive anaerobes if other conditions are favorable for growth. using Clostridium botulinum. reproducible results in all instances. 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. 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. only salt was used in sufficient concentration in commercial hams to prevent proteolytic activity at 37°C. putrificum. were not inhibitory to any of the strains. 44. 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. .000 µg/g. or 12. The authors concluded that nitrate at concentrations as low as 22. despite meat having a pH closer to 6.000 µg/g did not inhibit the 12 strains tested in either pork infusion or egg-meat media. found that sodium nitrate up to 22. Nitrate was believed to provide the antimicrobial effect.Nitrite 171 documentary of Cassens (1990). there has been a long. and the value of nitrate was relegated to serving merely as a reservoir for the generation of nitrite.000 µg/g of sodium nitrite in a pork infusion broth buffered to pH 7. Until the early 1940s. controversial history over whether nitrate and nitrite have antimicrobial properties. At levels between 22. the highest levels tested.6 and held at 37°C. By the time the nitrosamine controversy arose in 1969 and the early 1970s. Levels of 3100 µg/g in pork infusion and 3900 µg/g in egg-meat. data appeared that began to prove the opposite. RESEARCH HISTORY BEFORE 1950 Tucker (1930) studied the proteolytic activity of a Clostridium putrificum isolate from sour ham. During the 1940s and early 1950s. Considering that research continues to reveal new interactions among the chemical constituents of cured meat. irregular inhibition occurred among seven botulinal cultures.000 and 44.000 µg/g. putrificum. Research of the 1920s and 1930s focused on using nitrite to cure meat. solving a problem of sour hams. Tanner and Evans (1933). Unlike the other antimicrobial agents discussed in this text.0. Clever as the scientific community has been in more clearly defining the antimicrobial properties of nitrite. not nitrate. We are still ignorant of all the significant factors that influence the efficacy of nitrite or. 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. He concluded that. By the middle 1950s. This concept became widely accepted. He suggested that nitrite. and Clostridium sporogenes. is responsible for retarding proteolysis by C.0. 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. the value of nitrate. At 44.000 µg/g of sodium nitrate. for that matter. One of the messages from the 1920s is that nitrate serves as a potential reservoir for conversion to nitrite and thus to nitrous acid.0% sodium chloride (salt). This basic flaw in experimental design led to results that misguided and hardened opinions in the industry for 20 years. C. 6 of the 7 cultures were inhibited. The list of recognized factors influencing the antimicrobial efficacy of nitrite in cured meats continues to grow 60 years later. nitrite was considered to have no value in meat other than for flavor and color development. research had clearly demonstrated that nitrite has significant antimicrobial properties. 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. The same results were obtained with Clostridium putrefaciens at 23°C. Proteolysis was inhibited by 4. It is still difficult to design experiments that yield predictable.

1964). The authors concluded that reliance cannot be placed on nitrite alone to prevent spoilage by clostridia. From tests with various blends of salt. It is surprising that the fundamental importance of pH. A marked inhibition was noticed. sugar. 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. Brooks et al. the sole cause of the spoilage was found to be nitrate-reducing Bacillus species (Jensen et al. The characteristic cured flavor of bacon is primarily the result of the action of nitrite. 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.3).” Botulinal toxin was produced in the cooked meat and a few samples of raw meat.172 Antimicrobials in Food although the nitrite was added to the media (pH 7. but the research led to the following general conclusions. In acid solutions. Nitrite could not replace nitrate. 1934). It seemed that nitrate was especially valuable in preventing high degrees of acidity or souring of meat. so clearly stated and available to researchers at that time. spiced ham and luncheon meats containing nitrate. The conversion . They further believed that the claim of meat packers that small amounts of nitrate in the pickle produced better preservation of the meat.4). A medium was developed with nitrate.6°C in the meat. and of nitrous acid also. 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. in mixtures containing reducing substances.4) before autoclaving.. citing earlier work by MacNeal and Kerr.e. To obtain gas production in the medium. and cured meat to detect the gas-producing bacteria in raw materials and the plant environment. The sides were then immersed in a tank and cured using a pickle solution containing potassium nitrate. insufficiently heated to be stable at room temperature) canned. and nitrate in peptic digest broth (pH 7. Under these conditions. They stated that if nitrate were omitted from the product. as the more recent results suggest (Jacobs et al. 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. 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. 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. and sugar swelled in 1 to 90 days during temperature abuse at 37°C. 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. pork sides were held at ambient temperature overnight and then moved into refrigeration for 24 hours to achieve 5. it was used as a food. After the slaughtering process.49 to 5. however. It is curious that Tanner and Evans (1933).. state the following: [P]otassium nitrate in neutral or alkaline solutions exerted no special restrictive effect on bacterial activity. nitrite. Satisfactory bacon can be produced with the use of nitrite. Several interesting questions were raised. chopped. Examining 1000 cans representing all manufacturers at the time. nitrite. Perhaps the cured meat provides a source of heme. was borne out by their results. the results were quite different. (1940) described the contemporary method of making bacon in the United Kingdom. both nitrate and cured meat had to be present.. The reason for this specific combination of ingredients remains unknown. 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. raw pork. Perishable (i. Potassium nitrate was therefore considered to be particularly effective in restricting acid fermentation of organic substances that are already slightly acid. and cooked pork. The opinions expressed by Jensen et al. in 1934 laid the foundation for a new concept. was not pursued.

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

Salt was found to be the major factor retarding botulinal outgrowth in temperature-abused products. Adding 200 µg/g of sodium nitrite was considerably more inhibitory to toxin formation than the addition of 1000 µg/g of sodium nitrate. to some degree. and nitrite). a combination of both nitrite and nitrate was the most inhibitory.1 to 6. 1950–1960 Research of the 1950s clarified the relative significance of salt. and high heat resistance. increased inhibition of PA 3679 was obtained as sodium nitrite was increased from 400 to 800 µg/g.37% and a pH range of 6.0. A mixed cure of salt (3. even in the presence of salt or nitrite. An underlying factor in the research before 1940 was the unfavorable experience of 1928 and 1929 with temperature-abused perishable canned hams. pasteurized. . by nitrite. The relative roles of nitrate and nitrite as preservatives in cured meats were unclear. A mixed cure of all three (salt.” This experience may have led to improvements in meat handling and equipment sanitation. In the absence of added salt. yielded the maximum inhibition. (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.5. Although thermal destruction was not shown to be enhanced by the presence of curing salts. Halvorson (1955) later discussed the widespread problem of temperature abuse at the retail level. nitrate. 7. The question was raised whether the growth of spoilage organisms is influenced by the depletion of nitrite to noninhibitory levels. but the condition of the meat was a factor. and sterilized meat were of low.174 Antimicrobials in Food Vinton et al. especially if the product pH was below 7. however. 5. 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. During the 1930s some processors incubated perishable canned cured meat at 37°C as an index of “keeping quality. however. Salt at the levels used at that time was considered the cornerstone for the preservation of cured meats. SUMMARY FOR BEFORE 1950 A summary of the state of knowledge by 1950 might include the following: 1.5%) and nitrite (150 to 170 µg/g) was much more effective than either substance alone. and nitrite as preservative agents and expands this information to other microorganisms. Bulman and Ayres (1952) found that levels of salt in excess of 4.05% to 5. 3. Nitrate at the level permitted in meat (1250 µg/g) was without effect.000 µg/g were required to prevent spoilage from anaerobic spore formers in pork. The disappearance of nitrite during processing and storage was assumed to make it an unreliable preservative. intermediate. there was increased inhibition of outgrowth of surviving spores by salt and. The basic formulation resulted in a product having a moderately high brine of 5. Nitrate was without effect. Nitrite was clearly shown to be an effective antimicrobial agent. The concept of a combined effect of heating in the presence of curing salts was proposed. nitrate. 4. Steinke and Foster (1951) investigated the effect of packaging liver sausage in an oxygenimpermeable film (Saran™) that was gaining acceptance in the industry. Strong opinions were expressed that nitrite alone was ineffective at the levels used in commercial practice.4% or sodium nitrate in excess of 40. 6. Spores produced in raw. 2. respectively. It is perhaps for this reason that Jensen believed so strongly about the need for nitrate in perishable canned cured meats.

Halvorson (1955) cited a private communication from L. 1955) on semipreserved meats in hermetically sealed containers included a curious debate. Inst. the spores could germinate.05. The debated issue was the time and temperature for preenrichment. The brine requirement of 5. Nitrite was more inhibitory in the presence of ascorbate. found in the interior of cured meats. the growth of Bacillus species would occur. these existing products were not affected.5% would have caused consumer . 1 1/2 lb canned ham). This combination of ingredients was derived from the literature and unpublished results from Jensen. brine values traditionally have been calculated at % brine = (% salt/% salt + % water) × 100. growth would occur. At 6. Pasteur. As late as 1957. S. Department of Agriculture (USDA) issued a regulation to assure the safety of new perishable canned cured meat products (e.3 or below. causing the cans to swell and forewarn consumers of a potential hazard. A pH of 5. Additional tests showed that if the broth medium (pH 6. was unstable. The USDA concluded that the high brine would retard the growth of C. Jensen. At pH 5. a similar debate would include the time elapsed since manufacturing. The requirements imposed on new products included a brine level of 5. these observations leave no doubt inhibition by nitrite is at least a possibility. the control of salt concentration and resultant water activity was the most reliable bacteriostatic system for cured meats. who performed a series of tests with spiced ham inoculated with different strains of C.Nitrite 175 Henry et al. botulinum. In North America. and 1 ounce of sodium nitrate per 100 pounds of meat. aureus.5%. Today. (1954) reported that at pH 7. Jensen concluded that if the brine content were less than 5. nitrite rapidly disappeared and was ineffective. Scott (1955) concluded from the literature that because nitrate exhibited relatively poor antimicrobial inhibition and nitrite.8 was optimal for antibacterial efficacy. 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. On adding a filter-sterilized solution of nitrite to a broth medium. although effective. B.. They found S. aureus to die rather rapidly in ham-curing pickle.5 or above. and toxin would be produced. Tests in broth media and raw pork led them to conclude that S. aureus was killed in hams when heated to an internal temperature of 58.g. a very small amount of added nitrite prevented staphylococcal growth when incubated anaerobically. toxin formation was inhibited with a heat treatment as low as F0 = 0.55) was autoclaved with glucose. (1956) examined whether curing salts plus the anaerobiosis of a vacuum package would inhibit the growth of S. At that time European microbiologists commonly preincubated perishable canned cured meats before microbiological examination. They investigated why Staphylococcus aureus is rarely. and in the presence of nitrate. nitrate. 1/4 ounce sodium nitrate (156 µg/g). this cautious view of the inhibitory effect of nitrite was expressed by Eddy (1957): “Taken in their totality.5% sugar.27.3°C. the legal requirement for trichina destruction. A typical formula being used for large canned hams at that time included 0. as suggested by Castellani and Niven (1955).S.” An international symposium (Ann. Lechowich et al. 1/8 ounce sodium nitrite (78 µg/g).9 to 5. In the United Kingdom. During the mid-1950s the U. and a brine level of 3. who concluded that 1 ounce nitrate was optimal for gas formation by Bacillus species in temperature-abused perishable canned luncheon meat.0%.75% sugar. The authors postulated that nitrous acid was responsible for the bacteriostatic action of nitrite. 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) × 100.6 to 5.4%.5%. the addition of 1. Earlier. aureus growth can occur in any combination of salt. Also. less nitrite was necessary for inhibition as the pH was decreased from 6. nitrite enhanced bacterial growth in curing brine. botulinum.0% brine or above. This effect was reversed by adding sulfhydryl compounds. Germination was apparently prevented because viable spores could be recovered. Dack (1949) reported toxin inhibition in meat with brine levels above 6. Because the procedures traditionally used for large canned hams had a history of safety. if ever. unless protected by meat juices. and nitrite that is palatable and permissible.

Nitrate could serve as an electron acceptor. 7. salt. Greenberg et al.4°C to 37. Anaerobic intolerance to nitrite within the interior of ham. Several new reports of unstable commercially canned cured meats containing nitrate appeared (Hankins et al. At 5. was not confirmed. toxin was produced without obvious organoleptic spoilage. 1950.12%.176 Antimicrobials in Food complaints of excessive saltiness in the new products. This brought nitrate under renewed attack. The system providing stability consisted of the combined effect of nitrite. and thermal injury to the low indigenous level of anaerobic spores. the reasons for stability had not been defined. 2.09%. the primary effect of nitrite was to prevent germination and/or outgrowth of heat-injured spores. Brine level (0%. 1950. S. Nitrite was shown to play a significant role in the stability of shelf-stable canned meat.25%. There have been no reported attempts to duplicate this effect of brine at levels approaching total inhibition. 3.. Eddy and Ingram. as postulated by test tube experiments. the following became increasingly clear: 1.95% brine. A significant observation was that at brine levels of 6.14% brine or less.8°C for up to 90 days. At 8. SUMMARY FOR 1950–1960 As 1960 approached. and 7. thermal injury to the spores. had no antimicrobial effect.0% to 7. aureus was killed during the heating process given to hams.5%. Nitrate. This research and a subsequent review by Silliker (1959) established the future direction for research on nitrite. permitting the growth of aerobes. Although nitrite was recognized as an effective antimicrobial agent. its value as a preservative in perishable cured meats was still in doubt.0%) alone was not responsible for stability. 1956). Because viable spores could be recovered from stable commercial product. Sodium nitrite levels were 22 µg/g or less at the time the meat was inoculated. . per se. Verhoeven. although there have been several studies under other conditions in which toxin has been detected without obvious organoleptic spoilage. the product was organoleptically unacceptable — as the cans swelled — and toxin assays were performed. salt. The suspected role of nitrite was to prevent germination or outgrowth of surviving heat-injured spores. Although shelf-stable canned cured luncheon meat had been produced for years. 3. other than its possible influence on water activity. and a low indigenous spore level. such as micrococci and bacilli. 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. (1958) used this class of product to show that nitrate played no role in retarding putrid spoilage. At brine levels approaching the inhibitory level. toxin was not produced. toxin was produced without concomitant organoleptic spoilage. (1959) subsequently demonstrated botulinal spore outgrowth in perishable canned cured meat having brine levels from 3. 5. 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. As expected.125% and abused at 29. or 5. nitrate actively stimulated spoilage by aerobic spore formers. 6. 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. Brine content was further shown to influence botulinal outgrowth and toxin formation. 4.

It was postulated that nitrous acid reacts either with the cell itself or with various constituents of the medium. canned chopped ham was inoculated with botulinal spores and given 2 thermal processes. a 4.6 at the center of the can would yield 10-fold higher values near the surface of the can. the initial spore load in the product is too high. even if nitrate is present as an alternative electron acceptor. 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. development ceased before lysis or rupture of the spore walls. producers of shelf-stable canned cured meat was reported to range from F0 = 0. Immediately after germination. The thermal process used during the early 1960s by seven U. but at a reduced rate. Product inoculated with the lowest inoculum level (173 spores/g) had 0. Gould (1964) observed germination. and outgrowth of spores with unpublished data from the Danish Meat Research Institute. In the course of this research. Hemin had no effect on nitrate reduction or growth of Escherichia coli or Bacillus polymyxa.203) were compared.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. Shank et al. If the surviving spore counts between the enzyme-inactivating (74°C internal temperature) and the shelf-stable process (F0 = 0.36% did not prevent outgrowth. Another factor in spore destruction is that a thermal process of F0 = 0.0. making them unavailable for subsequent metabolism.. heat resistance. of spores of Bacillus species in the presence of less than 300 µg/g of sodium nitrite at pH 6. Germination was completely prevented by sodium nitrite levels of 750 µg/g or higher at pH 6. (1962) explored whether nitrite or the oxides of nitrogen were involved in the inhibition of Gram-negative bacteria in cured meats. 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. and nitrate is not reduced anaerobically. He concluded that most of the spores are killed during the thermal processing of shelf-stable canned cured meat. The impaired synthesis of heme prevents the formation of the required cytochrome systems.5. The surviving spores are subsequently inhibited by the salt and nitrite. If. Shelf-Stable Cured Meats Riemann (1963) supplemented existing information on the incidence. (1964). spoilage.1 to 0. however.to 5-log reduction occurred. They concluded that nitrite and nitrate have either neutral or stimulatory effects on bacteria and that nitric oxide has virtually no effect. Outbreaks of botulism from temperature-abused vacuum-packed smoked fish during the early 1960s added impetus to this research. and toxin formation from this low level of survivors during storage at 30°C to 37.2 (Greenberg et al. . inhibition.4 to 0. but not in others. 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. The increased use of vacuum packaging raised new questions about the microbiological safety of cured meats. The inhibited spores did not appear to become fully phase dark. Nitrate and oxygen were considered interchangeable as electron acceptors required for heme synthesis in some bacteria. Research was initiated on irradiation for shelf-stable canned cured meats. In a study comparing irradiation with conventional thermal processing. The presence of 25 µg/g of residual nitrite after processing and a relatively low brine level of 3.017 viable cells/g remaining after processing to F0 = 0. (1967) opened new possibilities. 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 toxicity of nitrite was 3 to 5 times greater at pH 6 than at pH 7. with a median of 0.7°C for 6 months.S. 1965). He estimated the thermal process caused a 4-log reduction of the indigenous spores.2. heme synthesis is impaired. the observations of Perigo et al.

the normal pH value of canned luncheon meat. (2) partial destruction of spores by the initial long thermal process but at the same time retaining a high level of residual nitrite. The meat mixture was then filled into cans. This process caused the surface of the meat in the can to reach 96°C to 107°C. Increased stability also resulted from improving the canning process to reduce or prevent voids. The refrigerated storage period was necessary to assure stability.g.0015 to 1.g. 1 psig). Curing salts interfered with some stage of germination and development of the surviving heated spores at concentrations that were ineffective with unheated spores. A nitric oxide-generating material. (3) destruction of spores on the surface of the meat by the short but high thermal process. The process had to be properly controlled to be successful (Kueck.. The effect of nitrite was pH dependent and led the authors to support the position that undissociated nitrous acid is the inhibitory agent.1 to 0.. but nitrite enhanced heat injury. Nitrate had no effect beyond that on water activity. The product was then heated for a short time at a high temperature (e. and a low incidence of clostridial spores (less than 1/g) were necessary for the safety and stability of shelf-stable canned cured meats. The process involved a unique sequence of adding the curing ingredients. They demonstrated that increased stability could be achieved by omitting sodium nitrate and sucrose from the formulation. Schack and Taylor (1966) described a procedure to produce shelf-stable canned cured meat with improved quality as a result of reduced thermal process. salt and sodium nitrate at 0. This process was tested extensively in at least one commercial plant.. and then thermally processed.5% to 1% (5000 to 10.5% to 4. such as sodium nitrite.4. At 2% to 4%. The thermal process required to achieve shelf stability of canned hams larger than 1 1/2 pounds caused excessive purge (e. 2 to 2 1/2 hours in 74°C water) to achieve an internal temperature of at least 71°C. nitrite.. When heated in the presence of these agents. First. A 3-pound meat portion was filled into each can. and nitrate than unheated spores. was then added and mixed into the meat while under a positive pressure (e. and the product was given a long thermal process at a low temperature (e.0. spores tended to be protected by salt and nitrate. This process was used commercially with the approval of the USDA. but the process was not dependable and was abandoned. 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.g. 15 minutes at 113°C to 118°C). When heated and cultured in the presence of the agents.3°C) before release as a shelf-stable product. . sealed. (1958). Kueck et al. personal communication). The two Bacillus species were rendered more sensitive to salt by lower heat treatments than clostridia. including (1) the destruction of vegetative cells by heating to the initial internal temperature of 71°C. (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. 30%) and poor meat texture.5 before placing them onto culture media containing salt. 30 days at 6°C to 8.g. and nitrate.178 Antimicrobials in Food Spencer (1966) concluded from the literature that 3. 75 to 150 µg/g of added sodium nitrite to provide about 20 µg/g after processing to F0 = 0.. 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). Nitrite was strongly inhibitory. Duncan and Foster (1968a) also reported that heated putrefactive anaerobe spores were less tolerant of salt. The success of the process was believed to be the result of a combination of factors. the meat was ground or chopped and then deaerated and backflushed with nitrogen to displace free oxygen. and appeared to be the chief preservative against putrefactive anaerobic spoilage. heat resistance was decreased. Their results confirmed the concept of spore injury and increased sensitivity to salt and nitrite proposed by Silliker et al.g. nitrite. The process was considered to greatly increase the amount of nitric oxide available for use as an antibacterial agent.0% brine. Roberts and Ingram (1966) heated spores in water from F0 = 0.000 µg/g) increased the apparent heat resistance of the spores. Ascorbic acid was then uniformly distributed throughout the meat. The product was then cooled in tap water and placed into refrigerated storage (e. These data reemphasized the importance of spore contamination level for the stability of shelf-stable canned cured meats. especially at pH 6.

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

and temperature on a Microbacterium species in all-purpose Tween (APT) broth.35% and 3. The effect of brine on type E toxin production was more .1% to 4. Of 25 lactobacilli from cured meat.4°C. Their findings that the type of cured meat influenced toxin production were particularly significant. toxin was produced at 20°C to 30°C but not at 15°C. From studies in thioglycollate medium containing salt. At the highest brine level (4.180 Antimicrobials in Food nitrogen or vacuum was organoleptically acceptable. Toxin was produced within 7 days at 30°C in bologna (3. In sliced ham.7 or less after 4 weeks. and nitrite. Nitrite was more inhibitory at 0°C than at the other temperatures tested (10°C and 25°C). type E toxin was not produced. the presence of nitrite caused very little or no inhibition.5% brine and in 4 days at 4. toxin was produced only at 30°C. A similar effect occurred with increasing salt from 0% to 4% during incubation at 7. 34 µg/g of residual nitrite). A total of 90% of the samples held at 25°C remained above pH 6. In jellied pork tongue. increasing the amount of nitrite from 25 to 200 µg/g caused progressively greater inhibition. In jellied ox tongue. botulinum in vacuum-packaged perishable cured meats were reported during this period. Christiansen and Foster (1965) found type A botulinal toxin production to occur at the same rate on bologna whether packaged with vacuum or without. 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. botulinum.6.4% brine at 30°C. the authors concluded that the levels used in ham would permit growth.8%. A decrease in pH likely occurred during storage and prevented toxin formation in the other variables.5 in the presence of 200 µg/g of sodium nitrite. The rate of toxin formation decreased as the brine level increased from 3.1% or higher. When S.0 and below. Another 25 strains of microbacteria failed to grow at pH 5.75%). even at 30°C.8%). Bologna was relatively resistant to type A toxin production but not ham. At pH 7. The combination of brine (3. On sliced bologna. No mention was made of pH and none of the agents were tested in combination. pH 6.8% brine). 21 were able to grow under the same conditions. toxin was detected in 2 days at 2. At pH 6.1. residual nitrite. nitrate.7% brine. This would help to explain the dominance of the lactobacilli as spoilage organisms in many perishable cured meats. 30°C. and 37°C). Brownlie (1966) demonstrated the combined inhibitory effect of sodium nitrite concentration. 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). and decreasing pH during temperature abuse were the suggested inhibitory factors. but not at 25°C if the brine was 5.0 to 4. Cooked sliced ham from five producers inoculated with type E spores yielded variable results at 30°C. type E toxin was produced at 10°C to 25°C but not at 5°C. but not at 29. a fermentable carbohydrate. but no attempt was made to determine whether they could multiply if the hams were subsequently temperature abused. Failure to obtain toxin production at 5°C may have been influenced by pH because 13 of 15 samples had a pH of 5. Pivnick and Bird (1965) found the oxygen permeability of packaging films to influence spoilage but not toxigenesis of sliced cured meats inoculated with C. which was thought to be the result of product differences in brine and residual nitrite levels. lower maximum population levels occurred in vacuum packages than in nonvacuum packages at 15°C and 22°C. Several studies on the growth of C. pH. the authors recommended that vacuum-packaged nonsterile processed foods be stored in the frozen state. The time required for toxin production decreased as the abuse temperature was increased (22°C. The four strains failed to produce toxin during 5 weeks at 10°C in sliced bologna (2.0. Similar results were obtained with jellied corned beef stored at 15°C and 25°C. This suggested that salt becomes more inhibitory as storage temperatures are decreased. Within 1 week the product pH had decreased from 6. aureus was inoculated onto sliced chopped ham.8°C or 10°C. perfringens inoculated into raw hams could survive the thermal process. Toxin was not produced at 10°C or lower.0. Gough and Alford (1965) showed that spores of C. 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.

botulinum.6% brine at pH 6. 1967). 9% brine at pH 5. The process of outgrowth was more sensitive than spore germination to nitrite.2 and 3% brine at all pH values.35% and after 8 days with 3.5% brine at pH 5. Although not mentioned. and more than 10% brine at pH 6. The effect of nitrite levels on types A and B botulinal toxin production in vacuum-packaged sliced ham (4.5 to 5. The concept of thermal injury to spores that survive the processing of shelf-stable canned cured meats was confirmed.5% brine at 20°C. and 30°C). which disappeared during the thermal process. during which time naturally occurring lactics drop the pH to 5.5.1. (1967) reported type E botulinal toxin production in ground beef held at 22. An additional factor influencing the apparent significance of nitrite is the growth of competitive flora.28%. aureus is inhibited by more than 4% brine at pH 5. He concluded that C. 25°C. an increase in the level of residual nitrite also occurred. and Lactobacillus viridescens. they determined that type B botulinal outgrowth was inhibited during 4 weeks at 30°C if the juice had a combined pH of 5. a brine level of 4. 3.. The authors concluded that heat injury of spores is not necessary for nitrite to prevent toxin production in cured meats.. considering the bacon had a brine level of 5. (1958).3% brine) was studied next (Pivnick et al. sporogenes. All three products (bologna. A new explanation for the shelf stability of canned cured meat was proposed. All of them reached the same general conclusions as those set forth by Silliker et al. perfringens.5% brine at pH 6.2°C. Toxin was also detected in bacon held at 12.9. The system responsible for the shelf stability of canned cured meats given less than a botulinal cooking was studied by several research groups. The growth of S. more importantly. Riemann (Anon.8°C and 22. . 5. and an input nitrite level of 500 µg/g or more. In both products a marked delay in toxin production occurred at 20°C. Kafel and Ayres (1969) demonstrated that the growth of enterococci in culture media and. Toxin was not produced in either bologna or country cured ham.8 or less. (1967) suggested an inhibitory substance was formed from nitrite. Warnecke et al. 2.3.8. 1968) emphasized maintaining adequate salt levels to assure the microbiological safety of perishable vacuum-packaged cured meats. In jellied pork tongue.1. increasing the nitrite level increased the time for toxin production. botulinum type A seemed to be completely inhibited by 4. 5. C. Toxin was not produced in product with 4. Type E botulinal growth is inhibited by 2% brine at pH 5. and 8. and jellied pork tongue) were formulated with commercial levels of nitrate and nitrite. as the brine level increased in the bologna and ham.8% to 4. Evidence was presented that the antimicrobial effect of nitrite is related to the formation of nitrous acid. The product had been changed to a shelf-stable fermented product precooked to 80°C in an oxygen-impermeable casing.2°C for 3 to 18 days.4% brine. ham. C. Perigo et al. SUMMARY FOR 1960–1970 Progress made during the 1960s toward defining the role of nitrite in cured meats included the following: 1.1% brine) and jellied pork tongue (3.5% or above.3. in perishable canned hams can inhibit the growth of C. toxin was detected after 4 days with 2.Nitrite 181 dramatic. The raw product was then sold at ambient temperature for cooking at home. but this is highly questionable. 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. At each temperature (20°C. 4. Using juices expressed from the newer sausage product.

but not all the results were directly applicable to commercially cured products. 7. dynamic system in which nitrite plays a role in cured meats has been virtually impossible to duplicate in microbiological media. (4) meat products produced and packaged in pilot plants. (1980) would later report growth at 8°C in liver sausage. The data were discussed in terms of applying the D concept to shelf-stable canned cured meat. and 120°C) and curing salts on the destruction of Clostridium subterminale spores.1%) affected thermal destruction when tested alone. The significance of decreasing pH on botulinal inhibition in cooked cured meats with sufficient fermentable carbohydrate was reported for the first time. Sodium nitrite alone caused increased destruction. nitrite at the level used commercially (200 µg/g) did not enhance destruction during heating or germination during subsequent storage. 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). the formation of nitrosamines in cured meats was an issue of immediate concern. Neither sodium chloride (3%) nor sodium nitrate (0. 1970–1980 From the outset of the 1970s.0% salt. Limited data indicated the lowest temperature reported for growth of type E on cured meats to be 10°C. The timely report of Perigo et al. 8. The complex. residual nitrite. Lücke et al. Factors influencing growth included brine level. and (4) these rapidly germinating spores are then inhibited by the nitrite remaining after processing and subsequently die before the nitrite has decreased appreciably. 9. (3) nitrite increases the germination rate of spores that survive heating.01% . 110°C. and (5) commercially produced meats. but their presence in the culture medium markedly reduced the recovery of the heated spores. Salt and nitrite had little effect on the degree of thermal destruction. Throughout the decade pressure increased to reduce or eliminate nitrate and/or nitrite from cured meat. (2) nitrite potentiates direct killing by heat without the need for prior germination. Shelf-Stable Cured Meat and the Perigo Factor Pivnick et al. and the germinated spores are killed by heat. Certain cured meats are more prone to supporting botulinal growth. pH. The chief value of nitrite appeared to be its ability to prevent growth from spores that survive and germinate following processing. Research during this period can be grouped into five categories by the approaches used. and inoculum level. 0. Considerable effort was applied toward isolating and characterizing the Perigo factor. They include tests in (1) test tubes or Petri plates using controlled media and conditions. (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.1% sodium nitrate. the test tube approach was productive. A combination of 3. The growth of enterococci in perishable canned hams was reported to inhibit several clostridial species. As in the past. Matsuda and Sekiguchi (1971) studied the combined effect of heat (105°C. in 1967 opened the possibility of a breakthrough on the mechanism of nitrite inhibition. and 0.182 Antimicrobials in Food 6. Of the four possible roles. (3) meat products processed in tubes or cans. storage temperature. Each of the approaches had its favorable and unfavorable features. with the goals of greater understanding and its possible use as a preservative to replace nitrite. (2) water slurries of meat processed in bottles. 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.

(1973) studied the effect of adding nitrite to the brine solution for canned Vienna sausages. Outgrowth was determined by blackening of the tubes as a result of ferric citrate having been added to the meat.966. held overnight for nitrite equilibration. respectively. thereby confirming earlier research. Ashworth and Spencer (1972) reported the equivalent levels to be . Considerable differences were noted in their redox potentials. A similar response with strain 3679 would not be unexpected. and then packed into cans.4). Reducing agents (e. A 1:1 extract was next prepared with 3% saline and ground. The cans were then filled with brine (3% salt).6% salt. and 50 µg/g of sodium nitrite. Preliminary data.5°C on the outgrowth of surviving spores was also examined by placing the heated tubes at 23°C for up to 1 year. sodium ascorbate. Small differences in the amount of sodium nitrite below 0. The magnitude of the effect was much less than was previously reported in culture media. residual nitrite levels remained above 125 µg/g throughout 80 days at 30°C.. The residual nitrite values of the four media and three meat suspensions were about the same after preparation.7 to 6. One of the sausages in each can was inoculated with C. These observations led to the conclusion that a Perigo-type effect was detected in the meat system. nitrite and salt levels had lesser effects. thioglycollate) enhanced the effect of nitrite.5% sodium tripolyphosphate was studied by Nordin et al. the liquid placed into tubes.3. 150. cooked. After a loss of 15% to 25% during processing and an initial decline. and 0. (1967) reported equivalent inhibition with 8 µg/g of nitrite added before heating the medium and 52 µg/g added after heating the medium. Reducing agents (e. as already discussed. A significant observation was that residual nitrite did not undergo normal depletion in the extract. In ground pork. The sausages were prepared with nitrite-containing curing salt. It was reported that less nitrite was required for inhibition when it was added before autoclaving. as opposed to perishable canned cured meats heated in hot water to a minimum internal temperature of 66°C. 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. Farkas et al... cysteine. although they were “clear and undisputable subsidiary effects. The inhibitory level of nitrite was not influenced by pH within the range 5.4. 115°C). and then inoculated with vegetative clostridial cells. pH. aw 0. and heated (F0 = 0. This emphasized the importance of medium selection when studying the antimicrobial activity of nitrite and relating the results to commercially produced meats. sporogenes spores to provide a level of 104 spores per can. botulinum.6. (1975). Perigo et al. and heated to varying degrees. 200 µg/g sodium nitrite. Ashworth and Spencer (1972) described a model system of ground pork (25 g) in 1-ounce bottles.g. and overheated (115°C for 90 minutes) canned luncheon meat required 500.g. showing a similar iron–nitrite interaction with C. but not at levels above 0. Aqueous meat suspensions prepared from raw. nitrite.01%. salt. Between 120 and 200 µg/g sodium nitrite provided stability through 5 weeks of storage.Nitrite 183 sodium nitrite caused the greatest destruction when heated. The extract had 2.01% strongly influenced the results. The pork was autoclaved before or after adding nitrite. personal communication).g. and. the solids removed. The major factor influencing outgrowth was pH. cooked sausages. Omitting any one or all three of the additives did not affect thermal destruction.8% salt. ascorbate and cysteine) enhanced the antibotulinal effect of the meat suspensions but did not induce Perigo factor formation. however. The effect of nitrite. and salt) appeared to be additive.. and pH 6. normal.35 were as stable as sausages in brine without nitrite and processed to F0 = 2. Sausages packed in brine with 400 µg/g of sodium nitrite and processed to F0 = 0.5°C in ground pork containing 2. to some degree. 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. inoculated (105/ml). The effect of heating spores of PA 3679 at 115. exist (Christiansen et al. Johnston and Loynes (1971) obtained Perigo factor formation in the Perigo et al. sealed. for inhibition. and heating at 115.” The effect of all three factors (pH. (1967) medium and reinforced clostridial medium but not in liver veal or Wynne fluid media. sporogenes strain PA 6982 in perishable canned cured meat as occurs with C. overlaid with paraffin oil.

Pivnick and Chang (1974) considered. In this case alone. The data obtained with the spore inoculum should be more relevant to shelf-stable canned cured meats.1 µg/g) were the residual nitrite levels after 0 and 2 weeks in product made with 200 µg/g 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. another inhibitory factor may be formed when nitrite is heated with meat. With excessive heating. Chang and Akhtar (1974) prepared buffered saline suspensions of canned luncheon meat made with 0. with prolonged storage (42 days at 37°C). less residual nitrite was required for inhibition when nitrite was added before heating. Ashworth et al. 100. outgrowth and toxin production decreased.1 µg/g. which indicates that residual nitrite influenced the results. which had survived heating to F0 = 0. such as perishable canned ham. The nitrite level of the suspensions was adjusted after heating to 53. on the basis of added nitrite. (1967) based their conclusions on the added level of nitrite. Johnston et al.4 in raw meat juice with 4. (1973) extended this research to the milder heating given to large perishable canned hams and obtained similar results. It was originally suggested (Perigo et al. the conclusion that a Perigo effect was demonstrated must be tempered with the observation that. that although the Perigo inhibitor may not be present in meat. more was required when added before heating the meat. concluded that a PTF was produced. and it may also play a role in the safety and stability of pasteurized cured meats. autoclaving. and 200 µg/g of sodium nitrite and processed to F0 = 0. The meat was then inoculated with botulinal spores.4. They concluded that although a PTF was formed. However. higher levels of added nitrite were required for inhibition. Shelf-stable canned cured luncheon meat was prepared with 0 to 300 µg/g of sodium nitrite and processed to F0 = 0. The levels selected (53. The amount of PTF formed was related to the amount of original nitrite added to the meat before heating. results similar to those just mentioned were obtained during the early stages of temperature abuse.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. per se.5 or 22. and inoculated. 150. If residual nitrite is a valid means for comparison.000/g) added after heating and spores (20/g) added before or after heating. (1967) to be inactivated when added to meat.. Residual nitrite.5 and 22.184 Antimicrobials in Food 60 to 70 and 90 to 100 µg/g. (1969) found the inhibitor of Perigo et al. 1967) that the nitrite that disappears during heating may be involved in the formation of the unknown inhibitor. 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. deaerated in boiling water.. thermal destruction of spores and inhibition of the survivors by the salt). the relative inhibitory effect was quantitatively small. When spores were used. Adding salt (3. Ashworth and Spencer based their conclusions on the residual level of nitrite. Similar residual nitrite levels were required to inhibit vegetative cells (160. higher initial residual nitrite levels were required for inhibition. The term Perigo-type factor (or PTF) was selected to describe the effect observed in their test. the Perigo effect was of low magnitude. rather than after (150 µg/g). The depletion rate of the nitrite added to the suspensions was not influenced by the amount of nitrite originally added to the meat. Botulinal . The additional nitrite provided comparable residual levels after the various heat treatments. Ashworth and Spencer (1972) found no relation between the amount of nitrite lost during heating and the inhibition obtained.g. Ashworth et al. Actually.4 The product was held until the highest level of nitrite declined to less than 2 µg/g. strongly influenced clostridial inhibition. In all cases. however.5% salt and 150 µg/g of sodium nitrite. This was considered evidence for an inhibitory factor other than residual nitrite. In summary. more added nitrite was required in the pork if the nitrite was added before (300 µg/g). Inhibition by nitrite diminished gradually over a period of 8 weeks at 37°C. Perigo et al. 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. The authors found that as the original input nitrite level increased.

(1967) had a comparable effect on the inhibition of clostridial spores. van Roon. This was later reassessed by Christiansen (1980). Tjaberg and Kvale (1972) studied the antibotulinal effect of nitrite (0 to 480 µg/g) added to canned raw meat. Hansen and Levin. Research on perishable canned cured meat or meat slurries is discussed first.. Additional studies were reported by 1980 that dealt with the Perigo factor (Mirna and Hofmann. Van Roon (1979a. 1976. 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. 1974. 1978. 1972. Botulinal outgrowth in product abused at 27°C was influenced by the spore inoculum level and the level of nitrite. At 22°C.. causes inhibition by reacting with enzymes containing functional sulfhydryl groups. heat injured. predictably. 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.e. At 4°C. perfringens in a chemically defined medium. Huhtanen and Wasserman. Additional data by O’Leary and Solberg (1976) suggested that inhibition of C. respectively. Asan and Solberg. Nitrate had a slight but statistically significant inhibitory effect that may have been caused by nitrite generated from nitrate. Decreasing inhibition occurred as the product was held for 0. They subsequently concluded (Riha and Solberg.. Thus. regardless of the inoculum level or whether the spores were untreated. They hypothesized that nitrite. shelf-stable canned meat. 200 µg/g) usually used in meat products.Nitrite 185 inhibition was related to the original amount of nitrite. Incze et al. Moran et al. The authors concluded that it was not possible to demonstrate any preservative action of nitrite at the levels (i. Neither swelling nor toxin occurred in some treatments having 240 or 480 µg/g of nitrite. Inoculation of the medium after heating with nitrite and aging for 3 months showed that it was less inhibitory. 1975. indicating the Perigo factor is not stable. Perishable canned cured pork was studied by Christiansen et al. the rate of swelling ranged from 11 to 48 days across all variables. There was no evidence of germination (i. 1974. as nitrous acid. Ashworth et al. van Roon. A second test using canned product held for 4 weeks at 35°C before preparing suspensions with 22.. and sulfhydryl compounds are involved in the formation of the Perigo factor in broth media may be significant. and 37°C. considerable effort was devoted to perishable cured meats. swelling and toxin production ranged from 3 to 17 days. 1969.1 µg/g of sodium nitrite yielded similar results. and perishable canned meat and held at 4°C. and 4 weeks before preparing the suspensions. common agreement that nitrite.. vegetative cells) in the media with inhibitory levels of nitrite. (1973). and fewer treatments proved stable. Riha and Solberg (1975a) studied the formation and effect of a PTF on seven strains of C. It was stated that botulinal inhibition was related to the level of nitrite added during formulation rather than residual nitrite. 2. . 22°C. iron.e. or irradiation injured. Lee et al. Roberts and Smart (1974) found that the inhibitor in the medium of Perigo et al. who concluded that residual nitrite is a significant factor influencing botulinal inhibition. perfringens may involve an interaction of nitrite. At 37°C.. 1975. 1975b) that inhibition was apparently at the cellular level. 1979a. age of product must be considered when investigating the presence or activity of PTF in commercial product or model meat systems. 1974. 1974. in some form.b). toxin was not detected. although the spores remained viable during prolonged incubation.. b) concluded that Perigo-type inhibitors do not contribute to clostridial inhibition in heated cured meats. The degree of inhibition was correlated with the amount of nitrite originally added to the product. 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. Debatable as the formation of a Perigo-type in meat may be. Wasserman and Huhtanen. 1974). Mirna and Coretti. Nontoxic spoilage occurred at 7°C in uninoculated product made without nitrite. with sulfhydryl-containing constituents of the bacterial cell. 1975.

neither salt level provided much inhibition. a lower concentration of L-ascorbic acid (0. 22. The number of strains showing growth in broth was found to decrease with increasing nitrite (50. On investigation (Tompkin et al. (1976).0%. The effect of nitrite in perishable canned cured meat was also reported by Tompkin et al.0%) markedly increased the effectiveness of nitrite with none of the 38 strains tested showing growth in broth containing 50 µg/g sodium nitrite.5°C.186 Antimicrobials in Food Baird-Parker and Baillie (1974) studied the growth of a large. a race occurs between death of germinated botulinal cells and depletion of residual nitrite.0.8% or 3. The antibotulinal effect of nitrite was considerably greater than that reported for the meat slurries of Roberts et al.0% w/v). 4.5% salt (on water phase). product pH. Adding L-ascorbic acid (1. 101). Although less effective.5%. a preliminary test in bacon led to the conclusion that L-ascorbic acid does not alter the antibotulinal properties of nitrite in bacon. The emulsions were filled into cans and heated. This agreed with an earlier observation (Tompkin et al. Christiansen et al. Isoascorbate had enhanced the antibotulinal efficacy of nitrite in the canned meat.5). 5. It was suggested that Bacillus species are less sensitive to nitrite than clostridia.4°C or 10°C for up to half a year before placing at 27°C. Jarvis et al. perishable canned cured meat is placed at 27°C. 3.5°C and 25°C. 1978b) involving product held at 4. nitrite. Sodium nitrite and L-ascorbic acid were added as filter-sterilized solutions.. less inhibition occurred. in the cooked pork medium. Furthermore. 15°C). The rate at which inoculated cans swelled at 27°C was inversely related to the level of nitrite added to the product. The longer the product was held at refrigeration temperatures. 1978a). Spores heated at lower temperatures (70°C or 80°C) were inhibited by 4. (1978) concluded that when inoculated. Jarvis et al. 103. This led to the pork slurry system of Rhodes and Jarvis (1976) and used by Roberts et al. 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 . and decreasing inoculum level (106. 100. a pronounced salt–nitrite interaction occurred. Autoclaving nitrite in the Perigo medium resulted in significantly more inhibition than autoclaving the nitrite in the meat slurry.5% salt. At 20°C. (1976) found that heating botulinal spores in an aqueous extract of lean pork at 85°C. pro or con. In another study. one reason was found to be the addition of sodium isoascorbate to the canned meat but not to the meat slurries. 6.1%) also increased the efficacy of nitrite in broth but had no effect. even with 300 µg/g of sodium nitrite. diverse collection of botulinal strains in a broth medium and a cooked pork medium. the greatest inhibition occurred with 1.5% or above. Botulinal outgrowth was dependent on the relative levels of residual nitrite and surviving botulinal cells. At 20°C. 150. 6. Temperature of abuse was the most significant factor influencing the rate of botulinal growth.. At 15°C. decreasing pH (7. (1976) to study the interactions of temperature of abuse. (1977). the more the residual nitrite levels declined and the less inhibitory the product became when placed at 27°C.5% but not 3. salt. level of clostridial spores. At the lower temperatures.5%. or 95°C decreased the number that could grow in a recovery medium containing 2. Stability was influenced by nitrite content.5°C. 20 µg/g of nitrite in the Perigo medium and 210 µg/g in the slurry were equally inhibitory. or 25°C. 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 22.0. 200 µg/g). and inoculum level on botulinal growth and toxin production at pH 6. recommended that large-scale tests be used to quantify the many interacting factors influencing the safety of perishable cured meats. increasing salt (1. Grever (1974) examined the stability of emulsions normally used for preparing cooked sausage and liver sausage. At 17. 20°C.5% salt.5. 90°C. The spores heated at 70°C and 95°C were equally sensitive to the autoclaved nitrite. and whether the products were treated by a perishable or shelf-stable thermal process.0. 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. 6. 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. decreasing temperature (25°C.

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

the relatively high brine. One toxic sample occurred at 100 µg/g and none at 150 µg/g of sodium nitrite. and omission of nitrate.. Toxic product occurred with 50 µg/g of sodium nitrite at all levels of ascorbate. The pH of product made with 50. .5% to 4. 100. The franks were produced in the same facilities and formulated as before (Hustad et al. beef. except for changes in the ratio of pork to beef. 40. Toxin production was slower. The decline in pH caused by a lactic fermentation was also the dominant factor preventing botulinal outgrowth in fermented sausage (Christiansen et al. and 4.83%. 1975). Under these circumstances.08 or lower within 1 week at 27°C. A significant delay in botulinal toxin production occurred in pork. and pork in their system. The rate of botulinal toxin production was slower in all soy protein formulations than in all meat formulations. reported by Bowen and Deibel (1974) and Bowen et al. The second test. botulinum spores in a chicken frank formulation to be unaffected by the addition of sodium nitrite (0. In a meat–soy mixture. the two studies indicate that the combination of 4. the addition of 156 µg/g of sodium nitrite was very effective in delaying botulinal toxin production. (1974). Sofos et al..52% brine.188 Antimicrobials in Food Sofos et al. and 156 µg/g). (1979c) evaluated various soy proteins in their system. (1979e) found the rate of germination of C. and the effect of ascorbate in franks should be restated or reexamined. The rate of nitrite depletion was influenced by the type of soy. In mildly heated (58.0 or less within 21 days. In that variable the pH was 5. Toxin was produced in all variables formulated without dextrose and more quickly in product having 0 or 50 µg/g of nitrite. 450 µg/g of nitrate gave a variable response. 1973). the levels of nitrite (0 to 150 µg/g) and ascorbate (9. 1973). the exception being one variable with 150 µg/g of nitrite.6.3°C) sausages containing dextrose. The rate of botulinal toxin production in formulations with soy protein was equal to or slower than in an all-meat formulation. 105. They concluded that the effect of nitrite was on delaying the development of the cells after germination and before toxin was produced. The type of soy protein influenced the results.0 or less within 14 days. The addition of 80 µg/g of sodium nitrite was relatively ineffective in chicken and beef. or 150 µg/g of nitrite declined to 5. 20. Vacuum-Packaged and Fermented Meats The effect of nitrite in vacuum-packaged franks was studied on two occasions. The strong inhibitory effect observed was likely the result of the combined effects of nitrite. In the first. 400 µg/g of sodium ascorbate.8% fermentable carbohydrate. (1979d) compared the efficacy of nitrite (0 and 80 µg/g) in chicken. Nitrite was ineffective in a formulation of soy isolate and pork fat. The depletion of residual nitrite was faster as the amount of beef was increased in a formulation containing soy. Although product made with 150 µg/g of sodium nitrite did not become toxic.. a fairly rapid decrease in product pH during abuse. Sofos et al. in both the presence and absence of nitrite. Hustad et al. and.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. only those variables without nitrite became toxic at 27°C. and 655 µg/g). and the products remained nontoxic. and then only 2 of the 55 franks became toxic. The finished franks had a brine of 4. Adding 50 µg/g of sodium nitrite delayed botulinal toxin formation for more than 4 weeks during abuse at 27°C. input versus residual nitrite. It was concluded that ascorbate did not affect the antibotulinal efficacy of nitrite. examined whether an increase in ascorbate over normal levels would alter the antibotulinal effect of nitrite. the conclusions in both studies concerning the inhibitory levels of nitrite. A later review of the pH data showed that all variables had a pH of 5. about 2. (1973) formulated franks with various levels of nitrite and nitrate. more importantly. and fewer toxic samples occurred in product with 100 or 150 µg/g of nitrite. The pH of the dextrosefree product remained above 5. Assuming a similar pH response in the first test (Hustad et al.

cereus was evidently more resistant to nitrite than C. Including nitrate in the bacon enhanced the growth of micrococci. The nitrate likely served as an electron acceptor for the growth of micrococci in the vacuum package. and 200 µg/g) and sodium erythorbate (0. medium 4.7% became toxic.80 and 0. Raevuori (1975) studied the combined effect of sodium nitrite (0. 42. Omitting nitrate led to higher numbers of Moraxella species in the cured bacon. 20°C. the sausages had relatively high pH and water activity values of 6. growth was faster at 25°C than at 15°C. Moraxella species. 67. There was a general trend toward reduced residual nitrite levels at the tune of inoculation when ascorbate was added.9% became toxic.g. Erythorbate alone was not inhibitory. Bacon Crowther et al. 19. mixed cure with 100 µg/g of nitrite. The data included botulinal challenge tests in frankfurters made with chicken meat and ham made with turkey meat. 1977). The predominant flora of the bacon after curing consisted of micrococci. 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. The addition of ascorbate enhanced the antibotulinal effect of 100 µg/g but not 200 µg/g of nitrite. A higher percentage of samples were toxic with the addition of 200 µg/g of nitrite than with 100 µg/g of nitrite. In vacuum packages. and brine level (mild. and 34.3%.4%. The naturally occurring B. and 25°C) on the rate of botulinal toxin formation. 500. (1977) that demonstrated the efficacy of sodium nitrite in poultry meat. Raevouri encouraged more widespread use of erythorbate for sausage production in Finland. If all samples with no mixed cure.65 to 6. Inoculation studies examined the effect of nitrite (0 and 100 µg/g) and storage temperature (15°C. 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. and 2000 µg/g). Food and Drug Administration (FDA) for approval of sodium nitrite as an additive in poultry products. and 1000 µg/g) on the growth of Bacillus cereus in cooked sausage placed at 20°C for 48 hours.7% to 6. When all samples having the mild and medium brine were compared. Because brine levels could not be held constant.. cereus tested. As expected. 35.S. It also was reported that S. 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. . A summary of the data showing the antibotulinal effect of nitrite in the two poultry products appears in Hauschild (1982).6%) on botulinal toxin production in vacuum-packaged back bacon. botulinum. The request included results by Christiansen et al. B. and Moraxella-like bacteria. 100.98.Nitrite 189 A request was made by the poultry industry to the U. aureus grew well in the medium-salted bacon. regardless of the level of nitrite or ascorbate. lower populations resulted and enterotoxin was not detected.. higher populations of S. aureus developed and enterotoxin A was produced. 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. In opened packages. and mixed cure with 200 µg/g of nitrite were compared. 2. respectively. After cooking in Saran™ casings.9% became toxic.9% to 4.8% and 21. cereus). (1976) in the United Kingdom studied the effects of a mixture of nitrite (100 or 200 µg/g) and nitrate (250 µg/g). respectively. ascorbate (0. 1000. Noninoculated product containing nitrite developed a slight unusual taste at 20°C owing to the growth of spore-forming spoilage organisms (e. Product with 100 µg/g of sodium nitrite remained nontoxic through 14 days at all three temperatures. botulinum. the interaction of nitrite and ascorbate is difficult to interpret. Erythorbate caused reductions in the residual nitrite and the redox potential of the product.7% and 18. respectively. Erythorbate was found to enhance the inhibitory effect of nitrite. Bacon that was sliced and vacuum packaged developed a flora mainly of micrococci and lactics.9%. If all samples containing mixed cure with and without ascorbate were compared. Shaw and Harding (1978) studied the effect of nitrate and nitrite on the microbial flora of Wiltshire bacon.

7. it was suggested that nitrite could be important in delaying the sour spoilage caused by the growth of lactics. The initial period of delay depends on the inoculum level. Sofos et al. Because phosphates were present in almost all the studies. The addition of ascorbate or isoascorbate can act in concert with residual nitrite to retard botulinal outgrowth in freshly produced bacon. and vacuum packaged (100 g per package). 8. 5. sucrose) were left to the discretion of the producing plant. phosphate.. This was likely the result of the interaction of the other components of the product (brine level. outgrowth is increasingly delayed. At least on the basis of the botulinal results... 1980b. phosphates do not appear to influence botulinal outgrowth in bacon. Three types of bacon were produced in four commercial plants: bacon with no preservative. 10 appear in the technical literature (Christiansen et al. 1980). All three bacons contained 550 µg/g of sodium ascorbate or sodium isoascorbate. 4. and fermentable carbohydrate (e. The data indicate the following: 1. 1974. the data supported the potential approved use of a nitrite–sorbate blend to produce bacon. The level of residual nitrite at the time the bacon is abused influences the extent of the delay in botulinal outgrowth. Ivey et al. The level of nitrite added to the product is not important.. There was considerable variation in toxin production in the bacon from the four plants. and bacon with 40 µg/g of sodium nitrite plus 0.0%. Brine levels below 4. These factors interact to influence botulinal inhibition.g. the combination of relatively higher brine and decreasing pH can prevent botulinal outgrowth. Although the studies were performed for a variety of reasons. Temperatures below 27°C or 30°C increase the delay in outgrowth and the inhibitory effects of the foregoing factors.. Too high a level of botulinal cells when the bacon is abused can overwhelm the inhibitory system. Botulinal outgrowth is influenced by the temperature of abuse. it is difficult to assess their effect. As the brine level exceeds 4.. fermentable carbohydrate. Within the levels and types used. The results demonstrated that the combination of nitrite–sorbate was equal to or better than nitrite alone. it is possible to glean the relative effect of nitrite by examining the nitrite-containing control variables from each test. 1979). and growth of a spoilage flora). Tanaka et al.7% sugar (sucrose) or more provides sufficient fermentable carbohydrate that naturally occurring lactics cause a decline in pH to inhibitory levels. inoculated with 1000 spores/g. 6. Vacuum-packaged bacon prepared with 0. If a lactic fermentation develops in the interim.0% are not inhibitory to botulinal outgrowth.26% potassium sorbate. The pH of the bacon during abuse significantly influences the potential for botulinal outgrowth. Collins-Thompson et al.190 Antimicrobials in Food Because higher numbers of lactics were present in bacon with the lowest initial nitrite concentration. A total of 30 botulinal studies were performed with vacuum-packaged bacon during the 1970s in the United States and Canada. They can be more readily compared because they were similar in processing and final product. The bacon was then incubated at 27°C for up to 56 days. aside from the fact that the amount of added nitrite partially determines the level of residual nitrite. Bowen and Deibel. 3. . 1974. bacon with 120 µg/g of sodium nitrite. ascorbate and isoascorbate can also have a negative effect by causing more rapid loss of residual nitrite during processing and storage. 1978. The bacon was shipped to a laboratory. 2. The levels of sodium chloride. Of the 30 tests. However. 1974. The USDA conducted a test to verify the antibotulinal effectiveness of a combined nitrite–sorbate mixture in bacon (Johnston.

Anaerobic toxin production was prevented with 0.58. growth of competitive flora.3) on the growth of S. 7. and Pediococcus cerevisiae (1 strain) were examined for their tolerance to nitrite in the presence and absence of 4. no residual nitrite.01% salt. aureus populations as . Buchanan and Solberg (1972) studied the effect of nitrite (0. 8 µg/g enhanced the growth of the lactobacilli and. future research might demonstrate the involvement of additional factors. Lactobacilli (78 strains). and 0. and product pH) contributes to this effect. Extensive tests were also reported for laboratory-cured ham. aureus is less acid tolerant under anaerobic conditions. low oxygen level.g. At pH 7.3 in two samples with negligible nitrite levels. anaerobic toxin production occurred at pH 5. 500. aureus and Other Bacteria A cluster of reports appeared during the early 1970s that dealt with the effect of nitrite on S.9% salt and 100 µg/g of sodium nitrite) incubated at 37°C in environments of 20%. the micrococci. The authors questioned the hypothesis that nitrite inhibition is dependent on the conversion of nitrite to nitrous acid. aureus under aerobic but not anaerobic conditions. to a slight extent. because high nitrous acid values (>0. Buchanan and Solberg suggested that nitrite inhibits the growth of S. but it remains debatable how much each of the possible factors (e.34. Nitrite was metabolized by S. but it is unclear whether nitrite level influenced anaerobic enterotoxin production.3. (1976) indicate that the growth of S. reduced pH.6%. At 40 µg/g growth was comparable to that in the control without nitrite. and 0% oxygen yielded lower S. it is uncertain whether high nitrous acid content. The commercially canned ham used for the test had a brine level of 3. however. aureus. aureus in brain heart infusion broth (BHI). Of significant importance was a report that the growth of S. The probability of toxin production at 10°C reportedly decreased as the level of nitrous acid increased. aureus. This suggested either that nitrite was not being used as an electron acceptor or the nitrite reductase system was blocked anaerobically.6 µg/g) were possible only at reduced pH levels (pH 5. Nitrous acid levels were calculated from the residual nitrite and pH values as described by Castellani and Niven (1955). the data along with the aforementioned results of Christiansen and Foster (1965) and Crowther et al.3. 5%. Inoculated raw sausage (2. 15%. 22 µg/g of nitrite.0). aureus and a review of the literature. 10%. nitrite was without effect in an aerobic environment and 500 µg/g or less was without effect anaerobically. A third sample with a pH of 5.159 µg/g of nitrous acid also became toxic. micrococci and staphylococci (24 strains). They also concluded that vacuum packaging should improve the safety of cured meats relative to S.6 or less). nitrite.3 and 200 µg/g. 200. aureus was found to be restricted to the periphery of commercially fermented sausage (Barber and Deibel. Understanding why this occurs would be very instructive. At pH 6. However. (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.3. and an abnormally low pH of 5. Reasons for the reduced growth and enterotoxin production are evident from the reports. Heterofermentative lactobacilli were more tolerant of salt than the homofermentative strains. One reason given was that S. At higher temperatures (22°C and 30°C).. 1972). Genigeorgis et al. Very high levels of nitrite (1000 and 5000 µg/g) either prevented or severely retarded the growth of all strains.6 µg/g or more nitrous acid and pH values below 5. Toxin was produced in certain variables having residual nitrite levels as high as 145 µg/g. brine. and 2000 µg/g). the increase in the adjustment phase was 10 hours or less aerobically and about 30 hours anaerobically. 1000. Collectively. and initial pH (6. or both prevented anaerobic toxin production. At 200 µg/g growth was delayed or slower. thus preventing the normal metabolism of pyruvate. aureus by blocking the sulfhydryl sites of either coenzyme A or α-lipoic acid. oxygen pressure. Nurmi and Turunen (1970) studied the effect of adding nitrite to a previously autoclaved broth medium (pH 6.Nitrite 191 Tests with S. After considering the effects of changing pH during the growth of S. aureus is reduced and enterotoxin production can be prevented by the environmental conditions existing within vacuum-packaged cured meats.

Morse and Mah (1973) studied the effect of glucose on enterotoxin B synthesis in a broth medium buffered to an alkaline pH (7. more available energy. and nitrite on the heat resistance and recovery of S. aureus. and nitrite increased (Tompkin et al. Markus and Silverman (1970) reported enterotoxin A production in broth media (pH 6. Bean and Roberts (1974. Nitrite did not affect glucose transport by S. enterotoxin was not detected through 5 days of storage in the absence of oxygen. salt increased. no synergistic effect occurred between salt. nitrite. The formation of the Perigo factor in commercially cured meat is highly debatable. and pH). both of which lack cytochromes. the percentage of lactic acid as an end product. and 7. However..0 but to a lesser degree than at pH 7. If manipulations of thermally processed meat were attempted to extract such an inhibitor. 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. 1973).5 or 5. the question could be raised whether the inhibitor would be altered or destroyed. 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. SUMMARY FOR 1970–1980 The Perigo Factor in Cured Meats 1. oxygen uptake. and oxidative phosphorylation in Pseudomonas aeruginosa.5. 4. The inhibitory effect of nitrite in the recovery medium increased with increasing salt content. There was a general pattern of increased heat resistance as salt level (0. salt level. decreasing incubation temperature. and decreasing pH. 6. cytochrome oxidase) to the ferric state. and 8%) increased at pH 6.. Glucose repression of enterotoxin B production was also reported to occur at pH 6. and nitrate on either growth or enterotoxin production. 1973). Adding glucose caused decreased toxin production as a result of the rapid oxidative decarboxylation of pyruvate.g. In another report on non-spore-forming bacteria.0. Rowe et al.7). 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. faecalis or Streptococcus lactis. increasing the level of nitrite had the general effect of reducing the recovery of both heat-damaged and undamaged cells.6 to 6. 1975) demonstrated the combined effect of pH. Contrasting results were subsequently reported that showed the production of enterotoxin A to decrease as pH decreased. and nitrate was converted to nitrite. Formation of some similar inhibitor(s) (PTF) during the thermal processing of shelfstable canned cured meat appears possible. (1979) reported that nitrite inhibits active transport. . residual nitrite. 2. presumably. Perhaps because of the atypically high pH. the sum of which influences stability. salt.0 but not at pH 5. Adding nitrite as an electron acceptor at the time of anaerobic shock prevented increased toxin production. and.192 Antimicrobials in Food the oxygen level decreased. 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. The effect of nitrite on heat resistance was inconsistent.8) to be unaffected by sodium nitrite (200 µg/ml) or sodium nitrate (1000 µg/ml). when added to the recovery medium. Although adequate growth occurred within 48 hours. The evidence strongly suggested that nitrite exerted its inhibitory effect by oxidizing ferrous ion of an electron carrier(s) (e..g.7 (Morse and Baldwin.

Nitrite was reported to inhibit active transport. ferredoxin) within the germinated cells. enterococci). 9. Inhibition seems to be influenced only by the amount of residual nitrite in excess of the asymptotic level on the depletion curve. It was suggested that nitrite inhibition of C. heating times. oxygen uptake. long. iron.. All the factors are interrelated. certain competitive flora are inhibitory to botulinal outgrowth (e. 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. Inhibition of C. 2.g. 3. The list of recognized factors influencing the antibotulinal efficacy of nitrite continued to grow and included the following: 1. 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. and perhaps injury. aeruginosa... 3. of the germinated cells as the temperature is increased.Nitrite 193 The Perigo Factor in Media The data appear conclusive that nitrite. 7. Whether phosphates can contribute to inhibition by some other mechanism (e. and oxidative phosphorylation in P. can provide the opportunity for germination followed by destruction. and sulfhydryl groups are involved in the formation of the inhibitor. Although the thermal process given to perishable cured meats does not appear to cause thermal injury to the spores. In addition. The effect appeared to be at the electron carrier level. Mechanism of Nitrite Inhibition 1. The disappearance of residual nitrite in cured meat is faster as product pH is decreased and isoascorbate level and storage temperature are increased. 11.g. sequestering iron) remained unclear. perfringens and S. 8.g. 6. The low level of residual nitrite that continues to be detected after prolonged storage does not appear to be inhibitory. 10. . 5. 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. aureus was suggested to involve the blocking of sulfhydryl sites within the bacterial cells. slow. PTFs do not appear to play any role in perishable cured meats. 4. but this effect was not clearly defined in meats. Glucose transport was not affected in two bacterial species that lack cytochromes. 2. as used in certain experiments with bacon and large-diameter sausages. botulinum may be the result of a reaction of nitric oxide with an essential iron-containing compound (e.

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

This information was reiterated by Hauschild and Simonsen (1986). . Hauschild and Simonsen (1985) conducted an extensive review of the technology for producing shelf-stable canned cured meats (Table 6. Reductions in the recommended levels should be based on extensive plant experience and/or experimental work and controls to assure minimal spore levels. carbohydrate. 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.0 5. The level of risk differed among the various products. existing government regulations. 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.5 0. selective reductions in nitrite could safely be made provided the levels of brine. further accelerating the trend of the past 40 years.2–0. Another major trend developed during this time. Bacon was considered a low-risk product. 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.3 3.7 4 2. The level of risk could also differ among products of the same type as a result of differences in their brine concentrations. They summarized research.0 0. Throughout the decade.1).5 Ham and shoulder Sausages Source: Hauschild and Simonsen (1985). whereas turkey ham was a high-risk product.0 0. there has been a very rapid growth in the volume and variety of cured poultry products in the United States.5 3. He proposed expressing botulinal inhibition as log I/P.5–1. the impact of further reductions in salt deserved close examination. Reductions in the level of nitrite can be compensated for by moderate increases in the brine level and/or thermoprocess. TABLE 6.1–0. In 1983.0–1. He concluded that significant reductions in nitrite should not be made for all cured meats.0–4.5 1.5 0. Their review was to assist in the development of a document for these products.3 0. This was used to quantitatively compare the data from the various challenge tests.0–5.5 Thermoprocess (F0 ) 1. the FDA acknowledged that prior sanction existed for the use of nitrite as an additive to poultry meat (Cassens. and commercial practices to arrive at guidelines to assure the microbiological safety of these products. The data provided estimates of the relative degree of botulinal risk for each product.3–0. where P is the probability of outgrowth and toxigenesis from one spore.Nitrite 195 for sodium intake in the diet brought new pressure to reduce the salt content of cured meats. the per capita consumption of red meats decreased while the consumption of poultry products increased. Since that time. However.1 Shelf Stable Canned Cured Meats: Minimal Brine and Thermoprocess with 150 mg/g Sodium Nitrite Product Luncheon meat % Brine 3–4 4. 1990).2 1.5 4.5–5. and erythorbate are controlled.

At brine levels of 3. with levels of at least below pH 6. Some consumers find this amount of salt unacceptable.2% brine. 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 antibotulinal efficacy of sorbic acid was pH dependent. The use of nitrite in smoked fish in the United States has been restricted. competitive flora could have been destroyed. Large bone-in hams were inoculated with nonproteolytic botulinal spores before curing in a solution of salt. Wagner and Busta (1983) compared the antibotulinal effect of combinations of sodium nitrite (40 and 120 µg/g). The tests involved vacuum-packaged smoked whitefish fillets prepared with about 2. After curing.24/kg. and 6.196 Antimicrobials in Food Botulinal Challenge Tests Tests in meat and fish products. The test system consisted of a frank emulsion prepared from mechanically deboned chicken meat that was extruded into test tubes. Similar results were obtained during the dry curing of hams at 8°C or 10°C. Toxin was detected after 9 to 11 days. nitrite. Perhaps the milder thermal process permitted the survival of a competitive flora that prevented toxigenesis.2. and potassium sorbate (0% and . botulinum. The pH of the fillets decreased during 27°C incubation from about pH 6. 1982). The nitrite (40 µg/g) also significantly increased the inhibitory effect of the sorbic acid. Storage at 8°C until a nearly complete loss of nitrite occurred did not diminish the effect of the nitrite. The number of botulinal spores was estimated to be about 0. By increasing the processing temperature and/or prolonging the time of processing. the inhibitory effect of the lactic fermentation and decreasing water activity values during fermentation and subsequent drying prevented botulinal toxicity. They concluded that nonproteolytic. Liver sausage was prepared with 83 µg/g of added sodium nitrite. The hams had a slight off-odor but no apparent putrefaction. and nitrate at 8°C. Another possibility could be offered for this observation. The total C. (1982) assessed the relative risk of botulism from temperature-abused liver sausage. 10°C. cooled. The presence of sorbic acid slowed the rate of nitrite depletion.2. and incubated at 27°C. (1982).3%. In fermented sausage.4 to as low as 5.8% to 4. Hauschild et al. nitrite in the range of 0 to 100 µg/g had little or no effect on toxigenesis. Hauschild and Hilsheimer (1983) examined 138 samples of liver sausage from retail outlets in Canada.44%. Cuppett et al. and hams was investigated by Liicke et al. The latter were suspected to be of particular importance as a cause of botulism from raw cured meat products in Europe (Lücke.2%. nitrite was more effective and an appreciable delay in toxicity was obtained with 50 and 100 µg/g of nitrite.2 without loss of the benefit of sorbic acid. a high level of salt (5% brine) has been required. By increasing the thermal process. At a higher brine level of 5. toxin production was increasingly more rapid. the pH of the formulation could be increased to pH 6. and 15°C. Product with low levels of toxin often appeared acceptable. but the recovery of viable botulinal cells became increasingly difficult. heated. and aw 0. Sofos et al. The rate of pH decrease was slower as the brine level increased. (1980).0 being required. Toxin was not produced at 5°C. (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. fermented sausage. botulinum was estimated to be about 0. overlaid with vaspar.15 spores/kg in the product. toxin was detectable 10 cm from the injection site. Methods and media were recommended to facilitate the recovery of both the proteolytic and the psychrotrophic nonproteolytic strains of C. Greater protection was obtained with 150 µg/g. thereby permitting the more heat resistant C. Tests in model systems. toxin levels remained high. botulinum to dominate and develop toxin. Additional data on the ham experiments were presented by Lücke et al.4%).98. 4. botulinum. pH 6. As the hams continued through the completion of curing. the inhibitory effect of the nitrite was reduced. (1980a) studied the significance of pH on the efficacy of nitrite and sorbic acid mixtures in a chicken frank emulsion. As temperature was increased to 8°C. Toxin production by a mixture of type E and nonproteolytic type B botulinal strains in liver sausage.4%. If a low level of nitrite (40 µg/g) was added. To prevent the growth of C. sodium acid pyrophosphate (0% and 0.

72). or thermal process.4 units in the lower pH slurries and about 0. and thermal process on botulinal outgrowth in pork slurries of two different pH ranges (pH 5.03. but the reason for the increased inhibition when adding polyphosphate to the higher pH meat slurries is less evident.19. (1981a. There appeared to be more botulinal inhibition with the addition of sodium acid pyrophosphate. It is unclear whether the target or analytical values for salt were used for statistical analysis. This increased inhibition was not observed with nitrite levels of 75 to 125 µg/g. (1982) examined the interaction of potassium sorbate in their cured pork slurry system. No systematic difference in inhibition was observed with the meat from three breeds of pig.2 units in the higher pH slurries.78 to 6.4%). (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.3 to 6. more like that of a reformed ham after heating. Tubes showing gas or other spoilage were preferentially selected for toxin assay. and 4. They found the nitrite to be less effective in slurries prepared with meat from the shoulder than meat from the leg. The combination of 40 µg/g of sodium nitrite.27 to 6. A separate formula was developed for slurries prepared with low-pH pork (pH 5. Of the polyphosphates tested.b) evaluated the interactions of salt. sodium acid pyrophosphate was more effective than sodium hexametaphosphate or sodium tripolyphosphate. nitrite. 1983).6 to 6. The pH of the four product variables was between 5. nitrite level. although the trends in the data may not change. The pH was important in all treatments tested. The effect of sorbate was greater as salt levels .4 and 5.7). 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. (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 increased botulinal inhibition when added to combinations of sorbic acid or potassium sorbate and a low level of nitrite (40 µg/g). This may alter the quantitative assessment of the individual and combined effects of the additives. Roberts et al.5%.1 to 0. They used the same test system as in Sofos et al. polyphosphate.26% potassium sorbate. One nitrite level (40 µg/g) was used throughout. (1983) assessed the antibotulinal effect of three phosphates in the test system just described (Wagner and Busta.4% sodium acid pyrophosphate caused the greatest delay in toxin production.65. Considerable variation occurred between the meat from different animals within each breed. Jarvis et al. The combination of nitrite (40 µg/g). nitrate. The initial pH for the various products was 5. Statistically significant reductions in toxin production occurred with increasing salt level.65 to P80°C = 6. They also found that the antibotulinal efficacy of nitrite could be increased by the addition of phosphate. Nelson et al. with certain phosphates being more effective than others.7 and 6. (1979) modified their pork slurry to a thicker consistency.27 to 6. The levels of salt found in the slurries were often as much as 0.5% salt (wt/vol water). Roberts et al. or decreasing the abuse temperature. The pH of the pork used for the lower pH slurries ranged from 5. (1979b–e.5%.36 and 6. and 0.56. The polyphosphates did not influence botulinal inhibition in the absence of nitrite. except for two outliers of 5. The addition of polyphosphate caused an increase of about 0.4 to 6. potassium sorbate (0. The frankfurters were made from chicken instead of beef and pork. 0. The pH of the pork used for the higher pH slurries ranged from 6. The increase in pH could explain the decreased inhibition in the lower pH slurries.26%) in frankfurters.0. Gibson et al.Nitrite 197 0. 3. and pH control of the emulsion was the most effective to achieve reduced nitrite levels and provide enhanced botulinal inhibition.54. sodium acid pyrophosphate (0.3) and high-pH pork (pH 6. The test system avoided the problem of recontamination that occurs when peeling and packaging franks. They found that increasing the heating from P80°C = 0. adding isoascorbate or nitrate.5 to 6.5% (wt/vol water) below the targeted values. 1980a). isoascorbate. although the conclusions were stated on the basis of the target values of 2.26%). potassium sorbate significantly decreased toxin production. At a given spore inoculum. Roberts et al.

The addition of nitrate led to higher residual nitrite levels for a longer period. responded similarly. 30. and the addition of isoascorbate. there was a marked decrease in the residual nitrite. Residual nitrite levels after irradiation were inversely related to the irradiation dose.K. 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. forms an inhibitor when autoclaved in the presence of nitrite. In another study of Perigo factor formation. The bacteriostatic action was reversed by the addition of cysteine. and stored at 30°C for 28 days. an iron-binding protein in casein. legislation was proposed to control the use of nitrite by monitoring the residual nitrite in cured meat products. Higher or lower levels prevented the inhibitory effect. The slurries were irradiated. unless ferric chloride was present and the pH was 6. botulinum. which otherwise prevent bacteriostatic activity. a similar protein in blood serum.198 Antimicrobials in Food increased (2. and 200 µg/g of sodium nitrite. They suggested that the bacteriostatic effect is the result of facilitated conversion of cysteine to Bactin and a concomitant removal of thio compounds. 50. 40. This led Gibson et al. The amount of iron required for inhibitor formation was reportedly similar to the level found in muscle tissue. then inoculated with botulinal spores. They found that the rate of depletion in their pork slurry system was influenced by many intrinsic and extrinsic factors. the addition of nitrate. Custer and Hansen (1980) reported that lactoferrin. and 50 kGy). (1984) to examine the effect of various factors on the rate of nitrite depletion and botulinal outgrowth. and as the storage temperature decreased.0. However. They concluded that the level of residual nitrite did not influence botulinal outgrowth in their experiments.5%). After higher doses of irradiation (10.5% to 3. as pH levels decreased to below 6. the . thioglycollate. the rate of nitrite depletion was influenced by storage temperature. The results are intriguing and leave relatively unexplored the underlying mechanisms influencing the results. Bactin was not produced when heating cysteine. As expected. A rather high level of isoascorbate (1000 µg/g) caused nitrite to disappear rapidly and delayed the outgrowth of C. 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). The probability of botulinal outgrowth decreased as the level of added nitrite increased above 50 µg/g. Pretreatment of the lactoferrin and transferrin with a protease was necessary for inhibitor formation. 20. 156. 100. 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. and iron in culture media. At lower dose levels of 0 to 9 kGy. For example. The significance of microbial growth on nitrite depletion in the slurries during storage was not addressed. 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. Transferrin. (1989) prepared ground pork slurries with 0. 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. sealed with vaspar. No suggestion was offered for the increased botulinal inhibition when nitrate was added. which resulted in rapid botulinal outgrowth. U. 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. The authors suggested that Bactin formation may be responsible for the inhibition observed in Perigo-type media. 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.

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

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

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

0.0. perfringens was enhanced with a decrease in pH. (1989) found the rate of death of L. more generally.202 Antimicrobials in Food The debate over whether clostridial ferredoxin or pyruvate-ferredoxin oxidoreductase is the site of nitrite inhibition for C. blisters at the surface of the cells were observed when viewed under the electron microscope. the efficacy of sodium nitroprusside was explored (Joannou et al. 120.5. It appears that the hypothesis proposed earlier is fairly close (Tompkin et al. and sodium nitrite on the inhibition of C. (1987). 5. They were unable to demonstrate a direct action of the compounds on the iron–sulfur proteins. The final pH of the products was about pH 4. Their results led to the conclusion that both are inactivated. Buchanan et al. At pH 6. Juntilla et al. botulinum was again addressed by Carpenter et al. (1989) tested the effect of nitrite on L. In a broth medium at pH 6. However. A high salt concentration and low incubation temperatures were the more important factors influencing the inhibition of these bacteria. perfringens foodborne illness involving cured meats. It can be concluded that factors (e. (1990a. it is fairly certain that the mechanism of nitrite involves the inactivation of iron–sulfur proteins. perfringens to 3% to 4% salt could explain why there have been few outbreaks in C. Miscellaneous Studies in Laboratory Media Botha and Holzapfel (1987) tested the effect of nitrite on Sporolactobacillus isolates from a variety of sources. The cell-wall surface was the primary point of attack for the nitrosyl complex associated with nitroprusside. After continued research on the mechanism of compounds associated with the Perigo factor. 1978). At the pH values (6. incubation temperature. (1980). One aspect that the more recent data suggests is that the interaction of nitric oxide with iron–sulfur complexes is irreversible. the data more strongly demonstrate that L.5%) and sodium nitrite (50. sporogenes. 28°C. which is more typical of cured meat and . leading them to doubt that the inhibitory effect of nitrite is on preformed iron–sulfur proteins of the bacterial cell. 1998).5 and 6. comparable to the inhibitory properties of the anion of Roussin’s black salt. and 5.0% and 3. and 37°C) had the strongest effect on growth rate and lag-phase duration. Research by Payne et al. monocytogenes in a broth medium at pH 7. and 200 µg/g). Gibson and Roberts (1986b) demonstrated the interactions of pH.. monocytogenes is quite resistant to the inhibitory effect of nitrite.b) examined the effect of nitrite and iron–sulfur–nitrosyl complexes on iron–sulfur clusters in C. The cell walls showed a 90% decrease in thiol content when compared with untreated cells. The incubation temperature (5°C.0. Nitroprusside was used as a model to investigate the inhibitory mechanism of sodium nitroprusside and. Sodium nitroprusside was found to be a very effective inhibitor of C.5. On the basis of the research conducted by these researchers and others. monocytogenes to be similar in fermented sausage manufactured with different levels of salt (3. the toxicity of nitrosyl compounds to bacteria. decreasing pH and aw) influenced the rate of death. Sodium nitrite was added to the broth before autoclaving. (1991) and Roberts and Dainty (1991).0) found in cured meats.5. Additional information and views on the mechanism of nitrite are available in Roberts et al.6. In addition. Most cured meats have traditionally had brine levels of 3% or greater. The relative sensitivity of C.. The inhibitory effect of nitrite against C. although the nitrite had been autoclaved with the broth. sporogenes cells and isolated proteins. In the research of Duncan and Foster (1968b) such blister formation was not reported following exposure to sodium nitrite. The fecal streptococci proved to be quite resistant to nitrite. such as ferredoxin and pyruvate oxidoreductase. sodium chloride. The effect of nitrite on the growth of Listeria monocytogenes was examined in trypticase soy broth by Shahamat et al.. nitrite was not an effective inhibitor at pH 7. Consistent with previous reports over the past 60 years. perfringens and fecal streptococci in broth media. 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. 19°C. and/or an increase in the salt concentration.g. some reduction in growth was observed with the addition of 200 µg/g of sodium nitrite. a decrease in temperature of incubation.

Subsequent tests demonstrated that a strain of L. viridescens was among the most active species. 1988). The significance of these observations was discussed. When comparing 0. manganese). viridescens. Thus.5 to 5.5). the canned hams were opened. and some interesting questions were posed. plantarum.Nitrite 203 poultry products.5% and 4. the lactic acid bacteria . Spoilage of Cured Meats Zeuthen (1980) studied the effect of meat pH on the rate of microbial growth on sliced ham. the rate of nitrite depletion in cured meats can be important. and L. The growth of Brochothrix thermosphacta was restricted when grown on sliced vacuum-packaged bologna with Lactobacillus brevis or L. The authors suggested that inhibitory strains produced specific antibiotic substances other than hydrogen peroxide or lactic acid. plantarum but not with L. acidophilus. 1985). Competitive inhibition can be a factor affecting the spoilage flora of cured meats.g. 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. the degree of inhibition was most strongly enhanced by reducing the incubation temperature. vacuum packaged.0%. Fresh pork was divided into two groups: low-pH meat (5. The lower pH meat resulted in a ham with a pH of about 6. nitrite was inhibitory. For example. CollinsThompson and Lopez (1982) reported the inhibition to be species dependent. can accelerate nitrite depletion. CollinsThompson and Lopez (1982) and Dodds and Collins-Thompson (1984) further demonstrated that lactic acid bacteria. L. 50.0 and a residual nitrite level of 38 µg/g after processing. lactis. The nitrite reductases generally are heme-containing enzymes.. Both products had a brine level of approximately 4. commonly involved in the spoilage of cured meats. 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. The hams were boned and pumped with a pickle solution containing sodium tripolyphosphate. sliced. During the 7 to 8 weeks of storage at 5°C.3 to 6.e. plantarum. it was suggested that in trace metal metabolism. L. leichmannii. lactis was able to reduce nitrite to N2O (Dodds and CollinsThompson.3 within 2 weeks at 5°C.5 within 4 weeks. Inhibitory species caused the pH of the bologna to decrease to 5. By 1988 it was established that nitrite reduction can occur in some strains of L. 100. The depletion of nitrite was attributed to enzymatic activity (i. The data of Grau (1980). and 200 µg/g of sodium nitrite. The meat was then used for processing as canned hams. Under certain circumstances the level of analytically detectable residual nitrite can influence microbial inhibition. the rate of microbial growth was considerably slower in the sliced ham prepared from the lower pH meat. and even less by increasing the salt level (0. both strains accumulated manganese in their cells. Collins-Thompson and Thomson (1986) found two strains of L. viridescens or Leuconostoc mesenteroides. The loss of residual nitrite in bologna as a result of the action of the lactic acid bacteria was estimated as 30%. Noninhibitory strains decreased the pH of the bologna to only 5. When grown anaerobically. monocytogenes will grow in processed meat and poultry products at refrigeration temperature. demonstrate that inhibition can be dependent on the concentration of undissociated lactic acid and the degree of anaerobiosis existing at the site of growth. Both cultures were sensitive to the inhibitory effect of nitrite under anaerobic but not under aerobic conditions. buchneri (Wolf and Hammes. L. to a lesser extent by anaerobiosis. The higher pH meat resulted in a ham with a pH of 6. particularly homofermentative lactobacilli. L. nitrite may stimulate the uptake and transport of ions (e. L. nitrite reductase) rather than lowering the pH of the medium.. that were resistant to the addition of 50 µg/g of sodium nitrite if grown aerobically in APT broth. however. 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.5%).35 and a residual nitrite level of 90 µg/g. After cooking and chilling. however. and placed at 5°C.7) and high-pH meat (6.

did not activate nitrite reductase or catalase activity. about 0. Kafel and Jozwik (1987) investigated the spoilage rates of commercially produced products from 6 meat processing plants in Poland. An industry survey found that liver sausage was commonly cooked to an internal temperature of 65°C. 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. Lactobacilli were the dominant flora in all four products at the time of spoilage. phosphate. sliced. as the storage temperature increased.. 10°C. cereus in liver sausage has been a problem in Finland. Again. Nielsen (1983a) studied the effect of sodium nitrite (0.e.e. 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. and 200 µg/g) on the spoilage flora of a sliced vacuum-packed bologna-type sausage. Enterobacteriaceae. and 10°C). hemoglobin. Additional additives were tested to reduce this risk. and Moraxella and Moraxella-like organisms were increasingly inhibited by increasing the nitrite concentration and/or decreasing the temperature of storage (2°C. If the product is temperature abused. Omitting nitrite resulted in a perfume-like odor that was associated with the growth of enterococci. gluconolactone (glucono delta lactone. and 200 µg/g) on the spoilage flora of cooked pork loin. Protoporphyrin. High levels of B.7 to 5. The Pediococcus isolate was not as heat resistant as the enterococcus. Wolf and Hammes (1988) found all the strains of L. being insensitive to the presence of nitrite. Nielsen (1983b) subsequently studied the effect of sodium nitrite (0. 156 µg/g of sodium nitrite. B. 68°C. 50. Gramnegative bacteria often constituted the major flora of the product without nitrite. Nitrite inhibited the growth of B. (1988) reported that the growth of B. 5°C. plantarum tested were able to reduce nitrite when hematin was provided to cell suspensions. faecalis (Chyr et al. plantarum and L.2 to 0. 100. an iron-free compound. 5°C. Polish regulations require the heating of perishable canned . cereus was delayed or prevented by including a combination of erythorbate. The enterococcus survived 65°C for 5 minutes or 60°C for 60 minutes with only 103 cells/ml broth. Chyr et al. A correlation was found between the nitrite concentration at the time of slicing and packaging and the time for spoilage to occur. The spoilage flora of the liver sausage prepared without nitrite also included Pediococcus pentosaceus. and sodium nitrite (120 µg/g). thermosphacta and Enterobacteriaceae at all temperatures of storage (2°C. 100. hematin) did activate the enzymes. the Gram-negative flora proliferated in product with nitrite. This led to the lactic acid bacteria becoming the dominant spoilage flora in the vacuum-packed product. dominated the spoilage flora in product with nitrite. and citric acid in the product.. yeasts. 100. The formulated raw meat was stuffed into a casing. In addition. Product with GDL had a pH value of 5. Adding GDL was considered the most important. The level of surviving enterococci decreased as the processing temperature increased (63°C. particularly in the summer. A marginal thermal process (i. cooked. sake tested were able to produce catalase when provided with hematin. chilled. whereas other iron-containing porphyrins (i. all the strains of L.8. the lactic acid bacteria. Asplund et al. and 150 µg/g) retarded microbial growth. (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. and 20°C). myoglobin. 1981). GDL). and 74°C). Grampositive cocci.204 Antimicrobials in Food were considered incapable of synthesizing heme compounds. thermosphacta may also have significantly influenced spoilage of two of the products. 63°C to 68°C) led to the growth of enterococci during storage at 7°C. B. The growth of B. Silla and Simonsen (1985) studied the rate and type of spoilage of four cured meat products packaged under vacuum and in modified atmospheres.3 units lower than with the other variables tested. and processing to 68°C resulted in a shelf life of more than 16 weeks at 5°C. Also. Using good quality raw materials. The spoilage of liver sausage was shown to be the result of S. thermosphacta. cereus can grow in the product even with the normal curing ingredients of salt. and lactic acid bacteria were inhibited to a limited extent. and then vacuum packed. Increasing the level of sodium nitrite (0..

This could be influenced by the fact that white meat is lower in pH and iron content than dark meat. The spoilage rate of this second group of cans was 4%. of naturally occurring spores in fairly large cans of commercial product. even a low level of nitrite (40 µg/g) caused some inhibition in white meat but not in dark meat.5%). the NAS was not able to conclude that nitrate can act as either a carcinogen or mutagen. phosphate. 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. additional cans (8290) were removed from storage at around 8°C and incubated at 37°C for 3 days. The pH of the white and dark meat was 5. high levels of iron (40 to 160 µg/g) increased the efficacy of nitrite. 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. 2. 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. respectively. However. 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. 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. These conclusions meant that neither nitrite nor nitrate would be banned as food additives. When spoilage first occurred in each lot. isoascorbate. pH. 980 cans. and thermal process on botulinal outgrowth. the efficacy of nitrite increased when the pH of dark meat was adjusted to 5. Adding EDTA enhanced the efficacy of nitrite and caused an increase in the lag phase for aerobic microbial growth. The ground meat was aerobically packaged and stored at 4°C to 5°C. The authors also investigated the effect of the inherent pH of the meat on the efficacy of nitrite. whether cooked or raw. psychrotrophic strains of C. From among 4322 cans of 8 different classes of perishable canned cured meats. Perhaps some adjustment occurred in the microbial population.6 and 6. Low levels of iron (5 to 15 µg/g) reduced the efficacy of nitrite. 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. Extensive studies in meat and meat slurries demonstrated the interactions of nitrite.Nitrite 205 meats to a minimum internal temperature of 68. Bushway and Serreze (1984) found that adding iron to raw white meat caused an increase in the rate of aerobic microbial growth. a concept . (1982). These data reflect the influence of a mixture. The antimicrobial effect of nitrite was blocked when the pH of the white meat was adjusted to 6.4.4. 1980). Nitrite was considerably more effective in white meat than in dark meat. The effect of nitrite on the aerobic spoilage flora of raw chicken patties was studied by Bushway et al. The time elapsed was normally 7 to 10 days after production. Likewise.8°C. Bushway and Jen (1984) found lower residual nitrite levels in chicken white meat than in dark meat. 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 effect of iron on aerobic microbial growth depended on the amount of iron added. 3. This led to the development of a model system for predicting botulinal outgrowth in food. Furthermore. or 23%. The reason for this effect on the aerobic spoilage flora was not known. If nitrite (130 µg/g) was also added. This could explain the high number of cases that have occurred during certain periods in Europe (Tompkin. swelled. Bushway and Serreze (1984) also studied the effect of adding EDTA to raw dark chicken meat. Adding EDTA alone had no significant impact on the rate of aerobic microbial growth. 4. Tests demonstrated that nonproteolytic. With the addition of sodium chloride (2. SUMMARY FOR 1980–1990 1. This provided a basis for assessing the risk associated with different cured meats.6.

At the time he stated. This probably reflects the effect of the higher salt levels and nitrite found in cured products compared with noncured products. monocytogenes in tryptose phosphate buffer under a wide variety of conditions (salt. but no evidence was generated to link the Perigo factor to the antimicrobial effect that has been demonstrated in perishable. Some research continued on the Perigo factor. Adding dried plasma. 200. monocytogenes in ready-to-eat foods. 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. in my opinion. The debate over the risk of C..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. The mechanism of nitrite inhibition differs in different bacterial species. 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. The potential impact of various blood fractions as ingredients in sausage was investigated (Miller and Menichello. Sausage formulated without sodium nitrite became toxic within 1 week compared with 3 weeks when nitrite was present. 400 µg/g). 1974). and the addition of plasma delayed the time for toxigenesis compared to the control sausage (Miller et al. Other available options have not received commercial acceptance. however. or whole blood reduced the time for botulinal outgrowth and toxin formation compared with the control with nitrite. 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. 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. At pH values of 6. Buchanan and Phillips (1990) studied the growth of L. but none have been adopted commercially. cured products that receive no heat treatment or are cooked to final internal temperatures in the range of 60°C to 75°C. 6. very slow growth did occur. Again. red blood cells. is not more inoculated pack experiments but a rationale for interpreting them” (Ingram. Adding hemoglobin. Despite this evidence. Autoclaving the sodium nitrite in the medium did not increase the degree of inhibition against L. Alternatives to nitrite continued to be investigated. 1993). pH. (1991). SINCE 1990 C. delayed the time for toxin to be detected. “What we need at the present time. monocytogenes to the degree that . 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. temperature. In the absence of nitrite. McClure et al. 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. 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. 1991). 8. 7.3. that has been expanded to include other pathogens and has gained acceptance throughout the world. Since 1990 there has been increased interest in control of L. 100.206 Antimicrobials in Food 5.0 and 5°C no growth was observed with any of the levels of sodium nitrite evaluated (50. using an automated tubidimetric system with multiwelled plates containing trypticase soy broth. At pH 6. the rate of toxin formation was inversely related to the level of iron. aerobic/anaerobic conditions) and found sodium nitrite to interact with combinations of the factors tested. In subsequent tests the effect of various decolorized blood fractions was evaluated. The database was incorporated into the USDA Pathogen Modeling Program. At 20°C and below no visible growth occurred even with 50 µg/g sodium nitrite and a pH of 5. found the efficacy of sodium nitrite was temperature and pH dependent.

sodium erythorbate (0. L. The effect of salt and nitrite on the growth of 24 lactics isolated from vacuum-packed cooked sausage was evaluated by Korkeala et al. Buchanan et al. heat. inoculum level. Applying the procedures described by Hauschild (1982).Nitrite 207 had been observed with clostridia (see Perigo Factor). sausages made with nitrite and having pH 5. Liver paté has been implicated in outbreaks of listeriosis in the United Kingdom. Since about 1990.7. and other factors for inactivating spores and preventing the outgrowth of survivors. 3%). McClure et al. Leuconostoc isolates seemed more sensitive to the effects of sodium nitrite and salt in comparison to the homofermentative lactobacilli. As in the case of earlier research with C. they estimated the various formulations and thermal processes provided the equivalent of 4 to 7 log10 destruction of C. 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. although sodium nitrite concentration was not varied. the effectiveness of sodium nitrite was significantly increased by the addition of sodium ascorbate. Fermented Meats and Dry Cured Hams Although removed from most cured meat products in the United States during the 1970s. but levels above 3% were inhibitory. Farber et al.2 with 200 µg/g. 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. smoked flavor can be found. and Enterococcus faecalis. and sodium nitrite in laboratory medium and a predictive model for the growth of L. sporogenes. 1997). respectively. and the United States.7 with 50 µg/g). Duffy et al. Australia. viridescens. Low levels of salt (1% to 2%) enhanced growth. Three µM of residual undissociated nitrite doubled the time taken for a 3 log10 increase in numbers. 550 µg/g). F0 = 0. botulinum. The procedures described show promise for estimating and comparing the combined effects of multiple treatments on pathogen survival and growth. (1995) examined the effect of sodium nitrite (0 and 200 ppm). and 15 days for L. monocytogenes by sodium nitrite is pH dependent and related to the concentration of undissociated nitrous acid (Buchanan and Golden. and temperature of storage (4°C. monocytogenes in the sliced vacuumpackaged product when stored at 4°C or 8°C. a combination of heat and sodium nitrite was necessary to prevent survival and growth (F0 = 0. An extensive series of experiments led to the conclusion that nonthermal inactivation of L. as had been reported earlier for C. 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. 10°C) on the rate of growth of L. If the broth had been adjusted to pH 6. 1992b).5% sodium chloride and 50 or 200 µg/g sodium nitrite inhibited growth during 125 days storage at 37°C. some .0 and a sour. 4. and additives to achieve shelf stability of canned luncheon meat (Farkas and Andrássy. 1995.. Perhaps the high iron content of liver pâté negated the effect of sodium nitrite and sodium erythorbate in this product. (1996) subsequently investigated the effects of salt. (1997) examined the effect of nitrite on three lactics in vacuum-packed cooked sausage made with and without nitrite. (1992).8 and containing 3. Sameshima et al. monocytogenes. salt (1%. Only temperature proved to be a significant factor. 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). they found that broth adjusted to pH 5. monocytogenes in liver pâté. Farkas and Andrássy (1992a) described a test system to evaluate the combined effects of formulation. botulinum. sporogenes (9 × 105/ml) in reinforced clostridial medium. In regions of Northern Europe. Using C. temperature. irradiation. mesenteroides. pH. The model test system was then used to evaluate various combinations of heat. 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. (1994) inoculated a variety of vacuum-packaged cooked sliced meats with L.

1994. Flores. (1994) found that sodium nitrite (80 µg/g) reduced the catalase activity of a strain of L. 1990. It is curious that very little information has been published during that period from researchers in North America. pentosus. consisting of L. In France. was hematin dependent for nitrite reduction and yielded ammonia as the sole product. High-Acid Sausage 22°C –25°C 15°C –18°C <5. One group.. Nychas and Arkoudelos. Tests by Mares et al. The basic parameters of raw fermented sausage processing in Europe are summarized from Incze (1992) in Table 6. Subsequent research found the lactic acid bacteria could be separated into heme-dependent and nonhemedependent catalase producers (Wolf et al. pentosus. Lücke. these traditional raw fermented sausages have had higher pH values (e. perhaps. In these processes micrococci convert the nitrate to nitrite. incapable of reducing nitrate or influencing flavor development. published since 1987. and 100 µg/g) in salami. 1989. strongly acidic flavor. Several reviews. Hungary. acidilactici or L. loss of the traditional flavor and increased acidity has led to a decrease in consumer acceptance and declining sales. Spain. Italy. L. 1987. and Pediococcus pentosaceus.. >5. Flores and Bermell. Tests on 70 strains of lactic acid bacteria using cell suspensions incubated anaerobically led to two groups.7% Nitrite and.. the rapid production of acid renders the cocci inactive and.3 0. In Europe. 1996.g. firm texture manufacturers in the Mediterranean countries have been shifting away from the more traditional. Scannell et al. and nonacidic. softer. Nagy et al. complex.208 Antimicrobials in Food TABLE 6. 50. The trend toward using starter cultures and slightly higher fermentation temperatures to reduce processing times is accompanied by certain negative features. rich. thus. nitrate has traditionally been used for dry sausages and hams requiring long ripening times. Wolf et al. The cocci also play an important role in flavor and aroma development.3°C –0. Specifically. 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. In Spain. rubbery or spreadable texture Low-Acid Sausage <12°C 14°C –16°C ~ 6.. nonacidic flavor. the rate of fermentation is faster and micrococci become inactive as the result of an accumulation of acid. plantarum. nitrate Yes Salty. A few of the studies pertaining to the manufacture of fermented meats will be summarized. 1997. and the Balkan countries. and 1992. (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 (20.2 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). In studies involving faster fermentation. more than 50% of the initial nitrite disappearance was attributed to conversion to . describe the procedures used and changes occurring in Europe (Incze. In pursuit of more effective starter cultures for use in manufacturing dry sausage. plantarum but not P. round. Fernandez et al. 1991).0 None Nitrate Not necessary Salty.2. 1989. (1990) conducted further investigations on nitrite reductase activity among the lactic acid bacteria. In more modern processes. which is responsible for the reddening and other effects. 2001).8) and flavors that are more balanced. The other group was able to reduce nitrite in the absence of hematin and yielded nitric oxide and N2O.

1998). Of the ten strains selected for further study. 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. Adding nitrate to the growth medium favored the synthesis of catalase. which may occur aerobically or anaerobically. caseolyticus isolated from fresh pork were able to convert metmyoglobin to red myoglobin derivatives. 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. Morita et al. 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.. 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. In assimilation. xylosus and found it to produce a red myoglobin derivative in the same meat system without any added nitrate or nitrite. A study of cell suspensions from seven strains of staphylococci isolated from dry sausage (six) and cheese (one) found all but one (S. . (1998) then evaluated a strain of S. nitrate is used as an alternative electron acceptor when oxygen is not present. are able to perform assimilatory nitrate reduction but cannot use nitrate as an electron acceptor. warneri. but nitrate alone is more common (Sanz et al. Spectral analysis and electron spin resonance studies determined the pigment was nitrosomyoglobin. Other bacteria. nitrate is taken up. Second. nitrate is reduced to ammonia that is then incorporated into the cell and used as a source of nitrogen. carnosus. Morita and coworkers investigated the red pigment found in Parma ham and its formation. in both... which may be further reduced to nitrogen. which is used commercially as starter cultures for fermented sausages and dry cured ham. Neubauer and Götz (1996) investigated the nitrate reducing system of Staphylococcus carnosus. carnosus and S. S. 1993). Morita et al. epidermidis. Members of Enterobacteriaceae reduce nitrate to nitrite. and S. 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. In contrast. Obligately respiring bacteria (e. the red pigment that is associated with products made with nitrate or nitrite.g. 1992). all were staphylococci (S. and Savoie ham made in France involve long ripening times and are made without any added nitrate or nitrite (Leistner. which is then either excreted or further reduced to ammonia. Two main forms have been described for respiration and. certain other bacteria use both assimilatory and respiratory nitrate reduction. 1986). and excreted. respectively. Color stability was mainly affected by the presence of residual added ascorbate (Alley et al. pseudomonads) reduce nitrate to gaseous oxides of nitric oxide and nitrous oxide. Morita et al. The mechanism for the formation of the pigment in the absence of nitrate and nitrite was not determined. warneri) was able to synthesize nitrate reductase (Talon et al. Certain bacteria. such as S.. Parma and San Daniele hams made in Italy. Subsequently. In respiration. First. Microbiological examination of 471 isolates from Parma ham found the majority were able to convert metmyoglobin to a red myoglobin derivative. S. lentus). Synthesis of the nitrate and nitrite reductases is inhibited by oxygen and induced to greater or lesser degrees by nitrate or nitrite. after nitrate is depleted the accumulated nitrite is taken up and reduced to ammonia. reduced to nitrite. Karst ham made in Yugoslavia. particularly in S.Nitrite 209 nitrate. do not assimilate nitrogen through nitrate reduction during aerobic growth but grow anaerobically with nitrate as the terminal acceptor. the pigment found in hams made with nitrate and/or nitrite. 1999). Neubauer and Götz (1996) summarized nitrate utilization as being either assimilation or respiration. carnosus reduces nitrate to ammonia in two steps. Typhimurium.(1994) found that S. such as strict aerobes. Tests with two strains of S.. They found S. (1996) demonstrated that the red pigment found in Parma ham was much more stable and different from nitrosomyoglobin.

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

depending on the conditions of processing. 1987. In many countries there is no legal requirement for the minimum amount of salt. First. and chub. This may reflect survival or contamination. sodium nitrite could be used as an additional control measure. In France. Following a review of the literature. a lower salt content is permitted (i. a panel of experts reached the following consensus for its report to the FDA (Gram. Adding potassium nitrate at 347 or 686 µg/g was equal to or more effective than sodium nitrite. that combination of salt and temperature. toxin was detected in 3 to 4 weeks.000 tons shipped internationally from 28 exporting countries to 52 importing countries (Duffes. In 1992 more than 32. Cuppett et al. It is possible. 1982.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. brined shrimp). The addition of sodium nitrite is limited to smoked salmon. if necessary. it is highly unlikely that sodium nitrite will be adopted as an additive in smoked fish in Europe. 2001b). with a production of 12. although a minimum of 3% salt in the water phase is strongly recommended (Huss et al.6% (Duffes. allowing for short periods of elevated temperatures up to 10°C. and with refrigeration this combination is effective in inhibiting growth of C. particularly in combination with controlled levels of salt (Pelroy et al. smoked fish to contain at least 3.5% salt in the water phase). monocytogenes (Gram. botulinum would be further inhibited. 2. sablefish. The addition of nitrite or nitrate increased the time the fish was considered acceptable by 3 or 4 weeks at 4°C.4°C) for at least 4 weeks..0% salt if combined with 200 µg/g sodium nitrite (Gram. although the numbers. 2001a): • • Psychrotrophic C.e. . 1999). Two options are available in the event commercially manufactured product is implicated in botulism. it would appear the risk of botulism is lower than the research has suggested. A level of 3% salt in the water phase will yield perfectly acceptable. Furthermore. a minimum concentration for salt in the water phase can be established. Analysis demonstrated that nitrite was produced during storage. but. but the panel did not consider the more recent risk assessments demonstrating the safety of dietary nitrite (see section on toxicology).5% in the water phase) and cold storage (<4. federal regulations were adopted requiring vacuumpacked. If packaged aerobically. if present. Controlling the salt in the water phase and temperature should be adequate. whereas with salt alone. 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. 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. Until there is evidence of a need.. 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.. shad. botulinum can be expected to occur in smoked fish. gravad fish.. will prevent toxin production in cold smoked fish for several weeks beyond its sensory shelf life when packaged under reduced oxygen conditions. however. The panel expressed concern for the potential carcinogenic effects of nitrosamines.. 1982.. 1997). 1987). are low. 1995).380 tons of cold smoked salmon in 1997. 2001a). monocytogenes is common in smoked fish. 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. the salt in the water phase ranged from 2% to 4. 1997). Surveys conducted before the year 2000 indicate L.5% salt in the water phase or 3. Toxin production can be controlled with a combination of a moderate level of salt (3. lightly preserved fish products (cold smoked fish. Cuppett et al. NOCs were not detected in two studies in which nitrite had been added to smoked fish (Pelroy et al. botulinum (Huss. Hyytiä et al.. however. 1999).

Cans inoculated with E. perfringens swelled within 2 days at 37°C. and held at room temperature. and 45 µg/g of sodium nitrate. Very erratic growth occurred on bologna at 18°C. Typhimurium in vacuum-packaged luncheon meats. Because of the relatively high tolerance of L. 1994a). 14 µg/g of sodium nitrite. Goepfert and Chung (1970) inoculated the surface of sliced bologna and liver cheese with S. Growth occurred in both products.. 1993. Mock chicken supported the best growth at both temperatures tested (24°C and 37°C). there was a gradual decline. the pH of mock chicken . Typhi remained after 20 months at 27°C to 38°C.5 to 11.. Kittaka and Greenberg (personal communication) obtained similar results in corned beef inoculated with three different S. Davidson and Webb (1973) also studied the growth of S. Pelroy et al. it uses the nitrate in the product in place of oxygen as an alternative hydrogen acceptor. At 5°C. nitrite accelerated the death of all four strains. Typhi has a nitrate reductase. 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. increasing the salt concentration above the normal range for smoked fish is an unacceptable option for control. In subsequent research. (1994b) found the addition of sodium nitrite (190 to 200 µg/g) to be very effective in slowing the growth of L. Viable S. one domestic and one imported. Typhimurium or Salmonella anatum.. tests were not performed to show whether the presence of nitrate influenced growth. and 720 µg/g of sodium nitrate at the time of opening and inoculation. 1994.212 Antimicrobials in Food with strict adherence to good manufacturing practices. The inocula died in wieners at both 18°C and 24°C. rewrapped in Saran™. Peterson et al. Typhimurium occurred more rapidly and to higher levels when the packages were opened. 15 µg/g of sodium nitrite. monocytogenes in salmon containing 3% salt in the water phase. because S. Similar results were obtained on ham. S. were used for testing. Survival of the inoculum in product held at 7°C varied with the product and inoculum level. (1993) found the rate of growth to be similar at 5°C for salt concentrations of 3% to 5% in the water phase. The domestic product contained 4. It is reasonable to assume the products were recently produced. This combination was more effective when a low inoculum level (10 cfu/g) was used as a challenge. 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. Typhi strains. Previous studies had found levels of 0. coli remained flat. Pelroy et al.6% salt. but a steady increase in numbers occurred at 24°C. The authors implied that. Anatum grew in bologna but not in liver cheese. After reaching 107/g. although not before some initial growth at 24°C. The initial level of contamination is an important factor influencing growth (Guyer and Jemmi. Sodium lactate (2%.13% brine. Dehydration prevented growth if the packages were opened and left exposed at room temperature. Peterson et al. Typhi in inoculated canned corned beef stored at room temperature and at 37°C. A lower maximum population (106 versus 108/g) and slower die-off occurred in the imported product. Typhimurium grew in both vacuumpackaged products when held at room temperature. the imported product had 4. including an isolate from the 1964 Aberdeen outbreak involving canned corned beef. In both products. to produce cold smoked salmon with low levels.7 cfu/g in positive samples of cold smoked fish from five plants. Chemical data were not provided for either luncheon meat. often less than 1 cfu/g and at a low prevalence. 2000).26% brine. nor were analytical data presented for the product tested. Rørvik. Meers and Goode (1965) reported the rapid growth of S. The growth of S. w/w) was much more effective and prevented growth through 50 days at 5°C when used in combination with 3% salt. Product from two sources. 1991. Typhimurium declined during the first 2 weeks at 5°C and then remained stable through 6 weeks. S. At the end of storage. however. cans inoculated with C. S. however. viable cells remained even after 6 months of storage. 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. monocytogenes to salt and the adverse effect on flavor.

Fermented sausage (teewurst) was prepared with four sodium nitrite levels (0. that of the ham was near 7. other than to say that fresh. whether the nitrite was added before or after autoclaving the medium of Perigo et al. The primary factors influencing growth were the temperature of storage and growth of competitive flora. commercially produced sliced ham and chopped ham did support the growth of the pathogens studied. aureus. coli. coli. and ammonia as the sole nitrogen source in a chemically defined minimal medium. A low-brine ham was prepared with 1. but pH changes were noted.0 by 160 µg/g of sodium nitrite. The pH of the chopped ham with 70% and 59% moisture decreased to 5. Nitrite and ammonia were assimilated at comparable rates. It is not possible to assess the influence of nitrite from the data. 65. Stiles and Ng (1979) studied ham and chopped ham obtained fresh from two processors.8% to 2. 50.000/g). The products were inoculated with five “enteropathogens” (C. (1967). 4 of 5 strains of Salmonella grew readily. respectively. The authors did not state whether the nitrite was added before or after autoclaving the medium.2% salt and 60 µg/g of potassium nitrite and a finished brine level of about 2. (1979) tested the same bacteria on sliced bologna from two processors. No other chemical data were provided. A similar response occurred with enteropathogenic E. pH values were followed through storage.6 to 6. Typhimurium was inhibited at pH 6. 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 6.5. but a mixture of Enterobacter. A high-brine ham had a brine level of about 6%. and Hafnia strains readily multiplied at all nitrite levels. The growth of Klebsiella and Enterobacter species on vacuum-packaged luncheon meats at 3°C was reported earlier by Hechelmann et al. Typhimurium.7 to 4. Enteritidis were then used to study the effect of nitrite and salt on growth in nutrient broth at 22°C. Aside from temperature of storage. The resistance of Salmonella to nitrite in culture media was demonstrated by Roberts and Garcia (1973). Leistner et al.58. In the high-brine ham held at 22°C.0. growth was reduced by only about 30%. At a high pH of 7.0 and 5% salt. the primary factor influencing growth was product pH. Castellani and Niven (1955) reported S. perfringens did not occur under any condition. The trend in the United Kingdom toward using less salt for processing ham was studied by Akman and Park (1974).47 and 5. nitrite values were not given. given the proper circumstances. E.0. Although salt and nitrite values were not reported. (1974).0. The rate of growth or death was influenced by the sodium nitrite content (0. Again. and B. and 100 µg/g) and the level of inoculum (100 or 100. and the pH for the wieners was 4. Stiles et al. (1973) inoculated vacuum-packaged luncheon meat with a mixture of Salmonella strains and held the product at 8°C for 22 days.8%. Klebsiella. The mean pH for the ham during storage decreased to no lower than 5. nitrite. viable counts of the 5 strains remained near the inoculum level. 98. S. The product from one supplier remained above pH 6.7. Under anaerobic conditions.000). nitrite was without effect.Nitrite 213 and bologna was about 6. Typhimurium to utilize nitrate. and 130 µg/g) and two Salmonella inoculum levels (100 and 100. the fastest generation times occurred in . Two strains of S. and 100 µg/g of sodium nitrite. including Salmonella. that of the chopped hams was 70% and 59%. Neither of two strains of S. This probably reflects the quantity of fermentable carbohydrate added to the product and/or the nature of the competitive flora. Page and Solberg (1980) found S. The moisture content of the hams was 70%. Death occurred after 3 days in the sausage with the lower inoculum and 130 µg/g of nitrite. Growth occurred in the sausage containing 0 and 65 µg/g of nitrite. It was concluded that cooked ham is a more likely source of salmonellosis if prepared with a low salt content. cereus) and then vacuum packaged. 75. All the enteropathogens grew in one or more of the products within 24 hours at 30°C. Salmonella Enteritidis was able to grow at 17°C but not at 10°C. In the low-brine ham at 22°C.0.8. Growth of C.78. whereas the other frequently decreased to below pH 5. 5% salt. S. no data were provided for brine or nitrite levels. Both genera were prevalent in commercially produced luncheon meats and fermented sausages. perfringens. The other four species were able to grow under certain conditions. At a low pH of 5.

Schillinger and Lücke (1989) measured Salmonella growth and/or survival in mettwurst. 100. coli. the effect of added phosphate was relatively minor. sporogenes. Rice and Pierson (1982) tested the effect of sodium nitrite (0. Hughes and McDermott (1989) used a procedure similar to that of Gibson and Roberts (1986a.8 versus 6. Typhimurium to be relatively resistant to sodium nitrite across the ranges of salt and pH values found in nonfermented and acidulated foods. 27°C. and the presence of lactic acid-producing bacteria. vacuum packaged. a cured raw spreadable sausage. Similar results were observed with S.2. 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. complete inhibition occurred. and incubated at 15°C or 27°C for 21 days. S. and 156 µg/g) in frankfurters on a mixture of two strains of Salmonella. (1991). 1991). 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). 50. This research was primarily concerned with preventing the growth of Salmonella. Collins-Thompson et al. The phosphates generally delayed the outgrowth of E. coli. and 6. The presence of nitrite did not influence the survival of salmonellae. and 100 µg/g sodium nitrite. (1985) identified a nitrite reductase in the cytoplasmic fraction of S. Gibson and Roberts (1986a) examined the interaction of salt (1% to 10% wt/vol).6 to 5. the inherent pH of the meat (normal pH 5. aureus and C. (1984) prepared fermented sausages with sodium nitrite (0.6.8. (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.8 and held at 24°C independent of sodium nitrite concentration. At pH 5. and 200 µg/g) and three strains of Salmonella. Isolates of Enterobacter and Serratia species were tolerant to nitrite.b) to determine the additional effect of eight different phosphates of E.8). pH (5.0. 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. 1977).0 to 6. None of the variables tested would likely assure the destruction of Salmonella in this product category. and 37°C (McClure and Roberts. Nitrite was only inhibitory with the highest level of nitrite and when the franks were incubated at 15°C. S. 6. coli was also apparent in tests involving two-dimensional gradient plates incubated at 20°C. 150. using a gradient gel plate system. but growth was delayed with 200 µg/g (Turanta and Ünlütürk.214 Antimicrobials in Food media containing both nitrate and ammonia. The presence of nitrite had no effect on aerobically grown cultures. coli. the addition of sugar. Factors influencing Salmonella growth in mettwurst that was cured and stored at 20°C include the amount of nitrite and salt.2). Typhimurium had shorter generation times and increased cell yields compared with nitrite-free cultures. At concentrations of 500 µg/g and higher. Typhimurium when grown anaerobically with nitrite as the sole nitrogen source. 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. Growth did not occur during fermentation or drying. 50. Collins-Thompson et al. found S. Typhimurium used nitrite as the sole nitrogen source at concentrations as high as 400 µg/g. Thomas et al. 50. When examined after 10 weeks of incubation. The reductase required NADH and was most active at pH 8. When grown anaerobically in a glucose-limited minimal medium with peptone and nitrite. Typhimurium numbers decreased in BHI broth adjusted to pH 4. He recommended that a starter culture and/or gluconolactone be added to provide optimum safety. 1987). except when the cultures were incubated at the lowest temperature tested (10°C). being able to use nitrite in broth culture under anaerobic conditions at 5°C. Page et al. The nitrite resistance of E. The franks were inoculated on the surface. In a traditional Turkish dry sausage that is fermented and dried at temperatures . numbers increased during 72 hours in the presence of 0.

1996).5. salt level. and incubation temperature (Chou and Tsai. The effect of sodium nitrite (0.5) and enhanced by lowering the incubation temperature (Buchanan and Bagi. and temperature. Inhibition was much greater at 28ºC than at 37ºC (Zaika et al. This is not true for the clostridia. Another study involving anaerobic conditions yielded similar trends (Zaika et al. the inhibitory effect of smoke. Riordan et al. Raccach and Henningsen (1997) reported sodium nitrite and salt to act synergistically in the reduction of Y. nitrite.. storage temperature. 1994). but there is no published information that nitrite or nitrate inhibits their growth.5% salt and 100 µg/g sodium nitrite. coli O157:H7 (Yu and Chou. That cured meats have been implicated in outbreaks of salmonellosis supports the available research that Salmonella can survive. These factors include water activity. pH 5.0. Preventing salmonellosis from cured meats is best accomplished by means other than relying on the level of nitrite in the product.g. 1994 and 1995). S. The data indicate that the nitrite content of cured meats can influence the growth and survival of Salmonella and E.8. 1993). enterocolitica when tested in a meat system.. The inhibitory effect of nitrite was much greater at pH 5. the addition of sodium nitrite (0. The highest survival involved a starter culture that produced the least amount of acid and yielded a relatively high final pH of 5. Inhibition of Shigella flexneri in broth incubated aerobically was significantly influenced by the combined effect of nitrite concentration. 1994). The effect of nitrite in Chinese-style sausage during drying at 50ºC to 60ºC for different time periods (2. (1998) found E. a fermented noncooked beef sausage.. coli O157:H7 when lower temperature and/or shorter time combinations (e. coli. This sausage is not fermented or cooked and relies on relatively rapid dehydration to achieve microbial stability. They found some increased death of E. the lowest pH tested and with increasing salt levels. 1997). 1996). and 120 µg/g) on Y. Inhibition of E. coli O157:H7 (Chikthimmah et al. coli O157:H7 in a broth system containing sodium nitrite was strongly pH dependent (i.Nitrite 215 not exceeding 24°C. Typhimurium was not affected by the addition of 200 µg/g sodium nitrite (Turanta and Ünlütürk.7 × 105/g to nondetectable levels by 35 days in the sausages formulated with 80 µg/g nitrite. Inhibition of Yersinia enterocolitica in broth incubated aerobically was significantly influenced by the combined effect of nitrite concentration. 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..5 to 6 hours) was next studied (Yu and Chou. if not multiply. 1996. 78. fermented to pH 4.5. and. salt level. in a variety of commercially cured products. the availability of oxygen.. Pin et al. coli O157:H7 at 37ºC were inhibited by 200 µg/g sodium nitrite at pH 5. 55ºC for 4 hours) were used. Effect of Nitrite on Yeasts and Molds A wide variety of yeasts and molds have been isolated from commercially cured meats. and then dried for 7 days. but even 1 g/L sodium nitrite was ineffective at higher pH values (Tsai and Chou. coli O157:H7 numbers decreased less than 1 log10 in pepperoni formulated with 2. 1996). (1993). The inoculum decreased from 1.e. 1991). and 156 µg/g) had no effect on the death rate of E.. pH. where . Typhimurium has a nitrate reductase to reduce nitrate to nitrite and can assimilate inorganic nitrogen from nitrate. 100. and temperature (Bhaduri et al.. In Lebanon bologna. 50. Adding sodium nitrite did not increase death of E. 80. pH. Inhibition of Aeromonas in broth has been found to be highly dependent on nitrite concentration. 2001). The data for Salmonella can be summarized as follows. particularly because salmonellae are destroyed during the manufacturing process for most cured meats. enterocolitica 0:3 to grow and survive during the manufacture of raw dry fermented sausage was examined by Asplund et al. sodium nitrite at 70 to 150 µg/g did not affect the growth or survival of E. pH. the death rate of S. In ground pork held at 5ºC or 25ºC. In tryptic soy broth 4 strains of E. or ammonia under anaerobic conditions. coli O157:H7 with the addition of curing agents and drying at time–temperature combinations that appear to cause stress of the inoculum.

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

172. A unique feature of the U. Germany reduced the permitted level of nitrite in nitrite-containing curing salt by 20% (Leistner. Other countries limit the amount of nitrite and nitrate on the basis of the total weight of the product formulation.S. The use of nitrite and nitrate in the processing of fish products is specified in 21 CFR 172. With the exception of bacon and smoked chubs.21-23 of the federal regulations (Code of Federal Regulations. 2002a). Nitrite is of relatively little value for controlling Gram-negative enteric pathogens in commercially prepared foods. U. 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. Braathen. The status of nitrite and nitrate in cured meats and cheese in the United Kingdom was reviewed in 1978 (Ministry of Agriculture. 1974. 1978). 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.175. This further reduces the nitrite level in the product formulation and.Nitrite 217 7. The regulations were summarized in two comprehensive surveys (Meester. 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. As the percentage of extenders and other nonmeat ingredients is increased in the products.S. The most recent summary of the regulations in different countries appears in Cassens (1990). the level of added nitrite is decreased. as proposed by the Expert Panel on Nitrites and Nitrosamines (Food Safety and Quality Service. 1978). 1981. regulations do not specify minimum nitrite levels for cured products. 1986). Gerhardt. The net effect of the U. 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.3.170. leads to lower residual nitrite levels after processing. and 172. 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 weight of this meat is subtracted from the fresh meat portion of the formulation and the amount of nitrite added is reduced accordingly.17 and 424. 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. the method of assay applicable to each country should be used. 1995). and Food. 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. 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.160. An example of rework is the round ends of sausages that are trimmed off before slicing the product. 1979). Because it has already been cured.177 (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 . The current USDA regulations controlling the use of nitrite and nitrate in meat and poultry products are specified in 9 CFR 317.S. 9. Fisheries. The use of nitrate or nitrite as additives in cheese and seafood products was clarified. meat and poultry regulations is that nitrite and nitrate are added on the basis of the uncured meat in the product formulation. 172. 2002b). ASSAY METHOD Because the levels of nitrite and nitrate permitted in foods are established by regulation. 8.

2002). estimates for the lethal dose have ranged from 2 to 9 g (about 33 to 250 mg/kg body weight). with the lower doses applying to infants who are more sensitive to methemoglobinemia. (AOAC international.06 mg/kg body weight. 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.7 and 0 to 0.. 1994). respectively (JECFA. Source: Based on formulations in Pearson and Tauber (1974). Cyanosis. The normal range of methemoglobin levels is 0. the criterion most commonly used for nitrite toxicity. 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 . Two years of rat studies on nitrate showed no evidence of carcinogenicity.3 mg sodium nitrite/kg body weight (Gangolli et al. 1994). For sodium nitrite. and it appears that nitrate per se is not genotoxic.5% to 2. TOXICOLOGY The acceptable daily intake for potassium nitrate and sodium nitrite have been reported to be 0 to 3. International agreement has been reached on the specifications and analytical methods for potassium nitrate and sodium nitrite (JECFA.. Data on reproductive toxicity for nitrate were limited. nonfat dry milk. 1995).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. 1994). whereas 30 to 60 g of sodium nitrate have been given for 2 months as an acidifying diuretic without manifestation of adverse effects. The no-adverseeffect dose level was estimated to be 2500 mg sodium nitrate/kg body weight/day (Gangolli et al.218 Antimicrobials in Food TABLE 6. estimates for the toxic dose range from 1 to 8. respectively. The values are expressed as nitrate and nitrite ions. 1995). occurs at methemoglobin levels in excess of 10%.. Using this criterion. estimates of the lethal dose have ranged from 4 to 30 g (about 70 to 500 mg/kg body weight). For potassium nitrate. as evidence of toxicity.0%. and apply to all sources of intake but do not apply to infants younger than age 3 months. the uppermost level being found in infants and pregnant women.

An assessment of NOCs by Hotchkiss et al. Sodium nitrite is now viewed in an entirely different light than during 1970 to 1990 (CAST. Gangolli et al. 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). 1981. nitrite. For humans the greatest exposure to NDMA is from tobacco smoke (Koppang. 1500. Assessments conducted during the past decade have led to the conclusion that nitrite. not to list sodium nitrite as a developmental toxicant under the state’s Proposition 65 law. 1996). 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%. An extensive review was conducted during the development of two reports by the NAS (Assembly of Life Sciences.Nitrite 219 and sheep. Endogenous synthesis of nitrate is an additional important source of nitrate. particularly among young children. Gangolli et al. human effects. per se. Several countries that monitor their foods for nitrate. summarizing studies conducted over a 2-year period on the carcinogenesis and genetic toxicology of sodium nitrite in drinking water. Murphy et al. 1998). More recently. 1982). Investigations for NDMA in foods led to cured meats. 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. including a review of epidemiologic studies seeking to establish a link between intake of nitrate and nitrite from food and water and cancer. Cassens (1995) has provided an update of the nitrite controversy. The report concluded that there was no evidence of carcinogenic activity for sodium nitrite in rats and mice exposed to 750. 1997). or 3000 µg/ml. an extensive review of the literature and testimony from experts led the California Developmental and Reproductive Toxicant Identification Committee to vote on June 2. and NOCs have concluded that dietary nitrite and NOCs represent a relatively small contribution to the total body burden of these compounds (Cassens. 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. 2000. and NOC and determined that vegetables provide more than 85% of the average daily human dietary intake of nitrate. Those reports provide the most complete assessment of the safety of nitrite as a food additive for that period. Even the American Cancer Society has concluded that “nitrites in foods are not a significant cause of cancer among Americans” (American Cancer Society. For example. This was followed by the final report from the National Toxicology Program in May 2001. 1999). is not carcinogenic. In addition. In 1963 it was proposed that nitrosodimethylamine formed during the herring meal process was the toxic agent. A review of animal toxicologic studies. 1995). Controls adopted by the food industry over the past 20 years have led to reductions in the risk of exposure to nitrosamines (Lijinsky. Endogenous formation of NOCs is influenced by dietary intake and a variety of other factors. (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. (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. 1997). nitrite. (1994) conducted a comprehensive risk assessment on nitrate. and beer from exposing the malt to nitrogen oxides. (1994) concluded that dietary sources of nitrite and NOCs contribute relatively small amounts to the body burden. There was equivocal evidence of carcinogenic activity in the forestomach of the female mice tested (National Toxicology Program. The major source of these compounds in the body is derived . 2001). 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. especially bacon when prepared and cooked under certain conditions.

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

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

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

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

DHA. 1985). this would usually be the result of poor-quality ingredients (Gibbs and Hurst.. Nisin is available commercially (e.1. 1976.. 1994). 2000). yeasts. It is classified as a Class I bacteriocin. The structure of nisin A. lanthionine.. molecules characterized by their unusual amino acids. nisin offers the possibility (within safety limits) of energy saving and consequent cost cutting. CHEMICAL AND PHYSICAL PROPERTIES The polypeptide can be prepared from culture fluids or the cells of the producer organism. Lee and Kim. three-dimensional structure.. as well as amino butyric acid (Abu). 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. Lipinska et al. Gross and Morell (1971) first elucidated the unusual structure of nisin. It can form dimers or even oligomers. 1991). The net charge of nisin is pH-dependent. Wilimowska-Pelc et al. Methods for its concentration and purification have been described (Cheeseman and Berridge. 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. It is amphipathic in character. Nisin will be less effective in a processed food that has a high spore count. Nisin contains the thioether amino acids lanthionine (Ala-S-Ala) and β-methyllanthionine (Abu-S-Ala). dehydroalanine (Dha). 1994. 1964). is shown in Figure 7. which is determined by its internal thioether rings. 1981b). 1988).. also known as β-methyldehydroalanine). the N-terminal contains several hydrophobic residues. The structure and synthesis of nisin have been reviewed (de Vuyst and Vandamme. and its synthetic production has been accomplished (Fukase et al. dehydroalanine. Apart from the advantages already mentioned. The natural variant nisin Z differs by a substitution of histidine for asparagine at position 27 (Mulders et al. dehydrobutyrine (β-methyldehydroalanine). aminobutyric acid. but this activity was highly dependent on pH and nisin concentration (Bani-Jaber et al. . The effectiveness of nisin is also dependent on the bacterial load (Scott and Taylor. ALA-S-ALA.. ABU. whereas the C-terminal is more hydrophilic. and dehydrobutyrine (Dhb. Kupke and Gotz. Nisin has a flexible. Nisaplin® produced by Danisco). which is a 34-amino acid polypeptide with a molecular mass of 3510 Daltons. with its distinctive five internal ring structures formed by the disulphide bridges. 1996). and molds. 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). 1973.g. 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. β-methyllanthionine. 1971.1 The structure of nisin.Nisin 239 Regulatory authorities and food producers acknowledge and appreciate that nisin does not hide poor manufacturing practice. It does not contain aspartate or glutamate. 1957. and extended shelf life can be achieved with a combination of nisin and a reduced heat treatment. Bailey and Hurst. Thermally injured spores are more sensitive to nisin. ABU-S-ALA. Nisin is not effective against Gram-negative bacteria. DHB.

1 to 6.25 mg/ml at pH 8. is the same value as the Reading Unit. Nisin is. Nisin is extracted from the test food by an acid-heat treatment. The nisin preparation Nisaplin® was developed by Aplin & Barrett (now Danisco) between 1962 and 1965. first developed by Tramer and Fowler (1964) and Fowler et al. In triangle taste trials conducted by Danisco. Liu and Hansen (1990) reported nisin solubility at pH 2. IU/g). research in Danisco laboratories has shown that care must be taken when preparing high-concentration nisin solutions from commercial preparations such as Nisaplin®.25% biologically active nisin.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. At high pH. International Unit. suggesting that the molecule is protected to some extent by food components (Delves-Broughton. 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. 1966). In this chapter nisin is expressed as levels of pure nisin (µg/ml or µg/g). dropping to 1.2 as approximately 56 mg/ml. To convert these units to equivalent Nisaplin® levels (mg/L. The horizontal agar diffusion test. 1 µg/g pure nisin is equivalent to 40 IU/g or 40 mg Nisaplin®/kg. Nisin has no significant taste and imparts no negative taste to foods in which it is used. and nisin activity was then defined in International Units.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. with levels of usage in food rarely above 0.5. mg/kg) or to International (Reading) Units (IU/ml.5. 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. Solubility in foods is never a problem because nisin levels are always very low. heating for 3 minutes at 250°F (121°C) destroyed 25% to 50% of the added nisin. Delves-Broughton et al. The effect of processing temperature and pH on the stability of nisin in different foods was studied by Heinemann et al. 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 a specification was set for commercial nisin concentrates containing at least 2. This was contrary to previous work by Tramer (1964. (1975). and this bioassay method contains .9. volunteers could not detect 200 mg/L Nisaplin® in mineral water (equivalent to 5 µg pure nisin/g). 1998).025 mg/ml (equivalent to 1000 mg/kg Nisaplin®). For example. 1964). is the most widely used nisin assay. 1969). the levels of pure nisin should be multiplied by 40. equivalent to 1 µg of this preparation. The current term. These may be intended as stock solutions for the preparation of test dilutions in smallscale laboratory experiments. however. 1992).. and contains 2. Both the solubility and stability of nisin in solution are affected by pH. successfully used in high-pH/heat-treated food products such as canned vegetables and pasteurized liquid egg. 1970). producing clearly distinguished and measurable zones of inhibition in the presence of nisin. However. (1965). Optimum heat stability occurs at pH 3 to 3. A similar degree of destruction was reported for highly acid foods having pH values of 3.3 to 4. It is recommended that such solutions should not exceed a maximum concentration of 250 µg/ml. This has a standard potency of 1 million International Units per g.5 mg/ml at pH 6 and 0. Their results show that in low-acid foods at pH 6. otherwise a loss of activity may occur as a result of salt precipitation or filtering.5 (Davies et al. 1990. Liu and Hansen (1990) suggested that the Dha and Dhb residues become more susceptible to modification as a result of the presence of nucleophiles. An international nisin reference preparation was later established by the WHO Committee on Biological Standardisation (Anonymous. Pure nisin was deemed too potent for use in food.

detectable by bioluminescence (Waites and Ogden. whereas . as its name suggests. 1990. again most probably because of detection of inactive degraded nisin or possibly biologically active nisin that was bound within the food (Bouksaim et al. wine and beer) that are often not heat processed. which was thought to result from the test measuring a partially degraded nisin molecule that had lost biological activity. The meat spoilage organism Brochothrix thermosphacta. This latter test does not measure biological activity. Nisin has a stronger bactericidal effect against energized cells. sauces. Joosten and Nunez.1. There have been several reported improvements to this test (Rogers and Montville. or alcoholic beverages (e.. Listeria monocytogenes. A relatively new species.. capable of transducing the signal into detectable bioluminescence (Wahlstrom and Saris. organisms that may cause spoilage of low pH food such as salad dressings. because nisin Z diffuses more rapidly than nisin A. first described in 1951. molds. 1987). was first described in 1985 and. Such heat treatments usually eliminate all vegetative cells so that such foods are vulnerable to spoilage only by these surviving spore-forming bacteria. lactis construct strain sensitive to nisin has been reported. ANTIMICROBIAL SPECTRUM Nisin has a broad spectrum of activity against Gram-positive bacteria. Wide variation in nisin sensitivities has been reported. Its activity range encompasses. 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. ensuring that nondegraded nisin only would be measured. nisin at 0. 2001). The growth of 104 cfu/ml of four different strains of this species was controlled for 7 days at 37°C by 0.125 µg nisin/ml (Thomas and DelvesBroughton. 1999). Rose et al. 1996). After 7 days at 25°C. this bacterium produces extremely heat-resistant spores capable of surviving ultra-high temperature (UHT) treatments. This species is extremely nisin sensitive. 1989a). Important pathogens in this group include Bacillus cereus and Clostridium botulinum.. 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. Bacillus sporothermodurans. the Gram-positive endospore-forming genera that include Bacillus and Clostridium. Nisin is also effective against another food-associated pathogen. The outcome of nisin activity can also vary from achieving total kill of the cells to a growth inhibitory effect (DelvesBroughton et al. 1999). is extremely nisin sensitive. 1996). or Gram-negative bacteria. nisin-sensitive organisms relevant to food are shown in Table 7. unpublished results). Falahee and Adams.2 shows the activity of nisin against three different strains of Bacillus.g. Wolf and Gibbons.625 to 2. Another important group of nisin-sensitive bacteria is lactic acid bacteria. most importantly for its use as a food preservative. (1999) described detection of nisin by matrix-assisted laser/desorption time-offlight spectrometry. 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. enabling it to be used as a preservative in pasteurized or heat-treated foods that are not fully sterilized. Hurst (1981) reported that lower nisin levels prevent spore outgrowth compared with the levels required for inhibition of vegetative cells. Comparative studies of processed cheese assayed by ELISA and the horizontal agar diffusion bioassay showed a lack of correlation (Aplin & Barrett Ltd. An L. 1992).. This could be corrected by developing an antibody that detected distal parts of the molecule. For example.. An ELISA system using nisin Z polyclonal antibodies in rabbits also suffered from similar problems. In normal circumstances nisin does not significantly inhibit yeasts.Nisin 241 numerous control procedures to ensure nisin activity is not confused with other antimicrobial agents that may be present in the food. It is active against both bacterial cells and their heat-resistant spores.5 µg/ml prevented the growth of 103 cells/ml (Thomas and Delves-Broughton. Figure 7. 1995. 2001). with considerable variation being observed even between strains of the same species (Gupta and Prasad. 1991.

bulgaricus. Heat-resistant aerobic and facultative anaerobic spore formers. Cause blackening of canned food. sporothermodurans. Aerobe/anaerobe. and food-poisoning pathogens. sporogenes. brevis. Varied nisin sensitivity. producing slime. Maisnier-Patin et al. Spore forming. butyricum. acidresistant. Aerobes/anaerobes.. Bacillus brevis. Aerobes/anaerobes Aerobes/anaerobes. coagulans. Spoilage organism of fresh/pasteurized fruit juice stored at ambient temperature. helveticus. wine. fermentum. damnosus. Heat-resistant spore-forming obligate anaerobes. Varied nisin sensitivity. Can grow at low pH. Aerobes/anaerobes. histolyticum. Aerobes. Growth at pH 2. quiescent cells may survive but their growth will be inhibited (Sahl. M. varians Spp. Cause spoilage of wine and beer. Varied sensitivity. tyrobutyricum nigrificans Enterococcus Lactobacillus faecalis. Cause spoilage. Cause spoilage of acid products. cider.