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

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

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

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

130 184. 182. The target pathogen or spoilage microorganisms must be identified first. 182. Sofos et al. 2002 to foods (Tables 1. dehydroacetic acid Benzoic acid. GRN 000067 184. Title 9. (1998).155 184. 184. 182. 182. cooked meat. meats.1021.160. wines Fruits. propionates Sorbic acid. molds. 184. 172.Food Antimicrobials — An Introduction 3 TABLE 1. bacteria Yeasts.145 Molds Yeasts. 2000). baked goods.3225. GRAS Notice No.133 184. Section 424.1207. diacetates.1550. 172. sorbates Sulfites a Clostridium botulinum Yeasts. Source: Davidson and Harrison. wines 184. sauces Beverages. Extensive reviews on natural antimicrobials may be found in Dillon and Board (1994). methyl.1005. 184. 184.175.1 Traditional or Regulatory-Approved (U. nitrate Parabens (alkyl esters (propyl.1081. GRN 000065 Nitrite. lactates Lactoferrin Lysozyme Microbial Target Yeasts.1784 182. and poultry products Cheese Cheese. casings for frankfurters. 172. SELECTION OF ANTIMICROBIALS It is not an easy process to select the appropriate preservation system for a particular food product.3795 Various For meat products. cooked meat.2). Food and Drug Administration) Food Antimicrobialsa (Title 21 of the Code of Federal Regulations) Compound(s) Acetic acid. margarine Beverages Meats. and then the possible preservation systems must be evaluated via model studies and studies in the food product in question. 172. and Davidson and Zivanovic (2003). a combination of chemical preservatives and other preservation methods is needed (Leistner.1185. dairy products Most foods.S.1768 GRAS Notice No.3640.1221. dairy products. 184.21 and 424.3089. 184.1639.170. fermented foods Meats Cheese.1721. bacteria Primary Food Applications Baked goods. Naidu (2000). GRN 000064 Yeasts. molds Yeasts Bacteria Bacteria Clostridium botulinum. other bacteria Natamycin Nisin Molds Clostridium botulinum. 184. and poultry products Cured meats Beverages. syrups.1490. beverages.1670.1733 172. casings for frankfurters. fruit products.6197. acetates. dry sausage CFR Designation 184. molds. 184. other bacteria 172. heptyl) of p-hydroxybenzoic acid) Propionic acid.1 and 1. condiments. . 184. GRAS Notice No. molds Bakery products.1538. confections. fruit products.22. food antimicrobials permitted by USDA Food Safety and Inspection Service are listed in the Code of Federal Regulations. 184. benzoates Dimethyl dicarbonate Lactic acid. potato products. fats/oils.1754.1061. 172. Generally. bacteria (Gram positive) 172.177 184.

For example. 2002. The antimicrobial spectrum should involve an evaluation of the compound against various types of microorganisms (e. Even species. the activity of very hydrophobic antimicrobials may be limited against Gram-negative bacteria. species. the chemical properties of the antimicrobial. Each of these factors is discussed in detail. which have the ability to screen the antimicrobials because of the outer membrane lipopolysaccharide layer. Selection of the proper antimicrobial depends on several primary factors. Quite often. negative) can have dramatic influences on apparent activity. strain. however. although desired. or strains of microorganism. spores). including the spectrum of antimicrobial activity. Source: Verbruggen. a broad spectrum of activity. Appropriate methods for in vitro evaluation of the activity of food antimicrobials are described in Chapter 21. and Gram reaction (positive vs. yeasts. molds) and forms of those microorganisms (vegetative cells vs. does growth in a synthetic .” Seldom. the physicochemical properties and composition of the food product in question. is not easy to achieve. ANTIMICROBIAL SPECTRUM The initial selection of the antimicrobial is normally based on an assessment of the overall microbial spectrum of the chemical in question.. bacteria. and the type of preservation or processing and storage systems used.4 Antimicrobials in Food TABLE 1. “Methods for Activity of Assay and Evaluation of Results. Few chemicals have the ability to inhibit several different types.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. The antimicrobial spectrum of a compound is generally determined by following the growth of organisms in the presence of various concentrations of the antimicrobial.g.

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

79 4.. the acid dissociates because the cell interior has a higher pH than the exterior. Protons generated from intracellular dissociation of the organic acid acidify the cytoplasm and must be extruded to the exterior. 1980. Generally.5 or greater. In addition. which exists in the majority only at a pH below the pKa of the compound.0 (Table 1. organic acids function at low concentrations only in high-acid foods (generally less than pH 4. All regulatory-approved organic acids used as antimicrobials have pKa values less than 5. lysozyme) to include Gram-negative bacteria. nisin. This is because the most effective antimicrobial form is the undissociated acid.75 4. which are not normally inhibited by the compounds alone (Cutter and Siragusa. organic acids can penetrate the cell membrane lipid bilayer more easily.6 Antimicrobials in Food TABLE 1. thus requiring an antimicrobial capable of limiting the growth and activity of these organisms. Although the undissociated form of a weak acid has most of the antimicrobial activity. 1995. In the undissociated form.3). For food products with a pH of 5. allowing the antimicrobials access to the inner cell membrane. For example. Branen and Davidson. nucleic acids. where microbial growth occurs (Branen et al. there are very few compounds that are effective at low concentrations. Vacuum or modified atmosphere packaging results in . Rico-Munoz Davidson. Because protons generated by the organic acid inside the cell must be extruded using energy in the form of adenosine triphosphate (ATP). Chelating compounds such as ethylenediamine tetraacetic acid (EDTA) or its salts have a potentiating effect on some antimicrobials. They expand the activity of certain antimicrobials (e.19 3.87 4. PROCESS FACTORS The type of preservation process used in conjunction with antimicrobials has a significant influence on the type and level of antimicrobial needed.. which means their maximum activity will be in high-acid foods. 2004). enzymes. Packaging can directly alter the environment of the food and thus influence the overall growth pattern and type of organisms in the food. pH of the food can result in ionization of an antimicrobial and a change in activity. Bacteria maintain internal pH near neutrality to prevent conformational changes to the cell structural proteins. Once inside the cell.g. some Gram-positive bacteria are more susceptible to certain antimicrobials in the presence of chelators including EDTA. thus indicating the need for a different antimicrobial. It is theorized that chelators may destabilize the LPS layer of the outer cell membrane of the Gram-negative bacteria. the constant influx of these protons will eventually deplete cellular energy. molds survive and yeasts can grow at a lower water activity than bacteria.6). A lowering of the water activity can select for those organisms that have the ability to survive and/or grow at lower water activity. Refrigeration generally selects for psychrotrophic Gram-negative microorganisms. Certain preservation processes may result in the need to control sporeformers that have the ability to survive the heating process. and phospholipids.3 pKa of Regulatory-Approved Organic Acids Compound or Group of Compounds Acetic acid Benzoic acid Lactic acid Propionic acid Sorbic acid pKa 4.5 to 4. 1983). Eklund (1983) demonstrated that the anion does contribute slightly to antimicrobial activity.75 into the lipid areas of the food and away from the water phase.

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

Extensive studies at a pilot-plant level are necessary on any food additive to prove its overall usefulness. 2003). would defeat the purpose of using a natural compound. . For that reason. or texture. they are not normally consumed in the concentrations necessary to achieve antimicrobial activity. less purification may be better. ACTIVITY VALIDATION METHODS Currently. Obviously. Therefore. ECONOMICS OF USE A food antimicrobial will not be useful to the food industry unless it is inexpensive enough and has the ability to pay for itself based on reducing spoilage and minimizing foodborne illness. This would involve very expensive and time-consuming toxicologic testing. it is much less likely to require complex regulatory approval for use (Davidson and Zivanovic. such as nisin. This. LABELING One of the alleged attractions of naturally occurring antimicrobials is their reduced negative impact on the labeling of foods.1550) and nisin (U. In addition to adverse effects on flavor. so should there be validation for the activity of food antimicrobials. In addition to lack of toxicity. odor. Many antimicrobials must be used at high concentrations to achieve activity against target microorganisms. they may need to be approved as food additives. it is known that peptides. Consumers are reportedly concerned about the presence of synthetic chemicals in their foods and would prefer natural compounds. it would be unacceptable for a food antimicrobial to mask spoilage because spoilage may protect consumers from ingesting foodborne pathogens. Code of Federal Regulations. SENSORY EFFECTS Another major factor that needs to be addressed when applying antimicrobials is their potential impact on the sensory characteristics of a food. Code of Federal Regulations. If a product is simply an “extract of” a commonly consumed plant or animal food product. there are methods for the determination of the activity of lysozyme (U. Finally. 21CFR 184. assays need to be developed that evaluate the activity of these compounds against the pathogen they are designed to kill. This is because. For example. 1993). 21CFR 184.1538). In many cases. the compound would probably have to be listed using a chemical name on a food label. The reason for these assays is that various conditions of process or storage could reduce the effectiveness of the compound. an additional 2 or 3 days of shelf life can significantly help to offset the cost of using an antimicrobial. there are no other activity assays specified or required for food antimicrobials. are susceptible to inactivation by enzymes in foods. just as thermal processes need validation. This is only possible if the product from which the extract is taken is known to be nontoxic. The efficiency of these compounds can be extremely important in determining the overall economics of their use. there are few standardized methods for validation of the activity of regulatory-approved food antimicrobials. they should be nonallergenic or bind or destroy important nutrients in a food product (Harlander. If antimicrobials are to be used exclusively as inhibitors of pathogens in food products. In the United States. compounds that negatively affect flavor and odor or contribute inappropriate flavors and odors would be unacceptable. of course.S. although spices have been consumed for centuries.8 Antimicrobials in Food common potential natural antimicrobials such as spice extracts. A potential problem with natural antimicrobials is that if they are highly purified.S. naturally occurring compounds must be able to be metabolized and excreted so as to not lead to residue buildup. In addition.

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

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

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

1 Structures of benzoic acid and sodium benzoate..2). peach. 18°C. Benzoic acid has also been identified as a major constituent in extracts of blackberries (Humpf and Schreier.. Several new types of low-molecular-mass compounds were also identified. (1995) surveyed and analyzed many types of cultured dairy products and cheeses for the natural occurrence of benzoic acid (Table 2. Benzoic acid has also been identified as a natural by-product in culture filtrates of Lactobacillus plantarum (Niku-Paavola et al. including derivatives of benzoic acid. It is much more soluble in water than benzoic acid (62. pear. 1984. also called phenylformic acid or benzenecarboxylic acid. and 75°C. depending on the variety (Abdullah et al. Its presence appears to occur as a by-product of the microbial degradation of either hippuric acid or phenylalanine in these products (Figure 2. may also contribute to benzoic acid generation.. pineapple. This may be of significance in countries like Switzerland.1. it is preferred for use in many cases. was found to have antimicrobial properties. 1929). MECHANISM OF ACTION It is the undissociated molecule of benzoic acid that is responsible for antimicrobial activity (Table 2.3). Teuber. Naturally occurring nonflavonoid phenolic compounds. .8. Benzoic acid occurs naturally in several foods and commodities (Table 2. occurs in pure form as colorless or white needles or leaflets. 1992). and 74. 1994). of mushrooms. 1989. and grape juices were analyzed to establish phenolic profiles unique to each type of juice.1). 0. A third pathway. Orange. 1992). 1990). 1986a). 1999). oxidation of benzaldehyde.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. p-hydroxybenzoic acid was detected at 0. apricot.2 g dissolves in 100 ml water at 0°C.1% and in neutral media at 0. In four of the seven juices. For this reason.18. It is soluble to a limited extent in water (0. In an extensive review. 66. respectively).1). and of fresh tomatoes (Marlatt et al.6 mg/l. Sieber et al. respectively). where benzoic acid has not been approved as a food additive (Sieber et al. and 2. although their solubility in water is less than that of the sodium salt.1) is a white granular or crystalline powder.2 to 2. Similar results were reported for fungi and yeasts (Cruess and Richert. Potassium benzoate and calcium benzoate have also been approved for use. Yogurts have been found to contain natural levels of benzoic acid (Stijve and Hischenhuber. Gabel (1921) was one of the first to demonstrate that benzoic acid was effective against bacteria in acid media at a level of 0. along with benzoic acid. 20°C.27. were used to characterize commercial fruit juices (Fernandez de Simon et al. apple. O OH Benzoic Acid O O− Na+ Sodium Benzoate FIGURE 2. Sodium benzoate (molecular weight 144. 1991). and a mixture of these.2% but inactive in alkaline media. and 100°C.. It accounted for approximately 16% of the growth inhibition of Saccharomyces bayanus and Pseudomonas fluorescens from ethanolic extracts of cranberries (Marwan and Nagel. Benzoic acid (molecular weight 122. 1995).2)..0.2 g dissolves in 100 ml water at 4°C.

e. smear-ripened Source: Adapted from Sieber et al.Sodium Benzoate and Benzoic Acid 13 TABLE 2. cultured Teas. fruit Sour cream Buttermilk Cottage cheese Cheese Cheese. black Wines Cinnamon Cloves. Range in Concentration (mg/kg) 9–56 5–39 10–18 10–19 2–18 0–200 0–41 0–622 . (1995).. blackberries) Blueberries Cherries Cranberries Grapes Plums. Source: Chipley (1993). TABLE 2. Fenaroli (1996).1 Natural Occurrence of Benzoic Acid Category Fruits and berries Product Apples Apricots Berries (i. greengage Prunes Strawberries Tomatoes Beers Dairy. non-smear-ripened Cheese.2 Benzoic Acid Content of Cultured Dairy Products Product Yogurts Yogurt. 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.

NaCl. sucrose. The combined effects of pH and benzoate on the growth of these strains are presented in Table 2. 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.3 12.14 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 2.8 1.44 0. Macris (1975) observed a rapid uptake of benzoic acid in Saccharomyces cerevisiae. Proteinaceous material was apparently involved in the uptake of this preservative. TABLE 2.2 Biosynthesis of benzoic acid by lactic acid bacteria in fermented dairy products. . As expected.3 Effect of pH on the Dissociation of Benzoic Acid pH 3 4 5 6 7 pK Undissociated Acid (%) 93. Sorbate was more inhibitory than benzoate on the strains that were tested. The strong dependence of uptake on pH was because of the relative distribution of undissociated and dissociated forms in solution. The effects of pH. Source: Adapted from Sieber et al. and that the toxicity of sodium benzoate in solution was a result of the undissociated benzoic acid molecule. not to the pH itself. 1997). Zygosaccharomyces bailii and Yarrowia lipolytica were the most resistant to the two preservatives at pH 5.4. increasing the pH of the culture medium decreased the effectiveness of this preservative.144 4. Saturation was reached in about 2 minutes and remained constant thereafter. The undissociated form was the only one taken up by cells. (1995). and sorbic and benzoic acids on the growth of 30 strains of food spoilage yeasts have been reported (Praphailong and Fleet. The effect of temperature on the uptake was similar to that observed in enzymatic reactions.0. that only the undissociated acid was antimicrobial.19 Source: From Baird-Parker (1980).5 59.

2. Microbial cells can pump protons out during extended lag phase and raise internal pH despite further influx of preservative. bailii (Warth. 1991a). The authors reported that these preservatives were among the first compounds shown to act as selective inhibitors of heat-induced protein expression in yeasts. . rouxii No. Source: Adapted from Praphailong and Fleet (1997). membranaefaciens Saccharomyces cerevisiae Kluyveromyces marxianus Kloeckera apiculata Zygosaccharomyces bailii Z. bailii was to cause a general energy loss (ATP depletion). The authors concluded that the metabolic effects of benzoate or sorbate were only marginally pH dependent. 1994). fermentation was inhibited. At concentrations up to 4 mM. Near the minimum inhibitory concentration (MIC) (10 mM). (1993) preincubated cells of S. 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. In yeast (S. Two modeling studies to predict the inhibitory effects of organic acid preservatives have been reported. cerevisiae (Cheng and Piper. cerevisiae with benzoate or sorbate at an extracellular pH of 6. 1991b). Inhibition depends more on the preservative concentration within cells rather than on undissociated acid concentration per se.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. no growth in the absence of benzoic acid. the glucose-induced switch from gluconeogenesis to glycolysis was prevented. Fermentation was inhibited as a result of high ATP usage rather than of lowered intracellular pH (Warth. Growth rates were substantially reduced by both preservatives even at an extracellular pH of 6. The authors stated that. then added glucose. The author concluded that the primary action of benzoic acid in Z. and benzoate was accumulated to higher levels. Burlini et al.Sodium Benzoate and Benzoic Acid 15 TABLE 2. cerevisiae). in general. Inhibition of several glycolytic enzymes was not observed. Lambert and Stratford (1999) demonstrated the following: 1.8. fermentation was stimulated and low levels of benzoate were accumulated. — no growth in the presence of 250 mg of benzoic acid/L. These effects were dependent on the pH of the culture medium. adenosine triphosphate (ATP) levels declined. The resulting metabolic effects included reduced glucose consumption and suppression or reduction of glucose-triggered reactions within cells.8. The effects of benzoic acid were studied in the preservative-resistant yeast Z. Sorbic and benzoic acids both affected the heat shock response and thermotolerance of S.

phosphofructokinase activity was inhibited (Francois et al. For example. 1976. 1997).. The production of lipase by P. coli. In yeast cells. The authors suggested that the potency of weak acids.16 Antimicrobials in Food 3. 1989. α-ketoglutarate and succinate dehydrogenases appear to be quite sensitive (Bosund. 1980).0) for 1 hour (Buazzi and Marth. Theoretical ATP consumption for proton pumping can be directly correlated with reduction in cell yield. 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. Corlett and Brown. 50% of the trimethylamineN-oxide reductase activity was inhibited by 1. One hypothesis is that they inhibit or kill microorganisms by interfering with the permeability of the microbial cell membrane. growth inhibition is the result predominantly of the undissociated acid. 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. Acid-susceptible bacteria (Bacillus and Alicyclobacillus) and acid-resistant bacteria (Lactobacillus and Escherichia coli) were grown in media containing organic acids. B. 2001). membrane transport of amino acids is inhibited. E. They also stated that although both the undissociated and dissociated forms of these acids cause the intracellular pH to fall. A pH-related increase in protein production was observed when cultures of E.2 mM benzoic acid (Kruk and Lee. resulting in nutritional starvation of cells (Gould et al. 1980..3). 4. Gould. Only those organic acids that are lipophilic. In addition to these mechanisms. and Pseudomonas aeruginosa (Freese et al. 1962). In Bacillus subtilis (Freese. Sheu et al.0 as well as pH 6. in many bacteria and yeasts. its pK value (Table 2. 1960. Duration of the lag phase can be predicted from the model. 1980). 1989). 1978).. subtilis. Eklund. In the citric acid cycle. 1975. Aflatoxin production by a toxigenic strain of Aspergillus flavus was greatly reduced by the presence of these compounds in synthetic media (Uraih and Chipley. yielding protons that acidify the alkaline interior of the cell. 1992). 1983.. Eklund. 1984). Synthesis of messenger ribonucleic acid (mRNA) was critical for restoration of salt tolerance. MICs were determined for each acid and used with principal components to produce models with good correlations. Davidson. fluorescens was completely inhibited by sodium benzoate (Andersson et al. At pH 6. coli. 1973). Uraih et al.. Similar effects have been found using a variety of microorganisms. benzoate addition resulted in an increased production of 33 proteins. and the effectiveness of undissociated acids as preservatives has been noted for many years (Ingram et al... Chipley and Uraih. 1966).. Hsiao and Siebert (1999) constructed a model based on physical and chemical properties of organic acids and principal components analysis.5% sodium benzoate (pH 7.. 1973). coli have been reported (Salmond et al. 1973. Increased permeability of cell membranes during the course of benzoate injury was not observed. enzymes involved in acetic acid metabolism and oxidative phosphorylation can be inhibited (Bosund. such as benzoic. as food preservatives is related to their capacity specifically to reduce the intracellular pH.. 1956. In resting cells of E. benzoic acid or sodium benzoate can also inhibit specific enzyme systems within cells (Webb. 1977. Production of 12 of these was induced at pH 8. 1982). Benzoate also inhibits amino acid uptake in Penicillium chrysogenum (Hunter and Segel. such as benzoic acid. 1986). coli were grown in liquid media with or without 20 mM benzoate (Lambert et al. 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. The inhibitory potency of this acid can be determined by its lipid/water partition coefficient. show antimicrobial activity. Reduction .5. The effects of organic acid preservatives on the growth and intracellular pH of E. 1980). causing uncoupling of both substrate transport and oxidative phosphorylation from the electron transport system (Freese et al. 1962).5. More than 99% of the viable cells of Listeria monocytogenes were injured after exposure to a solution of 8. 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. 1997).

This might result in inhibition of membrane-bound enzymes as a secondary effect. as an inhibitor of Damino acid oxidases (Quay and Massay.6. and sodium benzoate was determined (Manganelli and Casolari. as a weak inhibitor of poly(ADP ribose) polymerase (Oikawa et al. In general. 1983). Increasing the concentration of sodium benzoate increased the percentage of inhibition at the end of incubation (10 days). It has been reported to have a flower-inducing effect in plants (Watanabe and Takimoto. Sodium benzoate was found to control both growth and aflatoxin production by Aspergillus parasiticus in liquid media (El-Gazzar and Marth. potassium sorbate. 1974). prior exposure to the preservative.. Similar results were observed with 2.02% undissociated acid. 1979). 4-dinitrophenol and cell envelope-associated enzymes of E. with the greatest increase occurring when the medium contained 0. The scattering of MIC values was lower with potassium sorbate and benzoic acid and higher with sorbic acid and sodium benzoate. The sensitivity of 42 yeast cultures to sorbic and benzoic acids. coli and Salmonella Enteritidis (Chipley. Baird-Parker. benzoic acid cannot be relied on to effectively preserve foods capable of supporting bacterial growth (Chichester and Tanner. all treated cultures produced measurable levels of toxin 3 to 7 days later than controls. but many spoilage bacteria are much more resistant. 1997). Conversion could be prevented by addition of benzoic acid or sodium benzoate. In liquid media.3). 1977. Currently. Therefore.3% sodium benzoate. these compounds are used primarily as antimycotic agents. the use of benzoic acid or sodium benzoate as a food preservative has been limited to those products that are acid in nature. In benzoic acid-supplemented cultures. and as an inhibitor of nitrosamine formation (Sung et al.. and most yeasts and fungi are inhibited by 0. however. Fusarium species were more sensitive to benzoic acid (210 to 420 µg/ml) than were Penicillium species (250 to 3000 µg/ml). Addition of benzoate to chemostat cultures of S. The results suggested that these preservatives blocked an enzymatic step late in the biosynthetic pathway of aflatoxin B1.1% of the undissociated acid. aflatoxin B1 production was higher than in controls.. including pH. cerevisiae decreased the biomass and increased the specific oxygen uptake rate of cells (Verduyn et al. and environment from which the microorganism was originally isolated. . The effects of several preservatives and antimicrobial agents on aflatoxin B1 production by A. temperature. 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.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. ANTIMICROBIAL ACTIVITY SPECTRUM OF ACTION Because the quantity of undissociated acid decreases with increasing pH (Table 2. and fungi involved in food poisoning and food spoilage are given in Tables 2. 1980).. genus and species of the microorganism in question. yeasts. 1980). Several factors interact to determine the MIC. Food-poisoning and spore-forming bacteria are generally inhibited by 0. composition of the growth medium. 1972. 1992). 1981). flavus have been reported (Bauer et al. With the previously mentioned results in mind. MICs for some of the bacteria.01% to 0. 1987). 1980). as an inhibitor of passive anion transport (Lucas-Heron and Fontenaille. 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 0.5 and 2. 1988). Yagi et al. Benzoate may also serve as a scavenger for free radicals (Harvath. The authors concluded that subinhibitory concentrations of these compounds may stimulate toxin production in some cases.. 1979). The average accumulation of mycelial dry weight was greater in the presence of benzoate than in its absence.1979).

0 6.5 Antimicrobial Spectrum of Benzoic Acid against Selected Bacteria.0 5.0 5.0–5.0 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 4.2–5. Pseudomonas sp.0–5.0 4. (1999). Yeasts.6 6. and Fungi Microorganisms pH Bacteria 6.0 2.3 5.3–6.6 Yeasts 2. Pseudomonas aeruginosa Staphylococcus aureus Streptococcus sp.0 MICa Bacillus cereus Escherichia coli Klebsiella pneumoniae Lactobacillus sp.0 5. and Steels et al.0 5. 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).8 4. Zygosaccharomyces bailii Z.8 4.6 (4°C) 5. 1993).18 Antimicrobials in Food TABLE 2.0 4.0 4. Hansenula subpelliculosa Oospora lactis Pichia membranefaciens P. Source: Adapted from Chipley (1983.0 3.0 5. . cerevisiae Torulopsis sp.5 4.3 5.6 6.0 4.0 5.6 (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. Davidson and Juneja (1990).0 6. lentus Z.2–5.6–4.1 5. Aspergillus parasiticus Aspergillus niger Byssochlamys nivea Chaetomonium globosum Cladosporium herbarum Mucor racemosus Penicillium sp.0 4. rouxii 4. Russell (1991). Saccharomyces bayanus S.5 5. Listeria monocytogenes Micrococcus sp.6–5.0 6. pastori Rhodotorula sp.8 Fungi 5.5–5.0 5.0 3.

Ten generally regarded as safe (GRAS) substances. 1989). Growth of this fungus was accompanied by production of fumitremorgin mycotoxins. Both fungistatic and fungicidal properties have been attributed to benzoic acid. monocytogenes (El-Shenawy and Marth. Fungal growth was reduced by lowering the pH of laboratory media from 7. 1977). activities against cells in a liquid minimal medium. including benzoic acid. Yousef et al. 1988). 1995). 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. In a series of studies involving L.0 to 2. including benzoic (Rusul and Marth. Isolates were grown at 25°C in yeast extract medium containing 5% glucose (pH 3.5. enhanced aflatoxin production when present at low levels but became inhibitory at higher levels. Sodium benzoate has been suggested as an inhibitor of cellulose-decomposing bacteria and fungi (Sauer. Source: Adapted from Warth (1989c). and its survival . according to the results of a study involving several strains of Trichophyton and Microsporum (Pelayo. such as sodium and potassium chlorides and sodium nitrate. Eight of these had a lethal effect on both morphotypes of this bacterium. This organism was isolated from milk.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. Under appropriate conditions. but relatively modest bactericidal. 1988. 1979). it was found that benzoic acid at concentrations of approximately 1000 to 3000 ppm had strong bacteriostatic. The authors questioned the suitability of benzoic acid alone to control this pathogen in foods. Salts.Sodium Benzoate and Benzoic Acid 19 TABLE 2. were tested against both the opaque and translucent morphotypes of Vibrio vulnificus (Sun and Oliver. Small amounts (75 mg/L) of potassium sorbate or sodium benzoate completely inhibited germination of ascospores and subsequent outgrowth.5) without addition of 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. 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. Growth and aflatoxin production by toxigenic strains of Aspergillus were partially or completely inhibited by the undissociated form of six organic acid preservatives... 1989). bacteriostatic and bactericidal properties of benzoic acid can also be demonstrated. selected organic acids promoted growth and toxin production when added to the media. Most were isolated from spoiled foods that had contained preservative.

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

RE. Sodium benzoate prevented the growth of Staphylococcus aureus in an intermediate-moisture food (Boylan et al. such as heating. and 500 ppm sodium benzoate. or drying. 1990). Growth at 21°C.8 0.0. refrigeration. relative effectiveness (the time. The effects of pH and benzoate on the growth of yeasts in an experimental grape drink containing 1. Source: Marwan and Nagel (1986b).3 Effect of the medium on the inhibition of Saccharomyces bayanus by benzoic acid at 30°C and pH 4. 150 ppm potassium sorbate. benzoic acid can be solubilized with a surfactant to hold it in solution in its undissociated form (Lewis and Payne. However. an experimental bottled beverage made from papaw fruit was reported to have a shelf life of 80 weeks at 10°C and 30°C when pasteurized and supplemented with sodium benzoate (1250 ppm and pH 3. 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). 1984).6 1/RE 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. 1958).Sodium Benzoate and Benzoic Acid 21 1. respectively.0 0. 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. 1961). . Likewise.2 0. 1976). For example. cerevisiae was inoculated in a laboratory medium supplemented with several combinations of preservatives (Parish and Carroll.. Open circles: Trypticase® soy broth. 1988). Closed circles: yeast nitrogen base.9) (Okoli and Ezenweke. The presence of other chemicals may be beneficial or detrimental to the action of the preservative. the antimicrobial activity of benzoic acid is reduced by nonionic surfactants (de Navarre and Bailey. 1956). Sulfur dioxide was apparently antagonistic when used in combination with any of the other antimicrobial agents (including benzoate) tested in this study. Sulfur dioxide had the most significant effect on the rate of biomass and patulin production by this organism. and 37°C over a 25-day incubation period was significantly retarded by 75 ppm sulfur dioxide. 1975).5 volumes of CO2 have been reported (Kelly.0 50 100 150 ppm 200 250 300 350 FIGURE 2. irradiation. in hours. depending on the chemical in question. In addition to combining several preservatives together.4 0. 30°C. followed by potassium sorbate and sodium benzoate. it may also be advantageous to combine preservatives with physical methods of food preservation.b). Antagonistic as well as synergistic effects were observed when a wine yeast strain of S. A combination of benzoic acid and low temperatures was used effectively to reduce the numbers of yeasts in grape juice (Pederson et al.

the authors proposed that chitosan is highly polycationic and could react with the anionic components of the microbial surface. Carbonated and noncarbonated citrus drinks and fruit juices were involved. The concentrations of benzoic acid tested (in parts per million) are indicated. The effects of quinic acid on yeasts and molds were investigated (Kallio et al. cited by Lueck (1980). Growth of yeasts was unaltered. A combination of the polysaccharide chitosan (0.2 and 4.025% sodium benzoate acted synergistically against three food spoilage yeasts suspended in saline solutions at pH 6. However. coli after 14 passages in media containing benzoic acid.005%) and 0.22 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 2.0. Adding 1% quinic acid to media almost completely eliminated the inhibitory effect of 0. ACQUIRED RESISTANCE TO BENZOIC ACID There have been conflicting reports in the literature concerning the possibility of acquired resistance to benzoic acid by microorganisms. The resistance of a wide variety of yeasts to benzoic acid has been reported in several studies. In a German study of 1959. At these pH values. recent studies imply that this may no longer be the case. no resistance could be observed in cells of E. this might enhance the antimicrobial effects of benzoate. 2002).5 (Sagoo et al. This acid was antagonistic to the antifungal effects of both potassium sorbate and sodium benzoate.. but mold growth was accelerated in the presence of quinic acid. They had been supplemented with the maximum levels of benzoic acid permitted for use in these products. 1985). Source: Marwan and Nagel (1986b).02% sodium benzoate.4 Inhibition of Saccharomyces bayanus by benzoic acid in Trypticase® soy broth at 30°C and pH 4.01% to 0.. .

Three strains of T.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. were tested for tolerance to several organic acids during and after heating (Beuchat. 1988) it was determined that energy was required for the reduction in cytoplasmic benzoate concentration and for the maintenance of internal pH. and lethality was enhanced as the pH of the heating medium was reduced.25 mM benzoic acid (first five species) or 2 mM benzoic acid (last four species). Potassium sorbate and sodium benzoate prevented outgrowth.. In a later study (Warth.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 0.4 to 3. cells were resistant only when a sufficient energy source. Fumaric. the energy source was unavailable for growth. resulting in reduced growth yields and rates of cell production by yeasts. and benzoic acids were the most effective. TABLE 2. Resistance resulted primarily from an inducible energy-requiring system that transported benzoate from the cell. respectively (Ethiraj and Suresh. 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. The heat-resistant yeast Talaromyces flavus was isolated from fruit juice concentrates involved in spoilage of packaged reconstituted fruit juice (King and Halbrook. Heat resistance increased with age. isolated from spoiled fruit products.7).19). A strain of S. . flavus. was present. Thus. Because this system required energy. 1987). Source: Adapted from Warth (1988). The authors observed that preservative resistance may be strain specific. 1990). heat tolerance appeared to be related to the morphology of the ascospores. bailii grew in the presence of high concentrations (600 mg/L) of benzoic acid at pH values below the pK of this acid (4. However. 1988). this isolate was less resistant to potassium sorbate. sorbic. and in some cases.5. with the required concentrations lower when the pH of the growth media was reduced from 5.5. Several species of yeasts grown in the presence of benzoic acid tolerated 40% to 100% higher benzoic acid concentrations than yeasts grown in its absence (Table 2. In an inoculated peach-based system. cerevisiae was found to have multiple resistances to both physical treatment and chemical preservatives (Guerzoni et al. 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. such as glucose.0 and 4. Warth (1977) reported that cells of Z. 1988).

in which benzoic acid is continuously removed from the cell. A new osmophilic. Some yeasts are intrinsically resistant to relatively high concentrations of preservatives (Stratford and Lambert. Five strains of this yeast grew over a wide range of temperature (4°C to 25°C) and pH (2. or legally. Uptake of weak acid preservatives by passive diffusion is quite rapid. Spoilage resulted from a Z. the capacity of the cell to remove it. Three selective media were compared for detecting and enumerating nine strains of Z. Benzoic acid was taken up 27 times faster by cells than propanoic acid. 1985). bailii can grow in concentrations of food preservatives in great excess of those normally. Uptake rates of benzoic and propanoic acids in cells of Z.. results also suggested that the resistance mechanism. 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. 1999). Adaptation to higher preservative concentrations was demonstrated with benzoic acid. allowed in beverages and can adapt to concentrations even higher. The Zygosaccharomyces genus also contains some of the most osmotolerant species known. The principal mechanism of uptake appeared to be diffusion of undissociated acid. bailii and S. Growth of yeasts in the presence of benzoic acid reduced their subsequent permeability to this acid. cerevisiae.5 g/kg) and packaged in glass jars. benzoic. Z. 1988). However. bailii has been described (Buchta et al. The authors stated that microorganisms resist or adapt to weak acid preservatives (e. The nonselective medium supported higher recovery of all nine strains from the six food products compared to the three selective media. Glucose stimulated the rates of uptake and was also required for continuous elimination of benzoate from cells. 1989b). bailii were proportional to the concentration of undissociated acid (Warth. 300. It was preserved using benzoic acid (1.g. Eight tons of table mustard were exported to the United States. or an intrinsic sensitivity to benzoic acid or its anion (Warth. Mustard spoilage by Z.0).24 Antimicrobials in Food The genus Zygosaccharomyces is often regarded as being synonymous with food spoilage as a result of the remarkable resistance of its species to commonly used food preservatives such as sorbic. Growth in the presence of weak acid preservatives appeared to involve a common resistance mechanism. They were resistant to sorbic acid (≤400 mg/l) and benzoic acid (≤900 mg/l) at pH 4.2 to 7. 1996). 1996). Adaptation can result in rapid growth in moderate levels or slow growth in previously inhibitory levels. lentus was likely to be more significant than Z. Zygosaccharomyces lentus. The authors concluded that Z. Cells grown in syrup without sodium benzoate were more sensitive to selective media than were cells grown in syrup containing the preservative. benzoate) by one of the following three methods: 1. requiring only 5 to 15 minutes to reach equilibrium. Resistant species may be less permeable than sensitive ones to undissociated benzoic acid (Warth. preservative-resistant spoilage yeast. 1989c). Zygosaccharomyces and Saccharomyces are able to adapt to changing levels of preservatives such as benzoate. Removal or metabolism. bailii strain that was resistant to both benzoic and sorbic acids. Differences in resistance among yeast species could be the result of differences in the rate of penetration of the cell by benzoic acid. 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. bailii grown in six commercial food products (Makdesi and Beuchat. 1999). and most can grow at very low pH. In another study (Warth. and all strains were osmotolerant. is a common and major determinant of the preservative tolerance of yeast species (Table 2. 1989c).. . Resistance to dimethyldicarbonate was greater than that of Z. has been described (Steels et al. These media were also evaluated for enumerating two of these strains grown in blueberry syrup with or without sodium benzoate (0. and 600 µg/ml). Growth at 4°C was significant. bailii in the spoilage of refrigerated foods.0. 1989a). Foods at particular risk are acidic and contain relatively high levels of sugar.. and acetic acids and ethanol (Thomas and Davenport.6) (Warth. The minimum pH for growth was not related to this acquired resistance.

Holyoak et al. When G. 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. Reducing the pH of the media to 3. however. which is freely permeable across the plasma membrane and is thus able to enter the cell. Benzoic acid occurs naturally as a phytoalexin of immature apples (Seng et al. coli initially grown in media at pH 5. cerevisiae were able to extrude benzoic acid by an energy-dependent mechanism.. inhibition of microbial cell growth occurs because various key metabolic reactions are inhibited. 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. 1989). resulting in the release of charged anions and protons. Henriques et al. They included the acid tolerance responses of E. In an extensive review. However.0 survived exposures to inorganic acid or to acid pH plus organic acid that prevented subsequent growth of cells grown at pH 7. respectively. Whether efflux pumps are involved is open to debate. oxydans was grown in the presence of sublethal concentrations of either compound before the MIC was determined. 1985). (1997) found that cells of S.. the MIC of both antimicrobials increased substantially within 1 hour. 1996. Anions and protons accumulate inside the cell. In yeasts. For example. 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. 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. and adaptive mechanisms have been suggested. The pathogenicity of this fungus could be increased by growth on a medium supplemented with benzoic acid before inoculation into fruit. The net result is that the preservative diffuses into the cell until equilibrium is reached in accordance with the pH gradient across the membrane. earlier studies by Henriques et al. adaptation could involve enhanced removal of protons using H+ATPase. undissociated state of the molecule. Prevention of uptake. MICs of sorbic and benzoic acids for Gluconobacter oxydans were 1000 and 900 mg/L.Sodium Benzoate and Benzoic Acid 25 2.0 (Goodson .. Weak organic acid preservatives have maximum inhibitory activity at low pH. However. This appears unlikely because uptake occurs rapidly by passive diffusion. 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. 3. which cannot cross the plasma membrane. in media at pH 3. 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. coli O157:H7 and L. (1998) demonstrated the existence of a multidrug-resistance pump that actively extruded preservative anions from cells of this yeast. Piper et al. b. Therefore. which can infect apple tissues. This favors the uncharged.8. 1997. Bracey et al. Adaptation to or repair of damage. Mutants of the fungus Nectria galligena. were isolated and found to be resistant to benzoic acid. The inhibitory action is classically believed to occur when the molecule encounters the higher pH inside the cell and then dissociates.3 reduced the MIC of both antimicrobials by about 300 mg/L (Eyles and Warth. When this happens. 1998).. Cells of E. if damage is caused by low intracellular pH. monocytogenes and the role of membrane ATPase in the resistance of food spoilage yeasts. Several microbial-resistance mechanisms induced by weak organic acid preservatives were also reviewed by Brul and Coote (1999). The authors proposed that in yeasts. as cited previously (see “Mechanism of Action”). Acquired resistance to benzoic acid has been reported for other microorganisms. 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.

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

gentisate. including Pseudomonas. Pseudomonas solanacearum. Seventeen strains of aerobic bacteria were tested for their ability to degrade 15 aromatic substrates (Lang. The enzymes responsible for ring cleavage were induced depending on the substrate used for growth. In Aspergillus niger. 1996). one of the causal agents of wilt in banana plants. Kinetic models have been derived for the metabolism of [14C]-benzoate by Pseudomonas species (Simkins and Alexander. respectively (Shailubhai et al. involves monooxygenase and dioxygenase enzymes that catalyze the incorporation of molecular oxygen into the aromatic nucleus to form dihydroxylated intermediates (see also Heider and Fuchs. 1997). fumarylpyruvate. and fumarate + pyruvate. anaerobic pathways have been far less studied and reported. was also capable of metabolizing benzoate (Kumari and Mahadevan. foods.5). (3) introduction of a carbonyl group. 4-dioxygenase was induced. A permease-like protein involved in benzoate transport and degradation has now been isolated from cells of Acinetobacter (Collier et al. 1997). . putida (Jeffrey et al. a commonly occurring soil bacterium.. Active transport of benzoate may occur in this organism (Thayer and Wheelis.2-dioxygenase for ring fission. These results were confirmed by assaying key enzymes from benzoate-grown cells. end product of the β-ketoadipate pathway (Figure 2. This is followed by conversion of the remainder of the molecule to acetyl-CoA. Anaerobic metabolism can also occur but involves reductive saturation of the nucleus. 1988). Catechol.. 1995). One pathway proposed for the anaerobic microbial metabolism of benzoate is shown in Figure 2. and cosmetics. A mutant incapable of growth on benzoate was isolated and characterized.. Both chromosomal and plasmid-encoded pathways have been described for the catabolism of benzoate in P. 1988). Nitrate.6. In the presence of benzoate. Further metabolism proceeded via the catechol branch of the β-ketoadipate pathway with the occurrence of high specific activities of catechol 1.2-dioxygenase. 1995). cis. (4) ring opening.. In Alcaligenes eutrophus. He reported that benzoic acid was easily metabolized by all nine bacterial species tested. and with Micrococcus (Haribabu et al. 1994). The proposed intermediates included benzoyl-CoA. both aerobically and anaerobically. Compared to relatively well-characterized aerobic mechanisms for metabolism of benzoate and other aromatic compounds. and (5) a β-oxidation sequence. 1993. 1984). Harwood and Gibson (1986) stated that aerobic degradation of benzoate by various Gramnegative bacteria. the rate of benzoate metabolism may be regulated by the presence of succinate. 1990). 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. Benzoate was converted to catechol by the strains able to grow on this substrate. maleylpyruvate. and sulfate can be used as electron acceptors during metabolism (Colberg and Young. 3-hydroxybenzoyl-CoA. 1982). 1984). such as glucose..5) (Ampe et al. and β-ketoadipate were identified as intermediates of benzoate metabolism (Figure 2. 1992). cis-muconate. Hugo (1991) described the ability of nine species of Pseudomonas and Vibrio to degrade 18 aromatic compounds used as preservatives in medicines. benzoate and salicylate are metabolized via the protocatechuate and β-ketoadipate pathways. 1984). Niemetz et al. 1982).Sodium Benzoate and Benzoic Acid 27 β-ketoadipate pathway. with Rhodococcus (Janke et al.. which are central intermediates in the anaerobic metabolism of many aromatic compounds (Elder and Kelly. protocatechuate 3. 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.. Harwood and Gibson (1997) stated that this pathway could be divided into several phases: (1) formation of CoA thioesters. iron.. (2) ring reduction. whereas salicylate induced catechol 1. This isolate was able to oxidize benzoate completely to carbon dioxide. The enzymes of these pathways appeared to be under coordinate control and were repressed in the presence of a primary substrate.. Similar results have been reported with Bacillus stearothermophilus (Adams and Ribbons. 1997). Benzoate was metabolized by 16 of these strains when added as the sole carbon source to liquid mineral media. Outlines of the new aerobic pathway were deduced.

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

and other foods in several countries (Tables 2. A Food Technology special report (1986) reviewed the characteristics. Although the detectable yield was extremely low (<1 ppb). 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. generate detectable levels of benzene. Applications of these preservatives to foods have been reviewed by Chichester and Tanner (1972).1021 and 184. their main advantages include low price.1733) up to a maximum permitted level of 0.15% and 0. However. Subsequent studies indicated that soft drinks containing both ascorbic acid and sodium benzoate could produce very low levels of detectable benzene. 1994). Kimble (1977). bakery products. Regulatory agencies consider it to be GRAS to the same extent that sodium benzoate is GRAS as a preservative with the limitation of 0. the off-flavor they may impart to the foods they are to preserve. Title 21. Similar results were reported by McNeal et al. APPLICATIONS Benzoic acid and sodium benzoate are most suitable for foods and beverages that naturally are in a pH range below 4.8 and 2. Gardner and Lawrence (1993) demonstrated that low levels of detectable benzene could be produced in aqueous solutions of sodium benzoate and ascorbic acid in the presence of the transition metal catalysts iron (III) or copper (II). As food preservatives. Secs.4 g per 100 ml solution) than the sodium salt. Calcium benzoate has also been approved for use. and limitations of preservatives and antimicrobial agents. Inc. USE IN VARIOUS FOOD SYSTEMS Benzoic acid and sodium benzoate have been widely used to preserve beverages. In most other countries. They may also include foods from which benzoate is excluded in the United States. the authors recommended that the combination of ascorbic acid and sodium benzoate in foods and beverages be evaluated more carefully. fruit products. (1993) when they analyzed several foods and fruit-flavored soft drinks for benzene.). REGULATORY STATUS In the United States. Benzoate does not control the growth of high levels of microorganisms and therefore cannot be used to compensate for poor ingredients or processing.25%. benzene levels higher than the permissible levels for mineral waters prompted a recall in one state. and lack of color. the use of benzoic acid and other chemicals in the processing and freezing of shrimp has been reviewed (Chandrasekaran. 1990). . 1990). 1993). Potassium benzoate is now commercially available and apparently evolved in response to consumers’ interest in reduced sodium intake. 1977/1988. Product information for both salts is available (Miles Laboratories. benzoic acid and sodium benzoate are GRAS preservatives (Code of Federal Regulations. They may also be used in certain standardized foods.1%.5 or that can be brought into this range by acidification.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. Approximately 1. including benzoic acid. 184. Lueck (1980). the maximum permissible quantities generally range between 0.9). The potassium salt is less water soluble (42.1%. applications. under certain conditions. An ADI (average daily intake) value of 0 to 5 mg/kg of body weight has been specified for benzoic acid (Pollard. and Chipley (1983. ease of incorporation into products. the narrow pH range in which they are effective. In one case (a fruit juice–mineral water combination). although its solubility in water is much less than the sodium salt (Pollard.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. In addition.

Potassium sorbate was more effective than sodium benzoate. prevented spoilage of the product by Z. semipreserved Fruit juice Fruit pulp Jam. jellies Liquid egg white.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.S. Recent reports indicate that these preservatives continue to be of benefit in a variety of products.K. but A. ketchup Soft drinks containing fruit juice Soft drinks. niger.8 Maximum Permitted Levels (ppm) for Benzoic Acid and Its Salts in Foods in Selected Countries Food Fish products Fish. was most sensitive to propionic acid and sodium benzoate. were dominant. The effectiveness of sodium benzoate and potassium sorbate. was evaluated against the growth of Z. 1994). 1995).04%) inhibited growth of all three fungi. The shelf life of sardines stored in an acid brine containing 0. Halotolerant fungi were isolated from salted dried fish (Chakrabarti and Varma. d Good manufacturing practice. 2000). A maximum level of 2000 ppm may be used in orange juice for manufacturing. Aspergillus flavus. a maximum level of 1000 ppm benzoic acid or its salts may be used for all products listed.. salted Mayonnaise Mustard Pickles. separately or combined. separately or combined. Penicillium sp. and Penicillium sp. Several additives and preservatives were evaluated as inhibitors of melanosis (black spot) and microbial spoilage in prawns in chilled storage (Montero et al. However. 1996). relishes Salads. carbonated a Canada 1000 1000 1000 1000 1000 Denmark Norway Sweden U.3% sodium benzoate was three times longer than that of the controls (Ponce de Leon et al. then evaluated during shelf life (Efiuvwevwere and Ajiboye.02%) or sodium benzoate (0.30 Antimicrobials in Food TABLE 2. Orange juice not for manufacturing may not contain a preservative (federal standards of identity). Fresh catfish were dipped in various concentrations of sodium benzoate or potassium sorbate and smoked. yolk or whole Margarine. A. A combination of sodium benzoate and kojic acid was effective in inhibiting melanosis. 2001). bailii in an inoculated salsa mayonnaise stored at room temperature (Wind and Restaino. However. Sorbate treatment was more effective than benzoate and increased the shelf life by 8 days. neither preservative.a 1000 200 500 500 5000 1500 1500 1500 5000 2000 1000 1000 1000 10. flavus was most sensitive to potassium sorbate.06%) or potassium sorbate (0.. bailii. . Propionic acid (0. c Soft drinks for consumption without dilution. Source: Adapted from Chipley (1993). U. 4-hexylresorcinol was the most effective inhibitor of both melanosis and microbial spoilage in prawns. salad dressings Sauces. b Soft drinks for consumption after dilution.

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

1989).5% lactic acid combined with 0. Contamination of many of these foods frequently occurs after processing. low pH.. this antimicrobial was evaluated for control of postharvest diseases of various fruits and vegetables. and ethanol were very effective bactericidal treatments for L. coli O157:H7 in cider because of the severity of illness this pathogen causes. or S. However. monocytogenes (Barker and Park. monocytogenes usually attach following cooking and during slicing and packaging (Farber and Peterkin. A study was conducted to evaluate aqueous dipping solutions of organic acids or salts to control L. monocytogenes. 1995). 1995). However. Unpasteurized apple cider has been implicated in several foodborne disease outbreaks caused by pathogenic microorganisms. Washing the skin with solutions of either 0. Large outbreaks of infection have been linked to consumption of contaminated coleslaw.15% fumaric acid and 0.6) or adding potassium sorbate (0..05% sodium benzoate reduced the numbers of these pathogens compared to washing with water.32 Antimicrobials in Food temperature. Resistance to these preservatives was greater at 4°C than at 25°C (Fisher and Golden. These solutions could be used for dips to sanitize chickens intended for frying before presentation to consumers (Hathcox et al. 1994). 5% sodium diacetate. Most research efforts have been directed toward E. hot dogs.0) made from milk acidified to pH 5. especially in younger children and the elderly. coli O157:H7 in apple juice and apple cider with or without potassium sorbate or sodium benzoate has been investigated.3%) to cheese (pH 6.9 with propionic acid.05% sodium benzoate followed by holding at 25°C for 6 hours and at 4°C for 24 hours.. Benzoate was one of the most effective compounds tested in the presence or absence of ethanol. Sorbates and benzoates are currently approved in various countries for use as dipping solutions to prevent fungal growth in dry sausages (Sofos. 2002). Survival of E. vacuum-packaged bologna stored at 4°C for up to 120 days. At 8°C. coli O157:H7. The treatment that was successful included addition of 0. Both preservatives reduced the heat resistance of E.. or 5% potassium benzoate for 120 days (Samelis et al. respectively. growth was inhibited by benzoate or sorbate (Koodie and Dhople. 2001). Combinations of organic acids. C. The same preservatives added to cider resulted in a greater than 5-log reduction in less than 5 and 2 hours when held at 25°C and 35°C.. No 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. milk. 1999). several factors may influence the effectiveness of benzoic acid in the treatment of fresh . the combination of sorbate and benzoate was more effective than either preservative alone (Zhao et al. jejuni. 1996. and wash solutions were evaluated for effectiveness in decontaminating the skin (Hwang and Beuchat. 1998). 2001). coli O157:H7 in apple cider (Comes and Beelman. but benzoate was about two to eight times more effective than sorbate (Splittstoesser et al. 1999. and luncheon meats. Listeria monocytogenes is recognized as a foodborne pathogen of major concern to humans.. Dock et al.5% or 5% acetic acid. cheeses. USE AS A POSTHARVEST FUNGICIDE Because of the long and successful use of benzoic acid in the processed food industry. The antimicrobial can be applied directly onto the product surface where cells of L. 2000). coli failed to grow in either type of apple juice. Control strains of E. the growth of some O157:H7 strains in apple cider was not affected by the presence of either preservative (Miller and Kaspar.. No significant increase in the numbers of this pathogen occurred on bologna slices treated with 2.3% or 0. Growth was either prevented or delayed by adding sodium benzoate (0. Tompkin et al. L. aureus. A preservative treatment was developed that was capable of achieving the FDA mandate for a 5-log reduction of E. 2001). Chicken skin was inoculated with Salmonella spp.3%) to cheese (pH 6. monocytogenes on sliced. 1993). Ethanol-enhanced killing correlated with damage to the bacterial cytoplasmic membrane. Application of antimicrobials as sprays or mists to meats following processing may be more effective than their addition in the meat formulation. These strains grew well in unpasteurized and pasteurized apple juice.

1% to 0. respectively.. Significant antifungal properties were demonstrated against food spoilage fungi. rubber manufacturing.. 1989). 1980). 1976). A summary of the antimicrobial activities of 30 preservatives. A sodium benzoate–sorbic acid combination used in a pharmaceutical product for treating ulcers was effective against a wild strain of Pseudomonas cepacia.. their concentrations were high enough to inhibit growth of periodontal pathogens.025%.Sodium Benzoate and Benzoic Acid 33 fruits and vegetables. Although its use in pharmaceuticals has largely been displaced by other compounds (Grundy. 1994). have been used to preserve cosmetic formulations. saliva samples from volunteers who used a dentifrice containing these antimicrobials indicated that for 5 to 10 minutes after toothbrushing. Benzoic acid and its derivatives have been proposed for use as fungicides. 1968). in peanuts (Uraih and Offonry. The minimum inhibitory concentrations of sodium benzoate and dichlorobenzyl alcohol for 115 strains of dental plaque microorganisms were determined (Ostergaard. 1994). textiles. as a control for dentureborne Candida albicans (Lambert and Kolstad. However. . The chilling-induced production of ethylene in cucumbers was inhibited by the addition of sodium benzoate (Wang and Adams. liquid coolants. 1981). 1968). possible areas for specialized applications still exist — for example.. modified starch adhesives. A single oral rinse with a mouth rinse containing these antimicrobials did not affect plaque removal (Danielsen et al. concentrations of 0.5%.. adhesives for paper. It is currently used in animal feeds and in some tobacco products as a fungicide at levels of up to 0. 1997).. as a disinfectant for artificial kidneys (Kolmos. Sodium benzoate did not inhibit growth of any Gram-positive cocci. and leather. 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. These include animal feedstuffs. Hewala. 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. In the pharmaceutical industry. especially against toxigenic strains of A. 1986).. For example. and treatment for microbial infections in trees. Currently. 1967). Generally. cepacia grown under different conditions. insoles for shoes. 1996). A p-hydroxybenzoate-based system was effective in protecting the product against a variety of strains of P. OTHER APPLICATIONS Benzoic acid is one of the oldest preservatives in the cosmetic and pharmaceutical industries. 1996). including benzoic acid. wood preservatives.1% and 0. 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. this preservative system was ineffective against an adaptiveresistant strain.05% to 0. sealing gaskets for food containers. A benzoic acid-based polymer coating for apples has been developed (Ivanov et al. with perhaps the most pertinent consideration the pH of the superficial tissues (Eckert.. Methylcellulose was mixed with chitosan and 4% sodium benzoate or potassium sorbate to produce a food-grade film (Chen et al. used in cosmetics has been published (Bach et al. and as a preservative in certain brands of cough syrup (Chen et al. 1988. A mixture of fruit-coating polymers and potassium sorbate or sodium benzoate completely inhibited postharvest fungal growth on bananas (Al Zaemey et al. following official methods of analysis (Zani et al. benzoic acid was used for oral preparations in concentrations of 0. lubricants. 1994). incorporated as sodium benzoate. Patents have also been issued for the use of benzoic acid or sodium benzoate as an industrial fungicide in several products. However. Sodium benzoate has also been used to inhibit postharvest changes in fruits and vegetables.1%. flavus. varnishes. 1990). 1996).

7 (White et al.. Sax and Lewis (1989) reported data for several animal species based on the route of administration (Table 2.11).0 g/kg body weight 1.b rabbit.10. intramuscular. The first reaction is catalyzed by a synthetase enzyme. 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. In addition. Extensive human feeding trials conducted early in the twentieth century led Chittenden et al. The overall reaction sequence proceeds as shown in Figure 2.10 Toxicity of Benzoic Acid and Sodium Benzoate by Exposure Categorya Category Acute Species Tested Rat Guinea pig. Containers should be kept closed as much as possible. Its formation can be increased greatly by administration of benzoic acid. is then excreted in the urine (White et al.0 g/kg body weight Results 50% mortality 100% mortality Subchronic Chronic 80 mg/kg body weight 3% of diet 4% of diet (as sodium benzoate) 1 g/day 12 g per 14 days 0. the second is catalyzed by an acyltransferase enzyme. dry place.4–2.5% 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. synthesized in the liver.3–4.. However. cat.7–4. . both benzoic acid and sodium benzoate are moderately toxic by ingestive. Source: Adapted from Lueck (1980). This product is not corrosive. Hippuric acid. Excretion of benzoic TABLE 2. 1964). There is little danger of its being a toxicant or fire hazard under normal conditions of use. These have been summarized by exposure category in Table 2. 1964). 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. and intraperitoneal routes (Sax and Lewis.34 Antimicrobials in Food STORAGE AND HANDLING Sodium benzoate should be stored in a cool. Benzoate does not appear to be accumulated in the body.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. 1989). (1909) and Dakin (1909) to conclude that sodium benzoate was not deleterious to human health. Route of administration was per os. TOXICOLOGY Several toxicologic studies involving different animal species have been conducted using both benzoic acid and sodium benzoate.

lowest published toxic dose. b Source: Adapted from Sax and Lewis (1989). 35 . severe irritation effects.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. SEV. d Total dosage for 22 days to pregnant rats.Sodium Benzoate and Benzoic Acid TABLE 2. LDLo. Ipr. mild irritation effects. Ims. lowest published lethal dose. c Total dosage. subcutaneous. LD50. lethal dose 50 percent kill. TDLo. e Total dosage for 3 days to pregnant rats. MLD. intraperitoneal. intramuscular.

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

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

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

1989). The solubility of sorbic acid is also higher in alcohols. however. and inhibition of enzymes or transport functions (Sofos. 1989. especially potassium sorbate. alterations in cell membranes... stability. substrate properties. 1986. or both. 1989).50 Antimicrobials in Food activity against microbial growth and the potential application of the compound in additional food products (Sofos and Busta. as pharmaceuticals and cosmetics. 1989. changes in the genetic material. Sofos et al. sodium. Inhibition of bacterial spore germination (Sofos et al. including yeasts. The solubility of sorbic acid (Table 3. with a molecular weight of 112. 1989). 1980). in inhibition of microorganisms by sorbates.. The salts (e. 1986). the pH of the solution. 1981. sorbates are considered effective food preservatives when used under sanitary conditions and in products processed using good manufacturing practices.15 g per 100 ml. and in other industrial applications (Sofos. There is variation. depending on differences in microbial types. 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. molds. and it has a weak but characteristic acrid odor and acid taste. Sofos. Sofos et al. Robach and Sofos. especially ethanol. Sorbic acid forms colorless flakes or needles when crystallized. species and strains.2) in water at room temperature is only 0. especially in combination with reduced nitrite levels for a reduction in nitrosamine formation (Sofos et al. has an antimicrobial potency of 74%. and . Under certain conditions. trans-trans unsaturated fatty acid (2. 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. Inhibition of microbial metabolic function is probably the result of morphologic alterations in the structure of the cells. 1989). potassium sorbate) of sorbic acid may find more frequent applications in foods because of their greater solubility in water. which makes it valuable as a delayed-release form of sorbic acid (Sofos. but it increases with temperature. 1989. however. Sofos. as animal feeds.4-hexadienoic acid. Commercially available salts include calcium. The volatility of the compound in steam is useful in its isolation for quantitative detection in foods or other materials. Calcium sorbate has a water solubility of 1. 1981). Potassium sorbate may be manufactured as a powder or granules and.g.1). CHEMISTRY Sorbic acid is a straight-chain. and bacteria. CH3CH=CH-CH=CH-COOH).2% w/v at 20°C). and potassium sorbates. 1982). and environmental factors. 1982). The calcium salt is formed as a white odorless and tasteless powder. During the 1970s extensive research was performed on the potential for using sorbic acid and its salts as antibotulinal agents in meat products. 1981. while the health effects of sorbate were reexamined (Sofos. and in glacial acetic acid. on an equivalent weight basis with the acid. Currently. Good solubility (58.2% and is insoluble in fats.22. Robach and Sofos.. 1981. The molecular weight of potassium sorbate is 150.2). The extensive use of sorbates as preservatives is based on their ability to inhibit or delay growth of numerous microorganisms. Sofos. The sodium salt has a water solubility of 32% (wt/vol). it is available as a white freeflowing powder or as granules. probably through action on spore membranes or protease enzymes involved in germination (Blocher and Busta. 1985. are collectively known as sorbates and are used widely throughout the world as preservatives for various foods. sorbic acid and its more water-soluble salts. 1979a. 1979d) by sorbate is believed to occur at the connecting reactions of the germination process. In general.13 (Table 3. 1992). and it constitutes the most soluble form of sorbate (Table 3. some microbial strains are resistant to inhibition by sorbate or even metabolize the compound. The carboxyl group of sorbic acid is highly reactive and results in formation of various salts and esters.. In food systems the solubility of the compound is estimated as higher than 50%. The mechanisms of antimicrobial activity of sorbates are not fully defined (Sofos and Busta.

001 10 43 Decomposes 143 119 >98 0.4-Hexadienoic acid CH3-CH=CH-CH=CH-COOH 112.1 g — — — — — — >98 1. maximum ppm Arsenic content.5 10 3 0. malonic acid.13 126 1. Oxidation and losses of sorbic acid are inhibited by antioxidants. 1988. 1989). 1981). increasing the production of carbonyls. Sofos. acetaldehyde. % Water content. 1996). 1989). the partitioning of sorbic acid between the water and fat phases of foods depends on the pH of the food. Aqueous solutions of sorbates are unstable and degrade through oxidation. and anaerobic conditions (Sofos.4-Hexadienoic acid potassium salt CH3-CH=CH-CH=CH=COOK 150. Ethylenediamine tetraacetic acid (EDTA) enhances sorbate degradation probably as a result of iron scavenging from the packaging material (glass or polypropylene) through EDTA complexation.1 N HCL per 1. at 20°C 120°C 140°C Boiling point. Thus. such as sugars and salts (Sofos and Busta. 1992). maximum% Heavy metal content.0 10 3 — <0. °C 760 mm Hg 50 mm Hg 10 mm Hg Purity. . maximum% Ash. the type and amount of fat.. maximum% Source: From Sofos (1989).2 ease of manufacture make potassium sorbate the most widely used form in food systems (Sofos. which take part in nonenzymatic browning. malonaldehyde.22 — — 1. the higher iron content in the EDTA-containing systems might be responsible for the higher rates of sorbic acid degradation as well as for the increase in nonenzymatic browning observed (Campos et al. appropriate packaging materials. °C (COC ASTM D-92) Ionization constant at 25°C Density at 20°C g/ml Melting range. 1989.36 Decomposes higher than 270°C — ml 0. It is proposed that EDTA-Fe2+ complexes catalyze sorbic acid autoxidation.8 ml 0. 1995). Sorbic Acid 2. and β-carboxylactolein (Arya and Thakur. acrolein.73 ∞ 10-5 — 132–137 11.. The amount of sorbic acid in the aqueous phase is reduced in food systems of higher lipid content because the solubility of sorbic acid in fat is approximately three times that in water (Gooding et al. mm Hg. and other ingredients present (Sofos.1 N NaOH to 0.0 to 7. °C Heat of combustion at 25°C. 1992). Btu/Lb Alkalinity/acidity Vapor pressure. The partition quotient is increased from 3. such as crotonaldehyde. The rate of oxidation in aqueous solutions is increased at lower pH values by the presence of light and acids or increased temperatures.1 Physical and Chemical Properties of Sorbic Acid and Potassium Sorbate Property Chemical name Molecular formula Molecular weight Flash point. In general. Oxidation of sorbic acid yields various carbonyl compounds. formic acid. whereas in the pure dry form they are stable.Sorbic Acid and Sorbates 51 TABLE 3.927 — Potassium Sorbate 2.0 with increased levels (50%) of soluble food components.

moisture content..00 — — — <0.60 45.00 47. types of food materials present.00 0.50 12. processing conditions.34 8.0% enhanced sorbic acid destruction.00–5.25 0. 50% Ethanol. 1987.00 34.80 0.05 5.22 1.50–12.01 0.00 28.52 Antimicrobials in Food TABLE 3. 20% Ethanol.4) Water. 1992. 50°C Propylene glycol. 20°C (pH 5.03 — 0.50 2. 1983. 85. Gerschenson et al.00–15.90–14. Sofos. other additives present. 75°C Ethyl ether. 25°C Benzene.00 12. packaging material. 100°C Pentane.2 Solubility (%) of Sorbic Acid and Potassium Sorbate Solvent Water. 1994). 15% solution Acetic acid. 20°C (pH 3.30 Potassium Sorbate 58.12 0. 20°C Carbon tetrachloride.11 0.00 48. 20°C Acetone. 5% Ethanol. 95% Ethanol.20 12.01 Losses of sorbic acid during storage of foods depend on sorbate levels.b.15 0.15 0. 75°C Benzene.02 0. 1990). 20°C Sucrose. In general. 50°C Water. 50°C Benzene. 1986a.80 2.28 1. 1989. 20°C Corn oil.10 0.10 0.04 11.00 — — — — 57.01 — — 0.0% it had a protective effect (Guerrero et al..90 0.02 0. antioxidants. 85% Ethanol. 50°C Pentane.00 55. 20°C (pH 4.5% and 8.1) Water.30 2.00 64. 40% solution Sucrose.9) Water. 10% solution Sodium chloride. 5% solution Sodium chloride.30 6.. glacial Lactic acid.30 0.20 0. Sodium chloride at 3. 50% Phosphoric acid.26 0. 20°C Propylene glycol. 25°C Pentane. and storage temperature and time (Bolin et al. 20°C Methanol.60 1.00 5. 1984.20 — — 61. 50°C Soybean oil.15 0.00 0.14 24.5% Citric acid. 1980. 20°C Cottonseed oil.40 54. Sorbic Acid 0.00 0.00 0.00 — <0. 20°C Source: From Sofos (1989). light.07 0.00 45. 60% solution Sodium chloride. 100% Propylene glycol.60–14.10 16.52 1.01 58. metal ions.08 0.31 9. 20°C Glycerol.29 — 12. 10% solution Sucrose.08 2.55 4. oxidative degradation of . amino acids.16 0. 100°C Corn oil.50 0.00 20. 20°C Cyclohexane. the nature and pH of the food. but at 13. Vidyasagar and Arya.

Although the salts of sorbic acid have been commercially developed. suspensions. 1992). salt-. Sofos and Busta. and it is influenced by the nature of the food and its components and by processing.2) (Dudman. granules. 1989). Uptake and diffusion of sorbate into foods should be influenced by properties of the substrate. calcium or sodium (salts). such as composition. and storage conditions (Sofos. moisture. dry. sorbohydroxamic acid. Several procedures have been patented for the manufacture of sorbates (Sofos. ethyl sorbate. The lipid and protein fractions that appear to bind sorbic acid or its degradation products were identified with the help of 14C-sorbic acid. amine salts. Because the compounds are sensitive to heat. 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. 1994). was an effective mold inhibitor over a wide pH range (3. sorbamide. 1990). A proportion of the binding is with the low-molecular-weight components of these fractions (Jideani and Wedzicha. they are shipped and stored in fiber drums with a moisture barrier in the carton wall. 1976). strong offflavors and odors. other derivatives of sorbic acid have also been examined. physical state. and light. fura.. Commercial application of such derivatives. These include esters. alcohols. Commercial applications of sorbates include use of the free acid or its potassium and.6%).Sorbic Acid and Sorbates 53 sorbic acid in foods is less extensive than in aqueous solutions.9±0. and sorbic anhydride. 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. These cartons should be kept closed in cool. 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. 1963.5±0. 1992).6 to 9. 1989). 1995). methyl sorbate. with a dissociation constant (pKa value) of 8. and acetic acid-soluble fractions suggested that 14C activity tends to be associated with the low-molecular-weight components of these fractions. 1976). but commercial production of sorbic acid has mostly involved the methods of oxidation of . such as low solubility in water. Sorbamide was 1000 times more inhibitory than sorbic acid against yeast alcohol dehydrogenase (Martoadiprawito and Whitaker. or solutions. sorbohydroxamic acid. to a lesser extent. 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. Dialysis of the ethanol-. and potential health problems (Lück. the water-soluble (6. In general. aldehydes. A mixed anhydride of sorbic and palmitic acids called sorboyl palmitate has found some application in yeast-leavened bakery products.. the water-. and dark storage areas. moisture. 1980.8. some of these compounds are sorboyl palmitate. broadly tested.5±0.. and amide derivatives.7%). salt-. handling. 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. and sorbic aldehydes were also effective antimicrobials (Troller and Olsen.2 ± 0. In addition. This suggests that sorbic acid is indeed reacting with thiol groups in the flour protein. 2000.. and a further loss is apparent after the dough is cooked to give the traditional West African food. the salt-soluble (3. however. Specifically. The results show that sorbic acid binds with the lipid components. 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. the ethanol-soluble (4. 1986a. 2001). which are available as powders. and ethanol-soluble components of fura from pearl millet. Charvalos et al. When wheat flour doughs are treated with sorbic acid and heated.3%). Vojdani and Torres. structure. is hindered by disadvantages. 1989. and the acetic acid-soluble components (0.b.7%). Troller and Olsen. Sofos. and water activity (Giannakopoulos and Guilbert. New controlled-release. Fractionation of the lipids and proteins from fura showed the 14C to be associated with the petroleum spirit crude fat extract (28±5%). and widely used as antimicrobial agents. a significant amount of sorbic acid is not recovered by extraction with methanol. 1981). 1963).

Pullularia. Botrytis. Under this atmosphere and at aw values in the studied range (0. 1986. Sporotrichum. Zygosaccharomyces rouxii is considered resistant to sorbate treatments. 1970. and purification procedures follow the second method. Humicola.b. Torulopsis. 1979. and sorbic acid. Kaul et al. as well as against many bacteria (Sofos. 1983. Bhattacharya and Majumdar. potassium sorbate used at 150 ppm resulted in final counts. cottage cheese. syrups.. reaction conditions. Colletotrichum. Sorbates inhibit the formation of mycotoxins by various molds in culture media and in foods (Bullerman. 1957. Monilia. Matamoras-León.80 to 0. 1984. 1961. Rhizopus. Hansenula. 1999). 1985. whereas no growth was observed when the preservative concentration was increased to 220 ppm (El Halouat and Debevere. Gliocladium. Piper et al. were able to depress mycelial weight and aflatoxin production by Aspergillus flavus or Aspergillus parasiticus cultured in yeast extract-sucrose (YES) broth (Fan . 1998. Effective antimicrobial concentrations of sorbates in most foods are in the range of 0. Endomycopsis. Candida. Rosellinia. Sorbates also inhibit molds in butter. 1983. In addition to their effectiveness in fermented vegetables. 1984. Saccharomyces.. 1952. and chocolate syrup (Restaino et al. 1952. Pederson et al.. Geminder. Cunninghamella. 1998). 1989). 1985a. hydroxides. CO2 atmospheres. wines. Huang and Armstrong. Truncatella.b.. lower than the inoculum level (103 CFU/g).30%.54 Antimicrobials in Food 2. Fan and Chen. tomato products.. 1959.90). Kivanç.. Chaetomium. 1984. grains. 1954a. Skirdal and Eklund. 1984. Melnick and Luckmann. Gourama and Bullerman. Mucor. Aspergillus. and smoked fish (Liewen and Marth. Mold species inhibited by sorbates belong to the genera Alternaria. Marshall and Bullerman. Deuel et al.. 2000). Cephalosporium. Ascochyta. Rhizoctonia. the inhibitory amounts of sorbic acid were reduced by 40% to 50%. Yeasts inhibited by sorbates include species of the genera Brettanomyces. 1988). Aly. 1989). Helminthosporium. Fusarium. Baldock et al. Torulaspora. 1954a. El Halouat et al. jellies. 1985a). 1954a. Hurdles used in combination included aw. 1954a. Trichoderma. 1982. 1970. Liewen and Marth. Ulocladium. 1996). Debaryomyces. Penicillium. which is most widely used. 1998.. Geotrichum. neither sorbate nor propionate. applied at 10 mg/ml. sorbates inhibit yeasts in fruit juices. 1984. 1992. ANTIMICROBIAL ACTIVITY INHIBITION Sorbic acid is active against yeasts and molds. Sporobolomyces. candy. Melnick et al. 1996). Numerous studies have also documented the effectiveness of sorbates against molds (Emard and Vaughn. Rhodotorula.4-hexadienal and the condensation reaction of ketene and crotonaldehyde. The inhibitory action of sorbate against yeasts was first documented in the 1950s in fermented vegetable products. 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. Tsai et al. sausages. Ascosphaera..b. Pestalotiopsis. fruits and juices. 1972). 1985. Bracey et al. 1999. 1979. and others.. Under an 80% CO2 atmosphere. Cladosporium. Huang and Armstrong. Heterosporium. Ferguson and Powrie. and Zygosaccharomyces (Sofos. Although. Smith and Rollin. jams. 1993. 1993. dried fruits. Use of sorbates for inhibition of yeasts is especially important in low-pH and/or intermediate water activity (aw) products. Phoma.02% to 0. Tong and Draughon. Cryptococccus. salad dressings. Curvularia. Pichia. Lennox and McElory. A major application of sorbates in food products is their use for inhibition of molds in cheeses (Chichester and Tanner. use of a hurdle approach resulted in a synergistic effect (El Halouat and Debevere. However. Kloeckera. after 21 days. 1996. Pepularia. Sofos. Alkaline salts of sorbic acid are formed by its neutralization with alkali metal carbonates. Liewen and Marth. Numerous patents describing catalysts. such as carbonated beverages. The effectiveness of sorbates against yeasts has been documented by numerous studies (Emard and Vaughn. 1984. bread. Roland and Beuchat. Garza et al..b. and other suitable salts in the presence of necessary additives. and meat and fish products. cakes.

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

... Of 100 yeast strains isolated from spoiled foods and beverages. and concentration of sorbate. 1984. 1982. 1955. 1954. 1989). McMeekin et al. various species and strains of microorganisms exhibit different sensitivities to inhibition by sorbate. and two strains of Z. 1983. Piper et al. Saccharomyces.. 1982. Kondaiah 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. 1996. Yeast strains resistant to sorbate belong to the genera Zygosaccharomyces. Hansen and Appleman.. bailii tolerated 800 ppm of sorbic acid (Neves et al. Blocher et al. Varying sensitivities of bacterial species and strains to sorbate may lead to shifts in the microbial flora during storage of foods (Chung and Lee. potassium sorbate suppressed growth of Z. 1983. and Triganopsis (Warth. 1981. and previous exposure of the organism to low levels of sorbate (Sofos. a heat shock protein of S.. Exposure of Saccharomyces cerevisiae to sorbic acid caused strong induction of two plasma membrane proteins. 1995). or protection of enzyme systems from inhibition by sorbate through production of compatible solutes. 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. 2001). most tolerated 150 ppm sorbic acid. 1977. 1982. Lahellec et al. 1987). The induction of Hsp26. Exposure of S.. Lenovich et al. inoculum level. bailii in salsa mayonnaise more than sodium benzoate (Wind and Restaino.. which explains the usefulness of the compound as a preservative in vegetable fermentations.. Emard and Vaughn. Brettanomyces.... 2001). cerevisiae to 0.. When the yeast cells have been previously adapted to sorbate in media containing glucose or sucrose. 1971). 1994).9 mM sorbic acid at pH 4. Another bacterium that appears to be more resistant to inhibition by sorbate than other spore formers is Sporolactobacillus (Botha and Holzapfel. Torulopsis. 1951. 1987. 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. Cole et al. however. the inhibitory action against lactics by sorbate is less than that against yeasts. confers resistance to the inhibitory effects of sorbic acid (de Nobel et al. Functional categories of genes that are induced by sorbic acid stress included cell stress (particularly oxidative stress). 1950. Bills et al.. One proposed mechanism of resistance of osmotolerant yeasts has involved an inducible. Splittstoesser et al. Lynch and Potter. mating response.. 1983). Other proposed mechanisms of yeast resistance to sorbate at reduced aw have been related to yeast cell shrinkage and decreases in membrane pore size. York and Vaughn. 1952. 1985. 1978.. Candida. 1955. cerevisiae. Blocher and Busta.. . 1998). 1989). Variations and resistance to inhibition by sorbate may lead to failures in preservation and defective food products (Sofos. one of which was identified as adenosine triphosphate (ATP)-binding cassette transporter (Pdr12). which occurs during adaptation to sorbic acid. 1982. Restaino et al. sorbate concentration. 1985. Resistance of yeasts to inhibition by sorbate depends on species and strains. rouxii in high moisture prunes (El Halouat et al. 1985). 1982. 1988.5 resulted in the increased transcription and translation (upregulation) of genes encoding 10 different proteins and the downregulation of three proteins (de Nobel et al. storage temperature. retarding the flow of sorbate into the cell (Restaino et al. However. Vaughn and Emard. 1998). other studies have reported either no effect or inhibition of lactics and clostridia by sorbate (Costilow et al. Potassium sorbate or sodium benzoate resulted in complete inhibition of Z. Hamdan et al. Mihyar et al. 1988).56 Antimicrobials in Food of packaging. such as polyols (Bills et al.... subsequent exposure of the cells shows little effect of solute type on sorbate resistance (Lenovich et al. and energy generation. energy-requiring system that transports the preservative out of the cell (Warth. transposon function. 40% tolerated 500 ppm. 1982). Overall. pH. Resistance of osmotolerant yeasts to inhibition by sorbate was acquired by preconditioning the yeast to sorbate (Bills et al. 1989). 1977). 1955). In addition to bacteria. under certain conditions some species and strains of yeasts and molds are resistant to inhibition by sorbate. 1982).. 1981. In contrast. 1997). In general..

that there is no apparent relationship between sorbate resistance and the toxigenic properties of molds (Tsai et al.5-diene (Edinger and Splittstoesser. Other strains of molds that may degrade sorbate belong to the genera Aspergillus. a geranium-like odor is usually associated with wines treated with sorbate and contaminated with high microbial loads. 1975. 1954b. amount of sorbate present. The study showed that the resistance of Z. about a 7-fold increase in lethality (Splittstoesser et al. In addition to certain molds. Liewen and Marth.. Some mold strains can grow and metabolize sorbate under certain conditions as detected in cheeses and fruit products (Melnick et al. coli O157:H7 during storage .. some bacterial strains may also degrade sorbate under appropriate conditions. caused by ethyl sorbate. prior exposure to subinhibitory levels of sorbate. sorbate is completely oxidized to carbon dioxide and water. processing.3-pentadiene. and Geotrichum (Sofos. 1992).b). In general. 1954b. Sofos. sorbate yields 6. and type of substrate (Sofos. 1985a. The MIC of sorbic acid increases with the size of the inoculum. 2000). Lück.1%) reduced the D50°C of E. 2000). 1989. bailii resistance to sorbic acid activity. 1989). Fusarium. 1989.. 1989).b. It should be noted that 0. 1988). packaging. substantial metabolism of sorbic acid.Sorbic Acid and Sorbates 57 DEGRADATION Animals and certain microorganisms can metabolize sorbate. and other additives (Sofos and Busta. It appears that selection may occur in sorbate-treated cheeses for certain molds tolerant to the compound (Schroeder and Bullerman. Mucor. other ingredients. coli O157:H7 from 36 to 5. Sofos. 1986a. as a fatty acid through β-oxidation. Degradation of sorbate by molds depends on species and strains.. 1966. Products of sorbate metabolism by molds include 1. Sofos.6 kcal/g. 1985a). under normal conditions of alimentation. or binding of sorbic acid to dead cells. It appears. however. 1995). 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. Like caproic and butyric acids. Finol et al. level of inoculum. which is a volatile compound formed through a decarboxylation reaction and has a kerosene-like. This metabolism is mostly associated with lactic acid-producing bacterial strains present as high inocula in sublethal concentrations of sorbate (Crowell and Guymon. dehydration of cultures. of which 50% is biologically usable. Mold strains of the genus Penicillium isolated from cheese treated with sorbate were able to grow and metabolize high (0. 1-ethoxyhexa-2. 1966.18% to 1. such as concentration. Liewen and Marth. When sorbate levels are high. certain strains are resistant and can metabolize the compound. or hydrocarbon-like odor (Marth et al. Steels et al. Degradation of sorbate by lactic acid bacteria has been associated with geranium-type off-odors in wines and fermented vegetables. Increasing the concentration of sorbic acid from 200 to 1000 mg/L in apple cider decreased the D50°C value of E. In general. 1981. large inocula at high cell concentrations therefore require considerably higher concentrations of the inhibitor to prevent growth than do dilute cell suspensions. Sorbic acid (0. 1980). pH.. there is also evidence of some ω-oxidation (Deuel et al. inoculum size.2 min. 1977. and environmental factors. using it as a carbon source. temperature. aw... 2000). 4hexenoic acid. which was not an artifact caused by insufficient growth time. 1985a–c). gas atmosphere.20%) sorbate levels (Marth et al. 1989). These factors can act synergistically or be antagonistic and either enhance or negate the antimicrobial activity of sorbate (Sofos. 1981. Because it is metabolized like other fatty acids. A study found a pronounced positive inoculum effect of Z. 1989).. microbial flora. and 2-ethoxyhexa-3..1% sorbate is usually sufficient to inhibit sensitive molds (Liewen and Marth. 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..4-diene. under certain conditions. although many molds are sensitive to inhibition by sorbate. 1982). INTERACTIONS The antimicrobial activity of sorbates is influenced by compositional. Bullerman. plastic paint. 1985).. Horwood et al.

and it may inhibit the repair and growth of .b). it is believed that both undissociated and dissociated sorbic acids have antimicrobial activity (Skirdal and Eklund. In environments of pH higher than 6. 1982). the maximum pH for antimicrobial activity by most other common food preservatives is lower — for example. 1981. 1955. 1996). 1980. 1990).. Cerruti et al. In general. Studies have indicated that the dissociated sorbic acid also had antimicrobial activity..5 is advantageous because it allows for their use in foods of higher pH values in which preservatives. Statham and McMeekin. This popular theory has been questioned.. 1944. Robach and Stateler. 1955. sorbates have the advantage of being effective at pH values as high as 6. 1993). approaching its dissociation constant (pKa = 4.2 minutes. more than 50% of the inhibition was the result of dissociated sorbic acid (Eklund.64 min (Splittstoesser et al. Liewen and Marth. 1989. The antimicrobial action of sorbate is pH dependent and increases as the pH of the substrate decreases. Chung and Lee.. certain studies have indicated antimicrobial activity by sorbate at pH values as high as 7. 1979a.5 for propionate and benzoate. Lück. Preconditioning of Saccharomyces rouxii cells in 60% sucrose + 0. 1981. Beuchat. 1984. 1954a. Cerruti et al. 1987. supporting previous evidence that sorbic acid is effective in its undissociated form (Sun and Oliver. 1988). not as a substitute for appropriate sanitation and hygienic practices. as well as dormancy and recovery of heated microorganisms (Sofos. such as parabens. Sofos and Busta.0 to 5. Interactions of sorbate with heat may affect the rate and extent of microbial destruction during heating. 1983. which exhibited favorable antimicrobial properties against Vibrio vulnificus...0. Use of artificial saltwater prevented dissociation of the 1% sorbic acid. Sorbate may enhance heat activation and destruction of spores. Hoffman et al. have reduced the synergistic effect of sorbate and heat on thermal inactivation of microorganisms (Beuchat. whereas benzoic acid reduced it to 0. 1960). 1985a. Lück. Skirdal and Eklund. 1955.76) (Cowles..4 (Liu et al. and sodium chloride. 1983).58 Antimicrobials in Food of apple juice from 18 to 5. which is believed to be the effective antimicrobial form (Lück. where it is needed for microbial control (Oka.. The increased activity of sorbates at pH values higher than 5. salt and sugars) also reduce the concentration of sorbate in the aqueous phase (Gooding et al. Sorbic acid (0.. 1976. 1994). Sheneman and Costilow. 1985a). In contrast.g. 1981c). Sugar and salt. 1944.5 (Bell et al. 1956.0 to 4.1% sorbate rendered the cells more sensitive to inhibition by sorbate than preconditioning in 0% sucrose + 0. 1988). but increased levels of microbial contamination reduce antimicrobial activity. Sofos and Busta. Sofos. Nevertheless..1% sorbate (Bills et al. Sofos and Busta. respectively (Sofos and Busta. 1990). 1980. 1993).. Not only are certain strains of microorganisms resistant to inhibition by sorbate. In certain instances. Increased amounts of fat in a product reduce the concentration of sorbate in the water phase. Other food ingredients (e. 1982. 5. 1955. however. 1981. 1981a–c. however (Sofos.4 but not at pH 6.0 (Raevuori. sorbates should be used to preserve foods processed using good manufacturing practices. Although activity is greater at low pH values.1%) enhanced destruction of E. Sodium chloride (1. 1957. Rahn and Conn. however. 1976. coli O157:H7 cells at pH 3. Sofos et al. 1989).. glucose. however. 1981).5 and 4. 1997). Thus.. but it was 10 to 600 times less inhibitory than the dissociated acid (Eklund.5%) reduced the inhibition of Clostridium botulinum by sorbate in a nutrient broth (Wagner and Busta. 1989).. Sofos and Busta. 1983). Statham and McMeekin. The increased antimicrobial activity of sorbate at lower pH values has been attributed to the increased amount of undissociated acid present. 1976. 1959. 1988). 1989. 1976. however. Lund et al. 1981. Acott et al. Lowering the aw enhanced the resistance of the same organism to increasing concentrations of sorbate (Restaino et al. 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. Sucrose. 1982.. might not be effective owing to their increased solubility in fat. Gooding et al. act synergistically to enhance the antimicrobial activity of sorbate (Costilow et al. 1941..25 and 2. Cerruti et al. 1981). 1993).. solutes should increase the inhibitory activity of sorbate by reducing the aw of the substrate (Sofos. 1980.

Elliot and Gray. 1999).. 1989). Concentrations of sorbate as high as 0.. Tuncan and Martin. Huhtanen et al.. but growth of surviving spores that had been exposed to high temperatures was greatly inhibited by sorbate concentrations as low as 0. 1983.. 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.Sorbic Acid and Sorbates 59 thermally injured organisms (Beuchat. Inhibition of microbial growth by sorbate is more effective as storage temperature decreases. Sorbate may also eliminate the protective effect of sucrose against the thermal inactivation of yeasts and molds (Sofos.. 1961.. 1992). Myers et al. Although food acids may reduce the water solubility of sorbate. Cells grown in sucrose-supplemented media tolerated higher concentrations of sorbate than did those grown in glucose-supplemented media.. 1979. but the effect of sorbate on thermal inactivation and recovery of injured microorganisms is variable among species and strains. and propyl gallate (PG). cerevisiae depending on pH (Cheng and Piper. 1982. 1984). a true aerophilic mold involved in the spoilage of grape preserves (Splittstoesser et al.. the specific anion itself may contribute antimicrobial activity (Juven.5 it acted as a powerful inducer of thermotolerance in the absence of sublethal heat treatment. rouxii to sorbate was enhanced in cells preconditioned to elevated sorbate concentrations. Combinations of sorbate with antioxidants. cerevisiae (Cheng et al. Roland et al. Huhtanen and Feinberg.007% (Splittstoesser and Churey. Lopez et al.. Park and Marth. however. Sofos. 1988. 1999). McMeekin et al. The finding that plasmolysis increased in cells grown in sucrose. 1989). sorbate inhibited induction of thermotolerance by sublethal heat shock.. and types of acids (Restaino et al. 1996. Splittstoesser et al.. butylated hydroxytoluene (BHT)... Sorbic and benzoic acids affected the thermotolerance and heat shock response of S... Low concentrations of sorbic and fumaric acids in the heating medium had little effect on the heat resistance of Eurotium herbariorum. Another report also indicated that potassium sorbate inhibited the heat-resistant N. 1996).. fischeri (Nielsen et al. 1983). 1984. The antimicrobial activity of sorbate is usually enhanced under vacuum or modified gas atmosphere storage conditions. In addition. did not enhance the bactericidal activity of cellulose-based edible films against Salmonella Montevideo (Zhuang et al.. Gray et al. as indicated with several food items. Davidson .. Robach.4%). vary with substrates.. they can enhance its antimicrobial activity by increasing the concentration of undissociated sorbic acid. At low pH. It is of particular interest that 0. 1994. 1982.. but at pH 5. tertiary butyl hydroquinone (TBHQ). 1980. Sorbic acid (0. Park et al. 1981. Reductions of 5 to 10 log-units of E. 1983.as opposed to glucose-containing media provides evidence that cell morphology influences heat resistance (Golden and Beuchat.1% potassium sorbate did not have any significant effects on the heat resistance of Bacillus stearothermophilus spores in distilled water. Oloyede et al. Specific effects. 1980. 1995). Banks et al. Sofos. microorganisms. such as butylated hydroxyanisole (BHA). 1985). 1980. especially in media containing 0. indicating that the compound should be more useful as a preservative in refrigerated foods (Pederson et al. 1985). including meat (Wagner et al. 1973. 1981. 1982. 2000). McMeekin et al. 1981a–d.. Elliot et al. Heat resistance increased substantially when cells were grown in media containing sorbate. 1982.. 1996). Tolerance of Z. 1984) and fish products (Bremmer and Statham. 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. 1981. 1989). Statham et al.. however. Combinations of carbon dioxide and sorbate have also been reported as effective inhibitors of microbial growth (Danzinger et al. 1989). 1984. Roberts et al. 1982. 1989). 1985. 1989).2% to 0. 1984. 1970.1% sorbate. Lusher et al.1% had little effect on the thermal resistance of ascospores of Neosartorya fischeri. 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. Sorbic acid was found to induce thermotolerance without inducing the heat shock response through accumulation of trehalose in S. 1972.. Roland and Beuchat. had increased antimicrobial activity compared with individual components (Klindworth et al. 1994. 1984. and particularly in the presence of sucrose as opposed to glucose.. 1976.

aureus) using gradient gel plates (Thomas et al.8. 1995).. 1988). pH 3. Nelson et al. TellezGiron et al. Numerous publications and patents describe interactions of sorbate with many other factors.4 and addition of 250 ppm ascorbic acid.. 1990). 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. However. 1984. and Clostridium butyricum by adjustment of aw to 0. A study found that growth was not inhibited at pH 3.97 and pH to 3. and substrates. Bell and De Lacy.98. Kabara. 1989. Combinations of sorbate with benzoate or propionate may be used to expand the range of microorganisms inhibited with reduced concentrations of each preservative. pH. 1999).. 400 ppm sodium bisulfite. Miller and Brown.. and aw 0. certain amino acids. Marshall and Bullerman. 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.. 1994). however. At temperatures of 12°C and below. 1957. 1993. Variations existed. 1994). aureus. 1960. 1999). Lahellec et al. Deak and Beuchat. Sorbate was completely effective against E. In addition. Z. these concentrations of chemicals represented a greater than 50% reduction of the amounts needed to suppress growth when used individually (Matamoras-León et al. mixtures of sorbate with various antibiotics have demonstrated increased antimicrobial activity (Schmidt. 1985). 1986a. sodium chloride concentration. Amano et al. At <25°C.93. antioxidants. 1985. aureus when used with higher concentrations of NaCl (Thomas et al. Ough and Ingraham. The ability of C.. with types of microorganisms. These combinations offer the advantage of simultaneous inhibition of microbial growth and development of rancidity. 1993). 1986. 1982.06%) combined with aw values lower than 0.. 1981. Robach. glucose oxidase..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. and potassium sorbate was evaluated against growth of three foodborne bacterial pathogens (Bacillus cereus. 1979b. Robach et al. and other compounds (Ferguson and Powrie. Several studies have also indicated increased antimicrobial effects when sorbate was combined with various phosphates (Ivey and Robach. 1978. 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.. Clostridium pasteurianum.. 1983.60 Antimicrobials in Food et al. including sorbate. The effect of temperature. Potassium sorbate was found more inhibitory against yeasts than hydroxycinnamic acid (Stead. propylene glycol. Wagner and Busta.. as well as multifactorial interactions (Sofos. Thomas et al. 1983. 1994. cereus. Sofos. Poerschke and Cunningham. 1981. Seward et al... bailii is highly resistant to individual inhibitory factors. 1989). Guerrrero et al. 1984.06% potassium sorbate. Gourama and Bullerman. 1987. fatty acids. except nitrite when used against S. or at 10°C (Deak and Beuchat. at aw of 0. the log probability of growth of a single bacterium at 12°C after 60 d at pH 5.. molds. sucrose fatty acid esters.... After 14 days at 30°C in the presence of 280 mg undissociated sorbic acid/L. 1968. 1988). sulfur dioxide. 1987. 1994).5. 1981. verocytotoxigenic Escherichia coli. As indicated. 1960. coli at all temperature/pH/NaCl combinations and was the most effective preservative tested against B. 1990).95 (Deak and Beuchat. 1993). Bacillus coagulans. equivalent to a total sorbic acid concentration of 1300 mg/L. the probability of growth was approximately –6 (Lund et al. 1982. Morad et al. The microbial stability of shelf-stable banana puree was enhanced through inhibition of inoculated osmophilic and nonosmophilic yeasts. and S. 1994). sorbate was more effective than benzoate against S. and mild heat (Guerrero et al.. the log probability of growth of a single vegetative cell is approximately –4. 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.. Kadam et al. Interactions and increased antimicrobial effects have also been observed in combinations of sorbates with propionate. sorbic acid resulted in marked inhibition. 100 ppm potassium sorbate. ascorbate. Thomas and Wagner. in a medium containing 0. Mendonca et al.

Part 133 (2001)]. As yeast and mold inhibitors. objectives to be accomplished. pharmaceuticals.3%. the extent of the antimicrobial activity of sorbate under commercial conditions is difficult to predict because it is influenced by a variety of factors. In the United States. but levels as low as 0.02–0. cake mixes.F. Sofos.3). juices.10 0.25 0. as is the case with all food preservatives. and sugar and confectionery items (Sofos. icings.10 0. confectionary Source: From Sofos (1989).20 0. amounts of 0. APPLICATIONS Sorbates are widely used food preservatives throughout the world. 1981. doughnuts Vegetable products: fermented. relishes. dusting with a powder.4). mixes. fruit drinks.05–0.02–0. 1989. jellies.3 Common Applications of Sorbates as Antimicrobial Agents Products Dairy products: aged cheeses. product processing. 1989). animal feeds. sour cream. type of food. The most commonly used forms include sorbic acid and the potassium salt. olives. fresh salads Fruit products: dried fruit. certain meat and fish products. dry sausages Miscellaneous: dry sausage casings. and technical preparations that come in contact with foods or the human body. 1992). semimoist pet foods. edible fat emulsion products. spraying or immersing the food material in a solution. DAIRY PRODUCTS Cheese preservation is one of the most common applications of sorbates. cosmetic products.05–0. processed cheeses. cheese dips. higher levels may cause undesirable changes in the taste of most foods. .R. syrups. However.03 0. cheese spreads.03–0.30 In general. Although these concentrations have no major impact on food quality. Levels (%) 0. jams. packaging. 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. concentrates Beverages: still wines. federal standards of identity permit the use of sorbates as preservatives in more than 40 types of cheese. including dairy products. Application methods for sorbates include direct addition in the formulation.30 0. carbonated and noncarbonated beverages. toppings. cheese spreads. 1989) (Table 3. cottage cheese. and storage temperature (Sofos and Busta. yogurt Bakery products: cakes. 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.02–0. pickles. packaging materials. pies. presence of other ingredients and preservatives. sorbates have found wide application in various foods. fillings. fruit and vegetable products.Sorbic Acid and Sorbates 61 TABLE 3. Amounts of sorbate used in foods are in the range of 0. or addition in a coating or packaging material (Table 3. Selection of the most appropriate method depends on processing procedures. The compounds find application in human foods of all types. In general. In the United States. salad dressings Meat and fish products: smoked and salted fish. 1989).3% are tolerated. and convenience (Sofos.30 0. fruit salads. margarine. gas atmosphere.1% to 0. fudges. These factors include product pH and composition (fat and moisture). low-calorie drinks Food emulsions: mayonnaise.02% to 0. equipment available.05–0. purees.05–0. They should be considered as adjuncts to proper sanitation and hygiene. bakery items. sanitation and contamination.1% may be detectable in some foods. and cheese food [21 C.

3 0. Edam. blue.05–0.3 0. In these applications sorbates not only prevent cheese spoilage by mold. Caciocarallo.05–0. spreads Cottage cheese Bakery: icings.1–0. and domestic and imported cheeses.1–0. 1980).15 0.02–0. olives.1–0.02–0. 1980.075–0. cottage cheese).. and regulations.3 Spraying Immersion or spraying Dry mixing Direct addition Dry mixing or direct addition Source: From Sofos (1989). beverages. available facilities and equipment.4 0. Swiss cheese.05–0. 1981). however.1 0.05–0.03–0. the high activity of sorbates against molds.5–10 20–40 20–40 2.075 0.1 0.2 0. is limited to the surface of cheese. In certain products.e. Gouda Dry sausage or casings Dairy: Swiss.05–0. For application on . toppings. Addition of sorbate does not significantly affect the organoleptic properties of mozzarella cheese. Siciliano. where mold development usually occurs.3 0. and distribution. however.1 0. especially surface mold growth during storage. such as Roquefort or blue cheese and Camembert cheese. in which they can interfere with the desirable molds involved in the manufacture of these cheeses.3 0. fudges Vegetables: fresh salads (potato. 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. fillings. Monterey Jack. slaw.3 0. sauerkraut) Fruits: syrups. which may vary among countries (Lück.05–0.62 Antimicrobials in Food TABLE 3. and the improved action of sorbates at high pH values compared to other common preservatives (Lück.05–0.1 0.05–0. Muenster.1 0. Emmentaler Dairy: cheddar. 1976. relishes. 1996).3 0. Kivanç (1992) found that Penicillium was the major genus found on cheddar cheese.01–0. drinks Still wines Other: Margarine Semimoist pet food Bakery: pound cake Devil’s food cake Chocolate cake Solution (%) 20–40 2.15 0.5–5 5 2. sorbic acid may be incorporated into the cheese curd.05–0. in general.06 0.06 0. that sorbates should not be used in mold-ripened cheeses.1 0.05–0.1 0.1–0.3 0.1–0. pickles. It is obvious.1–0. aging. 1976. The method of application varies with the type of cheese.04 0.025–0. preservation objectives. Sofos and Busta. but they also protect human health by preventing the formation of toxic mold metabolites (mycotoxins). Sorbates are widely used in preserving cheese owing to the susceptibility of all types of cheese to microbial deterioration.25 0.5–5 5 Application Level (%) 0. there may develop a slight objectionable flavor (Aly.4 Methods of Application of Sorbates Methods Immersion Products Dairy: provolone.075 0. Gruyere. however (e. depending on the technique used to treat the cheese (i.15 0. vegetable.05–0.. pasta filata. Sorbate application.g. dipping or in brine salting). Colby.1–0.

0 with hydrochloric.6°C) temperatures (Kasrazadeh and Genigeorgis. Sofos and Busta. 1994). and it was initiated through extensive research performed during the early stages of sorbate testing. monocytogenes survival in two commercial cheese brines (Larson et al. 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. The appearance of Penicillium verrucosum var. Addition of potassium sorbate (≤6%) to mozzarella cheese inhibited growth of Streptococcus salivarius var. potassium sorbate (0. and 25°C after 30 days (Kivanç. including fresh. at the relatively high level of 1%. The compound is insoluble in fat and only slightly soluble in water. 1989. fermented. The use of sorbates in the processing of vegetables was one of the first applications. Weng and Chen.. These concentrations are in agreement with use of these preservatives in other dairy products. Inclusion of sorbic acid in cottage cheese was effective as a preservative but not against L. or propionic acid significantly increased the effectiveness of potassium sorbate. potassium sorbate inhibited L. soft cheese (Kasrazadeh and Genigeorgis. and (3) sorbic acid. 1981). or calcium sorbate impregnated into the packaging material or other coatings (e.. 1996).3%. VEGETABLE PRODUCTS The water-soluble salts of sorbic acid are used widely in the preservation of a variety of plant products.0) at levels of ≥0. ripened cheeses.1 to 0. 1995). bulgaricus. coli O157:H7 in queso fresco. respectively) extended the shelf life by 14 days. acetic. In addition to reducing cheese spoilage and the possibility of aflatoxin formation. In another study. thermophilus and Lactobacillus delbrueckii var. Concentrations applied to cheese surfaces range from 0.05% to 0. however. 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. when applied to the surface of hard. especially at lower (≤6. respectively) to maintain stability when kept at 5°C for 7 or 14 days. Amounts of sorbic acid permitted and applied in cheese preservation range from 0.3%) showed a significant effect in delaying growth of E.. 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. such as cottage cheese and yogurt (Mihyar et al.9. initial numbers of yeasts were low and relatively low concentrations of potassium sorbate and sodium benzoate (<150 and <250 mg/kg. use of sorbates in aged cheeses also reduces labor and other costs through reduction of the need for cheese washing and trimming. 1996). addition of potassium sorbate may increase the meltability and improve the fat leakage of certain cheeses (Aly. wax) used in cheese storage and marketing (Han et al. spray.. 15°C. 1980).Sorbic Acid and Sorbates 63 the surface of the cheese. Sofos. 1995). monocytogenes (Piccinin and Shelef. Labaneh with higher initial yeast counts needed higher concentrations of preservative (>300 and >400 mg/kg for K-sorbate and Na-benzoate. 1996). whereas when applied to packaging films.3%) or sodium benzoate (0. In labaneh (a yogurt product) produced under strict hygienic processing measures and effectively chilled. Levels of 0. Milk acidification to pH 6.07% sorbic acid are generally used in direct addition to cheese. (2) a sorbic acid powder for dusting. Treatment of mozzarella cheese with potassium sorbate in the kneading water or brine may be more effective than dipping (Aly. and pickled vegetables (Sofos. 1998. 1997). and the cheese was stored at ≤30°C (Kasrazadeh and Genigeorgis. Furthermore. which can be used to dip.05% to 0. Growth of Salmonella in soft cheese was inhibited if the cheese was prepared from milk that was acidified with propionic acid to pH 5. 1989). potassium sorbate was added to the cheese (pH 6. 1997).3 g/dm2. 1976). This research demonstrated the effectiveness of sorbates against undesirable organisms without interfering with the desirable . one of the following procedures may be followed: (1) potassium sorbate in solution or in a brine. potassium.g. 1999). amounts of 2 to 4 g/m2 are used (Lück. 1994).30% (Lück. respectively (Aly. or wash. and was especially effective against mold and yeast contamination by Penicillium roqueforti and Mucor miehi. 1980. 1992). Calcium sorbate may be preferred in cheese preservation over other forms of sorbate owing to its solubility properties.

Other fruit products that may be preserved with sorbates include maraschino cherries and strawberry puree.1% sorbate and 0.10% may be used to preserve refrigerated fresh salads. which may influence the survival of E. In sweet relishes. 1994). coli O157:H7 and result in spoilage of the product (Zhao et al. coli O157:H7 in apple cider. Because potassium may result in potassium bitartrate precipitation in some wines. At these concentrations the selective action of sorbic acid allows growth of the desirable lactic acidproducing organisms.02% to 0. fruit cocktails. Other vegetable products preserved with sorbates include tomato products and fermented Asian sauces (Chichester and Tanner. such as yeasts. 1980. preserves.g. Potassium sorbate and sodium benzoate were highly effective in reducing yeast and mold populations in tomato juice (Bizri and Wahem. prunes. sodium sorbate may be used to avoid the problem. sorbates are used mostly at the preprocessing stage. 1952. FRUIT PRODUCTS Fruit products preserved with sorbates throughout the world include dried fruits. fermentation).02% to 0. or bacteria. The higher moisture content in these products is preferred by the consumers. In these products. Also.02% to 0. cucumbers and olives).. Lück.. smaller quantities of sorbic acid are adequate for preservation because of the synergistic action of sorbate with sugar (Lück. sorbates are used to prevent refermentation (Parish and Carroll. raisins.b).. and wines.05% are adequate for preserving high-moisture dried fruits (e. the lower is the sorbate level needed for preservation. molds. but it makes them susceptible to mold and yeast spoilage. Good wine preservation is achieved with concentrations of 0.02% to 0. Sorbate concentrations as low as 0. coli O157:H7 growth was inhibited in both tryptic soy broth and in apple cider by sorbic acid applied at 0. jams and jellies). 2001).1% sodium benzoate and 0. sorbate acts as an inhibitor of yeasts.05% and as such may be an option that processors have to reduce contamination in their products (Koodie and Dhople. sorbate is more effective than benzoate owing to the pH of these products. fruit juices and syrups. In wine processing.05% sorbate to a 15% to 20% brine retards yeast scum formation. 1989). 1993). Depending on the particular product. Sorbate levels in the range of 0. dried fruits). however. In fruit juice and drink processing. sorbate is either added directly into the product or applied to the surface of the product or packaging material. Levels of 0.05% sorbates in the fermenting and holding tanks. Combinations of sorbic acid and sulfur dioxide are also used in the preservation of high-pulp fruit juices. and putrefactive bacteria. while additionally suppressing the growth of yeasts and molds. jams. Sofos. In these products sorbic acid acts as the microbial inhibitor and sulfur dioxide prevents oxidation and enzymatic spoilage (Lück.05% to 0. salt concentration).. the pH values of fermented green olives favor the use of 0.g. to inhibit chemical. jellies.64 Antimicrobials in Food fermentations. 1980).02% sorbic acid in combination with . 1980)..10% are adequate in improving the preservation of soft drinks. 1980). however.10% sorbate reduce the number of bloaters and poor seed cavities.. 1988a.20% inhibits growth of organisms not desired in vegetable fermentations.05% and 0. Potassium sorbate was found more effective than chitosan in inhibiting growth of Aspergillus niger on lowsugar candied “kumquat” (Fang et al. Selection of precise levels of sorbate.1% potassium sorbate substantially inhibited E. molds. Mold and yeast spoilage is also prevented by sorbates in pickled vegetable products (e. enzymatic.02% to 0. Potassium sorbate is preferred because of its solubility. in high-sugar products (e.g.g. 1972. 0. or microbiological deterioration (e. 1994).g.. Use of the combination of 0. depends on products and specific fermentation conditions (e. 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.5% acetic acid completely inhibit microbial spoilage at sucrose concentrations ranging from 2% to 40%. The lower the moisture content of the product (e. beverages. Concentrations as low as 0. Also. In sweet cucumber packs. together with sulfur dioxide and pasteurization. Use of sorbates at levels between 0.. Costilow et al. Addition of 0. Another study showed that E.g.. Lück. figs). 1955.

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

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

1%)–benzoate (0. garlic oil. This should assure mycotoxin-free animal feeds and thus eliminate any hazards associated with mycotoxin-contaminated feed. 1988). The literature contains reports on several other food products that may be preserved with sorbates (Sofos. 1980. prepeeled carrots. 1976. 1976. Sorbic acid levels in the range of 0. the flavors of both types of bacon were equally desirable (National Academy of Sciences. Sorbic acid at a concentration of 0. Several studies have reported improved preservation of various types of fish with sorbate (Geminder.Sorbic Acid and Sorbates 67 of bacon treated only with nitrite.. Application of potassium sorbate with bifidobacteria extended the shelf life of whole and peeled shrimp by 3 days (Al-Dagal and Bazaraa. 1983. Debevere and Voets. cucumbers. dried cod). 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). such as margarine. are also effective in preventing mold growth on animal feeds during storage.05% to 0. sorbic acid may be mixed with its potassium salt or with other preservatives. 1995). shredded cabbage. They include high-moisture marshmallow candy. 1959. tomato juice. 1991). Shaw et al.g. Lück. Bremner and Statham. at which they prevent growth of molds and osmophilic yeasts in items such as fillings for chocolate and pralines (Chichester and Tanner.30% can be effective mold inhibitors. MISCELLANEOUS FOOD PRODUCTS Sorbates are commonly used in the preservation of oil-in-water or water-in-oil emulsion-based products. potato chips. In addition. 1978. 1972. orange peels. Fey and Regenstein. 1978. Sorbates may be particularly important in intermediate-moisture (25% moisture content) pet foods. as well as fermented plant foods (Chichester and Tanner. mayonnaise. however. 1989).20%. and pancake batter (Sofos.. in which the emulsifiers may be inactivated by nonionic compounds (Chichester and Tanner.1%) solution preserved brined shrimp for 59 days. delicatessen items.g. thus. 1972. One study.. 1976). This optimal concentration of sorbic acid coincided with the lethal dosage for most of the associated fungal microorganisms during malting. diastatic powers. Chung and Lee. 1972. OTHER APPLICATIONS Other items that may be preserved by sorbic acid and its salts include pharmaceuticals. Lück. especially those of the emulsion type. 1986). ethyl and propyl) may find applications in cosmetics and pharmaceuticals. 1972. salad dressings. 1989).1%) was used (Fletcher et al. 1981). cut squash. and animal feeds (Sofos. which is longer than the preservation (31 days) achieved by the most effective bacteriocin (nisin) tested (Einarsson and Lauzon. cosmetic products. Robach and Sofos.. and similar products. 1982). Sofos and Busta. botulinum in shucked scallops developed slightly more rapidly at 27°C in vacuum packages when sorbate (0. and cold and hot water extracts of the malt samples. 1999). Bremner et al. such as benzoates.. Lynch and Potter. A sorbate (0. Regenstein. Sorbates are also used to prevent mold growth on the surface of dried and smoked fish (e. it may be desirable to apply sorbic acid during malting (Ogundiwin et al. Statham and Bremner.03% to 0. Sharp et al. 1982. 1980. In Asian countries sorbates are commonly used in combination with other preservatives to preserve fish sausages and similarly processed fish and meat products. Lück. Mexican-type hot sauces. 1983. Robach and Hickey.5% did not adversely affect the germination energies and germination capacities of sorghum grain and did not significantly affect malting loss. In certain instances and for better preservation. 1983. strawberry puree. 1982.10%.. Other food-related applications of sorbates include sugar-based and confectionery products at concentrations of 0. inhibiting their growth. pineapples. Sofos. tangerine sherbet base. 1989). 1983. sour cream. in which levels of up to 0. indicated that toxin production by C. 1989). 1981. Samples treated with sorbic acid lost biological activity within a few days of aerobic storage (confirmed by aerobic . depending on moisture content. the esters of sorbic acid (e.. 1982.

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

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

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

is smaller. Sorbic acid has been shown to inhibit microbial growth. (3) suppression of glucose-triggered peak of hexose monophosphates. (2) inhibition of glucose.2 to 10. and because it is a metabolizable fatty acid. sorbate is considered a GRAS compound. 1954a.or longterm exposure. the LD50 for common salt (NaCl) is 5 g/kg body weight. Neutralization of the PMF is probably not involved in growth inhibition in the presence of carbohydrates because they do not depend on this force for their transport (Sofos. On the whole. in such situations. Demaree et al. and environmental conditions (Sofos.. For comparison. Rat feeding studies have indicated that a dosage of 10% sorbic acid in the diet could be tolerated for 40 days (Lück. 1989. Deuel et al. 1980). Acute toxicity studies with rats have determined LD50 (mean lethal dose) values for sorbates in the range of 4. Lück. 1993). 3.. Thus. On extending the feeding period to 120 days. Overall. the World Health Organization has set the acceptable daily intake for sorbate at 25 mg/kg body weight per day. . Although dissociation of the acid inside the cell may eliminate the pH difference across the membrane.. and (5) block of catabolite inactivation of fructose-1. Thus. carcinogenicity. and teratogenicity after short. 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). Sofos. 1998. as well as influence on metabolism. 1980. 1989).Sorbic Acid and Sorbates 71 apparent after the subsequent addition of glucose. 1992). 1993). which is the difference in electrical potential. Additional studies with rats and dogs have shown no damage with feeding 5% sorbic acid for 90 days.. the mechanisms involved in inhibition of microbial metabolism and proliferation by sorbic and other similar lipophilic acid preservatives appear to be different. although less efficiently. unlike former assumptions. Overall.5 g/kg body weight (Smyth and Carpenter. TOXICOLOGY AND SAFETY In the United States.and fructose-phosphorylating activities. but 8% sorbic acid resulted in an increase in liver weight (Deuel et al. the growth rate and liver weight of the animals increased (Demaree et al. these studies demonstrated the relative harmlessness of sorbates and their relative superiority in safety compared to other chemical additives. 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. 2000). 2000). Reasons for this conclusion include the following: 1.6-bisphosphatase and phosphoenolpyruvate carboxy-kinase.. 1954a. but not of cytoplasmic malate dehydrogenase (Burlini et al. this pattern resulted in prevention of glucose-induced switch of metabolism from a gluconeogenic to a glycolytic state. The compounds were fed to various animal species for determination of acute toxicity. These effects can be summarized as follows: (1) reduced glucose consumption. depending on the type of microorganism. The relative nontoxicity of sorbates was established early in their testing as food preservatives. Bracey et al. 1998. Thus. 1973. 2. sorbate is considered one of the least harmful preservatives in use. even at pH values near neutrality. its effect on the other component of the PMF. Lück. based on acute toxicity. 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. uptake of the compound based on pH difference across the cell membrane would not be adequate to explain the observed antimicrobial activity.6-bisphosphate. 1954a).. it is believed to be adequate to energize the uptake of substances needed for cell maintenance and growth. substrates. 1955. (4) substantial reduction of glucose-triggered peak of fructose 2.. 1976). however. Food and Drug Research Laboratories. However. 1948. Sofos.

1981. to some extent. Food and Drug Research Laboratories. Some taste panelists reported certain “allergic-type” symptoms after tasting uninoculated experimental bacon (U. In general. and vegetable juices and is inactivated by heat (Kada. it may react with nitrite in products such as cured meats. 1981). In addition.. Thus. 1992). Khoudokormoff.. including lysine and glutamate. As preservatives for cosmetics and pharmaceutical products.S. (2) formation of the c-nitroso mutagens requires the presence of excess nitrite. 1989).. show skin irritations when exposed to sorbic acid (National Academy of Sciences. The literature is contradictory. probably because the acid environment of the stomach leads to the breakdown of the sorbic-protein adducts (Quattrucci and Masci. 1979. Lück.. This reaction was demonstrated in model systems but. 1979b). 1981. may undergo nonenzymatic browning reactions when stored with sorbic acid at a favorable range of aw and temperature (Quattrucci and Masci. of course. 1989). 1974. 1975. does not rule out the possibility that high concentrations of sorbate may act as irritants to certain susceptible individuals (Sofos. the potential for such irritations in commercial products is minor. 1980. Litton Biometrics. It has been regarded that amino-compounds. Walker (1990) concluded that sorbate use. and Robach and Adam (1980) were able to induce such symptoms from panelists consuming commercial bacon manufactured without sorbate. Sofos. 1979b. Osawa and Namiki.. which is the average pH of most meat products. 1980. 1968. not at 6. Although it was implied that sorbate might have been involved in producing those symptoms.0.10% to 0. Sorbic acid has a conjugated system of double bonds. 1981. which makes it susceptible to nucleophilic attack. 1970. 1981. Department of Agriculture. but it indicates that most people are not affected by sorbic acid applied to the skin. Osawa et al. 1980) and because sorbic acid is an unsaturated fatty acid. leading to decreased amino acid availability (Quattrucci and Masci. 1992). 1979. The products of such reactions have the potential of being mutagenic (Sofos. Considering the average use levels of 0. 1975. 1993. Gaunt et al. Sofos and Busta. does not appear to create toxicologic problems. in general. and some very sensitive individuals may show irritations even at lower levels (Lück. 1982. 1986. 2001).b). 1980. sorbates have been extensively examined for skin tolerance (Lück. Because there is evidence of reactions between nitrite and fatty acids (Benedict. National Academy of Sciences.S. Dejobert et al. Robach et al. sometimes giving mutagenic products (Ferrand et al. 1974. cysteine.. No abnormalities have been observed. 1992). 1981. Some sensitive individuals. forming high-molecular-weight products that do not seem to result in a nutritional loss. 2000a. no direct relation could be proved between symptoms and specific ingredients used in formulating the bacon (U. 1994). 1999). Shtenberg and Ignat’ev. 1982). botulinum. Berry and Blumer. various studies have indicated the following: (1) nitrite reacts with sorbic acid to form mutagens optimally at pH 3. 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.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. This. There has been some concern about possible sorbate–nitrite reactions when added to the same substrate (Sofos. however. Department of Agriculture. 1976.. 1982). 1977. 1976).. and (3) mutagen formation is inhibited by such ingredients as ascorbic acid. it may apply also to more complex systems such as foods. apparently because of an increased caloric intake resulting from the metabolizable sorbic acid.30% in food processing. 1978. Sorbates were found to react easily with proteins. 1980a. and in some instances the growth rate of sorbate-fed animals increased significantly.5. Under conditions typical of . No carcinogenic or mutagenic effects have been observed with sorbate alone (Dickens et al. Kito et al. 1982. Average sorbic acid concentrations for skin irritations are in the range of 1%. no such symptoms were observed by other individuals who tasted sorbate–nitrate experimental bacon from other studies. Namiki. however. 1976. 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. Hendy et al. 1989). Patrizi et al. 1983..

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

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

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

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

75 4. 1980). The pKa of most organic acids lies between pH 3 and 5 (Table 4.87 4.08 3.98 pK2 pK3 5. The inhibitory effect of acids has been compared based on pH. and protect color. it would be advantageous to use the acids near these values.44 5. The multiple-barrier concept (“hurdle” concept) has gained popularity for use in a variety of foods in recent years. EFFECTIVE USE Various approaches have been used to study the efficiency of antimicrobial effects on cells. chain length.41 4. This concept is based on the premise that foods can be preserved using several inhibitors concurrently rather than relying on a single factor. but also the decreased processing time would aid in preserving the palatability of the product (Corlett and Brown. gaseous atmosphere.75 5. Effective use of an acidulant depends on the dissociation constant (pKa) or the pH at which 50% of the total acid is dissociated. and chemical analyses can be found in the references by Ash and Ash (1995) and Doores (2002.34 6. Not only would this interaction ensure commercial sterility of the food.03 3. pK1 4.40 4. Hoffman et al. or molarity to achieve final pH.77 4. The use of the multiple-barrier concept results in lowering the concentrations of acidulants or inhibitors. This primary concern limits the use of acidulants to foods with pH values of less than 5. They can control pectin gel formation. (1939) observed that acids with fewer than 7 carbons were more effective at lower pH.Organic Acids 93 TABLE 4. In addition to any antimicrobial effects an acid possesses. Because some experiments express concentration in percent. 2003).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). Acids contribute to the taste and tartness of a product.14 3.89 3.0.27 4. water activity. aid in the inversion of sucrose. They can create a synergistic relationship with antioxidants by chelating metal ions.61 4. and degree of branching to inhibit or kill a wide variety of microorganisms. Some can be used . and acids with 8 to 12 carbons were more effective at neutrality and above. Given that this value lies at the lower limits of growth for many bacteria. Comprehensive texts describing applications. Because the undissociated portion of the molecule is believed to be responsible for the antimicrobial effect. Therefore. concentration. normality. type.16 2. prevent browning. the choice of an acidulant may depend on secondary effects.39 times.11 5. 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. and other inhibitory compounds. adjustments in pH levels can be coupled with changes in temperature.43 4. organic acids are usually more effective as antimycotic agents.1). physical parameters.

1966) The toxicology and safety of the acids must be considered when selecting an acid for use. At the levels and pH values in which these acids are normally used in foods. application. the same hazards would not be expected. 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.9). L. and molds. which do not. 1939) inhibited Bacillus cereus (pH 4. however. The long-term consumption of these acids.0 than lactic acid bacteria. many of these studies used high levels of the concentrated acids at low pH values. This association could explain the apparent stability that occurs in natural fermentations. all of the groups were similarly affected (Woolford. which exist naturally in many foods. and caprylic acid. The results of these studies are usually presented in the section describing the acid that gave the most antimicrobial effects. would further indicate that hazards are unlikely. are more tolerant to this acid than homofermentative lactic acid bacteria (homolactics). yeasts.0. propionic acid. ACETIC ACID Antimicrobial Properties Acetobacter and heterofermentative lactic acid bacteria (heterolactics) produce acetic acid as a byproduct of their metabolism and. 1975b). The salts of the acids are important in regulating the acidity of the foods (Gardner.4 to 5.9) and Aspergillus niger (pH 4. THE ACIDS Information on the toxicology. McDonald et al. 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. plantarum at pH 4. Adams and Hall (1988) showed a weakly synergistic inhibitory effect between acetic and lactic acids with Salmonella enteritidis and Escherichia coli.7. and regulatory status is presented here for each acid. These acids. The succession typically begins with growth of coliforms and natural microflora followed by Leuconostoc mesenteroides (heterolactic). the information appears separately for each acid. although they could also serve other functions in foods. Most of the acids appear safe for use in food products. Microorganisms vary in their susceptibility to acetic acid. Owen (1946) suggested that acetic acid inhibited Gram-positive organisms. Although these acid mixtures might be expected to occur naturally during fermentations by heterolactic bacteria. Gram-positive bacteria. as the name implies. In some instances. Concentrations of acid lower than those needed to inhibit Saccharomyces cerevisiae (pH 3.1) (Levine and Fellers. Numerous studies included in this chapter compare several organic acids for their antimicrobial effects. and Staphylococcus aureus (pH 5. all these acids can be metabolized by the body primarily through lipid oxidation and via the tricarboxylic acid cycle. as the pH decreased to 4. such as sauerkraut. The lipophilic acids include acetic acid and acetic acid derivatives. mesenteroides ceased when the internal pH of the cells reached 5. are relatively nonpolar compounds. however.0. . These organisms are found naturally associated with fermented products. 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. such as pickles. and vinegar.6 to 4.9). Salmonella Aertrycke (pH 4. a more acid-tolerant organism. as such. Several animal studies have indicated possible safety hazards. Bacillus and Clostridium species and Gram-negative bacteria were more inhibited at pH 6. In addition.94 Antimicrobials in Food as chemical leavening agents. (1990) found that growth of L.0). then by Lactobacillus plantarum (homolactic).8. sauerkraut. this apparent synergy has not been demonstrated in other foods. antimicrobial properties. They are usually added to foods for their antimicrobial properties.

0 with 0.7% acetic acid with reduction to undetectable levels in 1 and 2 weeks with 0. tartaric. Reduced-calorie mayonnaise (RCM) manufactured with 0. although more resistant than Salmonella. 1979). A 0. 1982).5. 1940). coli.0% lactic. monocytogenes. 1991). respectively. Buffered acidulant systems (brain heart infusion [BHI] broth. malic.7% acetic acid (Glass and Doyle. S.5%. respectively. coli O157:H7 is considered more acid tolerant than strains of E.7% acetic acid. malic.6.5 (Noda et al.4 to 4.5% and 0. most likely because of the absence of egg yolks in CFM and the presence of egg white. citric.1-M concentration of acetate and lactate completely inhibited multiplication at pH 6. 1988). 1997). aureus was most sensitive to acetic.3%. other ingredients can interact with acid to influence the degree of inhibitory action.8% reduced growth of Micrococcus and Bacillus species. cereus was inhibited at pH 5. E. respectively. pH 4. which has recognized antimicrobial properties.15 M acetic acid and with 0. In addition. monocytogenes than lactic and citric acids and increased inhibition as the temperature of incubation .0 (Conner and Kotrola. pH 4. B. Acetic acid exerted the most inhibition but had the lowest dissociation constant. whereas tartaric acid had one of the highest dissociation constants yet exerted the weakest inhibitory action. The effect of temperature-acid interactions is noteworthy for the psychrotrophic pathogens. some strains of S. Malic acid allowed growth at pH 4. or 0.6) at 20°C shifted to pH 7. 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. and Enterobacteriaceae in Japanese fermented soy sauce (Hayashi et al. Similar results were noted when various acids were compared at the pH level required for a 90% and 99% reduction in numbers of S. and HCl. Salmonella Blockley. 1988). in the aqueous phase of CFM and RCM would still produce a microbiologically sound product. monocytogenes was also reduced to undetectable levels in RCM within 10 days and in CFM within 14 days compared to 2 days for Salmonella.. L. Acetic acid was effective at pH 5. 1970). especially with the use of pasteurized egg products. and tartaric acid permitted growth at pH 4.0 buffer.0% acetic acid and 1. coli than unbuffered systems. followed by lactic. aureus in 12 hours (Minor and Marth.. Destruction of Salmonella occurred more quickly in CFM than RCM. Acetic acid caused greater inactivation of L. aureus. A concentration of 0.3.5.0.Organic Acids 95 S. pH 4. Conner et al. Both studies concluded that the antimicrobial effect was the result of the undissociated molecule. aureus preconditioned in acetate buffer (pH 4.2 and 4.1 and 5. tartaric. or citric acid were far more effective in reducing numbers of S.9 and 4. lactic. 0.0. but no injury occurred in cells not exposed to acidic conditions before heating (Smith et al. respectively. When E.6.3% acetic acid formulations. Traditionally acidic foods contain a single or multiple acids. citric. 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. L. followed by citric. 22°C) containing 1.3% and 0. pH 4.2 and 5. coli O157:H7 was inoculated into tryptic soy broth (TSB) containing acetic. pH 4. 1995.. It is unlikely that these products would contain such high initial levels of either of these organisms. and tartaric acids. E.4% to 0. and E. Streptococcus faecalis.0 with hydrochloric acid was compared to cholesterol-free (egg yolk-free) reduced-calorie mayonnaise (CFM) manufactured with 0. coli O157:H7 grew in all media at pH 5.5 to 3. added or developed through fermentation. 0. such as mayonnaise (Debevere. and caused 50% inhibition of spore germination at pH 4.1%.0. lactic.6 and 4.. and hydrochloric acids (Nunheimer and Fabian. This finding is important in the manufacture of medium acid foods containing more than one organic acid. and exposed to 40°C became injured. At 25°C and 37°C.8.33 M lactic acid (Wong and Chen. monocytogenes and Yersinia enterocolitica.9°C. L. 1984). Salmonella were inactivated within 48 hours in both RCM and CFM formulated with 0. was reduced by 4 logs in both mayonnaises within 72 hours in formulations containing 0. growth occurred at pH 5. malic. therefore the standard use of 0. aureus.0 with scant growth in TSB containing acetic acid. Gram-negative aerobic bacteria.5 for all acidified media at 10°C except for acetic acid.7 and 4.7% acetic acid in the aqueous phase and adjusted to pH 4.7% acetic acid.

. coli O157:H7. citric. 1990. Dorsa et al. coli O157:H7 to nondetectable levels by enrichment methods. The greatest antimicrobial effect occurred at 35°C with the greatest survival at 10°C. and hydrochloric acids at equal pH values and at all incubation times and temperatures (Sorrells et al.0 have been used to rinse pork. there can be adverse sensory effects including discoloration of meat.0. Parsley. Numerous organic acid combinations.9 log reduction for the nontreated seeds (Weissinger et al. 1992.7 log without affecting germination of seeds. monocytogenes irrespective of temperature. and pH 4.0 for 30 or 60 seconds (Biemuller et al. Depending on the treatments. When comparisons were based on equal molar concentrations. 1997. 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. A variety of organic acid treatments has been used to control microbial loads associated with meat carcasses and products (Dickson and Anderson. enterocolitica. seed germination was effected (Weissinger and Beuchat.. but resulted in changes in sensory characteristics. malic. Treatments spanned varying amounts of time with concomitant decrease in microbial loads up to 3 logs. Mung bean seeds inoculated with 3 to 5 logs of S. Acetic acid displayed the greatest antimicrobial action against L. Sorrells and Enigl (1990) also demonstrated interactive effects between sodium chloride. Smulders and Greer.33 and 5. 40%. and no reduction in microbial .6% to 3. 2000). Y.01 N for 75 min. enterocolitica was not detectable after exposure to 2% and 5% acetic acid or 40% and 50% vinegar for 30 minutes (Karapinar and Gönül. or 5%) or vinegar (30%. off-odors. Little et al. lamb. Typhimurium. 1991. Acid injury did not appear to involve damage to ribosomes or other nucleic acid material. A 90% reduction of the population required exposure to 0.96 Antimicrobials in Food decreased from 35°C to 13°C. containing from 0. monocytogenes was isolated from 2 of 10. However. In addition. and temperature. injury and death rates were not affected by refrigeration temperatures for Salmonella Bareilly. a twofold reduction at pH 2. Acetic acid was more effective than lactic or citric acids in producing this effect (Adams et al. Typhimurium and E.10 N for 5 min. monocytogenes when compared to citric. 0. citric acid caused greater injury than either lactic or acetic acids and injured organisms survived longer at low temperatures (Ahamad and Marth.5 to 3. and hydrochloric acids. 1998. a mesophilic organism. Similar growth inhibition was demonstrated for Y. 1992).8 to 5.5 min. This treatment reduced S. Pork carcasses inoculated with 106 CFU/ml S. 1997. 1989).4 for malic. 1981). vapor treatment also reduced levels of resident microflora on seed but did not affect seed germination rates (Delaquis et al. monocytogenes were exposed to acetic acid at a concentration of 242 µL/L of air for 12 hours at 45°C. Because produce is typically refrigerated with only minimal processing. respectively. 1973).. Brocklehurst and Lund. Treatment at low temperature (4 days at 10°C) with 200 and 500 mg/L of air was effective in reducing Salmonella by 2.05 N for 8.0 for acetic acid. and beef carcasses or meat tissue.4 to 4.. but L. Enteritidis were sprayed with acetic acid at pH 1.0% acetic acid at pH levels from 1. and off-flavors.. 25-g samples. however. 1998).5. but malic acid was more bacteriostatic than other acids at 10°C. 0.3% inhibited L. 1992b).5. pH 4. concentration ≥0. citric acid was more bactericidal than other acids at 25°C and 35°C. enterocolitica. but recovery required the presence of amino acids or peptones (Blankenship. and L. these foods can be the carriers of psychrotrophic pathogens. enterocolitica at lower pH levels as the temperature increased. 1992a.2 N for 4 min.. Dorsa. 1 log unit at pH 2. Total plate counts were reduced by 4 logs at pH 1. was dipped in acetic acid solutions (1%. and 0.72 logs. lactic. inoculated with approximately 107 CFU/g Y. Immersion of alfalfa seeds in mixtures containing 5% lactic or citric acid for 10 minutes resulted in several log reductions. Karapinar and Gönül. such as Y.6 for lactic acid. 1990). 1999). E. or 50%) for 15 or 30 minutes.. 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. In addition. Exposure at 50°C for 12 hours with lower levels (100 and 300 mg/L of air) reduced the population >1. Conversely. but acetic acid was less effective. 2001). 1989.5 or 2. 2%. The minimum inhibitory pH level was pH 4. acidulants.

vacuum packaged.. Unda et al. dirt.8 log CFU/cm2. Temperature played a greater role in reducing numbers than did the acid concentration. 1997).0. 1994).5. 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..4°C (Anderson et al.. Typhimurium. or manure. type of chemicals. 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. however. coli O157:H7. 1995). 1974).Organic Acids 97 numbers at pH 3. Hardin et al. organic acids can be used in preevisceration rinse systems consisting of a water rinse followed by an organic acid rinse.046% formic acids for 10 seconds caused discoloration from both treatments. As part of recently established guidelines by the U. Quartey-Papafio et al. 1983). 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. Cutter and Siragusa. resulted in less effective reduction of S.6% acetic and 0. 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.. At this concentration.S. Typhimurium was reduced by 0. Beef cubes treated with 1. Typhimurium. Typhimurium on beefsteaks (Tinney et al. respectively.5 and 11 of 72 for pH 2.2% acetic acid or 0. An 18% acetic acid or 12% lactic acid spray significantly reduced bacterial counts on lamb carcasses. They found that acetic acid was a superior sanitizer compared to hypochlorite and had a greater residual effect. S.8 to 4. the pH level decreased to 2. 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. Cutter et al. An increase in organic material. Typhimurium reductions were greater when the bacteria were attached to fatty tissue. (1987) used intermittent sprays of 1% acetic acid or lactic acids. Dorsa et al. Incorporation of pulsed power electricity and a 2% acetic acid spray led to improved reduction of inoculated E... and application rate and sequence in the process (Gorman et al. or manure slurry.. a final application of acid washes could provide some residual effect during storage (Brackett et al. (1977) reduced bacterial counts by 99.5% acetic. Anderson et al.6% on meat using a 3% concentration of acetic acid before washing. some discoloration occurred (Cacciarelli et al. S. 1994). Typhimurium followed by treatment with 2% acetic acid. The treatment effectively reduced recovery of S. 1986). Department of Agriculture Food Safety Inspection Service (FSIS). 1994. Dickson (1992) contaminated lean and beef tissue surfaces with S. possibly because this tissue retained less moisture (20%) than lean tissue (75%) and the reduced water activity could have enhanced the antimicrobial effects. but beef treated with the mixture of acids did not differ in flavor from untreated beef (Bell et al. 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.. citric. 1994. Enteritidis to 1 of 6 samples at pH 1. Hamby et al. and lactic acid bacteria counts. however.. L. however. 1995). 1989b). 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. Reductions in microbial counts depend on the water temperature. 1997. 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. coli. Meat was sprayed. Microbial loads on beef carcasses subjected to machine washing and sanitization with 1.. Although washing of pork carcasses with 1. coli O157:H7 and S. 1997. It was noted that the use of acetic acid as a rinse for beef tissue led to sublethal injury of bacterial cells.. and bleaching of the carcasses occurred (Ockerman et al. 1996). monocytogenes was detected in 69% of loins and 33% of chops..0.1 and 1. and stored for 28 days at 2°C in a high-oxygen barrier film. this was not significantly different from the controls. suggesting that acids had limited effectiveness (Fu et al. (1991) inoculated beef roasts by injection and on the . Lactobacillus species predominated as storage progressed.3 log/cm2). 1987).5% acetic acid were reduced more when treatments were conducted at 52°C than at 14. anaerobic.. S. (1980). such as rumen fluid. which resulted in significant reductions in aerobic plate count (1.5 to 0..

0% acetic acid caused instantaneous death. Kurita and Koike (1983) combined 3% ethanol. although the skin of the 0.0 on inhibition of L. 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. respectively. and tartaric acid adjusted to pH 5. Typhimurium using a model system.6% acetic acid-treated carcasses was darkened or yellowed (Dickens et al. 1987). and 0. Addition of 1.5 to 1. 1976). with and without the use of air injection to agitate the chill water. Poultry scald water containing 0. Other studies continued to explore the use of acetic acid in chill water.98 Antimicrobials in Food surface with approximately 103 CFU of Clostridium sporogenes and L. further poultry processing can reintroduce microorganisms onto carcasses.0% acetic acid compared to 2 days for 0.71 log most probable number (MPN)/ml (Dickens et al. Rubin (1978) found that acetic and lactic acid acted synergistically to retard growth of S. but changes in pH and concentration of acid were more successful.6% acetic acid under the same conditions.. 1 mM perillaldehyde—or 0.5 with acetic acid was the most effective antimicrobial treatment followed by other acids in descending order of effectiveness: adipic.5 logs (Okrend et al. The acetic acid in the brine inhibited anaerobic and aerobic bacteria. Despite the reduction in microbial numbers at this pH. Typhimurium and Campylobacter jejuni by 0.. greater than a 4% concentration of acid was needed to reduce levels by 2 logs (Tamblyn and Conner. aerobic plate counts were unaffected by the treatments.. (1985) found that mushrooms reached an equilibrium pH of 4. germination and outgrowth of Bacillus . 1986). see Chapter 7) coupled with 0. monocytogenes.5% concentrations of acetic.35 log MPN/ml for the 0. of the same components—with 2% salt to inhibit growth of contaminating microorganisms for more than 20 days at 27°C. monocytogenes. The acid level could be reduced when used in combination with brines and sugar. fumaric. Nisin (an approved bacteriocin for some foods. Products are now being preserved by a number of antimicrobial compounds using a multiplebarrier approach to preservation. citric.3% and 0.. succinic. Immersion of broilers for 10 minutes in 0. 20°C. 1994). Acetic and tartaric acids were more inhibitory than lactic and citric acids. lactic. Again.2% to 0. 1994). acetic acid was the most destructive acid at all combinations of parameters. citric.05% acetic acid. Levels of 0. Intervention steps incorporated into poultry processing have been effective in reducing microbial contamination. Air injection did not affect reduction of these counts. El-Shenawy and Marth (1989b) investigated the effect of acetic.24 log (Lillard et al. but Enterobacteriaceae counts were significantly reduced by 0. Stroup et al.5% acetic acid reduced Enterobacteriaceae counts by 2.6 depending on the concentration of acid in can brine within 1 day with 1. Dickens and Whittemore (1994) exposed broilers to 0.7% acetic and 4 days for the same concentration of citric acid. 1997).5%. As the incubation temperature to recover spores was lowered.9% acetic acid did not support growth or toxin formation of Clostridium botulinum (Ito et al.1% acetic acid at 52°C decreased levels of S.. Acidified media showed greater inhibitory activity at 35°C than at 13°C. 1994).0) did not result in a significant reduction of aerobic plate counts but did significantly reduce levels of Enterobacteriaceae by 0.05% and 0.86 log MPN/ml for the 0. and 30°C (Oscroft et al.7% citric acid. Expanding on their work. Because of the possible attachment of Salmonella to broiler chicken skin. and lactic (Mountney and O’Malley.3% acid and 2. 1990).05%. Adjustment of the chill water to pH 2. 0. Despite these treatments.. Although each individual acid influenced the heat resistance of Bacillus spores differently.6 or 5. There was no significant difference in texture or sensory characteristics between the treatments. 0. 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. Whole pickles preserved with brine at 12° Brix and 0. Water pockets occurred under the skin of chicken carcasses with air-agitated samples (Dickens and Whittemore. For pearl onions. 1965). acetic acid caused the skin of the poultry to be hard and leathery.6% acetic solution acid (pH 3.35% solution required 7 days for equilibration for acetic and citric acid but 1 day for 0.6% acid solutions.5 mM. lactic. a 0.

2% (0. All rats showed mucosa damage owing to the feeding method. 1971). Ingestion of acetic acid by humans has caused burned lips (Palmer.0.2% acid at pH 5.. aerobic bacteria.0 and below to inhibit growth (Kirby et al. gastric erosion (O’Keane et al. Johnson (1968) noted that rat gastric mucosa responded to a 100-mM concentration of acetic acid by releasing histamine into the interstitial fluid. Studies on nonmammalian species showed that acetic acid at a concentration of 0.001 M accelerated spindle activity and retarded anaphase in the mitotic cycle of the root tips of the onion. Damage from acetic acid resembled that from ionizing radiation or radiometric chemical damage (Manna and Mukherjee. chilled.5% level. Nisin. glucono-delta lactone. Although acetic acid is generally used as an inhibitor of bacteria and yeasts. a 1% concentration of acetic acid at pH 4. interference with blood coagulation (Fin'ko.2 or 5 mg/m3 but not at 0.5 inhibited Saccharomyces ellipsoideus and Penicillium glaucum. rats decreased their food intake level and lost 2. 1968). Other forms of acetic acid have antimicrobial uses. niger. Rats exposed to vapor levels of acetic acid showed muscle imbalance at 0.999% after exposure to a 1. On contact with organic substrates. Allium cepa (Makinen. 1969). 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.2). pneumonia (Gerhartz. Rats consuming acetic acid in drinking water in concentrations of 0. lactic.8% to 1. Rats fed 10% acetic acid and cats fed 20% acid developed ulcers in the gastric mucosa (Okabe et al.01% to 0. cabbage. ready-to-eat products. it is effective against the black bread molds. followed by glucono-delta lactone. 1949).04 M acetic acid at pH 3 to 4 showed chromosomal and chromatid breaks.01 mg/m3. Takhirov (1969) suggested limits of 0. Toxicology Acute toxicity data for acetic acid in mice and rats indicate varied tolerance levels by different routes of administration (Table 4. When used as a surface application.5 completely inhibited growth and aflatoxin production by Aspergillus parasiticus. Spore-forming bacteria are killed on surfaces at a concentration of 0. and Rhizopus nigrificans at pH 3. 1966).8% partially inhibited growth and decreased toxin formation by 70% and 90%. are especially susceptible to spoilage because of indigenous microbial populations. 1932). Parmeggiani and Sassi (1954) reported that workers . Populations of Pseudomonas aeruginosa were reduced by 99.5.06 mg/m3. and citric acids when temperatures were reduced to 65°C for 60 minutes.48 mg/m3 altered sensitivity to light. 2000). however... At the 0.0001 and 0. it decomposes to yield oxygen and acetic acid.0001 and 40% relative humidity within 10 minutes (Portner and Hoffman. A.8 and 0. lactic. 1976). Lower concentrations of 0. but a concentration greater than 4% was needed when the pH was at 7. renal failure (Paar et al.29 mg/m3 caused changes in electroencephalograms.Organic Acids 99 spores were more restricted. and coliforms. however. Peracetic acid is formed by an oxygen molecule bound to the carboxyl carbon atom of acetic acid. in combination with organic acids. suggesting its use as a decontamination agent for equipment (Hedberg and Miller.6% or 0. 1968).2 g/kg body weight) did not show any adverse effects. it was not possible to determine the actual quantity of acid consumed. Aspergillus fumigatus required 0. Grasshoppers treated with 0. 1969). Foods. and potatoes than peroxyacetic (80 ppm) acid alone (Hilgren and Salverda. and citric acid when spores were heated at 95°C for 15 minutes and acetic.0% concentration of acetic acid. It was found that acetic acid was the most effective acid. 1958). A combination of these parameters could be used to extend the shelf life of these products.4% acetic acid at pH 5. and death.0% acetic acid adjusted to pH 3. 1921). respectively (Buchanan and Ayres. Cruess and Irish (1932) showed that apple juice containing 0. 1937). This treatment could also be used in recycling of water to reduce levels of yeasts and molds.6% of their body weight (Sollman.. particularly precooked. Mori (1952) fed rats acetic acid ranging from 10 to 50 cm3 acid/kg of rice. Human olfactory thresholds to acetic acid vapor were 0. and 0.60 mg/m3. 1971). Levels of 0. displayed synergistic effects in increasing the destruction of the organism.

430–4. pharynx. although it is assumed that this derivative would be assimilated in a manner similar to that for acetic acid.020 5. No toxicologic data were available for peracetic acid.340 580–1. and tartaric acids.200 485 Reference Spector (1956) Orö and Wretland (1961) Spector (1956) Spector (1956) Spencer et al. sodium Dehydroacetic Adipic Rabbit Caprylic Citric Mouse Mouse Enders (1941) Orö and Wretland (1961) Yokotani et al.100 Antimicrobials in Food TABLE 4.65 mg/L. (1971) Rat Citrate.460 1. sodium Tartaric Spector (1956) Orö and Wretland (1961) Hara et al.700 725 884 5. and epidermal reactions (Weil and Rogers. Men having 7 to 25 years of exposure displayed blood abnormalities (Cesaro and Granata.790 940–960 42 203 961 2. (1971) Gruber and Halbeisen (1948) Horn et al. (1950) Horn et al.860 2.310–3. cold sensitivities (Wiseman and Adler.700 11.730 1. and lungs with an air concentration of 0. (1971) Gruber and Halbeisen (1948) Gruber and Halbeisen (1948) Lactic Propionic Propionate.960 525 3. as well as citric. eyes. calcium Propionate. 1955). (1957) Gruber and Halbeisen (1948) Yokotani et al.900 680 275 2. teeth.000 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. (1963) Horn et al. In rare instances acetic acid. (1963) Hara et al.2 to 0. 1956).530 380 1. 1956).210 338 3. sodium Rabbit Mouse Rat Rabbit Rat Guinea pig Mouse Rat Rat Mouse Gruber and Halbeisen (1948) Yokotani et al. lactic.430 600 5.100 1.380–3. .500 330 44 1.810 625 3. 1951).040–5. (1957) Acetate. (1957) exposed for 7 to 12 years to acetic acid vapors in the production of acetyl cellulose displayed corrosion of the hands. caused allergic reactions by producing canker sores (Tuft and Ettelson.

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

untreated samples reached the spoilage mark within 10 days. 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.. Waterhouse and Scott (1962) attributed the toxic effect to the sodium ion rather than the acid moiety.5% trisodium citrate with or without B. 1999). breve.25% acetate and 2. Treatment with SA alone or in combination with bifidobacteria extended shelf life at 4°C by 3 days (Kim et al. 1994).. the indicator of spoilage. A 22-year-old woman with sensitivity to acetic anhydride was not allergic to SA (Weil and Rogers. sodium lactate (3%). 1996). 1995b). potassium sorbate (1. 1997).75% and 1% significantly reduced the initial aerobic plate counts of catfish fillets by 0. sodium lactate. monocytogenes in vacuum-packaged turkey bologna during storage at 4°C and was similar to 0. Psychrotrophic counts from untreated whole shrimp increased to >107/g.to 6-week storage life (Blom et al. 1. Histologic findings showed hypertrophy of the kidneys and ureters.0% SA alone inhibited growth for more than 6 days at 4°C and counts increased only by 1. and 1.. Catfish fillets treated with 2% SA. but they did appear brownish and watery (Kim et al.8%.5% potassium sorbate. A combination of B. or trisodium citrate were used alone or in combination with Bifidobacterium breve (5%) as a dip for fresh camel meat (Al-Sheddy et al.25% potassium sorbate.. B. The combination of 0.3 or 1. and increased mortality. cremoris culture added to catfish fillets was an effective treatment against growth of Gram-negative spoilage bacteria at 4°C (Kim and Hearnsberger. 1976).5% SA.7% SA and 0. and 2.6% or 0. growth and toxin formation were decreased 70% and 90%. respectively (Buchanan and Ayres. 1994). breve culture and stored at 4°C for 9 days (Al-Dagal and Bazaraa. whereas potassium sorbate increased shelf life to 9 days.3% to 0. Chickens fed diets supplemented with 5.26% potassium sorbate or 2% sodium citrate in its efficacy (Wederquist et al.102 Antimicrobials in Food SA (10%). 1995a). over 6 days. depressed growth rate.. SA alone or SA-MKP-treated fillets were not significantly different in odor from fresh controls up to 9 days.5% lactic cultures or 0. Toxicology The LD50 (mean lethal dose) for SA in mice is presented in Table 4. although MKP alone had no effect. For peeled shrimp.5.. and held at 4°C for 12 days contained lower population levels of psychrotrophic and anaerobes than untreated samples (Zhuang et al.0% concentration of SA at pH 4. SA (0. 1963). SA in combination with potassium sorbate and Lactococcus lactis ssp. breve and SA exhibited an additive effect that extended shelf life still further. 1951).7 logs when stored at 4°C compared with the nontreated fillets and increased shelf life by 6 days. although a mild acetic acid odor was detected. 1999). .2. and trisodium citrate extended shelf life to 6 days.5%) was also effective in reducing populations of L. 0. Whole and peeled shrimp were immersed for 2 minutes in solutions containing 10% SA. 5% sodium lactate. At lower acid concentrations of 0.4% monopotassium phosphate (MKP) more effectively prolonged shelf life to 12 days compared with SA alone.5 to 1.44% SA showed decreased appetite.5%). parasiticus was inhibited by a 1.5% lactate was effective as an antimicrobial treatment in sliced servelat sausage over a 4.6 to 0. Color remained acceptable. SA extended shelf life to 12 days at 4°C. No toxicologic data were available for calcium acetate (Food and Agricultural Organization. but bifidobacteria did not grow within 9 days. SA at concentrations of 0.2 logs by day 12. A combination of 0. The combination of 0. but SA provided better sensory characteristics and more controlled growth of psychrotrophs than potassium sorbate with bifidobacteria. Growth and aflatoxin production of A. although it is assumed that this derivative would be assimilated in a manner similar to that for acetic acid. vacuum packaged.

1185) acetates are approved as GRAS substances for miscellaneous and general-purpose usage.55.5% at pH 4.5 inhibited Aspergillus flavus. L.5% and 3. monocytogenes was not significantly different in the presence of the individual inhibitors compared with control samples after 20 days at 10°C. 32 mM (pH 5. such as Bacillus mesentericus (subtilis). fumigatus.2% sodium diacetate or 1% buffered sodium citrate and within 3 and 5 weeks for products containing 2.3% sodium diacetate was effective in suppressing total aerobic counts. L. Sodium diacetate at concentrations of 0. SA can be used as a boiler additive for food-grade steam (Federal Register.1% to 0. enterocolitica. Sodium diacetate was more effective than acetic acid.4) at 20°C. monocytogenes and bactericidal . sodium diacetate was also effective against hemorrhagic E. vacuum-packaged.4% at pH 3.2% sodium diacetate.1% or 0.3% and sodium lactate at concentrations of 2% to 3% were equally effective as antilisterial compounds in meat with little effect on pH and sensory characteristics (Mbandi and Shelef. a concentration of 0. coli. L. in products containing buffered sodium citrate. Aspergillus glaucus. and 6.8% and 2. or 1% buffered sodium citrate (Stekelenburg and Kant-Muermans 2001).3 and incubated at 5°C. Saccharomyces species (Glabe and Maryanski. S.1721) and calcium (21 CFR 184. and B. Enteritidis were inoculated into a comminuted beef emulsion formulated with 0.5 inhibited these same strains and Mucor pusillus in feeds and silage. monocytogenes were inoculated into the cooked ham products at an initial level of 104 and 102/g of product. and the inhibitory effect was attributed to the diacetate moiety rather than pH alone. 1994).2% sodium diacetate.2% sodium diacetate.25) at 35°C. or S.1% and 0. 1981). monocytogenes and S.5% level in malt syrups. However. respectively.15% to 0. it prevented mold growth (Chichester and Tanner. A. Enteritidis. A concentration of 0. and when it was incorporated into butter wrappers. monocytogenes. 6. 2.Organic Acids 103 Application and Regulatory Status Sodium (21 CFR 184.25. Y. 5.0. Cooked-in-bag ham was formulated with additions of 0. Lactobacillus curvatus and L. Lactobacillus fermentans. L.0% delayed mold growth in cheese spreads. monocytogenes was inhibited by sodium lactate and 0.2% SA. Pseudomonas fluorescens.2% sodium diacetate and 2. In ground beef. Enterococcus faecalis. the addition of 0. In addition to L. 2001). The minimum inhibitory concentration (MIC) of sodium diacetate was determined for L. 1994).55) at 5°C. An interactive effect was noted between temperature and concentration of acid in that growth was inhibited with decreasing concentrations of acid and temperatures. Levels of 0. and 35°C (Shelef and Addala. Calcium acetate is classified as a stabilizer when migrating from food packaging materials (21 CFR 181.1% and 0. The addition of sodium diacetate affected the sensory qualities of the product precluding its use. A. 0. curvatus reached an end point of 107/g within 2 to 2. The MIC was determined to be 35 mM (pH 5.5 weeks in products containing 0. the combination 0. or 1.5% or 3. 1972).3% sodium lactate. 5. and 28 mM (pH 5. Sodium diacetate is equally effective in the baking industry where its inhibitory powers prevent the growth of bread mold and rope-forming bacteria.05% to 0. 20°C. However.5% sodium lactate was bacteriostatic to L. niger. 1962). cereus with no effect against Pseudomonas fragi. monocytogenes increased to approximately 106/g within 3 weeks. Shewanella putrefaciens. while having little effect on baker’s yeast. SODIUM DIACETATE Antimicrobial Properties Sodium diacetate was effective at a 0.4. and stored for up to 40 days at 4°C. monocytogenes grown in BHI broth adjusted to pH 5.5% sodium lactate or combinations of the acids and held at 5°C or 10°C.29). The growth of L. and Penicillium expansum. aureus (Shelef and Addala.1% to 2.3% sodium lactate.

Toxicology No toxicologic data were available for sodium diacetate (Food and Agriculture Organization. plantarum at levels of 0. niger (Banwart.54 log reduction). DEHYDROACETIC ACID Antimicrobial Properties Dehydroacetic acid (DHA) has one of the highest dissociation constants (Table 4.8.5 logs within 6 days. a fermentation by-product of lactic acid bacteria. Application and Regulatory Status Sodium diacetate is approved as a GRAS substance for miscellaneous and general-purpose usage (21 CFR 184.5%) was listericidal in combination with ALTA™. prepared with sodium diacetate (0. The powder dissolved in the surface moisture of the carcass to form acetic acid and SA and produced a surface pH of about 4.5%). 1993a). a more complex product. (1993b) used the multiple-barrier concept to limit growth of L.1% (Wolf. Sodium diacetate was determined to be listericidal over the 7-day period at 25°C only at the 0. In subsequent work (Mbandi and Shelef. . Sodium dehydroacetate was twice as effective as sodium benzoate at pH 5.. It is used in cheese spread. 1950).1) of the acidulants and remains effective at higher pH ranges.6 logs in crabmeat stored at 4°C for 6 days when treated with 2 M sodium diacetate. respectively. 0. populations increased approximately 0. At lower temperatures.5% sodium lactate and 0.8 logs (Degnan et al.4% (Glabe and Maryanski. monocytogenes was reduced by 2. 1962). In bread and cake products. As expected. the reduction of multiple strains of L.3% and 0.5%). cerevisiae and 25 times more effective against P. Schlyter et al.3% and 0. Enterobacteriaceae counts dropped 1000-fold over 6 days at 2°C. monocytogenes by 0.104 Antimicrobials in Food to S. Although 1 M sodium lactate solution reduced populations within 2 days. were pasteurized for 10 minutes at 68°C after which they were inoculated with L.005% to 0. Mixtures were incubated at 25°C or 4°C and sampled up to 7 and 42 days. Sensory characteristics of roasted chickens were not affected even at levels up to 8 g per chicken (Moye and Chambers.3%. butter. The growth of L.1%. producing a 2-log reduction after 7 days at 25°C (Schlyter et al. Sodium diacetate (0. Lactate and pediocin enhanced the listericidal properties of diacetate suggesting some type of synergistic activity. and wrapping material. stored aerobically at 5 and 10°C. use levels range from 0. 1994). monocytogenes at a level of approximately 4. 1991)..5% concentrations (0. but sodium diacetate reduced the pH slightly. glaucum and A. respectively. lower concentrations of the antimicrobials were equally as effective. Sodium lactate did not affect the pH of the meat. Enteritidis. 2002).5% concentration (0. monocytogenes and Salmonella species was demonstrated using 2.25% to 0. monocytogenes in turkey mixtures. L.5 logs CFU/ml slurry. malt syrups. Slurries of uncured turkey breast. 0. although it is fungistatic at concentrations of 0. the combination of acids achieved greater reductions of both organisms at both temperatures compared to individual acids and greatly reduced background spoilage microflora.59 log reduction). 1989). although it is assumed that this derivative would be assimilated in a manner similar to that for acetic acid.1754). Application of 4 M SA reduced the levels of L. and pediocin (5000 U/ml). 1981).0 against S.1%. 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. monocytogenes in mixtures held at 4°C for 42 days was inhibited at 0.2% sodium diacetate alone or in combination in beef bologna. This acid is effective against Enterobacter aerogenes and L. sodium lactate (2.

1953). mortality.02%. in addition to acidosis by the former route and intestinal adhesions by the latter route (Horn et al.1%. The 90-day feeding trials conducted on rats fed 0. 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. coli O157:H7 in broth and model meat products. appearance.2.130). but significant growth depression . benzoic acid. Furthermore. However. although a dose of 200 mg/kg led to growth and organ changes. Sorbate was bacteriostatic at half the MIC for E. It can also be used as a fungistat in cheese wrappers.. benzoic acid.0% adipic acid showed no effect at the two lowest levels. 1971). weight gains were lower and mortality was higher than in the controls (Lang and Bartsch. Enders (1941) showed similar symptoms in rabbits. thus extending marketability (Watada. The effects of potassium sorbate. treatment with sodium dehydroacetate was effective in reducing respiration rate and delaying ripening. Oral administration of the acid to mice caused hemorrhaging in the small intestine with distension of the stomach. and sodium dehydroacetate acid were investigated for three strains of E. altered behavior.. or 100 mg/kg of DHA per day for 34 days. produced diarrhea. 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. The sodium salt of DHA was nonirritating to rabbit’s skin. The MIC in broth at pH 6. resulting in the loss of up to 20% of body weight. Longer studies using male and female rats given 400 or 800 mg/day did not affect offspring. However. the 800 mg/day dosage depressed growth. 2000). 1950). which included marked obstruction in the liver and kidneys by the oral route and polyuria by intravenous administration. 1957). (1950) did not show any unusual effects in rats receiving 10. This acid at the appropriate concentration level would inhibit the growth of any microorganisms susceptible to low pH values.2).05%. hematology. Spencer et al. or histopathology. sodium p-hydroxybenzoate. 300 mg/kg led to severe weight loss and damage to internal organs. 1953). at the higher dosage levels. Intravenous and intraperitoneal injection produced hemorrhaging in the lungs. 1. 30.5 was 4 mg/ml for potassium sorbate. and 0. and caused slight histologic aberrations. 0. 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..Organic Acids 105 Strawberries dipped in or sprayed with 0.1% of the substance showed no changes in growth. Toxicology The LD50 for DHA in rats is given in Table 4. ADIPIC ACID Antimicrobial Properties Adipic acid has no antimicrobial properties other than those ascribed to its ability to reduce pH. Toxicology Acute toxicity data for adipic acid in mice and rabbits indicated varied tolerance levels by different routes of administration (Table 4. Application and Regulatory Status DHA or its sodium salt is GRAS when used in accordance with good manufacturing practices. and sodium dehydroacetate and 16 mg/ml for sodium p-hydroxybenzoate. even with prolonged contact (Spencer et al. However.0%. coli in homogenized beef but not in poultry (Yamamura et al.25% or 5% sodium dehydroacetate for 60 seconds were considered more marketable after 3 days of storage at 20°C than water-dipped berries. A 2-year feeding study with male and female rats fed 0. or 5.

288. 1943). saturated dicarboxylic acid having low water solubility.1%.04% for cheese.0.106 Antimicrobials in Food occurred at the 5. coli when grown in a chemically defined medium and was more effective than 1. It is used in gelatin desserts. It is used as a flavoring adjuvant in levels not to exceed 0. 0. 1980). Caprylic acid is approved as a GRAS substance for miscellaneous and general-purpose usage (21 CFR 184. 1957). oily liquid that has a slightly unpleasant odor and a burning. 1970). caprylic acid was ineffective at concentrations of up to 7. The 2-year feeding trials using male rats fed 0.005% for fats and oils.3% concentration inhibited E. 1972). 1973b). Inhalation of adipic acid for 6 hours at a rate of 126 µg/l produced no unusual findings (Gage. Teratologic studies using mice. respectively (Food and Drug Research Laboratory. 0.1009). 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. Application and Regulatory Status Caprylic acid is a colorless. mildly acid flavor. frozen dairy desserts. and 0..0% level although the food intake level was the same. Inc.0%. powdered fruit beverages. . candies. Caprylic acid at a 0. 1968). however. not to be used in amounts exceeding good manufacturing practice. and 205 mg/kg body weight. 0. 1. meat products.8% propionic acid.0%. At pH 6.0% acid did not show any abnormalities.5% acetic acid and 1. Toxicology Orö and Wretland (1961) determined the LD50 of caprylic acid for mice by intravenous administration (Table 4. Caprylic acid is also used as an antimicrobial agent for indirect use in cheese wraps. Application and Regulatory Status Adipic acid is a nonhygroscopic. The MIC of caprylic acid was 100 µg/ml in TSB for Vibrio parahaemolyticus (Beuchat. as a sequestrant in oils. 1972).8 µmol/ml (Kabara et al.2). refrigerated rolls. 1975b). baking powders.3. rancid taste. CAPRYLIC ACID Antimicrobial Properties When tested against a variety of Gram-positive and Gram-negative organisms and yeasts. gelatins and puddings.013% for baked goods. and 5% adipic acid and female rats fed 1. No limit has been set on the acceptable daily intake for humans.1025).0. It is slightly soluble in water. At pH 5. 3. 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.. however. the inhibitory concentrations were not dramatically lower (Woolford. weight gains for rats fed the two highest levels were significantly lower than those for control rats (Horn et al. and in improving the melting characteristics of processed cheese (Gardner. and soft candy. and biscuits because it imparts a smooth. caprylic acid inhibited bacteria and fungi at a lower concentration than either acetic or propionic acid..16% for snack foods. rats.001% or less for other food categories. The acceptable daily intake for humans is listed in Table 4. Caprylic acid was also more inhibitory to Shigella species than either acetic or propionic acids (Nakamura and Zangar. 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. It also serves a function in the canning of vegetables.

Organic Acids 107 CITRIC ACID Antimicrobial Properties Inhibition of L.5% sodium citrate and 1. and 1. citric.12 log/ml. Citric acid at a concentration of 0. Okra.0 with citric and hydrochloric acids (Conner et al.5% (pH 4.36 at 10°C. Additional interactive effects were also noted in the presence of salt. Similar pH-temperature relationships were noted by Cole et al. and 4. acetic. 4. Fabian and Graham. 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. monocytogenes was achieved in trypticase soy yeast extract broth (TSBYE) adjusted to pH 5. monocytogenes survived after 11 to 12 weeks in TSBYE adjusted with acetic.0. (1989) reported that on an equal molar basis.0. acetic. and propionic acids and for 6 weeks in media containing HCl or lactic acid.64 log/ml.19 at 5°C. Canned tomatoes acidified with citric. compared to counts in unacidified juice (6. monocytogenes after 4 weeks were pH 4. 1950).5% sodium lactate was equally inhibitory as sodium citrate alone. Acidification impaired the color but enhanced the flavor of canned okra. citric. Little bacteriostatic activity occurred at pH 5. malic. (1974).7 juice incubated at 20°C were 6. A comparison of equimolar concentrations identified citric acid as the most antimicrobial.0 at 5°C.66 at 30°C. When tomato juice was adjusted with citric acid to pH 4. propionic.0 with propionic acid. canned in a brine containing acetic. or malic acids did not change in physical or chemical attributes during processing compared with unacidified tomatoes (Schoenemann and Lopez. Interaction was demonstrated between citric acid and sodium citrate added to BHI broth adjusted to pH 4.3.8 and 2. The authors suggested the inhibitory activity might be the result of the chelating ability of citric acid.. or tartaric acid to achieve an equilibrium pH of 4.5) was more effective than lactic acid (pH 3. These experiments used citrate phosphate buffer in which the citric acid content was calculated at 1. . who determined that the minimum pH values that allowed survival of L. and degree of dissociation. Sodium citrate was also more effective than 2% sodium lactate. but inhibition increased with decreasing pH (Murdock. citric. pH was not considered a reliable indicator of preservation effectiveness (Fabian and Wadsworth. indicating that citrate was the more effective compound. (1990). Citric acid was particularly inhibitory to flat-sour organisms isolated from tomato juice. lactic. phosphoric. and hydrochloric. lactic. propionic. 6. and 7 (Buchanan and Golden.. enterocolitica based on concentration of acid. The effect was temperature dependent in that survival of L. Based on pH. L. 1997).1% at pH 4. and acetic (Brackett. monocytogenes decreased to undetectable levels within 1 to 3 weeks at 30°C.7 juice were 4. 1939. Citric acid was more inhibitory to thermophilic bacteria than acetic or lactic acids. respectively.83 at 10°C and 7.09. Schoenemann et al. aerobic counts for pH 3.22 log/ml). Yeast and mold counts were the reverse with counts of 2. and acetic. followed by acetic acid. 1987). 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. 1999).7% at pH 6. was processed for 30 minutes in boiling water. phosphoric.4 and 0. lactic. and 4.7 and stored for 7 days at 5°C.5 with acetic and lactic acids. followed by hydrochloric. The activity of the undissociated portion of the acid was hydrochloric followed by lactic propionic.4) in inhibiting Arcobacter butzleri in a broth system (Phillips. pH. All acids would be effective antibotulinal agents at that pH level (Nogueira et al. Rate of inactivation of L. 1973. and phosphoric acid was compared in TSB for Y. fumaric.0 and 3. respectively. 1994). lower aerobic plate counts of 3.6 log/ml were seen. 1994). The inhibitory capacity of hydrochloric. 4. 1990). however. the highest inhibitory activity was associated with propionic. 1953). 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. and the combination of 1. aerobic counts at 20°C for pH 3. then lactic.13. such as texture or appearance. but lower pH levels were toxic.42 log/ml (Bizri and Wahem. 2. whereas at 10°C. citric. 5. citric acid was more inhibitory than lactic acid. Sorrells et al.

31% CA–2.7 (control). Citric acid dips (0. Notermans et al. The addition of potassium sorbate did not enhance stability. Reduction in the thermal process time of canned liver paste was achieved through the addition of 0. The products were packed with or without modified atmosphere and stored at 4°C for 10 to 21 days.9% and spoilage was further reduced to 16.29% CA. and 4. (1989) developed an acid-blanch-chelate (ABC) process designed to reduce mesophilic and thermophilic bacterial spoilage in canned mushrooms. 0. 1992). but the shelf life was shortened at this higher temperature.14% CA (pH 5.05 led to more inactivation of S. but could grow at 8°C only when the pH was higher (5. Okereke et al. Growth in carrot puree was temperature dependent because B.0 at temperatures below 16°C. Only 2.8. leading the authors to conclude that the extracts have a protective effect on C. 1990b).108 Antimicrobials in Food Beelman et al. provided temperatures were at 16°C.5). Enteritidis PT4 at 22 than 5°C.5 were inoculated with C. cereus strains grown in model vegetable broths were conducted at typical refrigeration and abuse temperatures of 5°C.3 g/kg) for crawfish (Procambarus clarkii) stored at 4°C were not effective in retarding growth of L.4.0% TCA-0.. 6. Silla Santos et al. Microbial stability was achieved in liver paste formulated with 0. 118°C.05 M) solution buffered to pH 3. Mayonnaise made with >5% citric acid solution adjusted to pH below 4. botulinum type B when held for 70 days at 15°C or 14 days at 20°C. The preparation of these products included adjustment of the pH with acetic acid to 3... 1989. 1990).0% citric acid for 5 or 30 minutes. thereby leading to its survival regardless of the change in pH (Ocio et al.31% CA–2. Homemade mayonnaise has been a significant health hazard as a result of contamination by Salmonella from raw eggs (Membre et al. 1993). 0. this process reduced thermophilic spoilage from 68% to 23. 2003).14% citric acid (CA). 1992). Growth curves developed for B. monocytogenes (Dorsa et al. They found that acid blanching in a citric acid (0. Samples pretreated with 1% citric acid displayed significantly lower total numbers. coliforms.. B.3 when at least 20 ml of lemon juice (citric acid) per fresh egg yolk was used. Samples containing the reduced calorie dressing were even lower in microbial numbers than aciddipped vegetables (Eytan et al.4% of cans containing mushrooms vacuum hydrated by the ABC process spoiled (Beelman et al. 1999).22. Citric acid dips for foods not only retard some spoilage but also act as a chelator of metal ions responsible for enzymatic browning reactions. sporogenes. Mushroom extract acidified with citric acid to pH 6. 1990a). 5.1 and 5.5) at 12°C but did not grow in broth acidified to pH 5. 12°C. If the amount of lemon juice was increased to 20 to 35 ml per egg yolk.5 followed by canning in a solution containing 200 ppm ethylenediamine tetraacetic acid (EDTA) controlled the outgrowth of spores of C.9.34. 2000. and 0.4 to 4. Liver pastes inoculated with 106 to 107 Bacillus spores per g and 104 C. 115°C.4 and 5. 4.. Although the heat resistance of C. Both vegetable broths stimulated germination and growth of spores compared to nutrient broth. indicating a synergistic effect between pH and temperature. 5. Cabbage and carrots were pretreated with the addition of a reduced calorie dressing or with a dip of 0. 1997. sporogenes PA3679 after a sublethal heating process (Okereke et al..05. Extracts were then heated at 110°C.8% with the addition of EDTA.69) and processed with an F0 of 0.. sporogenes PA 3679 spores. This process improved quality and increased the microbial stability of canned mushrooms (Okereke and Beelman. The authors recommended that the final pH should be <3. and 4. By adding citric acid to can brine.1% potassium sorbate (Houben and Krol.. or 0. 1994. cereus could grow at pH 5. Xiong et al. 0.3.3. and 121°C. This process inhibited C.. and 16°C (Valero et al. the product should . 8°C. and lactic acid bacteria counts than samples dipped with the lower concentration of citric acid.0% trisodium citrate (TCA). sporogenes decreased with decreasing pH levels.2% or 1.1 with holding temperatures between 18°C and 20°C for up to 3 days before consumption. 1991).. 5. sporogenes spores per g were heated to an F0 of 0.1.2. cereus grew in zucchini broth (pH 6. heat resistance was not affected by higher processing temperatures.85.65 and asparagus with added citric acid or glucono-delta-lactone to pH levels of 5. (1985) dipped potatoes in a solution of 1% citric acid and 2% ascorbic acid for 2 minutes before vacuum packing and cooking.

1998). when 35 ml lemon juice per egg yolk was added. no growth occurred at pH 5.12 (Sinha et al.125% to 4% citric acid or 0.. However. suggesting that citrate was a chelator of ions essential for growth (Rammell. 1967).6.2.25% citric acid and 0. No toxin was detected in any of these foods over an 8-week shelf life (Post et al. whereas infected tissue was around 4. 2%. 1984). 1968). Sodium citrate in concentrations of 0. Concentrations of 12. Citrate inhibition of S.5 with citric. (1985) found no outgrowth or toxin production from spores of types A and B in TPGY medium acidified to pH 4.2% sodium benzoate were held for 30 days at 4°C or . Sodium citrate can also exhibit bacteriostatic activity.3% citric acid lowered the level of Salmonellae on poultry carcasses.. and 4%. 0.1% to 4. and rinsing with a citric acid solution adjusted to pH 4.75% for both citric and lactic acids had a slight growth-retarding effect on A. malic.75% concentration of either acid did not affect the growth of P. however. 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. Using the multiple-barrier concept. 1962). Similar destruction values were seen with potassium sorbate and sodium benzoate. the function of citrate appeared to be one of chelation of metal ions. When acetic acid was used. aureus was overcome by adding calcium and magnesium ions. The addition of garlic or mustard increased the death of S.5% sodium citrate were inhibitory to Salmonella anatum and Salmonella oranienburg (Davis and Barnes.Organic Acids 109 be held at 22°C for longer than 72 hours. Enteritidis. In concentrations of 12 to 18 µM/ml.75% lactic acid inhibited growth of Aspergillus versicolor. The metabolic effects of citrate on Arthrobacter species were twofold.1 or less. leading to enhanced growth. 1970). A level of 0. lactic. Typhimurium than lactic and hydrochloric acids (Subramanian and Marth. A 0. 80°C.0% concentrations of citric acid and 0. parasiticus. Sabouraud medium was adjusted with 0. with Arthrobacter simplex. a 0. The amount of sodium citrate had a dual effect on Lactobacillus casei. During ripening. Branen and Keenan (1970) also suggested that citrate chelation of metal ions inhibited L. the time decreased to 48 hours before use.25% concentration greatly reduced toxin production. type E grew and produced toxin at 26°C in media containing citric acid at pH 4. Thermal resistance studies were conducted in mango juice adjusted to pH 3. 1985). citrate appeared to inhibit glucose utilization (Imai et al. 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. expansum (Reiss. C.. Survival studies for Neosartorya fischeri ascospores were conducted in mango and grape juices and heated at 75°C. sodium citrate inhibited growth. botulinum showed no change in numbers when inoculated in tomato puree.0% was not as inhibitory to Streptococcus agalactiae when added to skim milk or fresh milk as citric acid at concentrations of 1%. Mangoes are subject to spoilage through infection with Penicillium cyclopium (Palejwala et al. As little as 0.0% to 12.08 to 4.5% lactic acid prevented toxin production. shrimp puree. fluorescens on beef carcasses (Thomson et al.. but salt had a protective effect on this organism.5%. 1984).. 1968). A level of 0. or tartaric acid. 1970). Ascospores were able to withstand more than 6 hours of heating at 75°C.0 reduced the numbers of P.6 and incubated at 35°C. even in media containing up to 1% citrate. The MIC of 0. Healthy tissue had a pH of 5. S.0. hard-cooked eggs allowed to equilibrate in 0.8. Tsang et al.5% citric or 0.2 or 4. however.016% to 1% malic acid as the sole carbon source because these acids predominate at these concentrations in a ripening mango. sodium citrate stimulated growth. and in concentrations greater than 40 µM/ml. 1976). 5 hours at 80°C.8% gave a pH of 4. or shrimp and tomato sauce acidified with citric or acetic acids to pH 4.75%. it did not appear to affect attachment (Appl and Marshall. the level of acidity decreased and sugars and nitrogenous compounds increased. With Arthrobacter pascens. but 0. 85°C. or 1. 1952). aureus showed inhibition both with increasing concentrations of citric acid and decreasing pH (Minor and Marth.8. and 3 to 4 hours at 85°C.. casei. Citric acid provided the maximal destruction of ascospores at 85°C. and 90°C. Skim milk acidified with citric acid was more inhibitory to S.

(1973) used a combination of 0. Long-term studies involving rats fed quantities up to 1. potassium (21 CFR 184. 1971). Typhimurium. or 12 g citric acid per kg of a noncariogenic diet to show the possible effect of citric acid on the development of teeth. (1982) used a combination of 0. and 4.. Cramer et al.5 depressed growth of L.75% citric acid alone for 21 days at 4°C. plantarum but did not affect P..02 mol sodium citrate and citric acid.75% glucono-deltalactone incorporated into sausage before fermentation.3. 1957) showed no abnormalities other than lowered weight gains and feed consumption in the 5% group. in the acid form (21 CFR 184. and rabbits indicated varied tolerance levels by different routes of administration (Table 4. the highest dosage contributed to enamel erosion.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. A concentration of 0. aeruginosa (Restaino et al.1625). 1944). Horn et al. Citric acid also acts synergistically with antioxidants to prevent rancidity by chelating metal ions (Gardner. It is highly water soluble and enhances the flavor of citrus-based foods. the urine contained calcium citrate. and jams and jellies. 1956. The authors concluded that citrate had a rachitogenic effect on the test animals. or sodium salt (21 CFR 184. It is approved for use in ice cream. beverages. enterocolitica. The number of cavities formed did not differ between the control and experimental groups. rats.05% potassium sorbate and citric acid at pH 5.. Potassium sorbate at 0. It is a precursor of diacetyl and therefore indirectly improves the flavor and aroma of a variety of cultured dairy products. Death by all routes and in all animals resulted from respiratory or cardiac failure and hemorrhaging of the gastric mucosa.7% sodium citrate (5% citric acid) fed to rabbits for 60 days produced no unusual effects (Packman et al.1751). Application and Regulatory Status Citric acid is a tricarboxylic acid having a pleasant sour taste.1195). Although there was no effect on weight gain.2% concentration in combination with citric or lactic acid at pH 5.2. Toxicology Acute toxicity data for citric acid in mice. Studies by Yokotani and coworkers (1971) compared toxicities of commercial citric acid and Takeda citric acid.0 to reduce the growth rate of S. 7.1033) or as the calcium (21 CFR 184. fruit preserves.5. Restaino et al..110 Antimicrobials in Food in 0.2%. (1957) evaluated dosage levels in mice given the acid intravenously. however.5. This combination inhibited the growth of S. coli. Daly et al. aureus (Fischer et al. or 5% citric acid (Bonting and Jansen. aureus early in the fermentation and the increased acidity of the meat mixture allowed the fermentation to proceed more quickly. Citric acid is approved as a GRAS substance for miscellaneous and general-purpose usage when used in accordance with good manufacturing practices. and S. sherbets and ices. 4. Deaths resulted from acute acidosis and lung hemorrhages. Dalderup (1960) fed rats 1. Acute toxicity data of sodium citrate are shown in Table 4. 1963).2%. 1981).4%. slight atrophy occurred in regions of the thymus and spleen (Yokotani et al. salad dressings. rouxii and Saccharomyces bisporus. Horn et al.2). 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. In another study.1% citric acid and 0. 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. 3%. 1985). The acceptable daily intake for humans is listed in Table 4. (1956) fed vitamin D-free diets containing low calcium levels but adequate levels of phosphorus supplemented with 0. a by-product of yeast fermentation.. . and it is used as an acidulant in canned vegetables and dairy products. Y.75% citric acid was sufficient to reduce inoculated populations of S.8% level. Short-term studies on rats fed 0. E. It can control the pH for optimum gel formation. 1972). 2.

and tartaric acids at a concentration of 2. monocytogenes and E..5% (pH 2.5%. 1982).Organic Acids 111 FUMARIC ACID Antimicrobial Properties Fumaric acid (0. Monomethyl and monoethyl (0. respectively. respectively. 1981.2%) and dimethyl and diethyl fumarates (0. fischeri (Conner and Beuchat. flavus ascospores (Beuchat. Medium chain-length alkyl esters had more activity than aliphatic acids (Dymicky and Trenchard. Fumaric acid was also more effective than lactic or acetic acid when used in meat products (Podolak et al. or 0. fumaric acid achieved the greatest reduction of aerobic.6 L in the vapor phase were also effective as a mold preventive (Huhtanen and Guy. rats showed an increased mortality rate and more atrophy of the liver (Fitzhugh and Nelson.2% showed considerable promise as a substitute for sodium nitrite in bacon. The 2-year studies using rats fed 0. Fumaric acid by itself was only slightly inhibitory.5% level.5.5% concentration of fumaric acid inactivated ascospores after 20 minutes of heating at 80°C.0% and 1.05%) or sodium propionate (0. botulinum types A and B in canned bacon.1%) controlled fungal growth in tomato juice. 1996a. or tartaric acid based on weight when added to the heating medium. The n-monoalkyl fumarates and maleates esterified with C13–C18 alcohols also possessed significant antibotulinal activity (Dymicky et al.1%. dimethyl. Cans packed with bacon and inoculated with C. Human feeding . 1975). Fumaric acid was more fungicidal against Talaromyces flavus ascospores than acetic. However. Monomethyl.05% sodium benzoate followed by holding at 25°C for 6 hours before refrigeration at 4°C for 24 hours achieved reduction.0 to 2. 1984).1% and 1. The addition of 0.b). but the dimethyl and diethyl esters at a concentration of 20 mg per 2.0) were not lethal to T. citric. In addition to reduction of E. whereas the presence of fumaric acid esters in the bacon prevented swelling for up to 8 weeks.9% (5% fumaric acid) showed no toxic effects (Packman et al.. and coliform bacteria. 1947).5% applied for 5 seconds at 55°C to the surface of lean beef inoculated with L. and ethyl esters of fumaric acid in concentrations of 0. The fumarate esters not only prevented mold growth on bread (Huhtanen et al. and di-n-alkyl fumarates were almost inactive (Dymicky et al.0% acid concentration at 82°C. It was speculated that the antimicrobial properties of fumaric acid were related to a lower pKa than lactic or acetic acids. coli O157:H7.15% fumaric acid coupled with 0. Fumaric acid at concentrations of 1. 1974) and inhibitor of malolactic fermentation in wine (Pilone.. total aerobic counts were reduced to levels below those achieved through pasteurization. When used in vacuum-packaged ground beef patties. These same preservatives provided a similar reduction in less than 5 hours and 2 hours when held at 25°C and 35°C.3 logs. 1988). Methyl n-alkyl fumarates demonstrated lower activity. 1987) heated in the presence of 2. Huhtanen (1983) and coworkers (1985) investigated the effect of various esters of fumaric acid on growth of C.. botulinum swelled in 4 to 6 days at 30°C. 1987). The use of fumaric acid to effect a 5-log reduction of E. At the 1. but malic acid did not. Fumaric.38% sodium fumarate or guinea pigs fed 1.5 to 5. coli O157:H7 inoculated into unpreserved apple cider was investigated by Comes and Beelman (2002). and tartaric acids increased heat inactivation.0% fumaric acid did not show any abnormalities. citric.8%.15%) served as an acidulant (Ough and Kunkee.. 1982). coli O157:H7 reduced levels by 1 and 1. and lethality increased as the pH decreased from 5. malic. malic. citric. Islam.15%. 0. A 0. psychrotrophic.125%. 0. Toxicology Weanling rats fed 0. 1963). acetic.2% fumaric acid or rabbits fed sodium fumarate at a concentration of 6.0% fumaric acid and 1. 0. and 1. 1987). Similar effects were noted with N.

vacuum packaging.0 but was ineffective against yeasts and molds (Woolford. an LD50 of 8. Fumaric acid and its salts are used for direct addition to food for human consumption (21 CFR 172. 1950). Destruction rates for S. (1984) found that spraying beef and sheep carcasses with a combination of 1% lactic. 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.25% lactic acid spray of beef carcasses followed by a treatment of hot-boned cuts with 2% acid. Similar reductions in microbial counts occurred with a 1.. pie fillings. lactic acid coupled with other antigrowth factors excreted by lactic acid-producing microorganisms inhibits competing microorganisms. but a 2% concentration led to discoloration of the carcass surface (Smulders and Woolthuis. 1985). The inhibitory capacity of this acid lies in its reduction of pH to levels below which bacteria cannot initiate growth. 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. Lactic acid sprays have been effective in limiting microbial growth on meat carcasses under a variety of storage conditions. propionic. fumaric acid does display some antioxidant properties in fat-containing foods (Gardner. LACTIC ACID Antimicrobial Properties Lactic acid has been used more extensively for its sensory qualities than its antimicrobial properties in the past. 1983. Enterobacteriaceae contaminated 50% of the untreated samples. Lactic acid was an excellent inhibitor of spore-forming bacteria at pH 5. 1991). Lactic acid was about four times more effective than malic. 1985).. citric. lowered microbial counts after storage for 14 days at 2°C. biscuit doughs. and 0. In fermented foods.6 to 2. Typhimurium. Acid treatment when combined with vacuum packaging was more effective in prolonging shelf life than vacuum packaging alone (Smulders and Woolthuis. 0.1% ascorbic acids selectively inhibited Enterobacteriaceae at 10°C.3. and storage for 10 days. . dicarboxylic acid having low water solubility. and chicken carcasses. Lactic acid was more inhibitory to Mycobacterium tuberculosis as the pH decreased (Dubos.0% to 1. A 2% L-lactic acid treatment at pH 2. Gill and Newton (1982) suggested that the inhibitory effect for psychrotrophic organisms was related to a decrease in pH rather than degree of dissociation. 1972). pork. It increases the strength of gelatin gels.350). followed by vacuum packaging. which was reduced to 10% after treatment. 1988). 2% acetic.3 of veal tongues. gelatin desserts. although more recently this acid has been used as a rinse for beef. 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. 1946). and wines. In addition to supplying acidity to a product.7 log CFU/cm2 (Visser et al. Woolthuis and Smulders.25% citric. A 1. Osthold et al.. and acetic acid in limiting growth of Bacillus coagulans. Fumaric acid is used in fruit drinks. unsaturated. Because most other studies chose refrigeration storage temperatures. Because of these dosage levels. 1970). Cold-pack cheese normally prepared with lactic acid was reformulated with acetic acid and inoculated with S. reduced aerobic mesophilic plate counts from 5. 1954).25% concentration of acid sprayed on veal carcasses.000 mg/kg was proposed (Levey et al.112 Antimicrobials in Food trials using a 500 mg/day oral dose for 1 year were also negative. 1988).. The acceptable daily intake for humans is listed in Table 4. the organism responsible for flat-sour spoilage in tomato juice (Rice and Pederson. combined with vacuum packaging and storage at 3°C. Although this additive has a strong acid taste. Application and Regulatory Status Fumaric acid is a nonhygroscopic. Typhimurium were similar for both acids (Park et al. 1975a).

As in beef trim. sub-primal meat was injected with one of three treatments: 0. 76°C.2%. There was little difference in bactericidal activity between the combination and either acid alone on sub-primal cuts of beef.. coli counts was detected for beef dipped in acetic or lactic acid solutions compared with a water-dipped control at day 1.3 M calcium chloride solution (CAL.8 to 7. Dixon et al. Kotula and Thelappurate (1994) compared acetic acid and lactic acid solutions applied at two concentrations. Typhimurium-inoculated beef trimmings.5) at a volume equal to 10% of the initial sub-primal weight. but odor was not affected (Ellebracht et al. In addition. The system alone produced a 3log reduction. 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.b). When sequential treatments were employed using pre-chill and postchill lactic acid sprays. 1990a. 0. moisture content. and E.0).3. Similar work was conducted using lean pork trim (LPT) and fat-covered pork trim (FPT) (Castelo et al.Organic Acids 113 Hot-boned.. psychrotrophic. coli O157:H7 or S. combinations of the treatments were examined. Beef trim was fabricated to make predominantly beef trim lean (BTL) and beef trim fat (BTF) products that were inoculated with bovine fecal samples (Kang et al. and at two time intervals. at 1°C to 2°C as a dip for rib eye steaks.. 482°C. (1987) compared a combination of 1% lactic. and 0. and sensory analysis were not affected by acid treatment. total fecal coliforms. The combination of acids performed as well as 1% lactic or acetic acids alone. the FPT supported lower microbial numbers than LPT. (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. but shear values. but this effect was not seen with acetic acidtreated tissue. Aerobic plate counts of meats injected with CAL were significantly higher than with LAC or COM. Greater reductions on all microbial loads were seen in BTF compared to BTL. 65 psi). Acid-treated samples were lighter in color as a result of leaching of the pigment during immersion. coli O157:H7 and S. hot water (30 psi) at 65°C.1). Lactic acid application as the final step in a multiple-hurdle process led to a residual effect during storage for 7 days at 4°C. The inoculated meat was subjected to sprays of tap water (15°C to 17°C. or hot air at 371°C.6 with either 1% lactic acid at pH 2. Minimal reduction in total plate and E.9 or 1% acetic acid at pH 3. but control cuts retained more desirable flavor profile (Eilers et al. 454°C. and presumptive lactic acid bacteria. 2001). b). 1999). Typhimurium were subjected to an automated wash water system (Castillo et al. Treatments containing lactic acid resulted in the lowest counts without affecting sensory qualities. coli biotype 1 counts. 71°C. 0. coli. coli was reduced by 6. reducing numbers by 2 logs and Enterobacteriaceae by 1. only suggesting that lactic acid can exert a continued antibacterial effect during the shelf life of the product. 2001a. Beef trim meat used to make ground beef is of particular concern because of its higher microbial load. Meat color was affected after application and through storage at 4°C. 1994). 2% acetic. 30 psi). All products were stored at 4°C and examined for aerobic.25% citric. . 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. Application of 95°C water alone or with 2% L-lactic acid was effective in reducing E.6% or 1.. Acuff et al. or a combination (1:1) CAL/LAC (COM. 20 or 120 seconds.b). Postchill beef rounds inoculated with E. 426°C. 1992). pH 5. Lactic acid was most effective against S. COM improved tenderness in muscle cuts.1% ascorbic acids adjusted to pH 2.2 logs. When temperature changed from 20°C to 70°C or lactic and acetic acid concentrations increased individually to 3%. coli (Anderson and Marshall. 2% lactic acid (12°C to 15°C.. <1 log for Enterobacteriaceae and 0. Typhimurium levels decreased approximately 1 log.. Furthermore. 1998. Typhimurium at 70°C. pH 2. 2001a. Significantly higher aerobic plate counts were obtained from hot-boned cuts over 17 days postmortem compared to cold-boned cuts. aerobic bacterial counts and S. or 510°C.5 logs for 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.5 logs (Anderson et al. E. or 82°C. 0. pH 3.3 M lactic acid solution (LAC.

The combination of acids and heat treatment led to reduction of spore-forming organisms in frankfurters.. lactic acid. Woolthuis et al. coagulans were heated to 121°C and 105°C or 110°C.. Gram-negative bacteria were the most sensitive to lactic acid. (1984) effectively reduced total plate counts. 2002). Therefore. . these treatments were not deemed usable in the control of E.8.6 log/6 cm2.. Beef inoculated with E. Frankfurter emulsions incorporated with acids to adjust pH to 5. coli O157:H7. Lactic acid (2%). Lactic acid treatments alone were only 50% as effective as acid/alginate treatments. and 0.. 1993). vacuum packaging.6 for Bacillus stearothermophilus and pH 4. and nisin (400 IU/ml) were used alone or in combination in beef to inhibit L. respectively (El-Khateib et al. malic. or 360 seconds to 2% lactic acid. 1993). All treatments significantly reduced the number of L. Although the gels reduced populations of L.. Numbers of S.2% lactic acid for 5 minutes. 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. A 1% concentration of lactic acid (pH 2. coli O157:H7 in beef burgers. These compounds reduced the level of L. nisin. Psychrotrophic. low-molecular-weight polylactic acid (2% PLA). whereas acetic acid was more effective than the acetic acid/gel treatment. Enterobacteriaceae.. Greater inactivation was noted for both organisms at the lower pH level when acetic or lactic acids were used but not with citric. 1.2 for B.5%). 2002). lactobacilli and yeasts were the least sensitive. monocytogenes. Lactic acid was effective in decreasing the number of Enterobacteriaceae as well as other Gram-negative mesophilic and psychrotrophic spoilage organisms. 55°C) had little effect on the aerobic plate counts taken from the surface of pork carcasses. coli O157:H7 was subjected to treatments of lowmolecular-weight polylactic acid. Elimination of these groups shifted the population to Gram-positive bacteria and yeasts. and nisin did not enhance the effects of lactic acid against E. The use of lactic acid for reduction of microbial numbers or pathogens has been similarly applied to pork products. 1999).2 or 4. After 42 days of storage the initial inoculum of 5 logs was reduced to <1 log after using lactic acid.000 IU/ml nisin. 180. and storing for 5 days at 3°C. and Lactobacillaceae counts by 2 to 3 logs. and 200 IU/ml of nisin (Mustapha et al. coli O157:H7 inoculated into beef trimmings (Bolton et al.7. or nisin and lactic acid. Aeromonas hydrophila. Beef cubes were inoculated with L. 240. and low-molecular-weight polylactic acid. followed by mesophilic Enterobacteriaceae. monocytogenes and treated with 2% lactic acid. Not only did the microbial counts decrease as the time of decontamination increased. but no differences were seen between polylactic acid and lactic acid. 1992). monocytogenes (Ariyapitipun et al. but the population of microorganisms shifted. enterocolitica. 1988). 2000). Beef was inoculated with L. 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. or sodium lactate (4%) alone or in combination with pulse electric field or freezing was ineffective in reducing numbers of E. and polylactic acid. 40. however. lactic acid.1. or hydrochloric acid (Lynch and Potter. monocytogenes. monocytogenes on beef tissue. numbers of Salmonella species and Campylobacter species were reduced immediately and remained lower 24 hours after slaughter (Epling et al.5 and 4. Salmonella species or Listeria species were not recovered nor were sensory characteristics affected (Prasai et al.. 1992. The use of these compounds was effective in reducing psychrotrophs and Enterobacteriaceae (Ariyapitipun et al. Typhimurium were reduced more by exposure to acetic acid/gel treatment than acetic acid alone. noncoated tissue became dehydrated during the 7-day study. by immersing porcine livers in 0. After 28 days of storage. 1993). and 3200 units/ml pediocin. A novel approach to shelf life extension was the use of lactic or acetic acid-impregnated calcium alginate gels applied to the surface of beef tissue (Siragusa and Dickson. monocytogenes before vacuum packaging and storage at 4°C. monocytogenes by 1.114 Antimicrobials in Food Lactic acid (2%). there was no significant difference between nisin. Y. acetic acid (0. By increasing the concentration of lactic acid to 2%. significant reductions were seen with all treatments. Five treatments were delivered to the pork skins: water-treated (control) or exposure at 21°C for 120. psychrotrophic Gram-positive bacteria.

1988). respectively (Sawaya et al.. E. fragi. Acid treatments of fat tissues led to the reduction of pathogen numbers to nondetectable numbers within 4 days. Lactic acid dips have also been used successfully in the poultry industry.25%. or formic acids. pH 2. 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. Higher concentrations of the acid did not ensure greater decontamination nor did repeated treatments. acidified media.9 to 2. thermosphacta. Hwang..000-fold). Although L. Lactic acid added to scald water alone had minimal effect on reducing the numbers of contaminated birds. 1995. The treatments did not affect sensory quality.23 in cells exposed to acid to 5.3 prolonged shelf life to 8 days (Zeitoun and Debevere. Chicken skin subjected to washing treatments using 1% lactic acid led to significant reductions of Salmonella species and L.000-fold. but no protective effect was observed if the sodium chloride was added to the medium 45 minutes after the inoculation. Levels of psychrotrophs decreased from 3. The spoilage organisms. tissue was dipped for 15 seconds in water or lactic acid and held for 15 days at 4°C. The interaction of sodium chloride (0. In an effort to eliminate carcass discoloration. 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. monocytogenes compared with monosodium phosphate. acetic.2) was examined for E. acidified with lactic acid. and Beuchat.3 to 2. .2 decreased from 5.5% lactic acid/0.62) were combined with propylene glycol (20%) in chill water.5 and 0.0 than cells grown at 37°C. discoloration still occurred and propylene glycol contributed an objectionable flavor (Izat et al. pH 2. hydrophila numbers declined by 5 logs within 11 days of storage. pH 2. Total microbial numbers from skin of birds immersed for 15 seconds at 19°C in 1% or 2% lactic acid at pH 2.6 log CFU/g.79 in acid-free media. coli decreased 10. 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 5.5% sodium benzoate dip concluded that sensory qualities were acceptable (Hathcox et al. After inoculation.2 logs. 1990a). however. E. 1995). although tissue treated with acid affected the growth rate.8) and scald water (54°C/2 minutes) reduced the bacterial level of broilers inoculated with S. produced similar reductions of approximately 1 log (Zhang and Farber. or Brochothrix thermosphacta (Greer and Dilts. such as Pseudomonas species. When treated broilers were packaged using modified atmospheres.2 to 3. None of the pathogens grew on lean tissue. Salmonellae were eliminated from broiler carcasses after a 1-hour exposure. P. Lactic acid was more effective than acetic acid when used as a 10-minute rinse for fresh-cut vegetables. other compounds. 1995). shelf life was lengthened to >36 and 35 days at 4°C and 7°C.2 in media containing lactic. 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. sodium tripolyphosphate. fragi and B. 1990). reduced levels of lactic acid (0. sodium acid pyrophosphate. 2002).5% to 4%) and pH (4. or sodium hexametaphosphate. 1995). E. 1983).1°C. coli O157:H7 rapidly died at pH 4. 1996).. however. and A. Typhimurium to almost nondetectable numbers.Organic Acids 115 P. S. coli O157:H7 grown in TSB at 37°C (Casey and Condon. Lactic acid added to broiler chill water resulted in the development of a brown coloration.5%. that contributed to spoilage.. These treatments inhibited hydrogen sulfide-producing bacteria. 1990b). Consumer evaluation of chicken treated with a 0..0) for chicken legs increased the shelf life from 6 to 12 days at 6°C. grew on both tissue types after acid treatment. This protective effect was even more pronounced with acetic acid (100. The number of Salmonella-positive birds was also reduced as a function of time of the dip (Izat et al. coli demonstrated a 10-fold decrease in numbers when grown in media containing sodium chloride. Lactic acid (1%) added to both chill water (0°C to 1. and a 2% lactic acid dip at pH 2. in sodium chloridefree. 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. A 10% lactic acid and sodium lactate-buffered acid spray (pH 3. such as chlorine or chlorine dioxide.88 or 0.7 log CFU/g and Enterobacteriaceae from 3. respectively. monocytogenes counts were reduced by 0.7 log CFU/g (van der Marel et al.

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

the rate of inactivation was dependent on the acid.5%.1. Sodium chloride at the same water activity as sodium lactate (0. 1. with Debaryomyces and Candida being the most resistant at 1161 and 1339 mM. spore germination. Calcium lactate was also more effective than sodium lactate in retarding growth of E.. 11 days for 1.. 1995) examined the effect of 3% sodium or calcium lactate added to pork liver sausage and various heat treatments. This level was listeriostatic at a moisture of >55%. As expected L. 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. monocytogenes per gram of sausage.0 at 4°C..5.5. monocytogenes as the target organisms. and 30°C (Houtsma et al. which might explain their prevalence in spoiled meat products.. 0.982) did not inhibit toxin production. however.5. and 7. Lactic acid/sodium lactate and acetic acid/SA were studied in a BHI broth at pH 7. At 30°C. The effect of sodium lactate on toxin production.0. toxin production was detected in media containing 4% sodium lactate within 11 days. At the abuse temperature of 20°C. and 4 using 0. calcium. innocua was modeled in broth adjusted to pH 5. 10°C. potassium. Sodium. Samples were stored up to 50 days at 5°C and up to 10 days at 20°C. 6. monocytogenes. 1996). The calcium form was found to be more inhibitory than the sodium form. and heat resistance was tested on proteolytic C. and the resultant pH. 1994b). Typhimurium when stored for 4 days at 20°C. 1% sodium lactate delayed growth for 10 days. 1995). Salts of lactic acid are equally as effective in muscle food systems.0. This model was then evaluated for use in a bologna-type sausage system (Houtsma et al. There did not appear to be a synergistic effect between a 4% concentration of lactate combined with 140 ppm . spore germination did not lead to growth after 7 days. The effect of sodium lactate and sodium chloride on the growth of L. monocytogenes was studied in a cooked meat model system containing varying moisture levels and 4% lactate. and 2 M concentrations (Buchanan et al. inhibition by sodium lactate was directly related to pH and temperature. comparable to a 3% concentration of calcium lactate. The pH level was influential in inhibition by sodium lactate but not by sodium chloride. respectively. or 4% and inoculated with 104 to 105 L.5%.0% sodium lactate. 20°C. Lactic acid was more effective at higher pH levels whereas acetic acid was more effective at lower pH levels and there was a correlation with the concentration of dissociated acid. At a higher temperature of 20°C. 3%. indicating that inhibition was not the result of a lowering of water activity. Addition of 2% sodium chloride enhanced the listericidal activity of the lactates (Weaver and Shelef. 1991. Using 108 CFU/g as a microbial end point.Organic Acids 117 their sensitivity. 6. Chen and Shelef (1992) suggested that in addition to pH change. As expected. A subsequent study (Shelef and Potluri. 4% sodium or potassium lactate achieved reductions in L. Pork liver sausage was formulated with sodium. Calcium lactate (2%) reduced populations below the inoculum level. 6. coli O157:H7 and S. no growth or toxin formation was detected after 32 days of incubation for media containing 4. L. 1994a). toxin formation was noted within 5 days for 0%. botulinum strains in peptone yeast extract medium (Houtsma et al. Survivor curves were developed using L. lactates also contributed to lowering the aw of a food. As expected. 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. 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. no toxin formation was detected after 49 days of incubation in media containing 3% sodium lactate. with lower amounts of sodium lactate needed as pH and temperature dropped. its concentration. but the combination of 2% or 3% lactate and 2% sodium chloride at the same moisture level was inhibitory. 1993). and 15 days for 2.. whereas a 2% level extended the time to 24 days. or calcium lactate at concentrations of 2%. 1993). 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. 5. Potassium lactate (4%) and sodium chloride (3%) reduced growth somewhat at 5°C. and potassium lactate were equally effective.

The microflora changed from predominantly staphylococci and micrococci at 0 days to coryneform. 1980). Frankfurters containing 2. rely on sodium nitrite to inhibit growth of C..0% potassium lactate were inoculated with a five-strain cocktail of L.7 to 8.5 reduced surface discoloration in chubs of fresh sausage and extended shelf life from 10 to 20 days. 1997). Pork sausages manufactured with 25% fat or 8% fat with and without 3. Retention of nitrite was linked to higher pH levels and was unaffected by the incorporation of 1.1% sodium diacetate (Glass et al. botulinum.c) injected beef top rounds with 0% to 4% sodium lactate. (1991a. or E. S. Red color was preserved by using 2% to 3% sodium lactate.5 log CFU/cm2 in the same period. 2. monocytogenes during storage at refrigerator and abuse temperatures.4% sodium lactate/0. 1993). Lactate increased cooking yields. sodium lactate permitted aerobic growth of B. enterocolitica or L. perfringens. hydrophila and aerobic growth of the aeromonads (Grau. The heat resistance of the two organisms was significantly lowered in beef containing either of the two concentrations of sodium lactate. Minced beef containing 0%. sodium lactate caused more rapid surface discoloration (Lamkey et al. 1999).. monocytogenes and heated to 55°C to simulate a sous vide process (McMahon et al. 2002). The growth of L. enterocolitica. Another study (Bedie et al.0 log CFU/cm2 after 84 days of storage. or 3. uncured bratwurst manufactured with 3. the plate counts of treated roasts increased to 6. Both concentrations of potassium lactate significantly inhibited or delayed the growth of L. monocytogenes and stored at 4°C and 10°C for up to 90 and 60 days.. Cooked beef top rounds containing 1%. Papadopoulos et al. but produced a mild throat irritation at a 4% concentration. At pH 6.5 in a meat system prevented anaerobic growth of Serratia liquefaciens. 2%. . Although the aerobic plate counts for control meat increased from 2.25% or 0. The pH of the product was unaffected by incorporation of sodium lactate. nitrite levels are reduced. Typhimurium. S. thermosphacta down to pH 5.1. 2002).. Y.2%.25% sodium diacetate were the most effective treatments throughout the 120-day shelf life conducted at 4°C with minimal change in pH. or 4% sodium lactate were inoculated with L.0% or 3. S. 3%.8% sodium lactate was inoculated with Y.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.b. Over the storage life of the product. A 3% concentration of sodium lactate at pH 7. 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. 2001) compared the use of 3% or 6% sodium lactate.6% sodium lactate into the product (Kilic et al.4%. When raised to 210 mM. 1981).5% SA or sodium diacetate as an antilisterial process for frankfurters... Enterobacter cloacae. or 4. monocytogenes. such as wieners. Sodium lactate at concentrations of 3% and 4% were generally effective in limiting growth of L. respectively (Porto et al. coli O157:H7 (Miller and Acuff. and A. Typhimurium. Papadopoulos et al. then cooked and vacuum-packaged the roasts. Levels of >3% sodium lactate or 1% each of lactate and diacetate were needed to prevented growth of Listeriae for 60 days at 4. Cured meats. Lactates inhibited the growth of psychrotrophic organisms and delayed the pH decline (Bradford et al. coli O157:H7 and stored at 10°C. Although Shelef and Yang (1991) suggested a possible aw effect for sodium lactate. 1991). 2.4%. In retail products.05 and 6. monocytogenes.. aureus. micrococci. and yeasts at 42 days to homofermentative and heterofermentative lactobacilli and streptococci at 84 days. 2002). monocytogenes was delayed for 4 and 12 weeks at 7°C and 3°C in unsmoked. the amount of L-lactate ordinarily found in muscle was not inhibitory to any of the strains.5. Sodium lactate at a 6% concentration or 0. A 100-mM concentration of sodium lactate buffered to pH 5. 0. enhanced flavor at the 1% level by adding a salty taste. (1991b) did not find this effect in their studies. and E. Sodium lactate was less effective against microorganisms when sausage contained soy concentrate.0% potassium lactate showed differences in spoilage rates. lactobacilli. 1994)..118 Antimicrobials in Food potassium nitrite. C.

7°C. monocytogenes increased by only 0. or 3. Maas et al. the 2. 7. and E only when lactic acid was used to reduce pH below 5. Modified atmosphere packaging (MAP) extended storage life for products without acid sprays to 17 days and even longer with sprays. 5%. 5%. thermosphacta and E. Typhimurium. 1993). or a combination of the two was inoculated with the two organisms and held at 5°C for 5 days. Notermans and Dufrenne (1981). sodium lactate was the most effective during an 8-week storage period at 3°C. Lactate contributed to water-holding capacity and increased cooking yields. pediocin (1400 U/g). and stored at 27°C. or 7 to 8 days for each concentration..56 log during 17 days of storage for the legs treated with 10% buffer combined with MAP. nisin (1400 U/g. The meat was cooked to an internal temperature of 71. Levels of L. found that glyceryl monolaurate at a concentration of 5 g/kg of a meat slurry inhibited C. glycerol monolaurin.5% concentration.8%.5% concentration was acceptable. (1991) also found that 2% lactate inhibited the growth of C.5% than sodium gluconate. 3%. B. (1989) suggested that a possible mechanism for the action of lactate was the inhibition of a major anaerobic energy metabolism pathway necessary for growth. which did not provide significant control (Stillmunkes et al.0) alone or combined with modified atmosphere packaging was examined on the survival of L. or 10% lactic acid/sodium lactate buffer rinses (pH 3. Organoleptically. Vacuumpackaged beef was treated with 2% sodium lactate. not sodium. 20°C. None of the treatments affected the growth of either organism. monocytogenes in comminuted chicken or beef when held at 35°C. (1989) demonstrated that sodium lactate was effective in retarding toxin formation by C. Typhimurium but did inhibit P. 10°C. whereas off-flavors developed in meat containing the 3. Sodium lactate (3.Organic Acids 119 Shelef and Yang (1991) found that a 5. however. Reduction in production of hydrogen sulfide and other sulfur-containing compounds resulted from the decreased populations of psychrotrophs. or 5°C. Beef roasts containing sodium lactate. sporogenes. Of all the treatments. or Microgard™ (2%) (Rozbeh et al. or glycerol monolaurate enhanced inhibition of S. Excised skin tissue was analyzed for Listeriae for up to 17 days posttreatment.1°C. or sodium gluconate and inoculated with L. 4 to 6. respectively. Nisaplin (500 IU/g). vacuum packaged.3%) was very effective in reducing numbers of B. calcium lactate up to 0. monocytogenes and the shelf life of chicken legs (Zeitoun and Debevere. Odor concomitant with growth of psychrotrophic spoilage organisms occurred in untreated samples within 8 days and within 9. They concluded that the two salts gave comparable results and could be used interchangeably. all were stored at 6°C.5%. Sodium lactate was more effective at reducing pathogen numbers at levels up to 3. Unda et al. (1991) looked at the interactive effects of sodium alginate and calcium or sodium lactate used in restructured meats on P. Lactic acid at 25 to 30 mEq in combination with sodium nitrite. Ground beef containing sodium alginate at concentrations up to 0. fragi and S. The influence of 2%.2.5%. 2002). fragi at even at the 4% level.0% concentration of sodium or potassium lactate delayed the growth of L. 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. monocytogenes and C. 2. 1993). Sodium lactate at the same concentration had no effect. . botulinum types A. 1980). aureus grown anaerobically (Smith and Palumbo. respectively.. 10. suggesting that the lactate moiety was the effective component. Some treated chicken was packaged without further modification. potassium sorbate. Harmayani et al. Product was considered spoiled when microbial numbers reached 7 to 8 log CFU/g. A 3% concentration of sodium lactate only minimally affected the growth of S. 1991). Lactates have been combined with other compounds in multiple-barrier studies. monocytogenes in TSB and 4% limited growth of L. and 13 days for the 2%. but other samples were placed into packages that were gas flushed with a 90% carbon dioxide/10% oxygen mixture. sporogenes were stored at 2°C.3%. Maas et al. botulinum in comminuted raw turkey using levels of 2%. Toxicity occurred within 3 days in turkey without lactate and within 4 to 5. and 25°C.. and 10% acid spray. They also noted that the inhibitory component was the lactate moiety.

Toxicology Malic acid fed to rats at levels of 500 and 5000 ppm in the basal diet had no effect. Chung and Goepfert (1970) compared various organic acids to determine the highest pH level that inhibited growth of S.. It is used in sherbets and ices. The acceptable daily intake for humans is listed in Table 4. 1994). vacuum packaged. and hydrolyzed vegetable protein and containing 1%.3. inoculated with L. Reproductive experiments using rats fed 1000 and 10. Dogs fed the same three levels of malic acid showed no effects using the same parameters (Hazelton Laboratories. MALIC ACID Antimicrobial Properties Malic acid is inhibitory to fungi and bacteria.1207). 2%. Comminuted salmon was mixed with sodium lactate. 1971a). anatum.. food consumption decreased and growth rate declined (Hazelton Laboratories. thus affecting the sensory qualities of the meat (Egbert et al. and beverages. 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. primarily for its flavoring and acidification characteristics (Gardner. monocytogenes. or 3% potassium lactate. 1970).000 ppm. Application and Regulatory Status Malic acid is a nonhygroscopic. sodium lactate was more effective than the other additives in inhibiting growth of L.1069). a 2% lactate and 3% sodium chloride inhibited L. Nunheimer and Fabian (1940) found that malic acid was inhibitory against S. Salmonella Senftenberg. 1992).3.1639). and an alginate binder.. 1% kappa carrageenan.0 or 4. monocytogenes and total aerobic plate counts (Harmayani et al. In general. potassium (21 CFR 184.1768) are also approved.8% sodium lactate. at pH 6. encapsulated salt. The products were inoculated with L. Carrageenan had no effect on bacterial loads. aureus at pH 3. and sodium nitrite. 0. subtilis). especially rope bacteria (B. the acid inhibited yeasts and molds but not to the same extent as bacteria (Woolford. Similar studies used low-fat ground beef patties formulated with carrageenan. dicarboxylic acid with high water solubility. Application and Regulatory Status Calcium (21 CFR 184. and . 1972).1% sodium erythorbate.0. PROPIONIC ACID Antimicrobial Properties Propionic acid inhibited spore-forming bacteria. however.0. Malic acid is approved as a GRAS substance for miscellaneous and general-purpose usage (21 CFR 184. Some discoloration and lipid oxidation occurred. but the addition of 2% or 3% potassium lactate reduced microbial numbers.000 ppm malic acid before mating showed no significant differences from the controls (Hazelton Laboratories. 1975b). It has a strong acid flavor but does not have the same buildup of acid taste as other acids. jams. probably as a direct effect of pH manipulation. monocytogenes for up to 50 days at 5°C.120 Antimicrobials in Food Raw and cooked ground beef were prepared with 1. jellies. As the pH decreased to 5. sodium chloride. and sodium lactates (21 CFR 184. at 50. 1993). and stored at 5°C or 10°C. fruit preserves. Sodium lactate displayed synergistic activity with nitrite and increasing concentrations of sodium chloride. The acceptable daily intake for humans is listed in Table 4.98. 1971b). monocytogenes and stored aerobically at 4°C.

botulinum.5. pH 5. fumaric and malic. and stored at 28°C. Exposure of L. Turkey breast meat was formulated with sodium salts of lactate. aeruginosa. Emphasizing the multiple-barrier concept in retarding growth. inoculated with C. and 0. Proteus vulgaris. succinic. monocytogenes to 8% sodium propionate for 60 minutes caused injury (Buazzi and Marth.000 IU/g) or calcium propionate (CP.1% to 5% delayed growth of S. ellipsoideus by as much as 5 days. and held at 28°C for 0 to 18 days.3..1.4. (1991) determined that 0. pH 4. The salts of propionic acid are also effective antimicrobial agents. A three-strain mixture of L. 2002). or 4°C. monocytogenes were undetectable.0 was more inhibitory in 60 minutes compared to formic acid in 3 hours for E.. 1. 1945). Reduction of pH to 5.0. plantarum.08% EDTA. 1992).7 M propionic acid at pH 5. pH 4. and greater than 18 days for propionate. 5.9% sodium propionate or SA. Calcium propionate prevented rope formation caused by B. which led to an increase in cell mass without cell division and a decrease in viability (Cherrington et al. little activity was ascribed to EDTA in combination with the acids. acetic. Citrate was most effective based on molarity (Miller et al. and Candida albicans. and berries but also prevented spoilage in vegetables having a more neutral pH. and 35°C and prevented growth at 4°C (El-Shenawy and Marth. 1989a). At a 6% concentration. tartaric. Cherrington et al. or 3. At a 5-mM concentration. Sodium propionate at levels of 0. monocytogenes (Janes et al.0.8. E. monocytogenes. pyruvate.0 with hydrochloric acid. 7. acetate. 1%) and coated onto ready-toeat chicken samples dipped in L. cherries. monocytogenes.6 (O’Leary and Kralovec. Propionic acid was more effective than acetic acid as an antimicrobial. monocytogenes grown at 13°C was extended by 7.0 and 13°C. 1993). L. pH 4. toxicity occurred after 7 days for pyruvate. The acids contributed to approximately 85% of the antimicrobial activity with 14% contributed by ascorbic acid. At pH 5.0 minimized growth at 13°C. pH 5. and 4 days. lipid. acetate.6 with acetic.3% concentration in tryptose broth adjusted to pH 5.156%. coli. and 35°C. respectively. Toxin production occurred after 2 days for pyruvate. Sodium propionate at a 0.3% concentration was also effective at pH 5. protein. aureus. the organism was able to grow at up to a 0. aureus.6. Propionic acid at pH 5. 1941). Eklund (1985) concluded that propionic acid was not as inhibitory as benzoic acid for P. Torula species. and pH. 21°C. pH levels. or lactic acids in media (El-Shenawy and Marth. 21°C.5. pH 4. or ZENCP.188%.3% sodium propionate was adjusted to pH 5. pH 5. pH 4. Zein (Z) film coatings were dissolved in propylene glycol (ZP) or ethanol (ZE) with and without nisin (N. 18 days for citrate. DNA (deoxyribonucleic acid). syrup. 1988). tartaric.. 3. the rate of RNA (ribonucleic acid). and S. (1995) examined the combination of sodium propionate and SA at a number of concentrations. 13°C. 0. applesauce. adjusted to pH 4.Organic Acids 121 Salmonella Tennessee when all other parameters were at their optimum. 1990). and temperatures. coli K12 and Salmonella. and cell-wall synthesis decreased. 4 for lactate and acetate.1. citrate. citric). 4. Nondetectable levels of the pathogen were found in products ZEN. Products were stored for up to 24 days at 4°C or 8°C. the lag phase of L. degree of dissociation of the acid. ZPNCP. Sarcina lutea. This substance not only postponed spoilage in fresh figs. and 5 with a 2% concentration. the levels of L. In laboratory studies examining the effect of sodium propionate on L. monocytogenes was the result of incubation temperature. adipic.6 and incubated at 4°C. S. Incorporation of calcium propionate into coatings for ready-to-eat products was effective in reducing problems with L. citric. lactic. monocytogenes was inoculated into BHI broth containing 0. At periodic intervals.5 inhibited the growth of Salmonellae at a higher pH level than other acids (pH 5. Much higher concentrations of propionic acid were bacteriostatic to B. and lactate. monocytogenes when used in conjunction with acetic acid in cold-pack cheese food (Ryser and Marth. subtilis. .02% ascorbic acid. 1992). or at a level of 0.0 in reducing numbers of L. 19°C. Golden et al. Inactivation of L.5 to 0.05. concentration of the organic acid. When media containing 0. mesentericus (subtilis) in bread dough at a level of 0. and propionate to a target level of pH 6. particularly at the higher pH level.4. such as lima beans and peas (Wolford and Anderson. samples were removed from storage and tested for production of neurotoxin. 3 for citrate.

rubrum. A 5% calcium propionate solution acidified to pH 5. Molds were inhibited by less calcium propionate by weight than sodium propionate.85. Propionic acids and their salts are primarily inhibitory to molds. 0. and repens were inoculated by needle into cake analogues prepared with calcium propionate. monocytogenes at 22% fat compared to 2.5 logs.2%. Ingle. 0. 6.8% sodium lactate. 1951. Increasing the fat content from 22% to 67% decreased the growth of L.03%. Toxicology Orö and Wretland (1961) determined the LD50 of propionic acid for mice. Pseudomonas species. Adding yeast extracts previously fermented by Propionibacterium freudenreichii provided a source of propionic acid in bread formulation.3% by weight. . and 0. Propionic acid at a concentration of 0. B. reduced growth and aflatoxin formation of A. pH 4. 2002a. no growth occurred. 1940). postprocess contamination from the atmosphere. and wrapping materials reintroduce molds. 1996). Mold spoilage is a significant economic problem in bakery products.8 log reductions were observed. amstelodami. 1952a). 1940). with sodium lactate.003%. rather than later (Ghosh and Häggblom. monocytogenes by only 1. Not only was the final pH of the substrate critical. The antimicrobial activity of sodium propionate was most affected at 4°C by fat concentration with a 1. This inhibitory effect was more pronounced with the addition of acid at the time of inoculation. 1942). A. potassium sorbate. (2001). rats. and potassium sorbate at a level of 0. and humans showed no toxic effects (Graham et al.5. Graham and Grice.3%) at pH 4.93 in a model agar system on the retardation of growth of E.. respectively. but also various organisms displayed different tolerances to the compounds (Olson and Macy. However. 1955. and 0. and Penicillium corylophilum.8. sodium benzoate.8 log reduction at 67% fat.0 and a water activity of 0. Eurotium amstelodami. 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. 1953). 0. 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. Bread produced with the extracts contained less ethanol and supported less spoilage by molds. Subsequent studies (Marin et al. Sausage batter was mixed with 0. Although mold spores are destroyed during baking. (1963) determined the LD50 of calcium and sodium propionate for rats (Table 4.03%) led to enhanced growth of Aspergillus and Penicillium isolates.5 log reduction of L. however.. at 0.5.. Suboptimal doses (0.1% sorbic acid or potassium sorbate. flavus and niger.8 to 0. herbariorum.5 with lactic acid was as effective as a 10% unacidified solution in preventing surface mold growth on butter (Olson and Macy. 2000).122 Antimicrobials in Food The effect of fat concentration on the efficacy of antimicrobial agents against L.b) examined several levels of calcium propionate. Hara.2% sodium propionate. 1946).0. 1985). 1. and sodium benzoate (0. 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. some species of Penicillium grew on media containing 5% propionic acid (Heseltine. and 7. Harshbarger. and Hara et al.1%. or Trichophyton mentagrophytes. coli. monocytogenes was studied in pork liver beaker sausage (Hu and Shelef. and rubrum. herbariorum. Small amounts of β-alanine could overcome bacteriostatic action of sodium propionate for E. flavus. 1945). 1965. 1952b). cooling surfaces. All preservatives were effective at pH 6.2). and 0. A novel use of propionic acid to retard spoilage of bread was investigated by Gardner et al.85 (Guynot et al. β-alanine could not reverse the inhibitory effect of propionic acid in Aspergillus clavatus..5 and water activity of 0. The inhibitory action could be the result of an interference with β-alanine synthesis (Wright and Skeggs. Spore germination was inhibited by 1402 ppm (Tzatzarakis et al. There is evidence that the sodium salt has some local antihistaminic activity (Heseltine. 1954. None of the preservatives were effective at neutral pH. subtilis. Additional studies using calcium and sodium propionate fed to mice.5 and 1. 2002).

Application and Regulatory Status Succinic acid is a nonhygroscopic.Organic Acids 123 Application and Regulatory Status Propionic acid is a monocarboxylic acid with a slightly pungent. . hair appearance. It has a slightly bitter taste and serves as a flavor enhancer. (1967) reduced the population of inoculated S. Chick embryos developed normally when comparable dosages were injected into the air sac (Dye et al. however. The acceptable daily intake for humans is listed in Table 4. It can be incorporated into gelatin deserts and cake flavorings (Gardner. Sodium propionate is recommended for use in chemically leavened products because the calcium ion interferes with the leavening action. or eye opening from the control group.38% in whole-wheat products. tooth eruption. which conform to standards of identity.5 mg succinic acid daily. gradually increasing to 2. and growth can be seen under the wrapper during storage.3. Swiss cheese contains up to 1% propionic acid because of the growth and metabolism of propionibacteria. rats received subcutaneous injections of 0. Calcium propionate is preferred. This naturally formed additive becomes a developed preservative that limits mold growth on Swiss cheese. 1980). dicarboxylic acid having low water solubility.23).32% can be used in flour and in white bread and rolls. 1974). however a 3% or 5% concentration used at 60°C impaired the appearance of the product (Cox et al. Thomson et al. The acid form is readily miscible with water.1221) and sodium propionate (21 CFR 184. but surface recontamination can occur during packaging. In addition. Salts of the acid have a slight cheeselike flavor. SUCCINIC ACID Antimicrobial Properties Succinic acid successfully lowered microbial loads on poultry carcasses (Mountney and O’Malley. A limit of 0.0% succinic acid. for use in bread and rolls because the calcium contributes to the enrichment of the product (Chichester and Tanner.0 mg/day at 4 weeks and continuing at this level for 100 days. 1944). 1965). which are associated with its manufacture and the characteristic Swiss cheese flavor. Propionic acid and its salts are used primarily as mold and rope inhibitors in bread. The test animals did not differ significantly in reproduction rates. Succinic acid is approved as a GRAS substance for miscellaneous and general-purpose usage (21 CFR 184. Succinic acid is used to modify the plasticity of bread doughs and in the production of edible fats. It is normally found in cheese and as a metabolite in the ruminant gastrointestinal tract. Baking destroys most molds. Propionic acid (21 CFR 184. and the sodium salt is more soluble than the calcium form. Propionates can be added to bread dough without interfering with leavening because there is little or no effect on yeast.3% in cheese products (Robach. calcium and sodium propionate are listed as antimycotics when migrating from food-packaging material (21 CFR 181. 1972)..1091). 1972). Toxicology In short-term studies. This additive is also used as a mold inhibitor in cheese foods and spreads. No upper limits are prescribed for use of this additive. No limit has been set on the acceptable daily intake for humans. disagreeable odor. except bread and rolls. Its antimicrobial effect is limited to most yeast and bacteria.. are approved as GRAS substances for miscellaneous and generalpurpose usage.1784). and 0. Typhimurium by 60% on chicken carcasses after spraying with 1.1081) and its salts. 0. calcium (21 CFR 184.

which disappeared after they left the work site (Barsotti et al.124 Antimicrobials in Food TARTARIC ACID Antimicrobial Properties Any antimicrobial property of tartaric acid is because of its ability to lower the pH.1099).25 to 1 g to rabbits produced abnormalities in blood chemistry. the most soluble of the solid acidulants. and yeasts. and phospholipids can be structurally altered by pH changes. growth. 0. proton donation. It is used in fruit jams. Inorganic acids. male rabbits consuming a diet containing 7. 1973a). The monopotassium salt (cream of tartar) is commonly found in baking powders.1%. and 215 mg/kg body weight. MODE OF ACTION The mode of action of organic acids in inhibiting microbial growth appears to be related to maintenance of acid–base equilibrium. 1978). Through the interaction of a series of chemical mechanisms. rats. The microbial cell normally reflects this equilibrium by attempting to maintain an internal pH near neutrality (Baird-Parker. respectively (Food and Drug Research Laboratory. and cholesterol levels. Application and Regulatory Status Tartaric acid. 1980). Changes resulting from pH can destroy bacteria. and 1. and the production of energy by the cells. nucleic acids. The workers also displayed skin eruptions. and it prevents discoloration in cheese (Gardner.2). 0.. jellies and preserves. and rabbits demonstrated normal offspring at levels of 274. 1972). 1963). this delicate balance is maintained and alteration of this balance causes destruction of the microbial cell. 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. hamsters. Biological and chemical systems depend on an interaction between acid and base systems. Rose’s (1924) work on subcutaneous administration of 0. These changes in membrane permeability can exert a dual effect by impairing transport of nutrients into the cell or by causing the leakage of internal metabolites to the outside. The 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.. including increased nonprotein nitrogen. sugar. The acceptable daily intake for humans is listed in Table 4.8%. such as hydrochloric. 1954). and grape-flavored beverages. or gross histology compared with controls (Packman et al.2% of the diet. mortality. However. Homeostasis is the tendency of a cell to sustain chemical equilibrium despite a fluctuation in the acid–base environment. Workers exposed to tartaric acid dust measuring up to 32 mg/m3 in a manufacturing plant that produced the acid experienced tooth erosion. Proteins. It is thus essential to understand each of these concepts before consideration of the actual mode of action of the organic acids. Tartaric acid acts synergistically with antioxidants to prevent rancidity. Fitzhugh and Nelson (1947) found similar results in long-term studies using 21-day-old weanling rats fed tartaric acid as 0. 225. . Tartaric acid is a GRAS substance for miscellaneous and general-purpose usage in accordance with good manufacturing practices (21 CFR 184. Studies measuring teratogenic activity in pregnant mice.3. although some microorganisms such as Acetobacter can exist at and even require extremes of acidity (Langworthy. 181.5%. molds. has a strong tart taste that enhances grapelike flavors.7% sodium tartrate (5% tartaric acid) did not show any changes in food consumption. Toxicology Acute toxicity data for tartaric acid by the intravenous route for mice indicates an LD50 of 485 mg/kg body weight (Table 4. sherbets.

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

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

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

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

. The pKa values for sulfur dioxide are 1. FORM AND SOLUBILITY In water solutions. 1928.1.76 and 7. 1968). their theoretical yields. A plot of the distribution of the three ionic forms can be calculated and is shown in Figure 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. The dry salts are easier to store and are less of a problem to handle than gaseous or liquid sulfur dioxide. they show increasing stability in the order sulfite > bisulfite > metabisulfite (Mason. Table 5. It is useful to have the sulfur dioxide in a salt form.1. The anhydrous sodium and potassium salts are prepared by precipitation and then dehydrated with the appropriate hydroxide.20. as well as by other methods. The metabisulfite is the anhydride of the acid sulfite: – → 2– 2 HSO3 ← S2O5 + H2O When these salts are exposed to air. and from reducing gypsum.144 Antimicrobials in Food Sulfur dioxide can be prepared commercially from burning sulfur (oxidation). Most of these salts are hygroscopic and easily hydrolyzed (Schroeter. 1928).2 lists the main forms of sulfur dioxide. and solubilities of each in water. 1966). Phillips. indicating a rather weak dibasic acid (Segal. 100 SO2 80 % of Total H2SO3 − HSO3 SO− 3 − 60 40 20 0 0 1 2 3 4 pH 5 6 7 8 FIGURE 5.1 Distribution of the ionized forms of sulfurous acid at various pHs. heating pyrites. Specific gravity for various water solutions of sulfur dioxide is shown in Table 5.

1970). the second equation listed previously is a key reaction (Heinmann et al.0001°F–1 TABLE 5.0292 1.003 1.4 53. If ascorbic acid is being used in combination with sulfur dioxide.028 1.Sulfur Dioxide and Sulfites 145 TABLE 5.0393 1.0091 1.5 61.at pH 3.56°C (60°F) 1..0040 1.037 — Corrections are approximately 0.018 1.008 1.6 57. much slower changes occurred in the sulfur dioxide content.40 and 15°C.. The sulfur dioxide stabilizes the dehydroascorbic acid by reacting with the ketone bonds (Wisser et al.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.0493 20°C (68°F) 1.7 ± 3. After the oxygen disappeared. .8 25.1 Specific Gravity of Various Sulfur Dioxide Water Solutions at Two Temperatures SO2 (g per 100 g) 1 2 3 6 8 10 a Specific Gravitya 15. This reaction is extremely rapid. The sulfur dioxide scavenges the hydrogen peroxide formed and keeps further oxidation of the dehydroascorbic acid and other products to a minimum.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 50.4 s-1 for H2O2 with SO32. Holt and Kumar (1986) found an observed k = 41. 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.0191 1.6 67. 1970).

1985).5-hexodiulose. only ketones with a methyl group adjacent to the carbonyl or carbonyls that are part of a four. Those reacting the most rapidly formed complexes that dissociated the least. 1973. He also noted that phosphoglyceraldehyde would likely react. Bisulfite addition products. 1964a. 1963). and arabinose reacted rapidly with bisulfite.3 (Burroughs and Whiting. 1954. and galacturonic acid were significant bisulfite binding forces (Burroughs and Sparks. and fructose and sucrose did not react at all. a Aerny (1986 a. L-xylosone. d-threo-2.to seven-member carbon ring system will react. In botrytized grapes made into wine. 1960. and the temperature. 1986 a. form rapidly with aldehydes: – – → HSO3 + R-COH ← R-CHOH-SO3 All aldehydes form the hydroxysulfonates.b). Lea et al. 3 = Navara (1985). Joslyn and Braverman. Aerny.12 — 2 99. Rhem (1964) noted that the rate of formation and the amount of sulfonate formed depended on the concentration of the reactive substances. Navara..5-diketogluconic acid.146 Antimicrobials in Food TABLE 5. 2-oxogluconic acid. and glucose reacted less rapidly.b. Otherwise. α-ketoglutarate.b). acetaldehyde. 2.7 × 102 — — — — 15. Relative percentages of aldehydes reacting with sulfur dioxide and their equilibrium constants are shown in Table 5.1 87 3 50 32 18 — — — — — 26 — 45 32 0. but not all ketones react.1 — 1.1 — 0. 5-oxofructose. and wines.0 0.9 6 × 10–5 Note: 1 = Burroughs and Whiting (1960). In moldy apples.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. pyruvate. fermented ciders.5 — 2. 1954). . 2 = Aerny (1986a. Glucose is by far the most abundant of the reactive aldehydes and ketones in most fruit juices. as much as 80% of the total sulfur dioxide may be bound by these types of carbonyls (Blouin.5 3 5 8 Ka × 10–6 × 10–4 × 10–4 × 10-6 — 2 × 10–4 5 × 10–2 1. 1944). 2000). natural fruit juices always bind more sulfur dioxide than would be calculated from the glucose present in the juice (Joslyn and Braverman. mannose.7 90 1. maltose. the pH. 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. Ingram and Vas (1950) found that galactose. raffinose reacted very slowly.1 0. lactose. 1954). Compared with model solutions. Diethyl ketone reacts slowly and to a limited extent. the hydroxysulfonic acids (Suter.5 72 44 98 — 66 1 2 — — — — 0.6 7.

Undoubtedly other reactions with sulfur dioxide take place that could reduce the amounts of available sulfur dioxide. Glories (1984) studied the equilibrium between SO2 and the anthocyanin–bisulfite complex. The reaction affected flavor of the mustard by reducing pungency. it is reduced by 80%. but these have been reported as the primary reactions.. 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. (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. In model solutions in the range of 30 to 50 mg/L of SO2 added. and at 50 mg/L of SO2 added.5 mg/L.g. (1983) tested 30 strains of Saccharomyces cerevisiae for production of SO2 binding materials.6 mg/L.. Jurd (1972) suggested the binding site for HSO3. Piracci and Spera (1983) reported similar results. and other SO2 binding substances limit the effective use of the added sulfite. but they found no increase in pyruvate or α-ketoglutarate. . pyruvate. sunset yellow. After approximately 100 days of storage. and αketoglutarate. 1998).5-diene (Crowell and Guymon. Traces of the compound could be found in stored commercial soft drinks that had been heated and contained sulfite. the following formula was used to calculate the equilibrium constant Ks. from winery trials. selected four yeasts that produced low levels of SO2 binding compounds. Bisulfites may also react with nucleotides such as nicotinamide adenine dinucleotide (Meyerhof et al. Damant et al. (1982). This treatment allows more effective use of SO2 in wine. Dittrich and Barth (1984) did find correlations between the SO2 binding substances and wineries and grape source. (1984) also showed that sulfite additions increased acetaldehyde. There is no evidence that these reactions occur in vivo.5 mg/L. Farris et al. (1985) found a reduction in the content of the SO2 binding materials by the addition of 0. Sulfur dioxide can loosely bind to anthocyanins.. The respective ranges and means for these components were 72 to 287 and 115. ˇ Farkas et al. 1938). 16 to 42 and 27. ketones. Shapiro and Weisgras (1970) also demonstrated that cytosine was transformed to cytidine by bisulfite. the color is reduced by about 25%. Margheri et al. (1989) found a food-coloring dye. reacted with bisulfite to form a lemon yellow compound. Lafon-Lafourcade (1985) reviewed the role of yeast and bacteria in the amounts of these components found in wines. and 15 to 57 and 31.5 mg/L of thiamine. This contributes to the difficulty in the measurement of free sulfur dioxide in highly colored wines.is on the 4-position rather than the 2-position (e.Sulfur Dioxide and Sulfites 147 The amounts of aldehydes. Farris et al. The addition product attached at the carbon 4 of the sunset yellow molecule. – 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. Allyl isothiocyanate in mustard was shown to react with sulfites used as antioxidants to form allylaminothiocarbonyl sulfonate (Cejpek et al. Heintz (1976) reported the occurrence of an addition product of sorbic acid and bisulfite. Uzuka et al. They measured acetaldehyde. 1975). 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.

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

3 log CFU/ml.. (1984) stated that the amounts of SO2 used in normal wine making are insufficient for acetic acid bacteria control. Oenococcus oeni grows preferentially at lower pH but seems less tolerant to sulfur dioxide (Mayer. Manca de Narda and Strosser de Saad (1987) investigated the tolerance of O. is very heat resistant and alcohol tolerant. They found that up to 1. Pediococcus.4 (Holt et al. Lactobacillus buchneri. 20 days was required to kill the bacteria when the same amount of aldehyde–bisulfite complex was used. Lafon-Lafourcade (1975) demonstrated the effectiveness of both free sulfur dioxide and bound sulfur dioxide in inhibiting Leuconostoc gracile in wine at pH 3. and Pediococcus pentosaceus. plantarum. for antimicrobial action at pH 6. (1980) reported that 30 mg/L of free sulfur dioxide is sufficient to inhibit malolactic fermentation in wine. They indicated A. Lactobacillus brevis. Leuconostoc mesenteroides subspecies dextranicum can also cause ropiness in beverages through the formation of dextrans. This organism is particularly intolerant to sulfur dioxide.0 in a buffered solution. respectively. 320 and 651 mg/L of sulfurous acid were required. red wine. 1983). Dupuy and Maugenet (1963) noted that even small doses of sulfur dioxide inhibited the activity of the cells. and ethanol. Cell growth was completely prevented. 1949) is described as causing a hairlike growth in fortified wines. with lower pH increasing the effectiveness of the sulfur dioxide. 1980). Rhem and Wittmann (1962) reported that 200 mg/L sulfur dioxide killed Acetobacter at pH 6. Liu and Gallander (1983) demonstrated that lower pH wines underwent .5. Several reports (Karova and Kircheva. to carbon dioxide and water. L. pH. oeni was the most sensitive to SO2. Levels above 120 mg/L of total sulfur dioxide (free and bound) decreased the incidence of malolactic fermentation. O. casei. 1983) indicated that around 50 mg/L of free sulfite could preserve wine vinegar for about half a year.. They are aerophilic and have pH optimum for growth of around 5.0 (Rhem and Wittmann. hilgardii. In model solutions containing Lactobacillus arabinosus and L.5 mg/L were bactericidal. 1994).5 mg SO2 per liter in the undissociated form was bacteriostatic to L. Carr and Davies (1971) noted that relatively high amounts of free sulfurous acid were present in ciders that contained viable lactobacilli. 1982. Lactobacillus trichodes (Fornachon et al. and Oenococcus.47 log and 0. 1962). 1982.49 log Acetobacter per ml exposed to 100 and 200 mg/L of total sulfur dioxide in grape juice were reduced to 2. respectively. however. Concentrations above 1. The juices high in insoluble solids ultimately had lower residual total sulfites. The homolactic species found in wines are Lactobacillus acidophilus. Cruess (1912) found that 5. Watanabe and Ino (1984) and Juven and Shomen (1985) reported that up to 100 mg/L of total sulfites for grape juice. Fornachon (1957) noted that sulfur dioxide and pH were important factors in controlling lactic bacteria in wines. and the heterolactic species are Lactobacillus fermentum.. Spirov et al. 1979). The bacterium. Lactobacillus leichmannii. Amerine et al. pentosaceus to SO2. but much larger doses were required for bactericidal action. after 36 hours. aceti can grow in red wine with 25 mg/L of unbound sulfur dioxide present.Sulfur Dioxide and Sulfites 149 oxidize ethanol in fermented beverages to acetic acid and. Lactobacillus delbrueckii. Bacteria common in acid fruits and beverages are the lactic acid-producing genera Lactobacillus. Lactobacillus trichodes. LafonLafourcade and Joyeux (1981) and Joyeux et al. Lactobacillus plantarum. As little as 30 mg/L of added sulfur dioxide may be lethal to the species. Lactobacillus casei. As low as 75 to 80 mg/L of total sulfur dioxide prevents growth and 100 mg/L kills this species. Pediococcus cerevisiae. Lactobacillus hilgardii. Leuconostoc. Addition of 30 mg/L of free sulfur dioxide killed the bacteria completely in 15 days. He found that this genus also fixed a certain amount of the sulfur dioxide. 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. and P. Additions of 100 mg/L bleached the color of the vinegar. and soft drinks were required to control acetic acid bacteria. 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. further. and Oenococcus oeni (Amerine et al.. oeni.

the main effect in meat appears to be the antioxidant properties (Roberts and McWeeney. 200–241. oeni strains were less tolerant to the sulfur dioxide than Pediococcus paroulus strains or the Lactobacillus species. were completely effective. In addition. 190–241.150 Antimicrobials in Food malolactic fermentation more slowly and that lower sulfur dioxide levels increased the fermentation rate of the O. coli were inhibited to a greater extent by sulfites than other bacteria. O.5 mg/L of molecular sulfite was sufficient to control malolactic bacteria growth. The microorganisms tested and the concentration of free sulfite (µg/ml) necessary to inhibit their growth at pH 7. oeni strains with unfavorable sensory results. The delay was proportional to the concentration of the bisulfite addition. Piracci (1984) found 0. This work confirms that wines with high total sulfur dioxide concentrations are more likely to undergo malolactic fermentation with other than O. E. They also noted that the interaction of sulfites with nitrites caused a lowering of the nitrites available for nitrosamine formation.0 were as follows: Salmonella. The Gram-positive bacteria remaining grew more slowly than the Gram-negative bacteria. 50–195. Enterobacter agglomerans. They noted that sulfites shifted the microflora of the meat to Gram-positive bacteria from the normal Gramnegative flora. Citrobacter freundii. The packaging used was a gas-permeable wrapping that allowed oxidative conditions (von Holy et al. and Hafnia alvei. All strains grew at pH 4.6 days at 0°C with the addition of 250 mg/kg of sulfur dioxide. (1983) determined that. (1987) found that vacuum packaging and a good oxygen barrier film decreased the . (1988) demonstrated that without sufficient SO2 and adjusted pH. in the Bordeaux area of France. The shelf life of ground beef was effectively increased from 1. such as Escherichia coli and Pseudomonas. Sensory tests showed no adverse results.. or in paired combinations. Banks et al. (1988) found significant delays in growth of O. The preservation of the color and odor of meats is improved by sulfite treatment and. 1988). 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. a rapid decline in cell numbers occurred. None of the compounds alone. (1988) tested 146 different strains of wild malolactic bacteria. oeni when SO2 was added and quite a variable response between strains. in their review. 83–142. Adams et al. Wibowo et al. This is demonstrated in the use of sulfites in meats. 65–136. although after sulfite addition. (1985) reviewed the use of sulfite as an additive to control microbiological changes occurring in meat products. although slowing or prevention of growth of surface bacteria is probably important. 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. Banks and Board (1982) tested several genera of Enterobacteriaceae isolated from sausage for their metabisulfite sensitivity. Tompkin et al. Yersinia enterocolitica. Roberts and McWeeny (1972). oeni PSU-1 used. Davis et al. Ough et al. than in inhibiting Gram-positive bacteria. They suggested that these microorganisms could cause spoilage in wines that were considered sterile using conventional counting techniques. The effect was inhibition of growth of the flora. 15–109. 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. O. There was some additive effect when used with dimethyl dicarbonate but not enough to warrant the use with SO2 for this purpose. oeni was the primary malolactic bacterium associated with wine.5 in beef broth and 20% tomato juice serum medium at 64 mg/L of total sulfur dioxide. 67–98. 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. Serratia marcescens. state that sulfur dioxide is more effective against the growth of Gram-negative rods. oeni in red table wine occurs readily. Salmonella and E. Lafon-Lafourcade et al. the growth of O. 1972). It was found to tolerate up to 100 mg/L of SO2. coli.8 days at 7°C storage with no treatment to 12. It survived alcoholic fermentation when others did not. (1980) found the addition of 100 mg/kg of SO2 as sodium metabisulfite to canned pork inoculated with Clostridium botulinum spores delayed cell growth.. The remaining cells later multiplied to significant numbers.

60 0. Any rule of thumb addition.2 mg/L of molecular sulfur dioxide were necessary at the finish of fermentation and during aging. According to Warth (1985). Thus the free sulfite. Carr and Davies (1971) found sulfur dioxide incorporated into growth medium (pH 3. Molecular sulfur dioxide is the most effective form for inhibiting yeast. phenylethylamine.05 M free sulfur dioxide for Kloeckera apiculata to 2.0 mg/L were required. In contrast. This was because of the lack of oxygen delaying yeast growth and the production of sulfite-binding substances. The yeast also showed increased resistance to the fungicide dimethyl dicarbonate. 1.0. bailii.81). with the pressure to reduce sulfur dioxide content. Sudraud and Chauvet (1985) found that to maintain yeast stability. Six strains could grow well at 1000 mg/L. or tryptamine.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. spoilage in sulfite-treated sausage. Rhodotorula. pH is extremely important in the effective use of sulfur dioxide. Saccharomycodes ludwigii was nearly as tolerant as Z. The amount of molecular SO2 (mg/L) in wine can be calculated using free SO2 mg/L/(1 + 10pH . Haznedari (1979) tested 30 strains of S.60 Rhem and Wittmann (1962). Ough et al. and five others were able to produce adequate amounts of ethanol. was maintained for a longer period. and several other genera were very susceptible. 2001). cerevisiae (105 CFU/ml) after 8 hours at 25°C.5 in tryptone yeast extract medium. its use to prevent the growth of yeast in sweet table wines is seldom ever contemplated.2–8. The concentration of tyramine and putrescine increased in the presence of sulfite.0 and 3. Pichia.20 0.0 and 3.40–0.10–20.20 0.4) at 25 mg/L was sufficient to kill a culture of S. which inhibited growth.8 mM for Z. cerevisiae that had been characterized as “SO2 resistant” for tolerance to high concentrations of sulfur dioxide. A 10-fold increase in molecular sulfur dioxide occurs between pH 4. (1988) found that with yeast acclimatized to sulfur dioxide.4).20 7.5 and 1. the growth rates and cell yield were similar. Delfini (1989) . bailii at 25°C under aerobic conditions at pH 3. resistance of yeast to sulfur dioxide ranges from 0.7 0. Sodium sulfite addition in sausage was shown to affect biogenic amines. Yeast The use of sulfur dioxide to deplete the wild yeast in grape juice is a standard practice dating back many years (Cruess.1.” for biological stability can be disastrous. Rhem and Wittmann (1962) determined the inactivation levels of sulfurous acid for a variety of yeast genera (Table 5. 1912). There was no effect on histamine.. such as “maintain the free SO2 at 20 mg/L.Sulfur Dioxide and Sulfites 151 TABLE 5. 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. whereas Kloeckera. but the level of cadaverine was reduced (Bover-Cid et al. respectively. 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. Once the inhibition was overcome. In fact. between 2.

1985.5). (1983). Fruit juices and preserves can be protected from microbial spoilage by sulfur dioxide addition. ludwigii. cerevisiae despite a reduction in the bacterial counts in the system at the same concentration. using sulfide salts before fermentation. ludwigii in a low-alcohol wine. (1977) determined the cause for sulfite production to be sulfate permease inhibition by methionine. In normal sweet table wines. however. cell numbers are depleted to very low levels at bottling. Spoilage yeast in dry wines are fairly rare. Suzzi et al. Generally. In high-sulfite-producing yeast. does not always inhibit growth and fermentation. Recommendations include cooling the fruit before crushing. Spoilage by Z. 1985. Although other agents may act as antimicrobial agents. 1985). Galassi and Mancini. Vernerova et al. (1989). The treatment was reported to be successful for juice. Even at low pH (2. bailii. substituting other preservatives at bottling. especially wines in barrels. ludwigii is also a noted spoiler in sweet grape juice containing high amounts of sulfur dioxide (Jakob. Brettanomyces can contaminate a winery. Yeast can form sulfite from sulfate via sulfate permease. Some yeast strains can form rather large amounts of sulfur dioxide during juice fermentation (Table 5. S.7 (Lloyd. 1986a. even at the legal limits. This shows that most wines will not be free from sulfite labeling requirements even if no sulfur dioxide is added. (1985) tested 1700 Saccharomyces. minimizing the air around the wine. (1985) used an initial 1000 mg/L of sulfur dioxide in the last stages of the manufacture . a late-harvest orange juice. none seem to be capable of replacing the antioxidant property of SO2. soft drinks. There have been numerous reports on techniques to minimize the amount of sulfur dioxide used in wines (Aerny. reducing the amount of sulfur dioxide before or during fermentation. Breweries have an interest in sulfur dioxide as an antioxidant. Valouyko et al. using yeast that produces minimum amounts of sulfur dioxide-binding components. and there was no correlation with the number of cells in the initial inoculum.152 Antimicrobials in Food reported the existence of very sulfite-resistant S.6). 1975). if proper sulfur dioxide levels are not maintained. In a cell recycled ethanol fermentation system. Ranote and Bains (1982) used 350 mg/L of sulfur dioxide to preserve Kinnow. Patel et al. excess amounts are used.. Schmitt et al. Asvany. bailii was becoming a problem in juices. 1982) as an alternative for pretreatment of grape juice before fermentation. Sethi and Anand (1984) had to use 692 mg/L in carrot preserves to inhibit Bacillus cereus. settling and racking the juices. alcohol. investigating the sulfur dioxide content of green beer. Dott et al. Proper selection of the yeast strain to avoid high levels of sulfite is an obvious choice. Chang et al. Strain differences caused variation in the sulfur dioxide produced from 11 to 26 mg/L.4). adjusting the pH downward if necessary. and eliminating oxygen from bottled wine. Minarik and Navara (1977) reported finding the spoilage yeast S. 1985. With modern filtration and sanitation technology. stated that the use of sulfide salts failed to protect wine and left negative sensory characteristics in the wine. bailii in comminuted orange drink was controlled by 230 mg/L of SO2 at pH 3. sulfur dioxide. 1978). This particular species was found to be very resistant to sulfurous acid.6 and 3. Angelino et al. 1985. Hydrogen sulfide (H2S) was used (Ubigli et al. Sand (1980) suggested that Z. Hernandez. Gomes and da Silva Babo. found no correlation with dissolved oxygen levels in wort and no correlation with ATP sulfurase or sulfite reductase. cerevisiae. and wines because of its tolerance to SO2. Very little of the sulfur dioxide formed by the yeast remains in the free state. which was at pH 3. the main spoilage yeast is Saccharomyces. and Schizosaccharomyces japonicus in the re-fermentation of sweet champagnes. They found a majority of the wines in the test to contain about 10 mg/L of total sulfur dioxide (Table 5.b.. Z. and low pH. With this genus. (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. because the pH is high. the sulfate permease was not repressed by methionine. (1997) showed that sulfite up to 400 mg/L had no effect on S.1 but not in the base material. Growth was found at the highest concentration of free sulfur dioxide used. Pearlstein (1988) found that extended aeration decreased sulfite production. S. 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.

170. uvarum S. (1976) Minarik (1975) Minarik (1975) Heinzel et al. (1976) Poulard and Brelet (1978) S. (1976) Delfini et al. 40 0 18. bayanus S. 150 0. 5. 100. bailii S. 18.5. rosei S. italicus S.5. 4. 100 5. 21. chevalieri S. pastorianus S. 35 20. (1976) Delfini et al. 56. (1976) Poulard and Brelet (1978) Minarik (1975) Poulard and Brelet (1978) Minarik (1975) Delfini et al. 1300 22. 76 17.5 14.5 Formation of Sulfur Dioxide by Various Yeasts during Grape Juice Fermentations Yeast Saccharomyces carlsbergensis Sulfur Dioxide (mg/L) 12 150 80. acidifacien S.6 Amounts of Sulfur Dioxide Found in Test Wines Fermented by 1700 Saccharomyces Yeasts (Suzzi et al. (1976) Delfini et al. 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 . cerevisiae S. 500 5. rouxii Schizosaccharomyces pombe Torulopsis stellata Kloeckera apiculata K. 18. (1976) Delfini et al.5. 3. 500 300. (1976) Poulard and Brelet (1978) Delfini et al. 44.5 0 0 0 1. 35. 160. 1. 5 18–23 85 0. 52. 20 1. 21. magna K. (1976) Delfini et al. (1976) Delfini et al. (1976) Delfini et al.5 12 0 9 Reference Minarik (1975) Eschenbruch and Bonish (1976) Heinzel et al.. (1976) Poulard and Brelet (1978) Delfini et al. (1976) Minarik (1975) Eschenbruch and Bonish (1976) Heinzel et al. 3. 20 26 27 0 0. (1976) Delfini et al.Sulfur Dioxide and Sulfites 153 TABLE 5. africana TABLE 5.

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

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

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

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

and fillings Jams and jellies Nonalcoholic beverages Sausage Sugar confectionary Vinegar Wine a Use Concentration (mg/kg SO2)a 10–30 100 50–1000 10–100 50–100 50–500 50–100 20–200 450 50 50–200 100–300 Sulfites are allowed only in certain foods in different countries. and .20 975.. purees. 2000) Food Beer Fresh fruits Fresh vegetables (onion. and concentration varies by country. The official methods of analysis for agricultural and food products are specific in their recommendations for analysis of sulfur dioxide (Table 5.02 961. horseradish) Fruit juices Fruit-based sauces and related products Fruit pulps..8 AOAC International Official Methods of Analysis for Sulfites (Warner et al. TABLE 5. A sample is placed in a distilling flask. acidified. Those sulfites that bind food matrices and are not converted to sulfur dioxide with heating or acidic conditions are irreversibly bound. garlic. Free sulfites are readily converted to sulfur dioxide upon acidification and can be quantitatively analyzed following distillation. 2000).16 963. Sulfites can be described as free.28a 990. reversibly bound.30 990. 2000) Method Number 892. Reversibly bound sulfites are converted to sulfur dioxide only after heat and acid or alkali treatments.158 Antimicrobials in Food TABLE 5.31 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.7 Application of Sulfites in Foods as Antimicrobials and the Concentrations Used (Gould. or irreversibly bound (Beck et al. One official method for measuring total sulfurous acid is the modified Monier-Williams procedure.29 990..32 980. 2000). Thiosulfonates are irreversibly bound (Beck et al.04 990.09 962.17 987.8).

Paul (1958).Sulfur Dioxide and Sulfites 159 heated. Schwedt (1986) considered the test strips satisfactory but noted that if dyes or natural pigments came in contact with the cellulose. the sulfate can be precipitated with barium and measured gravimetrically. 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. (1979) showed that sodium sulfide and allyl isothiocyanate gave positive results in the MonierWilliams tests. in addition. Basically the sample is sparged with gas for 12 to 15 minutes. leeks. 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. 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. Briefly. and the volatile sulfur dioxide is removed by a stream of nitrogen through a reflux condenser. This method is good for most products. 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. 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. The Ripper method for free sulfur dioxide is given by Ough and Amerine (1988). The reflux condenser must retain all volatile acids except the sulfurous acid.8) (Warner et al. Schneyder and Vlcek (1977) reported that iodate was superior to an iodine standard solution. Rankine and Pocock (1970). except dried onions. artificially high results occur. Free sulfur dioxide is easily measured in white wines by the Ripper method. Vahl and Converse (1980) made a collaborative study of the Ripper method for the Association of Official Analytical Chemists and concluded that the poor precision and large systematic error precluded it for adoption as an official method. Excess iodide is added to the sample. The optimized Monier-Williams method is used by the FDA to measure sulfites in official samples (Table 5. but if other oxidizable substances are present. 1988). and Ough and Amerine (1988) described the method and equipment needed.. The same errors are associated with this procedure as are associated with the Ripper procedure for total sulfur dioxide. Mitsuhashi et al. 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. Kielhofer and Aumann (1957). the sample is made basic to break the bisulfite addition products. Burroughs and Sparks (1964b). it is then acidified. . 2000). but determinations on red wines are more accurately done by this variation of the Monier-Williams method. and the hydrogen ion produced in the peroxide solution is titrated. and it is then titrated with the iodate solution to a starch end point. and cabbage. 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).

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

............... 2000) and the microbiologic.. 1971..... their use was to fulfill the basic need to preserve food..... 1981...... At the same time a considerable amount of research was directed toward identifying the risks associated with the use of nitrite in food. Reported outbreaks of botulism attributed to meat and poultry products and an assessment of nitrite for botulinal protection have been summarized (Tompkin.............. Lechowich et al. Roberts et al..217 Toxicology.......... By generally arranging the information in chronologic order....... For this reason.. 1963..... particularly in the United Kingdom and Europe............ 1979a..221 This review of the antimicrobial properties of nitrite is one among many.. It is a curious thought that if nitrite were disapproved.. the selection of the experimental procedure.... Binkerd and Kolari (1975) reviewed the history of nitrite and nitrate as curing agents for meats................ 1979)..... Duncan.............. 2000) and other components of meat (Cassens et al.... controlled processing conditions provide the benefits of food preservation with no measurable risk to consumers..................................... considerable effort was directed toward verifying the benefits of nitrite relative to control of certain pathogens and quality attributes....... would disappear from the market.... Extensive updates on the chemistry and impact of nitrite on the color and flavor of cured meats (Pegg and Shahidi. To better understand this complex issue.... and physiologic effects of nitrate and nitrite (Archer....... 1974... Holley...... It is tempting to delete a significant quantity of information from this third revision of the chapter..... With the new millennium a different perspective has evolved..... 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.............. as we know them...... insight is provided into why the research was performed......218 References ..... however.......... however..... in certain respects from other reviews..............216 Regulations Governing the Use of Nitrite and Nitrate . The review by Gray et al........................... this chapter would not exist in this book............................... Other comprehensive reviews are available that provide additional information and other perspectives (Anderton.. The use of nitrite and/or nitrate in foods has been influenced by a variety of factors.. 1991. Eklund (1982) reviewed the potential risk of botulism in fishery products and the role of nitrite as a preservative............................... Pegg and Shahidi.............. Cerveny (1980) described many of the changes in the production and marketing of cured meats in the United States since 1900............... 1980... 1978...... Ingram............. the reader should consider reactions with heme compounds (Fox. It differs.......... then cured meats............. Roberts et al.... the reader is encouraged to read the historical ........... Bard and Townsend............................ 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. (1981) and Pegg and Shahidi (2000) of the effect of nitrite on the stability of cured meat flavor provides additional possibilities.217 Assay Method . The time and resources expended to resolve the nitrite issue were enormous and worldwide. The many chemical reactions that occur when nitrite is added to meat offer clues to the reactions that may occur with microbial cells. The discovery in 1969 that nitrosamines can be formed in foods containing nitrite led to a series of events...... 2002) have been developed........................ Benedict........... 1966.......... 1991........ 1992)...... Sofos et al... Initially. toxicologic... 1970.. As questions were raised from about 1970 to 2002 over the toxicologic safety of nitrite.... This chapter concentrates on the antimicrobial research. is discussed in Hill (1991)............ Pivnick... 1980)..... The status of nitrate and nitrite in food and water until about 1990.... 1980... Current regulations for the use of nitrite and nitrate and modern................. Skovgaard... At the heart of the issue was whether nitrite would continue to be an approved food additive.... and the information that was available to influence the researchers’ conclusions......170 Antimicrobials in Food Summary for 1990–2002 ..... If nitrite were not available...............

solving a problem of sour hams. 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. nitrite was considered to have no value in meat other than for flavor and color development. Tanner and Evans (1933). 6 of the 7 cultures were inhibited. 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. controversial history over whether nitrate and nitrite have antimicrobial properties. Nitrate was believed to provide the antimicrobial effect. or 12. the highest levels tested. is responsible for retarding proteolysis by C. RESEARCH HISTORY BEFORE 1950 Tucker (1930) studied the proteolytic activity of a Clostridium putrificum isolate from sour ham. of the three. irregular inhibition occurred among seven botulinal cultures. By the time the nitrosamine controversy arose in 1969 and the early 1970s. 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. the value of nitrate. During the 1940s and early 1950s. Unlike the other antimicrobial agents discussed in this text.000 µg/g. and the value of nitrate was relegated to serving merely as a reservoir for the generation of nitrite. Considering that research continues to reveal new interactions among the chemical constituents of cured meat. 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. Proteolysis was inhibited by 4. 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. despite meat having a pH closer to 6. By the middle 1950s.6 and held at 37°C. He concluded that.0. putrificum.000 and 44.0. This concept became widely accepted. reproducible results in all instances. Most of the research on sour hams involved test tube experiments with culture media above pH 7. It is still difficult to design experiments that yield predictable.000 µg/g of sodium nitrate.Nitrite 171 documentary of Cassens (1990).0% sodium chloride (salt). were not inhibitory to any of the strains. C. One of the messages from the 1920s is that nitrate serves as a potential reservoir for conversion to nitrite and thus to nitrous acid. We are still ignorant of all the significant factors that influence the efficacy of nitrite or. At 44.000 µg/g of sodium nitrite in a pork infusion broth buffered to pH 7. The list of recognized factors influencing the antimicrobial efficacy of nitrite in cured meats continues to grow 60 years later. At levels between 22. He suggested that nitrite. The same results were obtained with Clostridium putrefaciens at 23°C. for that matter. Levels of 3100 µg/g in pork infusion and 3900 µg/g in egg-meat. 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. 44. using Clostridium botulinum. Clever as the scientific community has been in more clearly defining the antimicrobial properties of nitrite. Research of the 1920s and 1930s focused on using nitrite to cure meat. This basic flaw in experimental design led to results that misguided and hardened opinions in the industry for 20 years. only salt was used in sufficient concentration in commercial hams to prevent proteolytic activity at 37°C. Until the early 1940s. and Clostridium sporogenes. 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. The authors concluded that nitrate at concentrations as low as 22. .000 µg/g.000 µg/g did not inhibit the 12 strains tested in either pork infusion or egg-meat media. research had clearly demonstrated that nitrite has significant antimicrobial properties. there has been a long. putrificum. not nitrate. data appeared that began to prove the opposite. found that sodium nitrate up to 22.

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

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

6. 7. Nitrite was clearly shown to be an effective antimicrobial agent. and sterilized meat were of low. 1950–1960 Research of the 1950s clarified the relative significance of salt. Adding 200 µg/g of sodium nitrite was considerably more inhibitory to toxin formation than the addition of 1000 µg/g of sodium nitrate. 5.000 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. even in the presence of salt or nitrite.5%) and nitrite (150 to 170 µg/g) was much more effective than either substance alone.174 Antimicrobials in Food Vinton et al. Spores produced in raw. 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. increased inhibition of PA 3679 was obtained as sodium nitrite was increased from 400 to 800 µg/g. An underlying factor in the research before 1940 was the unfavorable experience of 1928 and 1929 with temperature-abused perishable canned hams. 4. intermediate. SUMMARY FOR BEFORE 1950 A summary of the state of knowledge by 1950 might include the following: 1. there was increased inhibition of outgrowth of surviving spores by salt and.37% and a pH range of 6. and nitrite as preservative agents and expands this information to other microorganisms. however. Salt at the levels used at that time was considered the cornerstone for the preservation of cured meats. however.4% or sodium nitrate in excess of 40. but the condition of the meat was a factor. Halvorson (1955) later discussed the widespread problem of temperature abuse at the retail level. and high heat resistance. The basic formulation resulted in a product having a moderately high brine of 5. The concept of a combined effect of heating in the presence of curing salts was proposed.1 to 6. A mixed cure of salt (3. Bulman and Ayres (1952) found that levels of salt in excess of 4. It is perhaps for this reason that Jensen believed so strongly about the need for nitrate in perishable canned cured meats. Steinke and Foster (1951) investigated the effect of packaging liver sausage in an oxygenimpermeable film (Saran™) that was gaining acceptance in the industry. respectively. During the 1930s some processors incubated perishable canned cured meat at 37°C as an index of “keeping quality. . yielded the maximum inhibition. Salt was found to be the major factor retarding botulinal outgrowth in temperature-abused products. and nitrite). by nitrite. nitrate. (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. to some degree. 3. pasteurized. especially if the product pH was below 7.0. Although thermal destruction was not shown to be enhanced by the presence of curing salts. Strong opinions were expressed that nitrite alone was ineffective at the levels used in commercial practice. Nitrate was without effect.” This experience may have led to improvements in meat handling and equipment sanitation. Nitrate at the level permitted in meat (1250 µg/g) was without effect. In the absence of added salt. The question was raised whether the growth of spoilage organisms is influenced by the depletion of nitrite to noninhibitory levels.05% to 5. 2. a combination of both nitrite and nitrate was the most inhibitory. nitrate. The relative roles of nitrate and nitrite as preservatives in cured meats were unclear. A mixed cure of all three (salt.5.

the legal requirement for trichina destruction. and nitrite that is palatable and permissible.3°C.9 to 5. On adding a filter-sterilized solution of nitrite to a broth medium. a very small amount of added nitrite prevented staphylococcal growth when incubated anaerobically. 1 1/2 lb canned ham). and in the presence of nitrate. this cautious view of the inhibitory effect of nitrite was expressed by Eddy (1957): “Taken in their totality. Scott (1955) concluded from the literature that because nitrate exhibited relatively poor antimicrobial inhibition and nitrite. At that time European microbiologists commonly preincubated perishable canned cured meats before microbiological examination. the spores could germinate. Halvorson (1955) cited a private communication from L. 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. the control of salt concentration and resultant water activity was the most reliable bacteriostatic system for cured meats. (1956) examined whether curing salts plus the anaerobiosis of a vacuum package would inhibit the growth of S. The brine requirement of 5. aureus growth can occur in any combination of salt.3 or below.4%. brine values traditionally have been calculated at % brine = (% salt/% salt + % water) × 100.6 to 5.75% sugar. aureus to die rather rapidly in ham-curing pickle.5%. nitrite enhanced bacterial growth in curing brine. This combination of ingredients was derived from the literature and unpublished results from Jensen. Because the procedures traditionally used for large canned hams had a history of safety. and 1 ounce of sodium nitrate per 100 pounds of meat. found in the interior of cured meats. A typical formula being used for large canned hams at that time included 0. and toxin would be produced. unless protected by meat juices. This effect was reversed by adding sulfhydryl compounds. The authors postulated that nitrous acid was responsible for the bacteriostatic action of nitrite.0% brine or above. botulinum. Nitrite was more inhibitory in the presence of ascorbate. aureus.5 or above.g. growth would occur. Pasteur. as suggested by Castellani and Niven (1955). a similar debate would include the time elapsed since manufacturing.05. causing the cans to swell and forewarn consumers of a potential hazard. B.5% would have caused consumer . the addition of 1. nitrate.Nitrite 175 Henry et al. although effective. 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. and a brine level of 3. who performed a series of tests with spiced ham inoculated with different strains of C. Inst. 1/8 ounce sodium nitrite (78 µg/g). Jensen concluded that if the brine content were less than 5. who concluded that 1 ounce nitrate was optimal for gas formation by Bacillus species in temperature-abused perishable canned luncheon meat. The debated issue was the time and temperature for preenrichment. Jensen. if ever. these existing products were not affected. Today. Lechowich et al. nitrite rapidly disappeared and was ineffective.S. At pH 5.27. Dack (1949) reported toxin inhibition in meat with brine levels above 6.5% sugar.8 was optimal for antibacterial efficacy. Earlier. 1955) on semipreserved meats in hermetically sealed containers included a curious debate. less nitrite was necessary for inhibition as the pH was decreased from 6.. The USDA concluded that the high brine would retard the growth of C. They found S.” An international symposium (Ann. Additional tests showed that if the broth medium (pH 6. In the United Kingdom. Also. the growth of Bacillus species would occur. was unstable.0%. At 6. They investigated why Staphylococcus aureus is rarely. A pH of 5. S. aureus was killed in hams when heated to an internal temperature of 58. As late as 1957. Germination was apparently prevented because viable spores could be recovered. Tests in broth media and raw pork led them to conclude that S. Department of Agriculture (USDA) issued a regulation to assure the safety of new perishable canned cured meat products (e.55) was autoclaved with glucose. (1954) reported that at pH 7. 1/4 ounce sodium nitrate (156 µg/g). In North America. The requirements imposed on new products included a brine level of 5.5%. botulinum. During the mid-1950s the U. these observations leave no doubt inhibition by nitrite is at least a possibility. toxin formation was inhibited with a heat treatment as low as F0 = 0.

The suspected role of nitrite was to prevent germination or outgrowth of surviving heat-injured spores. 5. 1956). Greenberg et al. salt. Brine content was further shown to influence botulinal outgrowth and toxin formation. . 6. Eddy and Ingram.95% brine. At 8. There have been no reported attempts to duplicate this effect of brine at levels approaching total inhibition. At 5. as postulated by test tube experiments. 3. toxin was produced without concomitant organoleptic spoilage.5%. A significant observation was that at brine levels of 6.4°C to 37. Although shelf-stable canned cured luncheon meat had been produced for years. 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. the primary effect of nitrite was to prevent germination and/or outgrowth of heat-injured spores.14% brine or less. the following became increasingly clear: 1. Nitrite was shown to play a significant role in the stability of shelf-stable canned meat. Anaerobic intolerance to nitrite within the interior of ham. was not confirmed. 4. 1950.. 1950. This brought nitrate under renewed attack. 2. Several new reports of unstable commercially canned cured meats containing nitrate appeared (Hankins et al. and a low indigenous spore level. As expected. (1958) used this class of product to show that nitrate played no role in retarding putrid spoilage. S. 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. Sodium nitrite levels were 22 µg/g or less at the time the meat was inoculated. had no antimicrobial effect. At brine levels approaching the inhibitory level.0% to 7. (1959) subsequently demonstrated botulinal spore outgrowth in perishable canned cured meat having brine levels from 3. The system providing stability consisted of the combined effect of nitrite.125% and abused at 29. SUMMARY FOR 1950–1960 As 1960 approached.0%) alone was not responsible for stability. the product was organoleptically unacceptable — as the cans swelled — and toxin assays were performed. other than its possible influence on water activity. This research and a subsequent review by Silliker (1959) established the future direction for research on nitrite. toxin was not produced. aureus was killed during the heating process given to hams. Although nitrite was recognized as an effective antimicrobial agent. and 7. 3. although there have been several studies under other conditions in which toxin has been detected without obvious organoleptic spoilage. Nitrate could serve as an electron acceptor. Brine level (0%. 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.176 Antimicrobials in Food complaints of excessive saltiness in the new products. the reasons for stability had not been defined. Verhoeven. Because viable spores could be recovered from stable commercial product. permitting the growth of aerobes.12%. salt.8°C for up to 90 days. nitrate actively stimulated spoilage by aerobic spore formers. such as micrococci and bacilli. per se. 7. or 5. Nitrate.25%. and thermal injury to the low indigenous level of anaerobic spores. toxin was produced without obvious organoleptic spoilage. its value as a preservative in perishable cured meats was still in doubt.09%. thermal injury to the spores. Silliker et al.

Germination was completely prevented by sodium nitrite levels of 750 µg/g or higher at pH 6. They concluded that nitrite and nitrate have either neutral or stimulatory effects on bacteria and that nitric oxide has virtually no effect.1 to 0. The thermal process used during the early 1960s by seven U. Shank et al. development ceased before lysis or rupture of the spore walls. 1965). Hemin had no effect on nitrate reduction or growth of Escherichia coli or Bacillus polymyxa. producers of shelf-stable canned cured meat was reported to range from F0 = 0. heat resistance.4 to 0. In a study comparing irradiation with conventional thermal processing. One reason given was that spores indigenous to meat emulsions have lower heat resistance than spores produced in laboratory media. Immediately after germination. inhibition. .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. (1964). Outbreaks of botulism from temperature-abused vacuum-packed smoked fish during the early 1960s added impetus to this research. but not in others. making them unavailable for subsequent metabolism.2 (Greenberg et al. (1967) opened new possibilities. but at a reduced rate. If the surviving spore counts between the enzyme-inactivating (74°C internal temperature) and the shelf-stable process (F0 = 0. of spores of Bacillus species in the presence of less than 300 µg/g of sodium nitrite at pH 6. He estimated the thermal process caused a 4-log reduction of the indigenous spores. Shelf-Stable Cured Meats Riemann (1963) supplemented existing information on the incidence. Gould (1964) observed germination. 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. and nitrate is not reduced anaerobically. It was postulated that nitrous acid reacts either with the cell itself or with various constituents of the medium.017 viable cells/g remaining after processing to F0 = 0. The surviving spores are subsequently inhibited by the salt and nitrite. The increased use of vacuum packaging raised new questions about the microbiological safety of cured meats. 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.6 at the center of the can would yield 10-fold higher values near the surface of the can. a 4. The presence of 25 µg/g of residual nitrite after processing and a relatively low brine level of 3. Research was initiated on irradiation for shelf-stable canned cured meats. The authors concluded that when oxygen is absent during the growth of some bacteria. The inhibited spores did not appear to become fully phase dark. with a median of 0.S. The impaired synthesis of heme prevents the formation of the required cytochrome systems.7°C for 6 months. the observations of Perigo et al.. spoilage.2. The toxicity of nitrite was 3 to 5 times greater at pH 6 than at pH 7. In the course of this research. He concluded that most of the spores are killed during the thermal processing of shelf-stable canned cured meat. heme synthesis is impaired. and outgrowth of spores with unpublished data from the Danish Meat Research Institute.to 5-log reduction occurred. If. (1962) explored whether nitrite or the oxides of nitrogen were involved in the inhibition of Gram-negative bacteria in cured meats. the initial spore load in the product is too high.0. Another factor in spore destruction is that a thermal process of F0 = 0. and toxin formation from this low level of survivors during storage at 30°C to 37. Product inoculated with the lowest inoculum level (173 spores/g) had 0. canned chopped ham was inoculated with botulinal spores and given 2 thermal processes.203) were compared.36% did not prevent outgrowth. even if nitrate is present as an alternative electron acceptor. Nitrous acid was considered primarily responsible for the quantitative as well as qualitative changes that occur in the bacterial flora when meat is cured. Nitrate and oxygen were considered interchangeable as electron acceptors required for heme synthesis in some bacteria.5. however.

Duncan and Foster (1968a) also reported that heated putrefactive anaerobe spores were less tolerant of salt. personal communication). but the process was not dependable and was abandoned. but nitrite enhanced heat injury. Curing salts interfered with some stage of germination and development of the surviving heated spores at concentrations that were ineffective with unheated spores. First. the meat was ground or chopped and then deaerated and backflushed with nitrogen to displace free oxygen.5% to 4. 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).3°C) before release as a shelf-stable product.g. When heated and cultured in the presence of the agents. (1958).000 µg/g) increased the apparent heat resistance of the spores. nitrite. and nitrate than unheated spores. When heated in the presence of these agents..5% to 1% (5000 to 10. The process was considered to greatly increase the amount of nitric oxide available for use as an antibacterial agent.1 to 0. Their results confirmed the concept of spore injury and increased sensitivity to salt and nitrite proposed by Silliker et al.g. 2 to 2 1/2 hours in 74°C water) to achieve an internal temperature of at least 71°C.. spores tended to be protected by salt and nitrate. The product was then cooled in tap water and placed into refrigerated storage (e. (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. including (1) the destruction of vegetative cells by heating to the initial internal temperature of 71°C. Ascorbic acid was then uniformly distributed throughout the meat. The thermal process required to achieve shelf stability of canned hams larger than 1 1/2 pounds caused excessive purge (e. A 3-pound meat portion was filled into each can. These data reemphasized the importance of spore contamination level for the stability of shelf-stable canned cured meats. and nitrate. At 2% to 4%. (3) destruction of spores on the surface of the meat by the short but high thermal process. salt and sodium nitrate at 0. heat resistance was decreased. Roberts and Ingram (1966) heated spores in water from F0 = 0.178 Antimicrobials in Food Spencer (1966) concluded from the literature that 3. They demonstrated that increased stability could be achieved by omitting sodium nitrate and sucrose from the formulation. 1 psig). . 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. Nitrite was strongly inhibitory. and appeared to be the chief preservative against putrefactive anaerobic spoilage. 75 to 150 µg/g of added sodium nitrite to provide about 20 µg/g after processing to F0 = 0. The refrigerated storage period was necessary to assure stability.g. The product was then heated for a short time at a high temperature (e. 15 minutes at 113°C to 118°C). nitrite.0% brine. This process was used commercially with the approval of the USDA.. The process had to be properly controlled to be successful (Kueck. and a low incidence of clostridial spores (less than 1/g) were necessary for the safety and stability of shelf-stable canned cured meats. Increased stability also resulted from improving the canning process to reduce or prevent voids. This process caused the surface of the meat in the can to reach 96°C to 107°C. was then added and mixed into the meat while under a positive pressure (e.0015 to 1. sealed. 30%) and poor meat texture. and then thermally processed. especially at pH 6. The meat mixture was then filled into cans.g. Kueck et al. A nitric oxide-generating material. 30 days at 6°C to 8. The success of the process was believed to be the result of a combination of factors. and the product was given a long thermal process at a low temperature (e.0. such as sodium nitrite..g.5 before placing them onto culture media containing salt. (2) partial destruction of spores by the initial long thermal process but at the same time retaining a high level of residual nitrite. The effect of nitrite was pH dependent and led the authors to support the position that undissociated nitrous acid is the inhibitory agent.4.. The two Bacillus species were rendered more sensitive to salt by lower heat treatments than clostridia. Nitrate had no effect beyond that on water activity. the normal pH value of canned luncheon meat. Schack and Taylor (1966) described a procedure to produce shelf-stable canned cured meat with improved quality as a result of reduced thermal process. This process was tested extensively in at least one commercial plant. The process involved a unique sequence of adding the curing ingredients.

their results supported only one: the inhibition of outgrowth of heat-injured spores.. The authors postulated that the unknown substance may be formed from the nitrite that disappears during heating. Perigo and Roberts (1968) subsequently expanded the observation of Perigo et al. they reasoned the inhibitor might play a complementary role in the stability of shelf-stable but not perishable canned cured meats. and the thermal process required for the safety of shelf-stable cured meats given less than a botulinal cooking. aureus were similar in back bacon packaged under nitrogen. Thus. Salt levels above 6% prevented germination. or Salmonella Typhimurium. the level of residual nitrite remaining after processing may be an inappropriate measurement of the inhibitory capacity of the product. The questions were raised as to what constitutes a safe process and at what point are the products underprocessed. nitrite. Of the four. The interrelation of thermal process. Perishable Cured Meats Thatcher et al. Because the bacon incubated under . air. sporogenes. (1967) offered a new possibility for the stability of shelf-stable canned cured meat. and enterotoxin was reportedly produced under all four packaging conditions. Sodium nitrate affected neither germination nor outgrowth at levels up to 20.1% alone. an unknown substance was formed that was extremely inhibitory to the growth of vegetative cells of C. levels within the range of brine in shelf-stable canned cured meat (i.e. Roberts and Garcia (1973) later reported 9 of 14 strains of Bacillus species and Streptococcus durans to be sensitive to the inhibitor. vacuum. Meat inoculated with 106 spores/g remained nontoxic after the same process if sufficient salt and nitrite were present. Ground pork inoculated with 1 botulinal spore/g and processed to F0 = 0. (1969).000 µg/g) did not prevent germination or swelling of the spores. Four possible mechanisms by which nitrite may inhibit heat-injured spores in shelf-stable canned cured meats were investigated by Labbe and Duncan (1970). one of 4. The newly emerged cells then lysed. and spore level in shelf-stable canned cured meat was further substantiated by Pivnick et al. to date there has been no agreement on the lower limits of brine.e. between 3% and 6%) allowed germination and vegetative cell formation.6 became toxic in the absence of salt and nitrite. but not Streptococcus faecium. When nitrite was heated in a laboratory medium. The significance of spore level on the thermal process required for preventing botulinal outgrowth was again demonstrated. salt. zymogenes. perfringens spores. 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.. the inhibitor did not prevent germination of most of the intact or heat-injured C. Despite the research and experience since the commercialization of these products. (1967) to 30 different clostridial strains. The bacon exposed to air or carbon dioxide in oxygen was obviously spoiled. Streptococcus faecalis. Streptococcus faecalis var. and 5% carbon dioxide in oxygen. (1962) questioned the safety of vacuum packaging. This required either a brine level of 6.000 µg/g.. Viable botulinal cells were recovered in stable inoculated product 18 months after processing. or intermediate levels of brine and nitrite. Perigo et al.6% with 300 µg/g of sodium nitrite.Nitrite 179 Duncan and Foster (1968b) also reported that nitrite levels up to 600 µg/g at pH 6. The stability of perishable cured meats was assigned to undissociated nitrous acid. 1969). Johnston et al. 40. in the case of shelf-stable canned cured meat. 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.0 allowed emergence and elongation of vegetative putrefactive anaerobe cells but blocked cell division. Even high nitrite levels (i. Because formation of the inhibitor required substantial heating. For this reason. nitrite. Growth levels of S. The first attempt to demonstrate the presence of a Perigo-type factor (PTF) in cured meat was unsuccessful (Johnston et al. When testing the PTF formed with nitrite levels up to 10 times greater than those commercially accepted. they selected a medium that enhanced the disappearance of nitrite during autoclaving.

Another 25 strains of microbacteria failed to grow at pH 5.8°C or 10°C. This would help to explain the dominance of the lactobacilli as spoilage organisms in many perishable cured meats. Similar results were obtained with jellied corned beef stored at 15°C and 25°C.35% and 3. pH. toxin was detected in 2 days at 2. When S. a fermentable carbohydrate.0. increasing the amount of nitrite from 25 to 200 µg/g caused progressively greater inhibition. In jellied ox tongue. The time required for toxin production decreased as the abuse temperature was increased (22°C. Within 1 week the product pH had decreased from 6. At pH 7. but no attempt was made to determine whether they could multiply if the hams were subsequently temperature abused. Of 25 lactobacilli from cured meat. Toxin was not produced at 10°C or lower. A decrease in pH likely occurred during storage and prevented toxin formation in the other variables. On sliced bologna. Nitrite was more inhibitory at 0°C than at the other temperatures tested (10°C and 25°C). Several studies on the growth of C. Bologna was relatively resistant to type A toxin production but not ham. and nitrite.4% brine at 30°C. but not at 29. Toxin was produced within 7 days at 30°C in bologna (3.1% to 4. and decreasing pH during temperature abuse were the suggested inhibitory factors. botulinum in vacuum-packaged perishable cured meats were reported during this period. The four strains failed to produce toxin during 5 weeks at 10°C in sliced bologna (2. Their findings that the type of cured meat influenced toxin production were particularly significant. the presence of nitrite caused very little or no inhibition. type E toxin was not produced. lower maximum population levels occurred in vacuum packages than in nonvacuum packages at 15°C and 22°C.180 Antimicrobials in Food nitrogen or vacuum was organoleptically acceptable. 30°C.1. From studies in thioglycollate medium containing salt.1% or higher. the authors concluded that the levels used in ham would permit growth.0 and below. A similar effect occurred with increasing salt from 0% to 4% during incubation at 7.8%).8%. Christiansen and Foster (1965) found type A botulinal toxin production to occur at the same rate on bologna whether packaged with vacuum or without. botulinum. 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. residual nitrite. At the highest brine level (4. In jellied pork tongue. Cooked sliced ham from five producers inoculated with type E spores yielded variable results at 30°C. In sliced ham. perfringens inoculated into raw hams could survive the thermal process.7% brine.75%). and temperature on a Microbacterium species in all-purpose Tween (APT) broth. 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). The rate of toxin formation decreased as the brine level increased from 3.7 or less after 4 weeks. Pivnick and Bird (1965) found the oxygen permeability of packaging films to influence spoilage but not toxigenesis of sliced cured meats inoculated with C. and 37°C).5 in the presence of 200 µg/g of sodium nitrite. which was thought to be the result of product differences in brine and residual nitrite levels.6.0 to 4. At pH 6. A total of 90% of the samples held at 25°C remained above pH 6. Gough and Alford (1965) showed that spores of C. The combination of brine (3. the authors recommended that vacuum-packaged nonsterile processed foods be stored in the frozen state. Failure to obtain toxin production at 5°C may have been influenced by pH because 13 of 15 samples had a pH of 5. even at 30°C. 21 were able to grow under the same conditions.8% brine). pH 6. toxin was produced at 20°C to 30°C but not at 15°C. aureus was inoculated onto sliced chopped ham. type E toxin was produced at 10°C to 25°C but not at 5°C. This suggested that salt becomes more inhibitory as storage temperatures are decreased.0. nitrate. but not at 25°C if the brine was 5. 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.5% brine and in 4 days at 4. Brownlie (1966) demonstrated the combined inhibitory effect of sodium nitrite concentration.4°C. No mention was made of pH and none of the agents were tested in combination. The effect of brine on type E toxin production was more . toxin was produced only at 30°C. 34 µg/g of residual nitrite).

but this is highly questionable. they determined that type B botulinal outgrowth was inhibited during 4 weeks at 30°C if the juice had a combined pH of 5.1. In both products a marked delay in toxin production occurred at 20°C. All of them reached the same general conclusions as those set forth by Silliker et al. and more than 10% brine at pH 6. increasing the nitrite level increased the time for toxin production.. considering the bacon had a brine level of 5. SUMMARY FOR 1960–1970 Progress made during the 1960s toward defining the role of nitrite in cured meats included the following: 1. in perishable canned hams can inhibit the growth of C. All three products (bologna. The growth of S. 5. 25°C. Type E botulinal growth is inhibited by 2% brine at pH 5. The effect of nitrite levels on types A and B botulinal toxin production in vacuum-packaged sliced ham (4. toxin was detected after 4 days with 2. during which time naturally occurring lactics drop the pH to 5. He concluded that C. 4. The raw product was then sold at ambient temperature for cooking at home. A new explanation for the shelf stability of canned cured meat was proposed. 5. a brine level of 4. In jellied pork tongue.3. Perigo et al. 2. 1967). (1967) suggested an inhibitory substance was formed from nitrite.3.28%.5% brine at pH 5. Warnecke et al..9.2°C.8% to 4. Kafel and Ayres (1969) demonstrated that the growth of enterococci in culture media and. an increase in the level of residual nitrite also occurred. and 30°C). which disappeared during the thermal process. Evidence was presented that the antimicrobial effect of nitrite is related to the formation of nitrous acid.1% brine) and jellied pork tongue (3.2°C for 3 to 18 days. . 3. more importantly.5 to 5. perfringens.35% and after 8 days with 3.Nitrite 181 dramatic.8 or less. and Lactobacillus viridescens. The product had been changed to a shelf-stable fermented product precooked to 80°C in an oxygen-impermeable casing. The process of outgrowth was more sensitive than spore germination to nitrite. Although not mentioned. At each temperature (20°C. Toxin was not produced in either bologna or country cured ham. The authors concluded that heat injury of spores is not necessary for nitrite to prevent toxin production in cured meats. 1968) emphasized maintaining adequate salt levels to assure the microbiological safety of perishable vacuum-packaged cured meats.5% brine at 20°C. ham. aureus is inhibited by more than 4% brine at pH 5.1.5. as the brine level increased in the bologna and ham. 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. botulinum type A seemed to be completely inhibited by 4.8°C and 22. Toxin was not produced in product with 4. C. and jellied pork tongue) were formulated with commercial levels of nitrate and nitrite. Toxin was also detected in bacon held at 12. and 8.8.5% or above. The system responsible for the shelf stability of canned cured meats given less than a botulinal cooking was studied by several research groups. The concept of thermal injury to spores that survive the processing of shelf-stable canned cured meats was confirmed. An additional factor influencing the apparent significance of nitrite is the growth of competitive flora. botulinum.5% brine at pH 6. sporogenes. Using juices expressed from the newer sausage product. (1958).6% brine at pH 6. 9% brine at pH 5. C.2 and 3% brine at all pH values. and an input nitrite level of 500 µg/g or more.3% brine) was studied next (Pivnick et al. Riemann (Anon.4% brine. (1967) reported type E botulinal toxin production in ground beef held at 22.

and (5) commercially produced meats. storage temperature. They include tests in (1) test tubes or Petri plates using controlled media and conditions. The significance of decreasing pH on botulinal inhibition in cooked cured meats with sufficient fermentable carbohydrate was reported for the first time. Salt and nitrite had little effect on the degree of thermal destruction. Research during this period can be grouped into five categories by the approaches used. nitrite at the level used commercially (200 µg/g) did not enhance destruction during heating or germination during subsequent storage. (3) nitrite increases the germination rate of spores that survive heating.1% sodium nitrate. (2) nitrite potentiates direct killing by heat without the need for prior germination. 110°C. Limited data indicated the lowest temperature reported for growth of type E on cured meats to be 10°C. Throughout the decade pressure increased to reduce or eliminate nitrate and/or nitrite from cured meat. (4) meat products produced and packaged in pilot plants. (2) water slurries of meat processed in bottles. and the germinated spores are killed by heat. 0. Matsuda and Sekiguchi (1971) studied the combined effect of heat (105°C. (3) meat products processed in tubes or cans. Shelf-Stable Cured Meat and the Perigo Factor Pivnick et al.1%) affected thermal destruction when tested alone. pH.182 Antimicrobials in Food 6. 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. The growth of enterococci in perishable canned hams was reported to inhibit several clostridial species. and 120°C) and curing salts on the destruction of Clostridium subterminale spores. 7. and inoculum level. The timely report of Perigo et al. 8. residual nitrite. Of the four possible roles. and (4) these rapidly germinating spores are then inhibited by the nitrite remaining after processing and subsequently die before the nitrite has decreased appreciably. dynamic system in which nitrite plays a role in cured meats has been virtually impossible to duplicate in microbiological media. Sodium nitrite alone caused increased destruction. (1980) would later report growth at 8°C in liver sausage. 1970–1980 From the outset of the 1970s. Lücke et al. The chief value of nitrite appeared to be its ability to prevent growth from spores that survive and germinate following processing. but not all the results were directly applicable to commercially cured products. and 0.01% . Considerable effort was applied toward isolating and characterizing the Perigo factor. The complex. 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. As in the past. the test tube approach was productive. Factors influencing growth included brine level. The data were discussed in terms of applying the D concept to shelf-stable canned cured meat. in 1967 opened the possibility of a breakthrough on the mechanism of nitrite inhibition.0% salt. with the goals of greater understanding and its possible use as a preservative to replace nitrite. A combination of 3. (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. but their presence in the culture medium markedly reduced the recovery of the heated spores. Each of the approaches had its favorable and unfavorable features. 9. Certain cured meats are more prone to supporting botulinal growth.

and.6. nitrite and salt levels had lesser effects. showing a similar iron–nitrite interaction with C.g. The extract had 2. sporogenes spores to provide a level of 104 spores per can.4). however. (1975). sealed. A significant observation was that residual nitrite did not undergo normal depletion in the extract. In ground pork.5°C on the outgrowth of surviving spores was also examined by placing the heated tubes at 23°C for up to 1 year.. Reducing agents (e. Between 120 and 200 µg/g sodium nitrite provided stability through 5 weeks of storage. A similar response with strain 3679 would not be unexpected. cooked sausages.5°C in ground pork containing 2. 150.5% sodium tripolyphosphate was studied by Nordin et al. as already discussed. botulinum. exist (Christiansen et al. overlaid with paraffin oil. Johnston and Loynes (1971) obtained Perigo factor formation in the Perigo et al. Considerable differences were noted in their redox potentials. The effect of heating spores of PA 3679 at 115. thioglycollate) enhanced the effect of nitrite. Sausages packed in brine with 400 µg/g of sodium nitrite and processed to F0 = 0. but not at levels above 0.7 to 6. (1967) reported equivalent inhibition with 8 µg/g of nitrite added before heating the medium and 52 µg/g added after heating the medium.8% salt. This emphasized the importance of medium selection when studying the antimicrobial activity of nitrite and relating the results to commercially produced meats. pH.” The effect of all three factors (pH. although they were “clear and undisputable subsidiary effects. The sausages were prepared with nitrite-containing curing salt.. The magnitude of the effect was much less than was previously reported in culture media. cysteine. cooked.3.01% strongly influenced the results.4.01%. for inhibition. and pH 6. Ashworth and Spencer (1972) described a model system of ground pork (25 g) in 1-ounce bottles. The pork was autoclaved before or after adding nitrite. normal. A 1:1 extract was next prepared with 3% saline and ground. salt. One of the sausages in each can was inoculated with C. Outgrowth was determined by blackening of the tubes as a result of ferric citrate having been added to the meat.966. sodium ascorbate. and then packed into cans. The major factor influencing outgrowth was pH.6% salt. Farkas et al. and heating at 115. The cans were then filled with brine (3% salt). and then inoculated with vegetative clostridial cells.. aw 0. It was reported that less nitrite was required for inhibition when it was added before autoclaving. to some degree.g. 200 µg/g sodium nitrite. After a loss of 15% to 25% during processing and an initial decline.35 were as stable as sausages in brine without nitrite and processed to F0 = 2. Preliminary data. inoculated (105/ml). sporogenes strain PA 6982 in perishable canned cured meat as occurs with C. as opposed to perishable canned cured meats heated in hot water to a minimum internal temperature of 66°C. 115°C). Reducing agents (e. Aqueous meat suspensions prepared from raw. the liquid placed into tubes. respectively. The effect of nitrite. and salt) appeared to be additive. Omitting any one or all three of the additives did not affect thermal destruction. These observations led to the conclusion that a Perigo-type effect was detected in the meat system. and heated (F0 = 0. and 0. the solids removed. and heated to varying degrees. The inhibitory level of nitrite was not influenced by pH within the range 5. residual nitrite levels remained above 125 µg/g throughout 80 days at 30°C. and 50 µg/g of sodium nitrite. personal communication). and overheated (115°C for 90 minutes) canned luncheon meat required 500. Perigo et al. 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. thereby confirming earlier research.. 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.g. Small differences in the amount of sodium nitrite below 0. nitrite. ascorbate and cysteine) enhanced the antibotulinal effect of the meat suspensions but did not induce Perigo factor formation. Ashworth and Spencer (1972) reported the equivalent levels to be . held overnight for nitrite equilibration. (1973) studied the effect of adding nitrite to the brine solution for canned Vienna sausages. (1967) medium and reinforced clostridial medium but not in liver veal or Wynne fluid media.Nitrite 183 sodium nitrite caused the greatest destruction when heated. The residual nitrite values of the four media and three meat suspensions were about the same after preparation.

Residual nitrite. higher levels of added nitrite were required for inhibition. They concluded that although a PTF was formed. The data obtained with the spore inoculum should be more relevant to shelf-stable canned cured meats. results similar to those just mentioned were obtained during the early stages of temperature abuse. Similar residual nitrite levels were required to inhibit vegetative cells (160. The amount of PTF formed was related to the amount of original nitrite added to the meat before heating. (1967) based their conclusions on the added level of nitrite. on the basis of added nitrite. and 200 µg/g of sodium nitrite and processed to F0 = 0.184 Antimicrobials in Food 60 to 70 and 90 to 100 µg/g. Ashworth and Spencer (1972) found no relation between the amount of nitrite lost during heating and the inhibition obtained. 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. Adding salt (3. more added nitrite was required in the pork if the nitrite was added before (300 µg/g). the conclusion that a Perigo effect was demonstrated must be tempered with the observation that. In summary. Pivnick and Chang (1974) considered. Chang and Akhtar (1974) prepared buffered saline suspensions of canned luncheon meat made with 0.. which indicates that residual nitrite influenced the results. However.000/g) added after heating and spores (20/g) added before or after heating. When spores were used.1 µg/g) were the residual nitrite levels after 0 and 2 weeks in product made with 200 µg/g of nitrite. strongly influenced clostridial inhibition. Ashworth et al. (1969) found the inhibitor of Perigo et al.4. Ashworth and Spencer based their conclusions on the residual level of nitrite. and it may also play a role in the safety and stability of pasteurized cured meats. Shelf-stable canned cured luncheon meat was prepared with 0 to 300 µg/g of sodium nitrite and processed to F0 = 0. higher initial residual nitrite levels were required for inhibition. It was originally suggested (Perigo et al. rather than after (150 µg/g).5 or 22. concluded that a PTF was produced.4 in raw meat juice with 4. the Perigo effect was of low magnitude.. (1973) extended this research to the milder heating given to large perishable canned hams and obtained similar results.g. Perigo et al. The depletion rate of the nitrite added to the suspensions was not influenced by the amount of nitrite originally added to the meat. Johnston 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. In this case alone. more was required when added before heating the meat. with prolonged storage (42 days at 37°C).5 and 22. Ashworth et al. The nitrite level of the suspensions was adjusted after heating to 53. the relative inhibitory effect was quantitatively small. another inhibitory factor may be formed when nitrite is heated with meat. The additional nitrite provided comparable residual levels after the various heat treatments. 100. Inhibition by nitrite diminished gradually over a period of 8 weeks at 37°C. Actually. outgrowth and toxin production decreased. which had survived heating to F0 = 0. 1967) that the nitrite that disappears during heating may be involved in the formation of the unknown inhibitor. With excessive heating. The term Perigo-type factor (or PTF) was selected to describe the effect observed in their test.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.5% salt and 150 µg/g of sodium nitrite. If residual nitrite is a valid means for comparison. that although the Perigo inhibitor may not be present in meat. The authors found that as the original input nitrite level increased.1 µg/g.4 The product was held until the highest level of nitrite declined to less than 2 µg/g. the levels of nitrite required to obtain inhibition were about the same or lower if added before heating than if added after heating. deaerated in boiling water. (1967) to be inactivated when added to meat. 150. This was considered evidence for an inhibitory factor other than residual nitrite. autoclaving. In all cases. The meat was then inoculated with botulinal spores. however. Botulinal . such as perishable canned ham. per se. and inoculated. thermal destruction of spores and inhibition of the survivors by the salt). The levels selected (53. less residual nitrite was required for inhibition when nitrite was added before heating.

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

.0. nitrite. Autoclaving nitrite in the Perigo medium resulted in significantly more inhibition than autoclaving the nitrite in the meat slurry. the greatest inhibition occurred with 1.5% salt. 101).5°C. decreasing pH (7. 6.8% or 3. 1978b) involving product held at 4.5% or above. one reason was found to be the addition of sodium isoascorbate to the canned meat but not to the meat slurries. (1976) to study the interactions of temperature of abuse. Sodium nitrite and L-ascorbic acid were added as filter-sterilized solutions. a race occurs between death of germinated botulinal cells and depletion of residual nitrite. 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 . It was suggested that Bacillus species are less sensitive to nitrite than clostridia. Jarvis et al. pro or con. In another study. 20 µg/g of nitrite in the Perigo medium and 210 µg/g in the slurry were equally inhibitory. perishable canned cured meat is placed at 27°C. or 25°C. level of clostridial spores. 3. a preliminary test in bacon led to the conclusion that L-ascorbic acid does not alter the antibotulinal properties of nitrite in bacon.5°C and 25°C. 90°C. At the lower temperatures. Botulinal outgrowth was dependent on the relative levels of residual nitrite and surviving botulinal cells. At 15°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. Stability was influenced by nitrite content. Although less effective. decreasing temperature (25°C. increasing salt (1.5% but not 3. The spores heated at 70°C and 95°C were equally sensitive to the autoclaved nitrite.0%) markedly increased the effectiveness of nitrite with none of the 38 strains tested showing growth in broth containing 50 µg/g sodium nitrite. At 17. (1976) found that heating botulinal spores in an aqueous extract of lean pork at 85°C.0%. or 95°C decreased the number that could grow in a recovery medium containing 2. The longer the product was held at refrigeration temperatures. (1977). recommended that large-scale tests be used to quantify the many interacting factors influencing the safety of perishable cured meats.5). and decreasing inoculum level (106. The emulsions were filled into cans and heated. (1976). the more the residual nitrite levels declined and the less inhibitory the product became when placed at 27°C. On investigation (Tompkin et al. 4.1%) also increased the efficacy of nitrite in broth but had no effect.4°C or 10°C for up to half a year before placing at 27°C. Grever (1974) examined the stability of emulsions normally used for preparing cooked sausage and liver sausage. neither salt level provided much inhibition.5%. At 20°C. 6.5% salt (on water phase). and inoculum level on botulinal growth and toxin production at pH 6.0% w/v). diverse collection of botulinal strains in a broth medium and a cooked pork medium. The antibotulinal effect of nitrite was considerably greater than that reported for the meat slurries of Roberts et al. 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. a lower concentration of L-ascorbic acid (0. Christiansen et al.5%. This led to the pork slurry system of Rhodes and Jarvis (1976) and used by Roberts et al.186 Antimicrobials in Food Baird-Parker and Baillie (1974) studied the growth of a large. The rate at which inoculated cans swelled at 27°C was inversely related to the level of nitrite added to the product. At 20°C. in the cooked pork medium. Spores heated at lower temperatures (70°C or 80°C) were inhibited by 4. 20°C.5°C. This agreed with an earlier observation (Tompkin et al. 15°C).. less inhibition occurred. 200 µg/g). even with 300 µg/g of sodium nitrite. Isoascorbate had enhanced the antibotulinal efficacy of nitrite in the canned meat. 150. (1978) concluded that when inoculated. 5. 1978a). Adding L-ascorbic acid (1. 100. The number of strains showing growth in broth was found to decrease with increasing nitrite (50.5% salt.0. The effect of nitrite in perishable canned cured meat was also reported by Tompkin et al. salt. Jarvis et al. a pronounced salt–nitrite interaction occurred. and whether the products were treated by a perishable or shelf-stable thermal process. 6.5. 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. product pH. Temperature of abuse was the most significant factor influencing the rate of botulinal growth.0. 103. Furthermore. 22.

. 1970). Suggested mechanisms were proposed for the relation of iron to the antibotulinal efficacy of nitrite (Tompkin. Adding 156 µg/g of sodium nitrite delayed botulinal toxin production..g. Adding hemoglobin. Substituting turkey thigh meat. (1978) studied the effect of nitrite on the stability of naturally contaminated perishable canned pork when temperature abused at 37°C. as can occur with nondenatured (i. cytochromes. and/or (2) repair through the formation of new unreacted ferredoxin by the vegetative cell. hemoglobin.. but as in other model systems receiving a mild heat treatment. (1979b–e). Wojton et al. ferredoxin) are essential for electron transport. and interference with energy metabolism in the vegetative cell to prevent outgrowth.5°C.. which resulted in a lower level of residual nitrite after processing. peroxidases. It was also shown that the type of meat could influence the degree of botulinal inhibition. a simplistic explanation for the inhibition of clostridia consisted of the uptake of undissociated nitrous acid. This avoided the problem of recontamination inherent in some studies after heating. 1979a). or pork hearts for fresh pork ham reduced the efficacy of nitrite.e. isoascorbate also reduces the efficacy of nitrite by causing more rapid depletion of residual nitrite (Tompkin et al. however.. no botulinal inhibition was observed. Clostridium bifermentans. 1978e). cysteine. energy production. beef round. 1978.. which later was demonstrated to be the sequestering of iron (Tompkin et al. and myoglobin). heating to 68. the degree of inhibition was less than that reported by others. and ethylenediaminetetraacetic acid (EDTA) were found to share a common function in meat.Nitrite 187 (Tompkin et al.. beef liver did not cause a substantial loss in the antibotulinal efficacy of nitrite. decreased botulinal inhibition. single-electron transfer sites consisting of four iron atoms. ascorbate. overlaying with vaspar. nonheated) heme compounds. 1978d).g. however. and enterococci. the rate at which the product spoiled increased. low levels of nitrite (20 and 40 µg/g) did not influence botulinal growth or toxin production (Sofos et al. nonheme iron–sulfur proteins (e. This was based on the rationale that in clostridia. 1979b). cytochrome oxidase. or 200 µg/g) and then processed to an internal temperature of 69°C. and then storing the tubes at 27°C. A third possibility is the germination of dormant spores after residual nitrite has declined to noninhibitory levels.. Spoiled cans yielded C. beef hearts. One possibility involved the reaction of nitrite with an essential iron-containing compound (e. Their test system mainly consisted of extruding a frankfurter emulsion into test tubes. Clostridial ferredoxin is believed to contain two independent.. sporogenes. C. When testing a frankfurter emulsion prepared with mechanically deboned chicken meat. The reason isoascorbate enhanced the antibotulinal effect of nitrite was pursued (Tompkin et al. Although isoascorbate enhances the antibotulinal effect of nitrite in freshly prepared perishable cured meat that is temperature abused. perfringens. ferredoxin) within the germinated cell. a background flora of thermodurics existed from the ingredients. nitric oxide)–iron reaction as in ferredoxin. The product was formulated with 2. The efficacy of nitrite in frankfurters was explored by Sofos et al. 1978c). Isoascorbate. . 1979b). 1978e). This made it possible to explain the differences in the relative efficacy of nitrite in various meats on the basis of their iron content. Subsequent research more clearly confirmed the relation of available iron to the antibotulinal efficacy of nitrite (Tompkin et al.. catalase. 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. 100. allowing the dissociation of the nitrogen oxide from the iron. 50. a nitrogen oxide (e. and enzyme activity. Subsequent outgrowth could occur when (1) residual nitrite depletes to noninhibitory levels. 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).. As the level of nitrite decreased.g. with one exception: beef liver.g. Bacillus species. cooling. Each active site is comprised of iron bonded to both cysteine and sulfur (Buchanan and Arnon. Despite its relatively high iron content (49 µg/g). Tompkin et al.. On the basis of the data existing in 1978.5% salt and sodium nitrite (0.

The type of soy protein influenced the results. Sofos et al. botulinum spores in a chicken frank formulation to be unaffected by the addition of sodium nitrite (0. Toxin was produced in all variables formulated without dextrose and more quickly in product having 0 or 50 µg/g of nitrite.. about 2. and the effect of ascorbate in franks should be restated or reexamined. 450 µg/g of nitrate gave a variable response. 400 µg/g of sodium ascorbate. It was concluded that ascorbate did not affect the antibotulinal efficacy of nitrite. (1979c) evaluated various soy proteins in their system. The pH of product made with 50. the levels of nitrite (0 to 150 µg/g) and ascorbate (9. examined whether an increase in ascorbate over normal levels would alter the antibotulinal effect of nitrite. Assuming a similar pH response in the first test (Hustad et al. except for changes in the ratio of pork to beef. the conclusions in both studies concerning the inhibitory levels of nitrite. a fairly rapid decrease in product pH during abuse. The addition of 80 µg/g of sodium nitrite was relatively ineffective in chicken and beef. 100. (1974). 1973). Although product made with 150 µg/g of sodium nitrite did not become toxic. (1973) formulated franks with various levels of nitrite and nitrate. The depletion of residual nitrite was faster as the amount of beef was increased in a formulation containing soy. and fewer toxic samples occurred in product with 100 or 150 µg/g of nitrite. The rate of nitrite depletion was influenced by the type of soy. 20.. The franks were produced in the same facilities and formulated as before (Hustad et al. Toxic product occurred with 50 µg/g of sodium nitrite at all levels of ascorbate.5% to 4. only those variables without nitrite became toxic at 27°C. Nitrite was ineffective in a formulation of soy isolate and pork fat. input versus residual nitrite. 40. or 150 µg/g of nitrite declined to 5. The second test. and the products remained nontoxic. (1979e) found the rate of germination of C. The rate of botulinal toxin production in formulations with soy protein was equal to or slower than in an all-meat formulation. Toxin production was slower. The strong inhibitory effect observed was likely the result of the combined effects of nitrite.188 Antimicrobials in Food Sofos et al. One toxic sample occurred at 100 µg/g and none at 150 µg/g of sodium nitrite.3°C) sausages containing dextrose. In the first. more importantly. The decline in pH caused by a lactic fermentation was also the dominant factor preventing botulinal outgrowth in fermented sausage (Christiansen et al. the addition of 156 µg/g of sodium nitrite was very effective in delaying botulinal toxin production. . (1979d) compared the efficacy of nitrite (0 and 80 µg/g) in chicken. In mildly heated (58. and 156 µg/g). reported by Bowen and Deibel (1974) and Bowen et al. The pH of the dextrosefree product remained above 5. A later review of the pH data showed that all variables had a pH of 5. Vacuum-Packaged and Fermented Meats The effect of nitrite in vacuum-packaged franks was studied on two occasions.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. 105. Under these circumstances. the exception being one variable with 150 µg/g of nitrite. A significant delay in botulinal toxin production occurred in pork.08 or lower within 1 week at 27°C. They concluded that the effect of nitrite was on delaying the development of the cells after germination and before toxin was produced. 1973). the relatively high brine.8% fermentable carbohydrate. and. and pork in their system. The rate of botulinal toxin production was slower in all soy protein formulations than in all meat formulations. In a meat–soy mixture. Hustad et al. In that variable the pH was 5. and omission of nitrate. The finished franks had a brine of 4.0 or less within 21 days.. the two studies indicate that the combination of 4. in both the presence and absence of nitrite.0 or less within 14 days. and then only 2 of the 55 franks became toxic. Adding 50 µg/g of sodium nitrite delayed botulinal toxin formation for more than 4 weeks during abuse at 27°C.83%. Sofos et al. and 655 µg/g).6. beef.52% brine. and 4. 1975).

Shaw and Harding (1978) studied the effect of nitrate and nitrite on the microbial flora of Wiltshire bacon. the interaction of nitrite and ascorbate is difficult to interpret. A combination of 200 µg/g of sodium nitrite and 500 µg/g of sodium erythorbate prevented the growth of the two strains of B. 100.80 and 0.4%. 2. As expected. cereus was evidently more resistant to nitrite than C. aureus developed and enterotoxin A was produced. and brine level (mild. and Moraxella-like bacteria. 20°C. and 200 µg/g) and sodium erythorbate (0. Moraxella species.6%) on botulinal toxin production in vacuum-packaged back bacon. If all samples containing mixed cure with and without ascorbate were compared. The addition of ascorbate enhanced the antibotulinal effect of 100 µg/g but not 200 µg/g of nitrite. Erythorbate caused reductions in the residual nitrite and the redox potential of the product. When all samples having the mild and medium brine were compared.8% and 21. (1976) in the United Kingdom studied the effects of a mixture of nitrite (100 or 200 µg/g) and nitrate (250 µg/g). and 2000 µg/g). 1000. cereus). and 25°C) on the rate of botulinal toxin formation.65 to 6. respectively. The request included results by Christiansen et al. Noninoculated product containing nitrite developed a slight unusual taste at 20°C owing to the growth of spore-forming spoilage organisms (e.9%. and 34. Erythorbate alone was not inhibitory.9% became toxic. regardless of the level of nitrite or ascorbate. and mixed cure with 200 µg/g of nitrite were compared. 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. lower populations resulted and enterotoxin was not detected. 67. 35. In vacuum packages.7% to 6. aureus grew well in the medium-salted bacon. It also was reported that S. Because brine levels could not be held constant. If all samples with no mixed cure. Bacon Crowther et al. In opened packages. Raevouri encouraged more widespread use of erythorbate for sausage production in Finland.9% became toxic.7% became toxic. 19. higher populations of S. Raevuori (1975) studied the combined effect of sodium nitrite (0.98. Including nitrate in the bacon enhanced the growth of micrococci. Omitting nitrate led to higher numbers of Moraxella species in the cured bacon. 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. medium 4. botulinum.. botulinum. The data included botulinal challenge tests in frankfurters made with chicken meat and ham made with turkey meat. The predominant flora of the bacon after curing consisted of micrococci. (1977) that demonstrated the efficacy of sodium nitrite in poultry meat. 1977). A summary of the data showing the antibotulinal effect of nitrite in the two poultry products appears in Hauschild (1982). . respectively. growth was faster at 25°C than at 15°C. respectively.. There was a general trend toward reduced residual nitrite levels at the tune of inoculation when ascorbate was added.g. Food and Drug Administration (FDA) for approval of sodium nitrite as an additive in poultry products. After cooking in Saran™ casings. the sausages had relatively high pH and water activity values of 6. Inoculation studies examined the effect of nitrite (0 and 100 µg/g) and storage temperature (15°C. Product with 100 µg/g of sodium nitrite remained nontoxic through 14 days at all three temperatures. mixed cure with 100 µg/g of nitrite. The nitrate likely served as an electron acceptor for the growth of micrococci in the vacuum package.Nitrite 189 A request was made by the poultry industry to the U. Bacon that was sliced and vacuum packaged developed a flora mainly of micrococci and lactics. 500. A higher percentage of samples were toxic with the addition of 200 µg/g of nitrite than with 100 µg/g of nitrite. Erythorbate was found to enhance the inhibitory effect of nitrite. ascorbate (0. cereus tested. 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. The naturally occurring B.3%. 42.S.7% and 18.9% to 4. B. and 1000 µg/g) on the growth of Bacillus cereus in cooked sausage placed at 20°C for 48 hours.

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

g. Very high levels of nitrite (1000 and 5000 µg/g) either prevented or severely retarded the growth of all strains. future research might demonstrate the involvement of additional factors. At 40 µg/g growth was comparable to that in the control without nitrite. anaerobic toxin production occurred at pH 5. Anaerobic toxin production was prevented with 0. One reason given was that S. At higher temperatures (22°C and 30°C). Understanding why this occurs would be very instructive. The probability of toxin production at 10°C reportedly decreased as the level of nitrous acid increased. and initial pH (6. because high nitrous acid values (>0. The authors questioned the hypothesis that nitrite inhibition is dependent on the conversion of nitrite to nitrous acid. aureus was found to be restricted to the periphery of commercially fermented sausage (Barber and Deibel. aureus under aerobic but not anaerobic conditions.01% salt.3. 1972).0). Nitrite was metabolized by S. and product pH) contributes to this effect.58.9% salt and 100 µg/g of sodium nitrite) incubated at 37°C in environments of 20%. low oxygen level. aureus by blocking the sulfhydryl sites of either coenzyme A or α-lipoic acid. Collectively. aureus and Other Bacteria A cluster of reports appeared during the early 1970s that dealt with the effect of nitrite on S. 10%. the data along with the aforementioned results of Christiansen and Foster (1965) and Crowther et al. 8 µg/g enhanced the growth of the lactobacilli and. At 200 µg/g growth was delayed or slower. Genigeorgis et al. and Pediococcus cerevisiae (1 strain) were examined for their tolerance to nitrite in the presence and absence of 4.3. Extensive tests were also reported for laboratory-cured ham. 500. growth of competitive flora. however.. Buchanan and Solberg (1972) studied the effect of nitrite (0. Lactobacilli (78 strains). and 2000 µg/g). Of significant importance was a report that the growth of S. This suggested either that nitrite was not being used as an electron acceptor or the nitrite reductase system was blocked anaerobically. (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.34. and 0.6 µg/g) were possible only at reduced pH levels (pH 5. At pH 6. After considering the effects of changing pH during the growth of S.3) on the growth of S. Buchanan and Solberg suggested that nitrite inhibits the growth of S.Nitrite 191 Tests with S. or both prevented anaerobic toxin production. oxygen pressure. aureus is reduced and enterotoxin production can be prevented by the environmental conditions existing within vacuum-packaged cured meats. A third sample with a pH of 5. They also concluded that vacuum packaging should improve the safety of cured meats relative to S. no residual nitrite. thus preventing the normal metabolism of pyruvate. Heterofermentative lactobacilli were more tolerant of salt than the homofermentative strains. 200. and an abnormally low pH of 5. it is uncertain whether high nitrous acid content.3. (1976) indicate that the growth of S. Inoculated raw sausage (2.3 in two samples with negligible nitrite levels. micrococci and staphylococci (24 strains).6 or less). aureus and a review of the literature. At pH 7. reduced pH.159 µg/g of nitrous acid also became toxic.6 µg/g or more nitrous acid and pH values below 5.6%. aureus. nitrite. aureus is less acid tolerant under anaerobic conditions. The commercially canned ham used for the test had a brine level of 3. the micrococci. 5%. Nurmi and Turunen (1970) studied the effect of adding nitrite to a previously autoclaved broth medium (pH 6. but it is unclear whether nitrite level influenced anaerobic enterotoxin production. aureus populations as . Nitrous acid levels were calculated from the residual nitrite and pH values as described by Castellani and Niven (1955). to a slight extent. brine. and 0% oxygen yielded lower S. 15%. but it remains debatable how much each of the possible factors (e.3 and 200 µg/g. aureus in brain heart infusion broth (BHI). 22 µg/g of nitrite. Reasons for the reduced growth and enterotoxin production are evident from the reports. 1000. 7. the increase in the adjustment phase was 10 hours or less aerobically and about 30 hours anaerobically. However. Toxin was produced in certain variables having residual nitrite levels as high as 145 µg/g. nitrite was without effect in an aerobic environment and 500 µg/g or less was without effect anaerobically. aureus.

and nitrite on the heat resistance and recovery of S. and nitrate was converted to nitrite. and nitrite increased (Tompkin et al. SUMMARY FOR 1970–1980 The Perigo Factor in Cured Meats 1.5 or 5. nitrite. If manipulations of thermally processed meat were attempted to extract such an inhibitor. Adding nitrite as an electron acceptor at the time of anaerobic shock prevented increased toxin production. 4.g.0 but to a lesser degree than at pH 7. enterotoxin was not detected through 5 days of storage in the absence of oxygen. 1973). cytochrome oxidase) to the ferric state. There was a general pattern of increased heat resistance as salt level (0. aureus. The inhibitory effect of nitrite in the recovery medium increased with increasing salt content. presumably. 1975) demonstrated the combined effect of pH. Markus and Silverman (1970) reported enterotoxin A production in broth media (pH 6.6 to 6. residual nitrite. salt level. oxygen uptake. the percentage of lactic acid as an end product. Nitrite did not affect glucose transport by S. (1979) reported that nitrite inhibits active transport.. and. 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. Bean and Roberts (1974. increasing the level of nitrite had the general effect of reducing the recovery of both heat-damaged and undamaged cells. salt.. In another report on non-spore-forming bacteria. Glucose repression of enterotoxin B production was also reported to occur at pH 6. The effect of nitrite on heat resistance was inconsistent. when added to the recovery medium.. and nitrate on either growth or enterotoxin production. salt increased. 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. and pH). and 8%) increased at pH 6. Morse and Mah (1973) studied the effect of glucose on enterotoxin B synthesis in a broth medium buffered to an alkaline pH (7.192 Antimicrobials in Food the oxygen level decreased. more available energy. Rowe et al. the sum of which influences stability. and oxidative phosphorylation in Pseudomonas aeruginosa.5.7 (Morse and Baldwin. Formation of some similar inhibitor(s) (PTF) during the thermal processing of shelfstable canned cured meat appears possible. . 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. Perhaps because of the atypically high pH. and decreasing pH.0 but not at pH 5.0. faecalis or Streptococcus lactis. 1973).g. the question could be raised whether the inhibitor would be altered or destroyed. However. Contrasting results were subsequently reported that showed the production of enterotoxin A to decrease as pH decreased. both of which lack cytochromes. no synergistic effect occurred between salt.7). Although adequate growth occurred within 48 hours. decreasing incubation temperature.8) to be unaffected by sodium nitrite (200 µg/ml) or sodium nitrate (1000 µg/ml). The evidence strongly suggested that nitrite exerted its inhibitory effect by oxidizing ferrous ion of an electron carrier(s) (e. 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. The formation of the Perigo factor in commercially cured meat is highly debatable. 6. and 7. Adding glucose caused decreased toxin production as a result of the rapid oxidative decarboxylation of pyruvate. 2.

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

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

1).7 4 2.1 Shelf Stable Canned Cured Meats: Minimal Brine and Thermoprocess with 150 mg/g Sodium Nitrite Product Luncheon meat % Brine 3–4 4.5–1. and commercial practices to arrive at guidelines to assure the microbiological safety of these products. and erythorbate are controlled.1–0. Hauschild and Simonsen (1985) conducted an extensive review of the technology for producing shelf-stable canned cured meats (Table 6. 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.5–5. carbohydrate. the FDA acknowledged that prior sanction existed for the use of nitrite as an additive to poultry meat (Cassens.5 0.0–5. However. there has been a very rapid growth in the volume and variety of cured poultry products in the United States.2 1. whereas turkey ham was a high-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. He concluded that significant reductions in nitrite should not be made for all cured meats. This information was reiterated by Hauschild and Simonsen (1986).5 3. Reductions in the level of nitrite can be compensated for by moderate increases in the brine level and/or thermoprocess. existing government regulations. TABLE 6. Since that time.5 4. Their review was to assist in the development of a document for these products. .0 0. where P is the probability of outgrowth and toxigenesis from one spore. 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. He proposed expressing botulinal inhibition as log I/P.3–0. Throughout the decade.Nitrite 195 for sodium intake in the diet brought new pressure to reduce the salt content of cured meats. This was used to quantitatively compare the data from the various challenge tests. Bacon was considered a low-risk product. They summarized research.0 0. The data provided estimates of the relative degree of botulinal risk for each product. selective reductions in nitrite could safely be made provided the levels of brine.5 0. the per capita consumption of red meats decreased while the consumption of poultry products increased.5 Thermoprocess (F0 ) 1. Another major trend developed during this time.3 3.2–0. the impact of further reductions in salt deserved close examination.5 Ham and shoulder Sausages Source: Hauschild and Simonsen (1985). 1990). The level of risk could also differ among products of the same type as a result of differences in their brine concentrations. Reductions in the recommended levels should be based on extensive plant experience and/or experimental work and controls to assure minimal spore levels.0–4.0 5. The level of risk differed among the various products. In 1983.0–1.3 0. further accelerating the trend of the past 40 years.5 1.

2 without loss of the benefit of sorbic acid. The rate of pH decrease was slower as the brine level increased. nitrite in the range of 0 to 100 µg/g had little or no effect on toxigenesis. As the hams continued through the completion of curing. fermented sausage. overlaid with vaspar. Sofos et al. (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. toxin production was increasingly more rapid. Wagner and Busta (1983) compared the antibotulinal effect of combinations of sodium nitrite (40 and 120 µg/g). pH 6.2% brine.2. Toxin was detected after 9 to 11 days. As temperature was increased to 8°C. botulinum. Another possibility could be offered for this observation. At brine levels of 3. and nitrate at 8°C.4%). Toxin was not produced at 5°C. The number of botulinal spores was estimated to be about 0. If a low level of nitrite (40 µg/g) was added. nitrite. Hauschild and Hilsheimer (1983) examined 138 samples of liver sausage from retail outlets in Canada. 10°C. botulinum was estimated to be about 0.2. but the recovery of viable botulinal cells became increasingly difficult. and incubated at 27°C. toxin was detectable 10 cm from the injection site. The latter were suspected to be of particular importance as a cause of botulism from raw cured meat products in Europe (Lücke. Toxin production by a mixture of type E and nonproteolytic type B botulinal strains in liver sausage. Hauschild et al. Large bone-in hams were inoculated with nonproteolytic botulinal spores before curing in a solution of salt. and 15°C.3%. the inhibitory effect of the lactic fermentation and decreasing water activity values during fermentation and subsequent drying prevented botulinal toxicity. Methods and media were recommended to facilitate the recovery of both the proteolytic and the psychrotrophic nonproteolytic strains of C. Similar results were obtained during the dry curing of hams at 8°C or 10°C. The total C. and potassium sorbate (0% and .8% to 4.24/kg. Product with low levels of toxin often appeared acceptable. 1982). Liver sausage was prepared with 83 µg/g of added sodium nitrite. To prevent the growth of C.4%. Additional data on the ham experiments were presented by Lücke et al. and aw 0.2%. The antibotulinal efficacy of sorbic acid was pH dependent. thereby permitting the more heat resistant C. cooled.15 spores/kg in the product. heated. the inhibitory effect of the nitrite was reduced.98. The tests involved vacuum-packaged smoked whitefish fillets prepared with about 2. nitrite was more effective and an appreciable delay in toxicity was obtained with 50 and 100 µg/g of nitrite.44%. The presence of sorbic acid slowed the rate of nitrite depletion.0 being required. The test system consisted of a frank emulsion prepared from mechanically deboned chicken meat that was extruded into test tubes. The hams had a slight off-odor but no apparent putrefaction. and 6. At a higher brine level of 5. Storage at 8°C until a nearly complete loss of nitrite occurred did not diminish the effect of the nitrite.4 to as low as 5. Tests in model systems. Some consumers find this amount of salt unacceptable. (1982) assessed the relative risk of botulism from temperature-abused liver sausage. (1980). Cuppett et al. The use of nitrite in smoked fish in the United States has been restricted. botulinum to dominate and develop toxin. By increasing the processing temperature and/or prolonging the time of processing. the pH of the formulation could be increased to pH 6. Perhaps the milder thermal process permitted the survival of a competitive flora that prevented toxigenesis. sodium acid pyrophosphate (0% and 0.196 Antimicrobials in Food Botulinal Challenge Tests Tests in meat and fish products. psychrotrophic strains can grow and produce toxin during the early stages of curing before the salt and nitrite levels penetrate and reach inhibitory levels. toxin levels remained high. botulinum. (1980a) studied the significance of pH on the efficacy of nitrite and sorbic acid mixtures in a chicken frank emulsion. and hams was investigated by Liicke et al. competitive flora could have been destroyed. Greater protection was obtained with 150 µg/g. a high level of salt (5% brine) has been required. They concluded that nonproteolytic. After curing. By increasing the thermal process. The pH of the fillets decreased during 27°C incubation from about pH 6. (1982). with levels of at least below pH 6. In fermented sausage. The nitrite (40 µg/g) also significantly increased the inhibitory effect of the sorbic acid. 4.

The pH of the pork used for the higher pH slurries ranged from 6.78 to 6. The polyphosphates did not influence botulinal inhibition in the absence of nitrite.0. (1983) assessed the antibotulinal effect of three phosphates in the test system just described (Wagner and Busta. and 4. 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. adding isoascorbate or nitrate. polyphosphate. potassium sorbate (0. The test system avoided the problem of recontamination that occurs when peeling and packaging franks.3 to 6. and 0. with certain phosphates being more effective than others.2 units in the higher pH slurries. They also found that the antibotulinal efficacy of nitrite could be increased by the addition of phosphate.7). The combination of nitrite (40 µg/g).5 to 6. (1979b–e. They used the same test system as in Sofos et al. The combination of 40 µg/g of sodium nitrite. and pH control of the emulsion was the most effective to achieve reduced nitrite levels and provide enhanced botulinal inhibition. Polyphosphate increased botulinal inhibition when added to combinations of sorbic acid or potassium sorbate and a low level of nitrite (40 µg/g).03.26%) in frankfurters. potassium sorbate significantly decreased toxin production. more like that of a reformed ham after heating. (1979) modified their pork slurry to a thicker consistency.1 to 0. The frankfurters were made from chicken instead of beef and pork. The increase in pH could explain the decreased inhibition in the lower pH slurries. The pH of the pork used for the lower pH slurries ranged from 5. The initial pH for the various products was 5.6 to 6. At a given spore inoculum.54. The pH was important in all treatments tested.b) evaluated the interactions of salt. and thermal process on botulinal outgrowth in pork slurries of two different pH ranges (pH 5. (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. It is unclear whether the target or analytical values for salt were used for statistical analysis.19.36 and 6.72). Of the polyphosphates tested.26% potassium sorbate.4 to 6.27 to 6. Jarvis et al.5% (wt/vol water) below the targeted values. This increased inhibition was not observed with nitrite levels of 75 to 125 µg/g. Roberts et al. Considerable variation occurred between the meat from different animals within each breed.4% sodium acid pyrophosphate caused the greatest delay in toxin production. No systematic difference in inhibition was observed with the meat from three breeds of pig.3) and high-pH pork (pH 6. sodium acid pyrophosphate (0. although the conclusions were stated on the basis of the target values of 2.5%. They found that increasing the heating from P80°C = 0. Roberts et al. The pH of the four product variables was between 5. Statistically significant reductions in toxin production occurred with increasing salt level.4 and 5. A separate formula was developed for slurries prepared with low-pH pork (pH 5. The addition of polyphosphate caused an increase of about 0. although the trends in the data may not change. The levels of salt found in the slurries were often as much as 0. or decreasing the abuse temperature. There appeared to be more botulinal inhibition with the addition of sodium acid pyrophosphate. 1980a). except for two outliers of 5. (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.65 to P80°C = 6.27 to 6. One nitrite level (40 µg/g) was used throughout.Nitrite 197 0. 1983). This may alter the quantitative assessment of the individual and combined effects of the additives.65. nitrite level. Roberts et al. They found the nitrite to be less effective in slurries prepared with meat from the shoulder than meat from the leg.5%. Gibson et al.5% salt (wt/vol water). The effect of sorbate was greater as salt levels . sodium acid pyrophosphate was more effective than sodium hexametaphosphate or sodium tripolyphosphate. nitrite.7 and 6. Tubes showing gas or other spoilage were preferentially selected for toxin assay. but the reason for the increased inhibition when adding polyphosphate to the higher pH meat slurries is less evident.4 units in the lower pH slurries and about 0.26%).4%).56. isoascorbate. (1981a. 3. (1982) examined the interaction of potassium sorbate in their cured pork slurry system. 0. Nelson et al. or thermal process. nitrate.

which resulted in rapid botulinal outgrowth. the addition of nitrate. and iron in culture media. The probability of botulinal outgrowth decreased as the level of added nitrite increased above 50 µg/g. They found that the rate of depletion in their pork slurry system was influenced by many intrinsic and extrinsic factors. an iron-binding protein in casein. 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. as pH levels decreased to below 6. The bacteriostatic action was reversed by the addition of cysteine. and as the storage temperature decreased. 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. 20. thioglycollate. U. Bactin was not produced when heating cysteine. a similar protein in blood serum. 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. At lower dose levels of 0 to 9 kGy. sealed with vaspar. This led Gibson et al. As expected. and stored at 30°C for 28 days. which otherwise prevent bacteriostatic activity. For example. No suggestion was offered for the increased botulinal inhibition when nitrate was added. 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. botulinum. 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. The significance of microbial growth on nitrite depletion in the slurries during storage was not addressed. legislation was proposed to control the use of nitrite by monitoring the residual nitrite in cured meat products. A rather high level of isoascorbate (1000 µg/g) caused nitrite to disappear rapidly and delayed the outgrowth of C. the rate of nitrite depletion was influenced by storage temperature. and the addition of isoascorbate. 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 another study of Perigo factor formation. 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). forms an inhibitor when autoclaved in the presence of nitrite.198 Antimicrobials in Food increased (2. 50. Custer and Hansen (1980) reported that lactoferrin. Pretreatment of the lactoferrin and transferrin with a protease was necessary for inhibitor formation.5%). The results are intriguing and leave relatively unexplored the underlying mechanisms influencing the results. 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.5% to 3. and 50 kGy). 40. After higher doses of irradiation (10. They concluded that the level of residual nitrite did not influence botulinal outgrowth in their experiments. The amount of iron required for inhibitor formation was reportedly similar to the level found in muscle tissue. (1984) to examine the effect of various factors on the rate of nitrite depletion and botulinal outgrowth. 30. unless ferric chloride was present and the pH was 6. The addition of nitrate led to higher residual nitrite levels for a longer period. 156. 100. The authors suggested that Bactin formation may be responsible for the inhibition observed in Perigo-type media. Higher or lower levels prevented the inhibitory effect.0. then inoculated with botulinal spores. (1989) prepared ground pork slurries with 0.K. Transferrin. the . However. responded similarly. there was a marked decrease in the residual nitrite. Residual nitrite levels after irradiation were inversely related to the irradiation dose. The slurries were irradiated.

Pediococcus acidilactici). Alternatives to Nitrite for Botulinal Protection A considerable amount of research has been devoted to finding an acceptable replacement for nitrite. The maximum inhibitory activity was achieved by heating the medium containing 4% tryptone and 0. 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. and microbial stability. . aureus. The second option in the USDA regulation is based on a three-plant study that verified the efficacy of this concept (Tanaka et al. The NAS report (Assembly of Life Sciences. and a USDA-approved partial quality-control program to assure compliance. It was learned that the addition of ascorbate or isoascorbate (erythorbate) can block nitrosamine formation during the frying process. Subsequently. There was evidence that the factor may be unstable toward oxygen.e. Thus. 2002a). a listing of some of the proposed chemical alternatives along with references was later provided by Roberts and Gibson (1986). a minimum of 0. and C.Nitrite 199 requirements of pretreatment with protease and autoclaving reduce the likelihood that this would occur under natural conditions in meats. 2002a). flavor. The second option is to manufacture the bacon using a combination of 40 to 80 µg/g of sodium nitrite. A PTF was produced when tryptone was heated with nitrite at 121°C for 20 minutes (Miwa and Matsuura. The system was developed to avoid the addition of nitrite. Hansen and coworkers published a series of papers devoted to the Perigo factor with the intent of learning the mechanism of nitrite inhibition. A key to this approach is to not eliminate the lactics from the environment through an overly aggressive sanitation program. Typhimurium. cereus was used throughout the research. The research and subsequent regulation ignore the significance of brine level as an important factor that should also be controlled. A new approach to replace nitrite was developed in Canada. The rationale for this approach is apparent from the summary outlined earlier in this chapter. in the bacon section for 1970–1980.2% thioglycollate with more than 50 µg/g of sodium nitrite.. antimicrobial agent. coli or S. The factor was inhibitory against S. or cured color and flavor development). Bacon was identified as the one cured meat in which unacceptable levels of nitrosamine could be produced. If the brine level is controlled. subtilis. In addition. The relevance of this information to the effect of nitrite on C.. None of the chemicals investigated have shown promise to date. antioxidant. 1985).7% sucrose). Inhibition was not observed when casein or an acid hydrolysate of tryptone was used. and a lactic acid-producing bacterial culture (i. B. 550 µg/g of sodium ascorbate or isoascorbate. 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. an adequate quantity of fermentable carbohydrate is present. the high temperature can result in the formation of NOCs. Nitrite is unique in being able to produce cured meats with the proper attributes of color. One option is to use a combination of 100 µg/g of sodium nitrite. and sufficient sodium nitrite is added to produce an acceptable cured product. When bacon is fried. botulinum and the stability of cured meats that are mildly heated remains to be established. special requirements were established by the USDA for the production of bacon and verifying compliance with a nitrosamine limit.g. The process also does not acknowledge the potential value of the natural contaminating flora associated with most bacon processing environments.e. They concluded that nitrite inhibits the germination of spores of B. botulinum but not against E. a readily fermentable carbohydrate (i.. The committee considered the different functions of nitrite as a food additive (e. 1983). B. The system consists of using nitric oxide to cure the heme portion of beef blood. This cured pigment is then further processed. two additional options were added to the USDA regulations (Code of Federal Regulations.. 1987). oxidative stability. cereus by inactivating membrane sulfhydryl groups (Buchman and Hansen. 1982) is the most complete assessment of the possible replacements and the issues involved in finding a replacement.

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

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

5. which is more typical of cured meat and . Consistent with previous reports over the past 60 years. sporogenes cells and isolated proteins. Additional information and views on the mechanism of nitrite are available in Roberts et al. and/or an increase in the salt concentration.0% and 3. Research by Payne et al. Nitroprusside was used as a model to investigate the inhibitory mechanism of sodium nitroprusside and. nitrite was not an effective inhibitor at pH 7. However.202 Antimicrobials in Food The debate over whether clostridial ferredoxin or pyruvate-ferredoxin oxidoreductase is the site of nitrite inhibition for C. One aspect that the more recent data suggests is that the interaction of nitric oxide with iron–sulfur complexes is irreversible.g. some reduction in growth was observed with the addition of 200 µg/g of sodium nitrite. incubation temperature. Their results led to the conclusion that both are inactivated. blisters at the surface of the cells were observed when viewed under the electron microscope.0. It appears that the hypothesis proposed earlier is fairly close (Tompkin et al. perfringens was enhanced with a decrease in pH. monocytogenes in a broth medium at pH 7. perfringens and fecal streptococci in broth media. monocytogenes is quite resistant to the inhibitory effect of nitrite. The final pH of the products was about pH 4. After continued research on the mechanism of compounds associated with the Perigo factor. perfringens foodborne illness involving cured meats. sporogenes. 1978). (1989) tested the effect of nitrite on L. such as ferredoxin and pyruvate oxidoreductase. The relative sensitivity of C. At pH 6. (1990a. 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. it is fairly certain that the mechanism of nitrite involves the inactivation of iron–sulfur proteins.0) found in cured meats. At the pH values (6. (1980). and 5. 5. The cell-wall surface was the primary point of attack for the nitrosyl complex associated with nitroprusside.0. 28°C..0.5 and 6. monocytogenes to be similar in fermented sausage manufactured with different levels of salt (3. 1998). Most cured meats have traditionally had brine levels of 3% or greater. In addition. and sodium nitrite on the inhibition of C. botulinum was again addressed by Carpenter et al. perfringens to 3% to 4% salt could explain why there have been few outbreaks in C.5. On the basis of the research conducted by these researchers and others. Buchanan et al. In the research of Duncan and Foster (1968b) such blister formation was not reported following exposure to sodium nitrite. Gibson and Roberts (1986b) demonstrated the interactions of pH. Sodium nitroprusside was found to be a very effective inhibitor of C.6. 19°C.b) examined the effect of nitrite and iron–sulfur–nitrosyl complexes on iron–sulfur clusters in C. The inhibitory effect of nitrite against C. A high salt concentration and low incubation temperatures were the more important factors influencing the inhibition of these bacteria. The incubation temperature (5°C. leading them to doubt that the inhibitory effect of nitrite is on preformed iron–sulfur proteins of the bacterial cell.5%) and sodium nitrite (50. In a broth medium at pH 6. the toxicity of nitrosyl compounds to bacteria. (1989) found the rate of death of L. Miscellaneous Studies in Laboratory Media Botha and Holzapfel (1987) tested the effect of nitrite on Sporolactobacillus isolates from a variety of sources. comparable to the inhibitory properties of the anion of Roussin’s black salt. a decrease in temperature of incubation. sodium chloride. Juntilla et al. The cell walls showed a 90% decrease in thiol content when compared with untreated cells. 120. Sodium nitrite was added to the broth before autoclaving.. (1987).. although the nitrite had been autoclaved with the broth. the data more strongly demonstrate that L. It can be concluded that factors (e. and 37°C) had the strongest effect on growth rate and lag-phase duration. They were unable to demonstrate a direct action of the compounds on the iron–sulfur proteins. (1991) and Roberts and Dainty (1991). more generally. decreasing pH and aw) influenced the rate of death.5. the efficacy of sodium nitroprusside was explored (Joannou et al. The effect of nitrite on the growth of Listeria monocytogenes was examined in trypticase soy broth by Shahamat et al. and 200 µg/g). The fecal streptococci proved to be quite resistant to nitrite.

When comparing 0. The authors suggested that inhibitory strains produced specific antibiotic substances other than hydrogen peroxide or lactic acid. plantarum but not with L. the canned hams were opened. 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.0 and a residual nitrite level of 38 µg/g after processing. CollinsThompson and Lopez (1982) reported the inhibition to be species dependent. the rate of microbial growth was considerably slower in the sliced ham prepared from the lower pH meat. buchneri (Wolf and Hammes. L. 1985). The significance of these observations was discussed.35 and a residual nitrite level of 90 µg/g. and placed at 5°C. The meat was then used for processing as canned hams. and some interesting questions were posed. and 200 µg/g of sodium nitrite. and L. can accelerate nitrite depletion. The growth of Brochothrix thermosphacta was restricted when grown on sliced vacuum-packaged bologna with Lactobacillus brevis or L. 1988). Spoilage of Cured Meats Zeuthen (1980) studied the effect of meat pH on the rate of microbial growth on sliced ham.3 within 2 weeks at 5°C. L. L. nitrite reductase) rather than lowering the pH of the medium. nitrite may stimulate the uptake and transport of ions (e. During the 7 to 8 weeks of storage at 5°C.g. Thus. the rate of nitrite depletion in cured meats can be important. particularly homofermentative lactobacilli. Inhibitory species caused the pH of the bologna to decrease to 5. The data of Grau (1980). The higher pH meat resulted in a ham with a pH of 6. Both cultures were sensitive to the inhibitory effect of nitrite under anaerobic but not under aerobic conditions. the lactic acid bacteria .Nitrite 203 poultry products. Collins-Thompson and Thomson (1986) found two strains of L. demonstrate that inhibition can be dependent on the concentration of undissociated lactic acid and the degree of anaerobiosis existing at the site of growth.. viridescens or Leuconostoc mesenteroides. Competitive inhibition can be a factor affecting the spoilage flora of cured meats. nitrite was inhibitory. plantarum.5%). 50.7) and high-pH meat (6. lactis was able to reduce nitrite to N2O (Dodds and CollinsThompson. The lower pH meat resulted in a ham with a pH of about 6. Noninhibitory strains decreased the pH of the bologna to only 5. L. it was suggested that in trace metal metabolism. Fresh pork was divided into two groups: low-pH meat (5. 100. Subsequent tests demonstrated that a strain of L. both strains accumulated manganese in their cells. 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. vacuum packaged. plantarum. After cooking and chilling.3 to 6. viridescens was among the most active species. The nitrite reductases generally are heme-containing enzymes. commonly involved in the spoilage of cured meats. however. The hams were boned and pumped with a pickle solution containing sodium tripolyphosphate. lactis.5). manganese). monocytogenes will grow in processed meat and poultry products at refrigeration temperature. When grown anaerobically.5 to 5. the degree of inhibition was most strongly enhanced by reducing the incubation temperature.0%. For example.. L. to a lesser extent by anaerobiosis. By 1988 it was established that nitrite reduction can occur in some strains of L. CollinsThompson and Lopez (1982) and Dodds and Collins-Thompson (1984) further demonstrated that lactic acid bacteria. acidophilus. Under certain circumstances the level of analytically detectable residual nitrite can influence microbial inhibition. The loss of residual nitrite in bologna as a result of the action of the lactic acid bacteria was estimated as 30%. sliced. leichmannii. 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.5 within 4 weeks. The depletion of nitrite was attributed to enzymatic activity (i.5% and 4. and even less by increasing the salt level (0. viridescens. that were resistant to the addition of 50 µg/g of sodium nitrite if grown aerobically in APT broth. however.e. Both products had a brine level of approximately 4.

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

SUMMARY FOR 1980–1990 1. (1982). The NAS concluded that the evidence available about nitrite does not indicate that nitrite can act directly as a carcinogen in animals. Bushway and Serreze (1984) also studied the effect of adding EDTA to raw dark chicken meat. the efficacy of nitrite increased when the pH of dark meat was adjusted to 5. 2. A test was not conducted in which the pH of the two meats was adjusted and iron was added along with the nitrite. isoascorbate. 980 cans. and thermal process on botulinal outgrowth. When spoilage first occurred in each lot. 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. pH. The spoilage rate of this second group of cans was 4%. Perhaps some adjustment occurred in the microbial population. even a low level of nitrite (40 µg/g) caused some inhibition in white meat but not in dark meat. Nitrite was considerably more effective in white meat than in dark meat. swelled. The authors also investigated the effect of the inherent pH of the meat on the efficacy of nitrite. This provided a basis for assessing the risk associated with different cured meats. This could explain the high number of cases that have occurred during certain periods in Europe (Tompkin. Low levels of iron (5 to 15 µg/g) reduced the efficacy of nitrite. A means to quantitatively compare the data from various challenge tests based on the probability of outgrowth and toxigenesis from one spore was proposed. Extensive studies in meat and meat slurries demonstrated the interactions of nitrite. These conclusions meant that neither nitrite nor nitrate would be banned as food additives. The antimicrobial effect of nitrite was blocked when the pH of the white meat was adjusted to 6. These data reflect the influence of a mixture. respectively. This could be influenced by the fact that white meat is lower in pH and iron content than dark meat.8°C. 4.5%). Adding EDTA alone had no significant impact on the rate of aerobic microbial growth. If nitrite (130 µg/g) was also added. 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. Tests demonstrated that nonproteolytic. With the addition of sodium chloride (2. Bushway and Jen (1984) found lower residual nitrite levels in chicken white meat than in dark meat. The reason for this effect on the aerobic spoilage flora was not known.4. Likewise. The ground meat was aerobically packaged and stored at 4°C to 5°C. 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. From among 4322 cans of 8 different classes of perishable canned cured meats.6 and 6. Bushway and Serreze (1984) found that adding iron to raw white meat caused an increase in the rate of aerobic microbial growth. This led to the development of a model system for predicting botulinal outgrowth in food. The effect of nitrite on the aerobic spoilage flora of raw chicken patties was studied by Bushway et al. 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. 3. a concept . The pH of the white and dark meat was 5. the effect of iron on aerobic microbial growth depended on the amount of iron added. 1980). the NAS was not able to conclude that nitrate can act as either a carcinogen or mutagen. high levels of iron (40 to 160 µg/g) increased the efficacy of nitrite. of naturally occurring spores in fairly large cans of commercial product. psychrotrophic strains of C.Nitrite 205 meats to a minimum internal temperature of 68. whether cooked or raw. However. 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. phosphate. Furthermore.4. Adding EDTA enhanced the efficacy of nitrite and caused an increase in the lag phase for aerobic microbial growth. additional cans (8290) were removed from storage at around 8°C and incubated at 37°C for 3 days.6. The time elapsed was normally 7 to 10 days after production. or 23%.

McClure et al. monocytogenes in tryptose phosphate buffer under a wide variety of conditions (salt. 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. is not more inoculated pack experiments but a rationale for interpreting them” (Ingram. 6. 1991). red blood cells. but no evidence was generated to link the Perigo factor to the antimicrobial effect that has been demonstrated in perishable. 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. This probably reflects the effect of the higher salt levels and nitrite found in cured products compared with noncured products. 7. Again. delayed the time for toxin to be detected. cured products that receive no heat treatment or are cooked to final internal temperatures in the range of 60°C to 75°C. 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.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. Alternatives to nitrite continued to be investigated. 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. “What we need at the present time. In subsequent tests the effect of various decolorized blood fractions was evaluated. however. 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. SINCE 1990 C. monocytogenes to the degree that . found the efficacy of sodium nitrite was temperature and pH dependent. The database was incorporated into the USDA Pathogen Modeling Program. The mechanism of nitrite inhibition differs in different bacterial species..206 Antimicrobials in Food 5. 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. in my opinion. Some research continued on the Perigo factor. the rate of toxin formation was inversely related to the level of iron. very slow growth did occur. and the addition of plasma delayed the time for toxigenesis compared to the control sausage (Miller et al. 1974). Adding dried plasma. 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. In the absence of nitrite. 200. At 20°C and below no visible growth occurred even with 50 µg/g sodium nitrite and a pH of 5. The debate over the risk of C.3. (1991). 100. Since 1990 there has been increased interest in control of L. Autoclaving the sodium nitrite in the medium did not increase the degree of inhibition against L. 1993).0 and 5°C no growth was observed with any of the levels of sodium nitrite evaluated (50. but none have been adopted commercially. aerobic/anaerobic conditions) and found sodium nitrite to interact with combinations of the factors tested. Adding hemoglobin. At pH values of 6. that has been expanded to include other pathogens and has gained acceptance throughout the world. At the time he stated. The potential impact of various blood fractions as ingredients in sausage was investigated (Miller and Menichello. Despite this evidence. temperature. 8. 400 µg/g). Sausage formulated without sodium nitrite became toxic within 1 week compared with 3 weeks when nitrite was present. monocytogenes in ready-to-eat foods. At pH 6. Buchanan and Phillips (1990) studied the growth of L. pH. using an automated tubidimetric system with multiwelled plates containing trypticase soy broth. or whole blood reduced the time for botulinal outgrowth and toxin formation compared with the control with nitrite. Other available options have not received commercial acceptance.

salt (1%.5% sodium chloride and 50 or 200 µg/g sodium nitrite inhibited growth during 125 days storage at 37°C. If the broth had been adjusted to pH 6. (1992). Farber et al. Sameshima et al. 1997).2 with 200 µg/g. An extensive series of experiments led to the conclusion that nonthermal inactivation of L. 10°C) on the rate of growth of L. and additives to achieve shelf stability of canned luncheon meat (Farkas and Andrássy. a combination of heat and sodium nitrite was necessary to prevent survival and growth (F0 = 0. the effectiveness of sodium nitrite was significantly increased by the addition of sodium ascorbate. sausages made with nitrite and having pH 5. Since about 1990. although sodium nitrite concentration was not varied. respectively. Liver paté has been implicated in outbreaks of listeriosis in the United Kingdom. Three µM of residual undissociated nitrite doubled the time taken for a 3 log10 increase in numbers. Buchanan et al. 550 µg/g). 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. 4. Farkas and Andrássy (1992a) described a test system to evaluate the combined effects of formulation. sodium erythorbate (0.. 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). 3%).8 and containing 3. and other factors for inactivating spores and preventing the outgrowth of survivors. monocytogenes by sodium nitrite is pH dependent and related to the concentration of undissociated nitrous acid (Buchanan and Golden. (1994) inoculated a variety of vacuum-packaged cooked sliced meats with L. 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. Australia. 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. some . and sodium nitrite in laboratory medium and a predictive model for the growth of L. The model test system was then used to evaluate various combinations of heat. sporogenes. Only temperature proved to be a significant factor. inoculum level. McClure et al. viridescens. pH. they found that broth adjusted to pH 5. Using C. monocytogenes in liver pâté. Leuconostoc isolates seemed more sensitive to the effects of sodium nitrite and salt in comparison to the homofermentative lactobacilli. 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. Duffy et al. The effect of salt and nitrite on the growth of 24 lactics isolated from vacuum-packed cooked sausage was evaluated by Korkeala et al. Applying the procedures described by Hauschild (1982).Nitrite 207 had been observed with clostridia (see Perigo Factor). and temperature of storage (4°C. as had been reported earlier for C. and the United States. 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. temperature. mesenteroides. and Enterococcus faecalis. 1992b). monocytogenes in the sliced vacuumpackaged product when stored at 4°C or 8°C.0 and a sour. smoked flavor can be found. (1995) examined the effect of sodium nitrite (0 and 200 ppm). (1996) subsequently investigated the effects of salt. irradiation. As in the case of earlier research with C. heat. 1995. sporogenes (9 × 105/ml) in reinforced clostridial medium. botulinum. Perhaps the high iron content of liver pâté negated the effect of sodium nitrite and sodium erythorbate in this product. monocytogenes. (1997) examined the effect of nitrite on three lactics in vacuum-packed cooked sausage made with and without nitrite. F0 = 0. The procedures described show promise for estimating and comparing the combined effects of multiple treatments on pathogen survival and growth.7. they estimated the various formulations and thermal processes provided the equivalent of 4 to 7 log10 destruction of C. Low levels of salt (1% to 2%) enhanced growth. botulinum.7 with 50 µg/g). and 15 days for L. In regions of Northern Europe. L.

incapable of reducing nitrate or influencing flavor development. which is responsible for the reddening and other effects. 1987. (1990) conducted further investigations on nitrite reductase activity among the lactic acid bacteria. Several reviews.3 0. loss of the traditional flavor and increased acidity has led to a decrease in consumer acceptance and declining sales.8) and flavors that are more balanced. One group. 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.3°C –0. firm texture manufacturers in the Mediterranean countries have been shifting away from the more traditional. nitrate Yes Salty. complex..2 Basics of Raw Fermented Sausage Processing in Europe Processing Characteristics: Fermentation Drying/aging Final pH Added carbohydrate Added nitrate/nitrite Added starter culture(s) Product characteristics: Source: Incze (1992). pentosus. published since 1987. Scannell et al. Subsequent research found the lactic acid bacteria could be separated into heme-dependent and nonhemedependent catalase producers (Wolf et al. nitrate has traditionally been used for dry sausages and hams requiring long ripening times. Wolf et al. 1991). The cocci also play an important role in flavor and aroma development. It is curious that very little information has been published during that period from researchers in North America. plantarum. Nychas and Arkoudelos. was hematin dependent for nitrite reduction and yielded ammonia as the sole product. Specifically.208 Antimicrobials in Food TABLE 6. strongly acidic flavor. 1996. (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. In studies involving faster fermentation.. perhaps. pentosus. In France. 50. the rate of fermentation is faster and micrococci become inactive as the result of an accumulation of acid. Fernandez et al. >5. Flores and Bermell. rich. consisting of L. In Spain. the rapid production of acid renders the cocci inactive and.. acidilactici or L. Tests on 70 strains of lactic acid bacteria using cell suspensions incubated anaerobically led to two groups. 1994. and nonacidic. Spain. 1997. Hungary.. more than 50% of the initial nitrite disappearance was attributed to conversion to . and 100 µg/g) in salami. In pursuit of more effective starter cultures for use in manufacturing dry sausage. Tests by Mares et al. thus. rubbery or spreadable texture Low-Acid Sausage <12°C 14°C –16°C ~ 6. The basic parameters of raw fermented sausage processing in Europe are summarized from Incze (1992) in Table 6. The other group was able to reduce nitrite in the absence of hematin and yielded nitric oxide and N2O. plantarum but not P. 1990. In these processes micrococci convert the nitrate to nitrite. (1994) found that sodium nitrite (80 µg/g) reduced the catalase activity of a strain of L. and Pediococcus pentosaceus. and the Balkan countries. these traditional raw fermented sausages have had higher pH values (e. L. Flores. 2001). A few of the studies pertaining to the manufacture of fermented meats will be summarized.2. round. Lücke. Italy.g.0 None Nitrate Not necessary Salty. High-Acid Sausage 22°C –25°C 15°C –18°C <5. Nagy et al. 1989. describe the procedures used and changes occurring in Europe (Incze. The trend toward using starter cultures and slightly higher fermentation temperatures to reduce processing times is accompanied by certain negative features. In Europe. nonacidic flavor. 1989. In more modern processes. and 1992.7% Nitrite and. softer.

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

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

The addition of sodium nitrite is limited to smoked salmon. 1982.380 tons of cold smoked salmon in 1997. although a minimum of 3% salt in the water phase is strongly recommended (Huss et al. Cuppett et al.. lightly preserved fish products (cold smoked fish. If packaged aerobically. In France.4°C) for at least 4 weeks. a minimum concentration for salt in the water phase can be established. Controlling the salt in the water phase and temperature should be adequate. First. sodium nitrite could be used as an additional control measure.e. 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. 1987).. 2. Following a review of the literature. Cuppett et al. botulinum would be further inhibited. The panel was reluctant to recommend expanded use of sodium nitrite despite evidence showing that psychrotrophic C. it is highly unlikely that sodium nitrite will be adopted as an additive in smoked fish in Europe. with a production of 12. and chub. are low.5% salt in the water phase or 3. Surveys conducted before the year 2000 indicate L.. Toxin production can be controlled with a combination of a moderate level of salt (3. 2001a).5% in the water phase) and cold storage (<4. and with refrigeration this combination is effective in inhibiting growth of C. toxin was detected in 3 to 4 weeks. 1987. but the panel did not consider the more recent risk assessments demonstrating the safety of dietary nitrite (see section on toxicology). Hyytiä et al. questions still remain about “aerobic packaging” and its value for controlling botulinal outgrowth. Furthermore. shad.. 2001b). brined shrimp). gravad fish. It is possible. 1999). botulinum (Huss. if necessary. Considering the lack of regulations and apparent variability in salt concentration in large quantities of cold smoked salmon distributed domestically and internationally. With the availability of lower cost farm-raised salmon the international market for cold smoked salmon has continued to increase. 1999). . but. This may reflect survival or contamination. Until there is evidence of a need. In 1992 more than 32. Analysis demonstrated that nitrite was produced during storage. that combination of salt and temperature. Two options are available in the event commercially manufactured product is implicated in botulism. whereas with salt alone. sablefish. the salt in the water phase ranged from 2% to 4.5% salt in the water phase).000 tons shipped internationally from 28 exporting countries to 52 importing countries (Duffes. The addition of nitrite or nitrate increased the time the fish was considered acceptable by 3 or 4 weeks at 4°C. monocytogenes (Gram. however. however. 2001a): • • Psychrotrophic C. depending on the conditions of processing. 1982. although the numbers. 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.. it would appear the risk of botulism is lower than the research has suggested.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. monocytogenes is common in smoked fish. In many countries there is no legal requirement for the minimum amount of salt.0% salt if combined with 200 µg/g sodium nitrite (Gram. The panel expressed concern for the potential carcinogenic effects of nitrosamines.6% (Duffes. 1995). if present. A level of 3% salt in the water phase will yield perfectly acceptable. smoked fish to contain at least 3.. particularly in combination with controlled levels of salt (Pelroy et al. botulinum can be expected to occur in smoked fish. federal regulations were adopted requiring vacuumpacked. NOCs were not detected in two studies in which nitrite had been added to smoked fish (Pelroy et al. 1997). 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. 1997). allowing for short periods of elevated temperatures up to 10°C.. will prevent toxin production in cold smoked fish for several weeks beyond its sensory shelf life when packaged under reduced oxygen conditions. a lower salt content is permitted (i.

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

A similar response occurred with enteropathogenic E. Growth of C. that of the ham was near 7. At a low pH of 5. In the high-brine ham held at 22°C. given the proper circumstances. that of the chopped hams was 70% and 59%. Leistner et al. including Salmonella. 50. cereus) and then vacuum packaged.8% to 2.0. and B. 75. Nitrite and ammonia were assimilated at comparable rates. aureus. and the pH for the wieners was 4. (1979) tested the same bacteria on sliced bologna from two processors. nitrite values were not given. 65.0 by 160 µg/g of sodium nitrite. The pH of the chopped ham with 70% and 59% moisture decreased to 5. respectively. pH values were followed through storage. Typhimurium.Nitrite 213 and bologna was about 6.8%. A low-brine ham was prepared with 1.0. (1974). The other four species were able to grow under certain conditions. coli.5. commercially produced sliced ham and chopped ham did support the growth of the pathogens studied. whether the nitrite was added before or after autoclaving the medium of Perigo et al. The products were inoculated with five “enteropathogens” (C. but pH changes were noted. Two strains of S. and 130 µg/g) and two Salmonella inoculum levels (100 and 100.2% salt and 60 µg/g of potassium nitrite and a finished brine level of about 2.58. and Hafnia strains readily multiplied at all nitrite levels. This probably reflects the quantity of fermentable carbohydrate added to the product and/or the nature of the competitive flora. no data were provided for brine or nitrite levels. perfringens did not occur under any condition. 98. coli. E. The growth of Klebsiella and Enterobacter species on vacuum-packaged luncheon meats at 3°C was reported earlier by Hechelmann et al.7 to 4. The product from one supplier remained above pH 6. S. The authors did not state whether the nitrite was added before or after autoclaving the medium.0 and 5% salt. growth was reduced by only about 30%. In the low-brine ham at 22°C. and 100 µg/g) and the level of inoculum (100 or 100. viable counts of the 5 strains remained near the inoculum level. (1967). Under anaerobic conditions. No other chemical data were provided. other than to say that fresh.47 and 5. Stiles et al. Again. Klebsiella. It is not possible to assess the influence of nitrite from the data. perfringens. Death occurred after 3 days in the sausage with the lower inoculum and 130 µg/g of nitrite.0.7. Fermented sausage (teewurst) was prepared with four sodium nitrite levels (0. nitrite.78. Neither of two strains of S. the fastest generation times occurred in . nitrite was without effect. Page and Solberg (1980) found S. but a mixture of Enterobacter. A high-brine ham had a brine level of about 6%. S. the primary factor influencing growth was product pH.0. At a high pH of 7. The mean pH for the ham during storage decreased to no lower than 5. 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. The primary factors influencing growth were the temperature of storage and growth of competitive flora. Growth occurred in the sausage containing 0 and 65 µg/g of nitrite. (1973) inoculated vacuum-packaged luncheon meat with a mixture of Salmonella strains and held the product at 8°C for 22 days. The trend in the United Kingdom toward using less salt for processing ham was studied by Akman and Park (1974). and ammonia as the sole nitrogen source in a chemically defined minimal medium. 5% salt.8. Salmonella Enteritidis was able to grow at 17°C but not at 10°C. 4 of 5 strains of Salmonella grew readily. The moisture content of the hams was 70%. and 100 µg/g of sodium nitrite. Typhimurium was inhibited at pH 6. Both genera were prevalent in commercially produced luncheon meats and fermented sausages. Although salt and nitrite values were not reported. The resistance of Salmonella to nitrite in culture media was demonstrated by Roberts and Garcia (1973). Enteritidis were then used to study the effect of nitrite and salt on growth in nutrient broth at 22°C. It was concluded that cooked ham is a more likely source of salmonellosis if prepared with a low salt content. The rate of growth or death was influenced by the sodium nitrite content (0.000/g). Castellani and Niven (1955) reported S.000).6 to 6. All the enteropathogens grew in one or more of the products within 24 hours at 30°C. Stiles and Ng (1979) studied ham and chopped ham obtained fresh from two processors. Typhimurium to utilize nitrate. whereas the other frequently decreased to below pH 5. Aside from temperature of storage.

At concentrations of 500 µg/g and higher. In a traditional Turkish dry sausage that is fermented and dried at temperatures . being able to use nitrite in broth culture under anaerobic conditions at 5°C. the inherent pH of the meat (normal pH 5. The nitrite resistance of E. the addition of sugar. complete inhibition occurred. (1985) identified a nitrite reductase in the cytoplasmic fraction of S. Page et al. Thomas et al.2. aureus and C. a cured raw spreadable sausage. but growth was delayed with 200 µg/g (Turanta and Ünlütürk. coli.8. Similar results were observed with S. He recommended that a starter culture and/or gluconolactone be added to provide optimum safety. Typhimurium to be relatively resistant to sodium nitrite across the ranges of salt and pH values found in nonfermented and acidulated foods. using a gradient gel plate system. S. found S.8 and held at 24°C independent of sodium nitrite concentration. and the presence of lactic acid-producing bacteria. pH (5. This research was primarily concerned with preventing the growth of Salmonella. Gibson and Roberts (1986a) examined the interaction of salt (1% to 10% wt/vol). the effect of added phosphate was relatively minor.0 to 6. Schillinger and Lücke (1989) measured Salmonella growth and/or survival in mettwurst. S. Isolates of Enterobacter and Serratia species were tolerant to nitrite. Nitrite was only inhibitory with the highest level of nitrite and when the franks were incubated at 15°C. At pH 5. (1984) prepared fermented sausages with sodium nitrite (0. When examined after 10 weeks of incubation. and 37°C (McClure and Roberts.214 Antimicrobials in Food media containing both nitrate and ammonia.0. The franks were inoculated on the surface. and 156 µg/g) in frankfurters on a mixture of two strains of Salmonella. 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. The presence of nitrite did not influence the survival of salmonellae.8 versus 6.6. S. Collins-Thompson et al. except when the cultures were incubated at the lowest temperature tested (10°C). (1991). 6. numbers increased during 72 hours in the presence of 0. Typhimurium had shorter generation times and increased cell yields compared with nitrite-free cultures. 1977). (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. and 200 µg/g) and three strains of Salmonella. 50. 1987). None of the variables tested would likely assure the destruction of Salmonella in this product category. 150.2). 100. sporogenes. 1991). Collins-Thompson et al. coli. and 6. 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. 50. 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. 50. 27°C.8). When grown anaerobically in a glucose-limited minimal medium with peptone and nitrite. The presence of nitrite had no effect on aerobically grown cultures.b) to determine the additional effect of eight different phosphates of E. coli was also apparent in tests involving two-dimensional gradient plates incubated at 20°C. Factors influencing Salmonella growth in mettwurst that was cured and stored at 20°C include the amount of nitrite and salt. coli. Growth did not occur during fermentation or drying. The reductase required NADH and was most active at pH 8. The phosphates generally delayed the outgrowth of E. Typhimurium used nitrite as the sole nitrogen source at concentrations as high as 400 µg/g. vacuum packaged. Rice and Pierson (1982) tested the effect of sodium nitrite (0.6 to 5. Typhimurium numbers decreased in BHI broth adjusted to pH 4. and 100 µg/g sodium nitrite. 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). Hughes and McDermott (1989) used a procedure similar to that of Gibson and Roberts (1986a. and incubated at 15°C or 27°C for 21 days.

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.Nitrite 215 not exceeding 24°C. The inhibitory effect of nitrite was much greater at pH 5. 2001). The effect of sodium nitrite (0. pH 5.g. if not multiply.0. pH. fermented to pH 4. sodium nitrite at 70 to 150 µg/g did not affect the growth or survival of E. Preventing salmonellosis from cured meats is best accomplished by means other than relying on the level of nitrite in the product.5 to 6 hours) was next studied (Yu and Chou. coli O157:H7 (Yu and Chou. or ammonia under anaerobic conditions. where . and temperature (Bhaduri et al. 1996).. This sausage is not fermented or cooked and relies on relatively rapid dehydration to achieve microbial stability. in a variety of commercially cured products. and incubation temperature (Chou and Tsai.. (1998) found E. 1996). coli O157:H7 with the addition of curing agents and drying at time–temperature combinations that appear to cause stress of the inoculum. 1997).. but there is no published information that nitrite or nitrate inhibits their growth.5% salt and 100 µg/g sodium nitrite. 55ºC for 4 hours) were used. a fermented noncooked beef sausage. salt level. 1996).5) and enhanced by lowering the incubation temperature (Buchanan and Bagi. 1991). and 156 µg/g) had no effect on the death rate of E. The data for Salmonella can be summarized as follows..5. Typhimurium has a nitrate reductase to reduce nitrate to nitrite and can assimilate inorganic nitrogen from nitrate. S. That cured meats have been implicated in outbreaks of salmonellosis supports the available research that Salmonella can survive.. 100. the inhibitory effect of smoke. and temperature.. Typhimurium was not affected by the addition of 200 µg/g sodium nitrite (Turanta and Ünlütürk. 78. Riordan et al. Effect of Nitrite on Yeasts and Molds A wide variety of yeasts and molds have been isolated from commercially cured meats.8. coli O157:H7 numbers decreased less than 1 log10 in pepperoni formulated with 2.e. coli. 80. the lowest pH tested and with increasing salt levels. particularly because salmonellae are destroyed during the manufacturing process for most cured meats. Adding sodium nitrite did not increase death of E.. Inhibition of Shigella flexneri in broth incubated aerobically was significantly influenced by the combined effect of nitrite concentration. Another study involving anaerobic conditions yielded similar trends (Zaika et al. coli O157:H7 when lower temperature and/or shorter time combinations (e. Pin et al. coli O157:H7 at 37ºC were inhibited by 200 µg/g sodium nitrite at pH 5. Inhibition of Yersinia enterocolitica in broth incubated aerobically was significantly influenced by the combined effect of nitrite concentration. The highest survival involved a starter culture that produced the least amount of acid and yielded a relatively high final pH of 5. nitrite.5. Inhibition was much greater at 28ºC than at 37ºC (Zaika et al. In ground pork held at 5ºC or 25ºC. This is not true for the clostridia. pH. and 120 µg/g) on Y. 1994). Inhibition of Aeromonas in broth has been found to be highly dependent on nitrite concentration. but even 1 g/L sodium nitrite was ineffective at higher pH values (Tsai and Chou. The effect of nitrite in Chinese-style sausage during drying at 50ºC to 60ºC for different time periods (2.7 × 105/g to nondetectable levels by 35 days in the sausages formulated with 80 µg/g nitrite. and. In tryptic soy broth 4 strains of E. The data indicate that the nitrite content of cured meats can influence the growth and survival of Salmonella and E. the addition of sodium nitrite (0. 1993). and then dried for 7 days. coli O157:H7 in a broth system containing sodium nitrite was strongly pH dependent (i. coli O157:H7 (Chikthimmah et al. pH. (1993). 50. They found some increased death of E. 1994). Inhibition of E. salt level. the death rate of S. 1994 and 1995). enterocolitica 0:3 to grow and survive during the manufacture of raw dry fermented sausage was examined by Asplund et al. Raccach and Henningsen (1997) reported sodium nitrite and salt to act synergistically in the reduction of Y. storage temperature. 1996. In Lebanon bologna. These factors include water activity. The inoculum decreased from 1. the availability of oxygen. enterocolitica when tested in a meat system.

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

1981. Within the United States the methods of assay appear in the Official Methods of Analysis of the Association of Official Analytical Chemists .175. 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. as proposed by the Expert Panel on Nitrites and Nitrosamines (Food Safety and Quality Service. The use of nitrite and nitrate in the processing of fish products is specified in 21 CFR 172.Nitrite 217 7. Nitrite and nitrate levels for curing meats in the United States were recommended by a USDA Expert Panel on Nitrites and Nitrosamines (Food Safety and Quality Service. ASSAY METHOD Because the levels of nitrite and nitrate permitted in foods are established by regulation. Examples of the effect of this regulation on the maximum allowable level of nitrite in several sausage and loaf products are listed in Table 6.177 (Code of Federal Regulations. As the percentage of extenders and other nonmeat ingredients is increased in the products. 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. 2002a). 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. and Food. Because it has already been cured. 1978). the weight of this meat is subtracted from the fresh meat portion of the formulation and the amount of nitrite added is reduced accordingly. depending on the circumstances.S. The status of nitrite and nitrate in cured meats and cheese in the United Kingdom was reviewed in 1978 (Ministry of Agriculture. the method of assay applicable to each country should be used. 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. leads to lower residual nitrite levels after processing.160. 1978). The net effect of the U.170. Germany reduced the permitted level of nitrite in nitrite-containing curing salt by 20% (Leistner. Fisheries. The regulations were summarized in two comprehensive surveys (Meester.17 and 424. 9. 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. With the exception of bacon and smoked chubs. This material is normally used as an ingredient at a level of about 5% in the manufacture of succeeding lots of the same product. The use of nitrate or nitrite as additives in cheese and seafood products was clarified. 1979). 1974. and 172. 172. An example of rework is the round ends of sausages that are trimmed off before slicing the product. meat and poultry regulations is that nitrite and nitrate are added on the basis of the uncured meat in the product formulation. 2002b). 8. the level of added nitrite is decreased. Nitrite is of relatively little value for controlling Gram-negative enteric pathogens in commercially prepared foods. The most recent summary of the regulations in different countries appears in Cassens (1990). regulations do not specify minimum nitrite levels for cured products.S. 1995). The current USDA regulations controlling the use of nitrite and nitrate in meat and poultry products are specified in 9 CFR 317. U.3. 1986).21-23 of the federal regulations (Code of Federal Regulations. 1978). 172. Other countries limit the amount of nitrite and nitrate on the basis of the total weight of the product formulation. Braathen. A unique feature of the U. It is also common to generate a certain amount of rework during the manufacture of sausage products.S. Gerhardt. This further reduces the nitrite level in the product formulation and.

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

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

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

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

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

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

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

A similar degree of destruction was reported for highly acid foods having pH values of 3. At high pH. These may be intended as stock solutions for the preparation of test dilutions in smallscale laboratory experiments. An international nisin reference preparation was later established by the WHO Committee on Biological Standardisation (Anonymous.025 mg/ml (equivalent to 1000 mg/kg Nisaplin®). suggesting that the molecule is protected to some extent by food components (Delves-Broughton. 1969). first developed by Tramer and Fowler (1964) and Fowler et al. In this chapter nisin is expressed as levels of pure nisin (µg/ml or µg/g). heating for 3 minutes at 250°F (121°C) destroyed 25% to 50% of the added nisin. 1964).1 to 6. Nisin is extracted from the test food by an acid-heat treatment. (1965). is the same value as the Reading Unit. For example. otherwise a loss of activity may occur as a result of salt precipitation or filtering. In triangle taste trials conducted by Danisco. equivalent to 1 µg of this preparation. is the most widely used nisin assay. The specifications for the purity and identity of nisin were set in 1969 by the FAO/WHO Joint Expert Committee on Food Additives (Anonymous.9. International Unit.2 as approximately 56 mg/ml.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. and a specification was set for commercial nisin concentrates containing at least 2. This was contrary to previous work by Tramer (1964. 1970). research in Danisco laboratories has shown that care must be taken when preparing high-concentration nisin solutions from commercial preparations such as Nisaplin®. Their results show that in low-acid foods at pH 6. 1966).5 mg/ml at pH 6 and 0. mg/kg) or to International (Reading) Units (IU/ml.25% biologically active nisin.5 (Davies et al. (1975). and this bioassay method contains . However. Optimum heat stability occurs at pH 3 to 3. Nisin has no significant taste and imparts no negative taste to foods in which it is used.3 to 4. Solubility in foods is never a problem because nisin levels are always very low. 1992). Nisin is. and nisin activity was then defined in International Units. IU/g). Liu and Hansen (1990) suggested that the Dha and Dhb residues become more susceptible to modification as a result of the presence of nucleophiles.5.5% 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. 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. Liu and Hansen (1990) reported nisin solubility at pH 2. It is recommended that such solutions should not exceed a maximum concentration of 250 µg/ml.. The horizontal agar diffusion test. the levels of pure nisin should be multiplied by 40. The nisin preparation Nisaplin® was developed by Aplin & Barrett (now Danisco) between 1962 and 1965. The current term. producing clearly distinguished and measurable zones of inhibition in the presence of nisin. volunteers could not detect 200 mg/L Nisaplin® in mineral water (equivalent to 5 µg pure nisin/g). however. Pure nisin was deemed too potent for use in food. 1998). This has a standard potency of 1 million International Units per g. 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. Both the solubility and stability of nisin in solution are affected by pH. The effect of processing temperature and pH on the stability of nisin in different foods was studied by Heinemann et al. dropping to 1. with levels of usage in food rarely above 0. successfully used in high-pH/heat-treated food products such as canned vegetables and pasteurized liquid egg. Delves-Broughton et al.5.25 mg/ml at pH 8. and contains 2. 1990. 1 µg/g pure nisin is equivalent to 40 IU/g or 40 mg Nisaplin®/kg. To convert these units to equivalent Nisaplin® levels (mg/L.

or Gram-negative bacteria. capable of transducing the signal into detectable bioluminescence (Wahlstrom and Saris. which was thought to result from the test measuring a partially degraded nisin molecule that had lost biological activity. enabling it to be used as a preservative in pasteurized or heat-treated foods that are not fully sterilized. Wide variation in nisin sensitivities has been reported. Joosten and Nunez. After 7 days at 25°C. 1987). again most probably because of detection of inactive degraded nisin or possibly biologically active nisin that was bound within the food (Bouksaim et al.125 µg nisin/ml (Thomas and DelvesBroughton. The growth of 104 cfu/ml of four different strains of this species was controlled for 7 days at 37°C by 0. Rose et al. is extremely nisin sensitive.2 shows the activity of nisin against three different strains of Bacillus. This latter test does not measure biological activity. ANTIMICROBIAL SPECTRUM Nisin has a broad spectrum of activity against Gram-positive bacteria. 2001). A relatively new species.1. It is active against both bacterial cells and their heat-resistant spores. 1995. 1991. 1996). because nisin Z diffuses more rapidly than nisin A. most importantly for its use as a food preservative.625 to 2. 1999). Nisin is also effective against another food-associated pathogen. Another important group of nisin-sensitive bacteria is lactic acid bacteria. Falahee and Adams. The meat spoilage organism Brochothrix thermosphacta.. Comparative studies of processed cheese assayed by ELISA and the horizontal agar diffusion bioassay showed a lack of correlation (Aplin & Barrett Ltd. This species is extremely nisin sensitive. Listeria monocytogenes.. or alcoholic beverages (e. An ELISA system using nisin Z polyclonal antibodies in rabbits also suffered from similar problems. first described in 1951. Bacillus sporothermodurans. nisin-sensitive organisms relevant to food are shown in Table 7. 1990. detectable by bioluminescence (Waites and Ogden. lactis construct strain sensitive to nisin has been reported. was first described in 1985 and. In normal circumstances nisin does not significantly inhibit yeasts. 1989a). nisin at 0. This could be corrected by developing an antibody that detected distal parts of the molecule. 1999). this bacterium produces extremely heat-resistant spores capable of surviving ultra-high temperature (UHT) treatments. whereas . unpublished results). 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. An L. 1992).. 2001). The outcome of nisin activity can also vary from achieving total kill of the cells to a growth inhibitory effect (DelvesBroughton et al. There have been several reported improvements to this test (Rogers and Montville. Its activity range encompasses. wine and beer) that are often not heat processed. 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.Nisin 241 numerous control procedures to ensure nisin activity is not confused with other antimicrobial agents that may be present in the food. with considerable variation being observed even between strains of the same species (Gupta and Prasad. 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. Hurst (1981) reported that lower nisin levels prevent spore outgrowth compared with the levels required for inhibition of vegetative cells. ensuring that nondegraded nisin only would be measured. Wolf and Gibbons..5 µg/ml prevented the growth of 103 cells/ml (Thomas and Delves-Broughton. Nisin has a stronger bactericidal effect against energized cells. the Gram-positive endospore-forming genera that include Bacillus and Clostridium. For example. molds.g. Important pathogens in this group include Bacillus cereus and Clostridium botulinum. sauces. Such heat treatments usually eliminate all vegetative cells so that such foods are vulnerable to spoilage only by these surviving spore-forming bacteria. Figure 7. organisms that may cause spoilage of low pH food such as salad dressings.. (1999) described detection of nisin by matrix-assisted laser/desorption time-offlight spectrometry. 1996). as its name suggests.

mesenteroides innocua. buchneri. Cause spoilage of wine and vegetables.. Aerobe/anaerobe.) and food-poisoning. UHT-resistant. 1991. sporogenes. curvatus. varians Spp. Heat-resistant spore-forming thermophiles. pentosaceus inulinus aureus Source: From Thomas et al. M. Cause blackening of canned food. brevis. As a result of the application of the hurdle theory of food preservation (Leistner and Gorris. cells will usually be nonenergized in foods because they may well be experiencing adverse conditions caused by low temperature. Aerobes/anaerobes. subtilis. stearothermophilus Brochothrix thermosphacta Clostridium Desulfotomaculum bifermentans. Some used for cheese production. Obligate anaerobes. Gram-negative bacteria are resistant to nisin because the antimicrobial is unable to penetrate their complex cell wall to reach its site of action: the cytoplasmic membrane. sordelli. producing slime. thermosaccharolyticum. 25°C–60°C. plantarum oenos. helveticus. beer. tertium. Maisnier-Patin et al. Bacillus brevis. wine. Heat-resistant aerobic and facultative anaerobic spore formers. pumilis. macerans.5–6. quiescent cells may survive but their growth will be inhibited (Sahl. Spo