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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2004062074

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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

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

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

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

182. 184. 184. 172. 182. wines 184. wines Fruits. 184.3089. sauces Beverages.22. 172. and then the possible preservation systems must be evaluated via model studies and studies in the food product in question. and poultry products Cured meats Beverages.175.1550.2). 184. molds. diacetates.1 and 1. meats. Section 424. acetates. 184. Generally.145 Molds Yeasts. bacteria Yeasts.3795 Various For meat products. baked goods. cooked meat. Food and Drug Administration) Food Antimicrobialsa (Title 21 of the Code of Federal Regulations) Compound(s) Acetic acid.6197.1670. (1998).1 Traditional or Regulatory-Approved (U. condiments.1005.1061. fermented foods Meats Cheese.Food Antimicrobials — An Introduction 3 TABLE 1.21 and 424.177 184. heptyl) of p-hydroxybenzoic acid) Propionic acid. lactates Lactoferrin Lysozyme Microbial Target Yeasts. dry sausage CFR Designation 184. cooked meat. syrups.130 184.1721. 2000). 182. 184. The target pathogen or spoilage microorganisms must be identified first.160. Title 9.1221.1185. casings for frankfurters. fruit products. Naidu (2000). GRN 000065 Nitrite.1639. 172. other bacteria 172. a combination of chemical preservatives and other preservation methods is needed (Leistner.3640.1538. GRAS Notice No. fats/oils. confections. Source: Davidson and Harrison. GRAS Notice No. 184.1207. GRN 000067 184. SELECTION OF ANTIMICROBIALS It is not an easy process to select the appropriate preservation system for a particular food product.1754. molds Bakery products. fruit products. molds. molds Yeasts Bacteria Bacteria Clostridium botulinum.133 184. 172.1784 182.170.1021. food antimicrobials permitted by USDA Food Safety and Inspection Service are listed in the Code of Federal Regulations. dairy products. .S. GRN 000064 Yeasts. 184. beverages. other bacteria Natamycin Nisin Molds Clostridium botulinum. Sofos et al. methyl. propionates Sorbic acid. dehydroacetic acid Benzoic acid. 182. 184. dairy products Most foods. 2002 to foods (Tables 1.3225.1768 GRAS Notice No. potato products.1733 172. margarine Beverages Meats. 184. bacteria (Gram positive) 172. sorbates Sulfites a Clostridium botulinum Yeasts. and poultry products Cheese Cheese. bacteria Primary Food Applications Baked goods. 172.1490. Extensive reviews on natural antimicrobials may be found in Dillon and Board (1994). nitrate Parabens (alkyl esters (propyl. and Davidson and Zivanovic (2003). casings for frankfurters.1081. benzoates Dimethyl dicarbonate Lactic acid.155 184.

. “Methods for Activity of Assay and Evaluation of Results. spores). Each of these factors is discussed in detail. although desired.” Seldom. Appropriate methods for in vitro evaluation of the activity of food antimicrobials are described in Chapter 21.4 Antimicrobials in Food TABLE 1. 2002. yeasts. and Gram reaction (positive vs. is not easy to achieve. The antimicrobial spectrum of a compound is generally determined by following the growth of organisms in the presence of various concentrations of the antimicrobial. the physicochemical properties and composition of the food product in question. Few chemicals have the ability to inhibit several different types. strain. Selection of the proper antimicrobial depends on several primary factors. molds) and forms of those microorganisms (vegetative cells vs. ANTIMICROBIAL SPECTRUM The initial selection of the antimicrobial is normally based on an assessment of the overall microbial spectrum of the chemical in question.g. species. and the type of preservation or processing and storage systems used. does growth in a synthetic . the chemical properties of the antimicrobial. Quite often. the activity of very hydrophobic antimicrobials may be limited against Gram-negative bacteria. a broad spectrum of activity. The antimicrobial spectrum should involve an evaluation of the compound against various types of microorganisms (e. including the spectrum of antimicrobial activity. Even species. negative) can have dramatic influences on apparent activity.2 Regulatory-Approved Food Antimicrobial Designations in the European Union (E Numbers) and in Codex Alimentarius (INS Numbers) Compound Sorbic acid K sorbate Ca sorbate Benzoic acid Na benzoate K benzoate Ca benzoate Ethyl paraben Na ethyl paraben Propyl paraben Na propyl paraben Methyl paraben Na methyl paraben Sulfur dioxide Na sulfite Na hydrogen sulfite Na metabisulfite K metabisulfite Ca sulfite Ca hydrogen sulfite K hydrogen sulfite Nisin a E or INS Number 200 202 203 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 226 227 228 234 Compound Natamycin DMDCa K nitrite Na nitrite Na nitrate K nitrate Acetic acid K acetate Na acetate Na diacetate Ca acetate Lactic acid Propionic acid Na propionate Ca propionate K propionate Boric acid Na tetraborate Na lactate K lactate Ca lactate E or INS Number 235 242 249 250 251 252 260 261 262 262 263 270 280 281 282 283 284 285 325 326 327 Dimethyl dicarbonate. For example. bacteria. Source: Verbruggen. which have the ability to screen the antimicrobials because of the outer membrane lipopolysaccharide layer. or strains of microorganism. however.

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

enzymes. For example. Chelating compounds such as ethylenediamine tetraacetic acid (EDTA) or its salts have a potentiating effect on some antimicrobials.19 3. They expand the activity of certain antimicrobials (e. All regulatory-approved organic acids used as antimicrobials have pKa values less than 5. In addition.. Refrigeration generally selects for psychrotrophic Gram-negative microorganisms. Bacteria maintain internal pH near neutrality to prevent conformational changes to the cell structural proteins. Protons generated from intracellular dissociation of the organic acid acidify the cytoplasm and must be extruded to the exterior. Branen and Davidson. the acid dissociates because the cell interior has a higher pH than the exterior. which exists in the majority only at a pH below the pKa of the compound.87 4. 1980. and phospholipids.5 or greater. nucleic acids. thus indicating the need for a different antimicrobial. Because protons generated by the organic acid inside the cell must be extruded using energy in the form of adenosine triphosphate (ATP).5 to 4. A lowering of the water activity can select for those organisms that have the ability to survive and/or grow at lower water activity. some Gram-positive bacteria are more susceptible to certain antimicrobials in the presence of chelators including EDTA. which means their maximum activity will be in high-acid foods. This is because the most effective antimicrobial form is the undissociated acid. nisin. where microbial growth occurs (Branen et al.75 4. Rico-Munoz Davidson. Packaging can directly alter the environment of the food and thus influence the overall growth pattern and type of organisms in the food.79 4. pH of the food can result in ionization of an antimicrobial and a change in activity. thus requiring an antimicrobial capable of limiting the growth and activity of these organisms.3 pKa of Regulatory-Approved Organic Acids Compound or Group of Compounds Acetic acid Benzoic acid Lactic acid Propionic acid Sorbic acid pKa 4. It is theorized that chelators may destabilize the LPS layer of the outer cell membrane of the Gram-negative bacteria.. PROCESS FACTORS The type of preservation process used in conjunction with antimicrobials has a significant influence on the type and level of antimicrobial needed. Vacuum or modified atmosphere packaging results in . allowing the antimicrobials access to the inner cell membrane. lysozyme) to include Gram-negative bacteria. Although the undissociated form of a weak acid has most of the antimicrobial activity.6 Antimicrobials in Food TABLE 1. organic acids can penetrate the cell membrane lipid bilayer more easily. Eklund (1983) demonstrated that the anion does contribute slightly to antimicrobial activity. organic acids function at low concentrations only in high-acid foods (generally less than pH 4. molds survive and yeasts can grow at a lower water activity than bacteria. the constant influx of these protons will eventually deplete cellular energy. Once inside the cell. 1995.0 (Table 1.75 into the lipid areas of the food and away from the water phase. 1983).g. there are very few compounds that are effective at low concentrations. Generally.6). In the undissociated form. which are not normally inhibited by the compounds alone (Cutter and Siragusa. 2004). Certain preservation processes may result in the need to control sporeformers that have the ability to survive the heating process.3). For food products with a pH of 5.

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

it is much less likely to require complex regulatory approval for use (Davidson and Zivanovic. such as nisin.1550) and nisin (U. This would involve very expensive and time-consuming toxicologic testing. it would be unacceptable for a food antimicrobial to mask spoilage because spoilage may protect consumers from ingesting foodborne pathogens. ACTIVITY VALIDATION METHODS Currently. . would defeat the purpose of using a natural compound. This is only possible if the product from which the extract is taken is known to be nontoxic. just as thermal processes need validation. 2003). In addition. Therefore. In the United States.1538). For example.8 Antimicrobials in Food common potential natural antimicrobials such as spice extracts. If antimicrobials are to be used exclusively as inhibitors of pathogens in food products. or texture. Consumers are reportedly concerned about the presence of synthetic chemicals in their foods and would prefer natural compounds. The reason for these assays is that various conditions of process or storage could reduce the effectiveness of the compound. the compound would probably have to be listed using a chemical name on a food label. In addition to adverse effects on flavor. are susceptible to inactivation by enzymes in foods. Many antimicrobials must be used at high concentrations to achieve activity against target microorganisms. assays need to be developed that evaluate the activity of these compounds against the pathogen they are designed to kill. 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. The efficiency of these compounds can be extremely important in determining the overall economics of their use. 21CFR 184. 1993). less purification may be better. SENSORY EFFECTS Another major factor that needs to be addressed when applying antimicrobials is their potential impact on the sensory characteristics of a food. it is known that peptides.S. This is because. so should there be validation for the activity of food antimicrobials. Obviously. If a product is simply an “extract of” a commonly consumed plant or animal food product. Code of Federal Regulations. compounds that negatively affect flavor and odor or contribute inappropriate flavors and odors would be unacceptable. there are methods for the determination of the activity of lysozyme (U. they should be nonallergenic or bind or destroy important nutrients in a food product (Harlander. naturally occurring compounds must be able to be metabolized and excreted so as to not lead to residue buildup. Extensive studies at a pilot-plant level are necessary on any food additive to prove its overall usefulness. For that reason.S. of course. LABELING One of the alleged attractions of naturally occurring antimicrobials is their reduced negative impact on the labeling of foods. they are not normally consumed in the concentrations necessary to achieve antimicrobial activity. In addition to lack of toxicity. they may need to be approved as food additives. although spices have been consumed for centuries. 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. 21CFR 184. A potential problem with natural antimicrobials is that if they are highly purified. odor. Code of Federal Regulations. there are few standardized methods for validation of the activity of regulatory-approved food antimicrobials. In many cases. Finally. This.

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

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

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

Benzoic acid (molecular weight 122.3). 1999).0. and 2. 1991). 1995). . 66. pear.18. of mushrooms. p-hydroxybenzoic acid was detected at 0. pineapple. 20°C. Potassium benzoate and calcium benzoate have also been approved for use.1). may also contribute to benzoic acid generation. MECHANISM OF ACTION It is the undissociated molecule of benzoic acid that is responsible for antimicrobial activity (Table 2. 1992). peach. and 75°C. were used to characterize commercial fruit juices (Fernandez de Simon et al. and 100°C. 1929). 1994). 18°C. 1990). Yogurts have been found to contain natural levels of benzoic acid (Stijve and Hischenhuber. 1992).2 g dissolves in 100 ml water at 0°C. Gabel (1921) was one of the first to demonstrate that benzoic acid was effective against bacteria in acid media at a level of 0. and of fresh tomatoes (Marlatt et al. also called phenylformic acid or benzenecarboxylic acid. Sieber et al.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. 0. O OH Benzoic Acid O O− Na+ Sodium Benzoate FIGURE 2.8. Orange.1% and in neutral media at 0. It is soluble to a limited extent in water (0. where benzoic acid has not been approved as a food additive (Sieber et al. although their solubility in water is less than that of the sodium salt. apple. 1986a).1 Structures of benzoic acid and sodium benzoate. Benzoic acid has also been identified as a natural by-product in culture filtrates of Lactobacillus plantarum (Niku-Paavola et al. In an extensive review.. occurs in pure form as colorless or white needles or leaflets. (1995) surveyed and analyzed many types of cultured dairy products and cheeses for the natural occurrence of benzoic acid (Table 2. 1989. Benzoic acid occurs naturally in several foods and commodities (Table 2. This may be of significance in countries like Switzerland. respectively)..2). It is much more soluble in water than benzoic acid (62.2% but inactive in alkaline media..1) is a white granular or crystalline powder. apricot.. Teuber. respectively). and grape juices were analyzed to establish phenolic profiles unique to each type of juice.2). it is preferred for use in many cases. along with benzoic acid. Benzoic acid has also been identified as a major constituent in extracts of blackberries (Humpf and Schreier.2 to 2. Several new types of low-molecular-mass compounds were also identified..2 g dissolves in 100 ml water at 4°C. It accounted for approximately 16% of the growth inhibition of Saccharomyces bayanus and Pseudomonas fluorescens from ethanolic extracts of cranberries (Marwan and Nagel. A third pathway. was found to have antimicrobial properties. Similar results were reported for fungi and yeasts (Cruess and Richert.6 mg/l.1. Its presence appears to occur as a by-product of the microbial degradation of either hippuric acid or phenylalanine in these products (Figure 2. Sodium benzoate (molecular weight 144. 1984. Naturally occurring nonflavonoid phenolic compounds. and a mixture of these. depending on the variety (Abdullah et al.27. oxidation of benzaldehyde. including derivatives of benzoic acid. and 74. In four of the seven juices. For this reason.1).

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

8 1. The effects of pH. that only the undissociated acid was antimicrobial. 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. . sucrose.3 12. TABLE 2. Proteinaceous material was apparently involved in the uptake of this preservative. not to the pH itself. The strong dependence of uptake on pH was because of the relative distribution of undissociated and dissociated forms in solution. increasing the pH of the culture medium decreased the effectiveness of this preservative.3 Effect of pH on the Dissociation of Benzoic Acid pH 3 4 5 6 7 pK Undissociated Acid (%) 93. 1997).144 4.0. As expected. Sorbate was more inhibitory than benzoate on the strains that were tested.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. The effect of temperature on the uptake was similar to that observed in enzymatic reactions. Saturation was reached in about 2 minutes and remained constant thereafter. and sorbic and benzoic acids on the growth of 30 strains of food spoilage yeasts have been reported (Praphailong and Fleet. Zygosaccharomyces bailii and Yarrowia lipolytica were the most resistant to the two preservatives at pH 5. The undissociated form was the only one taken up by cells. Source: Adapted from Sieber et al. NaCl. (1995).19 Source: From Baird-Parker (1980).2 Biosynthesis of benzoic acid by lactic acid bacteria in fermented dairy products.44 0. Macris (1975) observed a rapid uptake of benzoic acid in Saccharomyces cerevisiae.5 59. and that the toxicity of sodium benzoate in solution was a result of the undissociated benzoic acid molecule.4.

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

1982).16 Antimicrobials in Food 3. Theoretical ATP consumption for proton pumping can be directly correlated with reduction in cell yield. 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.. as food preservatives is related to their capacity specifically to reduce the intracellular pH. 1960. coli have been reported (Salmond et al. Similar effects have been found using a variety of microorganisms. α-ketoglutarate and succinate dehydrogenases appear to be quite sensitive (Bosund. Hsiao and Siebert (1999) constructed a model based on physical and chemical properties of organic acids and principal components analysis. 1989). subtilis. More than 99% of the viable cells of Listeria monocytogenes were injured after exposure to a solution of 8.. fluorescens was completely inhibited by sodium benzoate (Andersson et al. 1983. 1962).3). 1966). At pH 6. benzoic acid or sodium benzoate can also inhibit specific enzyme systems within cells (Webb. coli were grown in liquid media with or without 20 mM benzoate (Lambert et al. Uraih et al.5. 1962). membrane transport of amino acids is inhibited. Eklund. Aflatoxin production by a toxigenic strain of Aspergillus flavus was greatly reduced by the presence of these compounds in synthetic media (Uraih and Chipley. benzoate addition resulted in an increased production of 33 proteins. phosphofructokinase activity was inhibited (Francois et al.. 1997). 1992). in many bacteria and yeasts. Production of 12 of these was induced at pH 8. The authors suggested that the potency of weak acids. B. 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. 1973).0 as well as pH 6. its pK value (Table 2. 1997). such as benzoic acid.. Gould. For example.5. 1980). The inhibitory potency of this acid can be determined by its lipid/water partition coefficient.. In resting cells of E. such as benzoic. In the citric acid cycle. Reduction . coli. 1973). 1980).0) for 1 hour (Buazzi and Marth.2 mM benzoic acid (Kruk and Lee. Corlett and Brown. 4. coli. Davidson. In addition to these mechanisms. Synthesis of messenger ribonucleic acid (mRNA) was critical for restoration of salt tolerance. Duration of the lag phase can be predicted from the model.. resulting in nutritional starvation of cells (Gould et al. 1977. The production of lipase by P. and the effectiveness of undissociated acids as preservatives has been noted for many years (Ingram et al... 1980). and Pseudomonas aeruginosa (Freese et al.. 1984). MICs were determined for each acid and used with principal components to produce models with good correlations. E. 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. yielding protons that acidify the alkaline interior of the cell.. A pH-related increase in protein production was observed when cultures of E. Benzoate also inhibits amino acid uptake in Penicillium chrysogenum (Hunter and Segel. 1978). enzymes involved in acetic acid metabolism and oxidative phosphorylation can be inhibited (Bosund. They also stated that although both the undissociated and dissociated forms of these acids cause the intracellular pH to fall. 1989. Increased permeability of cell membranes during the course of benzoate injury was not observed. growth inhibition is the result predominantly of the undissociated acid. 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. Only those organic acids that are lipophilic. Sheu et al. In yeast cells. 1973. 1980. causing uncoupling of both substrate transport and oxidative phosphorylation from the electron transport system (Freese et al. show antimicrobial activity. Chipley and Uraih. 1976. 1956. 1986). One hypothesis is that they inhibit or kill microorganisms by interfering with the permeability of the microbial cell membrane. In Bacillus subtilis (Freese. Eklund. Acid-susceptible bacteria (Bacillus and Alicyclobacillus) and acid-resistant bacteria (Lactobacillus and Escherichia coli) were grown in media containing organic acids. 2001). The effects of organic acid preservatives on the growth and intracellular pH of E. 50% of the trimethylamineN-oxide reductase activity was inhibited by 1.5% sodium benzoate (pH 7. 1975.

benzoic acid cannot be relied on to effectively preserve foods capable of supporting bacterial growth (Chichester and Tanner. temperature.. 1997). 1977. In general.. as an inhibitor of Damino acid oxidases (Quay and Massay. 1983). This might result in inhibition of membrane-bound enzymes as a secondary effect. Therefore. In liquid media.1% of the undissociated acid. The results suggested that these preservatives blocked an enzymatic step late in the biosynthetic pathway of aflatoxin B1.01% to 0.5 and 2. as a weak inhibitor of poly(ADP ribose) polymerase (Oikawa et al. Food-poisoning and spore-forming bacteria are generally inhibited by 0. including pH. 1979).02% undissociated acid.1979). as an inhibitor of passive anion transport (Lucas-Heron and Fontenaille. Addition of benzoate to chemostat cultures of S. 1980). In benzoic acid-supplemented cultures.6. It has been reported to have a flower-inducing effect in plants (Watanabe and Takimoto..3).3% sodium benzoate. and environment from which the microorganism was originally isolated. 1988). 1980). genus and species of the microorganism in question.. 1974). MICs for some of the bacteria.. flavus have been reported (Bauer et al. The average accumulation of mycelial dry weight was greater in the presence of benzoate than in its absence. 4-dinitrophenol and cell envelope-associated enzymes of E. coli and Salmonella Enteritidis (Chipley. and sodium benzoate was determined (Manganelli and Casolari. yeasts. With the previously mentioned results in mind. Several factors interact to determine the MIC. cerevisiae decreased the biomass and increased the specific oxygen uptake rate of cells (Verduyn et al. prior exposure to the preservative. The effects of several preservatives and antimicrobial agents on aflatoxin B1 production by A. potassium sorbate. Baird-Parker. Currently. ANTIMICROBIAL ACTIVITY SPECTRUM OF ACTION Because the quantity of undissociated acid decreases with increasing pH (Table 2. composition of the growth medium. 1972. Benzoate may also serve as a scavenger for free radicals (Harvath. The sensitivity of 42 yeast cultures to sorbic and benzoic acids. 1979). with the greatest increase occurring when the medium contained 0. 1981). Fusarium species were more sensitive to benzoic acid (210 to 420 µg/ml) than were Penicillium species (250 to 3000 µg/ml). but many spoilage bacteria are much more resistant. Similar results were observed with 2. The scattering of MIC values was lower with potassium sorbate and benzoic acid and higher with sorbic acid and sodium benzoate. and most yeasts and fungi are inhibited by 0. all treated cultures produced measurable levels of toxin 3 to 7 days later than controls. and as an inhibitor of nitrosamine formation (Sung et al. and fungi involved in food poisoning and food spoilage are given in Tables 2. . these compounds are used primarily as antimycotic agents. 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.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. Yagi et al. Increasing the concentration of sodium benzoate increased the percentage of inhibition at the end of incubation (10 days). however. 1980). Conversion could be prevented by addition of benzoic acid or sodium benzoate. the use of benzoic acid or sodium benzoate as a food preservative has been limited to those products that are acid in nature. aflatoxin B1 production was higher than in controls. Sodium benzoate was found to control both growth and aflatoxin production by Aspergillus parasiticus in liquid media (El-Gazzar and Marth. 1987). The authors concluded that subinhibitory concentrations of these compounds may stimulate toxin production in some cases.05% to 0. 1992). 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.

0 5.8 Fungi 5. pastori Rhodotorula sp.2–5.0 5.6–5.0–5. Yeasts. and Fungi Microorganisms pH Bacteria 6.0 MICa Bacillus cereus Escherichia coli Klebsiella pneumoniae Lactobacillus sp.2–5.0 6.5–5.0 4.5 Antimicrobial Spectrum of Benzoic Acid against Selected Bacteria.6 Yeasts 2.6 (4°C) 5. Aspergillus parasiticus Aspergillus niger Byssochlamys nivea Chaetomonium globosum Cladosporium herbarum Mucor racemosus Penicillium sp. rouxii 4.0 2.18 Antimicrobials in Food TABLE 2. Source: Adapted from Chipley (1983.3 5. (1999).3 5.0 4.5 4. Pseudomonas aeruginosa Staphylococcus aureus Streptococcus sp. Saccharomyces bayanus S.0 6.0 4. 500 50–120 100–200 100–200 300–1800 3000 2000 50–100 200–480 200–500 50–100 200–400 Sporogenic yeasts Asporogenic yeasts Candida krusei Debaryomyces hansenii Hansenula sp.6–4.0 5.8 4.0 4.6 (21°C) 5.8 4. and Steels et al.0–5. Zygosaccharomyces bailii Z. Hansenula subpelliculosa Oospora lactis Pichia membranefaciens P. .0 5. lentus Z.1 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). cerevisiae Torulopsis sp.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. Russell (1991).5 5. 1993).0 3.0 4.0 5.0 4. Pseudomonas sp.0 6.6 6.3–6. Listeria monocytogenes Micrococcus sp.6 6.0 3. Davidson and Juneja (1990).

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

3). 1963).4.0) were used and required lower levels of sodium benzoate. they were very effective inhibitory agents for heatdamaged spores (Lopez et al. benzoic acid. Additive. aureus when used with higher concentrations of NaCl.. It was also the most effective against B. The growth patterns of S.. and antagonistic effects reported in several early studies have been extensively reviewed by Lueck (1980) for benzoic acid as well as for other preservatives. cereus. The relative effectiveness values of benzoic acid were established for S. 1972). when these organisms were grown in Trypticase® soy broth. A typical dose-response effect may be observed. At 35°C. A plot of the inhibitor concentration versus the reciprocal of relative effectiveness was linear (Figure 2. The infinite inhibition concentrations for S. 1986).. Combining glycerol monolaurate with benzoic acid gave greater inhibition of L. At <25°C. The x-axis intercept was the concentration of the inhibitor that gave infinite microbial inhibition.20 Antimicrobials in Food and growth were determined in media supplemented with organic acids and sodium benzoate (ElShenawy and Marth. This method was based on finding the relative effectiveness of an inhibitor. An interesting method to determine concentrations of antimicrobial agents that provide infinite microbial stability has been developed (Marwan and Nagel. For example. . 1988). sodium chloride. In theory. 1980). It should then be possible to achieve (1) a broader spectrum of action or (2) an increased antimicrobial action by using this combination (Lueck. synergistic. Increasing the acidity and/or NaCl generally improved the effect of all the preservatives. 1986b). or sucrose (Rehm. bayanus in the presence of different concentrations of benzoic acid are shown in Figure 2. sorbate was more effective than sodium benzoate against S. at various concentrations. like benzoic acid. bayanus and Hansenula species. Synergism between benzoic and sorbic acids was pH dependent. The anionic form of benzoic acid was shown to have a distinct effect on doubling time. carbon dioxide. bayanus and Hansenula species were 330 and 180 ppm benzoic acid. one should be able to combine various antimicrobials having different modes of action to compensate for this deficiency. 1996). and sorbic acid on the growth rate of the yeast Z. 1989). combinations of sorbic acid and benzoic acid inhibit several bacterial strains better than either antimicrobial alone (Rushing and Senn. Effectiveness increased as the pH of the recovery media decreased to 5. benzoate was the most effective preservative against S. boric acid. INFLUENCE OF OTHER CHEMICALS AND PHYSICAL ENVIRONMENT No antimicrobial is completely effective against all microorganisms present in a given foodstuff. Potassium sorbate and sodium benzoate did not have any significant effects on the heat resistance of four strains of B. Sodium nitrite was the least effective preservative tested. Chichester and Tanner. aureus. However. 1993). Individual and synergistic effects of pH. 1960. In general. Potassium sorbate was completely effective against Vero cytotoxigenic E. Infinite inhibition concentrations could be affected by the growth medium. stearothermophilus spores (Lopez et al. 1994)..0. bailii were determined and expressed in polynomial equations (Cole and Keenan. when these preservatives were added to recovery media. 1998). inactivation or inhibition of growth occurred in inoculated media when a lower incubation temperature (13°C) and pH (5. especially when used at pH <6. The effects of temperature. coli at all temperature/pH/NaCl combinations. monocytogenes than when each preservative was tested alone (Oh and Marshall. The influence of combinations of chemical antimicrobials on the growth of food-poisoning and spoilage microorganisms has been reported (Banks et al. and three preservatives on the growth of three foodborne bacterial pathogens were studied using gradient gel plates (Thomas et al. Synergistic effects have also been reported using combinations of benzoate and sulfur dioxide. pH. respectively. sodium chloride.

benzoic acid can be solubilized with a surfactant to hold it in solution in its undissociated form (Lewis and Payne. For example. depending on the chemical in question. 150 ppm potassium sorbate. it may also be advantageous to combine preservatives with physical methods of food preservation. Closed circles: yeast nitrogen base. followed by potassium sorbate and sodium benzoate. Sulfur dioxide was apparently antagonistic when used in combination with any of the other antimicrobial agents (including benzoate) tested in this study. Open circles: Trypticase® soy broth.b). Source: Marwan and Nagel (1986b). The presence of other chemicals may be beneficial or detrimental to the action of the preservative. 1961).. in hours. and 500 ppm sodium benzoate. irradiation. 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.0 0. 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 3.3 Effect of the medium on the inhibition of Saccharomyces bayanus by benzoic acid at 30°C and pH 4. refrigeration. respectively. 1956). Growth at 21°C.2 0. 1975). . cerevisiae was inoculated in a laboratory medium supplemented with several combinations of preservatives (Parish and Carroll.8 0. 1984). relative effectiveness (the time. 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. However. Likewise. 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). 1988). Antagonistic as well as synergistic effects were observed when a wine yeast strain of S. 1990). or drying.9) (Okoli and Ezenweke. Sulfur dioxide had the most significant effect on the rate of biomass and patulin production by this organism.6 1/RE 0. 1958). the antimicrobial activity of benzoic acid is reduced by nonionic surfactants (de Navarre and Bailey.Sodium Benzoate and Benzoic Acid 21 1.4 0. Sodium benzoate prevented the growth of Staphylococcus aureus in an intermediate-moisture food (Boylan et al. The effects of pH and benzoate on the growth of yeasts in an experimental grape drink containing 1. 1976)..0 50 100 150 ppm 200 250 300 350 FIGURE 2.5 volumes of CO2 have been reported (Kelly. 30°C. and 37°C over a 25-day incubation period was significantly retarded by 75 ppm sulfur dioxide. such as heating. In addition to combining several preservatives together. RE. A combination of benzoic acid and low temperatures was used effectively to reduce the numbers of yeasts in grape juice (Pederson et al.0.

The concentrations of benzoic acid tested (in parts per million) are indicated.5 (Sagoo et al. the authors proposed that chitosan is highly polycationic and could react with the anionic components of the microbial surface. At these pH values. Carbonated and noncarbonated citrus drinks and fruit juices were involved.025% sodium benzoate acted synergistically against three food spoilage yeasts suspended in saline solutions at pH 6. cited by Lueck (1980). Source: Marwan and Nagel (1986b).2 and 4. Adding 1% quinic acid to media almost completely eliminated the inhibitory effect of 0. no resistance could be observed in cells of E. this might enhance the antimicrobial effects of benzoate.. The effects of quinic acid on yeasts and molds were investigated (Kallio et al. A combination of the polysaccharide chitosan (0.4 Inhibition of Saccharomyces bayanus by benzoic acid in Trypticase® soy broth at 30°C and pH 4. 2002). ACQUIRED RESISTANCE TO BENZOIC ACID There have been conflicting reports in the literature concerning the possibility of acquired resistance to benzoic acid by microorganisms.005%) and 0. The resistance of a wide variety of yeasts to benzoic acid has been reported in several studies.02% sodium benzoate. . Growth of yeasts was unaltered.0.01% to 0. However. They had been supplemented with the maximum levels of benzoic acid permitted for use in these products. but mold growth was accelerated in the presence of quinic acid.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. recent studies imply that this may no longer be the case.. This acid was antagonistic to the antifungal effects of both potassium sorbate and sodium benzoate. In a German study of 1959. 1985). coli after 14 passages in media containing benzoic acid.

1987). heat tolerance appeared to be related to the morphology of the ascospores. were tested for tolerance to several organic acids during and after heating (Beuchat.7).. In a later study (Warth. The authors observed that preservative resistance may be strain specific. However. Resistance resulted primarily from an inducible energy-requiring system that transported benzoate from the cell. and in some cases.0 and 4. 1990). and lethality was enhanced as the pH of the heating medium was reduced. cells were resistant only when a sufficient energy source. 1988) it was determined that energy was required for the reduction in cytoplasmic benzoate concentration and for the maintenance of internal pH. 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. In an inoculated peach-based system. . this isolate was less resistant to potassium sorbate. respectively (Ethiraj and Suresh. cerevisiae was found to have multiple resistances to both physical treatment and chemical preservatives (Guerzoni et al. bailii grew in the presence of high concentrations (600 mg/L) of benzoic acid at pH values below the pK of this acid (4. 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. such as glucose.19). Three strains of T.5. Potassium sorbate and sodium benzoate prevented outgrowth. Because this system required energy. the energy source was unavailable for growth. 1988). flavus. A strain of S. Warth (1977) reported that cells of Z. Source: Adapted from Warth (1988). TABLE 2.4 to 3.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. 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. was present. sorbic. and benzoic acids were the most effective. Fumaric. resulting in reduced growth yields and rates of cell production by yeasts. the effectiveness of benzoic acid on this strain decreased as the water activity decreased and only a slight synergistic action with thermal treatments was observed.25 mM benzoic acid (first five species) or 2 mM benzoic acid (last four species).5. with the required concentrations lower when the pH of the growth media was reduced from 5. 1988). isolated from spoiled fruit products. Thus.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.

preservative-resistant spoilage yeast. results also suggested that the resistance mechanism. bailii and S. 1989c). In another study (Warth. Uptake rates of benzoic and propanoic acids in cells of Z. and most can grow at very low pH. Three selective media were compared for detecting and enumerating nine strains of Z. Growth at 4°C was significant. The authors concluded that Z.2 to 7.24 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. or legally. They were resistant to sorbic acid (≤400 mg/l) and benzoic acid (≤900 mg/l) at pH 4. Z. and acetic acids and ethanol (Thomas and Davenport. The nonselective medium supported higher recovery of all nine strains from the six food products compared to the three selective media. These media were also evaluated for enumerating two of these strains grown in blueberry syrup with or without sodium benzoate (0. Uptake of weak acid preservatives by passive diffusion is quite rapid. Some yeasts are intrinsically resistant to relatively high concentrations of preservatives (Stratford and Lambert. Eight tons of table mustard were exported to the United States. 1999). bailii strain that was resistant to both benzoic and sorbic acids. 300. Removal or metabolism. or an intrinsic sensitivity to benzoic acid or its anion (Warth. 1999). A new osmophilic. . The Zygosaccharomyces genus also contains some of the most osmotolerant species known. Differences in resistance among yeast species could be the result of differences in the rate of penetration of the cell by benzoic acid. Resistant species may be less permeable than sensitive ones to undissociated benzoic acid (Warth. benzoic. 1996). Glucose stimulated the rates of uptake and was also required for continuous elimination of benzoate from cells. Zygosaccharomyces lentus. 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. 1988). The authors stated that microorganisms resist or adapt to weak acid preservatives (e. Adaptation can result in rapid growth in moderate levels or slow growth in previously inhibitory levels. bailii were proportional to the concentration of undissociated acid (Warth. Resistance to dimethyldicarbonate was greater than that of Z. Foods at particular risk are acidic and contain relatively high levels of sugar. 1996). Benzoic acid was taken up 27 times faster by cells than propanoic acid. the capacity of the cell to remove it. cerevisiae. bailii in the spoilage of refrigerated foods. bailii grown in six commercial food products (Makdesi and Beuchat.0). bailii has been described (Buchta et al. has been described (Steels et al. Adaptation to higher preservative concentrations was demonstrated with benzoic acid.. 1989b). It was preserved using benzoic acid (1. lentus was likely to be more significant than Z. and all strains were osmotolerant. Cells grown in syrup without sodium benzoate were more sensitive to selective media than were cells grown in syrup containing the preservative.5 g/kg) and packaged in glass jars. However.g.0. Five strains of this yeast grew over a wide range of temperature (4°C to 25°C) and pH (2. bailii can grow in concentrations of food preservatives in great excess of those normally. requiring only 5 to 15 minutes to reach equilibrium. Spoilage resulted from a Z. in which benzoic acid is continuously removed from the cell. 1989a). allowed in beverages and can adapt to concentrations even higher. The minimum pH for growth was not related to this acquired resistance. and 600 µg/ml).. 1989c). is a common and major determinant of the preservative tolerance of yeast species (Table 2. 1985). benzoate) by one of the following three methods: 1. Zygosaccharomyces and Saccharomyces are able to adapt to changing levels of preservatives such as benzoate. Growth in the presence of weak acid preservatives appeared to involve a common resistance mechanism. The principal mechanism of uptake appeared to be diffusion of undissociated acid. Growth of yeasts in the presence of benzoic acid reduced their subsequent permeability to this acid. Mustard spoilage by Z.. 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.6) (Warth.

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

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

1990). Harwood and Gibson (1986) stated that aerobic degradation of benzoate by various Gramnegative bacteria. end product of the β-ketoadipate pathway (Figure 2. maleylpyruvate. and fumarate + pyruvate. The proposed intermediates included benzoyl-CoA. including Pseudomonas. Similar results have been reported with Bacillus stearothermophilus (Adams and Ribbons. which are central intermediates in the anaerobic metabolism of many aromatic compounds (Elder and Kelly. (2) ring reduction. 3-hydroxybenzoyl-CoA. 1988). 1997). (4) ring opening.. He reported that benzoic acid was easily metabolized by all nine bacterial species tested. 1996). Anaerobic metabolism can also occur but involves reductive saturation of the nucleus. 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. 1984). 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. putida (Jeffrey et al. 1992). Pseudomonas solanacearum. and with Micrococcus (Haribabu et al.. In Aspergillus niger. Hugo (1991) described the ability of nine species of Pseudomonas and Vibrio to degrade 18 aromatic compounds used as preservatives in medicines. Further metabolism proceeded via the catechol branch of the β-ketoadipate pathway with the occurrence of high specific activities of catechol 1.5). 1982). foods. These results were confirmed by assaying key enzymes from benzoate-grown cells. 1994). respectively (Shailubhai et al.5) (Ampe et al. 1993. and (5) a β-oxidation sequence.. Outlines of the new aerobic pathway were deduced. The enzymes responsible for ring cleavage were induced depending on the substrate used for growth. 1995). Nitrate. 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.. anaerobic pathways have been far less studied and reported. benzoate and salicylate are metabolized via the protocatechuate and β-ketoadipate pathways. One pathway proposed for the anaerobic microbial metabolism of benzoate is shown in Figure 2. both aerobically and anaerobically. gentisate. and sulfate can be used as electron acceptors during metabolism (Colberg and Young. The enzymes of these pathways appeared to be under coordinate control and were repressed in the presence of a primary substrate. iron. 1997).6. with Rhodococcus (Janke et al.. . the rate of benzoate metabolism may be regulated by the presence of succinate. 1995)... protocatechuate 3. This isolate was able to oxidize benzoate completely to carbon dioxide. (3) introduction of a carbonyl group. 1982). 1988). Harwood and Gibson (1997) stated that this pathway could be divided into several phases: (1) formation of CoA thioesters. Benzoate was metabolized by 16 of these strains when added as the sole carbon source to liquid mineral media.. Seventeen strains of aerobic bacteria were tested for their ability to degrade 15 aromatic substrates (Lang. and cosmetics.Sodium Benzoate and Benzoic Acid 27 β-ketoadipate pathway. This is followed by conversion of the remainder of the molecule to acetyl-CoA. one of the causal agents of wilt in banana plants. cis. Compared to relatively well-characterized aerobic mechanisms for metabolism of benzoate and other aromatic compounds. A mutant incapable of growth on benzoate was isolated and characterized. fumarylpyruvate. cis-muconate.2-dioxygenase for ring fission. a commonly occurring soil bacterium. 1997). Benzoate was converted to catechol by the strains able to grow on this substrate. In Alcaligenes eutrophus. whereas salicylate induced catechol 1. Catechol. 1984). was also capable of metabolizing benzoate (Kumari and Mahadevan. such as glucose. Active transport of benzoate may occur in this organism (Thayer and Wheelis. Niemetz et al. A permease-like protein involved in benzoate transport and degradation has now been isolated from cells of Acinetobacter (Collier et al. and β-ketoadipate were identified as intermediates of benzoate metabolism (Figure 2. 4-dioxygenase was induced. In the presence of benzoate.2-dioxygenase. 1984). Kinetic models have been derived for the metabolism of [14C]-benzoate by Pseudomonas species (Simkins and Alexander. Both chromosomal and plasmid-encoded pathways have been described for the catabolism of benzoate in P..

. Source: Adapted from Koch et al.. 1979).. were found to have this capability. jejuni could be developed. 1988).6 Proposed pathway for the anaerobic microbial metabolism of benzoic acid. 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. The N-benzoyl-glycine amidohydrolase (hippuricase) test is of paramount importance to differentiate C. For example. This enzyme cleaves hippuric acid into the constituent products glycine and benzoic acid. coli. coli. Note: This sequence of reactions is compiled from several studies. An equation was obtained to estimate the ratio of bound to unbound acid in food products based on their lipid and protein content. Microbial reduction of benzoate to benzyl alcohol under aerobic conditions has been reported (Kato et al. one species. making the models unacceptable under these conditions. They also showed that the gene was absent in other Campylobacter species. 1996). Nocardia asteroides and several fungi. In foods with high protein or lipid content. 4-diene carboxyl-CoA O OH 2-Hydroxycyclohexane carboxyl-CoA Cyclohex-1.28 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. 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. jejuni (hippuricase positive) from other species. jejuni from C.. jejuni and demonstrated that this gene was not present in 17 isolates of C. Based on these results. the authors concluded that it may not be limited to a small number of microorganisms. In the genus Campylobacter. C. most of which were fungi. has become recognized as a leading cause of human gastroenteritis (Hani and Chan. The authors isolated the gene responsible for the expression of hippuricase from 12 strains of C. including species of Rhizopus and Mucor. Harwood and Gibson (1997). jejuni. Ganzfried and McFeeters (1976) developed a model system to evaluate binding of benzoic acid by lipids and proteins. 1995). (1993). antilisterial activity was much lower than predicted. However. the hippuricase assay is the only reliable biochemical assay that can distinguish C. Models were tested in several food systems to determine the inhibition of 18 strains of L. 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. Heintze (1978) reported that benzoic acid accumulated in the lipid phase of herring salad. 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. the authors proposed that a species-specific diagnostic DNA probe for human gastrointestinal infection caused by C. Both bound benzoic acid to approximately the same extent. Solid arrows indicate intermediates detected in benzoate-grown cultures. Currently. although stability was not adversely affected with time. monocytogenes by benzoic acid and other compounds (Ramos-Nino et al. Although this appears to be a unique reaction occurring in microbial cells under aerobic conditions. Dashed arrows indicate proposed intermediates.

APPLICATIONS Benzoic acid and sodium benzoate are most suitable for foods and beverages that naturally are in a pH range below 4. benzene levels higher than the permissible levels for mineral waters prompted a recall in one state. the authors recommended that the combination of ascorbic acid and sodium benzoate in foods and beverages be evaluated more carefully. Product information for both salts is available (Miles Laboratories. Subsequent studies indicated that soft drinks containing both ascorbic acid and sodium benzoate could produce very low levels of detectable benzene.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. including benzoic acid. Potassium benzoate is now commercially available and apparently evolved in response to consumers’ interest in reduced sodium intake. The potassium salt is less water soluble (42. They may also be used in certain standardized foods. 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). and other foods in several countries (Tables 2.1021 and 184. Similar results were reported by McNeal et al. REGULATORY STATUS In the United States. Lueck (1980). the off-flavor they may impart to the foods they are to preserve. the use of benzoic acid and other chemicals in the processing and freezing of shrimp has been reviewed (Chandrasekaran. Kimble (1977). the maximum permissible quantities generally range between 0. 1977/1988. their main advantages include low price. applications. although its solubility in water is much less than the sodium salt (Pollard. fruit products. 1994). Applications of these preservatives to foods have been reviewed by Chichester and Tanner (1972). They may also include foods from which benzoate is excluded in the United States.1%. USE IN VARIOUS FOOD SYSTEMS Benzoic acid and sodium benzoate have been widely used to preserve beverages.15% and 0. ease of incorporation into products.8 and 2. bakery products.1%. Title 21. benzoic acid and sodium benzoate are GRAS preservatives (Code of Federal Regulations. In one case (a fruit juice–mineral water combination). Benzoate does not control the growth of high levels of microorganisms and therefore cannot be used to compensate for poor ingredients or processing. 1990). A Food Technology special report (1986) reviewed the characteristics. (1993) when they analyzed several foods and fruit-flavored soft drinks for benzene. As food preservatives. 184. However.9). In most other countries.). An ADI (average daily intake) value of 0 to 5 mg/kg of body weight has been specified for benzoic acid (Pollard. and lack of color.25%. 1993). under certain conditions. . generate detectable levels of benzene. Inc. 1990).1 times more potassium salt than sodium salt is required to obtain the same level of benzoic acid in solution and the same level of antimicrobial effect. Secs. and Chipley (1983. Regulatory agencies consider it to be GRAS to the same extent that sodium benzoate is GRAS as a preservative with the limitation of 0. and limitations of preservatives and antimicrobial agents. Approximately 1. In addition.5 or that can be brought into this range by acidification.1733) up to a maximum permitted level of 0. 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. Although the detectable yield was extremely low (<1 ppb). Calcium benzoate has also been approved for use. the narrow pH range in which they are effective.4 g per 100 ml solution) than the sodium salt.

bailii in an inoculated salsa mayonnaise stored at room temperature (Wind and Restaino. but A.04%) inhibited growth of all three fungi.S. bailii. However.02%) or sodium benzoate (0. was most sensitive to propionic acid and sodium benzoate. Sorbate treatment was more effective than benzoate and increased the shelf life by 8 days.K. c Soft drinks for consumption without dilution. salted Mayonnaise Mustard Pickles. Recent reports indicate that these preservatives continue to be of benefit in a variety of products. jellies Liquid egg white. relishes Salads. Halotolerant fungi were isolated from salted dried fish (Chakrabarti and Varma. U. Penicillium sp. 1996). Aspergillus flavus. then evaluated during shelf life (Efiuvwevwere and Ajiboye. A combination of sodium benzoate and kojic acid was effective in inhibiting melanosis. yolk or whole Margarine.8 Maximum Permitted Levels (ppm) for Benzoic Acid and Its Salts in Foods in Selected Countries Food Fish products Fish. separately or combined. were dominant. The effectiveness of sodium benzoate and potassium sorbate. salad dressings Sauces. A maximum level of 2000 ppm may be used in orange juice for manufacturing.3% sodium benzoate was three times longer than that of the controls (Ponce de Leon et al.06%) or potassium sorbate (0. d Good manufacturing practice. and Penicillium sp. . 2000). ketchup Soft drinks containing fruit juice Soft drinks. Source: Adapted from Chipley (1993). Propionic acid (0. separately or combined. niger. was evaluated against the growth of Z. The shelf life of sardines stored in an acid brine containing 0. 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. prevented spoilage of the product by Z.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. 2001).. However. Several additives and preservatives were evaluated as inhibitors of melanosis (black spot) and microbial spoilage in prawns in chilled storage (Montero et al. flavus was most sensitive to potassium sorbate. semipreserved Fruit juice Fruit pulp Jam.30 Antimicrobials in Food TABLE 2. neither preservative.. 4-hexylresorcinol was the most effective inhibitor of both melanosis and microbial spoilage in prawns. Potassium sorbate was more effective than sodium benzoate. A.a 1000 200 500 500 5000 1500 1500 1500 5000 2000 1000 1000 1000 10. 1994). carbonated a Canada 1000 1000 1000 1000 1000 Denmark Norway Sweden U. a maximum level of 1000 ppm benzoic acid or its salts may be used for all products listed. b Soft drinks for consumption after dilution. 1995).

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

monocytogenes on sliced.. 1999). and ethanol were very effective bactericidal treatments for L.0) made from milk acidified to pH 5.6) or adding potassium sorbate (0.32 Antimicrobials in Food temperature. monocytogenes usually attach following cooking and during slicing and packaging (Farber and Peterkin. These strains grew well in unpasteurized and pasteurized apple juice. However. or S. 1989). These solutions could be used for dips to sanitize chickens intended for frying before presentation to consumers (Hathcox et al. Application of antimicrobials as sprays or mists to meats following processing may be more effective than their addition in the meat formulation.05% sodium benzoate reduced the numbers of these pathogens compared to washing with water. Combinations of organic acids.9 with propionic acid. A preservative treatment was developed that was capable of achieving the FDA mandate for a 5-log reduction of E. Washing the skin with solutions of either 0. 2001). low pH. coli O157:H7 in apple cider (Comes and Beelman. 1993).3%) to cheese (pH 6.3%) to cheese (pH 6.15% fumaric acid and 0. several factors may influence the effectiveness of benzoic acid in the treatment of fresh . 2001). 2001)... this antimicrobial was evaluated for control of postharvest diseases of various fruits and vegetables. or 5% potassium benzoate for 120 days (Samelis et al. 1998). Growth was either prevented or delayed by adding sodium benzoate (0.. Ethanol-enhanced killing correlated with damage to the bacterial cytoplasmic membrane. monocytogenes (Barker and Park. Resistance to these preservatives was greater at 4°C than at 25°C (Fisher and Golden. 2000). the combination of sorbate and benzoate was more effective than either preservative alone (Zhao et al. 1999. Control strains of E. Sorbates and benzoates are currently approved in various countries for use as dipping solutions to prevent fungal growth in dry sausages (Sofos. Contamination of many of these foods frequently occurs after processing. The treatment that was successful included addition of 0. Large outbreaks of infection have been linked to consumption of contaminated coleslaw. Chicken skin was inoculated with Salmonella spp. 1995). aureus. monocytogenes. Dock et al. 2002).3% or 0. coli failed to grow in either type of apple juice.. 1996.. A study was conducted to evaluate aqueous dipping solutions of organic acids or salts to control L. 1995).5% or 5% acetic acid. and wash solutions were evaluated for effectiveness in decontaminating the skin (Hwang and Beuchat.05% sodium benzoate followed by holding at 25°C for 6 hours and at 4°C for 24 hours. hot dogs. However. cheeses. milk. USE AS A POSTHARVEST FUNGICIDE Because of the long and successful use of benzoic acid in the processed food industry. Most research efforts have been directed toward E.5% lactic acid combined with 0. but benzoate was about two to eight times more effective than sorbate (Splittstoesser et al. respectively. 1994). 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. Survival of E. 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.. jejuni. coli O157:H7 in apple juice and apple cider with or without potassium sorbate or sodium benzoate has been investigated. The antimicrobial can be applied directly onto the product surface where cells of L. L. Unpasteurized apple cider has been implicated in several foodborne disease outbreaks caused by pathogenic microorganisms. Tompkin et al. especially in younger children and the elderly. Listeria monocytogenes is recognized as a foodborne pathogen of major concern to humans. No significant increase in the numbers of this pathogen occurred on bologna slices treated with 2. growth was inhibited by benzoate or sorbate (Koodie and Dhople. 5% sodium diacetate. vacuum-packaged bologna stored at 4°C for up to 120 days. the growth of some O157:H7 strains in apple cider was not affected by the presence of either preservative (Miller and Kaspar. and luncheon meats. coli O157:H7. coli O157:H7 in cider because of the severity of illness this pathogen causes. At 8°C. Both preservatives reduced the heat resistance of E. Benzoate was one of the most effective compounds tested in the presence or absence of ethanol.

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

11). and intraperitoneal routes (Sax and Lewis.. 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. Source: Adapted from Lueck (1980). synthesized in the liver. . both benzoic acid and sodium benzoate are moderately toxic by ingestive.7 (White et al. the second is catalyzed by an acyltransferase enzyme.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. TOXICOLOGY Several toxicologic studies involving different animal species have been conducted using both benzoic acid and sodium benzoate. 1989). 1964). (1909) and Dakin (1909) to conclude that sodium benzoate was not deleterious to human health.3–4. 1964). Hippuric acid.4–2. The overall reaction sequence proceeds as shown in Figure 2. Excretion of benzoic TABLE 2. is then excreted in the urine (White et al.10.0 g/kg body weight Results 50% mortality 100% mortality Subchronic Chronic 80 mg/kg body weight 3% of diet 4% of diet (as sodium benzoate) 1 g/day 12 g per 14 days 0.34 Antimicrobials in Food STORAGE AND HANDLING Sodium benzoate should be stored in a cool. However. 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.10 Toxicity of Benzoic Acid and Sodium Benzoate by Exposure Categorya Category Acute Species Tested Rat Guinea pig. Sax and Lewis (1989) reported data for several animal species based on the route of administration (Table 2. There is little danger of its being a toxicant or fire hazard under normal conditions of use. intramuscular. cat. In addition.b rabbit. Extensive human feeding trials conducted early in the twentieth century led Chittenden et al.7–4.0 g/kg body weight 1. Its formation can be increased greatly by administration of benzoic acid. The first reaction is catalyzed by a synthetase enzyme.. dry place. Benzoate does not appear to be accumulated in the body. These have been summarized by exposure category in Table 2. This product is not corrosive.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. Route of administration was per os. Containers should be kept closed as much as possible.

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

(1964). They were also mutagenic to the yeast mitochondrial genome but only in the presence of oxygen. it was excreted almost completely as hippuric acid after systemic administration. acid by this particular pathway was first reported by Dakin (1909). This may be related to differences in the ability of liver and kidney cells to carry out glycine and glucuronic acid conjugation (for review. 1991). 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.9%) of the absorbed benzoic acid was conjugated with glycine to form hippuric acid. Since the late 1970s. Different animal species differ in their ability to metabolize benzoic acid. However. Cell sensitivity to these acids was enhanced by aerobic rather than anaerobic growth conditions.. 1993). (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. 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. S. 80. cerevisiae was used as a model system for testing antioxidant or prooxidant properties of benzoic acid and other weak organic acid food preservatives (Piper. 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.7 Metabolism of benzoic acid by mammals. Griffith (1929) found that this mechanism accounted for 66% to 95% of benzoic acid fed in studies in which the quantities were far in excess of those that might be ingested from foods preserved with permitted levels of benzoate.. 1991). the route of administration significantly affected the efficiency of benzoic acid metabolism (Nathan et al. or 160 mg/kg) were given at least 1 week apart to 6 healthy male volunteers. 1990). . A single oral dose of 250 mg of benzoic acid was quantitatively metabolized to hippuric acid and excreted in the urine within 4 hours. 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. These results have been confirmed in several animal studies.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. These authors analyzed plasma concentrations of benzoic and hippuric acids and urinary levels of hippuric acid after oral doses of sodium benzoate (40. 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. Source: White et al. 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. Akira et al. The food preservatives were shown to have a strong prooxidant effect on aerobically grown yeast cells. In guinea pigs. 1991).. only a small portion (6. Inhibition was caused by accumulation of benzoyl CoA with a resultant depletion of free CoA and acetyl CoA. When applied topically. 1999). see Chipley.

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

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

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

1989. sorbates are considered effective food preservatives when used under sanitary conditions and in products processed using good manufacturing practices. especially ethanol. changes in the genetic material.2% w/v at 20°C). 1989). 1992). depending on differences in microbial types. some microbial strains are resistant to inhibition by sorbate or even metabolize the compound. potassium sorbate) of sorbic acid may find more frequent applications in foods because of their greater solubility in water. Under certain conditions.13 (Table 3. CHEMISTRY Sorbic acid is a straight-chain. especially in combination with reduced nitrite levels for a reduction in nitrosamine formation (Sofos et al. Sofos. The salts (e. alterations in cell membranes. Potassium sorbate may be manufactured as a powder or granules and. 1981. The solubility of sorbic acid is also higher in alcohols. molds. CH3CH=CH-CH=CH-COOH). 1986. 1989). 1980). The sodium salt has a water solubility of 32% (wt/vol). The carboxyl group of sorbic acid is highly reactive and results in formation of various salts and esters. Sofos et al. and . 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. 1989). has an antimicrobial potency of 74%. The molecular weight of potassium sorbate is 150. with a molecular weight of 112. sorbic acid and its more water-soluble salts. 1981.2).22. while the health effects of sorbate were reexamined (Sofos. stability. sodium. and inhibition of enzymes or transport functions (Sofos. 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. including yeasts. The solubility of sorbic acid (Table 3. or both. species and strains. and in other industrial applications (Sofos. however. Sofos. 1989.4-hexadienoic acid. Robach and Sofos. 1979d) by sorbate is believed to occur at the connecting reactions of the germination process. Good solubility (58.g.50 Antimicrobials in Food activity against microbial growth and the potential application of the compound in additional food products (Sofos and Busta. 1982). and bacteria.2% and is insoluble in fats. probably through action on spore membranes or protease enzymes involved in germination (Blocher and Busta. Currently. Robach and Sofos. and potassium sorbates. are collectively known as sorbates and are used widely throughout the world as preservatives for various foods. on an equivalent weight basis with the acid. 1989). and it constitutes the most soluble form of sorbate (Table 3.15 g per 100 ml. Sorbic acid forms colorless flakes or needles when crystallized. Inhibition of microbial metabolic function is probably the result of morphologic alterations in the structure of the cells. Commercially available salts include calcium.. 1981). 1982).. 1986). it is available as a white freeflowing powder or as granules. The volatility of the compound in steam is useful in its isolation for quantitative detection in foods or other materials. In general.. 1981. During the 1970s extensive research was performed on the potential for using sorbic acid and its salts as antibotulinal agents in meat products. as pharmaceuticals and cosmetics. as animal feeds. 1979a. and it has a weak but characteristic acrid odor and acid taste. the pH of the solution. Inhibition of bacterial spore germination (Sofos et al. The calcium salt is formed as a white odorless and tasteless powder. and in glacial acetic acid.. Sofos.. in inhibition of microorganisms by sorbates. and environmental factors. however.1). The extensive use of sorbates as preservatives is based on their ability to inhibit or delay growth of numerous microorganisms. 1985. substrate properties. The mechanisms of antimicrobial activity of sorbates are not fully defined (Sofos and Busta.2) in water at room temperature is only 0. trans-trans unsaturated fatty acid (2. There is variation. Sofos et al. especially potassium sorbate. but it increases with temperature. which makes it valuable as a delayed-release form of sorbic acid (Sofos. In food systems the solubility of the compound is estimated as higher than 50%. Calcium sorbate has a water solubility of 1. 1989.

. the partitioning of sorbic acid between the water and fat phases of foods depends on the pH of the food.5 10 3 0.927 — Potassium Sorbate 2.4-Hexadienoic acid CH3-CH=CH-CH=CH-COOH 112. °C Heat of combustion at 25°C. maximum ppm Arsenic content. acetaldehyde. maximum% Ash.Sorbic Acid and Sorbates 51 TABLE 3. the type and amount of fat.22 — — 1. Thus. The rate of oxidation in aqueous solutions is increased at lower pH values by the presence of light and acids or increased temperatures. 1988.001 10 43 Decomposes 143 119 >98 0. Ethylenediamine tetraacetic acid (EDTA) enhances sorbate degradation probably as a result of iron scavenging from the packaging material (glass or polypropylene) through EDTA complexation. and β-carboxylactolein (Arya and Thakur. Btu/Lb Alkalinity/acidity Vapor pressure.1 Physical and Chemical Properties of Sorbic Acid and Potassium Sorbate Property Chemical name Molecular formula Molecular weight Flash point. 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.13 126 1. malonic acid. mm Hg. whereas in the pure dry form they are stable. It is proposed that EDTA-Fe2+ complexes catalyze sorbic acid autoxidation.8 ml 0. and other ingredients present (Sofos. Sofos. the higher iron content in the EDTA-containing systems might be responsible for the higher rates of sorbic acid degradation as well as for the increase in nonenzymatic browning observed (Campos et al. 1989. The partition quotient is increased from 3.1 g — — — — — — >98 1. 1989). such as sugars and salts (Sofos and Busta.36 Decomposes higher than 270°C — ml 0. such as crotonaldehyde. °C (COC ASTM D-92) Ionization constant at 25°C Density at 20°C g/ml Melting range. maximum% Heavy metal content. malonaldehyde.73 ∞ 10-5 — 132–137 11. 1989). Oxidation and losses of sorbic acid are inhibited by antioxidants.1 N HCL per 1. 1992). 1992). % Water content. 1996).0 10 3 — <0. maximum% Source: From Sofos (1989). In general. appropriate packaging materials.4-Hexadienoic acid potassium salt CH3-CH=CH-CH=CH=COOK 150. acrolein. Sorbic Acid 2. and anaerobic conditions (Sofos. °C 760 mm Hg 50 mm Hg 10 mm Hg Purity.. 1981).0 to 7. Oxidation of sorbic acid yields various carbonyl compounds.2 ease of manufacture make potassium sorbate the most widely used form in food systems (Sofos. at 20°C 120°C 140°C Boiling point. which take part in nonenzymatic browning. .0 with increased levels (50%) of soluble food components. increasing the production of carbonyls. 1995).1 N NaOH to 0. Aqueous solutions of sorbates are unstable and degrade through oxidation. formic acid.

60–14. oxidative degradation of .11 0.04 11.25 0. 1986a.50 2. the nature and pH of the food.00 0. 20°C Cyclohexane.00 64.00 48.00 0. 20°C Acetone.30 Potassium Sorbate 58.00 — — — <0.00–15. types of food materials present.5% and 8.60 45.05 5.01 — — 0.00 0. 20°C (pH 4.02 0. 100°C Pentane. 50°C Water.30 6. 25°C Pentane.00 47.80 2.10 16. packaging material. 20°C Methanol. 10% solution Sodium chloride. 50°C Pentane. 1983.00 55.b.60 1. 20°C Corn oil. 50°C Propylene glycol.07 0. other additives present. 40% solution Sucrose. 20°C Carbon tetrachloride.0% it had a protective effect (Guerrero et al.14 24.20 — — 61. Sorbic Acid 0.00 20. 75°C Ethyl ether.0% enhanced sorbic acid destruction. processing conditions.08 2. 20°C (pH 5.00 5.15 0. 75°C Benzene. 20°C Cottonseed oil. 5% Ethanol.30 2.16 0. 100% Propylene glycol. 50% Phosphoric acid. and storage temperature and time (Bolin et al. 20°C Source: From Sofos (1989). 5% solution Sodium chloride. 1992. 60% solution Sodium chloride.30 0.01 0. Gerschenson et al.10 0..9) Water.00 — <0. 50°C Soybean oil. 50% Ethanol.01 58.00 0.50–12. 20°C (pH 3. 50°C Benzene.00 12.22 1.01 Losses of sorbic acid during storage of foods depend on sorbate levels. In general.2 Solubility (%) of Sorbic Acid and Potassium Sorbate Solvent Water.52 Antimicrobials in Food TABLE 3.10 0. 1990).52 1.29 — 12. 95% Ethanol. 20°C Glycerol.03 — 0.90 0. 1980..12 0.28 1. glacial Lactic acid. light.15 0.08 0. 85. metal ions.00 28.90–14. 20°C Sucrose. 1987.20 12.50 12.50 0. 15% solution Acetic acid. 1989. 25°C Benzene. 20% Ethanol. 100°C Corn oil. moisture content.26 0.15 0. amino acids. 20°C Propylene glycol.55 4. 10% solution Sucrose. 85% Ethanol.1) Water.00 — — — — 57. 1994).00–5.20 0. antioxidants. 1984.34 8. Vidyasagar and Arya.00 34.4) Water. but at 13.00 45. Sofos..80 0.40 54.31 9.02 0.5% Citric acid. Sodium chloride at 3.

and acetic acid-soluble fractions suggested that 14C activity tends to be associated with the low-molecular-weight components of these fractions. 1986a. they are shipped and stored in fiber drums with a moisture barrier in the carton wall. 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. 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. and a further loss is apparent after the dough is cooked to give the traditional West African food. the water-soluble (6. 1963. Vojdani and Torres. Because the compounds are sensitive to heat. is hindered by disadvantages. and the acetic acid-soluble components (0. 1989. and sorbic aldehydes were also effective antimicrobials (Troller and Olsen. sorbohydroxamic acid. 1992). fura. and water activity (Giannakopoulos and Guilbert.6%). 1995). Sofos and Busta.2) (Dudman.b. some of these compounds are sorboyl palmitate. The lipid and protein fractions that appear to bind sorbic acid or its degradation products were identified with the help of 14C-sorbic acid.2 ± 0. 1989). These cartons should be kept closed in cool. Uptake and diffusion of sorbate into foods should be influenced by properties of the substrate. Troller and Olsen. and amide derivatives.5±0. and sorbic anhydride. 1990). moisture. 1976). salt-. 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. Several procedures have been patented for the manufacture of sorbates (Sofos. In addition. 1963). Commercial application of such derivatives. the ethanol-soluble (4.6 to 9.9±0. physical state. The results show that sorbic acid binds with the lipid components. or solutions. In general. other derivatives of sorbic acid have also been examined. A mixed anhydride of sorbic and palmitic acids called sorboyl palmitate has found some application in yeast-leavened bakery products. and light. 1992). and ethanol-soluble components of fura from pearl millet. was an effective mold inhibitor over a wide pH range (3. handling.7%). 2000. methyl sorbate.. and storage conditions (Sofos.7%). Specifically. Sofos. aldehydes. the water-. sorbamide. alcohols. ethyl sorbate. moisture.5±0. a significant amount of sorbic acid is not recovered by extraction with methanol. however. Fractionation of the lipids and proteins from fura showed the 14C to be associated with the petroleum spirit crude fat extract (28±5%). such as low solubility in water. salt-. Commercial applications of sorbates include use of the free acid or its potassium and. strong offflavors and odors.. calcium or sodium (salts). 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. Dialysis of the ethanol-.Sorbic Acid and Sorbates 53 sorbic acid in foods is less extensive than in aqueous solutions. dry. 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. 1989). broadly tested. Charvalos et al.. but commercial production of sorbic acid has mostly involved the methods of oxidation of . Sorbamide was 1000 times more inhibitory than sorbic acid against yeast alcohol dehydrogenase (Martoadiprawito and Whitaker. Although the salts of sorbic acid have been commercially developed. 1980. and it is influenced by the nature of the food and its components and by processing. sorbohydroxamic acid. the salt-soluble (3. 1994). A proportion of the binding is with the low-molecular-weight components of these fractions (Jideani and Wedzicha. suspensions. which are available as powders. granules. These include esters. When wheat flour doughs are treated with sorbic acid and heated.8.. and widely used as antimicrobial agents.3%). and potential health problems (Lück. such as composition. New controlled-release. 1976). to a lesser extent. 2001). amine salts. This suggests that sorbic acid is indeed reacting with thiol groups in the flour protein. 1981). structure. and dark storage areas. 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. with a dissociation constant (pKa value) of 8.

1984. Marshall and Bullerman. Pederson et al. Sofos. 1998). fruits and juices.. Melnick and Luckmann. Trichoderma. 1979. 1954a. Geminder. Bhattacharya and Majumdar. Liewen and Marth.. Phoma. Penicillium. bread. Rhizoctonia. 1984.. 2000). 1992. Use of sorbates for inhibition of yeasts is especially important in low-pH and/or intermediate water activity (aw) products. Cryptococccus. such as carbonated beverages. Numerous studies have also documented the effectiveness of sorbates against molds (Emard and Vaughn. Cladosporium. Aly. A major application of sorbates in food products is their use for inhibition of molds in cheeses (Chichester and Tanner. Truncatella. Sporobolomyces. Helminthosporium. Rosellinia. Saccharomyces. lower than the inoculum level (103 CFU/g). Colletotrichum. Cephalosporium. Tsai et al. CO2 atmospheres. cottage cheese.54 Antimicrobials in Food 2. and smoked fish (Liewen and Marth. wines. 1996). were able to depress mycelial weight and aflatoxin production by Aspergillus flavus or Aspergillus parasiticus cultured in yeast extract-sucrose (YES) broth (Fan . Zygosaccharomyces rouxii is considered resistant to sorbate treatments. sorbates inhibit yeasts in fruit juices. 1952. Torulaspora.30%. Ascosphaera. 1988). 1970.. and chocolate syrup (Restaino et al. Sorbates inhibit the formation of mycotoxins by various molds in culture media and in foods (Bullerman. and other suitable salts in the presence of necessary additives.. Huang and Armstrong.. Pepularia. Piper et al. and meat and fish products. 1985a). Ulocladium.b. Gliocladium. 1985a. Heterosporium. Rhizopus. as well as against many bacteria (Sofos. 1985.. Endomycopsis. 1954a. Under this atmosphere and at aw values in the studied range (0. 1996).. salad dressings. after 21 days. use of a hurdle approach resulted in a synergistic effect (El Halouat and Debevere. Kivanç. Mucor. Under an 80% CO2 atmosphere.b. 1984. reaction conditions.b. Liewen and Marth. 1984. 1993. 1986. 1954a. Hansenula. 1979. 1954a. Aspergillus. neither sorbate nor propionate. Bracey et al. grains.. Rhodotorula. Botrytis. Cunninghamella. Melnick et al. 1970. syrups. Sporotrichum. 1985. In addition to their effectiveness in fermented vegetables. Although. Torulopsis. Candida. Hurdles used in combination included aw.. the inhibitory amounts of sorbic acid were reduced by 40% to 50%. dried fruits. Skirdal and Eklund. Kaul et al.90). Kloeckera. potassium sorbate used at 150 ppm resulted in final counts. 1983. Ferguson and Powrie. Pestalotiopsis. El Halouat et al. Mold species inhibited by sorbates belong to the genera Alternaria. and others. Numerous patents describing catalysts. 1961. The effectiveness of sorbates against yeasts has been documented by numerous studies (Emard and Vaughn. Pullularia. Humicola. However.80 to 0.02% to 0. candy. 1957. Lennox and McElory. 1959. Effective antimicrobial concentrations of sorbates in most foods are in the range of 0. 1989). Pichia. Alkaline salts of sorbic acid are formed by its neutralization with alkali metal carbonates. 1983. Huang and Armstrong. Fan and Chen. Fusarium. 1996. 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. 1999). Garza et al. applied at 10 mg/ml. Deuel et al. jellies. Curvularia.. Monilia. tomato products. Tong and Draughon. and Zygosaccharomyces (Sofos. and sorbic acid. Baldock et al. cakes. Smith and Rollin. 1972). jams. hydroxides. Yeasts inhibited by sorbates include species of the genera Brettanomyces. ANTIMICROBIAL ACTIVITY INHIBITION Sorbic acid is active against yeasts and molds. Debaryomyces.b. 1982.4-hexadienal and the condensation reaction of ketene and crotonaldehyde. Chaetomium. 1984. sausages. and purification procedures follow the second method. Ascochyta. Roland and Beuchat. which is most widely used. Geotrichum. 1999. Gourama and Bullerman. The inhibitory action of sorbate against yeasts was first documented in the 1950s in fermented vegetable products. 1989). Matamoras-León. 1998. whereas no growth was observed when the preservative concentration was increased to 220 ppm (El Halouat and Debevere. 1998. Sorbates also inhibit molds in butter. 1993. 1952. 1984.

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

energy-requiring system that transports the preservative out of the cell (Warth. cerevisiae to 0. Hansen and Appleman. Lahellec et al. 1983. Cole et al. 1989). 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. 1981.. the inhibitory action against lactics by sorbate is less than that against yeasts. 1996. most tolerated 150 ppm sorbic acid. 1985. 1984. 1987). however. and energy generation.. 1955. 1997). bailii in salsa mayonnaise more than sodium benzoate (Wind and Restaino. In addition to bacteria. rouxii in high moisture prunes (El Halouat et al. Torulopsis. Varying sensitivities of bacterial species and strains to sorbate may lead to shifts in the microbial flora during storage of foods (Chung and Lee. 1982. 1994). potassium sorbate suppressed growth of Z. Exposure of S. 1951. One proposed mechanism of resistance of osmotolerant yeasts has involved an inducible.. and previous exposure of the organism to low levels of sorbate (Sofos. which explains the usefulness of the compound as a preservative in vegetable fermentations. retarding the flow of sorbate into the cell (Restaino et al. 1982). various species and strains of microorganisms exhibit different sensitivities to inhibition by sorbate. Vaughn and Emard. Another bacterium that appears to be more resistant to inhibition by sorbate than other spore formers is Sporolactobacillus (Botha and Holzapfel. mating response. 1955). pH. 1982. In general. 1955. When the yeast cells have been previously adapted to sorbate in media containing glucose or sucrose. Splittstoesser et al. Restaino et al. Potassium sorbate or sodium benzoate resulted in complete inhibition of Z. 1971). Hamdan et al. confers resistance to the inhibitory effects of sorbic acid (de Nobel et al. which occurs during adaptation to sorbic acid. 40% tolerated 500 ppm. 1995).. 1983). 1982. sorbate concentration. 1982). and concentration of sorbate.. 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. However. such as polyols (Bills et al. 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. Other proposed mechanisms of yeast resistance to sorbate at reduced aw have been related to yeast cell shrinkage and decreases in membrane pore size. 1950. under certain conditions some species and strains of yeasts and molds are resistant to inhibition by sorbate. Candida. 1954.. a heat shock protein of S. In contrast. Kondaiah et al. Exposure of Saccharomyces cerevisiae to sorbic acid caused strong induction of two plasma membrane proteins. 1981.. transposon function.. 1952. 2001).. and Triganopsis (Warth. 1978. 1985. or protection of enzyme systems from inhibition by sorbate through production of compatible solutes. 1982.56 Antimicrobials in Food of packaging. Yeast strains resistant to sorbate belong to the genera Zygosaccharomyces. 1987. McMeekin et al.. 1977). Resistance of osmotolerant yeasts to inhibition by sorbate was acquired by preconditioning the yeast to sorbate (Bills et al. 1998). . York and Vaughn. Mihyar et al. 1998). Brettanomyces. 1989). Of 100 yeast strains isolated from spoiled foods and beverages. Emard and Vaughn. 1983. Blocher et al. 1982. Lynch and Potter. Lenovich et al.. 2001). Bills et al.... The induction of Hsp26.. 1988). Functional categories of genes that are induced by sorbic acid stress included cell stress (particularly oxidative stress). 1988. 1989). Overall.. inoculum level. one of which was identified as adenosine triphosphate (ATP)-binding cassette transporter (Pdr12). Saccharomyces...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. subsequent exposure of the cells shows little effect of solute type on sorbate resistance (Lenovich et al. storage temperature. Variations and resistance to inhibition by sorbate may lead to failures in preservation and defective food products (Sofos. Resistance of yeasts to inhibition by sorbate depends on species and strains. cerevisiae. 1977. Blocher and Busta... bailii tolerated 800 ppm of sorbic acid (Neves et al. and two strains of Z. other studies have reported either no effect or inhibition of lactics and clostridia by sorbate (Costilow et al.9 mM sorbic acid at pH 4... 1985). Piper et al.

Steels et al. Because it is metabolized like other fatty acids. other ingredients.1%) reduced the D50°C of E. In general. 1989. Sofos. sorbate yields 6. 2000). caused by ethyl sorbate. large inocula at high cell concentrations therefore require considerably higher concentrations of the inhibitor to prevent growth than do dilute cell suspensions. The MIC of sorbic acid increases with the size of the inoculum. dehydration of cultures. aw. such as concentration. 1989). 1954b. Finol et al. and environmental factors.3-pentadiene. and 2-ethoxyhexa-3. 1985a. although many molds are sensitive to inhibition by sorbate. It appears. Products of sorbate metabolism by molds include 1. Lück. It should be noted that 0. Like caproic and butyric acids. inoculum size.Sorbic Acid and Sorbates 57 DEGRADATION Animals and certain microorganisms can metabolize sorbate. 1981. plastic paint. 1985a–c). 1-ethoxyhexa-2. that there is no apparent relationship between sorbate resistance and the toxigenic properties of molds (Tsai et al. Sorbic acid (0. 1966.. 1985a). 1988). It appears that selection may occur in sorbate-treated cheeses for certain molds tolerant to the compound (Schroeder and Bullerman. and type of substrate (Sofos. certain strains are resistant and can metabolize the compound. pH. under certain conditions. These factors can act synergistically or be antagonistic and either enhance or negate the antimicrobial activity of sorbate (Sofos. 1989). using it as a carbon source.1% sorbate is usually sufficient to inhibit sensitive molds (Liewen and Marth. of which 50% is biologically usable. sorbate is completely oxidized to carbon dioxide and water. INTERACTIONS The antimicrobial activity of sorbates is influenced by compositional. Sofos.6 kcal/g.20%) sorbate levels (Marth et al. Bullerman.. coli O157:H7 from 36 to 5. some bacterial strains may also degrade sorbate under appropriate conditions. In addition to certain molds. Increasing the concentration of sorbic acid from 200 to 1000 mg/L in apple cider decreased the D50°C value of E. 1981.4-diene. 1985)... Mucor. 1986a. When sorbate levels are high.2 min. packaging.b. processing. and Geotrichum (Sofos.b). microbial flora. Sofos.18% to 1. The study showed that the resistance of Z... 1982). 1966. or binding of sorbic acid to dead cells. bailii resistance to sorbic acid activity. substantial metabolism of sorbic acid. amount of sorbate present. coli O157:H7 during storage . Mold strains of the genus Penicillium isolated from cheese treated with sorbate were able to grow and metabolize high (0. as a fatty acid through β-oxidation.. however. which is a volatile compound formed through a decarboxylation reaction and has a kerosene-like. Liewen and Marth.. 1980). A study found a pronounced positive inoculum effect of Z. 2000). This metabolism is mostly associated with lactic acid-producing bacterial strains present as high inocula in sublethal concentrations of sorbate (Crowell and Guymon. 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. Liewen and Marth. temperature. gas atmosphere. and other additives (Sofos and Busta. 4hexenoic acid. prior exposure to subinhibitory levels of sorbate. 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.. 1995). Some mold strains can grow and metabolize sorbate under certain conditions as detected in cheeses and fruit products (Melnick et al. which was not an artifact caused by insufficient growth time. 2000). Horwood et al. about a 7-fold increase in lethality (Splittstoesser et al..5-diene (Edinger and Splittstoesser. a geranium-like odor is usually associated with wines treated with sorbate and contaminated with high microbial loads. Fusarium. under normal conditions of alimentation. or hydrocarbon-like odor (Marth et al. 1989). 1989. In general. Other strains of molds that may degrade sorbate belong to the genera Aspergillus. 1954b. 1977. there is also evidence of some ω-oxidation (Deuel et al. 1975. Degradation of sorbate by lactic acid bacteria has been associated with geranium-type off-odors in wines and fermented vegetables. 1989). 1992).. Degradation of sorbate by molds depends on species and strains. level of inoculum.

1976.0. more than 50% of the inhibition was the result of dissociated sorbic acid (Eklund.64 min (Splittstoesser et al. 1955. however.. Sugar and salt.b). 1994). have reduced the synergistic effect of sorbate and heat on thermal inactivation of microorganisms (Beuchat. 1985a). the maximum pH for antimicrobial activity by most other common food preservatives is lower — for example. 1982. respectively (Sofos and Busta. however. Other food ingredients (e. Studies have indicated that the dissociated sorbic acid also had antimicrobial activity. 1990). This popular theory has been questioned. 1954a.. 1989.1%) enhanced destruction of E. coli O157:H7 cells at pH 3. where it is needed for microbial control (Oka. 1979a. 1980.5 is advantageous because it allows for their use in foods of higher pH values in which preservatives. 1989). 1959. Sodium chloride (1. Gooding et al. Skirdal and Eklund. Robach and Stateler. Liewen and Marth. 1980. In general. Hoffman et al. 1988). 1976. 1983). Sorbic acid (0. 1955. 1988). sorbates should be used to preserve foods processed using good manufacturing practices. In environments of pH higher than 6. 1990). Not only are certain strains of microorganisms resistant to inhibition by sorbate.. sorbates have the advantage of being effective at pH values as high as 6. 1996). Increased amounts of fat in a product reduce the concentration of sorbate in the water phase. certain studies have indicated antimicrobial activity by sorbate at pH values as high as 7. 1983. and it may inhibit the repair and growth of . 1993). Chung and Lee. Cerruti et al.. but it was 10 to 600 times less inhibitory than the dissociated acid (Eklund. 1983). Cerruti et al.5 and 4. 1976.. 1982).0 to 5. Sofos et al. Sofos and Busta. 1981. Although activity is greater at low pH values.. 1984. 1981... Sofos and Busta. 5. 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. as well as dormancy and recovery of heated microorganisms (Sofos. Use of artificial saltwater prevented dissociation of the 1% sorbic acid.0 (Raevuori. 1960). 1955.. might not be effective owing to their increased solubility in fat. The increased activity of sorbates at pH values higher than 5. Lück. Interactions of sorbate with heat may affect the rate and extent of microbial destruction during heating. glucose. 1955. 1981. 1982. however. such as parabens. which exhibited favorable antimicrobial properties against Vibrio vulnificus. 1980. 1997). not as a substitute for appropriate sanitation and hygienic practices.5 (Bell et al. 1981a–c.2 minutes. Beuchat. supporting previous evidence that sorbic acid is effective in its undissociated form (Sun and Oliver. 1981....g.58 Antimicrobials in Food of apple juice from 18 to 5. In contrast. 1944.1% sorbate rendered the cells more sensitive to inhibition by sorbate than preconditioning in 0% sucrose + 0. Sucrose. 1993). Lück. 1989. 1976. 1956.0 to 4. Acott et al. Sofos and Busta. Sheneman and Costilow. act synergistically to enhance the antimicrobial activity of sorbate (Costilow et al. Sofos. Rahn and Conn. salt and sugars) also reduce the concentration of sorbate in the aqueous phase (Gooding et al.76) (Cowles.25 and 2. 1944. 1981).1% sorbate (Bills et al. Lowering the aw enhanced the resistance of the same organism to increasing concentrations of sorbate (Restaino et al. 1980. Sorbate may enhance heat activation and destruction of spores. The antimicrobial action of sorbate is pH dependent and increases as the pH of the substrate decreases.. In certain instances. Statham and McMeekin.4 but not at pH 6. solutes should increase the inhibitory activity of sorbate by reducing the aw of the substrate (Sofos. 1981c). and sodium chloride. however (Sofos.. however. Thus. whereas benzoic acid reduced it to 0.4 (Liu et al. 1941. 1988). but increased levels of microbial contamination reduce antimicrobial activity. Lund et al. Statham and McMeekin. 1987.. approaching its dissociation constant (pKa = 4. Preconditioning of Saccharomyces rouxii cells in 60% sucrose + 0. Cerruti et al.. 1981). it is believed that both undissociated and dissociated sorbic acids have antimicrobial activity (Skirdal and Eklund.5%) reduced the inhibition of Clostridium botulinum by sorbate in a nutrient broth (Wagner and Busta.. which is believed to be the effective antimicrobial form (Lück. 1993). 1985a. 1989). Nevertheless.5 for propionate and benzoate. 1957. The increased antimicrobial activity of sorbate at lower pH values has been attributed to the increased amount of undissociated acid present. Sofos and Busta.

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

.93.. Nelson et al. Ough and Ingraham.5. 1994). Wagner and Busta. Kadam et al. Several studies have also indicated increased antimicrobial effects when sorbate was combined with various phosphates (Ivey and Robach. 1978..4 and addition of 250 ppm ascorbic acid. As indicated.8..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. sorbate was more effective than benzoate against S. Robach.. and S. in a medium containing 0.. Sorbate was completely effective against E.. 1986a. and aw 0. 1995). 1981. 1994). certain amino acids. 1982. the log probability of growth of a single bacterium at 12°C after 60 d at pH 5. Interactions and increased antimicrobial effects have also been observed in combinations of sorbates with propionate. and mild heat (Guerrero et al. aureus. The ability of C. Mendonca et al. 1993). Amano et al.06%) combined with aw values lower than 0. At temperatures of 12°C and below. cereus.98. sorbic acid resulted in marked inhibition. 1960. Bell and De Lacy. mixtures of sorbate with various antibiotics have demonstrated increased antimicrobial activity (Schmidt. 1957... pH.. 1979b. interactions between two or more of these factors resulted in marked inhibition with the inhibitory effect of potassium sorbate occurring at the highest concentration (0. 1993). 1994). These combinations offer the advantage of simultaneous inhibition of microbial growth and development of rancidity. Marshall and Bullerman. 1985). except nitrite when used against S. However. and substrates. 1983. 1983. The microbial stability of shelf-stable banana puree was enhanced through inhibition of inoculated osmophilic and nonosmophilic yeasts.. Seward et al. 1989). A study found that growth was not inhibited at pH 3. at aw of 0. Numerous publications and patents describe interactions of sorbate with many other factors. sucrose fatty acid esters. Clostridium pasteurianum. 1982. sodium chloride concentration.. verocytotoxigenic Escherichia coli. 1988). Guerrrero et al. propylene glycol. 1981. 1988). and potassium sorbate was evaluated against growth of three foodborne bacterial pathogens (Bacillus cereus. Robach et al. Increase in the acidity and/or the NaCl concentration improved the effect of all the preservatives. After 14 days at 30°C in the presence of 280 mg undissociated sorbic acid/L. the probability of growth was approximately –6 (Lund et al. 1968. glucose oxidase. 1999).97 and pH to 3. Lahellec et al. molds. coli at all temperature/pH/NaCl combinations and was the most effective preservative tested against B. Morad et al. TellezGiron et al. and other compounds (Ferguson and Powrie. 1990). these concentrations of chemicals represented a greater than 50% reduction of the amounts needed to suppress growth when used individually (Matamoras-León et al.. Poerschke and Cunningham. however. 1984. Z.60 Antimicrobials in Food et al. pH 3. 1994). The effect of temperature. aureus) using gradient gel plates (Thomas et al. 1987. 1986. antioxidants.. sulfur dioxide.. fatty acids.. aureus when used with higher concentrations of NaCl (Thomas et al. the log probability of growth of a single vegetative cell is approximately –4. Sofos. with types of microorganisms. 1993. bailii is highly resistant to individual inhibitory factors. 1994. and Clostridium butyricum by adjustment of aw to 0. Deak and Beuchat. Miller and Brown.. At <25°C. 1989. Gourama and Bullerman.06% potassium sorbate. 1990). or at 10°C (Deak and Beuchat. 1987. 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. . Thomas and Wagner. 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. Variations existed.. including sorbate. 1985. 1960. 1999). 1981. 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. 1984.95 (Deak and Beuchat. Bacillus coagulans. Thomas et al.. Combinations of sorbate with benzoate or propionate may be used to expand the range of microorganisms inhibited with reduced concentrations of each preservative. ascorbate. Kabara. Potassium sorbate was found more inhibitory against yeasts than hydroxycinnamic acid (Stead. equivalent to a total sorbic acid concentration of 1300 mg/L. 100 ppm potassium sorbate. 400 ppm sodium bisulfite. In addition. as well as multifactorial interactions (Sofos.

purees. icings. fruit and vegetable products.3% are tolerated.05–0. and technical preparations that come in contact with foods or the human body. including dairy products. sanitation and contamination. sorbates have found wide application in various foods. juices. jellies. salad dressings Meat and fish products: smoked and salted fish. As yeast and mold inhibitors.1% to 0. pharmaceuticals. confectionary Source: From Sofos (1989). In general. spraying or immersing the food material in a solution. 1989.4). cake mixes. animal feeds. In the United States. processed cheeses.1% may be detectable in some foods. bakery items. In the United States. olives. cheese spreads. fudges.30 In general. mixes.3). semimoist pet foods. Levels (%) 0. and storage temperature (Sofos and Busta. fillings. and sugar and confectionery items (Sofos. low-calorie drinks Food emulsions: mayonnaise. They should be considered as adjuncts to proper sanitation and hygiene. packaging. presence of other ingredients and preservatives. equipment available. DAIRY PRODUCTS Cheese preservation is one of the most common applications of sorbates.3 Common Applications of Sorbates as Antimicrobial Agents Products Dairy products: aged cheeses. Selection of the most appropriate method depends on processing procedures. but levels as low as 0.Sorbic Acid and Sorbates 61 TABLE 3. edible fat emulsion products. gas atmosphere. amounts of 0.30 0. Although these concentrations have no major impact on food quality. higher levels may cause undesirable changes in the taste of most foods.F.10 0. federal standards of identity permit the use of sorbates as preservatives in more than 40 types of cheese. yogurt Bakery products: cakes. 1989) (Table 3.02–0. cosmetic products. margarine. These factors include product pH and composition (fat and moisture). packaging materials. fresh salads Fruit products: dried fruit. dusting with a powder. objectives to be accomplished. relishes. cheese dips. APPLICATIONS Sorbates are widely used food preservatives throughout the world. sour cream. Sofos. The compounds find application in human foods of all types.3%.30 0.05–0. cottage cheese.20 0. pies. pickles. doughnuts Vegetable products: fermented. syrups.02% to 0. . jams.05–0.02–0. fruit salads. However. and convenience (Sofos.05–0. concentrates Beverages: still wines. as is the case with all food preservatives. Amounts of sorbate used in foods are in the range of 0. dry sausages Miscellaneous: dry sausage casings. 1981. and cheese food [21 C. 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. toppings. or addition in a coating or packaging material (Table 3.10 0. 1989).R. Application methods for sorbates include direct addition in the formulation.03–0. 1992). fruit drinks.03 0. the extent of the antimicrobial activity of sorbate under commercial conditions is difficult to predict because it is influenced by a variety of factors. cheese spreads. product processing. certain meat and fish products. carbonated and noncarbonated beverages. 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. The most commonly used forms include sorbic acid and the potassium salt. 1989).02–0. type of food.25 0. Part 133 (2001)].

relishes.2 0.075 0. where mold development usually occurs. spreads Cottage cheese Bakery: icings. especially surface mold growth during storage.g. in general. Sorbates are widely used in preserving cheese owing to the susceptibility of all types of cheese to microbial deterioration. Gruyere. It is obvious. however (e. there may develop a slight objectionable flavor (Aly.04 0.05–0.1–0.15 0.05–0. Addition of sorbate does not significantly affect the organoleptic properties of mozzarella cheese.1–0. the high activity of sorbates against molds.05–0. For application on .02–0.3 0.06 0.1 0. and distribution. such as Roquefort or blue cheese and Camembert cheese. beverages. Kivanç (1992) found that Penicillium was the major genus found on cheddar cheese.. fillings. 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.1–0. Colby.1 0.05–0.1 0.05–0. toppings.075–0. In certain products.3 0. 1980. slaw.3 0. In these applications sorbates not only prevent cheese spoilage by mold. and regulations. and domestic and imported cheeses. pasta filata.25 0.1 0.05–0. depending on the technique used to treat the cheese (i. cottage cheese).62 Antimicrobials in Food TABLE 3.3 0.1–0.1 0.05–0.1–0. available facilities and equipment.1–0.15 0. is limited to the surface of cheese.5–10 20–40 20–40 2. Siciliano.5–5 5 2.1 0. however. Monterey Jack. pickles. vegetable. preservation objectives. dipping or in brine salting). Emmentaler Dairy: cheddar.4 Methods of Application of Sorbates Methods Immersion Products Dairy: provolone. but they also protect human health by preventing the formation of toxic mold metabolites (mycotoxins). in which they can interfere with the desirable molds involved in the manufacture of these cheeses. sorbic acid may be incorporated into the cheese curd.1 0. sauerkraut) Fruits: syrups.06 0. Edam.03–0. Sofos and Busta.05–0.1–0. Sorbate application. that sorbates should not be used in mold-ripened cheeses. olives. Caciocarallo.15 0.e.3 Spraying Immersion or spraying Dry mixing Direct addition Dry mixing or direct addition Source: From Sofos (1989). drinks Still wines Other: Margarine Semimoist pet food Bakery: pound cake Devil’s food cake Chocolate cake Solution (%) 20–40 2. and the improved action of sorbates at high pH values compared to other common preservatives (Lück.4 0.05–0. fudges Vegetables: fresh salads (potato..05–0. aging. 1976.02–0.3 0. 1996). Gouda Dry sausage or casings Dairy: Swiss.3 0. which may vary among countries (Lück. blue. Muenster. The method of application varies with the type of cheese.5–5 5 Application Level (%) 0.01–0.075 0. 1981).025–0. 1976.3 0. however. 1980). Swiss cheese.05–0.

Calcium sorbate may be preferred in cheese preservation over other forms of sorbate owing to its solubility properties. addition of potassium sorbate may increase the meltability and improve the fat leakage of certain cheeses (Aly. 1994). and was especially effective against mold and yeast contamination by Penicillium roqueforti and Mucor miehi. potassium sorbate was added to the cheese (pH 6. or wash. including fresh. one of the following procedures may be followed: (1) potassium sorbate in solution or in a brine. The use of sorbates in the processing of vegetables was one of the first applications. initial numbers of yeasts were low and relatively low concentrations of potassium sorbate and sodium benzoate (<150 and <250 mg/kg. amounts of 2 to 4 g/m2 are used (Lück. fermented. Treatment of mozzarella cheese with potassium sorbate in the kneading water or brine may be more effective than dipping (Aly. Growth of Salmonella in soft cheese was inhibited if the cheese was prepared from milk that was acidified with propionic acid to pH 5. Labaneh with higher initial yeast counts needed higher concentrations of preservative (>300 and >400 mg/kg for K-sorbate and Na-benzoate. Sofos and Busta. respectively) to maintain stability when kept at 5°C for 7 or 14 days.0 with hydrochloric. 1996).07% sorbic acid are generally used in direct addition to cheese. 1996). monocytogenes survival in two commercial cheese brines (Larson et al. respectively) extended the shelf life by 14 days. which can be used to dip. bulgaricus. This research demonstrated the effectiveness of sorbates against undesirable organisms without interfering with the desirable . Addition of potassium sorbate (≤6%) to mozzarella cheese inhibited growth of Streptococcus salivarius var.. 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. potassium sorbate inhibited L.3%) showed a significant effect in delaying growth of E. whereas when applied to packaging films. The appearance of Penicillium verrucosum var. or propionic acid significantly increased the effectiveness of potassium sorbate. and the cheese was stored at ≤30°C (Kasrazadeh and Genigeorgis. 1995). potassium sorbate (0.Sorbic Acid and Sorbates 63 the surface of the cheese. ripened cheeses. These concentrations are in agreement with use of these preservatives in other dairy products.. and 25°C after 30 days (Kivanç. and pickled vegetables (Sofos.30% (Lück. wax) used in cheese storage and marketing (Han et al.. 1997). 1989). Levels of 0. 15°C.3%.3 g/dm2. acetic. however. Therefore.3%) or sodium benzoate (0. Furthermore. monocytogenes (Piccinin and Shelef. In another study. coli O157:H7 in queso fresco. 1995). Concentrations applied to cheese surfaces range from 0. 1980. soft cheese (Kasrazadeh and Genigeorgis. Sofos. The compound is insoluble in fat and only slightly soluble in water. when applied to the surface of hard. 1996). 1998. or calcium sorbate impregnated into the packaging material or other coatings (e. 1980). 1994). and (3) sorbic acid. Milk acidification to pH 6.g. at the relatively high level of 1%.1 to 0. Absorption of the compound from the surface into the cheese is dependent on the porosity of the surface and the amount and distribution of fat in the cheese. such as cottage cheese and yogurt (Mihyar et al.. In labaneh (a yogurt product) produced under strict hygienic processing measures and effectively chilled. 1976).9. 1981). In addition to reducing cheese spoilage and the possibility of aflatoxin formation. Inclusion of sorbic acid in cottage cheese was effective as a preservative but not against L.6°C) temperatures (Kasrazadeh and Genigeorgis. Weng and Chen. especially at lower (≤6.0) at levels of ≥0.05% to 0. VEGETABLE PRODUCTS The water-soluble salts of sorbic acid are used widely in the preservation of a variety of plant products. 1999).05% to 0. 1992). 1997). 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. use of sorbates in aged cheeses also reduces labor and other costs through reduction of the need for cheese washing and trimming. thermophilus and Lactobacillus delbrueckii var. (2) a sorbic acid powder for dusting. spray. respectively (Aly. 1989. and it was initiated through extensive research performed during the early stages of sorbate testing. Amounts of sorbic acid permitted and applied in cheese preservation range from 0. potassium.

. Selection of precise levels of sorbate. preserves.05% to 0. 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. or microbiological deterioration (e. sorbate acts as an inhibitor of yeasts. 0. In fruit juice and drink processing. Use of the combination of 0.g.. 2001). fermentation).02% to 0. sorbate is either added directly into the product or applied to the surface of the product or packaging material. 1993). Other fruit products that may be preserved with sorbates include maraschino cherries and strawberry puree.1% sodium benzoate and 0.g. In sweet relishes. 1980). fruit cocktails. Costilow et al. Addition of 0.02% to 0. Combinations of sorbic acid and sulfur dioxide are also used in the preservation of high-pulp fruit juices. or bacteria.02% to 0. 1988a. sorbate is more effective than benzoate owing to the pH of these products. and putrefactive bacteria.10% sorbate reduce the number of bloaters and poor seed cavities. prunes. coli O157:H7 and result in spoilage of the product (Zhao et al. coli O157:H7 growth was inhibited in both tryptic soy broth and in apple cider by sorbic acid applied at 0.02% to 0.. in high-sugar products (e. 1952.g. together with sulfur dioxide and pasteurization.g. At these concentrations the selective action of sorbic acid allows growth of the desirable lactic acidproducing organisms.10% may be used to preserve refrigerated fresh salads. while additionally suppressing the growth of yeasts and molds.. Use of sorbates at levels between 0. 1980).05% and 0. The lower the moisture content of the product (e. molds. cucumbers and olives). Mold and yeast spoilage is also prevented by sorbates in pickled vegetable products (e. coli O157:H7 in apple cider.05% are adequate for preserving high-moisture dried fruits (e. and wines. which may influence the survival of E. salt concentration). Potassium sorbate and sodium benzoate were highly effective in reducing yeast and mold populations in tomato juice (Bizri and Wahem.64 Antimicrobials in Food fermentations. smaller quantities of sorbic acid are adequate for preservation because of the synergistic action of sorbate with sugar (Lück.05% and as such may be an option that processors have to reduce contamination in their products (Koodie and Dhople. 1980). 1972. Levels of 0.10% are adequate in improving the preservation of soft drinks. sorbates are used mostly at the preprocessing stage. jellies.02% to 0. the pH values of fermented green olives favor the use of 0. In these products. but it makes them susceptible to mold and yeast spoilage. 1955. Also.1% sorbate and 0.5% acetic acid completely inhibit microbial spoilage at sucrose concentrations ranging from 2% to 40%. jams. The higher moisture content in these products is preferred by the consumers. however. Other vegetable products preserved with sorbates include tomato products and fermented Asian sauces (Chichester and Tanner.. fruit juices and syrups.05% sorbate to a 15% to 20% brine retards yeast scum formation. 1989). dried fruits). sodium sorbate may be used to avoid the problem. enzymatic.. Potassium sorbate is preferred because of its solubility. Sorbate levels in the range of 0.1% potassium sorbate substantially inhibited E.g. Because potassium may result in potassium bitartrate precipitation in some wines. Sofos. Also. Another study showed that E.02% sorbic acid in combination with . FRUIT PRODUCTS Fruit products preserved with sorbates throughout the world include dried fruits. Depending on the particular product. Sorbate concentrations as low as 0. Good wine preservation is achieved with concentrations of 0.. such as yeasts. 1980. to inhibit chemical.b). molds. In wine processing. the lower is the sorbate level needed for preservation.g.20% inhibits growth of organisms not desired in vegetable fermentations. beverages. In these products sorbic acid acts as the microbial inhibitor and sulfur dioxide prevents oxidation and enzymatic spoilage (Lück. 1994). jams and jellies).05% sorbates in the fermenting and holding tanks. raisins. figs). 1994).. Lück. Lück. depends on products and specific fermentation conditions (e. Potassium sorbate was found more effective than chitosan in inhibiting growth of Aspergillus niger on lowsugar candied “kumquat” (Fang et al. sorbates are used to prevent refermentation (Parish and Carroll.. however. In sweet cucumber packs. Concentrations as low as 0.

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

Wagner and Busta. 1980b). eating quality. 1989. and the National Academy of Sciences (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. 1979a. Petaja et al. 1979a. 1974). Morad et al.b. comminuted pork products (Ivey and Robach. Amundson et al.. Serratia liquefaciens. 1987a. Elliot et al. 1985. Bacillus cereus. Myers et al.. 1989) and cooked pork chops (Prabhu et al. Chambers et al. 1980a. Nelson et al. 1982. 1964. Yersinia enterocolitica.. Robach and Ivey. Greer.. and increased carbon dioxide atmospheres (Sofos. that no other studies reported undesirable flavors or allergic reactions (Sofos. 1978. 1979. Cunningham. raw (Mendonca et al.. Vareltzis et al. Sofos et. uncured cooked sausage (Tompkin et al. Gray et al. 1984. 1979. 1981. 1981. 1977. 1978. Ivey et al. Products in which these microorganisms are inhibited include bacon and other cured meats.. Nitrite was reported to be a potential carcinogen. Clostridium sporogenes. 1983)... These studies have been thoroughly reviewed by Sofos and Busta (1980). 1980c. the effect of sorbates on overall product shelf life.. Perry et al. 1982). Pseudomonas. 1982. Robach et al. botulinum. botulinum in cured meat products. 1980. 1979b). 1983... 1981. To and Robach. Huhtanen et al. 1982. Tompkin et al. 1979a. aureus and delayed growth and toxin production by C. 1980a. and uncured poultry meat and carcasses. 1981a. 1961. 1981. Wagner et al. Lactobacillus. raw beef and pork. 1990). Department of Agriculture. 1974. Clostridium perfringens.. Brochothrix thermosphacta. uncured.. 1983. cured poultry products.. aureus. antioxidants. Bushway et al. 1974) was followed by extensive testing that examined the action of sorbates against clostridia and other pathogens in a variety of fresh... 1989). 1981. 1980b. Paquette et al. phosphates. Hall and Maurer. mesophiles. This work (Tompkin et al. Robach and Sofos. other pathogenic and spoilage bacteria inhibited by sorbate in various meat products include S. poultry products (Robach et al.S. beef steaks (Unda et al. 1983). Vareltzis and Buck. 1979b.b. botulinum spores during temperature abuse of the product. U. Based on the results of the various studies. Robach. 1981). low oxygen. National Academy of Sciences.. Draughon et al. such as color and flavor (Frank. pork slurries (Roberts et al. 1984). 1981).. 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. 1983. McMeekin et al. It should be noted. Robach and Sofos (1982). low storage temperature. 1978. 1978.b. Robach et al. 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. 1982. low pH. In addition. In addition..S. Wagner and Gehrke. 1986b. 1982.b). The antimicrobial activity of sorbate in these products has been enhanced when combined with nitrite.. National Academy of Sciences 1982). and it can react with amines to form carcinogenic nitrosamines during meat product processing and cooking (Sofos et al. 1983. 1979.66 Antimicrobials in Food showed that sorbate retarded growth of Salmonella species and S. Huhtanen and Feinberg. 1980. (1979a). Rice and Pierson.. 1980. Berry and Blumer. This proposal.. psychrotrophs. Kemp et al.. however. Escherichia coli... sodium chloride. did not take effect following a report that consumption of experimental bacon resulted in allergic-type reactions in certain individuals (Berry and Blumer. 1984. 1979. U.. 1982. 1978.. Sofos et al.c.. 1982.. Sorbate in meat products (<0. al. 1988). botulinum. Pierson et al.. 1978. 1979b. 1982. nitrosamine formation.. Zamora and Zaritzky.3%) had no major adverse effects on sensory qualities. Sofos et al. 1989). Sofos.. and lipolytic organisms (Kaloyereas et al. 2000). Sofos (1981. The antimicrobial activity of sorbate was demonstrated in bacon (Ivey et al. Hallerbach and Potter. 1979.. Salmonella. 1979b.. and processed cured meat products (Sofos. 1981.. In addition to C. Chang et al. Bauermann. Sofos et al. 1980b).. beef and pork frankfurters (Sofos et al. 1984. 1983).... 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. and hamburger beef (Yamamura et al. 1982).. and safety were examined.. however. 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 . Berry et al. Robach. acids. sliced bologna (Chang et al. Bacillus licheniformis.

1959. MISCELLANEOUS FOOD PRODUCTS Sorbates are commonly used in the preservation of oil-in-water or water-in-oil emulsion-based products. 1999). Robach and Hickey. such as benzoates. are also effective in preventing mold growth on animal feeds during storage. 1995). 1976. inhibiting their growth. In addition. and similar products.05% to 0. 1972. the esters of sorbic acid (e.. depending on moisture content. 1983. such as margarine. cut squash. Several studies have reported improved preservation of various types of fish with sorbate (Geminder. Sorbic acid levels in the range of 0. OTHER APPLICATIONS Other items that may be preserved by sorbic acid and its salts include pharmaceuticals. tangerine sherbet base. cucumbers. Mexican-type hot sauces. shredded cabbage. Bremner and Statham.g. potato chips. 1983. at which they prevent growth of molds and osmophilic yeasts in items such as fillings for chocolate and pralines (Chichester and Tanner. Statham and Bremner. Debevere and Voets. Sorbates may be particularly important in intermediate-moisture (25% moisture content) pet foods. Shaw et al. A sorbate (0. thus. diastatic powers. it may be desirable to apply sorbic acid during malting (Ogundiwin et al.30% can be effective mold inhibitors. 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). 1976. garlic oil. 1988). In certain instances and for better preservation. The literature contains reports on several other food products that may be preserved with sorbates (Sofos. and cold and hot water extracts of the malt samples. delicatessen items. Lynch and Potter. Sofos and Busta. 1989). salad dressings. This should assure mycotoxin-free animal feeds and thus eliminate any hazards associated with mycotoxin-contaminated feed. Sorbic acid at a concentration of 0. Robach and Sofos. Regenstein. Sofos.1%) was used (Fletcher et al.. They include high-moisture marshmallow candy. botulinum in shucked scallops developed slightly more rapidly at 27°C in vacuum packages when sorbate (0.10%. 1982). ethyl and propyl) may find applications in cosmetics and pharmaceuticals.5% did not adversely affect the germination energies and germination capacities of sorghum grain and did not significantly affect malting loss.20%. 1991). 1978. In Asian countries sorbates are commonly used in combination with other preservatives to preserve fish sausages and similarly processed fish and meat products. Fey and Regenstein. 1989).Sorbic Acid and Sorbates 67 of bacon treated only with nitrite. 1981).1%)–benzoate (0. Lück. This optimal concentration of sorbic acid coincided with the lethal dosage for most of the associated fungal microorganisms during malting. 1972. cosmetic products. 1978. Lück.1%) solution preserved brined shrimp for 59 days. 1983. 1989). 1982. orange peels. 1982. 1972. the flavors of both types of bacon were equally desirable (National Academy of Sciences. in which levels of up to 0. which is longer than the preservation (31 days) achieved by the most effective bacteriocin (nisin) tested (Einarsson and Lauzon. indicated that toxin production by C. however. 1980. as well as fermented plant foods (Chichester and Tanner.. Chung and Lee. Bremner et al. Other food-related applications of sorbates include sugar-based and confectionery products at concentrations of 0.g. and animal feeds (Sofos. 1986).. in which the emulsifiers may be inactivated by nonionic compounds (Chichester and Tanner. dried cod). pineapples. sorbic acid may be mixed with its potassium salt or with other preservatives. tomato juice. One study. Application of potassium sorbate with bifidobacteria extended the shelf life of whole and peeled shrimp by 3 days (Al-Dagal and Bazaraa. and pancake batter (Sofos. strawberry puree.03% to 0. Lück. Samples treated with sorbic acid lost biological activity within a few days of aerobic storage (confirmed by aerobic . 1976).. sour cream. 1980. Sharp et al. 1982. 1972. especially those of the emulsion type. 1981. prepeeled carrots.. mayonnaise. 1989). Sorbates are also used to prevent mold growth on the surface of dried and smoked fish (e.. 1983.

. 1982. 1976...b). molds. with bulbous formation and defective division (Seward et al. Death of microorganisms exposed to high concentrations of preservatives.. 1989). Ronning and Frank. and function of cell membranes and inhibition of transport functions and metabolic activity (Sofos. Published data have suggested that sorbate acts as a competitive and reversible inhibitor of amino acid-induced germination (Smoot and Pierson. the surviving microorganisms may resume growth and spoil the food. 1986.1% sorbic acid to citrus pulp were effective in inhibiting yeasts. integrity. Such changes have been observed in yeast cells as dense phosphoprotein granules. there was no detection of pectinolytic activities (Mayer and Hillebrandt. Several studies have indicated that sorbate inhibits bacterial spore germination (Sofos et al. irregular nuclei. 1981). 2001). Other reported uses of sorbates include preservation of tobacco products. 1989). 1986. when used individually or in combination as preservatives of an artificial diet for leafcutting bees. Sofos et al. strains. Blanching (80°C) and the application of 0. when present. There was also evidence of other membrane damage in sorbate-treated cells (Statham and McMeekin. 1988). the outer cell wall was absent in many areas (Ronning and Frank. Although the significance of such alterations is unknown. pH. 1981. 1979d. and not triggering of germination. 1986.. 1982. which could be overcome to some extent by addition of magnesium ions. 1978. Seward et al. 1985). Sofos et al. 1986). MECHANISMS OF ACTION Sorbate concentrations used in foods (<0. Treatment of Alteromonas putrefaciens with sorbate at pH 7. Freese and Levin. The preservatives sorbic acid and methyl paraben. type of substrate.. increased numbers and variable sizes of mitochondria. and vacuoles. 1989). reduced ensiling losses and increased the feed value characteristics of citrus pulp silages (Filya et al. Septation.. 1985. whereas higher amounts may result in death. and sorbate concentration (Sofos. Sofos. 1985a. and clostridia common in the spoilage of silage and. and the inner cell wall appeared to be thickened. It was also postulated that sorbate probably inhibits a postgerminant binding step in the process of germination. Sofos. such as sorbate. 1997). 1989). Sofos. environmental conditions. and the type of food processing..68 Antimicrobials in Food degradation of starch and triglycerides). therefore.0 increased cell hydrophobicity and cell wall lysis on exposure to lysozyme. resulted in minicells. Consequently. but the exact mechanisms of antimicrobial activity are not well defined (Freese et al. and it was thus concluded that it inhibits spore commitment to germination. 1989). Sorbate-treated cells of C. and it may be possible that several of them may be functional under various conditions. including types and species of microorganisms. and glues (Lück. has been attributed to generation of holes in the cell membrane . Although the presence of enzyme activities was shown. Another study reported that sorbate inhibited spores triggered to germinate or after germinant binding (Blocher and Busta. Under certain conditions sorbates have changed the morphology and appearance of microbial cells (Statham and McMeekin. sporogenes were usually filamentous and nonseptate but with distorted shapes characterized by numerous bends and bulges. botulinum were long. 1989). Sofos. adhesives. when the sorbate hurdle is reduced or removed. Sorbate inhibits cell growth and multiplication as well as germination and outgrowth of spore-forming bacteria. 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. Cells of C. did not influence the susceptibility of the bees to the fungus Ascosphaera aggregate (Goettel and Duke. 1973. 1996). Inhibition has involved various species of bacteria in laboratory culture media and in foods and has been influenced by species. 1985). enterobacteria. Other proposed mechanisms of inhibition of microbial growth by sorbate include alterations in the morphology. Wagner and Busta. Smoot and Pierson. Several mechanisms of inhibition of metabolic function by sorbate have been proposed. 1989. Blocher and Busta.3%) usually inhibit microorganisms. 1989). they may be the result of the incorporation of sorbate into specific cell structures and alteration of biosynthetic processes in the cell (Sofos et al. 1988.

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

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

even at pH values near neutrality. although less efficiently. 1998. as well as influence on metabolism.6-bisphosphatase and phosphoenolpyruvate carboxy-kinase. 2000).. 2. the growth rate and liver weight of the animals increased (Demaree et al. Thus. 2000).. the LD50 for common salt (NaCl) is 5 g/kg body weight. Although dissociation of the acid inside the cell may eliminate the pH difference across the membrane. The relative nontoxicity of sorbates was established early in their testing as food preservatives. 1980.2 to 10. the World Health Organization has set the acceptable daily intake for sorbate at 25 mg/kg body weight per day. Reasons for this conclusion include the following: 1.. sorbate is considered a GRAS compound. but 8% sorbic acid resulted in an increase in liver weight (Deuel et al.. 1954a). 1989. Lück. . Sofos. Additional studies with rats and dogs have shown no damage with feeding 5% sorbic acid for 90 days.. Deuel et al. 1989). On extending the feeding period to 120 days. but not of cytoplasmic malate dehydrogenase (Burlini et al. Overall. depending on the type of microorganism. 1998. it is believed to be adequate to energize the uptake of substances needed for cell maintenance and growth. these studies demonstrated the relative harmlessness of sorbates and their relative superiority in safety compared to other chemical additives. 1980). in such situations.. Thus. 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. Bracey et al. 1993). For comparison. 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.or longterm exposure.Sorbic Acid and Sorbates 71 apparent after the subsequent addition of glucose. (3) suppression of glucose-triggered peak of hexose monophosphates. Food and Drug Research Laboratories. which is the difference in electrical potential. substrates. and environmental conditions (Sofos. 1954a. sorbate is considered one of the least harmful preservatives in use. TOXICOLOGY AND SAFETY In the United States. These effects can be summarized as follows: (1) reduced glucose consumption. and (5) block of catabolite inactivation of fructose-1. (2) inhibition of glucose. the mechanisms involved in inhibition of microbial metabolism and proliferation by sorbic and other similar lipophilic acid preservatives appear to be different.. Sorbic acid has been shown to inhibit microbial growth.and fructose-phosphorylating activities. and teratogenicity after short. However. and because it is a metabolizable fatty acid. Sofos. 1955). 1992). its effect on the other component of the PMF. is smaller. Overall. Thus. uptake of the compound based on pH difference across the cell membrane would not be adequate to explain the observed antimicrobial activity. 1993). 1973. The compounds were fed to various animal species for determination of acute toxicity. based on acute toxicity. 1976).5 g/kg body weight (Smyth and Carpenter. 3. Demaree et al. Lück.6-bisphosphate. 1955. 1948. unlike former assumptions. (4) substantial reduction of glucose-triggered peak of fructose 2. it is believed that neutralization of the pH difference across the cell membrane is not the only mechanism of microbial inhibition by sorbate (Stratford and Anslow. however.. 1954a. On the whole. Acute toxicity studies with rats have determined LD50 (mean lethal dose) values for sorbates in the range of 4. this pattern resulted in prevention of glucose-induced switch of metabolism from a gluconeogenic to a glycolytic state. 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. Rat feeding studies have indicated that a dosage of 10% sorbic acid in the diet could be tolerated for 40 days (Lück. carcinogenicity.

Namiki. Food and Drug Research Laboratories.. and vegetable juices and is inactivated by heat (Kada. does not rule out the possibility that high concentrations of sorbate may act as irritants to certain susceptible individuals (Sofos. 1970. No carcinogenic or mutagenic effects have been observed with sorbate alone (Dickens et al. 1999). Khoudokormoff. not at 6. Berry and Blumer. 1992). 1982. 1994).30% in food processing. no direct relation could be proved between symptoms and specific ingredients used in formulating the bacon (U. 1976. (2) formation of the c-nitroso mutagens requires the presence of excess nitrite.. however. No abnormalities have been observed. 1975. The products of such reactions have the potential of being mutagenic (Sofos. 1981. 1979b). Shtenberg and Ignat’ev.5.. 1993.0. sorbates have been extensively examined for skin tolerance (Lück. 1982). Average sorbic acid concentrations for skin irritations are in the range of 1%.. 1975. the potential for such irritations in commercial products is minor. The literature is contradictory. It has been regarded that amino-compounds. In addition. it may react with nitrite in products such as cured meats. 1980. National Academy of Sciences. Litton Biometrics. Sofos. 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. 1976). apparently because of an increased caloric intake resulting from the metabolizable sorbic acid. 1981. Although it was implied that sorbate might have been involved in producing those symptoms. Gaunt et al. As preservatives for cosmetics and pharmaceutical products. Dejobert et al. Kito et al. 1977. 1983. 1981. in general. cysteine.b). Osawa and Namiki. Sorbic acid has a conjugated system of double bonds. which makes it susceptible to nucleophilic attack. 1974. 1980. Lück. This. 2000a. 1980) and because sorbic acid is an unsaturated fatty acid. 1981. There has been some concern about possible sorbate–nitrite reactions when added to the same substrate (Sofos. 1992). 1979b. however. and in some instances the growth rate of sorbate-fed animals increased significantly. 1992). may undergo nonenzymatic browning reactions when stored with sorbic acid at a favorable range of aw and temperature (Quattrucci and Masci. 1976. sometimes giving mutagenic products (Ferrand et al. leading to decreased amino acid availability (Quattrucci and Masci. 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. it may apply also to more complex systems such as foods. Department of Agriculture. 1981. Hendy et al. Osawa et al. botulinum. 1989). Sorbates were found to react easily with proteins. Because there is evidence of reactions between nitrite and fatty acids (Benedict. show skin irritations when exposed to sorbic acid (National Academy of Sciences. 1981). 1980. to some extent. In general. no such symptoms were observed by other individuals who tasted sorbate–nitrate experimental bacon from other studies. but it indicates that most people are not affected by sorbic acid applied to the skin. 1978. and Robach and Adam (1980) were able to induce such symptoms from panelists consuming commercial bacon manufactured without sorbate. Considering the average use levels of 0. 2001).S. and (3) mutagen formation is inhibited by such ingredients as ascorbic acid.. does not appear to create toxicologic problems. 1986.. Robach et al. 1989). Department of Agriculture. 1982). 1979. forming high-molecular-weight products that do not seem to result in a nutritional loss... of course. including lysine and glutamate. Some taste panelists reported certain “allergic-type” symptoms after tasting uninoculated experimental bacon (U. This reaction was demonstrated in model systems but. Some sensitive individuals.S. and some very sensitive individuals may show irritations even at lower levels (Lück. probably because the acid environment of the stomach leads to the breakdown of the sorbic-protein adducts (Quattrucci and Masci. Patrizi et al. 1968.. 1982. various studies have indicated the following: (1) nitrite reacts with sorbic acid to form mutagens optimally at pH 3. 1980a. 1989). Thus. 1974. which is the average pH of most meat products. Sofos and Busta. Walker (1990) concluded that sorbate use.10% to 0. 1979.72 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. Under conditions typical of .

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

HPLC was found to be adequate in obtaining accurate stability data for sorbic acid in creams (de Villiers and Bergh. In the United States. 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. and this status has been reaffirmed (U. 2000.S. 2000). Department of Agriculture. 1978) by a select committee of the U. 2000). especially for high sample throughputs (Hofer and Jenewein. and advantages over other preservatives. 2000). Solid extraction has been used as a cleanup procedure for the determination of sorbic acid by liquid chromatography in fruit products (Mandrou et al. caffeine. 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. 1977). however.. The use of reversed-phase highperformance liquid chromatography (RP-HPLC) was found to be a simple..16) for sorbic and benzoic acids in foods (AOAC Int. low toxicity. paper chromatography. 2000). 1999). 2000). method 980. and reliable and is thus well suited for routine determinations. 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. Thus. thin-layer chromatography. Inhibition of microorganisms has also been evaluated as a procedure for the qualitative detection of sorbate (Ellerman. Dobiasova et al. 1998). REGULATORY STATUS Several forms of sorbate are allowed for use in a variety of foods throughout the world (Sofos. sorbic acid and potassium sorbate are GRAS. The method was found to be fast. that in addition to sorbate. method 974.. wine. 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. This major use may expand in the future.S. In addition. The literature contains numerous reports of chromatographic methods used or evaluated for determination of sorbate in food products (Sofos. where the absorbance is measured at 300 nm and is specific for sorbyl CoA. have reported that cleanup procedures did not improve determination by liquid chromatography (Benassi and Cecchi. rapid method with high reproducibility for determining sorbic acid and other additives in fruit juices (Zou et al. such inhibition may be caused by a number of other inhibitors. 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. In addition. theobromine. Microdialysis was used to extract sorbic acid and benzoic acid from food to be separated and detected by HPLC (Mannino and Cosio. method 974. method 983.74 Antimicrobials in Food 1989). which is proportional to sorbic acid levels in the sample. precise. 1999. 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.. ion chromatography was effective in simultaneously determining artificial sweeteners. Moreover.17) and final action procedure for wines (AOAC Int. They include gas chromatography. Others. it can be used for the analysis of intensely colored juice samples. It is obvious.. 1996).10) (AOAC Int. 1998. Mercier et al.. Food and Drug Administration. 2001).08) and dairy products (AOAC Int. highperformance liquid chromatography (HPLC). 1995). 2001). preservatives. 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. and other substrates (Cancalon. and micellar electrokinetic capillary chromatography (MECC) and ion chromatography. The reaction is quantified by irreversibly hydrolyzing pyrophosphate to phosphate in the presence of inorganic pyrophosphatase. The method is an official first action procedure for ground beef (AOAC Int. however. Capillary electrophoresis was also applied for detection in citrus juices. In addition. 1989). 2002). 1990).. sorbates in the United States . A gas chromatographic method is a first action procedure (AOAC Int. considering their extensive testing. 1989). Castineira et al. and theophylline and as such may be a beneficial alternative to conventional HPLC.

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

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

concentration.16 2. Because some experiments express concentration in percent.27 4. and chemical analyses can be found in the references by Ash and Ash (1995) and Doores (2002. The multiple-barrier concept (“hurdle” concept) has gained popularity for use in a variety of foods in recent years. type. (1939) observed that acids with fewer than 7 carbons were more effective at lower pH.14 3.75 4. physical parameters. chain length. aid in the inversion of sucrose.1). 2003).44 5. Acids contribute to the taste and tartness of a product.98 pK2 pK3 5.41 4. In addition to any antimicrobial effects an acid possesses. and protect color. Comprehensive texts describing applications. Some can be used . The use of the multiple-barrier concept results in lowering the concentrations of acidulants or inhibitors.08 3.89 3. They can create a synergistic relationship with antioxidants by chelating metal ions.75 5.87 4. 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. organic acids are usually more effective as antimycotic agents.43 4. Because the undissociated portion of the molecule is believed to be responsible for the antimicrobial effect. They can control pectin gel formation.03 3. This primary concern limits the use of acidulants to foods with pH values of less than 5. The pKa of most organic acids lies between pH 3 and 5 (Table 4. water activity.34 6.39 times. but also the decreased processing time would aid in preserving the palatability of the product (Corlett and Brown. adjustments in pH levels can be coupled with changes in temperature. The inhibitory effect of acids has been compared based on pH. EFFECTIVE USE Various approaches have been used to study the efficiency of antimicrobial effects on cells. This concept is based on the premise that foods can be preserved using several inhibitors concurrently rather than relying on a single factor. Hoffman et al. normality.0. Not only would this interaction ensure commercial sterility of the food. pK1 4. 1980).77 4.Organic Acids 93 TABLE 4.1 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). it would be advantageous to use the acids near these values. or molarity to achieve final pH. the choice of an acidulant may depend on secondary effects. gaseous atmosphere. and degree of branching to inhibit or kill a wide variety of microorganisms. Effective use of an acidulant depends on the dissociation constant (pKa) or the pH at which 50% of the total acid is dissociated. 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.40 4.61 4. Therefore. prevent browning. and other inhibitory compounds.11 5.

propionic acid. (1990) found that growth of L. Adams and Hall (1988) showed a weakly synergistic inhibitory effect between acetic and lactic acids with Salmonella enteritidis and Escherichia coli.7. 1966) The toxicology and safety of the acids must be considered when selecting an acid for use. This association could explain the apparent stability that occurs in natural fermentations. L. The salts of the acids are important in regulating the acidity of the foods (Gardner. then by Lactobacillus plantarum (homolactic). many of these studies used high levels of the concentrated acids at low pH values. Microorganisms vary in their susceptibility to acetic acid. These acids. the same hazards would not be expected.8. plantarum at pH 4. are more tolerant to this acid than homofermentative lactic acid bacteria (homolactics). In addition.9). such as pickles.9). as the pH decreased to 4. however. THE ACIDS Information on the toxicology. mesenteroides ceased when the internal pH of the cells reached 5. . this apparent synergy has not been demonstrated in other foods. They are usually added to foods for their antimicrobial properties. and caprylic acid. The lipophilic acids include acetic acid and acetic acid derivatives. application. and regulatory status is presented here for each acid. and Staphylococcus aureus (pH 5. The results of these studies are usually presented in the section describing the acid that gave the most antimicrobial effects. Most of the acids appear safe for use in food products. ACETIC ACID Antimicrobial Properties Acetobacter and heterofermentative lactic acid bacteria (heterolactics) produce acetic acid as a byproduct of their metabolism and.0). Although these acid mixtures might be expected to occur naturally during fermentations by heterolactic bacteria. all these acids can be metabolized by the body primarily through lipid oxidation and via the tricarboxylic acid cycle.1) (Levine and Fellers. Concentrations of acid lower than those needed to inhibit Saccharomyces cerevisiae (pH 3. In some instances. Several animal studies have indicated possible safety hazards. are relatively nonpolar compounds.0. the information appears separately for each acid. Sauerkraut manufacture is a fermentation process brought about by a natural succession of microbes that inhabit the surface of cabbage leaves. and vinegar. The carboxylic acids are more polar than the lipophilic acids and are traditionally used in foods for secondary effects rather than for their ability to inhibit microbial growth. The long-term consumption of these acids. Salmonella Aertrycke (pH 4.0 than lactic acid bacteria. however. although they could also serve other functions in foods. a more acid-tolerant organism. antimicrobial properties.94 Antimicrobials in Food as chemical leavening agents. as the name implies.6 to 4. and molds. Gram-positive bacteria. sauerkraut. would further indicate that hazards are unlikely. which exist naturally in many foods. Owen (1946) suggested that acetic acid inhibited Gram-positive organisms. which do not. Numerous studies included in this chapter compare several organic acids for their antimicrobial effects. The succession typically begins with growth of coliforms and natural microflora followed by Leuconostoc mesenteroides (heterolactic). McDonald et al. whereas growth stopped in L. These organisms are found naturally associated with fermented products. At the levels and pH values in which these acids are normally used in foods. 1975b). plantarum maintained its pH gradient in the presence of either 160 mM sodium acetate (SA) or sodium lactate down to an external pH of 3.0.4 to 5. all of the groups were similarly affected (Woolford. as such. such as sauerkraut. yeasts. 1939) inhibited Bacillus cereus (pH 4. Bacillus and Clostridium species and Gram-negative bacteria were more inhibited at pH 6.9) and Aspergillus niger (pH 4.

3% and 0. 1991). malic. Buffered acidulant systems (brain heart infusion [BHI] broth. or 0. and tartaric acids. monocytogenes was also reduced to undetectable levels in RCM within 10 days and in CFM within 14 days compared to 2 days for Salmonella. respectively. tartaric. 1988). When E. citric.3.0 (Conner and Kotrola.5.0. citric. L. monocytogenes than lactic and citric acids and increased inhibition as the temperature of incubation . cereus was inhibited at pH 5.7% acetic acid.7% acetic acid.9 and 4.7% acetic acid in the aqueous phase and adjusted to pH 4.0 with 0.5 for all acidified media at 10°C except for acetic acid. and tartaric acid permitted growth at pH 4. lactic. 1984). Malic acid allowed growth at pH 4. coli than unbuffered systems. pH 4. A concentration of 0.5 to 3. followed by lactic.7% acetic acid (Glass and Doyle. 1940). in the aqueous phase of CFM and RCM would still produce a microbiologically sound product.3% acetic acid formulations.0 with hydrochloric acid was compared to cholesterol-free (egg yolk-free) reduced-calorie mayonnaise (CFM) manufactured with 0. 0. 1970). pH 4.7 and 4. respectively. malic. coli O157:H7 is considered more acid tolerant than strains of E. and hydrochloric acids (Nunheimer and Fabian.0 buffer. pH 4. respectively.15 M acetic acid and with 0. and caused 50% inhibition of spore germination at pH 4. respectively. Acetic acid exerted the most inhibition but had the lowest dissociation constant. L. 22°C) containing 1.0% acetic acid and 1. aureus. added or developed through fermentation. which has recognized antimicrobial properties. Acetic acid caused greater inactivation of L. 1982). 0. It is unlikely that these products would contain such high initial levels of either of these organisms.0 with scant growth in TSB containing acetic acid. L. followed by citric. lactic. and HCl. whereas tartaric acid had one of the highest dissociation constants yet exerted the weakest inhibitory action.5% and 0. such as mayonnaise (Debevere. coli O157:H7 was inoculated into tryptic soy broth (TSB) containing acetic.4% to 0. 1988). 1979). The effect of temperature-acid interactions is noteworthy for the psychrotrophic pathogens.9°C.1 and 5. Gram-negative aerobic bacteria. Reduced-calorie mayonnaise (RCM) manufactured with 0. coli O157:H7 grew in all media at pH 5. Streptococcus faecalis.1-M concentration of acetate and lactate completely inhibited multiplication at pH 6. Conner et al. therefore the standard use of 0. pH 4. other ingredients can interact with acid to influence the degree of inhibitory action. growth occurred at pH 5.2 and 5.4 to 4.2 and 4. monocytogenes and Yersinia enterocolitica. monocytogenes.5. In addition. and exposed to 40°C became injured. At 25°C and 37°C. aureus in 12 hours (Minor and Marth.0. aureus.. Salmonella were inactivated within 48 hours in both RCM and CFM formulated with 0. but no injury occurred in cells not exposed to acidic conditions before heating (Smith et al. some strains of S.. pH 4.5 (Noda et al.1%. 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 23.0% lactic.. Destruction of Salmonella occurred more quickly in CFM than RCM. Salmonella Blockley. Both studies concluded that the antimicrobial effect was the result of the undissociated molecule. S.7% acetic acid with reduction to undetectable levels in 1 and 2 weeks with 0.6.8. E.33 M lactic acid (Wong and Chen.5%. Traditionally acidic foods contain a single or multiple acids. especially with the use of pasteurized egg products.6.3%.6) at 20°C shifted to pH 7. aureus was most sensitive to acetic.0. E. A 0. Acetic acid was effective at pH 5. B. This finding is important in the manufacture of medium acid foods containing more than one organic acid. although more resistant than Salmonella. 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. most likely because of the absence of egg yolks in CFM and the presence of egg white.8% reduced growth of Micrococcus and Bacillus species. 1995.6 and 4. and E. aureus preconditioned in acetate buffer (pH 4. was reduced by 4 logs in both mayonnaises within 72 hours in formulations containing 0. and Enterobacteriaceae in Japanese fermented soy sauce (Hayashi et al.. tartaric. or citric acid were far more effective in reducing numbers of S.Organic Acids 95 S. coli. Similar results were noted when various acids were compared at the pH level required for a 90% and 99% reduction in numbers of S. 1997). malic.

1991. Enteritidis were sprayed with acetic acid at pH 1.0 have been used to rinse pork. 1981). pH 4. acidulants. inoculated with approximately 107 CFU/g Y.6 for lactic acid. injury and death rates were not affected by refrigeration temperatures for Salmonella Bareilly. 1992. Mung bean seeds inoculated with 3 to 5 logs of S. coli O157:H7.2 N for 4 min.3% inhibited L. 1 log unit at pH 2. monocytogenes were exposed to acetic acid at a concentration of 242 µL/L of air for 12 hours at 45°C..01 N for 75 min.0% acetic acid at pH levels from 1.72 logs. However. This treatment reduced S. 0. Typhimurium.0 for 30 or 60 seconds (Biemuller et al.6% to 3. 1989. Typhimurium and E. and no reduction in microbial . Little et al.5. Acetic acid was more effective than lactic or citric acids in producing this effect (Adams et al. and 0. monocytogenes was isolated from 2 of 10. Because produce is typically refrigerated with only minimal processing. 1997. Depending on the treatments.4 for malic. Conversely. 40%. 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. vapor treatment also reduced levels of resident microflora on seed but did not affect seed germination rates (Delaquis et al. Parsley. A variety of organic acid treatments has been used to control microbial loads associated with meat carcasses and products (Dickson and Anderson.5 min. these foods can be the carriers of psychrotrophic pathogens. Similar growth inhibition was demonstrated for Y. 1989). 1992a.05 N for 8. respectively.10 N for 5 min. enterocolitica at lower pH levels as the temperature increased... and temperature. 1998). citric acid caused greater injury than either lactic or acetic acids and injured organisms survived longer at low temperatures (Ahamad and Marth. Treatments spanned varying amounts of time with concomitant decrease in microbial loads up to 3 logs.9 log reduction for the nontreated seeds (Weissinger et al. 1990. In addition. Numerous organic acid combinations. Acetic acid displayed the greatest antimicrobial action against L. and off-flavors. E.0.5.. 1992b).5 to 3.5 or 2. seed germination was effected (Weissinger and Beuchat. Smulders and Greer.. citric acid was more bactericidal than other acids at 25°C and 35°C. or 5%) or vinegar (30%. 0. coli O157:H7 to nondetectable levels by enrichment methods. or 50%) for 15 or 30 minutes. 1998. enterocolitica. Immersion of alfalfa seeds in mixtures containing 5% lactic or citric acid for 10 minutes resulted in several log reductions. Pork carcasses inoculated with 106 CFU/ml S. containing from 0. Total plate counts were reduced by 4 logs at pH 1. but malic acid was more bacteriostatic than other acids at 10°C. monocytogenes irrespective of temperature. The greatest antimicrobial effect occurred at 35°C with the greatest survival at 10°C.4 to 4. Dorsa. 2000). Karapinar and Gönül.. and L. and beef carcasses or meat tissue. 2001). monocytogenes when compared to citric. 25-g samples. citric. but L.. 1990). such as Y. enterocolitica. Treatment at low temperature (4 days at 10°C) with 200 and 500 mg/L of air was effective in reducing Salmonella by 2. A 90% reduction of the population required exposure to 0. and hydrochloric acids at equal pH values and at all incubation times and temperatures (Sorrells et al.7 log without affecting germination of seeds. a twofold reduction at pH 2. 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. but resulted in changes in sensory characteristics. and hydrochloric acids. but recovery required the presence of amino acids or peptones (Blankenship. a mesophilic organism. In addition. there can be adverse sensory effects including discoloration of meat. Sorrells and Enigl (1990) also demonstrated interactive effects between sodium chloride. concentration ≥0. Exposure at 50°C for 12 hours with lower levels (100 and 300 mg/L of air) reduced the population >1. Y. Dorsa et al. The minimum inhibitory pH level was pH 4.8 to 5. and pH 4. 2%.96 Antimicrobials in Food decreased from 35°C to 13°C. enterocolitica was not detectable after exposure to 2% and 5% acetic acid or 40% and 50% vinegar for 30 minutes (Karapinar and Gönül. 1997.0 for acetic acid. lactic. however. When comparisons were based on equal molar concentrations. 1992). off-odors. Brocklehurst and Lund. but acetic acid was less effective. was dipped in acetic acid solutions (1%.33 and 5. Acid injury did not appear to involve damage to ribosomes or other nucleic acid material. lamb. 1999). 1973). malic.

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

and 30°C (Oscroft et al. acetic acid caused the skin of the poultry to be hard and leathery. 1 mM perillaldehyde—or 0. Stroup et al. (1985) found that mushrooms reached an equilibrium pH of 4. aerobic plate counts were unaffected by the treatments. Typhimurium and Campylobacter jejuni by 0. with and without the use of air injection to agitate the chill water. Kurita and Koike (1983) combined 3% ethanol. and lactic (Mountney and O’Malley. monocytogenes. 1997). The acetic acid in the brine inhibited anaerobic and aerobic bacteria. 20°C. Again.9% acetic acid did not support growth or toxin formation of Clostridium botulinum (Ito et al.5 mM. The acid level could be reduced when used in combination with brines and sugar.0% acetic acid caused instantaneous death.5%. As the incubation temperature to recover spores was lowered. Intervention steps incorporated into poultry processing have been effective in reducing microbial contamination. and 0. There was no significant difference in texture or sensory characteristics between the treatments.6 or 5.86 log MPN/ml for the 0. respectively. although the skin of the 0.05%. of the same components—with 2% salt to inhibit growth of contaminating microorganisms for more than 20 days at 27°C.5% acetic acid reduced Enterobacteriaceae counts by 2. Levels of 0. Immersion of broilers for 10 minutes in 0. Typhimurium using a model system.0% acetic acid compared to 2 days for 0.6 depending on the concentration of acid in can brine within 1 day with 1. Dickens and Whittemore (1994) exposed broilers to 0.5 with acetic acid was the most effective antimicrobial treatment followed by other acids in descending order of effectiveness: adipic. but Enterobacteriaceae counts were significantly reduced by 0. germination and outgrowth of Bacillus . 1994).5 logs (Okrend et al.05% and 0. further poultry processing can reintroduce microorganisms onto carcasses.. 1994). acetic acid was the most destructive acid at all combinations of parameters. and tartaric acid adjusted to pH 5. monocytogenes.71 log most probable number (MPN)/ml (Dickens et al.6% acetic acid-treated carcasses was darkened or yellowed (Dickens et al. greater than a 4% concentration of acid was needed to reduce levels by 2 logs (Tamblyn and Conner. citric.. but changes in pH and concentration of acid were more successful. 1987).2% to 0. 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. 1976).98 Antimicrobials in Food surface with approximately 103 CFU of Clostridium sporogenes and L. Nisin (an approved bacteriocin for some foods. lactic. Acetic and tartaric acids were more inhibitory than lactic and citric acids..05% acetic acid.6% acetic solution acid (pH 3.. Products are now being preserved by a number of antimicrobial compounds using a multiplebarrier approach to preservation.0 on inhibition of L. Addition of 1. a 0.7% citric acid. Expanding on their work. Whole pickles preserved with brine at 12° Brix and 0. 0. fumaric.5% concentrations of acetic. succinic. Because of the possible attachment of Salmonella to broiler chicken skin. Air injection did not affect reduction of these counts.6% acid solutions. 1994).3% and 0. 0.5 to 1.35 log MPN/ml for the 0. Although each individual acid influenced the heat resistance of Bacillus spores differently.0) did not result in a significant reduction of aerobic plate counts but did significantly reduce levels of Enterobacteriaceae by 0. Despite the reduction in microbial numbers at this pH. citric. 1990).24 log (Lillard et al. see Chapter 7) coupled with 0. For pearl onions.3% acid and 2.. 1986). El-Shenawy and Marth (1989b) investigated the effect of acetic. Acidified media showed greater inhibitory activity at 35°C than at 13°C. 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.35% solution required 7 days for equilibration for acetic and citric acid but 1 day for 0. 1965). Adjustment of the chill water to pH 2. Rubin (1978) found that acetic and lactic acid acted synergistically to retard growth of S. Other studies continued to explore the use of acetic acid in chill water. lactic. Despite these treatments.1% acetic acid at 52°C decreased levels of S. Water pockets occurred under the skin of chicken carcasses with air-agitated samples (Dickens and Whittemore..7% acetic and 4 days for the same concentration of citric acid. Poultry scald water containing 0.6% acetic acid under the same conditions.

pneumonia (Gerhartz. and potatoes than peroxyacetic (80 ppm) acid alone (Hilgren and Salverda. A combination of these parameters could be used to extend the shelf life of these products. Rats fed 10% acetic acid and cats fed 20% acid developed ulcers in the gastric mucosa (Okabe et al. 1932). Spore-forming bacteria are killed on surfaces at a concentration of 0. 1937). cabbage. Aspergillus fumigatus required 0.5% level. 1969). and 0. are especially susceptible to spoilage because of indigenous microbial populations. chilled. and citric acid when spores were heated at 95°C for 15 minutes and acetic. 1968).29 mg/m3 caused changes in electroencephalograms.0 and below to inhibit growth (Kirby et al. and citric acids when temperatures were reduced to 65°C for 60 minutes. respectively (Buchanan and Ayres. Human olfactory thresholds to acetic acid vapor were 0.. Ingestion of acetic acid by humans has caused burned lips (Palmer. Mori (1952) fed rats acetic acid ranging from 10 to 50 cm3 acid/kg of rice. ready-to-eat products.01% to 0. niger. Lower concentrations of 0. 1966). 1968).0.. rats decreased their food intake level and lost 2.2). lactic.48 mg/m3 altered sensitivity to light. On contact with organic substrates. however. When used as a surface application. displayed synergistic effects in increasing the destruction of the organism.2 g/kg body weight) did not show any adverse effects. it decomposes to yield oxygen and acetic acid. This treatment could also be used in recycling of water to reduce levels of yeasts and molds.01 mg/m3. Damage from acetic acid resembled that from ionizing radiation or radiometric chemical damage (Manna and Mukherjee.2% acid at pH 5. 1958). Rats consuming acetic acid in drinking water in concentrations of 0.5 completely inhibited growth and aflatoxin production by Aspergillus parasiticus. 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.6% of their body weight (Sollman. in combination with organic acids. glucono-delta lactone. At the 0.04 M acetic acid at pH 3 to 4 showed chromosomal and chromatid breaks.2% (0. followed by glucono-delta lactone.Organic Acids 99 spores were more restricted. and death.8% to 1. Parmeggiani and Sassi (1954) reported that workers . Allium cepa (Makinen. interference with blood coagulation (Fin'ko.6% or 0. 1969).5 inhibited Saccharomyces ellipsoideus and Penicillium glaucum. 1921).. Johnson (1968) noted that rat gastric mucosa responded to a 100-mM concentration of acetic acid by releasing histamine into the interstitial fluid. a 1% concentration of acetic acid at pH 4. lactic. but a concentration greater than 4% was needed when the pH was at 7. It was found that acetic acid was the most effective acid.0001 and 0. it is effective against the black bread molds. it was not possible to determine the actual quantity of acid consumed. aerobic bacteria. however. Cruess and Irish (1932) showed that apple juice containing 0. renal failure (Paar et al.2 or 5 mg/m3 but not at 0.0% concentration of acetic acid. Rats exposed to vapor levels of acetic acid showed muscle imbalance at 0. 1971).001 M accelerated spindle activity and retarded anaphase in the mitotic cycle of the root tips of the onion. Nisin. 1976). A. All rats showed mucosa damage owing to the feeding method. Although acetic acid is generally used as an inhibitor of bacteria and yeasts.999% after exposure to a 1. Grasshoppers treated with 0.0001 and 40% relative humidity within 10 minutes (Portner and Hoffman.8% partially inhibited growth and decreased toxin formation by 70% and 90%.5. Other forms of acetic acid have antimicrobial uses. suggesting its use as a decontamination agent for equipment (Hedberg and Miller. 2000). Takhirov (1969) suggested limits of 0. and coliforms. Studies on nonmammalian species showed that acetic acid at a concentration of 0.8 and 0. and Rhizopus nigrificans at pH 3. gastric erosion (O’Keane et al.0% acetic acid adjusted to pH 3. Toxicology Acute toxicity data for acetic acid in mice and rats indicate varied tolerance levels by different routes of administration (Table 4. Levels of 0. Peracetic acid is formed by an oxygen molecule bound to the carboxyl carbon atom of acetic acid.. Foods.06 mg/m3. particularly precooked. 1971). Populations of Pseudomonas aeruginosa were reduced by 99.4% acetic acid at pH 5.60 mg/m3. 1949).

eyes. caused allergic reactions by producing canker sores (Tuft and Ettelson.430–4. 1955).2 to 0. (1971) Rat Citrate. (1957) Gruber and Halbeisen (1948) Yokotani et al.210 338 3. In rare instances acetic acid.380–3. sodium Tartaric Spector (1956) Orö and Wretland (1961) Hara et al. pharynx. and epidermal reactions (Weil and Rogers. lactic.730 1. (1957) Acetate.530 380 1.020 5. and tartaric acids. calcium Propionate.860 2.100 Antimicrobials in Food TABLE 4.310–3.340 580–1.790 940–960 42 203 961 2. (1963) Hara et al.960 525 3. 1951). (1971) Gruber and Halbeisen (1948) Horn et al.000 1. Men having 7 to 25 years of exposure displayed blood abnormalities (Cesaro and Granata.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) 4.810 625 3.040–5.500 330 44 1. although it is assumed that this derivative would be assimilated in a manner similar to that for acetic acid.100 1.700 11. sodium Dehydroacetic Adipic Rabbit Caprylic Citric Mouse Mouse Enders (1941) Orö and Wretland (1961) Yokotani et al. 1956). as well as citric.700 725 884 5. teeth. 1956).900 680 275 2.65 mg/L.200 485 Reference Spector (1956) Orö and Wretland (1961) Spector (1956) Spector (1956) Spencer et al. sodium Rabbit Mouse Rat Rabbit Rat Guinea pig Mouse Rat Rat Mouse Gruber and Halbeisen (1948) Yokotani et al.460 1. No toxicologic data were available for peracetic acid. (1950) Horn et al.430 600 5. and lungs with an air concentration of 0. cold sensitivities (Wiseman and Adler. . (1957) exposed for 7 to 12 years to acetic acid vapors in the production of acetyl cellulose displayed corrosion of the hands. (1971) Gruber and Halbeisen (1948) Gruber and Halbeisen (1948) Lactic Propionic Propionate. (1963) Horn et al.

1005). It is highly soluble in water. Na+ Malic DL-malic Propionic Propionate. The data are scarce for the use of peracetic acid in food products. b Content of DL-lactic acid. NH4+.. K+. Acetic acid is generally regarded as safe (GRAS) for miscellaneous and general-purpose usage (21 CFR 184. The salt form.05%. 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. it has been added to infant feeding formulas to replace lactic acid (Seymour et al. 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. . which limits its use. c Content of DL-malic acid. and mayonnaise and is found in pickled products such as sausages and pigs’ feet. the malic acid content of malic acid should not exceed 0. salad dressings. 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. Mendonca et al. K+. It is the principal component of vinegars and as such is primarily used for its flavoring abilities. the estimated acceptable daily intakes listed here do not include amounts occurring naturally.Organic Acids 101 TABLE 4.. Ca+. K+. (1989a) used a dip of SA in combination with potassium sorbate (10%) and phosphates (10%) to extend the shelf life of pork chops.3 Acceptable Daily Intake for Humans Limitations (mg/kg body weight) Acid Acetic Acetate. catsup. Ca+. Ca+. K+. Because of cost and antimicrobial action. Na+ Sodium diacetate Adipic Citrica Citrate. Ca+. The acceptable daily intakes for humans for acetic acid and its derivatives are listed in Table 4.3. 1980). requires different handling and use procedures than the acid. 1954). It is used in condiments such as mustard.. K+. Na+ Fumaric Lactic DL-lactic Lactate. 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. however. Na+ Tartaric Tartrate. ACETATES Antimicrobial Properties Several derivatives of acetic acid are currently in use as antimicrobial agents. Application and Regulatory Status Acetic acid is a monocarboxylic acid with a pungent odor and taste. 1939).

102 Antimicrobials in Food SA (10%). Waterhouse and Scott (1962) attributed the toxic effect to the sodium ion rather than the acid moiety. 1951). SA in combination with potassium sorbate and Lactococcus lactis ssp.5% SA.4% monopotassium phosphate (MKP) more effectively prolonged shelf life to 12 days compared with SA alone.25% potassium sorbate. although a mild acetic acid odor was detected.5% potassium sorbate.3% to 0. No toxicologic data were available for calcium acetate (Food and Agricultural Organization. and 1. over 6 days. SA (0.to 6-week storage life (Blom et al.5% lactate was effective as an antimicrobial treatment in sliced servelat sausage over a 4. growth and toxin formation were decreased 70% and 90%.44% SA showed decreased appetite. 1996). untreated samples reached the spoilage mark within 10 days. 1963). respectively (Buchanan and Ayres. but they did appear brownish and watery (Kim et al. parasiticus was inhibited by a 1. although MKP alone had no effect. 1976). whereas potassium sorbate increased shelf life to 9 days. 5% sodium lactate. The combination of 0. . SA alone or SA-MKP-treated fillets were not significantly different in odor from fresh controls up to 9 days. breve and SA exhibited an additive effect that extended shelf life still further. and 2.2.75% and 1% significantly reduced the initial aerobic plate counts of catfish fillets by 0. For peeled shrimp. Growth and aflatoxin production of A.5.0% concentration of SA at pH 4.. Treatment with SA alone or in combination with bifidobacteria extended shelf life at 4°C by 3 days (Kim et al.7% SA and 0. although it is assumed that this derivative would be assimilated in a manner similar to that for acetic acid. but bifidobacteria did not grow within 9 days. SA at concentrations of 0.5%).5 to 1. B. 1997). Color remained acceptable.. 1999). breve culture and stored at 4°C for 9 days (Al-Dagal and Bazaraa. Psychrotrophic counts from untreated whole shrimp increased to >107/g. the indicator of spoilage. and trisodium citrate extended shelf life to 6 days.8%. potassium sorbate (1. 1994). or trisodium citrate were used alone or in combination with Bifidobacterium breve (5%) as a dip for fresh camel meat (Al-Sheddy et al. SA extended shelf life to 12 days at 4°C.. but SA provided better sensory characteristics and more controlled growth of psychrotrophs than potassium sorbate with bifidobacteria.0% SA alone inhibited growth for more than 6 days at 4°C and counts increased only by 1. Toxicology The LD50 (mean lethal dose) for SA in mice is presented in Table 4. 1999). 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. Whole and peeled shrimp were immersed for 2 minutes in solutions containing 10% SA. The combination of 0. monocytogenes in vacuum-packaged turkey bologna during storage at 4°C and was similar to 0. A combination of B.5%) was also effective in reducing populations of L. 1.25% acetate and 2. depressed growth rate. sodium lactate (3%). At lower acid concentrations of 0. sodium lactate.2 logs by day 12.. 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. 0. A 22-year-old woman with sensitivity to acetic anhydride was not allergic to SA (Weil and Rogers. Catfish fillets treated with 2% SA. Chickens fed diets supplemented with 5.3 or 1.6 to 0. breve. vacuum packaged.. 1994). 1995b).26% potassium sorbate or 2% sodium citrate in its efficacy (Wederquist et al.. Histologic findings showed hypertrophy of the kidneys and ureters. and held at 4°C for 12 days contained lower population levels of psychrotrophic and anaerobes than untreated samples (Zhuang et al. 1995a).6% or 0.5% lactic cultures or 0. A combination of 0.7 logs when stored at 4°C compared with the nontreated fillets and increased shelf life by 6 days.5% trisodium citrate with or without B.

the combination 0. The growth of L. Sodium diacetate is equally effective in the baking industry where its inhibitory powers prevent the growth of bread mold and rope-forming bacteria.25) at 35°C. a concentration of 0. while having little effect on baker’s yeast. A. S. However.2% SA. curvatus reached an end point of 107/g within 2 to 2. monocytogenes grown in BHI broth adjusted to pH 5. or 1% buffered sodium citrate (Stekelenburg and Kant-Muermans 2001). and 28 mM (pH 5. monocytogenes and bactericidal . However. 1994).1% to 2. A. niger. Enteritidis. sodium diacetate was also effective against hemorrhagic E.1185) acetates are approved as GRAS substances for miscellaneous and general-purpose usage. L. The addition of sodium diacetate affected the sensory qualities of the product precluding its use.05% to 0. In addition to L. An interactive effect was noted between temperature and concentration of acid in that growth was inhibited with decreasing concentrations of acid and temperatures. Enteritidis were inoculated into a comminuted beef emulsion formulated with 0. 32 mM (pH 5. respectively. 20°C.1% and 0.1% and 0. 2.2% sodium diacetate. Pseudomonas fluorescens.3% sodium diacetate was effective in suppressing total aerobic counts.2% sodium diacetate.5% at pH 4.Organic Acids 103 Application and Regulatory Status Sodium (21 CFR 184.55.1721) and calcium (21 CFR 184.0% delayed mold growth in cheese spreads. In ground beef. the addition of 0. 1972). 0. Calcium acetate is classified as a stabilizer when migrating from food packaging materials (21 CFR 181.5% and 3.2% sodium diacetate.4.3% sodium lactate. monocytogenes. L. and when it was incorporated into butter wrappers. Levels of 0.2% sodium diacetate and 2. L. monocytogenes increased to approximately 106/g within 3 weeks. it prevented mold growth (Chichester and Tanner. monocytogenes were inoculated into the cooked ham products at an initial level of 104 and 102/g of product. or 1.1% to 0. and stored for up to 40 days at 4°C. The MIC was determined to be 35 mM (pH 5.5% sodium lactate or combinations of the acids and held at 5°C or 10°C. 2001).5 inhibited these same strains and Mucor pusillus in feeds and silage. vacuum-packaged.3 and incubated at 5°C. Aspergillus glaucus. Y. 5. monocytogenes and S. L. 6.4) at 20°C. enterocolitica. monocytogenes was not significantly different in the presence of the individual inhibitors compared with control samples after 20 days at 10°C.3% sodium lactate. The minimum inhibitory concentration (MIC) of sodium diacetate was determined for L. Sodium diacetate at concentrations of 0. such as Bacillus mesentericus (subtilis).5% sodium lactate was bacteriostatic to L. and B.1% or 0. Lactobacillus fermentans. or S. and 35°C (Shelef and Addala. Enterococcus faecalis. coli. 1981). Lactobacillus curvatus and L. aureus (Shelef and Addala.25.5% level in malt syrups. Cooked-in-bag ham was formulated with additions of 0. SODIUM DIACETATE Antimicrobial Properties Sodium diacetate was effective at a 0.2% sodium diacetate or 1% buffered sodium citrate and within 3 and 5 weeks for products containing 2. 5. 1994).55) at 5°C. and 6. in products containing buffered sodium citrate. Saccharomyces species (Glabe and Maryanski.29).5 inhibited Aspergillus flavus.0. Sodium diacetate was more effective than acetic acid. cereus with no effect against Pseudomonas fragi. A concentration of 0. and Penicillium expansum. and the inhibitory effect was attributed to the diacetate moiety rather than pH alone. SA can be used as a boiler additive for food-grade steam (Federal Register. 1962).8% and 2. monocytogenes was inhibited by sodium lactate and 0.5 weeks in products containing 0. Shewanella putrefaciens.15% to 0. fumigatus.5% or 3.3% 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.4% at pH 3.

54 log reduction). and wrapping material. Toxicology No toxicologic data were available for sodium diacetate (Food and Agriculture Organization. prepared with sodium diacetate (0. 0.5 logs CFU/ml slurry. In subsequent work (Mbandi and Shelef. were pasteurized for 10 minutes at 68°C after which they were inoculated with L.1% (Wolf.5% sodium lactate and 0. the reduction of multiple strains of L. monocytogenes at a level of approximately 4. use levels range from 0. Enterobacteriaceae counts dropped 1000-fold over 6 days at 2°C.5%).104 Antimicrobials in Food to S. Slurries of uncured turkey breast. Application and Regulatory Status Sodium diacetate is approved as a GRAS substance for miscellaneous and general-purpose usage (21 CFR 184. and pediocin (5000 U/ml).59 log reduction). Sensory characteristics of roasted chickens were not affected even at levels up to 8 g per chicken (Moye and Chambers.8 logs (Degnan et al. 0. respectively. monocytogenes was reduced by 2.25% to 0. cerevisiae and 25 times more effective against P. 1981). lower concentrations of the antimicrobials were equally as effective.2% sodium diacetate alone or in combination in beef bologna. sodium lactate (2. plantarum at levels of 0. The growth of L.3%.. 1962).1754). monocytogenes and Salmonella species was demonstrated using 2. 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 turkey mixtures.1) of the acidulants and remains effective at higher pH ranges. Enteritidis. niger (Banwart. 2002).5%) was listericidal in combination with ALTA™. DEHYDROACETIC ACID Antimicrobial Properties Dehydroacetic acid (DHA) has one of the highest dissociation constants (Table 4. Although 1 M sodium lactate solution reduced populations within 2 days. the combination of acids achieved greater reductions of both organisms at both temperatures compared to individual acids and greatly reduced background spoilage microflora.5% concentrations (0.5%). butter. 1989). Sodium diacetate (0.4% (Glabe and Maryanski. monocytogenes in mixtures held at 4°C for 42 days was inhibited at 0. producing a 2-log reduction after 7 days at 25°C (Schlyter et al. a fermentation by-product of lactic acid bacteria. The powder dissolved in the surface moisture of the carcass to form acetic acid and SA and produced a surface pH of about 4. a more complex product. but sodium diacetate reduced the pH slightly. populations increased approximately 0. malt syrups. (1993b) used the multiple-barrier concept to limit growth of L.8. Sodium lactate did not affect the pH of the meat. respectively. This acid is effective against Enterobacter aerogenes and L.005% to 0. stored aerobically at 5 and 10°C.5 logs within 6 days. . Application of 4 M SA reduced the levels of L.. Mixtures were incubated at 25°C or 4°C and sampled up to 7 and 42 days. Schlyter et al. 1950).1%. At lower temperatures. Sodium dehydroacetate was twice as effective as sodium benzoate at pH 5.5% concentration (0. monocytogenes by 0. 1993a). 1991). It is used in cheese spread. As expected. L.0 against S. In bread and cake products.1%. glaucum and A.3% and 0. Lactate and pediocin enhanced the listericidal properties of diacetate suggesting some type of synergistic activity.6 logs in crabmeat stored at 4°C for 6 days when treated with 2 M sodium diacetate. 1994). although it is assumed that this derivative would be assimilated in a manner similar to that for acetic acid.3% and 0. Sodium diacetate was determined to be listericidal over the 7-day period at 25°C only at the 0. although it is fungistatic at concentrations of 0.

and sodium dehydroacetate acid were investigated for three strains of E. treatment with sodium dehydroacetate was effective in reducing respiration rate and delaying ripening. 0. The MIC in broth at pH 6. However.05%.1%. although a dose of 200 mg/kg led to growth and organ changes. Toxicology Acute toxicity data for adipic acid in mice and rabbits indicated varied tolerance levels by different routes of administration (Table 4. ADIPIC ACID Antimicrobial Properties Adipic acid has no antimicrobial properties other than those ascribed to its ability to reduce pH. Enders (1941) showed similar symptoms in rabbits.02%. 1953). Spencer et al.0%.5 was 4 mg/ml for potassium sorbate. or histopathology. The sodium salt of DHA was nonirritating to rabbit’s skin. in addition to acidosis by the former route and intestinal adhesions by the latter route (Horn et al. 1971).25% or 5% sodium dehydroacetate for 60 seconds were considered more marketable after 3 days of storage at 20°C than water-dipped berries. and 0.. 1953).. mortality. However. Oral administration of the acid to mice caused hemorrhaging in the small intestine with distension of the stomach. produced diarrhea. Toxicology The LD50 for DHA in rats is given in Table 4. Sorbate was bacteriostatic at half the MIC for E.2). hematology. 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. 1957). the 800 mg/day dosage depressed growth.2. 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..130).0% adipic acid showed no effect at the two lowest levels. 1950). (1950) did not show any unusual effects in rats receiving 10. coli O157:H7 in broth and model meat products. which included marked obstruction in the liver and kidneys by the oral route and polyuria by intravenous administration. thus extending marketability (Watada. coli in homogenized beef but not in poultry (Yamamura et al. even with prolonged contact (Spencer et al. resulting in the loss of up to 20% of body weight. benzoic acid. but significant growth depression . 1. However. Application and Regulatory Status DHA or its sodium salt is GRAS when used in accordance with good manufacturing practices. appearance. and caused slight histologic aberrations. sodium p-hydroxybenzoate. Longer studies using male and female rats given 400 or 800 mg/day did not affect offspring. 30. This acid at the appropriate concentration level would inhibit the growth of any microorganisms susceptible to low pH values. Intravenous and intraperitoneal injection produced hemorrhaging in the lungs. Furthermore. and sodium dehydroacetate and 16 mg/ml for sodium p-hydroxybenzoate. altered behavior. It can also be used as a fungistat in cheese wrappers. or 5. 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 effects of potassium sorbate. benzoic acid. at the higher dosage levels.Organic Acids 105 Strawberries dipped in or sprayed with 0. 300 mg/kg led to severe weight loss and damage to internal organs. 2000). The 90-day feeding trials conducted on rats fed 0.1% of the substance showed no changes in growth. A 2-year feeding study with male and female rats fed 0. weight gains were lower and mortality was higher than in the controls (Lang and Bartsch. or 100 mg/kg of DHA per day for 34 days.

Sodium adipate at the 5% level decreased the rate of weight gain (Informatics. candies. CAPRYLIC ACID Antimicrobial Properties When tested against a variety of Gram-positive and Gram-negative organisms and yeasts.0%. however.0. not to be used in amounts exceeding good manufacturing practice. Application and Regulatory Status Adipic acid is a nonhygroscopic.1009). The acceptable daily intake for humans is listed in Table 4. Application and Regulatory Status Caprylic acid is a colorless. and hamsters showed no significant difference from controls at levels of 263. It is used in gelatin desserts.16% for snack foods. respectively (Food and Drug Research Laboratory. The MIC of caprylic acid was 100 µg/ml in TSB for Vibrio parahaemolyticus (Beuchat. Teratologic studies using mice. 1973b). 1. caprylic acid inhibited bacteria and fungi at a lower concentration than either acetic or propionic acid. as a sequestrant in oils.8 µmol/ml (Kabara et al. 1943). Toxicology Orö and Wretland (1961) determined the LD50 of caprylic acid for mice by intravenous administration (Table 4.3. 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.001% or less for other food categories. and biscuits because it imparts a smooth.3% concentration inhibited E. It is used as a flavoring adjuvant in levels not to exceed 0. and 205 mg/kg body weight.005% for fats and oils. meat products. It also serves a function in the canning of vegetables. 1972).0%.5% acetic acid and 1. At pH 6. frozen dairy desserts.04% for cheese. however. 1968). and 5% adipic acid and female rats fed 1. . 0. and in improving the melting characteristics of processed cheese (Gardner. caprylic acid was ineffective at concentrations of up to 7. Caprylic acid is approved as a GRAS substance for miscellaneous and general-purpose usage (21 CFR 184. gelatins and puddings. weight gains for rats fed the two highest levels were significantly lower than those for control rats (Horn et al. 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. saturated dicarboxylic acid having low water solubility. rats. mildly acid flavor. coli when grown in a chemically defined medium and was more effective than 1. 288. refrigerated rolls.013% for baked goods. 3. 1972). It is slightly soluble in water. powdered fruit beverages. 1975b).2). rancid taste.1025).1%. oily liquid that has a slightly unpleasant odor and a burning.0.. 1980). No limit has been set on the acceptable daily intake for humans. Caprylic acid was also more inhibitory to Shigella species than either acetic or propionic acids (Nakamura and Zangar. and soft candy.. The 2-year feeding trials using male rats fed 0. 0. the inhibitory concentrations were not dramatically lower (Woolford.8% propionic acid. At pH 5. Inhalation of adipic acid for 6 hours at a rate of 126 µg/l produced no unusual findings (Gage. 1957). 1970).0% level although the food intake level was the same.. 0. baking powders. and 0.0% acid did not show any abnormalities. Caprylic acid at a 0. Inc. Caprylic acid is also used as an antimicrobial agent for indirect use in cheese wraps.106 Antimicrobials in Food occurred at the 5.

compared to counts in unacidified juice (6. pH was not considered a reliable indicator of preservation effectiveness (Fabian and Wadsworth. 1994).5) was more effective than lactic acid (pH 3. citric.5% sodium citrate and 1. 1987). and acetic. 4. 1994). 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. and phosphoric acid was compared in TSB for Y. but inhibition increased with decreasing pH (Murdock.. was processed for 30 minutes in boiling water. A comparison of equimolar concentrations identified citric acid as the most antimicrobial. These experiments used citrate phosphate buffer in which the citric acid content was calculated at 1. the highest inhibitory activity was associated with propionic.22 log/ml). 1973. aerobic counts at 20°C for pH 3. 1950). phosphoric. 1953).0 and 3. Rate of inactivation of L.7 and stored for 7 days at 5°C.0 with propionic acid. citric.83 at 10°C and 7.7 juice incubated at 20°C were 6. respectively. Similar pH-temperature relationships were noted by Cole et al. 2. acetic.4 and 0. The effect was temperature dependent in that survival of L. canned in a brine containing acetic.. Sorrells et al.0 at 5°C.13. and 4. All acids would be effective antibotulinal agents at that pH level (Nogueira et al.7 juice were 4.6 log/ml were seen. citric acid was more inhibitory than lactic acid. Schoenemann et al. enterocolitica based on concentration of acid. monocytogenes survived after 11 to 12 weeks in TSBYE adjusted with acetic. L.5% (pH 4. propionic. Citric acid was particularly inhibitory to flat-sour organisms isolated from tomato juice. followed by hydrochloric.42 log/ml (Bizri and Wahem. whereas at 10°C.1% at pH 4.0 with citric and hydrochloric acids (Conner et al.Organic Acids 107 CITRIC ACID Antimicrobial Properties Inhibition of L. and 7 (Buchanan and Golden.19 at 5°C. Citric acid was more inhibitory to thermophilic bacteria than acetic or lactic acids. 1939. malic. however. . (1990). The authors suggested the inhibitory activity might be the result of the chelating ability of citric acid. The inhibitory capacity of hydrochloric. monocytogenes was achieved in trypticase soy yeast extract broth (TSBYE) adjusted to pH 5. 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 propionic acids and for 6 weeks in media containing HCl or lactic acid. monocytogenes decreased to undetectable levels within 1 to 3 weeks at 30°C. phosphoric. followed by acetic acid. Citric acid at a concentration of 0. monocytogenes after 4 weeks were pH 4. respectively. pH. fumaric. Yeast and mold counts were the reverse with counts of 2. citric. Fabian and Graham. but lower pH levels were toxic. Sodium citrate was also more effective than 2% sodium lactate. lactic.36 at 10°C. and degree of dissociation. Based on pH. lower aerobic plate counts of 3. lactic. indicating that citrate was the more effective compound. The activity of the undissociated portion of the acid was hydrochloric followed by lactic propionic. (1974).7% at pH 6.3. 1999). citric. When tomato juice was adjusted with citric acid to pH 4. lactic. Little bacteriostatic activity occurred at pH 5. and 1. 5. or malic acids did not change in physical or chemical attributes during processing compared with unacidified tomatoes (Schoenemann and Lopez. or tartaric acid to achieve an equilibrium pH of 4. aerobic counts for pH 3.66 at 30°C.5% sodium lactate was equally inhibitory as sodium citrate alone.12 log/ml. such as texture or appearance.4) in inhibiting Arcobacter butzleri in a broth system (Phillips. 6. and acetic (Brackett.0.09. Acidification impaired the color but enhanced the flavor of canned okra.0.5 with acetic and lactic acids. and 4. Okra. and hydrochloric. Canned tomatoes acidified with citric. 4. who determined that the minimum pH values that allowed survival of L. 1990). 1997). propionic.8 and 2. Interaction was demonstrated between citric acid and sodium citrate added to BHI broth adjusted to pH 4. Additional interactive effects were also noted in the presence of salt. then lactic. and the combination of 1. acetic.64 log/ml. (1989) reported that on an equal molar basis. 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.

4 and 5.3. Mushroom extract acidified with citric acid to pH 6. Cabbage and carrots were pretreated with the addition of a reduced calorie dressing or with a dip of 0. The addition of potassium sorbate did not enhance stability. Notermans et al. or 0. Growth curves developed for B. 6. 0. and 16°C (Valero et al. Samples pretreated with 1% citric acid displayed significantly lower total numbers.65 and asparagus with added citric acid or glucono-delta-lactone to pH levels of 5. monocytogenes (Dorsa et al.22.1.0 at temperatures below 16°C. the product should . but the shelf life was shortened at this higher temperature.31% CA–2.0% citric acid for 5 or 30 minutes.. cereus strains grown in model vegetable broths were conducted at typical refrigeration and abuse temperatures of 5°C. 0.34.1 with holding temperatures between 18°C and 20°C for up to 3 days before consumption.14% citric acid (CA). and 4.05 M) solution buffered to pH 3. sporogenes spores per g were heated to an F0 of 0.0% trisodium citrate (TCA). and lactic acid bacteria counts than samples dipped with the lower concentration of citric acid. 0. They found that acid blanching in a citric acid (0.. Only 2.05. and 121°C. 4.3 g/kg) for crawfish (Procambarus clarkii) stored at 4°C were not effective in retarding growth of L. Enteritidis PT4 at 22 than 5°C. Reduction in the thermal process time of canned liver paste was achieved through the addition of 0. 1992).5) at 12°C but did not grow in broth acidified to pH 5.. 1999).3 when at least 20 ml of lemon juice (citric acid) per fresh egg yolk was used.3. Citric acid dips (0. Both vegetable broths stimulated germination and growth of spores compared to nutrient broth. leading the authors to conclude that the extracts have a protective effect on C. 5. cereus could grow at pH 5.29% CA. Okereke et al. 1990b). The authors recommended that the final pH should be <3.108 Antimicrobials in Food Beelman et al.85.. 118°C. Xiong et al.. 5. If the amount of lemon juice was increased to 20 to 35 ml per egg yolk.9% and spoilage was further reduced to 16. thereby leading to its survival regardless of the change in pH (Ocio et al. 1989. 1992). 1990). 2000.0% TCA-0. This process improved quality and increased the microbial stability of canned mushrooms (Okereke and Beelman. 8°C. (1989) developed an acid-blanch-chelate (ABC) process designed to reduce mesophilic and thermophilic bacterial spoilage in canned mushrooms. 12°C. sporogenes PA3679 after a sublethal heating process (Okereke et al.. Citric acid dips for foods not only retard some spoilage but also act as a chelator of metal ions responsible for enzymatic browning reactions..14% CA (pH 5.2% or 1. cereus grew in zucchini broth (pH 6. 1991).. 5. sporogenes. Mayonnaise made with >5% citric acid solution adjusted to pH below 4. Extracts were then heated at 110°C.4% of cans containing mushrooms vacuum hydrated by the ABC process spoiled (Beelman et al...8. 1994.05 led to more inactivation of S. botulinum type B when held for 70 days at 15°C or 14 days at 20°C.4. but could grow at 8°C only when the pH was higher (5. This process inhibited C. Silla Santos et al. Although the heat resistance of C.8% with the addition of EDTA. 1990a).7 (control). By adding citric acid to can brine. The products were packed with or without modified atmosphere and stored at 4°C for 10 to 21 days. provided temperatures were at 16°C. 115°C. heat resistance was not affected by higher processing temperatures. B. (1985) dipped potatoes in a solution of 1% citric acid and 2% ascorbic acid for 2 minutes before vacuum packing and cooking. Microbial stability was achieved in liver paste formulated with 0.69) and processed with an F0 of 0. sporogenes decreased with decreasing pH levels.4 to 4.1% potassium sorbate (Houben and Krol.31% CA–2. sporogenes PA 3679 spores.5). Samples containing the reduced calorie dressing were even lower in microbial numbers than aciddipped vegetables (Eytan et al. indicating a synergistic effect between pH and temperature. Homemade mayonnaise has been a significant health hazard as a result of contamination by Salmonella from raw eggs (Membre et al.1 and 5. and 4.2. coliforms. Liver pastes inoculated with 106 to 107 Bacillus spores per g and 104 C. 2003). and 0. 1993). Growth in carrot puree was temperature dependent because B.5 were inoculated with C.9. 1997. this process reduced thermophilic spoilage from 68% to 23. The preparation of these products included adjustment of the pH with acetic acid to 3.5 followed by canning in a solution containing 200 ppm ethylenediamine tetraacetic acid (EDTA) controlled the outgrowth of spores of C.

the function of citrate appeared to be one of chelation of metal ions. citrate appeared to inhibit glucose utilization (Imai et al. malic. aureus showed inhibition both with increasing concentrations of citric acid and decreasing pH (Minor and Marth.5%.2 or 4.. 1968).. Sodium citrate in concentrations of 0. hard-cooked eggs allowed to equilibrate in 0. sodium citrate inhibited growth. or shrimp and tomato sauce acidified with citric or acetic acids to pH 4.25% concentration greatly reduced toxin production. suggesting that citrate was a chelator of ions essential for growth (Rammell. 1962).3% citric acid lowered the level of Salmonellae on poultry carcasses.1% to 4. 1976).75% lactic acid inhibited growth of Aspergillus versicolor. Sabouraud medium was adjusted with 0. Enteritidis. but 0.0.75% concentration of either acid did not affect the growth of P.125% to 4% citric acid or 0. No toxin was detected in any of these foods over an 8-week shelf life (Post et al.0% concentrations of citric acid and 0.2% sodium benzoate were held for 30 days at 4°C or . (1985) found no outgrowth or toxin production from spores of types A and B in TPGY medium acidified to pH 4. sodium citrate stimulated growth. 2%.25% citric acid and 0. Using the multiple-barrier concept. Mangoes are subject to spoilage through infection with Penicillium cyclopium (Palejwala et al. 1968). C. shrimp puree. In concentrations of 12 to 18 µM/ml. With Arthrobacter pascens. Branen and Keenan (1970) also suggested that citrate chelation of metal ions inhibited L. and in concentrations greater than 40 µM/ml.5% sodium citrate were inhibitory to Salmonella anatum and Salmonella oranienburg (Davis and Barnes.. aureus was overcome by adding calcium and magnesium ions.5% lactic acid prevented toxin production. A level of 0. 80°C.75%. Survival studies for Neosartorya fischeri ascospores were conducted in mango and grape juices and heated at 75°C. Sodium citrate can also exhibit bacteriostatic activity.2. 1984). Healthy tissue had a pH of 5. fluorescens on beef carcasses (Thomson et al. Concentrations of 12. 1998).8. As little as 0.0% to 12. S. 1984). but salt had a protective effect on this organism. parasiticus. When acetic acid was used. or tartaric acid. a 0. however. even in media containing up to 1% citrate. 0. Citrate inhibition of S. expansum (Reiss. The amount of sodium citrate had a dual effect on Lactobacillus casei. 1952). Tsang et al.8% gave a pH of 4. and rinsing with a citric acid solution adjusted to pH 4. Ascospores were able to withstand more than 6 hours of heating at 75°C. when 35 ml lemon juice per egg yolk was added. 1970). whereas infected tissue was around 4. A 0. lactic. the time decreased to 48 hours before use. The MIC of 0. 1967).0 reduced the numbers of P. During ripening.016% to 1% malic acid as the sole carbon source because these acids predominate at these concentrations in a ripening mango. however. The metabolic effects of citrate on Arthrobacter species were twofold.12 (Sinha et al. The addition of garlic or mustard increased the death of S.5 with citric. However. type E grew and produced toxin at 26°C in media containing citric acid at pH 4.75% for both citric and lactic acids had a slight growth-retarding effect on A.Organic Acids 109 be held at 22°C for longer than 72 hours. no growth occurred at pH 5. and 90°C. A level of 0. leading to enhanced growth. 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. 1985).08 to 4.0% was not as inhibitory to Streptococcus agalactiae when added to skim milk or fresh milk as citric acid at concentrations of 1%. Typhimurium than lactic and hydrochloric acids (Subramanian and Marth. Similar destruction values were seen with potassium sorbate and sodium benzoate. 5 hours at 80°C.. the level of acidity decreased and sugars and nitrogenous compounds increased. Citric acid provided the maximal destruction of ascospores at 85°C. 85°C..1 or less..5% citric or 0. and 3 to 4 hours at 85°C. Skim milk acidified with citric acid was more inhibitory to S. casei.6. botulinum showed no change in numbers when inoculated in tomato puree. with Arthrobacter simplex.6 and incubated at 35°C. 1970).8. Thermal resistance studies were conducted in mango juice adjusted to pH 3. and 4%. Radford and Board (1993) found that acetic acid was more inhibitory than citric acid and recommended that mayonnaise be prepared with vinegar to a pH or 4. or 1. it did not appear to affect attachment (Appl and Marshall.

5 depressed growth of L.2% concentration in combination with citric or lactic acid at pH 5. Daly et al.110 Antimicrobials in Food in 0. Horn et al. Short-term studies on rats fed 0.0 to reduce the growth rate of S.75% citric acid was sufficient to reduce inoculated populations of S. E. Cramer et al. It is highly water soluble and enhances the flavor of citrus-based foods. and jams and jellies. 4.2%. plantarum but did not affect P. It is a precursor of diacetyl and therefore indirectly improves the flavor and aroma of a variety of cultured dairy products. sherbets and ices. and S. Although there was no effect on weight gain. In another study. slight atrophy occurred in regions of the thymus and spleen (Yokotani et al.5. Studies by Yokotani and coworkers (1971) compared toxicities of commercial citric acid and Takeda citric acid. and rabbits indicated varied tolerance levels by different routes of administration (Table 4. Toxicology Acute toxicity data for citric acid in mice.5.7% sodium citrate (5% citric acid) fed to rabbits for 60 days produced no unusual effects (Packman et al. rats. (1956) fed vitamin D-free diets containing low calcium levels but adequate levels of phosphorus supplemented with 0. and it is used as an acidulant in canned vegetables and dairy products. (1982) used a combination of 0. It can control the pH for optimum gel formation.1% citric acid and 0. or 5% citric acid (Bonting and Jansen. Citric acid is approved as a GRAS substance for miscellaneous and general-purpose usage when used in accordance with good manufacturing practices. beverages. 3%. 1956. The authors concluded that citrate had a rachitogenic effect on the test animals.05% potassium sorbate and citric acid at pH 5.1625).75% citric acid alone for 21 days at 4°C. Y. however. 1944). Acute toxicity data of sodium citrate are shown in Table 4. aeruginosa (Restaino et al.75% glucono-deltalactone incorporated into sausage before fermentation. in the acid form (21 CFR 184.3.02 mol sodium citrate and citric acid. Restaino et al. or sodium salt (21 CFR 184. 2.. a by-product of yeast fermentation. Typhimurium.1033) or as the calcium (21 CFR 184. The number of cavities formed did not differ between the control and experimental groups. Horn et al.1195). aureus (Fischer et al. and 4. 7. Death by all routes and in all animals resulted from respiratory or cardiac failure and hemorrhaging of the gastric mucosa..2).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. Long-term studies involving rats fed quantities up to 1. 1957) showed no abnormalities other than lowered weight gains and feed consumption in the 5% group. 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. or 12 g citric acid per kg of a noncariogenic diet to show the possible effect of citric acid on the development of teeth. Application and Regulatory Status Citric acid is a tricarboxylic acid having a pleasant sour taste. salad dressings.4%. A concentration of 0. Citric acid also acts synergistically with antioxidants to prevent rancidity by chelating metal ions (Gardner. aureus early in the fermentation and the increased acidity of the meat mixture allowed the fermentation to proceed more quickly. 1985). 1971). . The acceptable daily intake for humans is listed in Table 4. 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. the highest dosage contributed to enamel erosion.1751).. (1957) evaluated dosage levels in mice given the acid intravenously. enterocolitica.2. the urine contained calcium citrate.. 1972). It is approved for use in ice cream. (1973) used a combination of 0. Potassium sorbate at 0.2%. fruit preserves.. coli. potassium (21 CFR 184.8% level. 1981). 1963). rouxii and Saccharomyces bisporus. This combination inhibited the growth of S. Deaths resulted from acute acidosis and lung hemorrhages. Dalderup (1960) fed rats 1.

or tartaric acid based on weight when added to the heating medium. The fumarate esters not only prevented mold growth on bread (Huhtanen et al. Fumaric acid was also more effective than lactic or acetic acid when used in meat products (Podolak et al. 1988).5% (pH 2. botulinum swelled in 4 to 6 days at 30°C. The use of fumaric acid to effect a 5-log reduction of E. The addition of 0.5. Monomethyl and monoethyl (0. 1987). Similar effects were noted with N.15%) served as an acidulant (Ough and Kunkee. citric. Medium chain-length alkyl esters had more activity than aliphatic acids (Dymicky and Trenchard. 1996a. 1987).0) were not lethal to T. botulinum types A and B in canned bacon. In addition to reduction of E. At the 1. When used in vacuum-packaged ground beef patties. fischeri (Conner and Beuchat. respectively.15% fumaric acid coupled with 0. 1981. Islam. citric. 0. Fumaric acid was more fungicidal against Talaromyces flavus ascospores than acetic. 1982). The n-monoalkyl fumarates and maleates esterified with C13–C18 alcohols also possessed significant antibotulinal activity (Dymicky et al.0% fumaric acid and 1. Fumaric acid at concentrations of 1..38% sodium fumarate or guinea pigs fed 1. coli O157:H7 reduced levels by 1 and 1. flavus ascospores (Beuchat.6 L in the vapor phase were also effective as a mold preventive (Huhtanen and Guy. However. Fumaric. 1987) heated in the presence of 2..5% concentration of fumaric acid inactivated ascospores after 20 minutes of heating at 80°C. whereas the presence of fumaric acid esters in the bacon prevented swelling for up to 8 weeks.3 logs. 1975).0% fumaric acid did not show any abnormalities.1%) controlled fungal growth in tomato juice.. The 2-year studies using rats fed 0. total aerobic counts were reduced to levels below those achieved through pasteurization.0% acid concentration at 82°C..15%.b). psychrotrophic. These same preservatives provided a similar reduction in less than 5 hours and 2 hours when held at 25°C and 35°C. Methyl n-alkyl fumarates demonstrated lower activity.5 to 5. and 1. Cans packed with bacon and inoculated with C. but malic acid did not. or 0. 0. Monomethyl. coli O157:H7. and ethyl esters of fumaric acid in concentrations of 0. Human feeding .05% sodium benzoate followed by holding at 25°C for 6 hours before refrigeration at 4°C for 24 hours achieved reduction. monocytogenes and E.0 to 2. coli O157:H7 inoculated into unpreserved apple cider was investigated by Comes and Beelman (2002). It was speculated that the antimicrobial properties of fumaric acid were related to a lower pKa than lactic or acetic acids. acetic.5% applied for 5 seconds at 55°C to the surface of lean beef inoculated with L.2%) and dimethyl and diethyl fumarates (0.9% (5% fumaric acid) showed no toxic effects (Packman et al.5%. but the dimethyl and diethyl esters at a concentration of 20 mg per 2. 0.125%. 1982).5% level. citric.Organic Acids 111 FUMARIC ACID Antimicrobial Properties Fumaric acid (0.. fumaric acid achieved the greatest reduction of aerobic.1% and 1. rats showed an increased mortality rate and more atrophy of the liver (Fitzhugh and Nelson. malic. 1963). 1947). and tartaric acids increased heat inactivation. and tartaric acids at a concentration of 2. Toxicology Weanling rats fed 0. A 0. respectively.0% and 1.1%. 1974) and inhibitor of malolactic fermentation in wine (Pilone. and lethality increased as the pH decreased from 5.2% fumaric acid or rabbits fed sodium fumarate at a concentration of 6. and coliform bacteria.8%. and di-n-alkyl fumarates were almost inactive (Dymicky et al. malic. dimethyl.05%) or sodium propionate (0. 1984). Huhtanen (1983) and coworkers (1985) investigated the effect of various esters of fumaric acid on growth of C.2% showed considerable promise as a substitute for sodium nitrite in bacon. Fumaric acid by itself was only slightly inhibitory.

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

sub-primal meat was injected with one of three treatments: 0.1% ascorbic acids adjusted to pH 2. 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. 2% acetic. 1990a. 0.b). Typhimurium at 70°C. coli. In addition. coli O157:H7 and S. 1999). As in beef trim. 2001a. Lactic acid was most effective against S. or 82°C. coli counts was detected for beef dipped in acetic or lactic acid solutions compared with a water-dipped control at day 1. coli biotype 1 counts. Application of 95°C water alone or with 2% L-lactic acid was effective in reducing E. 71°C. b). coli (Anderson and Marshall. 426°C.3 M lactic acid solution (LAC. The inoculated meat was subjected to sprays of tap water (15°C to 17°C. coli was reduced by 6. (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..6 with either 1% lactic acid at pH 2. reducing numbers by 2 logs and Enterobacteriaceae by 1. pH 5. or a combination (1:1) CAL/LAC (COM. Treatments containing lactic acid resulted in the lowest counts without affecting sensory qualities. COM improved tenderness in muscle cuts. Minimal reduction in total plate and E.3 M calcium chloride solution (CAL.0). Kotula and Thelappurate (1994) compared acetic acid and lactic acid solutions applied at two concentrations. and sensory analysis were not affected by acid treatment. Furthermore.1).6% or 1. The combination of acids performed as well as 1% lactic or acetic acids alone. 2001a. hot water (30 psi) at 65°C. Typhimurium were subjected to an automated wash water system (Castillo et al. Significantly higher aerobic plate counts were obtained from hot-boned cuts over 17 days postmortem compared to cold-boned cuts. 2001). 1998. (1987) compared a combination of 1% lactic. 1994). 0.. combinations of the treatments were examined.. Similar work was conducted using lean pork trim (LPT) and fat-covered pork trim (FPT) (Castelo et al. 76°C. Beef trim meat used to make ground beef is of particular concern because of its higher microbial load. but control cuts retained more desirable flavor profile (Eilers et al. Acid-treated samples were lighter in color as a result of leaching of the pigment during immersion.8 to 7. 482°C..Organic Acids 113 Hot-boned.9 or 1% acetic acid at pH 3. 30 psi). and presumptive lactic acid bacteria. Greater reductions on all microbial loads were seen in BTF compared to BTL.3. When temperature changed from 20°C to 70°C or lactic and acetic acid concentrations increased individually to 3%. When sequential treatments were employed using pre-chill and postchill lactic acid sprays. Aerobic plate counts of meats injected with CAL were significantly higher than with LAC or COM. but shear values. 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. 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. psychrotrophic.2%. but odor was not affected (Ellebracht et al.5 logs for E.. and at two time intervals. and 0. coli O157:H7 or S. Postchill beef rounds inoculated with E. only suggesting that lactic acid can exert a continued antibacterial effect during the shelf life of the product. 65 psi). The system alone produced a 3log reduction. .25% citric. All products were stored at 4°C and examined for aerobic. 20 or 120 seconds. <1 log for Enterobacteriaceae and 0. E. and E. 0. at 1°C to 2°C as a dip for rib eye steaks. Dixon et al. but this effect was not seen with acetic acidtreated tissue.b).5 logs (Anderson et al. pH 3. or 510°C. Meat color was affected after application and through storage at 4°C. Acuff et al. There was little difference in bactericidal activity between the combination and either acid alone on sub-primal cuts of beef. Typhimurium-inoculated beef trimmings. moisture content.. 2% lactic acid (12°C to 15°C. aerobic bacterial counts and S. 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. 454°C. 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. the FPT supported lower microbial numbers than LPT. 1992). Typhimurium levels decreased approximately 1 log.5) at a volume equal to 10% of the initial sub-primal weight.2 logs. total fecal coliforms. or hot air at 371°C. pH 2.

Elimination of these groups shifted the population to Gram-positive bacteria and yeasts. 1988). significant reductions were seen with all treatments. Beef was inoculated with L. coli O157:H7. there was no significant difference between nisin. Gram-negative bacteria were the most sensitive to lactic acid. malic. 1992)... noncoated tissue became dehydrated during the 7-day study. and 0.5%). however. The use of lactic acid for reduction of microbial numbers or pathogens has been similarly applied to pork products. but no differences were seen between polylactic acid and lactic acid. whereas acetic acid was more effective than the acetic acid/gel treatment. 180. Beef cubes were inoculated with L. monocytogenes before vacuum packaging and storage at 4°C. 40.. monocytogenes. 1999). 2002). After 28 days of storage. psychrotrophic Gram-positive bacteria. lactic acid.. or 360 seconds to 2% lactic acid. acetic acid (0. monocytogenes on beef tissue. coli O157:H7 in beef burgers. 1993). 2000). 55°C) had little effect on the aerobic plate counts taken from the surface of pork carcasses.6 for Bacillus stearothermophilus and pH 4. 1993). Lactic acid (2%). lactic acid. These compounds reduced the level of L.6 log/6 cm2. 1992.2 for B.. lactobacilli and yeasts were the least sensitive. 1. 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. coli O157:H7 was subjected to treatments of lowmolecular-weight polylactic acid. Five treatments were delivered to the pork skins: water-treated (control) or exposure at 21°C for 120.1. but the population of microorganisms shifted. monocytogenes. these treatments were not deemed usable in the control of E.7. and nisin did not enhance the effects of lactic acid against E.. 2002). The combination of acids and heat treatment led to reduction of spore-forming organisms in frankfurters. coagulans were heated to 121°C and 105°C or 110°C..8. A 1% concentration of lactic acid (pH 2. Greater inactivation was noted for both organisms at the lower pH level when acetic or lactic acids were used but not with citric. Therefore. Lactic acid was effective in decreasing the number of Enterobacteriaceae as well as other Gram-negative mesophilic and psychrotrophic spoilage organisms. or nisin and lactic acid. and low-molecular-weight polylactic acid. by immersing porcine livers in 0. Enterobacteriaceae.114 Antimicrobials in Food Lactic acid (2%). Psychrotrophic. Y. coli O157:H7 inoculated into beef trimmings (Bolton et al. and storing for 5 days at 3°C. By increasing the concentration of lactic acid to 2%. and 200 IU/ml of nisin (Mustapha et al. Typhimurium were reduced more by exposure to acetic acid/gel treatment than acetic acid alone. The use of these compounds was effective in reducing psychrotrophs and Enterobacteriaceae (Ariyapitipun et al. 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. and Lactobacillaceae counts by 2 to 3 logs. monocytogenes and treated with 2% lactic acid.5 and 4. Numbers of S. . Lactic acid treatments alone were only 50% as effective as acid/alginate treatments. Aeromonas hydrophila.000 IU/ml nisin. Beef inoculated with E. After 42 days of storage the initial inoculum of 5 logs was reduced to <1 log after using lactic acid. enterocolitica. Frankfurter emulsions incorporated with acids to adjust pH to 5. Woolthuis et al. monocytogenes by 1. monocytogenes (Ariyapitipun et al. All treatments significantly reduced the number of L. (1984) effectively reduced total plate counts. 240. or hydrochloric acid (Lynch and Potter. Not only did the microbial counts decrease as the time of decontamination increased. and nisin (400 IU/ml) were used alone or in combination in beef to inhibit L.2 or 4. vacuum packaging. numbers of Salmonella species and Campylobacter species were reduced immediately and remained lower 24 hours after slaughter (Epling et al.2% lactic acid for 5 minutes. or sodium lactate (4%) alone or in combination with pulse electric field or freezing was ineffective in reducing numbers of E. Salmonella species or Listeria species were not recovered nor were sensory characteristics affected (Prasai et al. nisin. 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. Although the gels reduced populations of L. 1993). respectively (El-Khateib et al. low-molecular-weight polylactic acid (2% PLA). and polylactic acid. followed by mesophilic Enterobacteriaceae. and 3200 units/ml pediocin.

other compounds. sodium acid pyrophosphate. acidified with lactic acid. monocytogenes counts were reduced by 0. fragi. 1990b). Although L. pH 2.3 to 2. Lactic acid was more effective than acetic acid when used as a 10-minute rinse for fresh-cut vegetables. or formic acids. although tissue treated with acid affected the growth rate. E. Lactic acid added to scald water alone had minimal effect on reducing the numbers of contaminated birds.88 or 0. Lactic acid added to broiler chill water resulted in the development of a brown coloration. S. 1995). A 10% lactic acid and sodium lactate-buffered acid spray (pH 3. P. 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.Organic Acids 115 P. shelf life was lengthened to >36 and 35 days at 4°C and 7°C. This protective effect was even more pronounced with acetic acid (100. tissue was dipped for 15 seconds in water or lactic acid and held for 15 days at 4°C. 1995. Hwang.62) were combined with propylene glycol (20%) in chill water.25%. Chicken skin subjected to washing treatments using 1% lactic acid led to significant reductions of Salmonella species and L. E. 1995). produced similar reductions of approximately 1 log (Zhang and Farber. or sodium hexametaphosphate.5% lactic acid/0. The interaction of sodium chloride (0. E.7 log CFU/g (van der Marel et al.000-fold). thermosphacta. After inoculation. discoloration still occurred and propylene glycol contributed an objectionable flavor (Izat et al.2) was examined for E. however. pH 2.5 and 0.23 in cells exposed to acid to 5. coli O157:H7 rapidly died at pH 4. coli O157:H7 grown in TSB at 37°C (Casey and Condon. 1996). 1990). coli demonstrated a 10-fold decrease in numbers when grown in media containing sodium chloride.79 in acid-free media.. 1995). In an effort to eliminate carcass discoloration.. 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.2 logs. 2002).1°C.5%.2 in media containing lactic.0) for chicken legs increased the shelf life from 6 to 12 days at 6°C. however. respectively (Sawaya et al. Higher concentrations of the acid did not ensure greater decontamination nor did repeated treatments. monocytogenes compared with monosodium phosphate. pH 2. such as Pseudomonas species. These treatments inhibited hydrogen sulfide-producing bacteria. acidified media. Total microbial numbers from skin of birds immersed for 15 seconds at 19°C in 1% or 2% lactic acid at pH 2.8) and scald water (54°C/2 minutes) reduced the bacterial level of broilers inoculated with S.5% to 4%) and pH (4. Salmonellae were eliminated from broiler carcasses after a 1-hour exposure. fragi and B.7 log CFU/g and Enterobacteriaceae from 3. respectively.3 prolonged shelf life to 8 days (Zeitoun and Debevere. and A.6 log CFU/g. 1988). Lactic acid dips have also been used successfully in the poultry industry. hydrophila numbers declined by 5 logs within 11 days of storage. 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. Acid treatments of fat tissues led to the reduction of pathogen numbers to nondetectable numbers within 4 days. The spoilage organisms.. and a 2% lactic acid dip at pH 2. 1990a). in sodium chloridefree. Consumer evaluation of chicken treated with a 0.9 to 2. sodium tripolyphosphate. The treatments did not affect sensory quality. grew on both tissue types after acid treatment. 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. such as chlorine or chlorine dioxide.000-fold. When treated broilers were packaged using modified atmospheres. 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. coli decreased 10. acetic. .0 than cells grown at 37°C. Levels of psychrotrophs decreased from 3. Typhimurium to almost nondetectable numbers. 1983). but no protective effect was observed if the sodium chloride was added to the medium 45 minutes after the inoculation. None of the pathogens grew on lean tissue. and Beuchat. Lactic acid (1%) added to both chill water (0°C to 1. reduced levels of lactic acid (0.5% sodium benzoate dip concluded that sensory qualities were acceptable (Hathcox et al.. The number of Salmonella-positive birds was also reduced as a function of time of the dip (Izat et al. that contributed to spoilage.2 to 3.. or Brochothrix thermosphacta (Greer and Dilts.2 decreased from 5.

although Campylobacter was particularly sensitive with an MIC of 179 mM. dehydration.. retains color. syrupy liquid having a moderately strong acid taste. and beverages. (1987) found that A. lactis. and lactic acid concentration has been developed (Mellefont et al. Application and Regulatory Status Lactic acid is a hygroscopic. (1970) recommended that the L (+) form should be used in feeding premature infants. cultures decreased production of aflatoxin G1. Toxicology The LD50 of lactic acid for test animals varied according to animal species (Table 4. The Food and Agriculture Organization of the United Nations (Food and Agriculture Organization. improves juiciness.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. aureus and Lactobacillus coryniformis to 804 mM for Brochothrix.6 mg/100 g) did not differ in growth rate compared with control groups (Granados et al. 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. sherbets.. Ross et al. contributes to water-holding capacity.75% lactic acid adjusted to pH levels of 3. The MIC of sodium lactate was investigated under optimum growth conditions (pH 6. The autopsy revealed hemorrhaging and gangrenous gastritis (Young and Smith. Premature infants fed acidified milk containing the isomeric forms of lactic acid.. D (-) or DL. and in baking powders. The acceptable daily intake for humans is listed in Table 4. developed acidosis. Luchese and Harrigan (1990) also noted an increase in aflatoxin production adjusted to pH 4. Lactic acid is approved as a GRAS substance for miscellaneous or general-purpose usage (21 CFR 184. A predictive model for growth of E.5. infants suffered stricture of the esophagus following ingestion of milk acidified with 1 teaspoon of 85% (Pitkin. Aflatoxin production also increased during growth of A.5% and 0. Some enamel decalcification was apparent in the experimental groups. and vomiting. pH. In two other instances.5 or 4.116 Antimicrobials in Food El-Gazzar et al. coli in response to temperature. 2003. water activity. jellies.3.5% lactic acid (Trainer et al. Infants died after consuming milk acidified with an unknown quantity of lactic acid.. and extends the shelf life of products. Brochothrix. 20°C) for a number of bacteria and yeasts isolated from spoiled and unspoiled meat products (Houtsma et al. Gram-negative bacteria including Pseudomonas and Yersinia had a comparable range to Salmonella. Hamsters fed a cariogenic diet incorporating lactic acid in the drinking water (40 mg/100 ml) or in the feed (45. Bacillus. see Shelef (1994) and De Wit and Rombouts (1990). and Staphylococcus ranged from 268 for S. 1945). confectionery products.2 using HCl or lactic acid stimulated by substituting half of the carbon content by lactate and a reduction in production at pH 6.2).8. 1944). Carnobacterium. For an excellent review of the antimicrobial nature of sodium lactate. parasiticus in association with L.. 1949). 1973) supported this view. Ballabriga et al. weight loss. Lactic acid is used in the manufacture of jams. It is used to adjust acidity in brines for pickles and olives. monocytogenes and L. Yeasts were very broad in .. parasiticus NRRL 2999 grown in a laboratory medium containing 0. Lactococcus. MIC values for Salmonella species ranged from 714 to 982 mM and a narrower range for L. Therefore. Calcium lactate is used as a firming agent for apple slices. 2003). With increasing levels of lactic acid. appears naturally in meat products. increases meat yields. Presser et al. to prevent discoloration in fruit. 1998.1061). Gram-positive bacteria including lactic acid bacteria. 1993). 1935) or 87. innocua from 804 to 982 mM.

. The effect of sodium lactate on toxin production. A subsequent study (Shelef and Potluri.1. As expected L. Salts of lactic acid are equally as effective in muscle food systems. 20°C. The effect of sodium lactate and sodium chloride on the growth of L.Organic Acids 117 their sensitivity. and 4 using 0.5%.5%. 6. 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. 1994a). lactates also contributed to lowering the aw of a food. Using 108 CFU/g as a microbial end point. Typhimurium when stored for 4 days at 20°C. with Debaryomyces and Candida being the most resistant at 1161 and 1339 mM. monocytogenes per gram of sausage. The pH level was influential in inhibition by sodium lactate but not by sodium chloride. 0. This model was then evaluated for use in a bologna-type sausage system (Houtsma et al. monocytogenes as the target organisms. 1. 1994b). but the combination of 2% or 3% lactate and 2% sodium chloride at the same moisture level was inhibitory. 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. and 30°C (Houtsma et al. indicating that inhibition was not the result of a lowering of water activity.. monocytogenes was studied in a cooked meat model system containing varying moisture levels and 4% lactate. 1996).. inhibition by sodium lactate was directly related to pH and temperature. and 2 M concentrations (Buchanan et al. 1995) examined the effect of 3% sodium or calcium lactate added to pork liver sausage and various heat treatments. and potassium lactate were equally effective. Potassium lactate (4%) and sodium chloride (3%) reduced growth somewhat at 5°C. Lactic acid/sodium lactate and acetic acid/SA were studied in a BHI broth at pH 7. Calcium lactate (2%) reduced populations below the inoculum level. 1991. innocua was modeled in broth adjusted to pH 5. L. As expected. 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. At a higher temperature of 20°C. This level was listeriostatic at a moisture of >55%. At the abuse temperature of 20°C.0. its concentration. 6. 1% sodium lactate delayed growth for 10 days. 1993). Sodium chloride at the same water activity as sodium lactate (0. and the resultant pH. Samples were stored up to 50 days at 5°C and up to 10 days at 20°C. Survivor curves were developed using L.0. which might explain their prevalence in spoiled meat products. no toxin formation was detected after 49 days of incubation in media containing 3% sodium lactate. 10°C. and heat resistance was tested on proteolytic C. whereas a 2% level extended the time to 24 days. the rate of inactivation was dependent on the acid. potassium. 1995). Chen and Shelef (1992) suggested that in addition to pH change. calcium.982) did not inhibit toxin production. botulinum strains in peptone yeast extract medium (Houtsma et al. and 7. Pork liver sausage was formulated with sodium.0% sodium lactate. and 15 days for 2. There did not appear to be a synergistic effect between a 4% concentration of lactate combined with 140 ppm .5. no growth or toxin formation was detected after 32 days of incubation for media containing 4.5. with lower amounts of sodium lactate needed as pH and temperature dropped. At 30°C. spore germination did not lead to growth after 7 days. 1993). 6. 4% sodium or potassium lactate achieved reductions in L.. or 4% and inoculated with 104 to 105 L. 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. Addition of 2% sodium chloride enhanced the listericidal activity of the lactates (Weaver and Shelef. toxin formation was noted within 5 days for 0%. 5. spore germination.5. however. Sodium. or calcium lactate at concentrations of 2%.0 at 4°C. Calcium lactate was also more effective than sodium lactate in retarding growth of E. 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. respectively. As expected. 11 days for 1. The calcium form was found to be more inhibitory than the sodium form. coli O157:H7 and S. monocytogenes.. toxin production was detected in media containing 4% sodium lactate within 11 days. 3%. comparable to a 3% concentration of calcium lactate.

5 in a meat system prevented anaerobic growth of Serratia liquefaciens. or E. enterocolitica.05 and 6.2%. 2002). Lactates inhibited the growth of psychrotrophic organisms and delayed the pH decline (Bradford et al. Another study (Bedie et al. Minced beef containing 0%. Levels of >3% sodium lactate or 1% each of lactate and diacetate were needed to prevented growth of Listeriae for 60 days at 4.5% SA or sodium diacetate as an antilisterial process for frankfurters. enterocolitica or L. coli O157:H7 (Miller and Acuff. S. monocytogenes was delayed for 4 and 12 weeks at 7°C and 3°C in unsmoked.0% potassium lactate showed differences in spoilage rates. sodium lactate permitted aerobic growth of B.0% potassium lactate were inoculated with a five-strain cocktail of L.25% or 0. 1994). Over the storage life of the product. hydrophila and aerobic growth of the aeromonads (Grau. and E. The heat resistance of the two organisms was significantly lowered in beef containing either of the two concentrations of sodium lactate. monocytogenes. the amount of L-lactate ordinarily found in muscle was not inhibitory to any of the strains.4%. The pH of the product was unaffected by incorporation of sodium lactate.4%. . 2002). Cooked beef top rounds containing 1%. C. Papadopoulos et al.. 2%. 0. monocytogenes. In retail products. 2002). 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. At pH 6. 1999).8% sodium lactate was inoculated with Y. Papadopoulos et al. or 3.5. Cured meats.6% sodium lactate into the product (Kilic et al. 1981). Red color was preserved by using 2% to 3% sodium lactate. uncured bratwurst manufactured with 3.0% or 3. then cooked and vacuum-packaged the roasts.5 log CFU/cm2 in the same period. Although Shelef and Yang (1991) suggested a possible aw effect for sodium lactate. Enterobacter cloacae.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. lactobacilli. monocytogenes and heated to 55°C to simulate a sous vide process (McMahon et al.c) injected beef top rounds with 0% to 4% sodium lactate. or 4% sodium lactate were inoculated with L. micrococci. 1980).1% sodium diacetate (Glass et al. botulinum. 2001) compared the use of 3% or 6% sodium lactate.. nitrite levels are reduced. aureus. When raised to 210 mM. Typhimurium.4% sodium lactate/0. Both concentrations of potassium lactate significantly inhibited or delayed the growth of L. Sodium lactate at concentrations of 3% and 4% were generally effective in limiting growth of L. monocytogenes and stored at 4°C and 10°C for up to 90 and 60 days. Lactate increased cooking yields. 1997). Typhimurium. The growth of L. 2. Retention of nitrite was linked to higher pH levels and was unaffected by the incorporation of 1. Although the aerobic plate counts for control meat increased from 2. 1991).. enhanced flavor at the 1% level by adding a salty taste.7 to 8.. thermosphacta down to pH 5.5 reduced surface discoloration in chubs of fresh sausage and extended shelf life from 10 to 20 days. S. S. The microflora changed from predominantly staphylococci and micrococci at 0 days to coryneform. A 3% concentration of sodium lactate at pH 7.118 Antimicrobials in Food potassium nitrite. sodium lactate caused more rapid surface discoloration (Lamkey et al. Sodium lactate was less effective against microorganisms when sausage contained soy concentrate.. monocytogenes during storage at refrigerator and abuse temperatures. the plate counts of treated roasts increased to 6.. Pork sausages manufactured with 25% fat or 8% fat with and without 3. coli O157:H7 and stored at 10°C. rely on sodium nitrite to inhibit growth of C.25% sodium diacetate were the most effective treatments throughout the 120-day shelf life conducted at 4°C with minimal change in pH. Frankfurters containing 2. and yeasts at 42 days to homofermentative and heterofermentative lactobacilli and streptococci at 84 days. (1991b) did not find this effect in their studies. perfringens. Y. or 4. 3%. Sodium lactate at a 6% concentration or 0.0 log CFU/cm2 after 84 days of storage. but produced a mild throat irritation at a 4% concentration. respectively (Porto et al. such as wieners.b. 1993). and A.1. (1991a. A 100-mM concentration of sodium lactate buffered to pH 5. 2...

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

0 or 4. Salmonella Senftenberg. food consumption decreased and growth rate declined (Hazelton Laboratories. It has a strong acid flavor but does not have the same buildup of acid taste as other acids.. 1971b).0. probably as a direct effect of pH manipulation. primarily for its flavoring and acidification characteristics (Gardner. As the pH decreased to 5. It is used in sherbets and ices. a 2% lactate and 3% sodium chloride inhibited L.3.1069).0. 2%. dicarboxylic acid with high water solubility. 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. Malic acid is approved as a GRAS substance for miscellaneous and general-purpose usage (21 CFR 184. sodium chloride. anatum. The acceptable daily intake for humans is listed in Table 4.1207). at pH 6. and sodium lactates (21 CFR 184. 1971a). however. 1970). PROPIONIC ACID Antimicrobial Properties Propionic acid inhibited spore-forming bacteria. and . vacuum packaged. Reproductive experiments using rats fed 1000 and 10. encapsulated salt.1% sodium erythorbate. Application and Regulatory Status Calcium (21 CFR 184. monocytogenes for up to 50 days at 5°C. 1992). 1994). 1972). 1993). MALIC ACID Antimicrobial Properties Malic acid is inhibitory to fungi and bacteria. jams. jellies. and stored at 5°C or 10°C. and hydrolyzed vegetable protein and containing 1%. monocytogenes and total aerobic plate counts (Harmayani et al. Sodium lactate displayed synergistic activity with nitrite and increasing concentrations of sodium chloride. Chung and Goepfert (1970) compared various organic acids to determine the highest pH level that inhibited growth of S. aureus at pH 3. Toxicology Malic acid fed to rats at levels of 500 and 5000 ppm in the basal diet had no effect.1639).8% sodium lactate. sodium lactate was more effective than the other additives in inhibiting growth of L.120 Antimicrobials in Food Raw and cooked ground beef were prepared with 1.. potassium (21 CFR 184. subtilis). and sodium nitrite. Nunheimer and Fabian (1940) found that malic acid was inhibitory against S. 1975b). 0. monocytogenes.1768) are also approved.3. Application and Regulatory Status Malic acid is a nonhygroscopic. Dogs fed the same three levels of malic acid showed no effects using the same parameters (Hazelton Laboratories. Some discoloration and lipid oxidation occurred. The acceptable daily intake for humans is listed in Table 4. inoculated with L. at 50.000 ppm. 1% kappa carrageenan. monocytogenes and stored aerobically at 4°C.000 ppm malic acid before mating showed no significant differences from the controls (Hazelton Laboratories. the acid inhibited yeasts and molds but not to the same extent as bacteria (Woolford. The products were inoculated with L.98. fruit preserves. Similar studies used low-fat ground beef patties formulated with carrageenan. especially rope bacteria (B. and beverages. In general. or 3% potassium lactate. thus affecting the sensory qualities of the meat (Egbert et al. but the addition of 2% or 3% potassium lactate reduced microbial numbers. Carrageenan had no effect on bacterial loads.. Comminuted salmon was mixed with sodium lactate. and an alginate binder.

and lactate. (1991) determined that 0. pH 4. monocytogenes was the result of incubation temperature. concentration of the organic acid. little activity was ascribed to EDTA in combination with the acids.3% concentration in tryptose broth adjusted to pH 5. and berries but also prevented spoilage in vegetables having a more neutral pH. fumaric and malic. Incorporation of calcium propionate into coatings for ready-to-eat products was effective in reducing problems with L. citric. or at a level of 0. pH levels. 1%) and coated onto ready-toeat chicken samples dipped in L. Nondetectable levels of the pathogen were found in products ZEN.5 to 0.3% concentration was also effective at pH 5. tartaric. 4. ZPNCP. and 0. Turkey breast meat was formulated with sodium salts of lactate. lactic.0 and 13°C. and stored at 28°C. monocytogenes were undetectable. Sarcina lutea. Toxin production occurred after 2 days for pyruvate. Calcium propionate prevented rope formation caused by B.6 with acetic. ellipsoideus by as much as 5 days. Zein (Z) film coatings were dissolved in propylene glycol (ZP) or ethanol (ZE) with and without nisin (N. the organism was able to grow at up to a 0. applesauce.4. Inactivation of L. The acids contributed to approximately 85% of the antimicrobial activity with 14% contributed by ascorbic acid. pH 4. and S. and held at 28°C for 0 to 18 days. 1989a). and cell-wall synthesis decreased. 1. 21°C.1% to 5% delayed growth of S. pH 5. the levels of L. acetic. acetate.5. Reduction of pH to 5. or 3. toxicity occurred after 7 days for pyruvate. . At pH 5.. mesentericus (subtilis) in bread dough at a level of 0. 18 days for citrate. In laboratory studies examining the effect of sodium propionate on L. citric). When media containing 0. inoculated with C. and temperatures. 5.0 minimized growth at 13°C. 1990). the lag phase of L. the rate of RNA (ribonucleic acid). aureus. or ZENCP.6. 1992)... L.08% EDTA. syrup. monocytogenes grown at 13°C was extended by 7. citrate. (1995) examined the combination of sodium propionate and SA at a number of concentrations. A three-strain mixture of L. monocytogenes when used in conjunction with acetic acid in cold-pack cheese food (Ryser and Marth. Cherrington et al.7 M propionic acid at pH 5. monocytogenes. 1992). 13°C. and 5 with a 2% concentration.0 with hydrochloric acid. 19°C.1. 2002). 1941).0. and 4 days.0 in reducing numbers of L. 4 for lactate and acetate. coli. This substance not only postponed spoilage in fresh figs. lipid. pyruvate. aureus.3% sodium propionate was adjusted to pH 5. DNA (deoxyribonucleic acid).000 IU/g) or calcium propionate (CP. At a 6% concentration. S.188%. cherries. 21°C. or 4°C. Sodium propionate at levels of 0. protein. such as lima beans and peas (Wolford and Anderson. Emphasizing the multiple-barrier concept in retarding growth. 7. which led to an increase in cell mass without cell division and a decrease in viability (Cherrington et al. pH 4. monocytogenes. and Candida albicans. Much higher concentrations of propionic acid were bacteriostatic to B. plantarum. Golden et al. subtilis. botulinum. pH 5.6 and incubated at 4°C. and pH.5 inhibited the growth of Salmonellae at a higher pH level than other acids (pH 5. At a 5-mM concentration. The salts of propionic acid are also effective antimicrobial agents.02% ascorbic acid. At periodic intervals. samples were removed from storage and tested for production of neurotoxin. and greater than 18 days for propionate. acetate. monocytogenes to 8% sodium propionate for 60 minutes caused injury (Buazzi and Marth. pH 4. respectively. monocytogenes (Janes et al. pH 4.8. succinic. Propionic acid was more effective than acetic acid as an antimicrobial. Eklund (1985) concluded that propionic acid was not as inhibitory as benzoic acid for P. or lactic acids in media (El-Shenawy and Marth. adipic. aeruginosa.5. Exposure of L.0. Propionic acid at pH 5. 1993). Sodium propionate at a 0. 1988). and 35°C. Citrate was most effective based on molarity (Miller et al. tartaric. degree of dissociation of the acid.3.05.1. 3 for citrate.4.156%. 3.Organic Acids 121 Salmonella Tennessee when all other parameters were at their optimum. pH 5. adjusted to pH 4. particularly at the higher pH level. Torula species. 0.0 was more inhibitory in 60 minutes compared to formic acid in 3 hours for E. monocytogenes was inoculated into BHI broth containing 0. Products were stored for up to 24 days at 4°C or 8°C. Proteus vulgaris. E.9% sodium propionate or SA. 1945). coli K12 and Salmonella. and propionate to a target level of pH 6. and 35°C and prevented growth at 4°C (El-Shenawy and Marth.6 (O’Leary and Kralovec.

5 and water activity of 0. Graham and Grice. coli. The antimicrobial activity of sodium propionate was most affected at 4°C by fat concentration with a 1.03%. A 5% calcium propionate solution acidified to pH 5. 1. (1963) determined the LD50 of calcium and sodium propionate for rats (Table 4. herbariorum.5 with lactic acid was as effective as a 10% unacidified solution in preventing surface mold growth on butter (Olson and Macy. flavus and niger.85 (Guynot et al. Molds were inhibited by less calcium propionate by weight than sodium propionate. 1953).8% sodium lactate.. rather than later (Ghosh and Häggblom. Pseudomonas species. monocytogenes was studied in pork liver beaker sausage (Hu and Shelef.2% sodium propionate.8. .8 log reduction at 67% fat. no growth occurred. Hara. and Penicillium corylophilum. 2002). Propionic acids and their salts are primarily inhibitory to molds. and repens were inoculated by needle into cake analogues prepared with calcium propionate. B. Subsequent studies (Marin et al. 0.85.0. Toxicology Orö and Wretland (1961) determined the LD50 of propionic acid for mice.8 to 0.5 log reduction of L.b) examined several levels of calcium propionate.0 and a water activity of 0. rubrum. Although mold spores are destroyed during baking. 1952a). 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. A.3%) at pH 4. reduced growth and aflatoxin formation of A. Mold spoilage is a significant economic problem in bakery products. However. Harshbarger. and sodium benzoate (0. Adding yeast extracts previously fermented by Propionibacterium freudenreichii provided a source of propionic acid in bread formulation. 1945). and humans showed no toxic effects (Graham et al. and potassium sorbate at a level of 0. None of the preservatives were effective at neutral pH. A novel use of propionic acid to retard spoilage of bread was investigated by Gardner et al. This inhibitory effect was more pronounced with the addition of acid at the time of inoculation. Suboptimal doses (0. Bread produced with the extracts contained less ethanol and supported less spoilage by molds. monocytogenes at 22% fat compared to 2. cooling surfaces. 1965. β-alanine could not reverse the inhibitory effect of propionic acid in Aspergillus clavatus. 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. (2001).8 log reductions were observed.2). potassium sorbate.03%) led to enhanced growth of Aspergillus and Penicillium isolates. amstelodami. and wrapping materials reintroduce molds.. at 0.122 Antimicrobials in Food The effect of fat concentration on the efficacy of antimicrobial agents against L.5.5 and 1.5. 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. Increasing the fat content from 22% to 67% decreased the growth of L. Spore germination was inhibited by 1402 ppm (Tzatzarakis et al. sodium benzoate. The inhibitory action could be the result of an interference with β-alanine synthesis (Wright and Skeggs. 1951. respectively.1%. and 7. subtilis.2%. 1996). pH 4. 1940). 0. 1946). and Hara et al. and rubrum.3% by weight. with sodium lactate.1% sorbic acid or potassium sorbate. 2000).. 1952b). monocytogenes by only 1. Sausage batter was mixed with 0. but also various organisms displayed different tolerances to the compounds (Olson and Macy. 1954. and 0. 0.93 in a model agar system on the retardation of growth of E. and 0.5 logs. There is evidence that the sodium salt has some local antihistaminic activity (Heseltine. some species of Penicillium grew on media containing 5% propionic acid (Heseltine. 2002a. All preservatives were effective at pH 6. however. Small amounts of β-alanine could overcome bacteriostatic action of sodium propionate for E. or Trichophyton mentagrophytes. postprocess contamination from the atmosphere. Propionic acid at a concentration of 0..003%. 1985). 1955. Ingle. 1942). 6. herbariorum. flavus. and 0. rats. Additional studies using calcium and sodium propionate fed to mice. Not only was the final pH of the substrate critical. Eurotium amstelodami. 1940).

1965). tooth eruption. gradually increasing to 2. Succinic acid is approved as a GRAS substance for miscellaneous and general-purpose usage (21 CFR 184. . It can be incorporated into gelatin deserts and cake flavorings (Gardner. hair appearance.1221) and sodium propionate (21 CFR 184. for use in bread and rolls because the calcium contributes to the enrichment of the product (Chichester and Tanner. The acceptable daily intake for humans is listed in Table 4. and 0. Salts of the acid have a slight cheeselike flavor. Swiss cheese contains up to 1% propionic acid because of the growth and metabolism of propionibacteria. Propionic acid (21 CFR 184. 1972).32% can be used in flour and in white bread and rolls. Baking destroys most molds.0 mg/day at 4 weeks and continuing at this level for 100 days. 1974).. Sodium propionate is recommended for use in chemically leavened products because the calcium ion interferes with the leavening action. The acid form is readily miscible with water. Propionates can be added to bread dough without interfering with leavening because there is little or no effect on yeast. 0. Toxicology In short-term studies.3% in cheese products (Robach. SUCCINIC ACID Antimicrobial Properties Succinic acid successfully lowered microbial loads on poultry carcasses (Mountney and O’Malley. disagreeable odor.. except bread and rolls. It has a slightly bitter taste and serves as a flavor enhancer. or eye opening from the control group. In addition. Propionic acid and its salts are used primarily as mold and rope inhibitors in bread. It is normally found in cheese and as a metabolite in the ruminant gastrointestinal tract. which conform to standards of identity.0% succinic acid.Organic Acids 123 Application and Regulatory Status Propionic acid is a monocarboxylic acid with a slightly pungent. and growth can be seen under the wrapper during storage. Application and Regulatory Status Succinic acid is a nonhygroscopic.1081) and its salts. No upper limits are prescribed for use of this additive.23). calcium (21 CFR 184. calcium and sodium propionate are listed as antimycotics when migrating from food-packaging material (21 CFR 181.1784).5 mg succinic acid daily. 1944). 1972).3. Succinic acid is used to modify the plasticity of bread doughs and in the production of edible fats. Calcium propionate is preferred. A limit of 0. This additive is also used as a mold inhibitor in cheese foods and spreads. rats received subcutaneous injections of 0. This naturally formed additive becomes a developed preservative that limits mold growth on Swiss cheese. however a 3% or 5% concentration used at 60°C impaired the appearance of the product (Cox et al. Its antimicrobial effect is limited to most yeast and bacteria.38% in whole-wheat products. which are associated with its manufacture and the characteristic Swiss cheese flavor. (1967) reduced the population of inoculated S. are approved as GRAS substances for miscellaneous and generalpurpose usage.1091). Typhimurium by 60% on chicken carcasses after spraying with 1. No limit has been set on the acceptable daily intake for humans. however. and the sodium salt is more soluble than the calcium form. The test animals did not differ significantly in reproduction rates. Chick embryos developed normally when comparable dosages were injected into the air sac (Dye et al. but surface recontamination can occur during packaging. 1980). Thomson et al. dicarboxylic acid having low water solubility.

sugar. Workers exposed to tartaric acid dust measuring up to 32 mg/m3 in a manufacturing plant that produced the acid experienced tooth erosion. It is used in fruit jams. or gross histology compared with controls (Packman et al. Proteins. Biological and chemical systems depend on an interaction between acid and base systems. 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. and yeasts. It is thus essential to understand each of these concepts before consideration of the actual mode of action of the organic acids. Rose’s (1924) work on subcutaneous administration of 0.7% sodium tartrate (5% tartaric acid) did not show any changes in food consumption. Studies measuring teratogenic activity in pregnant mice. The acceptable daily intake for humans is listed in Table 4. 1963). this delicate balance is maintained and alteration of this balance causes destruction of the microbial cell. 0.3. 181. Toxicology Acute toxicity data for tartaric acid by the intravenous route for mice indicates an LD50 of 485 mg/kg body weight (Table 4. The monopotassium salt (cream of tartar) is commonly found in baking powders.1099). and it prevents discoloration in cheese (Gardner. 225.8%. including increased nonprotein nitrogen. molds. MODE OF ACTION The mode of action of organic acids in inhibiting microbial growth appears to be related to maintenance of acid–base equilibrium. growth. sherbets.124 Antimicrobials in Food TARTARIC ACID Antimicrobial Properties Any antimicrobial property of tartaric acid is because of its ability to lower the pH. The workers also displayed skin eruptions. has a strong tart taste that enhances grapelike flavors. However. the most soluble of the solid acidulants. which disappeared after they left the work site (Barsotti et al.. although some microorganisms such as Acetobacter can exist at and even require extremes of acidity (Langworthy. respectively (Food and Drug Research Laboratory. Homeostasis is the tendency of a cell to sustain chemical equilibrium despite a fluctuation in the acid–base environment. 1973a). Tartaric acid is a GRAS substance for miscellaneous and general-purpose usage in accordance with good manufacturing practices (21 CFR 184. and 215 mg/kg body weight. mortality. and 1. such as hydrochloric. Fitzhugh and Nelson (1947) found similar results in long-term studies using 21-day-old weanling rats fed tartaric acid as 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.2).5%. male rabbits consuming a diet containing 7. and cholesterol levels. Inorganic acids. and grape-flavored beverages. nucleic acids. The microbial cell normally reflects this equilibrium by attempting to maintain an internal pH near neutrality (Baird-Parker. 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. 1980). jellies and preserves. . and the production of energy by the cells. rats. hamsters.25 to 1 g to rabbits produced abnormalities in blood chemistry. Changes resulting from pH can destroy bacteria.1%. Through the interaction of a series of chemical mechanisms. 1978). Tartaric acid acts synergistically with antioxidants to prevent rancidity. and rabbits demonstrated normal offspring at levels of 274. 1954). 1972).2% of the diet. Application and Regulatory Status Tartaric acid. and phospholipids can be structurally altered by pH changes.. 0. proton donation.

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

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

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

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

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

56°C (60°F) 1.0493 20°C (68°F) 1.0091 1. Holt and Kumar (1986) found an observed k = 41. much slower changes occurred in the sulfur dioxide content.Sulfur Dioxide and Sulfites 145 TABLE 5.037 — Corrections are approximately 0. The sulfur dioxide stabilizes the dehydroascorbic acid by reacting with the ketone bonds (Wisser et al.003 1..6 57.4 53.6 67.0001°F–1 TABLE 5.028 1.. 1970).4 s-1 for H2O2 with SO32. This reaction is extremely rapid. .5 61.0292 1. The sulfur dioxide scavenges the hydrogen peroxide formed and keeps further oxidation of the dehydroascorbic acid and other products to a minimum.8 25.008 1.0191 1. 1970).7 ± 3.0040 1.018 1. the second equation listed previously is a key reaction (Heinmann et al. After the oxygen disappeared.0393 1. The oxidizability of the sulfurous acid salts is indicated by the following equations: 2– 2 SO3 + O2 → 2 SO2– 4 2– 2– SO3 + H2O2 → SO4 + H2O Jacobs (1976) showed that the amount of sulfur dioxide that reacted (oxidized) after 60 days of storage in bottled red wine was proportional to the original dissolved oxygen content of the wine.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 50.40 and 15°C. If ascorbic acid is being used in combination with sulfur dioxide.1 Specific Gravity of Various Sulfur Dioxide Water Solutions at Two Temperatures SO2 (g per 100 g) 1 2 3 6 8 10 a Specific Gravitya 15.4 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.at pH 3.

lactose. Navara.1 — 1. fermented ciders. 1944). 2000). In moldy apples. the pH.9 6 × 10–5 Note: 1 = Burroughs and Whiting (1960).5 72 44 98 — 66 1 2 — — — — 0.5 — 2. and glucose reacted less rapidly. . 1960. Lea et al. raffinose reacted very slowly.1 87 3 50 32 18 — — — — — 26 — 45 32 0. pyruvate.0 0. Ingram and Vas (1950) found that galactose. 3 = Navara (1985). 5-oxofructose. and fructose and sucrose did not react at all. Glucose is by far the most abundant of the reactive aldehydes and ketones in most fruit juices. 2 = Aerny (1986a. a Aerny (1986 a.6 7. Bisulfite addition products.b. Rhem (1964) noted that the rate of formation and the amount of sulfonate formed depended on the concentration of the reactive substances. 1964a.7 × 102 — — — — 15. and the temperature. Compared with model solutions. 1985).1 0.. and arabinose reacted rapidly with bisulfite.1 — 0.5-hexodiulose. form rapidly with aldehydes: – – → HSO3 + R-COH ← R-CHOH-SO3 All aldehydes form the hydroxysulfonates. as much as 80% of the total sulfur dioxide may be bound by these types of carbonyls (Blouin. natural fruit juices always bind more sulfur dioxide than would be calculated from the glucose present in the juice (Joslyn and Braverman. the hydroxysulfonic acids (Suter. Otherwise.146 Antimicrobials in Food TABLE 5. Aerny. 1986 a. and galacturonic acid were significant bisulfite binding forces (Burroughs and Sparks. Diethyl ketone reacts slowly and to a limited extent. 1954.7 90 1.3 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. acetaldehyde. Those reacting the most rapidly formed complexes that dissociated the least. 1963). He also noted that phosphoglyceraldehyde would likely react. Relative percentages of aldehydes reacting with sulfur dioxide and their equilibrium constants are shown in Table 5.b). maltose. L-xylosone. 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. 2-oxogluconic acid.b). d-threo-2. 1954). 2.to seven-member carbon ring system will react. 1973.5-diketogluconic acid. only ketones with a methyl group adjacent to the carbonyl or carbonyls that are part of a four.3 (Burroughs and Whiting.12 — 2 99.5 3 5 8 Ka × 10–6 × 10–4 × 10–4 × 10-6 — 2 × 10–4 5 × 10–2 1. α-ketoglutarate. In botrytized grapes made into wine. mannose. but not all ketones react. and wines. Joslyn and Braverman. 1954).

(1984) also showed that sulfite additions increased acetaldehyde. and 15 to 57 and 31.is on the 4-position rather than the 2-position (e. 1938). Traces of the compound could be found in stored commercial soft drinks that had been heated and contained sulfite.. Glories (1984) studied the equilibrium between SO2 and the anthocyanin–bisulfite complex. pyruvate. and at 50 mg/L of SO2 added. 1975).. reacted with bisulfite to form a lemon yellow compound. Jurd (1972) suggested the binding site for HSO3. Bisulfites may also react with nucleotides such as nicotinamide adenine dinucleotide (Meyerhof et al. from winery trials. (1982). Dittrich and Barth (1984) did find correlations between the SO2 binding substances and wineries and grape source. (1989) found a food-coloring dye. malvidin monoglucoside). 1998). Shapiro and Weisgras (1970) also demonstrated that cytosine was transformed to cytidine by bisulfite.5 mg/L of thiamine. This contributes to the difficulty in the measurement of free sulfur dioxide in highly colored wines. selected four yeasts that produced low levels of SO2 binding compounds. Allyl isothiocyanate in mustard was shown to react with sulfites used as antioxidants to form allylaminothiocarbonyl sulfonate (Cejpek et al. . In model solutions in the range of 30 to 50 mg/L of SO2 added. Lafon-Lafourcade (1985) reviewed the role of yeast and bacteria in the amounts of these components found in wines. This treatment allows more effective use of SO2 in wine. the color is reduced by about 25%. The reaction affected flavor of the mustard by reducing pungency. After approximately 100 days of storage. There is no evidence that these reactions occur in vivo.5-diene (Crowell and Guymon.5 mg/L. Piracci and Spera (1983) reported similar results. Margheri et al.6 mg/L. ketones. Farris et al. Undoubtedly other reactions with sulfur dioxide take place that could reduce the amounts of available sulfur dioxide. The respective ranges and means for these components were 72 to 287 and 115. (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. and other SO2 binding substances limit the effective use of the added sulfite. Damant et al. but they found no increase in pyruvate or α-ketoglutarate. ˇ Farkas et al. They measured acetaldehyde. it is reduced by 80%. the following formula was used to calculate the equilibrium constant Ks. 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. Uzuka et al. (1985) found a reduction in the content of the SO2 binding materials by the addition of 0.5 mg/L. (1983) tested 30 strains of Saccharomyces cerevisiae for production of SO2 binding materials.. but these have been reported as the primary reactions.g. and αketoglutarate. The addition product attached at the carbon 4 of the sunset yellow molecule. Heintz (1976) reported the occurrence of an addition product of sorbic acid and bisulfite. 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. sunset yellow. Sulfur dioxide can loosely bind to anthocyanins. 16 to 42 and 27.Sulfur Dioxide and Sulfites 147 The amounts of aldehydes. Farris et al. – 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.

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

. Watanabe and Ino (1984) and Juven and Shomen (1985) reported that up to 100 mg/L of total sulfites for grape juice. oeni was the most sensitive to SO2. 320 and 651 mg/L of sulfurous acid were required. 20 days was required to kill the bacteria when the same amount of aldehyde–bisulfite complex was used. respectively. Levels above 120 mg/L of total sulfur dioxide (free and bound) decreased the incidence of malolactic fermentation. In model solutions containing Lactobacillus arabinosus and L. 1962). Rhem and Wittmann (1962) reported that 200 mg/L sulfur dioxide killed Acetobacter at pH 6. and ethanol. This organism is particularly intolerant to sulfur dioxide. for antimicrobial action at pH 6.3 log CFU/ml. 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. (1984) stated that the amounts of SO2 used in normal wine making are insufficient for acetic acid bacteria control.49 log Acetobacter per ml exposed to 100 and 200 mg/L of total sulfur dioxide in grape juice were reduced to 2. Bacteria common in acid fruits and beverages are the lactic acid-producing genera Lactobacillus. Cruess (1912) found that 5. pH. Lactobacillus casei. Spirov et al. and Oenococcus oeni (Amerine et al. Lactobacillus trichodes (Fornachon et al.Sulfur Dioxide and Sulfites 149 oxidize ethanol in fermented beverages to acetic acid and. Lactobacillus hilgardii. They are aerophilic and have pH optimum for growth of around 5.4 (Holt et al. further. Addition of 30 mg/L of free sulfur dioxide killed the bacteria completely in 15 days. however. O. Lactobacillus plantarum. 1994). Fornachon (1957) noted that sulfur dioxide and pH were important factors in controlling lactic bacteria in wines. The juices high in insoluble solids ultimately had lower residual total sulfites. Pediococcus cerevisiae.0 (Rhem and Wittmann. pentosaceus to SO2. He found that this genus also fixed a certain amount of the sulfur dioxide. to carbon dioxide and water. but much larger doses were required for bactericidal action. casei. aceti can grow in red wine with 25 mg/L of unbound sulfur dioxide present. Lactobacillus brevis. 1982.. Amerine et al. Lactobacillus leichmannii.5 mg/L were bactericidal. is very heat resistant and alcohol tolerant.. with lower pH increasing the effectiveness 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. Leuconostoc mesenteroides subspecies dextranicum can also cause ropiness in beverages through the formation of dextrans. 1983). and Pediococcus pentosaceus. red wine. Dupuy and Maugenet (1963) noted that even small doses of sulfur dioxide inhibited the activity of the cells. The homolactic species found in wines are Lactobacillus acidophilus. Lactobacillus buchneri. and P. Concentrations above 1. Leuconostoc. plantarum. Liu and Gallander (1983) demonstrated that lower pH wines underwent . and the heterolactic species are Lactobacillus fermentum. 1983) indicated that around 50 mg/L of free sulfite could preserve wine vinegar for about half a year. Several reports (Karova and Kircheva. after 36 hours. Additions of 100 mg/L bleached the color of the vinegar. Oenococcus oeni grows preferentially at lower pH but seems less tolerant to sulfur dioxide (Mayer. LafonLafourcade and Joyeux (1981) and Joyeux et al. 1949) is described as causing a hairlike growth in fortified wines. oeni.5. (1980) reported that 30 mg/L of free sulfur dioxide is sufficient to inhibit malolactic fermentation in wine. 1979). Lafon-Lafourcade (1975) demonstrated the effectiveness of both free sulfur dioxide and bound sulfur dioxide in inhibiting Leuconostoc gracile in wine at pH 3. Pediococcus. respectively. 1982.47 log and 0. hilgardii.0 in a buffered solution. They indicated A. Lactobacillus trichodes. L. and Oenococcus. The bacterium.5 mg SO2 per liter in the undissociated form was bacteriostatic to L. As little as 30 mg/L of added sulfur dioxide may be lethal to the species. They found that up to 1. 1980). Cell growth was completely prevented. Lactobacillus delbrueckii. Carr and Davies (1971) noted that relatively high amounts of free sulfurous acid were present in ciders that contained viable lactobacilli. and soft drinks were required to control acetic acid bacteria. Manca de Narda and Strosser de Saad (1987) investigated the tolerance of O.. As low as 75 to 80 mg/L of total sulfur dioxide prevents growth and 100 mg/L kills this species.

(1988) demonstrated that without sufficient SO2 and adjusted pH. Enterobacter agglomerans. In addition. Millet and Lonvaud-Funel (2000) reported that sulfites cause a portion of lactic acid and acetic acid bacterial populations to enter a viable but nonculturable (VBNC) state. the growth of O. The shelf life of ground beef was effectively increased from 1. 1988). coli were inhibited to a greater extent by sulfites than other bacteria. the main effect in meat appears to be the antioxidant properties (Roberts and McWeeney. a rapid decline in cell numbers occurred. 65–136. oeni in red table wine occurs readily. 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. and Hafnia alvei. (1988) tested 146 different strains of wild malolactic bacteria. Wibowo et al. oeni strains with unfavorable sensory results. E. They suggested that these microorganisms could cause spoilage in wines that were considered sterile using conventional counting techniques. (1987) found that vacuum packaging and a good oxygen barrier film decreased the . The remaining cells later multiplied to significant numbers. 1972). Sensory tests showed no adverse results. 200–241. were completely effective. The Gram-positive bacteria remaining grew more slowly than the Gram-negative bacteria. 50–195. in their review. 190–241. Davis et al. Ough et al. The cells could not be cultured on nutrient agar plates but demonstrated metabolic activity through hydrolysis of fluorescent esters and were countable using direct epifluorescence microscopy. Serratia marcescens. They noted that sulfites shifted the microflora of the meat to Gram-positive bacteria from the normal Gramnegative flora. The packaging used was a gas-permeable wrapping that allowed oxidative conditions (von Holy et al.8 days at 7°C storage with no treatment to 12. The preservation of the color and odor of meats is improved by sulfite treatment and. This work confirms that wines with high total sulfur dioxide concentrations are more likely to undergo malolactic fermentation with other than O.0 were as follows: Salmonella. Roberts and McWeeny (1972). oeni when SO2 was added and quite a variable response between strains. The delay was proportional to the concentration of the bisulfite addition. The effect was inhibition of growth of the flora. (1985) reviewed the use of sulfite as an additive to control microbiological changes occurring in meat products. Tompkin et al. (1983) determined that. This is demonstrated in the use of sulfites in meats. than in inhibiting Gram-positive bacteria. 15–109. (1988) found significant delays in growth of O. oeni PSU-1 used.150 Antimicrobials in Food malolactic fermentation more slowly and that lower sulfur dioxide levels increased the fermentation rate of the O. Piracci (1984) found 0. They also noted that the interaction of sulfites with nitrites caused a lowering of the nitrites available for nitrosamine formation. 67–98. O. All strains grew at pH 4.6 days at 0°C with the addition of 250 mg/kg of sulfur dioxide. or in paired combinations. Salmonella and E. oeni strains were less tolerant to the sulfur dioxide than Pediococcus paroulus strains or the Lactobacillus species.5 mg/L of molecular sulfite was sufficient to control malolactic bacteria growth.. Banks and Board (1982) tested several genera of Enterobacteriaceae isolated from sausage for their metabisulfite sensitivity.5 in beef broth and 20% tomato juice serum medium at 64 mg/L of total sulfur dioxide. Lafon-Lafourcade et al. There was some additive effect when used with dimethyl dicarbonate but not enough to warrant the use with SO2 for this purpose. Yersinia enterocolitica. 83–142. O. 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. coli. state that sulfur dioxide is more effective against the growth of Gram-negative rods. Adams et al. It was found to tolerate up to 100 mg/L of SO2. The microorganisms tested and the concentration of free sulfite (µg/ml) necessary to inhibit their growth at pH 7. (1980) found the addition of 100 mg/kg of SO2 as sodium metabisulfite to canned pork inoculated with Clostridium botulinum spores delayed cell growth. oeni was the primary malolactic bacterium associated with wine. Banks et al. Citrobacter freundii. in the Bordeaux area of France. such as Escherichia coli and Pseudomonas.. None of the compounds alone. although slowing or prevention of growth of surface bacteria is probably important. It survived alcoholic fermentation when others did not. although after sulfite addition.

Sodium sulfite addition in sausage was shown to affect biogenic amines. 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. There was no effect on histamine. bailii. its use to prevent the growth of yeast in sweet table wines is seldom ever contemplated. whereas Kloeckera.. such as “maintain the free SO2 at 20 mg/L. 1912). Six strains could grow well at 1000 mg/L.10–20.60 0. but the level of cadaverine was reduced (Bover-Cid et al. 2001). The amount of molecular SO2 (mg/L) in wine can be calculated using free SO2 mg/L/(1 + 10pH . (1988) found that with yeast acclimatized to sulfur dioxide. Rhodotorula. 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. Sudraud and Chauvet (1985) found that to maintain yeast stability. and five others were able to produce adequate amounts of ethanol. A 10-fold increase in molecular sulfur dioxide occurs between pH 4. 1. cerevisiae that had been characterized as “SO2 resistant” for tolerance to high concentrations of sulfur dioxide. the growth rates and cell yield were similar.8 mM for Z.4). Rhem and Wittmann (1962) determined the inactivation levels of sulfurous acid for a variety of yeast genera (Table 5. Molecular sulfur dioxide is the most effective form for inhibiting yeast.2 mg/L of molecular sulfur dioxide were necessary at the finish of fermentation and during aging.81). spoilage in sulfite-treated sausage.0 and 3. respectively.0. Haznedari (1979) tested 30 strains of S. Any rule of thumb addition. phenylethylamine.60 Rhem and Wittmann (1962). Pichia.7 0.5 in tryptone yeast extract medium.20 0. In fact.05 M free sulfur dioxide for Kloeckera apiculata to 2.20 0. The yeast also showed increased resistance to the fungicide dimethyl dicarbonate. Yeast The use of sulfur dioxide to deplete the wild yeast in grape juice is a standard practice dating back many years (Cruess.5 and 1.20 7. The concentration of tyramine and putrescine increased in the presence of sulfite. In contrast. pH is extremely important in the effective use of sulfur dioxide. resistance of yeast to sulfur dioxide ranges from 0. cerevisiae (105 CFU/ml) after 8 hours at 25°C. which inhibited growth.0 mg/L were required.Sulfur Dioxide and Sulfites 151 TABLE 5. with the pressure to reduce sulfur dioxide content. was maintained for a longer period. Carr and Davies (1971) found sulfur dioxide incorporated into growth medium (pH 3.0 and 3. Ough et al. or tryptamine.40–0.1. Delfini (1989) . According to Warth (1985). between 2.2–8.4) at 25 mg/L was sufficient to kill a culture of S. bailii at 25°C under aerobic conditions at pH 3. Saccharomycodes ludwigii was nearly as tolerant as Z. Once the inhibition was overcome. This was because of the lack of oxygen delaying yeast growth and the production of sulfite-binding substances. and several other genera were very susceptible.” for biological stability can be disastrous. Thus the free sulfite.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.

none seem to be capable of replacing the antioxidant property of SO2. a late-harvest orange juice. cerevisiae despite a reduction in the bacterial counts in the system at the same concentration. sulfur dioxide. Pearlstein (1988) found that extended aeration decreased sulfite production. bailii. (1989). Chang et al. 1985. using yeast that produces minimum amounts of sulfur dioxide-binding components. Hernandez. S. Schmitt et al. ludwigii. reducing the amount of sulfur dioxide before or during fermentation. bailii was becoming a problem in juices. Even at low pH (2. Asvany. With this genus. alcohol. Dott et al. Although other agents may act as antimicrobial agents. Proper selection of the yeast strain to avoid high levels of sulfite is an obvious choice. Ranote and Bains (1982) used 350 mg/L of sulfur dioxide to preserve Kinnow. S.b.. 1975). and Schizosaccharomyces japonicus in the re-fermentation of sweet champagnes. Recommendations include cooling the fruit before crushing. Vernerova et al. Patel et al. cerevisiae. 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. Z. Some yeast strains can form rather large amounts of sulfur dioxide during juice fermentation (Table 5. the main spoilage yeast is Saccharomyces. and eliminating oxygen from bottled wine. using sulfide salts before fermentation. They found a majority of the wines in the test to contain about 10 mg/L of total sulfur dioxide (Table 5. (1985) used an initial 1000 mg/L of sulfur dioxide in the last stages of the manufacture . substituting other preservatives at bottling. Valouyko et al. if proper sulfur dioxide levels are not maintained. 1978). settling and racking the juices. The treatment was reported to be successful for juice.1 but not in the base material. In high-sulfite-producing yeast. bailii in comminuted orange drink was controlled by 230 mg/L of SO2 at pH 3. (1985) tested 1700 Saccharomyces. In normal sweet table wines. adjusting the pH downward if necessary. Yeast can form sulfite from sulfate via sulfate permease. (1997) showed that sulfite up to 400 mg/L had no effect on S. Sethi and Anand (1984) had to use 692 mg/L in carrot preserves to inhibit Bacillus cereus. Breweries have an interest in sulfur dioxide as an antioxidant. minimizing the air around the wine. Minarik and Navara (1977) reported finding the spoilage yeast S. Hydrogen sulfide (H2S) was used (Ubigli et al.4). excess amounts are used. especially wines in barrels. Brettanomyces can contaminate a winery. This shows that most wines will not be free from sulfite labeling requirements even if no sulfur dioxide is added. even at the legal limits. 1985. 1985. In a cell recycled ethanol fermentation system. (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. and wines because of its tolerance to SO2. Very little of the sulfur dioxide formed by the yeast remains in the free state. the sulfate permease was not repressed by methionine. stated that the use of sulfide salts failed to protect wine and left negative sensory characteristics in the wine. cell numbers are depleted to very low levels at bottling. however.. investigating the sulfur dioxide content of green beer. and there was no correlation with the number of cells in the initial inoculum.7 (Lloyd. which was at pH 3. With modern filtration and sanitation technology. There have been numerous reports on techniques to minimize the amount of sulfur dioxide used in wines (Aerny. Fruit juices and preserves can be protected from microbial spoilage by sulfur dioxide addition.152 Antimicrobials in Food reported the existence of very sulfite-resistant S. Galassi and Mancini. does not always inhibit growth and fermentation. 1985). Generally. 1985.6 and 3. soft drinks. Gomes and da Silva Babo. ludwigii in a low-alcohol wine. This particular species was found to be very resistant to sulfurous acid. (1977) determined the cause for sulfite production to be sulfate permease inhibition by methionine. found no correlation with dissolved oxygen levels in wort and no correlation with ATP sulfurase or sulfite reductase. Spoilage by Z. Sand (1980) suggested that Z.6). Growth was found at the highest concentration of free sulfur dioxide used. ludwigii is also a noted spoiler in sweet grape juice containing high amounts of sulfur dioxide (Jakob. and low pH.5). 1986a. Spoilage yeast in dry wines are fairly rare. Suzzi et al. (1983). because the pH is high. 1982) as an alternative for pretreatment of grape juice before fermentation. Angelino et al. Strain differences caused variation in the sulfur dioxide produced from 11 to 26 mg/L.

18.. 100 5. 1985) Range of SO2 Found in Synthetic Medium (mg/L) <10 10–20 20–30 30–40 >40 Average Total SO2 in Wine (mg/L) 10 17 22 34 58 Number of Strains 1292 354 50 2 2 . 3. (1976) Poulard and Brelet (1978) S. 170.5. (1976) Poulard and Brelet (1978) Delfini et al.5. 21. (1976) Delfini et al.5 12 0 9 Reference Minarik (1975) Eschenbruch and Bonish (1976) Heinzel et al. uvarum S. (1976) Poulard and Brelet (1978) Minarik (1975) Poulard and Brelet (1978) Minarik (1975) Delfini et al. (1976) Delfini et al. 500 300. 100. 52. 76 17. (1976) Delfini et al. cerevisiae S. (1976) Delfini et al.5 0 0 0 1. 4. 1300 22. 3. 18. 44. 21. (1976) Delfini et al. chevalieri S. 40 0 18. 35. (1976) Minarik (1975) Minarik (1975) Heinzel et al. (1976) Delfini et al. bailii S. 150 0. 56. (1976) Minarik (1975) Eschenbruch and Bonish (1976) Heinzel et al. 500 5. pastorianus S. 5.5 14. rouxii Schizosaccharomyces pombe Torulopsis stellata Kloeckera apiculata K.5 Formation of Sulfur Dioxide by Various Yeasts during Grape Juice Fermentations Yeast Saccharomyces carlsbergensis Sulfur Dioxide (mg/L) 12 150 80. 5 18–23 85 0.6 Amounts of Sulfur Dioxide Found in Test Wines Fermented by 1700 Saccharomyces Yeasts (Suzzi et al.5. (1976) Poulard and Brelet (1978) Delfini et al. 35 20. 20 26 27 0 0.Sulfur Dioxide and Sulfites 153 TABLE 5. (1976) Delfini et al. italicus S. 1. (1976) Delfini et al. acidifacien S. africana TABLE 5. 20 1. bayanus S. magna K. 160. rosei S.

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

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

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

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

32 980.17 987. Reversibly bound sulfites are converted to sulfur dioxide only after heat and acid or alkali treatments. garlic. One official method for measuring total sulfurous acid is the modified Monier-Williams procedure. Sulfites can be described as free.16 963.8).7 Application of Sulfites in Foods as Antimicrobials and the Concentrations Used (Gould.02 961.. purees.. horseradish) Fruit juices Fruit-based sauces and related products Fruit pulps.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.28a 990. and concentration varies by country.20 975.04 990. Thiosulfonates are irreversibly bound (Beck et al. and fillings Jams and jellies Nonalcoholic beverages Sausage Sugar confectionary Vinegar Wine a Use Concentration (mg/kg SO2)a 10–30 100 50–1000 10–100 50–100 50–500 50–100 20–200 450 50 50–200 100–300 Sulfites are allowed only in certain foods in different countries. 2000).8 AOAC International Official Methods of Analysis for Sulfites (Warner et al. Those sulfites that bind food matrices and are not converted to sulfur dioxide with heating or acidic conditions are irreversibly bound. Free sulfites are readily converted to sulfur dioxide upon acidification and can be quantitatively analyzed following distillation. 2000) Method Number 892.158 Antimicrobials in Food TABLE 5. 2000).30 990. A sample is placed in a distilling flask. or irreversibly bound (Beck et al. The official methods of analysis for agricultural and food products are specific in their recommendations for analysis of sulfur dioxide (Table 5..09 962. and . TABLE 5. reversibly bound.29 990. acidified. 2000) Food Beer Fresh fruits Fresh vegetables (onion.

2000). and cabbage. the sample is made basic to break the bisulfite addition products. Kielhofer and Aumann (1957). and it is then titrated with the iodate solution to a starch end point. This is a direct iodine titration of the substance at acid pH. 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. it is then acidified. Schwedt (1986) considered the test strips satisfactory but noted that if dyes or natural pigments came in contact with the cellulose. 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. The Ripper method for free sulfur dioxide is given by Ough and Amerine (1988). Schneyder and Vlcek (1977) reported that iodate was superior to an iodine standard solution.8) (Warner et al.. and the volatile sulfur dioxide is removed by a stream of nitrogen through a reflux condenser. Briefly. 1988). leeks. Burroughs and Sparks (1964b). and the freed sulfurous acid is titrated directly with iodine to a starch end point: I3 + SO2 + H2O → SO3 + 3I– + 2H+ – 2– SO3 + H2O → SO4 + 2H+ This is satisfactory for certain materials. 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. but determinations on red wines are more accurately done by this variation of the Monier-Williams method. Rankine and Pocock (1970). The optimized Monier-Williams method is used by the FDA to measure sulfites in official samples (Table 5. (1979) showed that sodium sulfide and allyl isothiocyanate gave positive results in the MonierWilliams tests. . 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. Paul (1958). 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. but if other oxidizable substances are present. False-negative and false-positive results were found by both groups. Free sulfur dioxide is easily measured in white wines by the Ripper method. The same errors are associated with this procedure as are associated with the Ripper procedure for total sulfur dioxide. and Ough and Amerine (1988) described the method and equipment needed. Joslyn and Braverman (1954) reviewed the errors associated with iodine titrations. in addition. Mitsuhashi et al. except dried onions. (1988) and Wanderer and Solomons (1987). This method is good for most products. Excess iodide is added to the sample.Sulfur Dioxide and Sulfites 159 heated. and the hydrogen ion produced in the peroxide solution is titrated. The reflux condenser must retain all volatile acids except the sulfurous acid. The Monier-Williams method can be modified to determine free sulfurous acid. the sulfate can be precipitated with barium and measured gravimetrically. Basically the sample is sparged with gas for 12 to 15 minutes. false results were recorded. artificially high results occur.

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

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

Research of the 1920s and 1930s focused on using nitrite to cure meat. Until the early 1940s. of the three. This concept became widely accepted. the highest levels tested. only salt was used in sufficient concentration in commercial hams to prevent proteolytic activity at 37°C. putrificum. research had clearly demonstrated that nitrite has significant antimicrobial properties. 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. RESEARCH HISTORY BEFORE 1950 Tucker (1930) studied the proteolytic activity of a Clostridium putrificum isolate from sour ham. 44. reproducible results in all instances. it remains to be seen whether wisdom has prevailed with the voluntary abandonment of nitrate as an additive to many meat products in the United States. the value of nitrate. By the time the nitrosamine controversy arose in 1969 and the early 1970s.000 µg/g did not inhibit the 12 strains tested in either pork infusion or egg-meat media. putrificum.000 µg/g. using Clostridium botulinum. controversial history over whether nitrate and nitrite have antimicrobial properties. Levels of 3100 µg/g in pork infusion and 3900 µg/g in egg-meat. At levels between 22. found that sodium nitrate up to 22. or 12. is responsible for retarding proteolysis by C.000 µg/g of sodium nitrite in a pork infusion broth buffered to pH 7. C. solving a problem of sour hams. He concluded that. 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.000 µg/g cannot be relied on to inhibit putrefactive anaerobes if other conditions are favorable for growth. One of the messages from the 1920s is that nitrate serves as a potential reservoir for conversion to nitrite and thus to nitrous acid. Proteolysis was inhibited by 4.Nitrite 171 documentary of Cassens (1990). Tanner and Evans (1934) reported that 5900 µg/g of sodium nitrite inhibited 9 of 12 strains of clostridia in nutrient broth and all 12 strains in dextrose broth. The same results were obtained with Clostridium putrefaciens at 23°C. At 44.6 and held at 37°C. Considering that research continues to reveal new interactions among the chemical constituents of cured meat. The authors concluded that nitrate at concentrations as low as 22. for that matter. there has been a long. Clever as the scientific community has been in more clearly defining the antimicrobial properties of nitrite. and Clostridium sporogenes. This basic flaw in experimental design led to results that misguided and hardened opinions in the industry for 20 years.000 µg/g of sodium nitrate. 6 of the 7 cultures were inhibited. irregular inhibition occurred among seven botulinal cultures. During the 1940s and early 1950s. Most of the research on sour hams involved test tube experiments with culture media above pH 7. Unlike the other antimicrobial agents discussed in this text. were not inhibitory to any of the strains.000 µg/g. data appeared that began to prove the opposite. Tanner and Evans (1933). It is still difficult to design experiments that yield predictable.0% sodium chloride (salt). 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.0. and dealing with perishable canned hams that were being temperature abused in the marketplace. and the value of nitrate was relegated to serving merely as a reservoir for the generation of nitrite. We are still ignorant of all the significant factors that influence the efficacy of nitrite or.000 and 44. . not nitrate. He suggested that nitrite. The list of recognized factors influencing the antimicrobial efficacy of nitrite in cured meats continues to grow 60 years later.0. Nitrate was believed to provide the antimicrobial effect. 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. By the middle 1950s. despite meat having a pH closer to 6. nitrite was considered to have no value in meat other than for flavor and color development.

After the slaughtering process. 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. 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. both nitrate and cured meat had to be present. 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. They stated that if nitrate were omitted from the product. but the research led to the following general conclusions.49 to 5. The authors concluded that reliance cannot be placed on nitrite alone to prevent spoilage by clostridia. insufficiently heated to be stable at room temperature) canned.e. in mixtures containing reducing substances. pork sides were held at ambient temperature overnight and then moved into refrigeration for 24 hours to achieve 5. 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. From tests with various blends of salt. the results were quite different. Under these conditions.. Brooks et al. the sole cause of the spoilage was found to be nitrate-reducing Bacillus species (Jensen et al. It seemed that nitrate was especially valuable in preventing high degrees of acidity or souring of meat. citing earlier work by MacNeal and Kerr.4). It is surprising that the fundamental importance of pH. It is curious that Tanner and Evans (1933). and sugar swelled in 1 to 90 days during temperature abuse at 37°C. 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. 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.. Repression of toxin formation in the raw meat was a result of other bacteria that caused a decline in pH to inhibitory levels (pH 4.6°C in the meat. however. sugar. 1934). The sides were then immersed in a tank and cured using a pickle solution containing potassium nitrate. was not pursued. To obtain gas production in the medium. A marked inhibition was noticed. Potassium nitrate was therefore considered to be particularly effective in restricting acid fermentation of organic substances that are already slightly acid. The reason for this specific combination of ingredients remains unknown. and of nitrous acid also. so clearly stated and available to researchers at that time. Several interesting questions were raised. Perhaps the cured meat provides a source of heme. The opinions expressed by Jensen et al. and nitrate in peptic digest broth (pH 7.172 Antimicrobials in Food although the nitrite was added to the media (pH 7. it was used as a food. Perishable (i. A medium was developed with nitrate. as the more recent results suggest (Jacobs et al. nitrite.” Botulinal toxin was produced in the cooked meat and a few samples of raw meat. In acid solutions. chopped. state the following: [P]otassium nitrate in neutral or alkaline solutions exerted no special restrictive effect on bacterial activity. (1940) described the contemporary method of making bacon in the United Kingdom. The characteristic cured flavor of bacon is primarily the result of the action of nitrite.4) before autoclaving. Satisfactory bacon can be produced with the use of nitrite. The conversion . They further believed that the claim of meat packers that small amounts of nitrate in the pickle produced better preservation of the meat. 1964). spiced ham and luncheon meats containing nitrate. nitrite. raw pork. Examining 1000 cans representing all manufacturers at the time.. was borne out by their results. in 1934 laid the foundation for a new concept.3). 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. and cooked pork. Nitrite could not replace nitrate. and cured meat to detect the gas-producing bacteria in raw materials and the plant environment.

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

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. even in the presence of salt or nitrite. respectively. especially if the product pH was below 7. Nitrate at the level permitted in meat (1250 µg/g) was without effect. intermediate. The basic formulation resulted in a product having a moderately high brine of 5.4% or sodium nitrate in excess of 40. a combination of both nitrite and nitrate was the most inhibitory. pasteurized. and sterilized meat were of low. Strong opinions were expressed that nitrite alone was ineffective at the levels used in commercial practice. 6. Although thermal destruction was not shown to be enhanced by the presence of curing salts. to some degree. The concept of a combined effect of heating in the presence of curing salts was proposed.5. and nitrite as preservative agents and expands this information to other microorganisms. 3. and high heat resistance. SUMMARY FOR BEFORE 1950 A summary of the state of knowledge by 1950 might include the following: 1. . 2. Spores produced in raw. increased inhibition of PA 3679 was obtained as sodium nitrite was increased from 400 to 800 µg/g. A mixed cure of all three (salt. Steinke and Foster (1951) investigated the effect of packaging liver sausage in an oxygenimpermeable film (Saran™) that was gaining acceptance in the industry. The disappearance of nitrite during processing and storage was assumed to make it an unreliable preservative. nitrate. Adding 200 µg/g of sodium nitrite was considerably more inhibitory to toxin formation than the addition of 1000 µg/g of sodium nitrate. A mixed cure of salt (3. 5. In the absence of added salt. however. and nitrite). but the condition of the meat was a factor. Nitrite was clearly shown to be an effective antimicrobial agent. Bulman and Ayres (1952) found that levels of salt in excess of 4. yielded the maximum inhibition. It is perhaps for this reason that Jensen believed so strongly about the need for nitrate in perishable canned cured meats. by nitrite.1 to 6. Salt was found to be the major factor retarding botulinal outgrowth in temperature-abused products.5%) and nitrite (150 to 170 µg/g) was much more effective than either substance alone. During the 1930s some processors incubated perishable canned cured meat at 37°C as an index of “keeping quality. Halvorson (1955) later discussed the widespread problem of temperature abuse at the retail level. however. Nitrate was without effect.” This experience may have led to improvements in meat handling and equipment sanitation.0. 4. (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.174 Antimicrobials in Food Vinton et al.000 µg/g were required to prevent spoilage from anaerobic spore formers in pork.37% and a pH range of 6. nitrate. The relative roles of nitrate and nitrite as preservatives in cured meats were unclear. 7. The question was raised whether the growth of spoilage organisms is influenced by the depletion of nitrite to noninhibitory levels. 1950–1960 Research of the 1950s clarified the relative significance of salt. there was increased inhibition of outgrowth of surviving spores by salt and.05% to 5. Salt at the levels used at that time was considered the cornerstone for the preservation of cured meats. An underlying factor in the research before 1940 was the unfavorable experience of 1928 and 1929 with temperature-abused perishable canned hams.

8 was optimal for antibacterial efficacy. a very small amount of added nitrite prevented staphylococcal growth when incubated anaerobically. Pasteur.S. and a brine level of 3. unless protected by meat juices.g. and 1 ounce of sodium nitrate per 100 pounds of meat. Because the procedures traditionally used for large canned hams had a history of safety. At that time European microbiologists commonly preincubated perishable canned cured meats before microbiological examination.Nitrite 175 Henry et al. (1954) reported that at pH 7. A pH of 5. Germination was apparently prevented because viable spores could be recovered. the growth of Bacillus species would occur. botulinum.4%. Nitrite was more inhibitory in the presence of ascorbate. Department of Agriculture (USDA) issued a regulation to assure the safety of new perishable canned cured meat products (e. aureus to die rather rapidly in ham-curing pickle. botulinum. They found S. The authors postulated that nitrous acid was responsible for the bacteriostatic action of nitrite. (1956) examined whether curing salts plus the anaerobiosis of a vacuum package would inhibit the growth of S.3°C. who concluded that 1 ounce nitrate was optimal for gas formation by Bacillus species in temperature-abused perishable canned luncheon meat. 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. toxin formation was inhibited with a heat treatment as low as F0 = 0. At pH 5.3 or below. if ever.. Also. nitrite rapidly disappeared and was ineffective. found in the interior of cured meats.5% sugar. and toxin would be produced.5% would have caused consumer .9 to 5.5 or above. less nitrite was necessary for inhibition as the pH was decreased from 6. B.0%. 1955) on semipreserved meats in hermetically sealed containers included a curious debate. Lechowich et al. these existing products were not affected. this cautious view of the inhibitory effect of nitrite was expressed by Eddy (1957): “Taken in their totality.5%. the addition of 1. Today. This combination of ingredients was derived from the literature and unpublished results from Jensen. The USDA concluded that the high brine would retard the growth of C. growth would occur.55) was autoclaved with glucose. Tests in broth media and raw pork led them to conclude that S.05. the spores could germinate. aureus growth can occur in any combination of salt. the control of salt concentration and resultant water activity was the most reliable bacteriostatic system for cured meats.27. Earlier. was unstable. and in the presence of nitrate. causing the cans to swell and forewarn consumers of a potential hazard. At 6. 1/8 ounce sodium nitrite (78 µg/g). This effect was reversed by adding sulfhydryl compounds. although effective. Dack (1949) reported toxin inhibition in meat with brine levels above 6. The brine requirement of 5. these observations leave no doubt inhibition by nitrite is at least a possibility. 1 1/2 lb canned ham). 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. aureus. A typical formula being used for large canned hams at that time included 0. 1/4 ounce sodium nitrate (156 µg/g). On adding a filter-sterilized solution of nitrite to a broth medium. nitrate. the legal requirement for trichina destruction.6 to 5. a similar debate would include the time elapsed since manufacturing. The requirements imposed on new products included a brine level of 5.75% sugar. Jensen concluded that if the brine content were less than 5.” An international symposium (Ann. As late as 1957. Halvorson (1955) cited a private communication from L. nitrite enhanced bacterial growth in curing brine. In the United Kingdom. as suggested by Castellani and Niven (1955). Inst. aureus was killed in hams when heated to an internal temperature of 58. During the mid-1950s the U. S. brine values traditionally have been calculated at % brine = (% salt/% salt + % water) × 100. and nitrite that is palatable and permissible.5%. who performed a series of tests with spiced ham inoculated with different strains of C.0% brine or above. The debated issue was the time and temperature for preenrichment. In North America. Scott (1955) concluded from the literature that because nitrate exhibited relatively poor antimicrobial inhibition and nitrite. Jensen. They investigated why Staphylococcus aureus is rarely. Additional tests showed that if the broth medium (pH 6.

4. A significant observation was that at brine levels of 6. At 5. They concluded that the stability of shelf-stable canned cured meat given less than a botulinal cook was the result of the combined effect of nitrite.95% brine. SUMMARY FOR 1950–1960 As 1960 approached. Nitrate.09%. There have been no reported attempts to duplicate this effect of brine at levels approaching total inhibition. This research and a subsequent review by Silliker (1959) established the future direction for research on nitrite. 2. salt.4°C to 37. nitrate actively stimulated spoilage by aerobic spore formers. 1950.0% to 7. Several new reports of unstable commercially canned cured meats containing nitrate appeared (Hankins et al. Because viable spores could be recovered from stable commercial product. Nitrite was shown to play a significant role in the stability of shelf-stable canned meat. its value as a preservative in perishable cured meats was still in doubt. salt. 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.8°C for up to 90 days.25%. 3. 1956). toxin was produced without concomitant organoleptic spoilage. and 7. 3. had no antimicrobial effect. the product was organoleptically unacceptable — as the cans swelled — and toxin assays were performed. As expected. At brine levels approaching the inhibitory level. and a low indigenous spore level. 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.125% and abused at 29. toxin was not produced. Greenberg et al. Verhoeven. S. the primary effect of nitrite was to prevent germination and/or outgrowth of heat-injured spores. other than its possible influence on water activity.5%. aureus was killed during the heating process given to hams. such as micrococci and bacilli. permitting the growth of aerobes. thermal injury to the spores. 5. 1950. Brine content was further shown to influence botulinal outgrowth and toxin formation. (1959) subsequently demonstrated botulinal spore outgrowth in perishable canned cured meat having brine levels from 3. and thermal injury to the low indigenous level of anaerobic spores. Nitrate could serve as an electron acceptor. Although nitrite was recognized as an effective antimicrobial agent. Anaerobic intolerance to nitrite within the interior of ham. At 8. 6.0%) alone was not responsible for stability. The suspected role of nitrite was to prevent germination or outgrowth of surviving heat-injured spores. (1958) used this class of product to show that nitrate played no role in retarding putrid spoilage. or 5. Eddy and Ingram.14% brine or less. Silliker et al. Although shelf-stable canned cured luncheon meat had been produced for years. Brine level (0%. although there have been several studies under other conditions in which toxin has been detected without obvious organoleptic spoilage. toxin was produced without obvious organoleptic spoilage.176 Antimicrobials in Food complaints of excessive saltiness in the new products. Sodium nitrite levels were 22 µg/g or less at the time the meat was inoculated. This brought nitrate under renewed attack.12%. as postulated by test tube experiments. . was not confirmed.. The system providing stability consisted of the combined effect of nitrite. 7. the reasons for stability had not been defined. the following became increasingly clear: 1. per se.

Another factor in spore destruction is that a thermal process of F0 = 0. of spores of Bacillus species in the presence of less than 300 µg/g of sodium nitrite at pH 6.4 to 0. The increased use of vacuum packaging raised new questions about the microbiological safety of cured meats. 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. 1965).2 (Greenberg et al. (1967) opened new possibilities. the initial spore load in the product is too high. He estimated the thermal process caused a 4-log reduction of the indigenous spores. Outbreaks of botulism from temperature-abused vacuum-packed smoked fish during the early 1960s added impetus to this research. Research was initiated on irradiation for shelf-stable canned cured meats.0. They concluded that nitrite and nitrate have either neutral or stimulatory effects on bacteria and that nitric oxide has virtually no effect. In a study comparing irradiation with conventional thermal processing.7°C for 6 months. The presence of 25 µg/g of residual nitrite after processing and a relatively low brine level of 3. Germination was completely prevented by sodium nitrite levels of 750 µg/g or higher at pH 6. Shank et al.5. heme synthesis is impaired. and toxin formation from this low level of survivors during storage at 30°C to 37. the observations of Perigo et al. (1962) explored whether nitrite or the oxides of nitrogen were involved in the inhibition of Gram-negative bacteria in cured meats. Gould (1964) observed germination. but not in others. One reason given was that spores indigenous to meat emulsions have lower heat resistance than spores produced in laboratory media.2.1 to 0. however. 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. but at a reduced rate.017 viable cells/g remaining after processing to F0 = 0. Nitrous acid was considered primarily responsible for the quantitative as well as qualitative changes that occur in the bacterial flora when meat is cured. heat resistance.36% did not prevent outgrowth. and nitrate is not reduced anaerobically. Shelf-Stable Cured Meats Riemann (1963) supplemented existing information on the incidence.to 5-log reduction occurred. Product inoculated with the lowest inoculum level (173 spores/g) had 0. development ceased before lysis or rupture of the spore walls. making them unavailable for subsequent metabolism. producers of shelf-stable canned cured meat was reported to range from F0 = 0. The toxicity of nitrite was 3 to 5 times greater at pH 6 than at pH 7. If. The thermal process used during the early 1960s by seven U. 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. even if nitrate is present as an alternative electron acceptor. (1964). The authors concluded that when oxygen is absent during the growth of some bacteria. The surviving spores are subsequently inhibited by the salt and nitrite. He concluded that most of the spores are killed during the thermal processing of shelf-stable canned cured meat. a 4. It was postulated that nitrous acid reacts either with the cell itself or with various constituents of the medium.S. If the surviving spore counts between the enzyme-inactivating (74°C internal temperature) and the shelf-stable process (F0 = 0. Nitrate and oxygen were considered interchangeable as electron acceptors required for heme synthesis in some bacteria. In the course of this research. with a median of 0. canned chopped ham was inoculated with botulinal spores and given 2 thermal processes. and outgrowth of spores with unpublished data from the Danish Meat Research Institute.6 at the center of the can would yield 10-fold higher values near the surface of the can..203) were compared. . Hemin had no effect on nitrate reduction or growth of Escherichia coli or Bacillus polymyxa. Immediately after germination. The impaired synthesis of heme prevents the formation of the required cytochrome systems. spoilage. The inhibited spores did not appear to become fully phase dark.

This process caused the surface of the meat in the can to reach 96°C to 107°C. heat resistance was decreased. This process was used commercially with the approval of the USDA.g.5% to 1% (5000 to 10... The meat mixture was then filled into cans. Nitrate had no effect beyond that on water activity..0% brine. (3) destruction of spores on the surface of the meat by the short but high thermal process. 15 minutes at 113°C to 118°C). Kueck et al.. and a low incidence of clostridial spores (less than 1/g) were necessary for the safety and stability of shelf-stable canned cured meats. 1 psig). (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. and then thermally processed. Schack and Taylor (1966) described a procedure to produce shelf-stable canned cured meat with improved quality as a result of reduced thermal process.1 to 0. the meat was ground or chopped and then deaerated and backflushed with nitrogen to displace free oxygen. Increased stability also resulted from improving the canning process to reduce or prevent voids. A nitric oxide-generating material. and nitrate than unheated spores.g. The process had to be properly controlled to be successful (Kueck. the normal pH value of canned luncheon meat.g. sealed. The two Bacillus species were rendered more sensitive to salt by lower heat treatments than clostridia. nitrite. Ascorbic acid was then uniformly distributed throughout the meat. personal communication). When heated and cultured in the presence of the agents. including (1) the destruction of vegetative cells by heating to the initial internal temperature of 71°C. First. spores tended to be protected by salt and nitrate.000 µg/g) increased the apparent heat resistance of the spores.4. but nitrite enhanced heat injury. 30%) and poor meat texture. and nitrate. and the product was given a long thermal process at a low temperature (e. and appeared to be the chief preservative against putrefactive anaerobic spoilage.0. They demonstrated that increased stability could be achieved by omitting sodium nitrate and sucrose from the formulation. especially at pH 6.5 before placing them onto culture media containing salt. 75 to 150 µg/g of added sodium nitrite to provide about 20 µg/g after processing to F0 = 0. This process was tested extensively in at least one commercial plant. 2 to 2 1/2 hours in 74°C water) to achieve an internal temperature of at least 71°C. Duncan and Foster (1968a) also reported that heated putrefactive anaerobe spores were less tolerant of salt. The product was then cooled in tap water and placed into refrigerated storage (e. (1958).g. Nitrite was strongly inhibitory. The process involved a unique sequence of adding the curing ingredients. Their results confirmed the concept of spore injury and increased sensitivity to salt and nitrite proposed by Silliker et al. The product was then heated for a short time at a high temperature (e.178 Antimicrobials in Food Spencer (1966) concluded from the literature that 3. The thermal process required to achieve shelf stability of canned hams larger than 1 1/2 pounds caused excessive purge (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. salt and sodium nitrate at 0. nitrite. The refrigerated storage period was necessary to assure stability. but the process was not dependable and was abandoned.g. 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. Curing salts interfered with some stage of germination and development of the surviving heated spores at concentrations that were ineffective with unheated spores. 30 days at 6°C to 8. Roberts and Ingram (1966) heated spores in water from F0 = 0. When heated in the presence of these agents.0015 to 1. 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). The process was considered to greatly increase the amount of nitric oxide available for use as an antibacterial agent. A 3-pound meat portion was filled into each can. .5% to 4. At 2% to 4%.3°C) before release as a shelf-stable product. The success of the process was believed to be the result of a combination of factors. was then added and mixed into the meat while under a positive pressure (e. These data reemphasized the importance of spore contamination level for the stability of shelf-stable canned cured meats. such as sodium nitrite..

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

0 and below. The effect of brine on type E toxin production was more . The four strains failed to produce toxin during 5 weeks at 10°C in sliced bologna (2.1% or higher. lower maximum population levels occurred in vacuum packages than in nonvacuum packages at 15°C and 22°C. 21 were able to grow under the same conditions. the authors recommended that vacuum-packaged nonsterile processed foods be stored in the frozen state. and 37°C). No mention was made of pH and none of the agents were tested in combination. the presence of nitrite caused very little or no inhibition. The time required for toxin production decreased as the abuse temperature was increased (22°C. Toxin was not produced at 10°C or lower. pH 6. Their findings that the type of cured meat influenced toxin production were particularly significant. Of 25 lactobacilli from cured meat. Pivnick and Bird (1965) found the oxygen permeability of packaging films to influence spoilage but not toxigenesis of sliced cured meats inoculated with C. nitrate. perfringens inoculated into raw hams could survive the thermal process. In jellied pork tongue. Bologna was relatively resistant to type A toxin production but not ham.7 or less after 4 weeks.180 Antimicrobials in Food nitrogen or vacuum was organoleptically acceptable.8% brine). toxin was produced only at 30°C. A total of 90% of the samples held at 25°C remained above pH 6. Similar results were obtained with jellied corned beef stored at 15°C and 25°C. even at 30°C. Schmidt and Segner (1964) concluded that factors or a combination of factors provided a much longer shelf life than might be expected from salt content alone.0. In sliced ham. aureus was inoculated onto sliced chopped ham. When S. This suggested that salt becomes more inhibitory as storage temperatures are decreased. botulinum.8%. residual nitrite.8°C or 10°C. In jellied ox tongue. A similar effect occurred with increasing salt from 0% to 4% during incubation at 7. At pH 6. and decreasing pH during temperature abuse were the suggested inhibitory factors. but no attempt was made to determine whether they could multiply if the hams were subsequently temperature abused.1. Failure to obtain toxin production at 5°C may have been influenced by pH because 13 of 15 samples had a pH of 5. Cooked sliced ham from five producers inoculated with type E spores yielded variable results at 30°C.0.4°C. and temperature on a Microbacterium species in all-purpose Tween (APT) broth. Brownlie (1966) demonstrated the combined inhibitory effect of sodium nitrite concentration. type E toxin was not produced. 30°C. pH.5 in the presence of 200 µg/g of sodium nitrite. toxin was detected in 2 days at 2. which was thought to be the result of product differences in brine and residual nitrite levels.0 to 4. At the highest brine level (4. The combination of brine (3. This would help to explain the dominance of the lactobacilli as spoilage organisms in many perishable cured meats. increasing the amount of nitrite from 25 to 200 µg/g caused progressively greater inhibition. but not at 25°C if the brine was 5.1% to 4. Several studies on the growth of C. 34 µg/g of residual nitrite).7% brine. Christiansen and Foster (1965) found type A botulinal toxin production to occur at the same rate on bologna whether packaged with vacuum or without. the authors concluded that the levels used in ham would permit growth. From studies in thioglycollate medium containing salt.6. At pH 7. and nitrite. The rate of toxin formation decreased as the brine level increased from 3.75%). botulinum in vacuum-packaged perishable cured meats were reported during this period. 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. but not at 29. 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). Within 1 week the product pH had decreased from 6.4% brine at 30°C. toxin was produced at 20°C to 30°C but not at 15°C. A decrease in pH likely occurred during storage and prevented toxin formation in the other variables. a fermentable carbohydrate.35% and 3. type E toxin was produced at 10°C to 25°C but not at 5°C. On sliced bologna. Another 25 strains of microbacteria failed to grow at pH 5. Gough and Alford (1965) showed that spores of C.8%).5% brine and in 4 days at 4. Toxin was produced within 7 days at 30°C in bologna (3. Nitrite was more inhibitory at 0°C than at the other temperatures tested (10°C and 25°C).

8°C and 22. C.1. The authors concluded that heat injury of spores is not necessary for nitrite to prevent toxin production in cured meats. considering the bacon had a brine level of 5. 25°C. Warnecke et al.6% brine at pH 6. (1967) suggested an inhibitory substance was formed from nitrite. An additional factor influencing the apparent significance of nitrite is the growth of competitive flora. The concept of thermal injury to spores that survive the processing of shelf-stable canned cured meats was confirmed. and Lactobacillus viridescens. 5. Riemann (Anon. Toxin was not produced in product with 4. aureus is inhibited by more than 4% brine at pH 5. 5. Perigo et al. and more than 10% brine at pH 6.5% or above. 1967). He concluded that C. The effect of nitrite levels on types A and B botulinal toxin production in vacuum-packaged sliced ham (4. Using juices expressed from the newer sausage product.8.5% brine at pH 5.5% brine at pH 6. In jellied pork tongue.2°C for 3 to 18 days. 3.8 or less.5. Evidence was presented that the antimicrobial effect of nitrite is related to the formation of nitrous acid.2 and 3% brine at all pH values. Type E botulinal growth is inhibited by 2% brine at pH 5..3. an increase in the level of residual nitrite also occurred. C. 9% brine at pH 5.3% brine) was studied next (Pivnick et al. The process of outgrowth was more sensitive than spore germination to nitrite. and 8. Although not mentioned.4% brine. botulinum. perfringens. 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. Toxin was also detected in bacon held at 12. but this is highly questionable.9.. All of them reached the same general conclusions as those set forth by Silliker et al.Nitrite 181 dramatic. 1968) emphasized maintaining adequate salt levels to assure the microbiological safety of perishable vacuum-packaged cured meats. and 30°C). A new explanation for the shelf stability of canned cured meat was proposed. The growth of S. which disappeared during the thermal process. a brine level of 4.2°C. . Kafel and Ayres (1969) demonstrated that the growth of enterococci in culture media and.1. increasing the nitrite level increased the time for toxin production. SUMMARY FOR 1960–1970 Progress made during the 1960s toward defining the role of nitrite in cured meats included the following: 1. more importantly. In both products a marked delay in toxin production occurred at 20°C. and jellied pork tongue) were formulated with commercial levels of nitrate and nitrite. (1967) reported type E botulinal toxin production in ground beef held at 22. and an input nitrite level of 500 µg/g or more. The system responsible for the shelf stability of canned cured meats given less than a botulinal cooking was studied by several research groups.5 to 5. 2. At each temperature (20°C. botulinum type A seemed to be completely inhibited by 4.1% brine) and jellied pork tongue (3. in perishable canned hams can inhibit the growth of C. Toxin was not produced in either bologna or country cured ham. 4. as the brine level increased in the bologna and ham. sporogenes. All three products (bologna. during which time naturally occurring lactics drop the pH to 5.35% and after 8 days with 3. toxin was detected after 4 days with 2. they determined that type B botulinal outgrowth was inhibited during 4 weeks at 30°C if the juice had a combined pH of 5.28%.8% to 4. The product had been changed to a shelf-stable fermented product precooked to 80°C in an oxygen-impermeable casing. The raw product was then sold at ambient temperature for cooking at home. (1958).5% brine at 20°C. ham.3.

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

but not at levels above 0. as opposed to perishable canned cured meats heated in hot water to a minimum internal temperature of 66°C. Perigo et al. Small differences in the amount of sodium nitrite below 0. 115°C). sporogenes spores to provide a level of 104 spores per can.5°C on the outgrowth of surviving spores was also examined by placing the heated tubes at 23°C for up to 1 year. Ashworth and Spencer (1972) reported the equivalent levels to be . and 0. the liquid placed into tubes. personal communication). inoculated (105/ml).6% salt. for inhibition. The inhibitory level of nitrite was not influenced by pH within the range 5. respectively. Omitting any one or all three of the additives did not affect thermal destruction. (1973) studied the effect of adding nitrite to the brine solution for canned Vienna sausages. The cans were then filled with brine (3% salt).g. exist (Christiansen et al. The effect of heating spores of PA 3679 at 115. Considerable differences were noted in their redox potentials. cysteine. It was reported that less nitrite was required for inhibition when it was added before autoclaving.5°C in ground pork containing 2.8% salt.” The effect of all three factors (pH. normal.4). aw 0. 150. and. and then inoculated with vegetative clostridial cells. One of the sausages in each can was inoculated with C. however. cooked sausages. as already discussed.7 to 6. The extract had 2. sporogenes strain PA 6982 in perishable canned cured meat as occurs with C. and salt) appeared to be additive. A 1:1 extract was next prepared with 3% saline and ground. the solids removed. Preliminary data. residual nitrite levels remained above 125 µg/g throughout 80 days at 30°C.5% sodium tripolyphosphate was studied by Nordin et al. The magnitude of the effect was much less than was previously reported in culture media.6. 200 µg/g sodium nitrite. Aqueous meat suspensions prepared from raw. showing a similar iron–nitrite interaction with C. Sausages packed in brine with 400 µg/g of sodium nitrite and processed to F0 = 0. A significant observation was that residual nitrite did not undergo normal depletion in the extract.3. nitrite. (1967) reported equivalent inhibition with 8 µg/g of nitrite added before heating the medium and 52 µg/g added after heating the medium. 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. pH. (1967) medium and reinforced clostridial medium but not in liver veal or Wynne fluid media. and heating at 115. These observations led to the conclusion that a Perigo-type effect was detected in the meat system. nitrite and salt levels had lesser effects. Outgrowth was determined by blackening of the tubes as a result of ferric citrate having been added to the meat. sealed. thereby confirming earlier research. overlaid with paraffin oil. cooked. The effect of nitrite.4. Between 120 and 200 µg/g sodium nitrite provided stability through 5 weeks of storage. and heated (F0 = 0. After a loss of 15% to 25% during processing and an initial decline. The major factor influencing outgrowth was pH. to some degree.. The residual nitrite values of the four media and three meat suspensions were about the same after preparation. A similar response with strain 3679 would not be unexpected. ascorbate and cysteine) enhanced the antibotulinal effect of the meat suspensions but did not induce Perigo factor formation. Ashworth and Spencer (1972) described a model system of ground pork (25 g) in 1-ounce bottles. The sausages were prepared with nitrite-containing curing salt.g. 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.01% strongly influenced the results. held overnight for nitrite equilibration.01%. This emphasized the importance of medium selection when studying the antimicrobial activity of nitrite and relating the results to commercially produced meats. and heated to varying degrees.. and overheated (115°C for 90 minutes) canned luncheon meat required 500. Farkas et al. and then packed into cans. and pH 6.35 were as stable as sausages in brine without nitrite and processed to F0 = 2. salt. Johnston and Loynes (1971) obtained Perigo factor formation in the Perigo et al. (1975)..Nitrite 183 sodium nitrite caused the greatest destruction when heated. The pork was autoclaved before or after adding nitrite. Reducing agents (e. although they were “clear and undisputable subsidiary effects.g. thioglycollate) enhanced the effect of nitrite.. Reducing agents (e. botulinum. In ground pork.966. sodium ascorbate. and 50 µg/g of sodium nitrite.

(1969) found the inhibitor of Perigo et al. Inhibition by nitrite diminished gradually over a period of 8 weeks at 37°C. Johnston et al. In this case alone. that although the Perigo inhibitor may not be present in meat. more was required when added before heating the meat. with prolonged storage (42 days at 37°C). Similar residual nitrite levels were required to inhibit vegetative cells (160. Ashworth et al. which had survived heating to F0 = 0.000/g) added after heating and spores (20/g) added before or after heating. Botulinal .5% salt and 150 µg/g of sodium nitrite. such as perishable canned ham. The authors found that as the original input nitrite level increased.g. (1973) extended this research to the milder heating given to large perishable canned hams and obtained similar results. autoclaving. Adding salt (3.. and inoculated. The data obtained with the spore inoculum should be more relevant to shelf-stable canned cured meats. the Perigo effect was of low magnitude. higher levels of added nitrite were required for inhibition. The meat was then inoculated with botulinal spores. Ashworth and Spencer based their conclusions on the residual level of nitrite.4 The product was held until the highest level of nitrite declined to less than 2 µg/g. results similar to those just mentioned were obtained during the early stages of temperature abuse. more added nitrite was required in the pork if the nitrite was added before (300 µg/g).5 and 22. and 200 µg/g of sodium nitrite and processed to F0 = 0. Perigo et al. outgrowth and toxin production decreased. rather than after (150 µg/g). however. Shelf-stable canned cured luncheon meat was prepared with 0 to 300 µg/g of sodium nitrite and processed to F0 = 0. It was originally suggested (Perigo et al. and it may also play a role in the safety and stability of pasteurized cured meats. Ashworth et al. another inhibitory factor may be formed when nitrite is heated with meat.184 Antimicrobials in Food 60 to 70 and 90 to 100 µg/g. The levels selected (53. (1967) to be inactivated when added to meat. 100. The term Perigo-type factor (or PTF) was selected to describe the effect observed in their test. If residual nitrite is a valid means for comparison. Chang and Akhtar (1974) prepared buffered saline suspensions of canned luncheon meat made with 0. When spores were used. 150. strongly influenced clostridial inhibition.1 µg/g) were the residual nitrite levels after 0 and 2 weeks in product made with 200 µg/g of nitrite. 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.4 in raw meat juice with 4. 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. In summary. (1967) based their conclusions on the added level of nitrite. Residual nitrite. concluded that a PTF was produced. thermal destruction of spores and inhibition of the survivors by the salt). the conclusion that a Perigo effect was demonstrated must be tempered with the observation that. The amount of PTF formed was related to the amount of original nitrite added to the meat before heating. The additional nitrite provided comparable residual levels after the various heat treatments. higher initial residual nitrite levels were required for inhibition.5 or 22. on the basis of added nitrite. deaerated in boiling water.1 µg/g. They concluded that although a PTF was formed. In all cases. the levels of nitrite required to obtain inhibition were about the same or lower if added before heating than if added after heating. With excessive heating. the relative inhibitory effect was quantitatively small. This was considered evidence for an inhibitory factor other than residual nitrite. less residual nitrite was required for inhibition when nitrite was added before heating. Pivnick and Chang (1974) considered. 1967) that the nitrite that disappears during heating may be involved in the formation of the unknown inhibitor. per se.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.. Actually. However. Ashworth and Spencer (1972) found no relation between the amount of nitrite lost during heating and the inhibition obtained. The depletion rate of the nitrite added to the suspensions was not influenced by the amount of nitrite originally added to the meat.4. The nitrite level of the suspensions was adjusted after heating to 53. which indicates that residual nitrite influenced the results.

e... . indicating the Perigo factor is not stable. They subsequently concluded (Riha and Solberg. 22°C. There was no evidence of germination (i. the rate of swelling ranged from 11 to 48 days across all variables. 200 µg/g) usually used in meat products. 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. Asan and Solberg. and fewer treatments proved stable. Inoculation of the medium after heating with nitrite and aging for 3 months showed that it was less inhibitory. 1978. predictably. 1975. heat injured. They hypothesized that nitrite. 1975. Hansen and Levin. Nitrate had a slight but statistically significant inhibitory effect that may have been caused by nitrite generated from nitrate. Mirna and Coretti. age of product must be considered when investigating the presence or activity of PTF in commercial product or model meat systems.. iron. 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. Decreasing inhibition occurred as the product was held for 0. 2. The degree of inhibition was correlated with the amount of nitrite originally added to the product. van Roon. Ashworth et al. and 37°C. toxin was not detected. Additional data by O’Leary and Solberg (1976) suggested that inhibition of C. The authors concluded that it was not possible to demonstrate any preservative action of nitrite at the levels (i. as nitrous acid. or irradiation injured. 1974. respectively. van Roon. in some form.. Moran et al.. and perishable canned meat and held at 4°C. Wasserman and Huhtanen. Van Roon (1979a. and 4 weeks before preparing the suspensions. This was later reassessed by Christiansen (1980). 1974). Thus. perfringens may involve an interaction of nitrite. Botulinal outgrowth in product abused at 27°C was influenced by the spore inoculum level and the level of nitrite.. with sulfhydryl-containing constituents of the bacterial cell. considerable effort was devoted to perishable cured meats. Roberts and Smart (1974) found that the inhibitor in the medium of Perigo et al. b) concluded that Perigo-type inhibitors do not contribute to clostridial inhibition in heated cured meats. (1973).. vegetative cells) in the media with inhibitory levels of nitrite. common agreement that nitrite.b). 1976. 1972. 1974. Neither swelling nor toxin occurred in some treatments having 240 or 480 µg/g of nitrite. who concluded that residual nitrite is a significant factor influencing botulinal inhibition. Riha and Solberg (1975a) studied the formation and effect of a PTF on seven strains of C. 1974.e. Incze et al. At 22°C. Tjaberg and Kvale (1972) studied the antibotulinal effect of nitrite (0 to 480 µg/g) added to canned raw meat. 1969. Nontoxic spoilage occurred at 7°C in uninoculated product made without nitrite. Huhtanen and Wasserman. At 4°C. It was stated that botulinal inhibition was related to the level of nitrite added during formulation rather than residual nitrite.Nitrite 185 inhibition was related to the original amount of nitrite. regardless of the inoculum level or whether the spores were untreated. although the spores remained viable during prolonged incubation. At 37°C. (1967) had a comparable effect on the inhibition of clostridial spores. 1975.1 µg/g of sodium nitrite yielded similar results. Lee et al. causes inhibition by reacting with enzymes containing functional sulfhydryl groups. 1979a. Perishable canned cured pork was studied by Christiansen et al. Additional studies were reported by 1980 that dealt with the Perigo factor (Mirna and Hofmann. swelling and toxin production ranged from 3 to 17 days. perfringens in a chemically defined medium. and sulfhydryl compounds are involved in the formation of the Perigo factor in broth media may be significant. shelf-stable canned meat. 1975b) that inhibition was apparently at the cellular level. Perishable Canned Cured Meat and Slurries of Meat In addition to research on shelf-stable canned cured meats and the composition and role of the Perigo factor in commercial products. Debatable as the formation of a Perigo-type in meat may be. Research on perishable canned cured meat or meat slurries is discussed first. A second test using canned product held for 4 weeks at 35°C before preparing suspensions with 22.

(1977). perishable canned cured meat is placed at 27°C. On investigation (Tompkin et al. The emulsions were filled into cans and heated. Spores heated at lower temperatures (70°C or 80°C) were inhibited by 4.8% or 3.5°C. At 17. 4.5% salt. 101). Adding L-ascorbic acid (1. The rate at which inoculated cans swelled at 27°C was inversely related to the level of nitrite added to the product. level of clostridial spores.0. a preliminary test in bacon led to the conclusion that L-ascorbic acid does not alter the antibotulinal properties of nitrite in bacon. less inhibition occurred.5% or above. In another study. The antibotulinal effect of nitrite was considerably greater than that reported for the meat slurries of Roberts et al.0%) markedly increased the effectiveness of nitrite with none of the 38 strains tested showing growth in broth containing 50 µg/g sodium nitrite.1%) also increased the efficacy of nitrite in broth but had no effect. 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. pro or con. The spores heated at 70°C and 95°C were equally sensitive to the autoclaved nitrite. 100. Christiansen et al. Although less effective. 1978b) involving product held at 4.5°C. one reason was found to be the addition of sodium isoascorbate to the canned meat but not to the meat slurries.4°C or 10°C for up to half a year before placing at 27°C. a lower concentration of L-ascorbic acid (0.186 Antimicrobials in Food Baird-Parker and Baillie (1974) studied the growth of a large. (1976).5°C and 25°C. At 20°C. 6. 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. product pH. Jarvis et al. 3. Stability was influenced by nitrite content. 1978a). or 25°C. Jarvis et al. 15°C). 20°C.5% but not 3.5% salt (on water phase).5).. 22. It was suggested that Bacillus species are less sensitive to nitrite than clostridia.5.. and decreasing inoculum level (106. 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 . Temperature of abuse was the most significant factor influencing the rate of botulinal growth.0. 5.0% w/v). (1976) to study the interactions of temperature of abuse. a race occurs between death of germinated botulinal cells and depletion of residual nitrite. and inoculum level on botulinal growth and toxin production at pH 6. 6. even with 300 µg/g of sodium nitrite.0. 150. 20 µg/g of nitrite in the Perigo medium and 210 µg/g in the slurry were equally inhibitory. neither salt level provided much inhibition. Sodium nitrite and L-ascorbic acid were added as filter-sterilized solutions.5%. Autoclaving nitrite in the Perigo medium resulted in significantly more inhibition than autoclaving the nitrite in the meat slurry. salt. the more the residual nitrite levels declined and the less inhibitory the product became when placed at 27°C. diverse collection of botulinal strains in a broth medium and a cooked pork medium. 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. in the cooked pork medium. (1978) concluded that when inoculated. and whether the products were treated by a perishable or shelf-stable thermal process. Botulinal outgrowth was dependent on the relative levels of residual nitrite and surviving botulinal cells. nitrite. This agreed with an earlier observation (Tompkin et al. 6. Grever (1974) examined the stability of emulsions normally used for preparing cooked sausage and liver sausage. a pronounced salt–nitrite interaction occurred. decreasing temperature (25°C. At 20°C.0%. recommended that large-scale tests be used to quantify the many interacting factors influencing the safety of perishable cured meats. decreasing pH (7. The longer the product was held at refrigeration temperatures. or 95°C decreased the number that could grow in a recovery medium containing 2. 103. the greatest inhibition occurred with 1. Isoascorbate had enhanced the antibotulinal efficacy of nitrite in the canned meat. This led to the pork slurry system of Rhodes and Jarvis (1976) and used by Roberts et al. At 15°C. 200 µg/g).5%. 90°C. increasing salt (1. The effect of nitrite in perishable canned cured meat was also reported by Tompkin et al.5% salt. The number of strains showing growth in broth was found to decrease with increasing nitrite (50. (1976) found that heating botulinal spores in an aqueous extract of lean pork at 85°C. At the lower temperatures. Furthermore.

Suggested mechanisms were proposed for the relation of iron to the antibotulinal efficacy of nitrite (Tompkin. heating to 68. This was based on the rationale that in clostridia. Each active site is comprised of iron bonded to both cysteine and sulfur (Buchanan and Arnon. Subsequent research more clearly confirmed the relation of available iron to the antibotulinal efficacy of nitrite (Tompkin et al. and then storing the tubes at 27°C. sporogenes.g. cysteine. Clostridium bifermentans. 1979b). with one exception: beef liver. Clostridial ferredoxin is believed to contain two independent. ascorbate. One possibility involved the reaction of nitrite with an essential iron-containing compound (e. On the basis of the data existing in 1978. however. It was also shown that the type of meat could influence the degree of botulinal inhibition.5°C. When testing a frankfurter emulsion prepared with mechanically deboned chicken meat. hemoglobin. and myoglobin). 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). a simplistic explanation for the inhibition of clostridia consisted of the uptake of undissociated nitrous acid. 1978. no botulinal inhibition was observed. and enterococci. This made it possible to explain the differences in the relative efficacy of nitrite in various meats on the basis of their iron content. as can occur with nondenatured (i.. overlaying with vaspar. but as in other model systems receiving a mild heat treatment. A third possibility is the germination of dormant spores after residual nitrite has declined to noninhibitory levels. the degree of inhibition was less than that reported by others. 1978e). Isoascorbate. As the level of nitrite decreased. cooling.g. 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. or pork hearts for fresh pork ham reduced the efficacy of nitrite. 1978d). C. 1979a). Tompkin et al.g. The efficacy of nitrite in frankfurters was explored by Sofos et al. which later was demonstrated to be the sequestering of iron (Tompkin et al. cytochrome oxidase. and interference with energy metabolism in the vegetative cell to prevent outgrowth. ferredoxin) within the germinated cell. This avoided the problem of recontamination inherent in some studies after heating. however. or 200 µg/g) and then processed to an internal temperature of 69°C. 1978e). the rate at which the product spoiled increased.. 50.5% salt and sodium nitrite (0.Nitrite 187 (Tompkin et al. Subsequent outgrowth could occur when (1) residual nitrite depletes to noninhibitory levels. beef liver did not cause a substantial loss in the antibotulinal efficacy of nitrite.e. cytochromes. . which resulted in a lower level of residual nitrite after processing.. a nitrogen oxide (e. and enzyme activity. 1979b). Their test system mainly consisted of extruding a frankfurter emulsion into test tubes. 100. (1979b–e)... allowing the dissociation of the nitrogen oxide from the iron.. Bacillus species.. single-electron transfer sites consisting of four iron atoms.. (1978) studied the effect of nitrite on the stability of naturally contaminated perishable canned pork when temperature abused at 37°C. ferredoxin) are essential for electron transport. a background flora of thermodurics existed from the ingredients. Wojton et al.. nitric oxide)–iron reaction as in ferredoxin. nonheated) heme compounds. and ethylenediaminetetraacetic acid (EDTA) were found to share a common function in meat. beef round. decreased botulinal inhibition. beef hearts. nonheme iron–sulfur proteins (e.. catalase. Substituting turkey thigh meat. Although isoascorbate enhances the antibotulinal effect of nitrite in freshly prepared perishable cured meat that is temperature abused. Adding 156 µg/g of sodium nitrite delayed botulinal toxin production. energy production. peroxidases. Adding hemoglobin. 1970). perfringens. and/or (2) repair through the formation of new unreacted ferredoxin by the vegetative cell. 1978c). low levels of nitrite (20 and 40 µg/g) did not influence botulinal growth or toxin production (Sofos et al. Despite its relatively high iron content (49 µg/g). isoascorbate also reduces the efficacy of nitrite by causing more rapid depletion of residual nitrite (Tompkin et al. The product was formulated with 2.g... Spoiled cans yielded C. The reason isoascorbate enhanced the antibotulinal effect of nitrite was pursued (Tompkin et al.

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

aureus developed and enterotoxin A was produced. Food and Drug Administration (FDA) for approval of sodium nitrite as an additive in poultry products. Bacon that was sliced and vacuum packaged developed a flora mainly of micrococci and lactics.7% and 18. growth was faster at 25°C than at 15°C.7% to 6.7% became toxic. A summary of the data showing the antibotulinal effect of nitrite in the two poultry products appears in Hauschild (1982). respectively. ascorbate (0. The predominant flora of the bacon after curing consisted of micrococci. 20°C. cereus tested. Erythorbate was found to enhance the inhibitory effect of nitrite. . If all samples containing mixed cure with and without ascorbate were compared. aureus grew well in the medium-salted bacon. 1977). 67. the interaction of nitrite and ascorbate is difficult to interpret. Erythorbate caused reductions in the residual nitrite and the redox potential of the product. Noninoculated product containing nitrite developed a slight unusual taste at 20°C owing to the growth of spore-forming spoilage organisms (e. medium 4.Nitrite 189 A request was made by the poultry industry to the U. Product with 100 µg/g of sodium nitrite remained nontoxic through 14 days at all three temperatures. and 2000 µg/g). Shaw and Harding (1978) studied the effect of nitrate and nitrite on the microbial flora of Wiltshire bacon. (1976) in the United Kingdom studied the effects of a mixture of nitrite (100 or 200 µg/g) and nitrate (250 µg/g).98. The nitrate likely served as an electron acceptor for the growth of micrococci in the vacuum package. The naturally occurring B. the sausages had relatively high pH and water activity values of 6.80 and 0.. botulinum.3%. 500. A higher percentage of samples were toxic with the addition of 200 µg/g of nitrite than with 100 µg/g of nitrite. It also was reported that S. The request included results by Christiansen et al. The data included botulinal challenge tests in frankfurters made with chicken meat and ham made with turkey meat. higher populations of S. respectively. respectively.8% and 21. B. and mixed cure with 200 µg/g of nitrite were compared. The addition of ascorbate enhanced the antibotulinal effect of 100 µg/g but not 200 µg/g of nitrite. lower populations resulted and enterotoxin was not detected. If all samples with no mixed cure. Inoculation studies examined the effect of nitrite (0 and 100 µg/g) and storage temperature (15°C. 2. When all samples having the mild and medium brine were compared. and 1000 µg/g) on the growth of Bacillus cereus in cooked sausage placed at 20°C for 48 hours. regardless of the level of nitrite or ascorbate.. There was a general trend toward reduced residual nitrite levels at the tune of inoculation when ascorbate was added.9% became toxic. 1000. 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. Raevouri encouraged more widespread use of erythorbate for sausage production in Finland. In vacuum packages. Including nitrate in the bacon enhanced the growth of micrococci. Omitting nitrate led to higher numbers of Moraxella species in the cured bacon. Because brine levels could not be held constant. 35.6%) on botulinal toxin production in vacuum-packaged back bacon. Moraxella species. mixed cure with 100 µg/g of nitrite. 19. 100. In opened packages.9% became toxic. As expected.9%. 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. and 25°C) on the rate of botulinal toxin formation. After cooking in Saran™ casings.9% to 4. and 34. Raevuori (1975) studied the combined effect of sodium nitrite (0. cereus was evidently more resistant to nitrite than C.65 to 6. botulinum. cereus). Bacon Crowther et al. and brine level (mild. and 200 µg/g) and sodium erythorbate (0.4%.g. 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. Erythorbate alone was not inhibitory.S. These values raise a question concerning the conclusions that (1) protection was greater if the level of nitrite was increased to 200 µg/g and (2) sodium ascorbate at a level up to 2000 µg/g did not reduce the protection afforded by nitrite against C. and Moraxella-like bacteria. (1977) that demonstrated the efficacy of sodium nitrite in poultry meat. 42.

The initial period of delay depends on the inoculum level. The addition of ascorbate or isoascorbate can act in concert with residual nitrite to retard botulinal outgrowth in freshly produced bacon. Too high a level of botulinal cells when the bacon is abused can overwhelm the inhibitory system. ascorbate and isoascorbate can also have a negative effect by causing more rapid loss of residual nitrite during processing and storage. 4. The bacon was then incubated at 27°C for up to 56 days. Ivey et al. Temperatures below 27°C or 30°C increase the delay in outgrowth and the inhibitory effects of the foregoing factors. 1974. The USDA conducted a test to verify the antibotulinal effectiveness of a combined nitrite–sorbate mixture in bacon (Johnston. 1978. 6. Botulinal outgrowth is influenced by the temperature of abuse. it is difficult to assess their effect.. 1979). phosphate.0%.0% are not inhibitory to botulinal outgrowth.. it was suggested that nitrite could be important in delaying the sour spoilage caused by the growth of lactics.7% sugar (sucrose) or more provides sufficient fermentable carbohydrate that naturally occurring lactics cause a decline in pH to inhibitory levels. Three types of bacon were produced in four commercial plants: bacon with no preservative. 8. fermentable carbohydrate. The levels of sodium chloride. Brine levels below 4. There was considerable variation in toxin production in the bacon from the four plants.. Of the 30 tests. These factors interact to influence botulinal inhibition. Because phosphates were present in almost all the studies. 7. This was likely the result of the interaction of the other components of the product (brine level. 1980). bacon with 120 µg/g of sodium nitrite. They can be more readily compared because they were similar in processing and final product.g. 1980b. 3. . All three bacons contained 550 µg/g of sodium ascorbate or sodium isoascorbate. phosphates do not appear to influence botulinal outgrowth in bacon. A total of 30 botulinal studies were performed with vacuum-packaged bacon during the 1970s in the United States and Canada. Within the levels and types used. The data indicate the following: 1. If a lactic fermentation develops in the interim. and bacon with 40 µg/g of sodium nitrite plus 0. Vacuum-packaged bacon prepared with 0. and vacuum packaged (100 g per package). 1974. Although the studies were performed for a variety of reasons. 1974. and growth of a spoilage flora). aside from the fact that the amount of added nitrite partially determines the level of residual nitrite. the combination of relatively higher brine and decreasing pH can prevent botulinal outgrowth. The level of residual nitrite at the time the bacon is abused influences the extent of the delay in botulinal outgrowth. Collins-Thompson et al. sucrose) were left to the discretion of the producing plant. However. Tanaka et al. Bowen and Deibel.. The bacon was shipped to a laboratory. Sofos et al.190 Antimicrobials in Food Because higher numbers of lactics were present in bacon with the lowest initial nitrite concentration. the data supported the potential approved use of a nitrite–sorbate blend to produce bacon. 10 appear in the technical literature (Christiansen et al. At least on the basis of the botulinal results. 2.26% potassium sorbate. and fermentable carbohydrate (e. 5. The pH of the bacon during abuse significantly influences the potential for botulinal outgrowth.. inoculated with 1000 spores/g. it is possible to glean the relative effect of nitrite by examining the nitrite-containing control variables from each test. The level of nitrite added to the product is not important. The results demonstrated that the combination of nitrite–sorbate was equal to or better than nitrite alone. As the brine level exceeds 4.. outgrowth is increasingly delayed.

Buchanan and Solberg (1972) studied the effect of nitrite (0. Lactobacilli (78 strains). aureus under aerobic but not anaerobic conditions.. anaerobic toxin production occurred at pH 5. 1000. future research might demonstrate the involvement of additional factors. because high nitrous acid values (>0. brine. aureus by blocking the sulfhydryl sites of either coenzyme A or α-lipoic acid. A third sample with a pH of 5.34. however.3) on the growth of S. (1969) found the production of type B staphylococcal enterotoxin to occur earlier on ham at 30°C than at 22°C to 24°C and earlier under aerobic than anaerobic conditions. aureus in brain heart infusion broth (BHI). 5%. and product pH) contributes to this effect. and initial pH (6.g. the data along with the aforementioned results of Christiansen and Foster (1965) and Crowther et al. aureus. The probability of toxin production at 10°C reportedly decreased as the level of nitrous acid increased. it is uncertain whether high nitrous acid content. to a slight extent.3 and 200 µg/g. Heterofermentative lactobacilli were more tolerant of salt than the homofermentative strains. 10%. and 2000 µg/g). aureus was found to be restricted to the periphery of commercially fermented sausage (Barber and Deibel. Nitrous acid levels were calculated from the residual nitrite and pH values as described by Castellani and Niven (1955). nitrite was without effect in an aerobic environment and 500 µg/g or less was without effect anaerobically.6 µg/g) were possible only at reduced pH levels (pH 5.3. the increase in the adjustment phase was 10 hours or less aerobically and about 30 hours anaerobically. They also concluded that vacuum packaging should improve the safety of cured meats relative to S. micrococci and staphylococci (24 strains). growth of competitive flora. Extensive tests were also reported for laboratory-cured ham. 8 µg/g enhanced the growth of the lactobacilli and. At pH 6. 500. This suggested either that nitrite was not being used as an electron acceptor or the nitrite reductase system was blocked anaerobically. Nurmi and Turunen (1970) studied the effect of adding nitrite to a previously autoclaved broth medium (pH 6. low oxygen level. The authors questioned the hypothesis that nitrite inhibition is dependent on the conversion of nitrite to nitrous acid. At 40 µg/g growth was comparable to that in the control without nitrite. aureus is less acid tolerant under anaerobic conditions. 22 µg/g of nitrite. but it is unclear whether nitrite level influenced anaerobic enterotoxin production. and an abnormally low pH of 5. Of significant importance was a report that the growth of S. oxygen pressure.58. Understanding why this occurs would be very instructive. Buchanan and Solberg suggested that nitrite inhibits the growth of S. 15%. However. thus preventing the normal metabolism of pyruvate. After considering the effects of changing pH during the growth of S. Very high levels of nitrite (1000 and 5000 µg/g) either prevented or severely retarded the growth of all strains.6 or less).3 in two samples with negligible nitrite levels. aureus and a review of the literature. and 0. One reason given was that S. At pH 7. Anaerobic toxin production was prevented with 0. (1976) indicate that the growth of S. the micrococci. reduced pH. nitrite. Reasons for the reduced growth and enterotoxin production are evident from the reports.6 µg/g or more nitrous acid and pH values below 5. 1972). aureus and Other Bacteria A cluster of reports appeared during the early 1970s that dealt with the effect of nitrite on S. At higher temperatures (22°C and 30°C). Nitrite was metabolized by S.01% salt.Nitrite 191 Tests with S. Genigeorgis et al.6%.3. and 0% oxygen yielded lower S. but it remains debatable how much each of the possible factors (e. aureus is reduced and enterotoxin production can be prevented by the environmental conditions existing within vacuum-packaged cured meats.0).3. At 200 µg/g growth was delayed or slower. 200. no residual nitrite. 7. Toxin was produced in certain variables having residual nitrite levels as high as 145 µg/g.9% salt and 100 µg/g of sodium nitrite) incubated at 37°C in environments of 20%. aureus. Collectively. The commercially canned ham used for the test had a brine level of 3. or both prevented anaerobic toxin production.159 µg/g of nitrous acid also became toxic. and Pediococcus cerevisiae (1 strain) were examined for their tolerance to nitrite in the presence and absence of 4. aureus populations as . Inoculated raw sausage (2.

Perhaps because of the atypically high pH. and 8%) increased at pH 6. The inhibitory effect of nitrite in the recovery medium increased with increasing salt content..6 to 6. both of which lack cytochromes. Morse and Mah (1973) studied the effect of glucose on enterotoxin B synthesis in a broth medium buffered to an alkaline pH (7. decreasing incubation temperature.0. and nitrate was converted to nitrite. If manipulations of thermally processed meat were attempted to extract such an inhibitor. more available energy. (1979) reported that nitrite inhibits active transport. Although adequate growth occurred within 48 hours. Glucose repression of enterotoxin B production was also reported to occur at pH 6. and 7. Subjecting the cells to anaerobic shock (sparging with 95% N2 and 5% CO2) to inhibit pyruvate decarboxylation caused an increase in the rate and quantity of toxin produced.g. the sum of which influences stability. salt..0 but to a lesser degree than at pH 7.g. and..0 but not at pH 5. residual nitrite. However. cytochrome oxidase) to the ferric state. increasing the level of nitrite had the general effect of reducing the recovery of both heat-damaged and undamaged cells. the percentage of lactic acid as an end product. 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. the question could be raised whether the inhibitor would be altered or destroyed. It has been difficult to design tests that prove the presence or absence of an unknown inhibitor that is likely unstable and is apparently formed in low quantity and functions within the midst of a complex system (e. and nitrite increased (Tompkin et al. 1973). aureus.192 Antimicrobials in Food the oxygen level decreased. and oxidative phosphorylation in Pseudomonas aeruginosa. The effect of nitrite on heat resistance was inconsistent. . and decreasing pH. presumably. Formation of some similar inhibitor(s) (PTF) during the thermal processing of shelfstable canned cured meat appears possible. Adding glucose caused decreased toxin production as a result of the rapid oxidative decarboxylation of pyruvate. Nitrite did not affect glucose transport by S. In another report on non-spore-forming bacteria. 1975) demonstrated the combined effect of pH. Contrasting results were subsequently reported that showed the production of enterotoxin A to decrease as pH decreased.7). The formation of the Perigo factor in commercially cured meat is highly debatable.7 (Morse and Baldwin. SUMMARY FOR 1970–1980 The Perigo Factor in Cured Meats 1. 1973). Rowe et al. Adding nitrite as an electron acceptor at the time of anaerobic shock prevented increased toxin production.5 or 5. 2. 6. Markus and Silverman (1970) reported enterotoxin A production in broth media (pH 6. when added to the recovery medium. and pH). The evidence strongly suggested that nitrite exerted its inhibitory effect by oxidizing ferrous ion of an electron carrier(s) (e. oxygen uptake. enterotoxin was not detected through 5 days of storage in the absence of oxygen. faecalis or Streptococcus lactis. Bean and Roberts (1974.8) to be unaffected by sodium nitrite (200 µg/ml) or sodium nitrate (1000 µg/ml). There was a general pattern of increased heat resistance as salt level (0. salt level. salt increased. They instead rely on glycolysis for generating adenosine triphosphate (ATP) when grown on glucose and transport glucose via phosphoenolpyruvate-phosphotransferase rather than by active transport. and nitrate on either growth or enterotoxin production. and nitrite on the heat resistance and recovery of S. nitrite. 4.5. no synergistic effect occurred between salt.

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

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

This information was reiterated by Hauschild and Simonsen (1986).2–0. carbohydrate.5–1. Since that time. Hauschild and Simonsen (1985) conducted an extensive review of the technology for producing shelf-stable canned cured meats (Table 6.0 0. 1990).5 4. Bacon was considered a low-risk product.5 0.5 Ham and shoulder Sausages Source: Hauschild and Simonsen (1985). This was used to quantitatively compare the data from the various challenge tests. and erythorbate are controlled.5 0.0 5. and commercial practices to arrive at guidelines to assure the microbiological safety of these products. . However.0 0.2 1. TABLE 6.0–1. He concluded that significant reductions in nitrite should not be made for all cured meats.5 3. where P is the probability of outgrowth and toxigenesis from one spore.7 4 2. Throughout the decade.1).0–5.3 3. there has been a very rapid growth in the volume and variety of cured poultry products in the United States.5–5.1 Shelf Stable Canned Cured Meats: Minimal Brine and Thermoprocess with 150 mg/g Sodium Nitrite Product Luncheon meat % Brine 3–4 4. They recommended that the bacterial spore levels in the raw product be rigidly controlled and that 150 µg/g of sodium nitrite be used to produce products with the following brine and thermoprocess values. the per capita consumption of red meats decreased while the consumption of poultry products increased. In 1983. existing government regulations.3–0. selective reductions in nitrite could safely be made provided the levels of brine. Reductions in the recommended levels should be based on extensive plant experience and/or experimental work and controls to assure minimal spore levels.5 Thermoprocess (F0 ) 1. the impact of further reductions in salt deserved close examination. 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. Reductions in the level of nitrite can be compensated for by moderate increases in the brine level and/or thermoprocess.3 0.0–4. 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. The level of risk differed among the various products.5 1. The data provided estimates of the relative degree of botulinal risk for each product. further accelerating the trend of the past 40 years.1–0. Their review was to assist in the development of a document for these products. the FDA acknowledged that prior sanction existed for the use of nitrite as an additive to poultry meat (Cassens. The level of risk could also differ among products of the same type as a result of differences in their brine concentrations.Nitrite 195 for sodium intake in the diet brought new pressure to reduce the salt content of cured meats. They summarized research. whereas turkey ham was a high-risk product. Another major trend developed during this time. He proposed expressing botulinal inhibition as log I/P.

competitive flora could have been destroyed. with levels of at least below pH 6. As the hams continued through the completion of curing. (1980a) studied the significance of pH on the efficacy of nitrite and sorbic acid mixtures in a chicken frank emulsion. Greater protection was obtained with 150 µg/g. and aw 0. To prevent the growth of C. The presence of sorbic acid slowed the rate of nitrite depletion.2%. fermented sausage. botulinum. (1982). Toxin was detected after 9 to 11 days. Some consumers find this amount of salt unacceptable. The tests involved vacuum-packaged smoked whitefish fillets prepared with about 2. botulinum. By increasing the processing temperature and/or prolonging the time of processing. thereby permitting the more heat resistant C. toxin levels remained high. Liver sausage was prepared with 83 µg/g of added sodium nitrite.2% brine.15 spores/kg in the product. Another possibility could be offered for this observation. The nitrite (40 µg/g) also significantly increased the inhibitory effect of the sorbic acid. the inhibitory effect of the nitrite was reduced. Large bone-in hams were inoculated with nonproteolytic botulinal spores before curing in a solution of salt. By increasing the thermal process. The number of botulinal spores was estimated to be about 0. and 15°C. (1987) demonstrated that adding sodium nitrite to obtain an initial level of 156 µg/g could reduce the amount of salt needed for botulinal protection. botulinum to dominate and develop toxin. The test system consisted of a frank emulsion prepared from mechanically deboned chicken meat that was extruded into test tubes.4%. a high level of salt (5% brine) has been required. Additional data on the ham experiments were presented by Lücke et al. psychrotrophic strains can grow and produce toxin during the early stages of curing before the salt and nitrite levels penetrate and reach inhibitory levels. cooled. and potassium sorbate (0% and . Methods and media were recommended to facilitate the recovery of both the proteolytic and the psychrotrophic nonproteolytic strains of C. The rate of pH decrease was slower as the brine level increased. Perhaps the milder thermal process permitted the survival of a competitive flora that prevented toxigenesis.196 Antimicrobials in Food Botulinal Challenge Tests Tests in meat and fish products. Toxin production by a mixture of type E and nonproteolytic type B botulinal strains in liver sausage. heated. overlaid with vaspar. Product with low levels of toxin often appeared acceptable. pH 6. At brine levels of 3.2.8% to 4. Similar results were obtained during the dry curing of hams at 8°C or 10°C. Cuppett et al. The total C. the inhibitory effect of the lactic fermentation and decreasing water activity values during fermentation and subsequent drying prevented botulinal toxicity. Wagner and Busta (1983) compared the antibotulinal effect of combinations of sodium nitrite (40 and 120 µg/g). The antibotulinal efficacy of sorbic acid was pH dependent. After curing. nitrite. They concluded that nonproteolytic. the pH of the formulation could be increased to pH 6.44%. The use of nitrite in smoked fish in the United States has been restricted. Tests in model systems. Hauschild et al. Hauschild and Hilsheimer (1983) examined 138 samples of liver sausage from retail outlets in Canada.98. nitrite in the range of 0 to 100 µg/g had little or no effect on toxigenesis.0 being required. If a low level of nitrite (40 µg/g) was added. The latter were suspected to be of particular importance as a cause of botulism from raw cured meat products in Europe (Lücke. The pH of the fillets decreased during 27°C incubation from about pH 6. (1980). nitrite was more effective and an appreciable delay in toxicity was obtained with 50 and 100 µg/g of nitrite. and hams was investigated by Liicke et al. toxin was detectable 10 cm from the injection site. In fermented sausage. and incubated at 27°C. 1982). toxin production was increasingly more rapid. botulinum was estimated to be about 0. (1982) assessed the relative risk of botulism from temperature-abused liver sausage. Toxin was not produced at 5°C. Sofos et al. The hams had a slight off-odor but no apparent putrefaction. and nitrate at 8°C.4%). As temperature was increased to 8°C. 10°C.2 without loss of the benefit of sorbic acid.3%. but the recovery of viable botulinal cells became increasingly difficult. Storage at 8°C until a nearly complete loss of nitrite occurred did not diminish the effect of the nitrite. and 6. At a higher brine level of 5. sodium acid pyrophosphate (0% and 0.24/kg.2. 4.4 to as low as 5.

Statistically significant reductions in toxin production occurred with increasing salt level.5% (wt/vol water) below the targeted values. (1979) modified their pork slurry to a thicker consistency. (1983) assessed the antibotulinal effect of three phosphates in the test system just described (Wagner and Busta. polyphosphate. 0. although the conclusions were stated on the basis of the target values of 2. Polyphosphate increased botulinal inhibition when added to combinations of sorbic acid or potassium sorbate and a low level of nitrite (40 µg/g). Nelson et al. (1982) examined the interaction of potassium sorbate in their cured pork slurry system.26%) in frankfurters.7 and 6. They found that increasing the heating from P80°C = 0. or decreasing the abuse temperature. They also found that the antibotulinal efficacy of nitrite could be increased by the addition of phosphate. 1980a).7).Nitrite 197 0.72). They used the same test system as in Sofos et al. sodium acid pyrophosphate was more effective than sodium hexametaphosphate or sodium tripolyphosphate.54. Tubes showing gas or other spoilage were preferentially selected for toxin assay.27 to 6. The addition of polyphosphate caused an increase of about 0.27 to 6. (1979b–e. although the trends in the data may not change.65 to P80°C = 6. The pH of the pork used for the higher pH slurries ranged from 6.03. The combination of 40 µg/g of sodium nitrite. One nitrite level (40 µg/g) was used throughout. and pH control of the emulsion was the most effective to achieve reduced nitrite levels and provide enhanced botulinal inhibition.6 to 6.56. The test system avoided the problem of recontamination that occurs when peeling and packaging franks. The pH of the four product variables was between 5. potassium sorbate significantly decreased toxin production.4 units in the lower pH slurries and about 0. and thermal process on botulinal outgrowth in pork slurries of two different pH ranges (pH 5.65. This increased inhibition was not observed with nitrite levels of 75 to 125 µg/g. potassium sorbate (0. The effect of sorbate was greater as salt levels .26% potassium sorbate. This may alter the quantitative assessment of the individual and combined effects of the additives. adding isoascorbate or nitrate.5%. The combination of nitrite (40 µg/g). (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. The pH of the pork used for the lower pH slurries ranged from 5. more like that of a reformed ham after heating. Jarvis et al.4 and 5. The increase in pH could explain the decreased inhibition in the lower pH slurries. No systematic difference in inhibition was observed with the meat from three breeds of pig. (1981a. The polyphosphates did not influence botulinal inhibition in the absence of nitrite. but the reason for the increased inhibition when adding polyphosphate to the higher pH meat slurries is less evident.3) and high-pH pork (pH 6. or thermal process.0. Roberts et al.5%. It is unclear whether the target or analytical values for salt were used for statistical analysis. Roberts et al. and 4. nitrite level.4% sodium acid pyrophosphate caused the greatest delay in toxin production.1 to 0. isoascorbate. except for two outliers of 5. The initial pH for the various products was 5.4 to 6. Gibson et al. Roberts et al. 1983). nitrate. The levels of salt found in the slurries were often as much as 0. The frankfurters were made from chicken instead of beef and pork. At a given spore inoculum.b) evaluated the interactions of salt. with certain phosphates being more effective than others. 3.78 to 6. A separate formula was developed for slurries prepared with low-pH pork (pH 5.5 to 6. The pH was important in all treatments tested.26%).5% salt (wt/vol water). nitrite in the range of 175 to 300 µg/g was even more inhibitory to botulinal outgrowth as measured by the percentage of slurries that became toxic. Considerable variation occurred between the meat from different animals within each breed.19. sodium acid pyrophosphate (0. They found the nitrite to be less effective in slurries prepared with meat from the shoulder than meat from the leg.36 and 6.2 units in the higher pH slurries.4%). Of the polyphosphates tested. (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. and 0. There appeared to be more botulinal inhibition with the addition of sodium acid pyrophosphate.3 to 6. nitrite.

The results are intriguing and leave relatively unexplored the underlying mechanisms influencing the results. sealed with vaspar. 50. as pH levels decreased to below 6. The authors suggested that Bactin formation may be responsible for the inhibition observed in Perigo-type media.198 Antimicrobials in Food increased (2. The addition of nitrate led to higher residual nitrite levels for a longer period. and 50 kGy). (1989) prepared ground pork slurries with 0. and iron in culture media. 40. 156. At lower dose levels of 0 to 9 kGy. there was a marked decrease in the residual nitrite. 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. an iron-binding protein in casein. 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. The amount of iron required for inhibitor formation was reportedly similar to the level found in muscle tissue. 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). They concluded that the level of residual nitrite did not influence botulinal outgrowth in their experiments. They concluded that lactoferrin and transferrin are precursors of the Perigo factor and that it is likely that nitrite-modified transferrin accounts for some of the antimicrobial effects of nitrite in cured meats. The probability of botulinal outgrowth decreased as the level of added nitrite increased above 50 µg/g. then inoculated with botulinal spores. As expected. the addition of nitrate. The significance of microbial growth on nitrite depletion in the slurries during storage was not addressed.K. which otherwise prevent bacteriostatic activity. Residual nitrite levels after irradiation were inversely related to the irradiation dose. This led Gibson et al. U. No suggestion was offered for the increased botulinal inhibition when nitrate was added. the rate of nitrite depletion was influenced by storage temperature. Custer and Hansen (1980) reported that lactoferrin. a similar protein in blood serum. Pretreatment of the lactoferrin and transferrin with a protease was necessary for inhibitor formation. 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. botulinum. 20. After higher doses of irradiation (10. 30. A rather high level of isoascorbate (1000 µg/g) caused nitrite to disappear rapidly and delayed the outgrowth of C. 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. Bactin was not produced when heating cysteine. They suggested that the bacteriostatic effect is the result of facilitated conversion of cysteine to Bactin and a concomitant removal of thio compounds. legislation was proposed to control the use of nitrite by monitoring the residual nitrite in cured meat products. The bacteriostatic action was reversed by the addition of cysteine. responded similarly. unless ferric chloride was present and the pH was 6. However. and 200 µg/g of sodium nitrite. which resulted in rapid botulinal outgrowth. 100. They found that the rate of depletion in their pork slurry system was influenced by many intrinsic and extrinsic factors. 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. and as the storage temperature decreased. Transferrin. thioglycollate. forms an inhibitor when autoclaved in the presence of nitrite. In another study of Perigo factor formation.5% to 3.5%). and stored at 30°C for 28 days. and the addition of isoascorbate. For example. (1984) to examine the effect of various factors on the rate of nitrite depletion and botulinal outgrowth. the . The slurries were irradiated.0. Higher or lower levels prevented the inhibitory effect.

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

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

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

5 and 6. Research by Payne et al. the efficacy of sodium nitroprusside was explored (Joannou et al. Juntilla et al. Additional information and views on the mechanism of nitrite are available in Roberts et al. The cell-wall surface was the primary point of attack for the nitrosyl complex associated with nitroprusside. botulinum was again addressed by Carpenter et al. (1987). The final pH of the products was about pH 4. The inhibitory effect of nitrite against C. sporogenes cells and isolated proteins. Buchanan et al. They were unable to demonstrate a direct action of the compounds on the iron–sulfur proteins.5. It appears that the hypothesis proposed earlier is fairly close (Tompkin et al. At pH 6. perfringens foodborne illness involving cured meats. 1978). One aspect that the more recent data suggests is that the interaction of nitric oxide with iron–sulfur complexes is irreversible. and sodium nitrite on the inhibition of C. However. the toxicity of nitrosyl compounds to bacteria.5%) and sodium nitrite (50.5. The effect of nitrite on the growth of Listeria monocytogenes was examined in trypticase soy broth by Shahamat et al.0.0) found in cured meats.g. In addition. Sodium nitrite was added to the broth before autoclaving. and 5. sodium chloride.b) examined the effect of nitrite and iron–sulfur–nitrosyl complexes on iron–sulfur clusters in C. perfringens and fecal streptococci in broth media. In a broth medium at pH 6. 120. (1989) tested the effect of nitrite on L. A high salt concentration and low incubation temperatures were the more important factors influencing the inhibition of these bacteria.0% and 3.0. 28°C. The cell walls showed a 90% decrease in thiol content when compared with untreated cells. (1980). nitrite was not an effective inhibitor at pH 7. although the nitrite had been autoclaved with the broth. Gibson and Roberts (1986b) demonstrated the interactions of pH. On the basis of the research conducted by these researchers and others. Most cured meats have traditionally had brine levels of 3% or greater. incubation temperature. blisters at the surface of the cells were observed when viewed under the electron microscope.. monocytogenes in a broth medium at pH 7. Miscellaneous Studies in Laboratory Media Botha and Holzapfel (1987) tested the effect of nitrite on Sporolactobacillus isolates from a variety of sources. Sodium nitroprusside was found to be a very effective inhibitor of C. The relative sensitivity of C.6. and/or an increase in the salt concentration. and 200 µg/g). monocytogenes is quite resistant to the inhibitory effect of nitrite.5. 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. leading them to doubt that the inhibitory effect of nitrite is on preformed iron–sulfur proteins of the bacterial cell. (1990a. it is fairly certain that the mechanism of nitrite involves the inactivation of iron–sulfur proteins.202 Antimicrobials in Food The debate over whether clostridial ferredoxin or pyruvate-ferredoxin oxidoreductase is the site of nitrite inhibition for C. such as ferredoxin and pyruvate oxidoreductase.0.. perfringens was enhanced with a decrease in pH. After continued research on the mechanism of compounds associated with the Perigo factor. Their results led to the conclusion that both are inactivated. The incubation temperature (5°C. decreasing pH and aw) influenced the rate of death. (1991) and Roberts and Dainty (1991). which is more typical of cured meat and . 1998). monocytogenes to be similar in fermented sausage manufactured with different levels of salt (3. 19°C. (1989) found the rate of death of L. In the research of Duncan and Foster (1968b) such blister formation was not reported following exposure to sodium nitrite. a decrease in temperature of incubation. Nitroprusside was used as a model to investigate the inhibitory mechanism of sodium nitroprusside and. the data more strongly demonstrate that L. The fecal streptococci proved to be quite resistant to nitrite. and 37°C) had the strongest effect on growth rate and lag-phase duration. comparable to the inhibitory properties of the anion of Roussin’s black salt. Consistent with previous reports over the past 60 years. At the pH values (6.. 5. It can be concluded that factors (e. more generally. some reduction in growth was observed with the addition of 200 µg/g of sodium nitrite. sporogenes. perfringens to 3% to 4% salt could explain why there have been few outbreaks in C.

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

being insensitive to the presence of nitrite. 100. 5°C. Omitting nitrite resulted in a perfume-like odor that was associated with the growth of enterococci. 100. 156 µg/g of sodium nitrite.204 Antimicrobials in Food were considered incapable of synthesizing heme compounds. Grampositive cocci.e. Protoporphyrin. The level of surviving enterococci decreased as the processing temperature increased (63°C. Nielsen (1983a) studied the effect of sodium nitrite (0. B. 10°C. Also. 68°C. and lactic acid bacteria were inhibited to a limited extent. faecalis (Chyr et al. 1981).. and 74°C). cereus can grow in the product even with the normal curing ingredients of salt. the Gram-negative flora proliferated in product with nitrite. Using good quality raw materials. yeasts.7 to 5. plantarum tested were able to reduce nitrite when hematin was provided to cell suspensions. Polish regulations require the heating of perishable canned . Again. gluconolactone (glucono delta lactone. The spoilage flora of the liver sausage prepared without nitrite also included Pediococcus pentosaceus. sake tested were able to produce catalase when provided with hematin. Increasing the level of sodium nitrite (0. chilled.. and 200 µg/g) on the spoilage flora of cooked pork loin.8. and 10°C). Nielsen (1983b) subsequently studied the effect of sodium nitrite (0. and Moraxella and Moraxella-like organisms were increasingly inhibited by increasing the nitrite concentration and/or decreasing the temperature of storage (2°C. and 20°C). If the product is temperature abused. Silla and Simonsen (1985) studied the rate and type of spoilage of four cured meat products packaged under vacuum and in modified atmospheres. dominated the spoilage flora in product with nitrite. hemoglobin. all the strains of L. This led to the lactic acid bacteria becoming the dominant spoilage flora in the vacuum-packed product. as the storage temperature increased. about 0. The spoilage of liver sausage was shown to be the result of S. and citric acid in the product. GDL). phosphate. The Pediococcus isolate was not as heat resistant as the enterococcus. plantarum and L. 63°C to 68°C) led to the growth of enterococci during storage at 7°C. (1980) found the most important factors influencing the rate of microbial spoilage of liver sausage to be the degree of cooking and the level of nitrite. and 150 µg/g) retarded microbial growth. the lactic acid bacteria. did not activate nitrite reductase or catalase activity. and 200 µg/g) on the spoilage flora of a sliced vacuum-packed bologna-type sausage. Kafel and Jozwik (1987) investigated the spoilage rates of commercially produced products from 6 meat processing plants in Poland. The formulated raw meat was stuffed into a casing.2 to 0. Chyr et al. In addition. Asplund et al. Adding GDL was considered the most important. particularly in the summer. The growth of B. 100. An industry survey found that liver sausage was commonly cooked to an internal temperature of 65°C. Product with GDL had a pH value of 5. A correlation was found between the nitrite concentration at the time of slicing and packaging and the time for spoilage to occur.. cereus was delayed or prevented by including a combination of erythorbate. Gramnegative bacteria often constituted the major flora of the product without nitrite. Lactobacilli were the dominant flora in all four products at the time of spoilage. sliced. myoglobin. High levels of B. whereas other iron-containing porphyrins (i. 5°C. thermosphacta and Enterobacteriaceae at all temperatures of storage (2°C. Enterobacteriaceae. thermosphacta. and sodium nitrite (120 µg/g). Wolf and Hammes (1988) found all the strains of L. The enterococcus survived 65°C for 5 minutes or 60°C for 60 minutes with only 103 cells/ml broth. (1988) reported that the growth of B. an iron-free compound. A marginal thermal process (i. thermosphacta may also have significantly influenced spoilage of two of the products. and then vacuum packed. cereus in liver sausage has been a problem in Finland.e. and processing to 68°C resulted in a shelf life of more than 16 weeks at 5°C. 50.3 units lower than with the other variables tested. Additional additives were tested to reduce this risk. 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. cooked. hematin) did activate the enzymes. 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. B. Nitrite inhibited the growth of B.

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

1974). Again. 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. Buchanan and Phillips (1990) studied the growth of L. Sausage formulated without sodium nitrite became toxic within 1 week compared with 3 weeks when nitrite was present. monocytogenes in tryptose phosphate buffer under a wide variety of conditions (salt.. In subsequent tests the effect of various decolorized blood fractions was evaluated. red blood cells. Since 1990 there has been increased interest in control of L.0 and 5°C no growth was observed with any of the levels of sodium nitrite evaluated (50. but no evidence was generated to link the Perigo factor to the antimicrobial effect that has been demonstrated in perishable. found the efficacy of sodium nitrite was temperature and pH dependent. and the addition of plasma delayed the time for toxigenesis compared to the control sausage (Miller et al. delayed the time for toxin to be detected. however. McClure et al. Despite this evidence. cured products that receive no heat treatment or are cooked to final internal temperatures in the range of 60°C to 75°C.206 Antimicrobials in Food 5. The mechanism of nitrite inhibition differs in different bacterial species. Alternatives to nitrite continued to be investigated. that has been expanded to include other pathogens and has gained acceptance throughout the world. 1993). 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. very slow growth did occur. The database was incorporated into the USDA Pathogen Modeling Program.3. Some research continued on the Perigo factor. At pH values of 6. but none have been adopted commercially. in my opinion. (1991). 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. pH. 7. Autoclaving the sodium nitrite in the medium did not increase the degree of inhibition against L. is not more inoculated pack experiments but a rationale for interpreting them” (Ingram. “What we need at the present time. aerobic/anaerobic conditions) and found sodium nitrite to interact with combinations of the factors tested. 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. 6. monocytogenes to the degree that . Adding hemoglobin. The potential impact of various blood fractions as ingredients in sausage was investigated (Miller and Menichello. At 20°C and below no visible growth occurred even with 50 µg/g sodium nitrite and a pH of 5. SINCE 1990 C. 400 µg/g). the rate of toxin formation was inversely related to the level of iron. In the absence of nitrite. monocytogenes in ready-to-eat foods. 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. At pH 6. Adding dried plasma. 200. temperature. 8. This probably reflects the effect of the higher salt levels and nitrite found in cured products compared with noncured products. The debate over the risk of C. 100. Other available options have not received commercial acceptance.0 sodium nitrite had little effect in delaying the time to detect visible growth except at the highest level tested (200 ppm) and a temperature of 15°C or below. 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. 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. 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. 1991). At the time he stated.

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. they estimated the various formulations and thermal processes provided the equivalent of 4 to 7 log10 destruction of C. 1995.7 with 50 µg/g).5% sodium chloride and 50 or 200 µg/g sodium nitrite inhibited growth during 125 days storage at 37°C. sporogenes. (1994) inoculated a variety of vacuum-packaged cooked sliced meats with L. and additives to achieve shelf stability of canned luncheon meat (Farkas and Andrássy. F0 = 0. sausages made with nitrite and having pH 5. Applying the procedures described by Hauschild (1982). The model test system was then used to evaluate various combinations of heat. Buchanan et al. 10°C) on the rate of growth of L. Using C. respectively. 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. Farber et al. botulinum. inoculum level. mesenteroides. 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). Australia. Sameshima et al. Perhaps the high iron content of liver pâté negated the effect of sodium nitrite and sodium erythorbate in this product. An extensive series of experiments led to the conclusion that nonthermal inactivation of L.0 and a sour. and temperature of storage (4°C. a combination of heat and sodium nitrite was necessary to prevent survival and growth (F0 = 0. (1997) examined the effect of nitrite on three lactics in vacuum-packed cooked sausage made with and without nitrite. heat. viridescens.7. The procedures described show promise for estimating and comparing the combined effects of multiple treatments on pathogen survival and growth. 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. monocytogenes in the sliced vacuumpackaged product when stored at 4°C or 8°C. Liver paté has been implicated in outbreaks of listeriosis in the United Kingdom.8 and containing 3. and Enterococcus faecalis. If the broth had been adjusted to pH 6. Fermented Meats and Dry Cured Hams Although removed from most cured meat products in the United States during the 1970s. (1995) examined the effect of sodium nitrite (0 and 200 ppm). and 15 days for L. temperature. 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. 3%).Nitrite 207 had been observed with clostridia (see Perigo Factor). some . monocytogenes. irradiation. smoked flavor can be found. although sodium nitrite concentration was not varied. salt (1%. and the United States. 1997). The effect of salt and nitrite on the growth of 24 lactics isolated from vacuum-packed cooked sausage was evaluated by Korkeala et al. and other factors for inactivating spores and preventing the outgrowth of survivors. 4. sodium erythorbate (0. Farkas and Andrássy (1992a) described a test system to evaluate the combined effects of formulation. Low levels of salt (1% to 2%) enhanced growth. Leuconostoc isolates seemed more sensitive to the effects of sodium nitrite and salt in comparison to the homofermentative lactobacilli. pH. monocytogenes by sodium nitrite is pH dependent and related to the concentration of undissociated nitrous acid (Buchanan and Golden. the effectiveness of sodium nitrite was significantly increased by the addition of sodium ascorbate. Three µM of residual undissociated nitrite doubled the time taken for a 3 log10 increase in numbers. but levels above 3% were inhibitory. 550 µg/g). McClure et al. (1996) subsequently investigated the effects of salt. L. Only temperature proved to be a significant factor.. 1992b). sporogenes (9 × 105/ml) in reinforced clostridial medium. As in the case of earlier research with C. and sodium nitrite in laboratory medium and a predictive model for the growth of L.2 with 200 µg/g. In regions of Northern Europe. botulinum. (1992). they found that broth adjusted to pH 5. monocytogenes in liver pâté. as had been reported earlier for C. Since about 1990. Duffy et al.

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

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

For more than 1500 years the rice has been dried. Assembly of Life Sciences (1981).. (1997) found the addition of sodium nitrite delayed or prevented toxin production by C. and 6 kg sucrose (Knøchel and Huss. The reduction in spoilage was not considered to be the result of microbial inhibition by nitrate or its reaction products (nitrite. 8. Two microbial hazards of concern in ready-to-eat fish products are C. and 2 barrels. or 50 g of potassium nitrate was added to each of 200 barrels containing 90 to 100 kg fish. with 0. In one study with 166 µg/g sodium nitrite. The 600 barrels were topped off with brine solution. Cuppett et al. ground.. 1995). 2001a. Klijn et al.000 cfu/kg) and after 4 weeks with a low inoculum (800 cfu/kg). cold-smoked trout having 3. They conducted an extensive study in which 0. In a second .4% salt in the water phase. Gouda and El-Zayat. flavor. in this form (called Angkak).3%. The incidence of spoilage was 13. 1988. particularly in China. 1999. sweet/sour. Although not permitted in the United States. 1981). In Denmark. When sodium nitrite (156 µg/g) and sodium ascorbate (550 µg/g) were added. 1991). 25 g. As the amount of sodium nitrate increased. toxin was detected at 4 weeks at 4°C with a high inoculum level (20.4% salt in the water phase. late fermentation and gassiness resulting from Clostridium tyrobutyricum or C. 1984. 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. for example. 1980). respectively. and keeping quality (Leistner et al.. Fink-Gremmels et al. Galesloot and Hassing. butyricum). and evaluated over 18 months storage at 4°C to 6°C. and. 16 kg salt. Among the options considered was the use of a red pigment producing mold used to ferment rice. The reader may recall that during the 1950s nitrate was found to be an electron acceptor in certain cured meat products.. nitrous acid) because a high percentage of the isolates from all three variables were able to reduce nitrate. 1991. Ariga et al. The additives provided no additional protection when the salt level in the fish was reduced to about 2. This chapter will not review the extensive literature on the use of nitrate to prevent cheese defects (e. Cheese Nitrate has been added during the manufacture of certain European cheeses for more than 150 years (Pivnick. In the Far East. pork. fewer barrels were judged to be spoiled with sour. added to a variety of foods (e. but no differences were noted for flavor or texture. 1983. Monascus purpureus (Leistner et al. fish. rice wine) to improve color. 1991.. botulinum type E in vacuum-packed. 1984a. Nitrate was found to delay the depletion of trimethylamineoxide (TMAO). and 50 g of sodium nitrate. 2000).g. Seafood The comprehensive review by Jarvis (1950) provides very few examples for the use of nitrate (i. Park et al. Knøchel and Huss (1984b) state that nitrate traditionally has been added to the salt used in producing sugar salted fish in Scandinavia. and virtually no mention is made of its value as a preservative. or putrid off-odors and offflavors. Gilles and Fryer. Adding nitrate caused the herring to be redder in color. fruity.. the allowable level has been 500 mg potassium nitrate per kg fish to provide about 300 µg/g nitrate.b). nitric oxide. Canadian regulations permit this practice (Assembly of Life Sciences. poultry. 1983.e.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. (1987) found that toxin production by C.g. monocytogenes (Gram. toxin production was inhibited for 56 days... rice is fermented with molds of the genus Monascus to form a pleasant red color. saltpeter) in the curing of fishery products prior to 1950 throughout the world. botulinum type E (103 spores/g) at 27°C was inhibited for 35 days in smoked whitefish containing 4.b).. botulinum and L.. 25 g. and others (Mayenobe et al. Tseng et al. tofu. Hyytiä et al. An opening to the literature on this subject can be obtained through references cited in Langsrud and Reinbold (1974). sealed.. 1984.

Adding potassium nitrate at 347 or 686 µg/g was equal to or more effective than sodium nitrite. Toxin production can be controlled with a combination of a moderate level of salt (3. 1999). questions still remain about “aerobic packaging” and its value for controlling botulinal outgrowth. particularly in combination with controlled levels of salt (Pelroy et al. 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. Furthermore. In 1992 more than 32. Surveys conducted before the year 2000 indicate L. a panel of experts reached the following consensus for its report to the FDA (Gram. it is highly unlikely that sodium nitrite will be adopted as an additive in smoked fish in Europe. The addition of sodium nitrite is limited to smoked salmon. with a production of 12. botulinum would be further inhibited. will prevent toxin production in cold smoked fish for several weeks beyond its sensory shelf life when packaged under reduced oxygen conditions. and with refrigeration this combination is effective in inhibiting growth of C.5% salt in the water phase or 3. Cuppett et al. Hyytiä et al.. shad. 1987). the salt in the water phase ranged from 2% to 4. NOCs were not detected in two studies in which nitrite had been added to smoked fish (Pelroy et al. smoked fish to contain at least 3. 2001b). lightly preserved fish products (cold smoked fish... gravad fish.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. Two options are available in the event commercially manufactured product is implicated in botulism. Analysis demonstrated that nitrite was produced during storage. A level of 3% salt in the water phase will yield perfectly acceptable. and chub.000 tons shipped internationally from 28 exporting countries to 52 importing countries (Duffes. It is possible. it would appear the risk of botulism is lower than the research has suggested. 1982. Considering the lack of regulations and apparent variability in salt concentration in large quantities of cold smoked salmon distributed domestically and internationally.. but. 1999). The panel was reluctant to recommend expanded use of sodium nitrite despite evidence showing that psychrotrophic C. botulinum (Huss. 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. a minimum concentration for salt in the water phase can be established. 1997).380 tons of cold smoked salmon in 1997. Controlling the salt in the water phase and temperature should be adequate. 2001a).4°C) for at least 4 weeks. Cuppett et al. federal regulations were adopted requiring vacuumpacked.. whereas with salt alone.. If packaged aerobically. toxin was detected in 3 to 4 weeks.0% salt if combined with 200 µg/g sodium nitrite (Gram. however. although a minimum of 3% salt in the water phase is strongly recommended (Huss et al. sablefish. brined shrimp). 2001a): • • Psychrotrophic C. The panel expressed concern for the potential carcinogenic effects of nitrosamines. allowing for short periods of elevated temperatures up to 10°C. With the availability of lower cost farm-raised salmon the international market for cold smoked salmon has continued to increase. are low. a lower salt content is permitted (i. if present. Until there is evidence of a need. . monocytogenes (Gram. In France. 1995). 2.5% salt in the water phase).. that combination of salt and temperature. This may reflect survival or contamination.e. 1982. although the numbers. however. 1987. sodium nitrite could be used as an additional control measure. Following a review of the literature. if necessary. monocytogenes is common in smoked fish.6% (Duffes. depending on the conditions of processing. 1997). but the panel did not consider the more recent risk assessments demonstrating the safety of dietary nitrite (see section on toxicology). The addition of nitrite or nitrate increased the time the fish was considered acceptable by 3 or 4 weeks at 4°C. botulinum can be expected to occur in smoked fish.5% in the water phase) and cold storage (<4. In many countries there is no legal requirement for the minimum amount of salt. First.

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

All the enteropathogens grew in one or more of the products within 24 hours at 30°C. A low-brine ham was prepared with 1. (1967).7. The pH of the chopped ham with 70% and 59% moisture decreased to 5. and Hafnia strains readily multiplied at all nitrite levels.47 and 5. Neither of two strains of S. and 130 µg/g) and two Salmonella inoculum levels (100 and 100. whereas the other frequently decreased to below pH 5. Page and Solberg (1980) found S.58. Leistner et al. S. and 100 µg/g) and the level of inoculum (100 or 100. It was concluded that cooked ham is a more likely source of salmonellosis if prepared with a low salt content. Salmonella Enteritidis was able to grow at 17°C but not at 10°C. Fermented sausage (teewurst) was prepared with four sodium nitrite levels (0. The rate of growth or death was influenced by the sodium nitrite content (0. E. nitrite values were not given. The other four species were able to grow under certain conditions. respectively. and 100 µg/g of sodium nitrite. viable counts of the 5 strains remained near the inoculum level.78. A similar response occurred with enteropathogenic E. the fastest generation times occurred in . that of the ham was near 7.6 to 6. whether the nitrite was added before or after autoclaving the medium of Perigo et al. Typhimurium to utilize nitrate. In the high-brine ham held at 22°C. and B. Castellani and Niven (1955) reported S. (1973) inoculated vacuum-packaged luncheon meat with a mixture of Salmonella strains and held the product at 8°C for 22 days. Enteritidis were then used to study the effect of nitrite and salt on growth in nutrient broth at 22°C. Growth of C. In the low-brine ham at 22°C.0. 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. Nitrite and ammonia were assimilated at comparable rates. Death occurred after 3 days in the sausage with the lower inoculum and 130 µg/g of nitrite. S. Growth occurred in the sausage containing 0 and 65 µg/g of nitrite. coli. At a low pH of 5. 50.0 and 5% salt. The authors did not state whether the nitrite was added before or after autoclaving the medium. Stiles and Ng (1979) studied ham and chopped ham obtained fresh from two processors.000).7 to 4. The product from one supplier remained above pH 6. 4 of 5 strains of Salmonella grew readily. This probably reflects the quantity of fermentable carbohydrate added to the product and/or the nature of the competitive flora.8. The growth of Klebsiella and Enterobacter species on vacuum-packaged luncheon meats at 3°C was reported earlier by Hechelmann et al. 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. Klebsiella. Although salt and nitrite values were not reported. coli. A high-brine ham had a brine level of about 6%. nitrite. pH values were followed through storage. other than to say that fresh. (1979) tested the same bacteria on sliced bologna from two processors. Typhimurium was inhibited at pH 6. The resistance of Salmonella to nitrite in culture media was demonstrated by Roberts and Garcia (1973). and the pH for the wieners was 4. Two strains of S.8% to 2. 5% salt. Stiles et al. growth was reduced by only about 30%. Again.2% salt and 60 µg/g of potassium nitrite and a finished brine level of about 2.0. Under anaerobic conditions.000/g). no data were provided for brine or nitrite levels. The mean pH for the ham during storage decreased to no lower than 5. No other chemical data were provided. 65. 75. the primary factor influencing growth was product pH.8%. but a mixture of Enterobacter. Both genera were prevalent in commercially produced luncheon meats and fermented sausages. 98.0. cereus) and then vacuum packaged. (1974). The products were inoculated with five “enteropathogens” (C. given the proper circumstances. It is not possible to assess the influence of nitrite from the data. nitrite was without effect. At a high pH of 7. aureus. but pH changes were noted. perfringens.Nitrite 213 and bologna was about 6. that of the chopped hams was 70% and 59%.0. The primary factors influencing growth were the temperature of storage and growth of competitive flora. including Salmonella.0 by 160 µg/g of sodium nitrite.5. Aside from temperature of storage. The moisture content of the hams was 70%. Typhimurium. commercially produced sliced ham and chopped ham did support the growth of the pathogens studied. perfringens did not occur under any condition.

At concentrations of 500 µg/g and higher. 1987). and 100 µg/g sodium nitrite. but growth was delayed with 200 µg/g (Turanta and Ünlütürk. 27°C. coli. The death rate was slower with potassium nitrate (150 or 300 µg/g) or a combination of nitrate and a low level of nitrite (75 µg/g of potassium nitrate and 50 µg/g of sodium nitrite). The phosphates generally delayed the outgrowth of E.214 Antimicrobials in Food media containing both nitrate and ammonia. Typhimurium when grown anaerobically with nitrite as the sole nitrogen source. Typhimurium to be relatively resistant to sodium nitrite across the ranges of salt and pH values found in nonfermented and acidulated foods. He recommended that a starter culture and/or gluconolactone be added to provide optimum safety. sporogenes. 100. When examined after 10 weeks of incubation. Similar results were observed with S. (1984) prepared fermented sausages with sodium nitrite (0. (1985) identified a nitrite reductase in the cytoplasmic fraction of S. Gibson and Roberts (1986a) examined the interaction of salt (1% to 10% wt/vol). 1977). the addition of sugar. and 156 µg/g) in frankfurters on a mixture of two strains of Salmonella. Factors influencing Salmonella growth in mettwurst that was cured and stored at 20°C include the amount of nitrite and salt. The presence of nitrite did not influence the survival of salmonellae. 50.8). Nitrite was only inhibitory with the highest level of nitrite and when the franks were incubated at 15°C. Isolates of Enterobacter and Serratia species were tolerant to nitrite.8. Collins-Thompson et al. Collins-Thompson et al. In a traditional Turkish dry sausage that is fermented and dried at temperatures . 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 nitrite resistance of E. 50. vacuum packaged. Typhimurium had shorter generation times and increased cell yields compared with nitrite-free cultures. S. Thomas et al. Schillinger and Lücke (1989) measured Salmonella growth and/or survival in mettwurst. and 6. and incubated at 15°C or 27°C for 21 days. the inherent pH of the meat (normal pH 5. coli. The presence of nitrite had no effect on aerobically grown cultures. (1991). numbers increased during 72 hours in the presence of 0. and sodium nitrite (0 to 400 µg/g) in BHI and incubation at 10°C to 35°C on the growth of Salmonella and E. and 37°C (McClure and Roberts. The franks were inoculated on the surface. coli was also apparent in tests involving two-dimensional gradient plates incubated at 20°C.0. S. and 200 µg/g) and three strains of Salmonella. When grown anaerobically in a glucose-limited minimal medium with peptone and nitrite. Page et al. 50.0 to 6. 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. (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. Hughes and McDermott (1989) used a procedure similar to that of Gibson and Roberts (1986a.2). Growth did not occur during fermentation or drying.6. aureus and C. 150. S.8 versus 6. This research was primarily concerned with preventing the growth of Salmonella. 1991). The reductase required NADH and was most active at pH 8. Typhimurium used nitrite as the sole nitrogen source at concentrations as high as 400 µg/g. a cured raw spreadable sausage.2. the effect of added phosphate was relatively minor. Rice and Pierson (1982) tested the effect of sodium nitrite (0. None of the variables tested would likely assure the destruction of Salmonella in this product category.8 and held at 24°C independent of sodium nitrite concentration. 6. At pH 5. coli. found S. using a gradient gel plate system.6 to 5. pH (5.b) to determine the additional effect of eight different phosphates of E. Typhimurium numbers decreased in BHI broth adjusted to pH 4. and the presence of lactic acid-producing bacteria. except when the cultures were incubated at the lowest temperature tested (10°C). being able to use nitrite in broth culture under anaerobic conditions at 5°C. complete inhibition occurred.

The highest survival involved a starter culture that produced the least amount of acid and yielded a relatively high final pH of 5. and temperature. Inhibition of Shigella flexneri in broth incubated aerobically was significantly influenced by the combined effect of nitrite concentration.. The inoculum decreased from 1. They found some increased death of E. coli O157:H7 when lower temperature and/or shorter time combinations (e. the death rate of S. and incubation temperature (Chou and Tsai. Effect of Nitrite on Yeasts and Molds A wide variety of yeasts and molds have been isolated from commercially cured meats. 50. where .8.. (1998) found E. Another study involving anaerobic conditions yielded similar trends (Zaika et al. and then dried for 7 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. coli O157:H7 in a broth system containing sodium nitrite was strongly pH dependent (i. coli.7 × 105/g to nondetectable levels by 35 days in the sausages formulated with 80 µg/g nitrite. The data for Salmonella can be summarized as follows.e. pH. Pin et al. In Lebanon bologna. These factors include water activity. 1996.g.. particularly because salmonellae are destroyed during the manufacturing process for most cured meats. pH 5. This sausage is not fermented or cooked and relies on relatively rapid dehydration to achieve microbial stability. (1993). 1996).0. Preventing salmonellosis from cured meats is best accomplished by means other than relying on the level of nitrite in the product. sodium nitrite at 70 to 150 µg/g did not affect the growth or survival of E. Raccach and Henningsen (1997) reported sodium nitrite and salt to act synergistically in the reduction of Y. The inhibitory effect of nitrite was much greater at pH 5. nitrite.. 1996). or ammonia under anaerobic conditions. S. coli O157:H7 (Chikthimmah et al. enterocolitica 0:3 to grow and survive during the manufacture of raw dry fermented sausage was examined by Asplund et al. 80.5. coli O157:H7 (Yu and Chou. 1997).5.5% salt and 100 µg/g sodium nitrite. but there is no published information that nitrite or nitrate inhibits their growth. pH. the addition of sodium nitrite (0. 1991). The effect of sodium nitrite (0.. coli O157:H7 numbers decreased less than 1 log10 in pepperoni formulated with 2. coli O157:H7 at 37ºC were inhibited by 200 µg/g sodium nitrite at pH 5. the lowest pH tested and with increasing salt levels. Inhibition of Yersinia enterocolitica in broth incubated aerobically was significantly influenced by the combined effect of nitrite concentration. 55ºC for 4 hours) were used. storage temperature. 1994). 100.Nitrite 215 not exceeding 24°C. 2001). in a variety of commercially cured products. but even 1 g/L sodium nitrite was ineffective at higher pH values (Tsai and Chou. In ground pork held at 5ºC or 25ºC. Adding sodium nitrite did not increase death of E. Inhibition of Aeromonas in broth has been found to be highly dependent on nitrite concentration. The effect of nitrite in Chinese-style sausage during drying at 50ºC to 60ºC for different time periods (2. a fermented noncooked beef sausage.5 to 6 hours) was next studied (Yu and Chou. Inhibition of E. That cured meats have been implicated in outbreaks of salmonellosis supports the available research that Salmonella can survive. if not multiply. the inhibitory effect of smoke. the availability of oxygen. salt level. 78. 1996). The data indicate that the nitrite content of cured meats can influence the growth and survival of Salmonella and E. 1994). and temperature (Bhaduri et al.. enterocolitica when tested in a meat system. 1993).. Typhimurium was not affected by the addition of 200 µg/g sodium nitrite (Turanta and Ünlütürk. fermented to pH 4. pH. In tryptic soy broth 4 strains of E. and 120 µg/g) on Y. 1994 and 1995). and. salt level. Inhibition was much greater at 28ºC than at 37ºC (Zaika et al. Typhimurium has a nitrate reductase to reduce nitrate to nitrite and can assimilate inorganic nitrogen from nitrate.5) and enhanced by lowering the incubation temperature (Buchanan and Bagi. Riordan et al. coli O157:H7 with the addition of curing agents and drying at time–temperature combinations that appear to cause stress of the inoculum. and 156 µg/g) had no effect on the death rate of E. This is not true for the clostridia.

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

With the exception of bacon and smoked chubs. This further reduces the nitrite level in the product formulation and. The most recent summary of the regulations in different countries appears in Cassens (1990).177 (Code of Federal Regulations. The use of nitrate or nitrite as additives in cheese and seafood products was clarified. 1986). 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. the method of assay applicable to each country should be used. as proposed by the Expert Panel on Nitrites and Nitrosamines (Food Safety and Quality Service.S. meat and poultry regulations is that nitrite and nitrate are added on the basis of the uncured meat in the product formulation. 8. ASSAY METHOD Because the levels of nitrite and nitrate permitted in foods are established by regulation. 1981. regulations do not specify minimum nitrite levels for cured products. A unique feature of the U. Nitrite is of relatively little value for controlling Gram-negative enteric pathogens in commercially prepared foods.3. 172. Other countries limit the amount of nitrite and nitrate on the basis of the total weight of the product formulation.17 and 424. Also listed is the actual amount of nitrite that could be added to the total formulation of the products if the permitted level of nitrite was reduced from 156 to 100 µg/g.175. An example of rework is the round ends of sausages that are trimmed off before slicing the product. the weight of this meat is subtracted from the fresh meat portion of the formulation and the amount of nitrite added is reduced accordingly. Germany reduced the permitted level of nitrite in nitrite-containing curing salt by 20% (Leistner. Fisheries.170. 2002a). U. The current USDA regulations controlling the use of nitrite and nitrate in meat and poultry products are specified in 9 CFR 317. 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. 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 nitrite and nitrate in the processing of fish products is specified in 21 CFR 172.160. The status of nitrite and nitrate in cured meats and cheese in the United Kingdom was reviewed in 1978 (Ministry of Agriculture. 2002b). It is also common to generate a certain amount of rework during the manufacture of sausage products. 1995).21-23 of the federal regulations (Code of Federal Regulations.S. leads to lower residual nitrite levels after processing. 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. 1979). 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. and Food. 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. The regulations were summarized in two comprehensive surveys (Meester. Gerhardt. 1978). As the percentage of extenders and other nonmeat ingredients is increased in the products. Within the United States the methods of assay appear in the Official Methods of Analysis of the Association of Official Analytical Chemists .S. 1978). 1974.Nitrite 217 7. 1978). depending on the circumstances. Braathen. Because it has already been cured. and 172. 172. The net effect of the U. Examples of the effect of this regulation on the maximum allowable level of nitrite in several sausage and loaf products are listed in Table 6. the level of added nitrite is decreased. 9.

respectively (JECFA. occurs at methemoglobin levels in excess of 10%. the criterion most commonly used for nitrite toxicity. The values are expressed as nitrate and nitrite ions. 1994). The normal range of methemoglobin levels is 0. 2002). 1994). as evidence of toxicity. nonfat dry milk. with the lower doses applying to infants who are more sensitive to methemoglobinemia.. International agreement has been reached on the specifications and analytical methods for potassium nitrate and sodium nitrite (JECFA.. the uppermost level being found in infants and pregnant women. The no-adverseeffect dose level was estimated to be 2500 mg sodium nitrate/kg body weight/day (Gangolli et al. 1995).. estimates for the toxic dose range from 1 to 8.0%. whereas 30 to 60 g of sodium nitrate have been given for 2 months as an acidifying diuretic without manifestation of adverse effects. respectively. TOXICOLOGY The acceptable daily intake for potassium nitrate and sodium nitrite have been reported to be 0 to 3.3 mg sodium nitrite/kg body weight (Gangolli et al. For potassium nitrate.06 mg/kg body weight. Using this criterion. Data on reproductive toxicity for nitrate were limited. (AOAC international. 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. and apply to all sources of intake but do not apply to infants younger than age 3 months. 1995). Two years of rat studies on nitrate showed no evidence of carcinogenicity. and it appears that nitrate per se is not genotoxic. Source: Based on formulations in Pearson and Tauber (1974).218 Antimicrobials in Food TABLE 6. For sodium nitrite.7 and 0 to 0. estimates for the lethal dose have ranged from 2 to 9 g (about 33 to 250 mg/kg body weight).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. estimates of the lethal dose have ranged from 4 to 30 g (about 70 to 500 mg/kg body weight). Cyanosis. 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 .5% to 2. 1994).

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

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

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

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

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

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

Optimum heat stability occurs at pH 3 to 3. is the same value as the Reading Unit. The nisin preparation Nisaplin® was developed by Aplin & Barrett (now Danisco) between 1962 and 1965. In triangle taste trials conducted by Danisco. 1966). equivalent to 1 µg of this preparation. 1964).1 to 6. successfully used in high-pH/heat-treated food products such as canned vegetables and pasteurized liquid egg.2 as approximately 56 mg/ml. and nisin activity was then defined in International Units. IU/g).3 to 4. first developed by Tramer and Fowler (1964) and Fowler et al. This was contrary to previous work by Tramer (1964.25 mg/ml at pH 8. Both the solubility and stability of nisin in solution are affected by pH. Liu and Hansen (1990) reported nisin solubility at pH 2. dropping to 1. The horizontal agar diffusion test. The effect of processing temperature and pH on the stability of nisin in different foods was studied by Heinemann et al. Solubility in foods is never a problem because nisin levels are always very low. with levels of usage in food rarely above 0. Their results show that in low-acid foods at pH 6. is the most widely used nisin assay.025 mg/ml (equivalent to 1000 mg/kg Nisaplin®). suggesting that the molecule is protected to some extent by food components (Delves-Broughton. International Unit. An international nisin reference preparation was later established by the WHO Committee on Biological Standardisation (Anonymous. (1965). 1 µg/g pure nisin is equivalent to 40 IU/g or 40 mg Nisaplin®/kg.5. In this chapter nisin is expressed as levels of pure nisin (µg/ml or µg/g). Nisin is. Liu and Hansen (1990) suggested that the Dha and Dhb residues become more susceptible to modification as a result of the presence of nucleophiles. heating for 3 minutes at 250°F (121°C) destroyed 25% to 50% of the added nisin. However. however. The specifications for the purity and identity of nisin were set in 1969 by the FAO/WHO Joint Expert Committee on Food Additives (Anonymous. These may be intended as stock solutions for the preparation of test dilutions in smallscale laboratory experiments. Nisin has no significant taste and imparts no negative taste to foods in which it is used. ANTIMICROBIAL ACTIVITY UNITS OF MEASUREMENT AND ASSAY The activity unit for nisin was first defined as a Reading Unit: the minimum amount of nisin required to inhibit the growth of one cell of Streptococcus agalactiae in 1 ml of broth (Tramer and Fowler.25% biologically active nisin.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.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. A similar degree of destruction was reported for highly acid foods having pH values of 3. and contains 2. 1970). At high pH. To convert these units to equivalent Nisaplin® levels (mg/L. (1975). It is recommended that such solutions should not exceed a maximum concentration of 250 µg/ml. and a specification was set for commercial nisin concentrates containing at least 2. the levels of pure nisin should be multiplied by 40.. 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.9. 1969). research in Danisco laboratories has shown that care must be taken when preparing high-concentration nisin solutions from commercial preparations such as Nisaplin®. The current term. For example.5. 1998). and this bioassay method contains . Nisin is extracted from the test food by an acid-heat treatment. This has a standard potency of 1 million International Units per g. 1990.5 mg/ml at pH 6 and 0. mg/kg) or to International (Reading) Units (IU/ml. volunteers could not detect 200 mg/L Nisaplin® in mineral water (equivalent to 5 µg pure nisin/g). 1992). Delves-Broughton et al. Pure nisin was deemed too potent for use in food. otherwise a loss of activity may occur as a result of salt precipitation or filtering.5 (Davies et al. producing clearly distinguished and measurable zones of inhibition in the presence of nisin.

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

242 Antimicrobials in Food TABLE 7. wine. botulinum. Nisin activity against spores is predominantly bacteriostatic. faecium Spp. Growth at pH 2. Heat-resistant spore-forming thermophiles. Causes food poisoning. damnosus. 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. Cause spoilage of wine and vegetables. mesenteroides innocua. coagulans. pasteurianum. quiescent cells may survive but their growth will be inhibited (Sahl.. Varied nisin sensitivity. curvatus. cured meat products. pumilis. Includes psychrotrophs. Heat-resistant aerobic and facultative anaerobic spore formers. low water activity. perfringens. Varied sensitivity. tertium. Some used for cheese production. licheniformis. and food-poisoning pathogens. pentosaceus inulinus aureus Source: From Thomas et al. sporogenes.) and food-poisoning. Aerobes/anaerobes. butyricum. low pH. 1995). brevis. Cause spoilage (blowing. thermophiles. Heat-resistant spore-forming obligate anaerobes. Cause spoilage. and the l