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

Third Edition

Edited by

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

Boca Raton London New York Singapore

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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

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.

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

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

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

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

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

1490.22.6197. molds Bakery products. heptyl) of p-hydroxybenzoic acid) Propionic acid. dairy products Most foods. 182. Extensive reviews on natural antimicrobials may be found in Dillon and Board (1994). fats/oils.1185. 184.133 184. sorbates Sulfites a Clostridium botulinum Yeasts. (1998). cooked meat.S. syrups. dehydroacetic acid Benzoic acid.160.1207. diacetates.3225. fermented foods Meats Cheese.3089. baked goods. a combination of chemical preservatives and other preservation methods is needed (Leistner. 182.1733 172. benzoates Dimethyl dicarbonate Lactic acid. GRN 000065 Nitrite. molds. Sofos et al. Title 9.1061. bacteria Primary Food Applications Baked goods. . beverages. Source: Davidson and Harrison.1639. fruit products.1005. methyl.155 184.1021. other bacteria Natamycin Nisin Molds Clostridium botulinum.21 and 424. condiments.1538. fruit products. 184.1 and 1. 172. and Davidson and Zivanovic (2003). GRN 000067 184.1670.1221. and poultry products Cured meats Beverages. Naidu (2000).1 Traditional or Regulatory-Approved (U.1550. food antimicrobials permitted by USDA Food Safety and Inspection Service are listed in the Code of Federal Regulations. 172. and then the possible preservation systems must be evaluated via model studies and studies in the food product in question. 184. 184.177 184. nitrate Parabens (alkyl esters (propyl. casings for frankfurters. wines Fruits.Food Antimicrobials — An Introduction 3 TABLE 1. 172. cooked meat. 184.145 Molds Yeasts.130 184. 184. 184. meats. sauces Beverages. other bacteria 172. SELECTION OF ANTIMICROBIALS It is not an easy process to select the appropriate preservation system for a particular food product. 172. margarine Beverages Meats. Section 424. acetates. potato products. confections. The target pathogen or spoilage microorganisms must be identified first. GRAS Notice No. dry sausage CFR Designation 184. Food and Drug Administration) Food Antimicrobialsa (Title 21 of the Code of Federal Regulations) Compound(s) Acetic acid. 184. 2000).175. and poultry products Cheese Cheese.1081. molds. lactates Lactoferrin Lysozyme Microbial Target Yeasts.170. bacteria Yeasts. 172.3640. bacteria (Gram positive) 172. dairy products.1768 GRAS Notice No. GRAS Notice No. 2002 to foods (Tables 1. 182. 184. molds Yeasts Bacteria Bacteria Clostridium botulinum.2). 184.1754. propionates Sorbic acid. casings for frankfurters.1721.1784 182. wines 184.3795 Various For meat products. 182. Generally. GRN 000064 Yeasts.

which have the ability to screen the antimicrobials because of the outer membrane lipopolysaccharide layer. a broad spectrum of activity. Each of these factors is discussed in detail. Even species. however. 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 antimicrobial spectrum should involve an evaluation of the compound against various types of microorganisms (e. yeasts. spores). including the spectrum of antimicrobial activity. Selection of the proper antimicrobial depends on several primary factors. Quite often. and Gram reaction (positive vs. the chemical properties of the antimicrobial. Few chemicals have the ability to inhibit several different types. or strains of microorganism.4 Antimicrobials in Food TABLE 1. the physicochemical properties and composition of the food product in question.2 Regulatory-Approved Food Antimicrobial Designations in the European Union (E Numbers) and in Codex Alimentarius (INS Numbers) Compound Sorbic acid K sorbate Ca sorbate Benzoic acid Na benzoate K benzoate Ca benzoate Ethyl paraben Na ethyl paraben Propyl paraben Na propyl paraben Methyl paraben Na methyl paraben Sulfur dioxide Na sulfite Na hydrogen sulfite Na metabisulfite K metabisulfite Ca sulfite Ca hydrogen sulfite K hydrogen sulfite Nisin a E or INS Number 200 202 203 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 226 227 228 234 Compound Natamycin DMDCa K nitrite Na nitrite Na nitrate K nitrate Acetic acid K acetate Na acetate Na diacetate Ca acetate Lactic acid Propionic acid Na propionate Ca propionate K propionate Boric acid Na tetraborate Na lactate K lactate Ca lactate E or INS Number 235 242 249 250 251 252 260 261 262 262 263 270 280 281 282 283 284 285 325 326 327 Dimethyl dicarbonate. the activity of very hydrophobic antimicrobials may be limited against Gram-negative bacteria. ANTIMICROBIAL SPECTRUM The initial selection of the antimicrobial is normally based on an assessment of the overall microbial spectrum of the chemical in question. does growth in a synthetic . 2002. negative) can have dramatic influences on apparent activity. “Methods for Activity of Assay and Evaluation of Results. although desired. Source: Verbruggen.” Seldom. For example.. bacteria. species.g. strain. molds) and forms of those microorganisms (vegetative cells vs. Appropriate methods for in vitro evaluation of the activity of food antimicrobials are described in Chapter 21. and the type of preservation or processing and storage systems used.

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

79 4. Vacuum or modified atmosphere packaging results in . Rico-Munoz Davidson.19 3. 1995. which exists in the majority only at a pH below the pKa of the compound. molds survive and yeasts can grow at a lower water activity than bacteria. and phospholipids. allowing the antimicrobials access to the inner cell membrane. Once inside the cell. 2004). PROCESS FACTORS The type of preservation process used in conjunction with antimicrobials has a significant influence on the type and level of antimicrobial needed. All regulatory-approved organic acids used as antimicrobials have pKa values less than 5. the acid dissociates because the cell interior has a higher pH than the exterior. some Gram-positive bacteria are more susceptible to certain antimicrobials in the presence of chelators including EDTA. Bacteria maintain internal pH near neutrality to prevent conformational changes to the cell structural proteins.75 4. In the undissociated form. Branen and Davidson. organic acids can penetrate the cell membrane lipid bilayer more easily.6). lysozyme) to include Gram-negative bacteria. Certain preservation processes may result in the need to control sporeformers that have the ability to survive the heating process.0 (Table 1. pH of the food can result in ionization of an antimicrobial and a change in activity. thus indicating the need for a different antimicrobial.3 pKa of Regulatory-Approved Organic Acids Compound or Group of Compounds Acetic acid Benzoic acid Lactic acid Propionic acid Sorbic acid pKa 4.. For example. Generally. where microbial growth occurs (Branen et al. In addition. enzymes.3). A lowering of the water activity can select for those organisms that have the ability to survive and/or grow at lower water activity. 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. Although the undissociated form of a weak acid has most of the antimicrobial activity. Because protons generated by the organic acid inside the cell must be extruded using energy in the form of adenosine triphosphate (ATP). the constant influx of these protons will eventually deplete cellular energy. there are very few compounds that are effective at low concentrations. For food products with a pH of 5. 1983). This is because the most effective antimicrobial form is the undissociated acid. which means their maximum activity will be in high-acid foods. Packaging can directly alter the environment of the food and thus influence the overall growth pattern and type of organisms in the food.5 or greater. Chelating compounds such as ethylenediamine tetraacetic acid (EDTA) or its salts have a potentiating effect on some antimicrobials. They expand the activity of certain antimicrobials (e.5 to 4.g. which are not normally inhibited by the compounds alone (Cutter and Siragusa.75 into the lipid areas of the food and away from the water phase. Protons generated from intracellular dissociation of the organic acid acidify the cytoplasm and must be extruded to the exterior. nucleic acids. nisin.6 Antimicrobials in Food TABLE 1. Refrigeration generally selects for psychrotrophic Gram-negative microorganisms. It is theorized that chelators may destabilize the LPS layer of the outer cell membrane of the Gram-negative bacteria.. thus requiring an antimicrobial capable of limiting the growth and activity of these organisms.87 4. 1980.

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

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

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

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

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

respectively). 1929). Potassium benzoate and calcium benzoate have also been approved for use. Its presence appears to occur as a by-product of the microbial degradation of either hippuric acid or phenylalanine in these products (Figure 2..2% but inactive in alkaline media.1 Structures of benzoic acid and sodium benzoate..1). Sodium benzoate (molecular weight 144.27. Benzoic acid (molecular weight 122. depending on the variety (Abdullah et al. Gabel (1921) was one of the first to demonstrate that benzoic acid was effective against bacteria in acid media at a level of 0. 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. apple. 0. 1986a). Sieber et al. respectively). 66. oxidation of benzaldehyde. Teuber. 1999). apricot. it is preferred for use in many cases.1) is a white granular or crystalline powder. 1991).6 mg/l...8. and 74.12 Antimicrobials in Food PHYSICAL AND CHEMICAL PROPERTIES AND NATURAL OCCURRENCES Benzoic acid (C6H5COOH) and sodium benzoate (C6H5COONa) have the structural formulas shown in Figure 2.3). O OH Benzoic Acid O O− Na+ Sodium Benzoate FIGURE 2. Benzoic acid has also been identified as a natural by-product in culture filtrates of Lactobacillus plantarum (Niku-Paavola et al. occurs in pure form as colorless or white needles or leaflets. A third pathway. and of fresh tomatoes (Marlatt et al. In four of the seven juices. and grape juices were analyzed to establish phenolic profiles unique to each type of juice.0. . It is much more soluble in water than benzoic acid (62. 1989. For this reason. 1994). was found to have antimicrobial properties.18. also called phenylformic acid or benzenecarboxylic acid. It accounted for approximately 16% of the growth inhibition of Saccharomyces bayanus and Pseudomonas fluorescens from ethanolic extracts of cranberries (Marwan and Nagel. 1992). It is soluble to a limited extent in water (0.. 1992). (1995) surveyed and analyzed many types of cultured dairy products and cheeses for the natural occurrence of benzoic acid (Table 2. Similar results were reported for fungi and yeasts (Cruess and Richert. and 75°C. and a mixture of these. were used to characterize commercial fruit juices (Fernandez de Simon et al. pineapple. where benzoic acid has not been approved as a food additive (Sieber et al. of mushrooms.2). Yogurts have been found to contain natural levels of benzoic acid (Stijve and Hischenhuber.2 g dissolves in 100 ml water at 0°C.2 to 2. Benzoic acid has also been identified as a major constituent in extracts of blackberries (Humpf and Schreier.2 g dissolves in 100 ml water at 4°C. and 100°C. This may be of significance in countries like Switzerland. In an extensive review. including derivatives of benzoic acid. 20°C. 1995).1). Orange. peach. Naturally occurring nonflavonoid phenolic compounds. and 2. 1990). p-hydroxybenzoic acid was detected at 0. 18°C. 1984. although their solubility in water is less than that of the sodium salt.1. pear. Several new types of low-molecular-mass compounds were also identified.2). Benzoic acid occurs naturally in several foods and commodities (Table 2.1% and in neutral media at 0. along with benzoic acid.

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

Saturation was reached in about 2 minutes and remained constant thereafter. not to the pH itself. 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. and that the toxicity of sodium benzoate in solution was a result of the undissociated benzoic acid molecule. (1995). The effects of pH. sucrose. The strong dependence of uptake on pH was because of the relative distribution of undissociated and dissociated forms in solution. and sorbic and benzoic acids on the growth of 30 strains of food spoilage yeasts have been reported (Praphailong and Fleet. 1997).3 12. Proteinaceous material was apparently involved in the uptake of this preservative.4.19 Source: From Baird-Parker (1980). NaCl.8 1. Source: Adapted from Sieber et al. increasing the pH of the culture medium decreased the effectiveness of this preservative.0. The effect of temperature on the uptake was similar to that observed in enzymatic reactions. Macris (1975) observed a rapid uptake of benzoic acid in Saccharomyces cerevisiae.144 4.5 59. Sorbate was more inhibitory than benzoate on the strains that were tested. that only the undissociated acid was antimicrobial. .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 2.2 Biosynthesis of benzoic acid by lactic acid bacteria in fermented dairy products.3 Effect of pH on the Dissociation of Benzoic Acid pH 3 4 5 6 7 pK Undissociated Acid (%) 93. TABLE 2. As expected. Zygosaccharomyces bailii and Yarrowia lipolytica were the most resistant to the two preservatives at pH 5. The combined effects of pH and benzoate on the growth of these strains are presented in Table 2.44 0. The undissociated form was the only one taken up by cells.

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

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

Baird-Parker. Sodium benzoate was found to control both growth and aflatoxin production by Aspergillus parasiticus in liquid media (El-Gazzar and Marth. as an inhibitor of Damino acid oxidases (Quay and Massay.5 and 2. 1979). with the greatest increase occurring when the medium contained 0. Several factors interact to determine the MIC. as an inhibitor of passive anion transport (Lucas-Heron and Fontenaille. . coli and Salmonella Enteritidis (Chipley. 1974). It has been reported to have a flower-inducing effect in plants (Watanabe and Takimoto. 1972. The authors concluded that subinhibitory concentrations of these compounds may stimulate toxin production in some cases. 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. In liquid media. cerevisiae decreased the biomass and increased the specific oxygen uptake rate of cells (Verduyn et al. as a weak inhibitor of poly(ADP ribose) polymerase (Oikawa et al. The average accumulation of mycelial dry weight was greater in the presence of benzoate than in its absence. 1987). yeasts. potassium sorbate. and most yeasts and fungi are inhibited by 0. Fusarium species were more sensitive to benzoic acid (210 to 420 µg/ml) than were Penicillium species (250 to 3000 µg/ml).6. Conversion could be prevented by addition of benzoic acid or sodium benzoate.. and sodium benzoate was determined (Manganelli and Casolari. 1980). Yagi et al. composition of the growth medium. In benzoic acid-supplemented cultures. genus and species of the microorganism in question. 1997). 1981). This might result in inhibition of membrane-bound enzymes as a secondary effect. In general. ANTIMICROBIAL ACTIVITY SPECTRUM OF ACTION Because the quantity of undissociated acid decreases with increasing pH (Table 2. prior exposure to the preservative. aflatoxin B1 production was higher than in controls. 1980). 1992). all treated cultures produced measurable levels of toxin 3 to 7 days later than controls.3% sodium benzoate. The results suggested that these preservatives blocked an enzymatic step late in the biosynthetic pathway of aflatoxin B1. 1988). flavus have been reported (Bauer et al. Therefore.. 1977. and environment from which the microorganism was originally isolated. 1979). 1980). Similar results were observed with 2.02% undissociated acid.01% to 0.. 4-dinitrophenol and cell envelope-associated enzymes of E. 1983). including pH. Food-poisoning and spore-forming bacteria are generally inhibited by 0.05% to 0. The sensitivity of 42 yeast cultures to sorbic and benzoic acids. MICs for some of the bacteria. Benzoate may also serve as a scavenger for free radicals (Harvath. the use of benzoic acid or sodium benzoate as a food preservative has been limited to those products that are acid in nature.. The scattering of MIC values was lower with potassium sorbate and benzoic acid and higher with sorbic acid and sodium benzoate. benzoic acid cannot be relied on to effectively preserve foods capable of supporting bacterial growth (Chichester and Tanner. Addition of benzoate to chemostat cultures of S. 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.3). these compounds are used primarily as antimycotic agents.. The effects of several preservatives and antimicrobial agents on aflatoxin B1 production by A. Currently. With the previously mentioned results in mind. temperature.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. and fungi involved in food poisoning and food spoilage are given in Tables 2.1979). but many spoilage bacteria are much more resistant.1% of the undissociated acid. however. Increasing the concentration of sodium benzoate increased the percentage of inhibition at the end of incubation (10 days). and as an inhibitor of nitrosamine formation (Sung et al.

Hansenula subpelliculosa Oospora lactis Pichia membranefaciens P.5 5. Aspergillus parasiticus Aspergillus niger Byssochlamys nivea Chaetomonium globosum Cladosporium herbarum Mucor racemosus Penicillium sp. Pseudomonas aeruginosa Staphylococcus aureus Streptococcus sp.0 4.5 Antimicrobial Spectrum of Benzoic Acid against Selected Bacteria. and Steels et al. Davidson and Juneja (1990).6 (21°C) 5. (1999).0 5.5–5.0 2. Pseudomonas sp. lentus Z.18 Antimicrobials in Food TABLE 2.0 5. Source: Adapted from Chipley (1983. 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.8 4. Yeasts. Russell (1991).3 5.8 Fungi 5.6 (4°C) 5.0 5.0 5.5 4. .0 4.0 3.6–4. and Fungi Microorganisms pH Bacteria 6.0–5.3–6. Saccharomyces bayanus S.6–5.0 3.6 6.0 MICa Bacillus cereus Escherichia coli Klebsiella pneumoniae Lactobacillus sp.1 5.0 5.0 5.6 Yeasts 2.2–5.0–5. cerevisiae Torulopsis sp. Listeria monocytogenes Micrococcus sp.0 6.0 6.0 4.0 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. Zygosaccharomyces bailii Z.8 4. pastori Rhodotorula sp. 1993).0 4.0 5.2–5. 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).0 4.3 5.0 4. rouxii 4.6 6.0 6.

activities against cells in a liquid minimal medium. In a series of studies involving L. Sodium benzoate has been suggested as an inhibitor of cellulose-decomposing bacteria and fungi (Sauer. according to the results of a study involving several strains of Trichophyton and Microsporum (Pelayo. 1977). Growth and aflatoxin production by toxigenic strains of Aspergillus were partially or completely inhibited by the undissociated form of six organic acid preservatives.5) without addition of benzoic acid. were tested against both the opaque and translucent morphotypes of Vibrio vulnificus (Sun and Oliver. Beuchat (1980) reported that sodium benzoate (300 µg/ml) inhibited the growth of Vibrio parahaemolyticus in laboratory media and enhanced the rate of thermal inactivation of this organism at slightly higher concentrations. Most were isolated from spoiled foods that had contained preservative. 1989). including benzoic (Rusul and Marth. Ten generally regarded as safe (GRAS) substances.0 to 2. Isolates were grown at 25°C in yeast extract medium containing 5% glucose (pH 3. 1988). Growth of this fungus was accompanied by production of fumitremorgin mycotoxins. and its survival .Sodium Benzoate and Benzoic Acid 19 TABLE 2. Source: Adapted from Warth (1989c). Incubation of cells in minimal media caused injury that depended on the temperature of incubation but not on the presence of benzoic acid. but relatively modest bactericidal.. monocytogenes (El-Shenawy and Marth. enhanced aflatoxin production when present at low levels but became inhibitory at higher levels. Salts. The authors questioned the suitability of benzoic acid alone to control this pathogen in foods. including benzoic acid. it was found that benzoic acid at concentrations of approximately 1000 to 3000 ppm had strong bacteriostatic. Eight of these had a lethal effect on both morphotypes of this bacterium. 1995). Yousef et al. 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. 1989). Both fungistatic and fungicidal properties have been attributed to benzoic acid. Fungal growth was reduced by lowering the pH of laboratory media from 7. This organism was isolated from milk.6 Minimum Inhibitory Concentration of Benzoic Acid for Food Spoilage Yeasts Isolatea Kluveromyces fragilis Kloeckera apiculata Pichia ohmeri Hansenula anomala Saccharomyces cerevisiae Zygosaccharomyces rouxii Zygosaccharomyces bisporus Candida krusei Saccharomycodes ludwigii Schizosaccharomyces pombe Zygosaccharomyces bailii a MIC (ppm) 173 188 200 223 170–450 242–330 200–350 440 500–600 500–567 600–1300 A total of 23 isolates were tested.. such as sodium and potassium chlorides and sodium nitrate. 1979). bacteriostatic and bactericidal properties of benzoic acid can also be demonstrated. Small amounts (75 mg/L) of potassium sorbate or sodium benzoate completely inhibited germination of ascospores and subsequent outgrowth. selected organic acids promoted growth and toxin production when added to the media. 1988.5. Under appropriate conditions.

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

3 Effect of the medium on the inhibition of Saccharomyces bayanus by benzoic acid at 30°C and pH 4. the antimicrobial activity of benzoic acid is reduced by nonionic surfactants (de Navarre and Bailey. such as heating. Sulfur dioxide had the most significant effect on the rate of biomass and patulin production by this organism. refrigeration. 1961). and 37°C over a 25-day incubation period was significantly retarded by 75 ppm sulfur dioxide. The effects of pH and benzoate on the growth of yeasts in an experimental grape drink containing 1.0 0. followed by potassium sorbate and sodium benzoate. 1976).0 50 100 150 ppm 200 250 300 350 FIGURE 2. 150 ppm potassium sorbate. irradiation.. 1956). benzoic acid can be solubilized with a surfactant to hold it in solution in its undissociated form (Lewis and Payne. 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). cerevisiae was inoculated in a laboratory medium supplemented with several combinations of preservatives (Parish and Carroll.9) (Okoli and Ezenweke.2 0. 1984). In addition to combining several preservatives together. Sulfur dioxide was apparently antagonistic when used in combination with any of the other antimicrobial agents (including benzoate) tested in this study.0. Open circles: Trypticase® soy broth.b). 1975). relative effectiveness (the time.6 1/RE 0. 30°C. A combination of benzoic acid and low temperatures was used effectively to reduce the numbers of yeasts in grape juice (Pederson et al. in hours. and 500 ppm sodium benzoate. or drying. Sodium benzoate prevented the growth of Staphylococcus aureus in an intermediate-moisture food (Boylan et al. For example. RE. Growth at 21°C. . Closed circles: yeast nitrogen base. Antagonistic as well as synergistic effects were observed when a wine yeast strain of S. 1958). depending on the chemical in question. 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.4 0.. respectively. Likewise. 1990). Source: Marwan and Nagel (1986b). it may also be advantageous to combine preservatives with physical methods of food preservation.8 0. Synergistic effects on yeasts and fungi using a combination of benzoic acid and heat have been reviewed by Lueck (1980) and have also been reported by Beuchat and Jones (1979) and Beuchat (1981a.Sodium Benzoate and Benzoic Acid 21 1.5 volumes of CO2 have been reported (Kelly. 1988). However. The presence of other chemicals may be beneficial or detrimental to the action of the preservative. 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.

5 (Sagoo et al. At these pH values. A combination of the polysaccharide chitosan (0. This acid was antagonistic to the antifungal effects of both potassium sorbate and sodium benzoate. 1985). but mold growth was accelerated in the presence of quinic acid. Carbonated and noncarbonated citrus drinks and fruit juices were involved. . 2002). They had been supplemented with the maximum levels of benzoic acid permitted for use in these products. cited by Lueck (1980).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. this might enhance the antimicrobial effects of benzoate. the authors proposed that chitosan is highly polycationic and could react with the anionic components of the microbial surface. The resistance of a wide variety of yeasts to benzoic acid has been reported in several studies. However.02% sodium benzoate. recent studies imply that this may no longer be the case. Source: Marwan and Nagel (1986b).005%) and 0. Growth of yeasts was unaltered.01% to 0.0. The concentrations of benzoic acid tested (in parts per million) are indicated. In a German study of 1959... coli after 14 passages in media containing benzoic acid.025% sodium benzoate acted synergistically against three food spoilage yeasts suspended in saline solutions at pH 6. no resistance could be observed in cells of E.4 Inhibition of Saccharomyces bayanus by benzoic acid in Trypticase® soy broth at 30°C and pH 4.2 and 4. ACQUIRED RESISTANCE TO BENZOIC ACID There have been conflicting reports in the literature concerning the possibility of acquired resistance to benzoic acid by microorganisms. The effects of quinic acid on yeasts and molds were investigated (Kallio et al. Adding 1% quinic acid to media almost completely eliminated the inhibitory effect of 0.

and in some cases. cells were resistant only when a sufficient energy source.25 mM benzoic acid (first five species) or 2 mM benzoic acid (last four species). were tested for tolerance to several organic acids during and after heating (Beuchat. was present. Three strains of T. 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.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. this isolate was less resistant to potassium sorbate. Thus. 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. 1988) it was determined that energy was required for the reduction in cytoplasmic benzoate concentration and for the maintenance of internal pH. flavus. 1990). A strain of S.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. such as glucose. In a later study (Warth. 1987).0 and 4. isolated from spoiled fruit products. resulting in reduced growth yields and rates of cell production by yeasts.4 to 3.5. respectively (Ethiraj and Suresh.7). and benzoic acids were the most effective. In an inoculated peach-based system. Potassium sorbate and sodium benzoate prevented outgrowth. . and lethality was enhanced as the pH of the heating medium was reduced. The heat-resistant yeast Talaromyces flavus was isolated from fruit juice concentrates involved in spoilage of packaged reconstituted fruit juice (King and Halbrook. Source: Adapted from Warth (1988). Because this system required energy. Heat resistance increased with age.5. Warth (1977) reported that cells of Z. bailii grew in the presence of high concentrations (600 mg/L) of benzoic acid at pH values below the pK of this acid (4. cerevisiae was found to have multiple resistances to both physical treatment and chemical preservatives (Guerzoni et al. 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. However..19). with the required concentrations lower when the pH of the growth media was reduced from 5. 1988). TABLE 2. heat tolerance appeared to be related to the morphology of the ascospores. 1988). Fumaric. The authors observed that preservative resistance may be strain specific. the energy source was unavailable for growth. sorbic. Resistance resulted primarily from an inducible energy-requiring system that transported benzoate from the cell.

0). Eight tons of table mustard were exported to the United States. and all strains were osmotolerant. bailii grown in six commercial food products (Makdesi and Beuchat.5 g/kg) and packaged in glass jars. 1996). Z. Five strains of this yeast grew over a wide range of temperature (4°C to 25°C) and pH (2. Growth in the presence of weak acid preservatives appeared to involve a common resistance mechanism. the capacity of the cell to remove it. and 600 µg/ml). However. Three selective media were compared for detecting and enumerating nine strains of Z. 1985). or an intrinsic sensitivity to benzoic acid or its anion (Warth. . Adaptation to higher preservative concentrations was demonstrated with benzoic acid.. Zygosaccharomyces lentus. has been described (Steels et al.. Benzoic acid was taken up 27 times faster by cells than propanoic acid. 1989c). 1996). Cells grown in syrup without sodium benzoate were more sensitive to selective media than were cells grown in syrup containing the preservative. Foods at particular risk are acidic and contain relatively high levels of sugar. Zygosaccharomyces and Saccharomyces are able to adapt to changing levels of preservatives such as benzoate. cerevisiae. They were resistant to sorbic acid (≤400 mg/l) and benzoic acid (≤900 mg/l) at pH 4. The minimum pH for growth was not related to this acquired resistance. Growth of yeasts in the presence of benzoic acid reduced their subsequent permeability to this acid. 1999). In another study (Warth. The nonselective medium supported higher recovery of all nine strains from the six food products compared to the three selective media. The principal mechanism of uptake appeared to be diffusion of undissociated acid. The Zygosaccharomyces genus also contains some of the most osmotolerant species known. 1989a). Removal or metabolism. 1999). Uptake rates of benzoic and propanoic acids in cells of Z. Spoilage resulted from a Z. 1989b). Resistance to dimethyldicarbonate was greater than that of 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. allowed in beverages and can adapt to concentrations even higher.g. The authors stated that microorganisms resist or adapt to weak acid preservatives (e. or legally. Growth at 4°C was significant. is a common and major determinant of the preservative tolerance of yeast species (Table 2. Resistant species may be less permeable than sensitive ones to undissociated benzoic acid (Warth. bailii in the spoilage of refrigerated foods. benzoate) by one of the following three methods: 1.6) (Warth.0. and acetic acids and ethanol (Thomas and Davenport. Differences in resistance among yeast species could be the result of differences in the rate of penetration of the cell by benzoic acid. benzoic. It was preserved using benzoic acid (1. bailii can grow in concentrations of food preservatives in great excess of those normally. 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. bailii were proportional to the concentration of undissociated acid (Warth. 1989c). in which benzoic acid is continuously removed from the cell. requiring only 5 to 15 minutes to reach equilibrium. Glucose stimulated the rates of uptake and was also required for continuous elimination of benzoate from cells. bailii strain that was resistant to both benzoic and sorbic acids. bailii has been described (Buchta et al. Some yeasts are intrinsically resistant to relatively high concentrations of preservatives (Stratford and Lambert. 1988). 300. A new osmophilic.. These media were also evaluated for enumerating two of these strains grown in blueberry syrup with or without sodium benzoate (0. and most can grow at very low pH.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. Adaptation can result in rapid growth in moderate levels or slow growth in previously inhibitory levels. Uptake of weak acid preservatives by passive diffusion is quite rapid. bailii and S. Mustard spoilage by Z.2 to 7. results also suggested that the resistance mechanism. The authors concluded that Z. lentus was likely to be more significant than Z. preservative-resistant spoilage yeast.

The inhibitory action is classically believed to occur when the molecule encounters the higher pH inside the cell and then dissociates. 1989). 1985). However. as cited previously (see “Mechanism of Action”). undissociated state of the molecule. The pathogenicity of this fungus could be increased by growth on a medium supplemented with benzoic acid before inoculation into fruit. MICs of sorbic and benzoic acids for Gluconobacter oxydans were 1000 and 900 mg/L. Weak organic acid preservatives have maximum inhibitory activity at low pH. if damage is caused by low intracellular pH. This appears unlikely because uptake occurs rapidly by passive diffusion. resulting in the release of charged anions and protons. (1998) demonstrated the existence of a multidrug-resistance pump that actively extruded preservative anions from cells of this yeast. The net result is that the preservative diffuses into the cell until equilibrium is reached in accordance with the pH gradient across the membrane. They included the acid tolerance responses of E. and adaptive mechanisms have been suggested. however. The authors proposed that some yeast adaptation to weak acid preservatives might simply be the result of accumulation of anions. When G. Mutants of the fungus Nectria galligena. Piper et al. 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. Anions and protons accumulate inside the cell. Holyoak et al. This favors the uncharged. For example. in media at pH 3. Prevention of uptake. 1996. 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.8. which can infect apple tissues. monocytogenes and the role of membrane ATPase in the resistance of food spoilage yeasts. oxydans was grown in the presence of sublethal concentrations of either compound before the MIC was determined.. 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. adaptation could involve enhanced removal of protons using H+ATPase. Whether efflux pumps are involved is open to debate. 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. b. cerevisiae were able to extrude benzoic acid by an energy-dependent mechanism. However. Several microbial-resistance mechanisms induced by weak organic acid preservatives were also reviewed by Brul and Coote (1999). In yeasts. coli initially grown in media at pH 5..0 survived exposures to inorganic acid or to acid pH plus organic acid that prevented subsequent growth of cells grown at pH 7. The authors proposed that in yeasts. coli O157:H7 and L. Adaptation to or repair of damage. respectively. 1997.3 reduced the MIC of both antimicrobials by about 300 mg/L (Eyles and Warth. (1997) found that cells of S... Therefore.0 (Goodson . Cells of E. earlier studies by Henriques et al. 1998). which cannot cross the plasma membrane. Benzoic acid occurs naturally as a phytoalexin of immature apples (Seng et al. 3. which is freely permeable across the plasma membrane and is thus able to enter the cell. Bracey et al. When this happens. were isolated and found to be resistant to benzoic acid. Several causes of damage by weak acid preservatives have been proposed. 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. the MIC of both antimicrobials increased substantially within 1 hour. 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. Reducing the pH of the media to 3. Henriques et al. In an extensive review.Sodium Benzoate and Benzoic Acid 25 2. inhibition of microbial cell growth occurs because various key metabolic reactions are inhibited. Acquired resistance to benzoic acid has been reported for other microorganisms.

Russell (1991) examined mechanisms whereby bacteria resist the effects of preservatives and nonantibiotic antibacterial agents. Resistance to benzoic and sorbic acids is well documented in yeasts (Warth 1977.26 Antimicrobials in Food and Rowbury. Moscoso-Vizcarra and Popoff (1977) found that Enterobacteriaceae could be divided into two groups with respect to benzoic acid metabolism.. none of these acids inhibited the subsequent growth of acid-adapted cells. The authors proposed that acid-adapted organisms might survive in acid foods. cis-Muconic Acid CO2H CO2H O β-Ketoadipic Acid Succinic Acid + CoA-SH Acetyl-CoA FIGURE 2. acid-resistance systems remained active for prolonged periods of cold storage at 4°C. He stated that there were two types of bacterial resistance to preservatives. benzoate could be metabolized through the β-ketoadipate pathway.5 Proposed pathway for the aerobic microbial metabolism of benzoic acid. including benzoic acid. 1988).5). They also found that once induced. were evaluated. an acid-induced arginine-dependent system. When considering ways of overcoming resistance or of preventing it.0. Metabolism of benzoate occurred by the O OH OH OH Benzoic Acid Catechol CO2H CO2H cis. Six organic acids. including benzoic. coli in the gastrointestinal tract. The enzymes of the pathway are inducible. 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). coli against the bactericidal effects of a variety of weak acids. . First. monocytogenes against lethal chemicals after adaptation to sublethal environmental stress (Lou and Yousef. The authors suggested that several acid-resistance systems might contribute to the survival of pathogenic E. coli were tested for their ability to survive extreme acid exposures (Lin et al. and their synthesis is elicited by primary substrates or metabolic intermediates in the pathway. Three previously reported acid-resistance systems were tested. Thayer and Wheelis (1976) characterized an inducible benzoate permease system involved in the uptake of benzoate by cells of Pseudomonas putida. this mechanism has not yet been reported for bacteria. 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. Russell (1991) noted that choosing a suitable preservative or a combination of preservatives might be the most effective. 1989). the glutamate-dependent system provided better protection than the arginine system and appeared equally effective in all strains. However. However. In the first group. 1996) and protection of L. the second group consisted of those organisms that did not metabolize benzoate. When challenged at pH 2. Others include the induction of acid resistance in E. and a glutamate-dependent system. Resistance to preservatives caused by chromosomal gene mutation is an example. Source: Adapted from Zeyer and Kearney (1984). Eleven E. 1997). coli O157:H7 (Lin et al. Both of these systems were effective in protecting E. 1996). Examples of this type would include basic cell wall structure/organization and the possession of constitutive enzymes that enable bacteria to metabolize preservatives. In a taxonomic study. Second. This is the result of an enhanced ability of adapted cells to eliminate these acids by energy-dependent extrusion. coli O157:H7 strains and four commensal strains of E. They included an acid-induced oxidative system.. intrinsic resistance could result from a natural chromosomally controlled property of a bacterial cell.

Seventeen strains of aerobic bacteria were tested for their ability to degrade 15 aromatic substrates (Lang. 1994). 3-hydroxybenzoyl-CoA. (4) ring opening. which are central intermediates in the anaerobic metabolism of many aromatic compounds (Elder and Kelly.2-dioxygenase. These results were confirmed by assaying key enzymes from benzoate-grown cells. (2) ring reduction. and (5) a β-oxidation sequence. A permease-like protein involved in benzoate transport and degradation has now been isolated from cells of Acinetobacter (Collier et al. whereas salicylate induced catechol 1. In Aspergillus niger. gentisate. The enzymes of these pathways appeared to be under coordinate control and were repressed in the presence of a primary substrate. This isolate was able to oxidize benzoate completely to carbon dioxide. Benzoate was converted to catechol by the strains able to grow on this substrate. 1982).Sodium Benzoate and Benzoic Acid 27 β-ketoadipate pathway. and fumarate + pyruvate. Similar results have been reported with Bacillus stearothermophilus (Adams and Ribbons. end product of the β-ketoadipate pathway (Figure 2. putida (Jeffrey et al. 4-dioxygenase was induced. 1997). cis. Harwood and Gibson (1986) stated that aerobic degradation of benzoate by various Gramnegative bacteria.2-dioxygenase for ring fission. 1984). one of the causal agents of wilt in banana plants. cis-muconate. This is followed by conversion of the remainder of the molecule to acetyl-CoA. such as glucose. foods. The enzymes responsible for ring cleavage were induced depending on the substrate used for growth. Nitrate.6.5) (Ampe et al. 1995). and sulfate can be used as electron acceptors during metabolism (Colberg and Young. the rate of benzoate metabolism may be regulated by the presence of succinate. He reported that benzoic acid was easily metabolized by all nine bacterial species tested. In Alcaligenes eutrophus. Both chromosomal and plasmid-encoded pathways have been described for the catabolism of benzoate in P. (3) introduction of a carbonyl group. 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. Kinetic models have been derived for the metabolism of [14C]-benzoate by Pseudomonas species (Simkins and Alexander. 1995). The proposed intermediates included benzoyl-CoA. 1997). Compared to relatively well-characterized aerobic mechanisms for metabolism of benzoate and other aromatic compounds. 1997). and cosmetics. 1996). including Pseudomonas. and with Micrococcus (Haribabu et al. anaerobic pathways have been far less studied and reported. Benzoate was metabolized by 16 of these strains when added as the sole carbon source to liquid mineral media. In the presence of benzoate. 1982). benzoate and salicylate are metabolized via the protocatechuate and β-ketoadipate pathways.. 1988). 1993. 1990). . Further metabolism proceeded via the catechol branch of the β-ketoadipate pathway with the occurrence of high specific activities of catechol 1. with Rhodococcus (Janke et al. Hugo (1991) described the ability of nine species of Pseudomonas and Vibrio to degrade 18 aromatic compounds used as preservatives in medicines.. iron. fumarylpyruvate. Catechol. Active transport of benzoate may occur in this organism (Thayer and Wheelis. 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. respectively (Shailubhai et al.. 1984). 1992). 1988). 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. Pseudomonas solanacearum. and β-ketoadipate were identified as intermediates of benzoate metabolism (Figure 2. both aerobically and anaerobically. One pathway proposed for the anaerobic microbial metabolism of benzoate is shown in Figure 2. Niemetz et al. maleylpyruvate. protocatechuate 3. a commonly occurring soil bacterium. 1984)..... was also capable of metabolizing benzoate (Kumari and Mahadevan. A mutant incapable of growth on benzoate was isolated and characterized... Harwood and Gibson (1997) stated that this pathway could be divided into several phases: (1) formation of CoA thioesters. Outlines of the new aerobic pathway were deduced. Anaerobic metabolism can also occur but involves reductive saturation of the nucleus.5).

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

Title 21.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. Lueck (1980). They may also be used in certain standardized foods. and other foods in several countries (Tables 2. 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. benzene levels higher than the permissible levels for mineral waters prompted a recall in one state. and lack of color.15% and 0. 1993).1%. 1994). 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.5 or that can be brought into this range by acidification. Regulatory agencies consider it to be GRAS to the same extent that sodium benzoate is GRAS as a preservative with the limitation of 0. Similar results were reported by McNeal et al. the off-flavor they may impart to the foods they are to preserve. In most other countries.). Benzoate does not control the growth of high levels of microorganisms and therefore cannot be used to compensate for poor ingredients or processing. the narrow pH range in which they are effective. .9). Secs. bakery products. their main advantages include low price. fruit products. Subsequent studies indicated that soft drinks containing both ascorbic acid and sodium benzoate could produce very low levels of detectable benzene.1733) up to a maximum permitted level of 0. Inc.1021 and 184. (1993) when they analyzed several foods and fruit-flavored soft drinks for benzene. benzoic acid and sodium benzoate are GRAS preservatives (Code of Federal Regulations. USE IN VARIOUS FOOD SYSTEMS Benzoic acid and sodium benzoate have been widely used to preserve beverages. REGULATORY STATUS In the United States. although its solubility in water is much less than the sodium salt (Pollard. Applications of these preservatives to foods have been reviewed by Chichester and Tanner (1972). 1990). applications. 184. and limitations of preservatives and antimicrobial agents.4 g per 100 ml solution) than the sodium salt. 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). An ADI (average daily intake) value of 0 to 5 mg/kg of body weight has been specified for benzoic acid (Pollard. As food preservatives. APPLICATIONS Benzoic acid and sodium benzoate are most suitable for foods and beverages that naturally are in a pH range below 4.1 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. the maximum permissible quantities generally range between 0. including benzoic acid. Although the detectable yield was extremely low (<1 ppb). In one case (a fruit juice–mineral water combination). Approximately 1. ease of incorporation into products. They may also include foods from which benzoate is excluded in the United States. under certain conditions. the use of benzoic acid and other chemicals in the processing and freezing of shrimp has been reviewed (Chandrasekaran. 1990). 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. In addition.1%. Calcium benzoate has also been approved for use. A Food Technology special report (1986) reviewed the characteristics. Kimble (1977). 1977/1988.8 and 2.25%. and Chipley (1983. generate detectable levels of benzene. However.

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

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

Combinations of organic acids.05% sodium benzoate reduced the numbers of these pathogens compared to washing with water. The treatment that was successful included addition of 0. 1998). or S. Benzoate was one of the most effective compounds tested in the presence or absence of ethanol.. A study was conducted to evaluate aqueous dipping solutions of organic acids or salts to control L. the growth of some O157:H7 strains in apple cider was not affected by the presence of either preservative (Miller and Kaspar.. Unpasteurized apple cider has been implicated in several foodborne disease outbreaks caused by pathogenic microorganisms.5% lactic acid combined with 0. monocytogenes (Barker and Park. or 5% potassium benzoate for 120 days (Samelis et al. this antimicrobial was evaluated for control of postharvest diseases of various fruits and vegetables. coli O157:H7. Control strains of E.05% sodium benzoate followed by holding at 25°C for 6 hours and at 4°C for 24 hours.15% fumaric acid and 0. Growth was either prevented or delayed by adding sodium benzoate (0. several factors may influence the effectiveness of benzoic acid in the treatment of fresh . 2001). cheeses. Large outbreaks of infection have been linked to consumption of contaminated coleslaw. Contamination of many of these foods frequently occurs after processing. coli O157:H7 in apple cider (Comes and Beelman. Most research efforts have been directed toward E. and wash solutions were evaluated for effectiveness in decontaminating the skin (Hwang and Beuchat. 1993). 1994). 1989).3%) to cheese (pH 6. Listeria monocytogenes is recognized as a foodborne pathogen of major concern to humans. 1996. and ethanol were very effective bactericidal treatments for L. aureus.. coli failed to grow in either type of apple juice. C. monocytogenes usually attach following cooking and during slicing and packaging (Farber and Peterkin. Resistance to these preservatives was greater at 4°C than at 25°C (Fisher and Golden. Washing the skin with solutions of either 0. However. the combination of sorbate and benzoate was more effective than either preservative alone (Zhao et al. The antimicrobial can be applied directly onto the product surface where cells of L.32 Antimicrobials in Food temperature. jejuni. 2002). especially in younger children and the elderly. 1995). respectively. 1999. Dock et al. coli O157:H7 in cider because of the severity of illness this pathogen causes. and luncheon meats. monocytogenes on sliced. 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.. 2001). Chicken skin was inoculated with Salmonella spp. low pH. Application of antimicrobials as sprays or mists to meats following processing may be more effective than their addition in the meat formulation. but benzoate was about two to eight times more effective than sorbate (Splittstoesser et al. hot dogs. 1995). 2001). At 8°C.3%) to cheese (pH 6.. These solutions could be used for dips to sanitize chickens intended for frying before presentation to consumers (Hathcox et al. Ethanol-enhanced killing correlated with damage to the bacterial cytoplasmic membrane. Sorbates and benzoates are currently approved in various countries for use as dipping solutions to prevent fungal growth in dry sausages (Sofos. However. 1999). A preservative treatment was developed that was capable of achieving the FDA mandate for a 5-log reduction of E.6) or adding potassium sorbate (0. 2000). No significant increase in the numbers of this pathogen occurred on bologna slices treated with 2.3% or 0. growth was inhibited by benzoate or sorbate (Koodie and Dhople.9 with propionic acid.5% or 5% acetic acid. 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.0) made from milk acidified to pH 5. USE AS A POSTHARVEST FUNGICIDE Because of the long and successful use of benzoic acid in the processed food industry. Both preservatives reduced the heat resistance of E.. coli O157:H7 in apple juice and apple cider with or without potassium sorbate or sodium benzoate has been investigated. Tompkin et al. monocytogenes. These strains grew well in unpasteurized and pasteurized apple juice. milk.. vacuum-packaged bologna stored at 4°C for up to 120 days. 5% sodium diacetate. L. Survival of E.

1981). In the pharmaceutical industry. However.025%. respectively. 1990).. 1968). 1997). 1994). 1994). as a disinfectant for artificial kidneys (Kolmos. the cosmetic applications for benzoic acid have largely been taken over by more potent antimicrobial agents and compounds that are active over a wider pH range (Manowitz. this preservative system was ineffective against an adaptiveresistant strain. It is currently used in animal feeds and in some tobacco products as a fungicide at levels of up to 0.1%. their concentrations were high enough to inhibit growth of periodontal pathogens. textiles.Sodium Benzoate and Benzoic Acid 33 fruits and vegetables. and as a preservative in certain brands of cough syrup (Chen et al. modified starch adhesives. liquid coolants.1% to 0.5%. The chilling-induced production of ethylene in cucumbers was inhibited by the addition of sodium benzoate (Wang and Adams. used in cosmetics has been published (Bach et al. The minimum inhibitory concentrations of sodium benzoate and dichlorobenzyl alcohol for 115 strains of dental plaque microorganisms were determined (Ostergaard.. incorporated as sodium benzoate. A sodium benzoate–sorbic acid combination used in a pharmaceutical product for treating ulcers was effective against a wild strain of Pseudomonas cepacia. benzoic acid was used for oral preparations in concentrations of 0. Generally. in peanuts (Uraih and Offonry.05% to 0. However. OTHER APPLICATIONS Benzoic acid is one of the oldest preservatives in the cosmetic and pharmaceutical industries. 1989). A p-hydroxybenzoate-based system was effective in protecting the product against a variety of strains of P. A single oral rinse with a mouth rinse containing these antimicrobials did not affect plaque removal (Danielsen et al. and leather. 1996). 1996). rubber manufacturing. following official methods of analysis (Zani et al. have been used to preserve cosmetic formulations. 1988... 1976). Currently.. sealing gaskets for food containers. as a control for dentureborne Candida albicans (Lambert and Kolstad. concentrations of 0.. Patents have also been issued for the use of benzoic acid or sodium benzoate as an industrial fungicide in several products. 1980).. 1968). insoles for shoes. saliva samples from volunteers who used a dentifrice containing these antimicrobials indicated that for 5 to 10 minutes after toothbrushing. Methylcellulose was mixed with chitosan and 4% sodium benzoate or potassium sorbate to produce a food-grade film (Chen et al.. varnishes. cepacia grown under different conditions. A mixture of fruit-coating polymers and potassium sorbate or sodium benzoate completely inhibited postharvest fungal growth on bananas (Al Zaemey et al. 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. with perhaps the most pertinent consideration the pH of the superficial tissues (Eckert. A summary of the antimicrobial activities of 30 preservatives. possible areas for specialized applications still exist — for example. . A benzoic acid-based polymer coating for apples has been developed (Ivanov et al.1% and 0. lubricants. These include animal feedstuffs. especially against toxigenic strains of A. 1986). 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. 1967). flavus. Although its use in pharmaceuticals has largely been displaced by other compounds (Grundy. Benzoic acid and its derivatives have been proposed for use as fungicides. Sodium benzoate did not inhibit growth of any Gram-positive cocci. and treatment for microbial infections in trees. Hewala. 1996). adhesives for paper. Significant antifungal properties were demonstrated against food spoilage fungi. For example. 1994). including benzoic acid. wood preservatives. Sodium benzoate has also been used to inhibit postharvest changes in fruits and vegetables.

and intraperitoneal routes (Sax and Lewis.7 (White et al. 1989).10. Sax and Lewis (1989) reported data for several animal species based on the route of administration (Table 2. TOXICOLOGY Several toxicologic studies involving different animal species have been conducted using both benzoic acid and sodium benzoate. Source: Adapted from Lueck (1980). This product is not corrosive.. 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.34 Antimicrobials in Food STORAGE AND HANDLING Sodium benzoate should be stored in a cool. synthesized in the liver. both benzoic acid and sodium benzoate are moderately toxic by ingestive. In addition. . The overall reaction sequence proceeds as shown in Figure 2.4–2.0 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. 1964).. Hippuric acid. Excretion of benzoic TABLE 2. dry place. Its formation can be increased greatly by administration of benzoic acid. (1909) and Dakin (1909) to conclude that sodium benzoate was not deleterious to human health. Benzoate does not appear to be accumulated in the body.10 Toxicity of Benzoic Acid and Sodium Benzoate by Exposure Categorya Category Acute Species Tested Rat Guinea pig. Extensive human feeding trials conducted early in the twentieth century led Chittenden et al. intramuscular. 1964). However.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.b rabbit. is then excreted in the urine (White et al. 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. the second is catalyzed by an acyltransferase enzyme. Containers should be kept closed as much as possible. These have been summarized by exposure category in Table 2.11). There is little danger of its being a toxicant or fire hazard under normal conditions of use. Route of administration was per os. cat.7–4. The first reaction is catalyzed by a synthetase enzyme.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) 1.0 g/kg body weight 1.3–4.

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

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. 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. or 160 mg/kg) were given at least 1 week apart to 6 healthy male volunteers. These results have been confirmed in several animal studies. However. 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. 1991). S. Cell sensitivity to these acids was enhanced by aerobic rather than anaerobic growth conditions. 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. only a small portion (6. 1990). 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.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. Akira et al.. 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. see Chipley. Different animal species differ in their ability to metabolize benzoic acid. 1993). acid by this particular pathway was first reported by Dakin (1909). The food preservatives were shown to have a strong prooxidant effect on aerobically grown yeast cells. the route of administration significantly affected the efficiency of benzoic acid metabolism (Nathan et al. . The authors reported that both the biotransformation of benzoic acid to hippuric acid and the urinary excretion of hippuric acid followed first-order reaction rates. cerevisiae was used as a model system for testing antioxidant or prooxidant properties of benzoic acid and other weak organic acid food preservatives (Piper. 80. A single oral dose of 250 mg of benzoic acid was quantitatively metabolized to hippuric acid and excreted in the urine within 4 hours.. it was excreted almost completely as hippuric acid after systemic administration. (1994) used deuterated benzoic acid and nuclear magnetic resonance spectroscopy to monitor metabolism in a healthy male volunteer. 1999). When applied topically. 1991).7 Metabolism of benzoic acid by mammals. (1964). This may be related to differences in the ability of liver and kidney cells to carry out glycine and glucuronic acid conjugation (for review. Griffith (1929) found that this mechanism accounted for 66% to 95% of benzoic acid fed in studies in which the quantities were far in excess of those that might be ingested from foods preserved with permitted levels of benzoate. 1991). Inhibition was caused by accumulation of benzoyl CoA with a resultant depletion of free CoA and acetyl CoA.. In guinea pigs.9%) of the absorbed benzoic acid was conjugated with glycine to form hippuric acid. Source: White et al. Since the late 1970s. These authors analyzed plasma concentrations of benzoic and hippuric acids and urinary levels of hippuric acid after oral doses of sodium benzoate (40. They were also mutagenic to the yeast mitochondrial genome but only in the presence of oxygen.

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

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

The salts (e. Sofos et al. 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. Potassium sorbate may be manufactured as a powder or granules and. 1989). sorbic acid and its more water-soluble salts.g. Commercially available salts include calcium. 1989. including yeasts. The molecular weight of potassium sorbate is 150. sodium. on an equivalent weight basis with the acid. it is available as a white freeflowing powder or as granules. as animal feeds. CH3CH=CH-CH=CH-COOH). Sofos. Currently. The solubility of sorbic acid (Table 3. 1992). and .50 Antimicrobials in Food activity against microbial growth and the potential application of the compound in additional food products (Sofos and Busta. 1989.2) in water at room temperature is only 0. Sorbic acid forms colorless flakes or needles when crystallized. the pH of the solution. 1981. are collectively known as sorbates and are used widely throughout the world as preservatives for various foods. 1989). as pharmaceuticals and cosmetics. Under certain conditions. In general.2% and is insoluble in fats. The solubility of sorbic acid is also higher in alcohols. There is variation.. however. changes in the genetic material. has an antimicrobial potency of 74%. 1979a. Calcium sorbate has a water solubility of 1. while the health effects of sorbate were reexamined (Sofos. and bacteria.. 1985. molds. and it constitutes the most soluble form of sorbate (Table 3. alterations in cell membranes. and in other industrial applications (Sofos. Good solubility (58. probably through action on spore membranes or protease enzymes involved in germination (Blocher and Busta. Robach and Sofos. 1981.2). The carboxyl group of sorbic acid is highly reactive and results in formation of various salts and esters. In food systems the solubility of the compound is estimated as higher than 50%. 1980). The volatility of the compound in steam is useful in its isolation for quantitative detection in foods or other materials. or both. Sofos. During the 1970s extensive research was performed on the potential for using sorbic acid and its salts as antibotulinal agents in meat products. The sodium salt has a water solubility of 32% (wt/vol).. species and strains. and in glacial acetic acid. Robach and Sofos. Inhibition of bacterial spore germination (Sofos et al. 1989). CHEMISTRY Sorbic acid is a straight-chain. 1982).1). substrate properties. Sofos. 1986). 1986. The extensive use of sorbates as preservatives is based on their ability to inhibit or delay growth of numerous microorganisms.4-hexadienoic acid. Sofos et al. The mechanisms of antimicrobial activity of sorbates are not fully defined (Sofos and Busta. trans-trans unsaturated fatty acid (2. 1982). and it has a weak but characteristic acrid odor and acid taste.2% w/v at 20°C). depending on differences in microbial types.. sorbates are considered effective food preservatives when used under sanitary conditions and in products processed using good manufacturing practices.. 1981. which makes it valuable as a delayed-release form of sorbic acid (Sofos. 1989. some microbial strains are resistant to inhibition by sorbate or even metabolize the compound. and potassium sorbates. The calcium salt is formed as a white odorless and tasteless powder. in inhibition of microorganisms by sorbates. Inhibition of microbial metabolic function is probably the result of morphologic alterations in the structure of the cells. with a molecular weight of 112. but it increases with temperature. however. 1979d) by sorbate is believed to occur at the connecting reactions of the germination process. 1981).22. and environmental factors.15 g per 100 ml. 1989). especially in combination with reduced nitrite levels for a reduction in nitrosamine formation (Sofos et al. potassium sorbate) of sorbic acid may find more frequent applications in foods because of their greater solubility in water.13 (Table 3. and inhibition of enzymes or transport functions (Sofos. especially ethanol. 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. especially potassium sorbate. stability.

°C 760 mm Hg 50 mm Hg 10 mm Hg Purity. and other ingredients present (Sofos. Oxidation and losses of sorbic acid are inhibited by antioxidants. °C (COC ASTM D-92) Ionization constant at 25°C Density at 20°C g/ml Melting range.8 ml 0. 1981).4-Hexadienoic acid potassium salt CH3-CH=CH-CH=CH=COOK 150. Sorbic Acid 2. The rate of oxidation in aqueous solutions is increased at lower pH values by the presence of light and acids or increased temperatures.. The partition quotient is increased from 3. In general. maximum ppm Arsenic content.13 126 1. Ethylenediamine tetraacetic acid (EDTA) enhances sorbate degradation probably as a result of iron scavenging from the packaging material (glass or polypropylene) through EDTA complexation.22 — — 1. . increasing the production of carbonyls. 1988. It is proposed that EDTA-Fe2+ complexes catalyze sorbic acid autoxidation. maximum% Source: From Sofos (1989). whereas in the pure dry form they are stable. 1989). and anaerobic conditions (Sofos. the type and amount of fat. 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. such as crotonaldehyde. malonaldehyde. 1992).4-Hexadienoic acid CH3-CH=CH-CH=CH-COOH 112. acrolein. Thus. Sofos. Btu/Lb Alkalinity/acidity Vapor pressure. and β-carboxylactolein (Arya and Thakur.001 10 43 Decomposes 143 119 >98 0. 1995).1 Physical and Chemical Properties of Sorbic Acid and Potassium Sorbate Property Chemical name Molecular formula Molecular weight Flash point. 1992).36 Decomposes higher than 270°C — ml 0.1 N HCL per 1. 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. °C Heat of combustion at 25°C.2 ease of manufacture make potassium sorbate the most widely used form in food systems (Sofos.1 N NaOH to 0.73 ∞ 10-5 — 132–137 11. 1996).0 10 3 — <0. malonic acid.Sorbic Acid and Sorbates 51 TABLE 3.5 10 3 0.0 to 7. maximum% Ash. mm Hg.927 — Potassium Sorbate 2.1 g — — — — — — >98 1. such as sugars and salts (Sofos and Busta.. appropriate packaging materials. acetaldehyde. 1989. maximum% Heavy metal content. 1989). which take part in nonenzymatic browning. formic acid. Oxidation of sorbic acid yields various carbonyl compounds. % Water content.0 with increased levels (50%) of soluble food components. at 20°C 120°C 140°C Boiling point. the partitioning of sorbic acid between the water and fat phases of foods depends on the pH of the food. Aqueous solutions of sorbates are unstable and degrade through oxidation.

50% Phosphoric acid.15 0.. 20°C Cyclohexane. 20°C Glycerol.4) Water.01 Losses of sorbic acid during storage of foods depend on sorbate levels.16 0.07 0.52 1. 40% solution Sucrose. 20°C (pH 3. 75°C Benzene.00 20. 1994). Sofos.00 28. 1987. 20°C (pH 5.80 2.00–15.02 0. metal ions.30 Potassium Sorbate 58.60–14.00–5.25 0.00 — <0. 50°C Propylene glycol.26 0.55 4.11 0.9) Water.00 34.20 — — 61.01 0. 5% solution Sodium chloride. 20°C Carbon tetrachloride. 1980.14 24.50–12. In general.. 20°C Acetone. 1983.00 5.29 — 12.90–14. 15% solution Acetic acid.60 1. other additives present. 10% solution Sucrose. and storage temperature and time (Bolin et al.30 0.02 0.00 55.00 64. oxidative degradation of . 1992.28 1. 1990).08 0.00 47. 25°C Benzene.00 — — — — 57.52 Antimicrobials in Food TABLE 3.10 16.00 12. the nature and pH of the food.90 0.05 5. 20°C Cottonseed oil. 20°C Methanol.00 45. but at 13. 20°C Corn oil. 5% Ethanol.34 8.5% Citric acid.00 48..5% and 8.01 — — 0. 50°C Benzene. packaging material. 100°C Pentane. 50% Ethanol. glacial Lactic acid.0% it had a protective effect (Guerrero et al. 85. 20°C Propylene glycol.01 58. antioxidants.20 0.1) Water. 100°C Corn oil.20 12. 20°C Source: From Sofos (1989). 100% Propylene glycol. 85% Ethanol. 25°C Pentane. 1986a. 20°C (pH 4.b. 1984. Vidyasagar and Arya.00 — — — <0. Sodium chloride at 3.00 0.00 0.50 0.22 1.30 6.08 2.04 11.12 0.31 9. 95% Ethanol. light. amino acids.60 45.30 2.00 0.15 0. Sorbic Acid 0. 50°C Soybean oil. processing conditions. 75°C Ethyl ether.50 2.00 0.15 0. 20% Ethanol. 50°C Water. 50°C Pentane.10 0.10 0.80 0. 20°C Sucrose.50 12. moisture content.40 54. 60% solution Sodium chloride.03 — 0. 1989. types of food materials present.0% enhanced sorbic acid destruction. Gerschenson et al. 10% solution Sodium chloride.2 Solubility (%) of Sorbic Acid and Potassium Sorbate Solvent Water.

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

90). 1984. 1983. 1952. Rosellinia. 1985a). Numerous patents describing catalysts. A major application of sorbates in food products is their use for inhibition of molds in cheeses (Chichester and Tanner. Bracey et al. Although. 1985. Liewen and Marth. applied at 10 mg/ml. Candida. 1993. Hansenula. which is most widely used. Monilia. Baldock et al. 1996). Debaryomyces. Pederson et al. 1985a. Endomycopsis.54 Antimicrobials in Food 2. 1972). 1984. and other suitable salts in the presence of necessary additives.30%. 1999. Hurdles used in combination included aw.b. 1954a. Sporotrichum. 1979. Rhizoctonia. Kivanç. and sorbic acid.. Kloeckera. Phoma. Pullularia. sausages. Aly. use of a hurdle approach resulted in a synergistic effect (El Halouat and Debevere. 1989). and chocolate syrup (Restaino et al. Colletotrichum. The inhibitory action of sorbate against yeasts was first documented in the 1950s in fermented vegetable products. Mold species inhibited by sorbates belong to the genera Alternaria. Garza et al. the inhibitory amounts of sorbic acid were reduced by 40% to 50%. Gourama and Bullerman.4-hexadienal and the condensation reaction of ketene and crotonaldehyde. Ferguson and Powrie.. such as carbonated beverages. potassium sorbate used at 150 ppm resulted in final counts. Zygosaccharomyces rouxii is considered resistant to sorbate treatments. Huang and Armstrong. Melnick et al. 1993. Saccharomyces. jellies. cakes. 1998. and meat and fish products. 1999). Curvularia. Marshall and Bullerman. reaction conditions. 1970. 1984. Under this atmosphere and at aw values in the studied range (0. Humicola. and others. cottage cheese. Sporobolomyces.. Chaetomium. Melnick and Luckmann.b. 1957. Torulaspora. and smoked fish (Liewen and Marth.80 to 0. Kaul et al. CO2 atmospheres. wines. sorbates inhibit yeasts in fruit juices. Matamoras-León. 1984. grains.b. 1985. Skirdal and Eklund. neither sorbate nor propionate. El Halouat et al. 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. Trichoderma. 1952. 2000).. as well as against many bacteria (Sofos. lower than the inoculum level (103 CFU/g). fruits and juices. Pichia. Huang and Armstrong. Penicillium.. Under an 80% CO2 atmosphere. syrups. 1984. 1959. jams.. Pepularia.b.. whereas no growth was observed when the preservative concentration was increased to 220 ppm (El Halouat and Debevere. Geminder. Smith and Rollin. Roland and Beuchat. Tsai et al. Use of sorbates for inhibition of yeasts is especially important in low-pH and/or intermediate water activity (aw) products. Cephalosporium. Ulocladium. 1998). were able to depress mycelial weight and aflatoxin production by Aspergillus flavus or Aspergillus parasiticus cultured in yeast extract-sucrose (YES) broth (Fan . Alkaline salts of sorbic acid are formed by its neutralization with alkali metal carbonates. 1992. The effectiveness of sorbates against yeasts has been documented by numerous studies (Emard and Vaughn. Bhattacharya and Majumdar. Lennox and McElory. Helminthosporium. 1983. Heterosporium. Deuel et al. Sorbates also inhibit molds in butter. Sofos. Effective antimicrobial concentrations of sorbates in most foods are in the range of 0. Sorbates inhibit the formation of mycotoxins by various molds in culture media and in foods (Bullerman. Mucor. 1989). 1954a. ANTIMICROBIAL ACTIVITY INHIBITION Sorbic acid is active against yeasts and molds. Cladosporium. Cunninghamella. Botrytis. 1961. Fusarium. Cryptococccus. hydroxides. In addition to their effectiveness in fermented vegetables. 1988). after 21 days. Ascosphaera. and purification procedures follow the second method.. Tong and Draughon. Torulopsis. 1986. 1979.02% to 0. Rhizopus. Rhodotorula. salad dressings. Numerous studies have also documented the effectiveness of sorbates against molds (Emard and Vaughn. However. 1970. Liewen and Marth. Fan and Chen. 1998. Gliocladium. 1984. Aspergillus. Pestalotiopsis. Truncatella. Yeasts inhibited by sorbates include species of the genera Brettanomyces. Piper et al. and Zygosaccharomyces (Sofos. Ascochyta. dried fruits. 1954a.. candy. 1996). tomato products. Geotrichum.. bread.. 1954a. 1982.

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

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

This metabolism is mostly associated with lactic acid-producing bacterial strains present as high inocula in sublethal concentrations of sorbate (Crowell and Guymon. 1992).2 min. under normal conditions of alimentation.. of which 50% is biologically usable. Sofos. A study found a pronounced positive inoculum effect of Z.5-diene (Edinger and Splittstoesser. 1954b. however. In general. Mold strains of the genus Penicillium isolated from cheese treated with sorbate were able to grow and metabolize high (0.. or hydrocarbon-like odor (Marth et al. that there is no apparent relationship between sorbate resistance and the toxigenic properties of molds (Tsai et al. Steels et al. a geranium-like odor is usually associated with wines treated with sorbate and contaminated with high microbial loads. sorbate yields 6. level of inoculum. 1981. In general. Other strains of molds that may degrade sorbate belong to the genera Aspergillus.b). there is also evidence of some ω-oxidation (Deuel et al. It appears. and environmental factors. prior exposure to subinhibitory levels of sorbate. under certain conditions. about a 7-fold increase in lethality (Splittstoesser et al..Sorbic Acid and Sorbates 57 DEGRADATION Animals and certain microorganisms can metabolize sorbate. processing. microbial flora. bailii resistance to sorbic acid activity. These factors can act synergistically or be antagonistic and either enhance or negate the antimicrobial activity of sorbate (Sofos.. inoculum size.1% sorbate is usually sufficient to inhibit sensitive molds (Liewen and Marth. 1977. certain strains are resistant and can metabolize the compound. coli O157:H7 from 36 to 5. 1966. plastic paint. INTERACTIONS The antimicrobial activity of sorbates is influenced by compositional. Because it is metabolized like other fatty acids. Increasing the concentration of sorbic acid from 200 to 1000 mg/L in apple cider decreased the D50°C value of E. 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. Some mold strains can grow and metabolize sorbate under certain conditions as detected in cheeses and fruit products (Melnick et al. 4hexenoic acid. Liewen and Marth. gas atmosphere. Products of sorbate metabolism by molds include 1.18% to 1. It appears that selection may occur in sorbate-treated cheeses for certain molds tolerant to the compound (Schroeder and Bullerman. Mucor. 1985a).4-diene. The study showed that the resistance of Z.. coli O157:H7 during storage . using it as a carbon source. 1988). 1985a–c). Sofos. 2000). 1995). which was not an artifact caused by insufficient growth time. 1989. such as concentration.20%) sorbate levels (Marth et al.3-pentadiene. 1989). Degradation of sorbate by lactic acid bacteria has been associated with geranium-type off-odors in wines and fermented vegetables. large inocula at high cell concentrations therefore require considerably higher concentrations of the inhibitor to prevent growth than do dilute cell suspensions. 1954b. amount of sorbate present. It should be noted that 0.b. caused by ethyl sorbate.. 1966. 1989). The MIC of sorbic acid increases with the size of the inoculum.. 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. Finol et al. 1985a. some bacterial strains may also degrade sorbate under appropriate conditions. In addition to certain molds. Fusarium. and type of substrate (Sofos. 1989).6 kcal/g. Sofos. Lück.. Horwood et al. 2000).1%) reduced the D50°C of E. 1-ethoxyhexa-2. Degradation of sorbate by molds depends on species and strains. 1989. 1989). 1986a.. 1985). which is a volatile compound formed through a decarboxylation reaction and has a kerosene-like. and Geotrichum (Sofos. 2000). Bullerman. temperature. dehydration of cultures. 1982). aw. although many molds are sensitive to inhibition by sorbate. When sorbate levels are high. Sorbic acid (0. 1980). 1981.. sorbate is completely oxidized to carbon dioxide and water. Like caproic and butyric acids. as a fatty acid through β-oxidation.. or binding of sorbic acid to dead cells. other ingredients. and other additives (Sofos and Busta. pH. 1975. packaging. substantial metabolism of sorbic acid. Liewen and Marth. and 2-ethoxyhexa-3.

. 1976. 1983). respectively (Sofos and Busta.64 min (Splittstoesser et al... Not only are certain strains of microorganisms resistant to inhibition by sorbate. Gooding et al.25 and 2. Chung and Lee. Skirdal and Eklund. Other food ingredients (e.1%) enhanced destruction of E. Lück. Sheneman and Costilow.0 to 5. Nevertheless. whereas benzoic acid reduced it to 0.. 1976. Studies have indicated that the dissociated sorbic acid also had antimicrobial activity. might not be effective owing to their increased solubility in fat.. 1955....0 to 4. 1981a–c. 1954a. 1980. sorbates have the advantage of being effective at pH values as high as 6. sorbates should be used to preserve foods processed using good manufacturing practices. 1989). Increased amounts of fat in a product reduce the concentration of sorbate in the water phase. Cerruti et al.0. as well as dormancy and recovery of heated microorganisms (Sofos. 1993).. 1985a).1% sorbate (Bills et al. glucose. 1982. 1979a. This popular theory has been questioned. Lund et al.. Robach and Stateler. 1981). but increased levels of microbial contamination reduce antimicrobial activity.0 (Raevuori.58 Antimicrobials in Food of apple juice from 18 to 5. Liewen and Marth. not as a substitute for appropriate sanitation and hygienic practices. 1981c). 1981. which exhibited favorable antimicrobial properties against Vibrio vulnificus. Cerruti et al. approaching its dissociation constant (pKa = 4. where it is needed for microbial control (Oka. 1993). however. 1955... Acott et al. however. such as parabens. Although activity is greater at low pH values. 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.5%) reduced the inhibition of Clostridium botulinum by sorbate in a nutrient broth (Wagner and Busta.. 1955.5 for propionate and benzoate. 1985a.. Sucrose. 1987. Thus. 1944. 1941.5 and 4. 1976. 1988). act synergistically to enhance the antimicrobial activity of sorbate (Costilow et al. Beuchat. 1993). Cerruti et al. 1996).76) (Cowles. 1984.g. Sugar and salt. Sofos and Busta. Sofos et al. 1980. Sorbic acid (0. the maximum pH for antimicrobial activity by most other common food preservatives is lower — for example. 1944. Lowering the aw enhanced the resistance of the same organism to increasing concentrations of sorbate (Restaino et al. however. which is believed to be the effective antimicrobial form (Lück. 1990).4 but not at pH 6. 1955. certain studies have indicated antimicrobial activity by sorbate at pH values as high as 7.1% sorbate rendered the cells more sensitive to inhibition by sorbate than preconditioning in 0% sucrose + 0. 1989. supporting previous evidence that sorbic acid is effective in its undissociated form (Sun and Oliver. salt and sugars) also reduce the concentration of sorbate in the aqueous phase (Gooding et al. 1959. In certain instances. 1982). however. solutes should increase the inhibitory activity of sorbate by reducing the aw of the substrate (Sofos. 1981. 1956. Preconditioning of Saccharomyces rouxii cells in 60% sucrose + 0. and it may inhibit the repair and growth of . Statham and McMeekin. 1982.5 (Bell et al. 1994). 5. 1988). The increased activity of sorbates at pH values higher than 5. Sofos and Busta. Sofos and Busta. more than 50% of the inhibition was the result of dissociated sorbic acid (Eklund. The increased antimicrobial activity of sorbate at lower pH values has been attributed to the increased amount of undissociated acid present. but it was 10 to 600 times less inhibitory than the dissociated acid (Eklund.. 1957. In environments of pH higher than 6. and sodium chloride. 1983. Sodium chloride (1. Interactions of sorbate with heat may affect the rate and extent of microbial destruction during heating. Use of artificial saltwater prevented dissociation of the 1% sorbic acid. coli O157:H7 cells at pH 3. Sofos and Busta.4 (Liu et al. have reduced the synergistic effect of sorbate and heat on thermal inactivation of microorganisms (Beuchat. Lück. Sorbate may enhance heat activation and destruction of spores. Rahn and Conn. 1980. In general. In contrast. 1989). 1990). Sofos. Hoffman et al. 1989. 1960). 1976.b). 1997). Statham and McMeekin. 1981. it is believed that both undissociated and dissociated sorbic acids have antimicrobial activity (Skirdal and Eklund. 1981). The antimicrobial action of sorbate is pH dependent and increases as the pH of the substrate decreases. 1983).2 minutes. 1988).5 is advantageous because it allows for their use in foods of higher pH values in which preservatives.. however (Sofos. 1980.. 1981.

and particularly in the presence of sucrose as opposed to glucose. Sofos. 1985). Park and Marth. a true aerophilic mold involved in the spoilage of grape preserves (Splittstoesser et al.. and propyl gallate (PG). Sorbate may also eliminate the protective effect of sucrose against the thermal inactivation of yeasts and molds (Sofos.. 1982. 1982. Splittstoesser et al. Heat resistance increased substantially when cells were grown in media containing sorbate. however. 1989). In addition. Sorbic acid was found to induce thermotolerance without inducing the heat shock response through accumulation of trehalose in S.. Inhibition of microbial growth by sorbate is more effective as storage temperature decreases. however. 1982.. 1982. Sorbic acid (0. but at pH 5. 1989). Myers et al. 1982. Tolerance of Z. 1984).5 it acted as a powerful inducer of thermotolerance in the absence of sublethal heat treatment.. Reductions of 5 to 10 log-units of E. 1980. At low pH. but growth of surviving spores that had been exposed to high temperatures was greatly inhibited by sorbate concentrations as low as 0. McMeekin et al. they can enhance its antimicrobial activity by increasing the concentration of undissociated sorbic acid.1% had little effect on the thermal resistance of ascospores of Neosartorya fischeri. 1996). Oloyede et al. 1994.. 1970. Lopez et al. including meat (Wagner et al. 1981. McMeekin et al. The antimicrobial activity of sorbate is usually enhanced under vacuum or modified gas atmosphere storage conditions.. cerevisiae (Cheng et al. 1981. but the effect of sorbate on thermal inactivation and recovery of injured microorganisms is variable among species and strains. microorganisms.. 1989). sorbate inhibited induction of thermotolerance by sublethal heat shock. Although food acids may reduce the water solubility of opposed to glucose-containing media provides evidence that cell morphology influences heat resistance (Golden and Beuchat. Combinations of sorbate with antioxidants. 1996). Roberts et al.. 1994..Sorbic Acid and Sorbates 59 thermally injured organisms (Beuchat.. Specific effects.2% to 0. 1961. did not enhance the bactericidal activity of cellulose-based edible films against Salmonella Montevideo (Zhuang et al. Elliot et al. Roland et al. Roland and Beuchat. The finding that plasmolysis increased in cells grown in sucrose. 1984.4%). It is of particular interest that 0. 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. Combinations of carbon dioxide and sorbate have also been reported as effective inhibitors of microbial growth (Danzinger et al.. 1972.. Huhtanen and Feinberg. 2000). 1983. Banks et al. 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. Another report also indicated that potassium sorbate inhibited the heat-resistant N. 1989). 1995).1% potassium sorbate did not have any significant effects on the heat resistance of Bacillus stearothermophilus spores in distilled water. 1996.007% (Splittstoesser and Churey. 1980. such as butylated hydroxyanisole (BHA).. rouxii to sorbate was enhanced in cells preconditioned to elevated sorbate concentrations. 1999). 1979.. 1983). 1976. Sorbic and benzoic acids affected the thermotolerance and heat shock response of S. 1973. Davidson .. cerevisiae depending on pH (Cheng and Piper. 1984) and fish products (Bremmer and Statham.. 1999).. 1992). tertiary butyl hydroquinone (TBHQ).1% sorbate. 1989). 1980. Low concentrations of sorbic and fumaric acids in the heating medium had little effect on the heat resistance of Eurotium herbariorum. 1984. Elliot and Gray. the specific anion itself may contribute antimicrobial activity (Juven. especially in media containing 0.. 1983.. 1984. coli O157:H7 or Salmonella typhimurium DT104 in apple cider were achieved through freeze-thaw treatments in the presence of sorbic acid (Uljas and Ingham. 1988. indicating that the compound should be more useful as a preservative in refrigerated foods (Pederson et al. 1984. Huhtanen et al. and types of acids (Restaino et al.. Cells grown in sucrose-supplemented media tolerated higher concentrations of sorbate than did those grown in glucose-supplemented media. vary with substrates. Sofos. 1981a–d. Lusher et al.. 1981. as indicated with several food items. butylated hydroxytoluene (BHT). Concentrations of sorbate as high as 0.. Robach. had increased antimicrobial activity compared with individual components (Klindworth et al. Park et al. Gray et al. 1985. fischeri (Nielsen et al.. Tuncan and Martin. 1985). Statham et al. 1989)..

.. Interactions and increased antimicrobial effects have also been observed in combinations of sorbates with propionate. At <25°C. Thomas et al.06% potassium sorbate. cereus. 1984. Bacillus coagulans. Gourama and Bullerman. aureus.. 1968. or at 10°C (Deak and Beuchat.. equivalent to a total sorbic acid concentration of 1300 mg/L. Numerous publications and patents describe interactions of sorbate with many other factors.98. 1995). verocytotoxigenic Escherichia coli. 1990)..93. Wagner and Busta. sorbic acid resulted in marked inhibition. except nitrite when used against S.. 1982. Marshall and Bullerman. 1993. . 1981. Mendonca et al. The ability of C. Several studies have also indicated increased antimicrobial effects when sorbate was combined with various phosphates (Ivey and Robach. 1999). The effect of temperature. 1985. sulfur dioxide. 400 ppm sodium bisulfite.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. 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. antioxidants. 1986. As indicated.. In addition. bailii is highly resistant to individual inhibitory factors.97 and pH to 3.5.. with types of microorganisms. 1960. molds. and S. pH 3. 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. 1994). 1960. 1999). and mild heat (Guerrero et al. 1994). 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. 1990). 1984. 1988). Combinations of sorbate with benzoate or propionate may be used to expand the range of microorganisms inhibited with reduced concentrations of each preservative. However. 1985). as well as multifactorial interactions (Sofos.. Morad et al. These combinations offer the advantage of simultaneous inhibition of microbial growth and development of rancidity. propylene glycol. Guerrrero et al. fatty acids. 1982. 1986a..4 and addition of 250 ppm ascorbic acid. Miller and Brown.60 Antimicrobials in Food et al. Clostridium pasteurianum. 1993). aureus) using gradient gel plates (Thomas et al. coli at all temperature/pH/NaCl combinations and was the most effective preservative tested against B. aureus when used with higher concentrations of NaCl (Thomas et al. 1988). Kabara.06%) combined with aw values lower than 0. 1983. TellezGiron et al.. Variations existed. At temperatures of 12°C and below.. and substrates. the log probability of growth of a single bacterium at 12°C after 60 d at pH 5. Sorbate was completely effective against E. the probability of growth was approximately –6 (Lund et al. and potassium sorbate was evaluated against growth of three foodborne bacterial pathogens (Bacillus cereus. sucrose fatty acid esters. Kadam et al. Robach. glucose oxidase. Nelson et al. Deak and Beuchat. Lahellec et al. pH. sorbate was more effective than benzoate against S. 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... Ough and Ingraham. Seward et al. The microbial stability of shelf-stable banana puree was enhanced through inhibition of inoculated osmophilic and nonosmophilic yeasts. 1983. 1993). 1981. in a medium containing 0.. certain amino acids. Amano et al. however.. 1981. Poerschke and Cunningham... at aw of 0. Bell and De Lacy.. A study found that growth was not inhibited at pH 3.8. and aw 0. the log probability of growth of a single vegetative cell is approximately –4. these concentrations of chemicals represented a greater than 50% reduction of the amounts needed to suppress growth when used individually (Matamoras-León et al. 1957. 1979b. Sofos. Robach et al. 1989. and other compounds (Ferguson and Powrie.95 (Deak and Beuchat. 1994). Thomas and Wagner. 100 ppm potassium sorbate. sodium chloride concentration. 1987. After 14 days at 30°C in the presence of 280 mg undissociated sorbic acid/L. 1978. Z. 1994. Increase in the acidity and/or the NaCl concentration improved the effect of all the preservatives. 1994). and Clostridium butyricum by adjustment of aw to 0. including sorbate. 1989). ascorbate. mixtures of sorbate with various antibiotics have demonstrated increased antimicrobial activity (Schmidt. Potassium sorbate was found more inhibitory against yeasts than hydroxycinnamic acid (Stead.

05–0. pharmaceuticals. APPLICATIONS Sorbates are widely used food preservatives throughout the world. cake mixes. but levels as low as 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. Although these concentrations have no major impact on food quality.20 0.10 0. jams. Part 133 (2001)]. sanitation and contamination. However. carbonated and noncarbonated beverages. cheese spreads. objectives to be accomplished. 1992). olives. Amounts of sorbate used in foods are in the range of 0. product processing.03 0. processed cheeses. 1989). cottage cheese. semimoist pet foods. Selection of the most appropriate method depends on processing procedures.03–0. 1989). and convenience (Sofos.3%. icings.02–0. These factors include product pH and composition (fat and moisture). fruit drinks.10 0. cheese dips. . higher levels may cause undesirable changes in the taste of most foods. cosmetic products. and sugar and confectionery items (Sofos. 1989) (Table 3.30 0. Levels (%) 0. doughnuts Vegetable products: fermented. 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. DAIRY PRODUCTS Cheese preservation is one of the most common applications of sorbates. 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.02–0. margarine. spraying or immersing the food material in a solution. mixes. cheese spreads. sour cream. and cheese food [21 C. fruit and vegetable products. sorbates have found wide application in various foods.05–0. type of food. edible fat emulsion products. packaging materials.1% may be detectable in some foods. dry sausages Miscellaneous: dry sausage casings. dusting with a powder. They should be considered as adjuncts to proper sanitation and hygiene.02% to 0. equipment available. animal feeds. 1981.Sorbic Acid and Sorbates 61 TABLE 3. purees.05–0. salad dressings Meat and fish products: smoked and salted fish. 1989.02–0. In the United States. jellies. As yeast and mold inhibitors. The most commonly used forms include sorbic acid and the potassium salt.F. amounts of 0. as is the case with all food preservatives. Sofos. gas atmosphere. In general.30 In general. fillings. fruit salads. pickles. toppings. juices. federal standards of identity permit the use of sorbates as preservatives in more than 40 types of cheese. fudges. certain meat and fish products.05–0. Application methods for sorbates include direct addition in the formulation.1% to 0.R. relishes. The compounds find application in human foods of all types. and storage temperature (Sofos and Busta. presence of other ingredients and preservatives. syrups.3 Common Applications of Sorbates as Antimicrobial Agents Products Dairy products: aged cheeses. In the United States. concentrates Beverages: still wines. confectionary Source: From Sofos (1989).4).3). low-calorie drinks Food emulsions: mayonnaise. or addition in a coating or packaging material (Table 3. packaging. and technical preparations that come in contact with foods or the human body. yogurt Bakery products: cakes.3% are tolerated. pies. including dairy products.25 0. fresh salads Fruit products: dried fruit.30 0. bakery items.

drinks Still wines Other: Margarine Semimoist pet food Bakery: pound cake Devil’s food cake Chocolate cake Solution (%) 20–40 2. depending on the technique used to treat the cheese (i. 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.15 0.1 0. Kivanç (1992) found that Penicillium was the major genus found on cheddar cheese. 1980. 1976.025–0.62 Antimicrobials in Food TABLE 3.1 0.075–0. blue.3 0. and distribution.1–0.1–0.04 0. Caciocarallo.2 0.05–0.5–5 5 Application Level (%) 0. It is obvious. Muenster.1 0. however (e.3 0. aging. 1981). fillings.05–0. Sorbate application. olives. The method of application varies with the type of cheese. and domestic and imported cheeses.3 0. preservation objectives.05–0.4 0.05–0.02–0. Monterey Jack. that sorbates should not be used in mold-ripened cheeses. such as Roquefort or blue cheese and Camembert cheese. Siciliano.05–0. dipping or in brine salting).e.15 0. slaw. Emmentaler Dairy: cheddar. however. however.. toppings.3 0. pickles.05–0.5–5 5 2. Gouda Dry sausage or casings Dairy: Swiss. sauerkraut) Fruits: syrups.3 Spraying Immersion or spraying Dry mixing Direct addition Dry mixing or direct addition Source: From Sofos (1989). fudges Vegetables: fresh salads (potato.3 0.05–0. Colby.1 0. and regulations. available facilities and equipment. 1996). In certain products.02–0.05–0. in which they can interfere with the desirable molds involved in the manufacture of these cheeses.1–0.3 0.1–0. is limited to the surface of cheese.03–0.06 0. beverages. Sorbates are widely used in preserving cheese owing to the susceptibility of all types of cheese to microbial deterioration.1 0.5–10 20–40 20–40 2.1–0. vegetable. Swiss cheese. Addition of sorbate does not significantly affect the organoleptic properties of mozzarella cheese. the high activity of sorbates against molds. spreads Cottage cheese Bakery: icings.15 0.05–0. 1980). 1976. cottage cheese). pasta filata.3 0.05–0.1–0.1 0. In these applications sorbates not only prevent cheese spoilage by mold.25 0.05–0. sorbic acid may be incorporated into the cheese curd. and the improved action of sorbates at high pH values compared to other common preservatives (Lück.4 Methods of Application of Sorbates Methods Immersion Products Dairy: provolone. Sofos and Busta. in general.1 0.06 0. For application on .075 0.075 0. especially surface mold growth during storage. but they also protect human health by preventing the formation of toxic mold metabolites (mycotoxins). where mold development usually occurs.01–0. Gruyere.g.1–0. which may vary among countries (Lück. relishes. Edam.. there may develop a slight objectionable flavor (Aly.

whereas when applied to packaging films.. use of sorbates in aged cheeses also reduces labor and other costs through reduction of the need for cheese washing and trimming. 1995). (2) a sorbic acid powder for dusting. when applied to the surface of hard. at the relatively high level of 1%. such as cottage cheese and yogurt (Mihyar et al. 1996). 1980. The compound is insoluble in fat and only slightly soluble in water. ripened cheeses. Amounts of sorbic acid permitted and applied in cheese preservation range from 0. Levels of 0.3 g/dm2. Calcium sorbate may be preferred in cheese preservation over other forms of sorbate owing to its solubility properties. 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. 1997). In addition to reducing cheese spoilage and the possibility of aflatoxin formation. Weng and Chen. Furthermore. In labaneh (a yogurt product) produced under strict hygienic processing measures and effectively chilled. one of the following procedures may be followed: (1) potassium sorbate in solution or in a brine.07% sorbic acid are generally used in direct addition to cheese. bulgaricus. or wash.3%) or sodium benzoate (0. potassium sorbate (0. The use of sorbates in the processing of vegetables was one of the first applications. 1997).. Labaneh with higher initial yeast counts needed higher concentrations of preservative (>300 and >400 mg/kg for K-sorbate and Na-benzoate. wax) used in cheese storage and marketing (Han et al. however. Growth of Salmonella in soft cheese was inhibited if the cheese was prepared from milk that was acidified with propionic acid to pH 5.3%. or calcium sorbate impregnated into the packaging material or other coatings (e. thermophilus and Lactobacillus delbrueckii var. Inclusion of sorbic acid in cottage cheese was effective as a preservative but not against L.g. respectively (Aly. 15°C. which can be used to dip. and pickled vegetables (Sofos. In another study. including fresh.0 with hydrochloric. coli O157:H7 in queso fresco. 1996). Treatment of mozzarella cheese with potassium sorbate in the kneading water or brine may be more effective than dipping (Aly. 1995). acetic. 1989. 1976). Sofos. soft cheese (Kasrazadeh and Genigeorgis. 1981). and (3) sorbic acid.1 to 0.30% (Lück. fermented. addition of potassium sorbate may increase the meltability and improve the fat leakage of certain cheeses (Aly. especially at lower (≤6.9. The appearance of Penicillium verrucosum var. Milk acidification to pH 6. VEGETABLE PRODUCTS The water-soluble salts of sorbic acid are used widely in the preservation of a variety of plant products. potassium. spray.Sorbic Acid and Sorbates 63 the surface of the cheese. 1992). and it was initiated through extensive research performed during the early stages of sorbate testing.. potassium sorbate inhibited L. Addition of potassium sorbate (≤6%) to mozzarella cheese inhibited growth of Streptococcus salivarius var.05% to 0. Sofos and Busta. 1989). and the cheese was stored at ≤30°C (Kasrazadeh and Genigeorgis. and was especially effective against mold and yeast contamination by Penicillium roqueforti and Mucor miehi. respectively) extended the shelf life by 14 days.0) at levels of ≥0. These concentrations are in agreement with use of these preservatives in other dairy products. potassium sorbate was added to the cheese (pH 6. and 25°C after 30 days (Kivanç. This research demonstrated the effectiveness of sorbates against undesirable organisms without interfering with the desirable . 1996). 1980).6°C) temperatures (Kasrazadeh and Genigeorgis. monocytogenes (Piccinin and Shelef. 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. Therefore. Concentrations applied to cheese surfaces range from 0. 1994). 1994).05% to 0. 1999). monocytogenes survival in two commercial cheese brines (Larson et al. or propionic acid significantly increased the effectiveness of potassium sorbate. 1998. amounts of 2 to 4 g/m2 are used (Lück.3%) showed a significant effect in delaying growth of E. initial numbers of yeasts were low and relatively low concentrations of potassium sorbate and sodium benzoate (<150 and <250 mg/kg. respectively) to maintain stability when kept at 5°C for 7 or 14 days. 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..

or bacteria.02% to 0.1% sodium benzoate and 0. Other vegetable products preserved with sorbates include tomato products and fermented Asian sauces (Chichester and Tanner... Use of sorbates at levels between 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. Levels of 0. such as yeasts. raisins. Depending on the particular product. prunes. jams and jellies). Sofos. 1994).02% sorbic acid in combination with . Potassium sorbate is preferred because of its solubility. 1980).g.. 1955. Sorbate levels in the range of 0. In wine processing. In these products.64 Antimicrobials in Food fermentations.10% sorbate reduce the number of bloaters and poor seed cavities. 1989).g.g. 1980). Other fruit products that may be preserved with sorbates include maraschino cherries and strawberry puree.10% are adequate in improving the preservation of soft drinks. Lück.02% to 0..10% may be used to preserve refrigerated fresh salads.. molds. Because potassium may result in potassium bitartrate precipitation in some wines. The lower the moisture content of the product (e. fermentation). however.05% to 0.g. Addition of 0. jellies.5% acetic acid completely inhibit microbial spoilage at sucrose concentrations ranging from 2% to 40%. Another study showed that E. cucumbers and olives). Also. sorbates are used mostly at the preprocessing stage. Potassium sorbate was found more effective than chitosan in inhibiting growth of Aspergillus niger on lowsugar candied “kumquat” (Fang et al.20% inhibits growth of organisms not desired in vegetable fermentations. Selection of precise levels of sorbate. 1972.b). together with sulfur dioxide and pasteurization. Also. Sorbate concentrations as low as 0. jams. and putrefactive bacteria. FRUIT PRODUCTS Fruit products preserved with sorbates throughout the world include dried fruits. enzymatic. coli O157:H7 growth was inhibited in both tryptic soy broth and in apple cider by sorbic acid applied at 0.. sorbates are used to prevent refermentation (Parish and Carroll. Combinations of sorbic acid and sulfur dioxide are also used in the preservation of high-pulp fruit juices.02% to 0.1% sorbate and 0. 1980).05% sorbate to a 15% to 20% brine retards yeast scum formation. Good wine preservation is achieved with concentrations of 0.02% to 0. dried fruits). the lower is the sorbate level needed for preservation. 1952.05% and as such may be an option that processors have to reduce contamination in their products (Koodie and Dhople. salt concentration). depends on products and specific fermentation conditions (e. in high-sugar products (e. or microbiological deterioration (e. Lück. 1993). sorbate acts as an inhibitor of yeasts. 1980. beverages. At these concentrations the selective action of sorbic acid allows growth of the desirable lactic acidproducing organisms. coli O157:H7 in apple cider. In sweet cucumber packs.. which may influence the survival of E. 2001). smaller quantities of sorbic acid are adequate for preservation because of the synergistic action of sorbate with sugar (Lück.05% sorbates in the fermenting and holding tanks. The higher moisture content in these products is preferred by the consumers.05% and 0. Costilow et al.g.. Mold and yeast spoilage is also prevented by sorbates in pickled vegetable products (e.1% potassium sorbate substantially inhibited E. fruit cocktails. the pH values of fermented green olives favor the use of 0. but it makes them susceptible to mold and yeast spoilage. 1994). to inhibit chemical. however.. 1988a.05% are adequate for preserving high-moisture dried fruits (e. sorbate is either added directly into the product or applied to the surface of the product or packaging material. figs). molds. sodium sorbate may be used to avoid the problem. while additionally suppressing the growth of yeasts and molds.02% to 0. Potassium sorbate and sodium benzoate were highly effective in reducing yeast and mold populations in tomato juice (Bizri and Wahem. Concentrations as low as 0. fruit juices and syrups. In these products sorbic acid acts as the microbial inhibitor and sulfur dioxide prevents oxidation and enzymatic spoilage (Lück. 0.g. In sweet relishes. Use of the combination of 0. coli O157:H7 and result in spoilage of the product (Zhao et al. sorbate is more effective than benzoate owing to the pH of these products. preserves. In fruit juice and drink processing. and wines.

Yeast-leavened products may be protected with sorbate spraying after baking. 1989). in general. figs).004% sulfur dioxide. For this purpose a solution of up to 10% potassium sorbate may be used to protect unrefrigerated dry sausages.g. 2000).. To avoid the effect of sorbates on desirable yeasts in yeast-leavened products. 1952. unless the amount of yeast and the leavening time are increased. Sofos. Lück. Sorbates should be valuable in baking powder-raised products. however. the dipping method of application is more effective (Sofos.. and fillings. 1989). Often. aureus. 1976. muffins. jams. 1972. In addition to undesirable molds. Tompkin et al. pizza shells and toppings. Sorbates. the compound is chemically degraded and releases sorbic acid after the yeast-leavened dough is raised.. mold inhibition may be achieved by sprinkling sorbate on the surface of the product. especially when used in combination with calcium propionate (Tellez-Giron et al. sorbate also inhibits rope-forming bacteria and pathogens.30% sorbic acid are adequate for preserving bakery products. 1954). In 1974. 1988). 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. 1999). York and Vaughn. and doughnut mixes (Sofos. In fruit preserves (e. Sorbate can also be added before bottling the wine and is very valuable in the preservation of sweet wines. 1980). sorbates are used in combination with benzoate in preserving fruit juices and drinks. which is especially important in preservation of juices. 1989. 1980). A few sporadic studies in the 1950s and 1960s also indicated its potential as a preservative for meat products (Sofos..03% to 0. (1974) . including cakes and confectionery. which is a mixed anhydride of sorbic and palmitic acids that has no direct antimicrobial activity. sorbates may also be useful in preventing aflatoxin formation (Lück. 1989). Use of this compound has the advantage of preserving yeast-leavened bread without interfering with the fermentation process (Lück. sorbates are also valuable because they are active at higher pH values and effective against molds (Chichester and Tanner. and marmalades). such as S. However. In addition to preventing mold spoilage. It has also been used in champagne preservation (Sofos.. In such applications sorbate may be used in the coating oil or the pan grease (e. 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. Sorbate levels above 0.05% to 0. Potassium sorbate increased the shelf life of acidified tortillas. toppings. may also be used. upon heating in the baking process. 1989). refrigerated dough products. Sorbates are also used in the preservation of pie crusts and fillings. such as Japan and Korea (Sofos.g. in cream pies (Schmidt et al. Use of sorbates must be avoided in products raised by yeasts.03% are not recommended in wine preservation because they may impart an off-flavor to the delicate taste of wine.. may be more suitable for use in fruit products than other common preservatives because of their milder organoleptic properties and their neutral taste. Sorbate acts as the inhibitor of yeast refermentation during storage. 1989). Concentrations of 0. 1980).Sorbic Acid and Sorbates 65 0. and sulfur dioxide protects against enzymatic and oxidative changes and bacterial fermentation. In dried products with an irregular surface conformation (e.. 1969.10% sorbate inhibit the spoilage of cake icings. cooked sausage product. sorbates are more common meat preservatives in other countries. examined its activity against various pathogenic bacteria in an uncured. brown-and-serve products). jellies. BAKERY PRODUCTS Although propionic acid-based preservatives are the most widely used compounds in preserving bakery goods. soft drinks. the derivative sorboyl palmitate. and other beverages. Concentrations of 0. 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.g.002% to 0. Tompkin et al.

low storage temperature. phosphates. National Academy of Sciences 1982). aureus. 1980a. 1982. cured poultry products. uncured cooked sausage (Tompkin et al. 1974). Sofos et al. 1982). 1980. Department of Agriculture. Gray et al. Robach. Draughon et al. 1987a.. 1978. sodium chloride. such as color and flavor (Frank.... botulinum. Bacillus licheniformis. and lipolytic organisms (Kaloyereas et al. 1980a.. Bauermann. Clostridium perfringens. and processed cured meat products (Sofos. The antimicrobial activity of sorbate in these products has been enhanced when combined with nitrite. nitrosamine formation. Nelson et al. Robach and Sofos (1982). Bushway et al. the effect of sorbates on overall product shelf life. Yersinia enterocolitica. 1979a. Brochothrix thermosphacta. and increased carbon dioxide atmospheres (Sofos. 1982. Hallerbach and Potter. The antimicrobial activity of sorbate was demonstrated in bacon (Ivey et al.. 1989. 1984. low oxygen.. Cunningham. 1979b). Department of Agriculture. 1982. pork slurries (Roberts et al. 1982.. 1983). did not take effect following a report that consumption of experimental bacon resulted in allergic-type reactions in certain individuals (Berry and Blumer. Salmonella. Robach et al. Lactobacillus. 1978. Based on the results of the various studies. 1981. 2000). 1978. Greer. 1982.. beef and pork frankfurters (Sofos et al.. 1985. Huhtanen et al... Pseudomonas.b. Huhtanen and Feinberg. (1979a). Myers et al.. This work (Tompkin et al. 1981. 1977.. 1980b). 1979. botulinum in cured meat products. 1984). McMeekin et al... Sofos.. 1980b).. 1982). al. 1979. 1981. 1982. 1979. 1978. Chang et al. beef steaks (Unda et al... Berry and Blumer.. It should be noted. Robach et al. 1974.3%) had no major adverse effects on sensory qualities. U. 1979. raw beef and pork. Vareltzis et al. 1981). Robach and Ivey. Serratia liquefaciens. 1979a. Sofos et. 1978.. uncured.. 1981. 1983). 1980c. 1981. botulinum. Wagner and Gehrke.. 1984. Ivey et al.. 1964.. 1982. 1980.. Escherichia coli. Nitrite was reported to be a potential carcinogen. acids. Robach and Sofos. 1983. 1989) and cooked pork chops (Prabhu et al. 1980b. In addition to C.. botulinum spores during temperature abuse of the product. Rice and Pierson... Sofos (1981.. antioxidants.. 1979b. 1986b. Robach.b. aureus and delayed growth and toxin production by C. however.. 1979a. 1981). Pierson et al. sliced bologna (Chang et al. 1961. other pathogenic and spoilage bacteria inhibited by sorbate in various meat products include S. Sofos et al. In addition. 1983. that no other studies reported undesirable flavors or allergic reactions (Sofos. Hall and Maurer.S.66 Antimicrobials in Food showed that sorbate retarded growth of Salmonella species and S. 1984. 1979.. 1983. Elliot et al. Kemp et al. U. and uncured poultry meat and carcasses.. Tompkin et al. Products in which these microorganisms are inhibited include bacon and other cured meats. Amundson et al. 1989). sorbate was proposed as a means of reducing nitrite and nitrosamine levels in bacon while maintaining antimicrobial activity and inhibition of C. Clostridium sporogenes. 1981. Bacillus cereus. mesophiles. Perry et al. Extensive studies published in the 1970s and the 1980s established the antibotulinal and overall antimicrobial activity of sorbates in various cured and uncured meat and poultry products.S. 1981a. 1988). 1990). and it can react with amines to form carcinogenic nitrosamines during meat product processing and cooking (Sofos et al. Wagner et al. 1989). 1982. eating quality. and the National Academy of Sciences (1981. Petaja et al. Zamora and Zaritzky. 1974) was followed by extensive testing that examined the action of sorbates against clostridia and other pathogens in a variety of fresh. Vareltzis and Buck. poultry products (Robach et al. comminuted pork products (Ivey and Robach. National Academy of Sciences. raw (Mendonca et al.. and safety were examined.. This proposal.. 1979b. 1978. Sorbate in meat products (<0.. Paquette et al. Chambers et al. These studies have been thoroughly reviewed by Sofos and Busta (1980). psychrotrophs.c. 1980. Morad et al. low pH. however.. To and Robach.b).b. Sofos et al.. 1983. 1983). 1979b. inclusion of sorbate and reduction of nitrite levels in bacon-pumping brine solutions resulted in reduced levels of nitrosamines in the fried product (Ivey et al. 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. Wagner and Busta. 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. In addition.

1959.1%) solution preserved brined shrimp for 59 days. 1976. cut squash. Sofos and Busta. 1983. are also effective in preventing mold growth on animal feeds during storage.. Samples treated with sorbic acid lost biological activity within a few days of aerobic storage (confirmed by aerobic . Sorbates may be particularly important in intermediate-moisture (25% moisture content) pet foods. 1983.1%) was used (Fletcher et al. garlic oil. indicated that toxin production by C. and similar products. pineapples. 1989). 1989). Lynch and Potter. salad dressings.g. 1986). delicatessen items. botulinum in shucked scallops developed slightly more rapidly at 27°C in vacuum packages when sorbate (0. depending on moisture content. however. thus. The literature contains reports on several other food products that may be preserved with sorbates (Sofos. 1978.03% to 0. 1991). 1982). strawberry puree. such as margarine. 1982. the flavors of both types of bacon were equally desirable (National Academy of Sciences.. ethyl and propyl) may find applications in cosmetics and pharmaceuticals. Debevere and Voets. 1981. cucumbers. Statham and Bremner. Shaw et al. Application of potassium sorbate with bifidobacteria extended the shelf life of whole and peeled shrimp by 3 days (Al-Dagal and Bazaraa. 1980. Robach and Hickey. shredded cabbage. 1989). Lück. tangerine sherbet base. One study. and pancake batter (Sofos. Lück.1%)–benzoate (0.g... at which they prevent growth of molds and osmophilic yeasts in items such as fillings for chocolate and pralines (Chichester and Tanner. Bremner and Statham. Sorbic acid levels in the range of 0. 1972. 1976. 1982. Chung and Lee. it may be desirable to apply sorbic acid during malting (Ogundiwin et al. Sharp et al. MISCELLANEOUS FOOD PRODUCTS Sorbates are commonly used in the preservation of oil-in-water or water-in-oil emulsion-based products. 1978. 1995). Sorbates are also used to prevent mold growth on the surface of dried and smoked fish (e. A sorbate (0. 1982. in which levels of up to 0. dried cod). Fey and Regenstein. Lück. This should assure mycotoxin-free animal feeds and thus eliminate any hazards associated with mycotoxin-contaminated feed.20%. as well as fermented plant foods (Chichester and Tanner. Sorbic acid at a concentration of 0. In addition. orange peels. 1988).5% did not adversely affect the germination energies and germination capacities of sorghum grain and did not significantly affect malting loss.Sorbic Acid and Sorbates 67 of bacon treated only with nitrite. 1989). cosmetic products. sorbic acid may be mixed with its potassium salt or with other preservatives. This optimal concentration of sorbic acid coincided with the lethal dosage for most of the associated fungal microorganisms during malting. inhibiting their growth. They include high-moisture marshmallow candy.. OTHER APPLICATIONS Other items that may be preserved by sorbic acid and its salts include pharmaceuticals.. In Asian countries sorbates are commonly used in combination with other preservatives to preserve fish sausages and similarly processed fish and meat products.30% can be effective mold inhibitors.. which is longer than the preservation (31 days) achieved by the most effective bacteriocin (nisin) tested (Einarsson and Lauzon. potato chips. the esters of sorbic acid (e. 1981). Mexican-type hot sauces. 1972. sour cream. 1980. Several studies have reported improved preservation of various types of fish with sorbate (Geminder. tomato juice. Sofos. In certain instances and for better preservation. prepeeled carrots. Other food-related applications of sorbates include sugar-based and confectionery products at concentrations of 0. 1972. diastatic powers. especially those of the emulsion type. 1983. 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).05% to 0. in which the emulsifiers may be inactivated by nonionic compounds (Chichester and Tanner. 1972. such as benzoates. 1976). Regenstein. and cold and hot water extracts of the malt samples.10%. mayonnaise. 1983. 1999). and animal feeds (Sofos. Bremner et al. Robach and Sofos.

but the exact mechanisms of antimicrobial activity are not well defined (Freese et al. 1985a. Cells of C. and it may be possible that several of them may be functional under various conditions. 1979d. including types and species of microorganisms. 1973. when the sorbate hurdle is reduced or removed. therefore. integrity. 1986. Published data have suggested that sorbate acts as a competitive and reversible inhibitor of amino acid-induced germination (Smoot and Pierson. 1989). botulinum were long. 1982. Death of microorganisms exposed to high concentrations of preservatives. 1986). 2001). There was also evidence of other membrane damage in sorbate-treated cells (Statham and McMeekin. 1997). 1986.3%) usually inhibit microorganisms. Sofos et al. 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. pH.b). 1989). Although the significance of such alterations is unknown. and glues (Lück. Sofos et al. and vacuoles. Septation. enterobacteria. 1978. type of substrate. and not triggering of germination. irregular nuclei. 1989. 1988). such as sorbate. and clostridia common in the spoilage of silage and.68 Antimicrobials in Food degradation of starch and triglycerides). with bulbous formation and defective division (Seward et al. which could be overcome to some extent by addition of magnesium ions..0 increased cell hydrophobicity and cell wall lysis on exposure to lysozyme. Another study reported that sorbate inhibited spores triggered to germinate or after germinant binding (Blocher and Busta. Inhibition has involved various species of bacteria in laboratory culture media and in foods and has been influenced by species. 1985. Sorbate-treated cells of C. sporogenes were usually filamentous and nonseptate but with distorted shapes characterized by numerous bends and bulges. Sorbate inhibits cell growth and multiplication as well as germination and outgrowth of spore-forming bacteria. has been attributed to generation of holes in the cell membrane . Blocher and Busta. did not influence the susceptibility of the bees to the fungus Ascosphaera aggregate (Goettel and Duke. and sorbate concentration (Sofos. 1985). and it was thus concluded that it inhibits spore commitment to germination. Sofos. Treatment of Alteromonas putrefaciens with sorbate at pH 7. Ronning and Frank. It was also postulated that sorbate probably inhibits a postgerminant binding step in the process of germination. the outer cell wall was absent in many areas (Ronning and Frank.. 1985). environmental conditions. Freese and Levin.. 1976. Wagner and Busta. Other reported uses of sorbates include preservation of tobacco products. Other proposed mechanisms of inhibition of microbial growth by sorbate include alterations in the morphology. Under certain conditions sorbates have changed the morphology and appearance of microbial cells (Statham and McMeekin. 1989). the surviving microorganisms may resume growth and spoil the food. 1989).. and function of cell membranes and inhibition of transport functions and metabolic activity (Sofos. when present. whereas higher amounts may result in death. Blanching (80°C) and the application of 0. 1986. adhesives.. reduced ensiling losses and increased the feed value characteristics of citrus pulp silages (Filya et al.1% sorbic acid to citrus pulp were effective in inhibiting yeasts. Sofos. and the inner cell wall appeared to be thickened. 1981). Sofos. 1996). 1989). Such changes have been observed in yeast cells as dense phosphoprotein granules.. molds. Although the presence of enzyme activities was shown. and the type of food processing. 1988. there was no detection of pectinolytic activities (Mayer and Hillebrandt.. MECHANISMS OF ACTION Sorbate concentrations used in foods (<0. Consequently. Smoot and Pierson. Seward et al.. increased numbers and variable sizes of mitochondria. Several mechanisms of inhibition of metabolic function by sorbate have been proposed. 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. strains. Several studies have indicated that sorbate inhibits bacterial spore germination (Sofos et al. 1982. when used individually or in combination as preservatives of an artificial diet for leafcutting bees. The preservatives sorbic acid and methyl paraben. Sofos. 1989). 1981. 1989). resulted in minicells.

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

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

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

Dejobert et al.. 1976. 1976. in general. Food and Drug Research Laboratories. Considering the average use levels of 0. Some sensitive individuals. and Robach and Adam (1980) were able to induce such symptoms from panelists consuming commercial bacon manufactured without sorbate. 1981. Because there is evidence of reactions between nitrite and fatty acids (Benedict. 1982).. 1974. Sorbates were found to react easily with proteins. does not rule out the possibility that high concentrations of sorbate may act as irritants to certain susceptible individuals (Sofos. National Academy of Sciences. Walker (1990) concluded that sorbate use. not at 6. Litton Biometrics.b). however. 1979. but it indicates that most people are not affected by sorbic acid applied to the skin. 1981. Department of Agriculture. 2001). Khoudokormoff. Under conditions typical of . 1999).. does not appear to create toxicologic problems. apparently because of an increased caloric intake resulting from the metabolizable sorbic acid. Osawa et al. and vegetable juices and is inactivated by heat (Kada. various studies have indicated the following: (1) nitrite reacts with sorbic acid to form mutagens optimally at pH 3. Shtenberg and Ignat’ev.30% in food processing. 1981. Berry and Blumer. Sorbic acid has a conjugated system of double bonds. Gaunt et al. 1981. it may apply also to more complex systems such as foods. the potential for such irritations in commercial products is minor. The products of such reactions have the potential of being mutagenic (Sofos. 1994). it may react with nitrite in products such as cured meats. may undergo nonenzymatic browning reactions when stored with sorbic acid at a favorable range of aw and temperature (Quattrucci and Masci. No carcinogenic or mutagenic effects have been observed with sorbate alone (Dickens et al. 1975. 1977.0. Osawa and Namiki. however. botulinum. show skin irritations when exposed to sorbic acid (National Academy of Sciences. which is the average pH of most meat products. leading to decreased amino acid availability (Quattrucci and Masci. Robach et al. including lysine and glutamate. The literature is contradictory. 1989). and some very sensitive individuals may show irritations even at lower levels (Lück. There has been some concern about possible sorbate–nitrite reactions when added to the same substrate (Sofos. probably because the acid environment of the stomach leads to the breakdown of the sorbic-protein adducts (Quattrucci and Masci. no direct relation could be proved between symptoms and specific ingredients used in formulating the bacon (U. 2000a.. 1980. Sofos and Busta. 1980) and because sorbic acid is an unsaturated fatty acid. This. In general. It has been regarded that amino-compounds.. Kito et al. 1974. and in some instances the growth rate of sorbate-fed animals increased significantly. 1981).S. which makes it susceptible to nucleophilic attack. 1992). 1992). 1992).S. and (3) mutagen formation is inhibited by such ingredients as ascorbic acid.. 1980.5. 1980a. Average sorbic acid concentrations for skin irritations are in the range of 1%. of course. Lück. 1993. Some taste panelists reported certain “allergic-type” symptoms after tasting uninoculated experimental bacon (U. 1989). An allergic-type response has been reported for sorbic acid in one study in which bacon samples with sorbate–nitrite combinations were tested against C. 1983. 1979b. (2) formation of the c-nitroso mutagens requires the presence of excess nitrite. 1975. cysteine.. forming high-molecular-weight products that do not seem to result in a nutritional loss. Sofos. Hendy et al. Patrizi et al..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. 1978. 1981. 1986. 1968. Thus. 1980. This reaction was demonstrated in model systems but. 1982). to some extent.. 1979. Although it was implied that sorbate might have been involved in producing those symptoms. 1982. 1970.10% to 0. As preservatives for cosmetics and pharmaceutical products. 1976). 1982. 1979b). sometimes giving mutagenic products (Ferrand et al. no such symptoms were observed by other individuals who tasted sorbate–nitrate experimental bacon from other studies. 1989). 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. sorbates have been extensively examined for skin tolerance (Lück. In addition. Department of Agriculture. Namiki. No abnormalities have been observed.

. dichloromethane. 1995. and sausage products (Maxstadt and Karasz. After development by Melnick and Luckman (1954a). Wilamowski. 1997). and double distillation and ether extraction (Tjan and Konter. wine. the method was modified by Alderton and Lewis (1958). Several modifications have been proposed as useful in avoiding interference and in improving the accuracy of the steam distillation procedure (Luckmann and Melnick.. dialysis and solvent extraction. 1980). bakery items.. 1973. 2000)..10) by the color reaction with αthiobarbituric acid (Roy et al. Rao et al. DETECTION AND ANALYSIS Analytic methods used or tested for qualitative and quantitative detection of sorbates in foods include acidimetry.. 1962.15 and 975. Sofos. 1982.. 1980). successive extractions with ether.. polarography. Mutagenesis studies. Overall. Sofos. 1954a). 1981.b).. cheese (AOAC Int. 1988). and solvent extension using ethyl or petroleum ether. did not protect against the toxic effects of other substances (Daoud and Griffin. 1989). 1995. The formation of new products by addition reactions using ethyl sorbate and various amines led. Sorbic acid. showed that none of the products studied presented mutagenic or genotoxic activities (Ferrand et al. sorbates appear to be one of the safest food preservatives available. method 975. Montano et al. It should be noted.. AOAC Int. dialysis. however. Noda et al. method 975. 1989). at 20°C. Sofos. in cyclic interaction products. methods 971. 1955.. 1982.. 1983.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. 1989). 1976. and propionate (Sofos. spectrophotometry. sodium hydroxide. 1960. and isooctane. 1983. 2000a. to linear monoadducts and. Extraction methods include acid-steam distillation. 1999. Stafford.b). 2000). colorimetry. selective gas diffusion. The most widely used methods. Extraction by steam distillation has been used extensively. including parabens.. 1972.. although chromatographic methods have gained acceptance in recent years. 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. however. however. cheese. The spectrophotometric (ultraviolet/UV. Holley and Millard. In some foods. 1989). . Larsson. to cyclic derivatives resulting from double addition (Ferrand et al. Such combinations have involved extraction under acid conditions from the steam distillate and reextraction with sodium hydroxide. The derivatives of sorbic acid are also relatively nontoxic. however. Puttermans et al. Mandrou et al. or direct analysis has been used (Sofos.b. Detection methods require quantitative extraction and separation of sorbic acid from the food material without food ingredient interference (DeLuca et al. The method is simple and usually very specific (Lück. No synergism in acute toxicity has been detected for combinations of sorbic acid with various other additives. it involves measurement of absorbance at 260 nm (250 to 290 nm). 1989). involving the Ames test and genotoxicity studies with HeLa cells and on plasmid DNA. Sofos. whereas combinations of various treatments have also been used to improve extraction and reduce interference (Sofos. Sorbate.. have been colorimetric (Schmidt. had no effect on nitrosamine formation from aminopyrine and sodium nitrite in animal stomachs (Kawanishi et al. Chung. that certain studies have reported inhibition by sorbate of carcinogenic nitrosamine formation in model systems (Tanaka et al. 1989). 1974.31). bromometry. 1962) and spectrophotometric (Melnick and Luckman. 1989). 2000a. including fruit products. 1981a. at 50°C. 1976.. and wine (AOAC Int. 1978. and chromatography. 1981. Yamamoto et al.. Special extraction and purification steps have been proposed in the literature to reduce interference problems (Sofos. and methylene chloride. 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. enzymatic. Potential injury in cell hepatocytes induced by potassium sorbate was prevented by antioxidants (Sugihara et al. benzoate. Lathia and Schellhoeh. Sofos.22). Massey et al. 1972. absorption) procedure has also been used in many foods. 1980). filtration.

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

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

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

normality.0.14 3.Organic Acids 93 TABLE 4. type. chain length.98 pK2 pK3 5.41 4.87 4. or molarity to achieve final pH. Because the undissociated portion of the molecule is believed to be responsible for the antimicrobial effect. The pKa of most organic acids lies between pH 3 and 5 (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).08 3. In addition to any antimicrobial effects an acid possesses.27 4.77 4. (1939) observed that acids with fewer than 7 carbons were more effective at lower pH. Not only would this interaction ensure commercial sterility of the food. 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. prevent browning. Comprehensive texts describing applications.40 4. Effective use of an acidulant depends on the dissociation constant (pKa) or the pH at which 50% of the total acid is dissociated.39 times. The multiple-barrier concept (“hurdle” concept) has gained popularity for use in a variety of foods in recent years. organic acids are usually more effective as antimycotic agents.75 4. Hoffman et al. and protect color.75 5. Acids contribute to the taste and tartness of a product. This concept is based on the premise that foods can be preserved using several inhibitors concurrently rather than relying on a single factor. adjustments in pH levels can be coupled with changes in temperature. aid in the inversion of sucrose.43 4. pK1 4. This primary concern limits the use of acidulants to foods with pH values of less than 5. They can create a synergistic relationship with antioxidants by chelating metal ions. water activity. Given that this value lies at the lower limits of growth for many bacteria. 2003). The inhibitory effect of acids has been compared based on pH. Because some experiments express concentration in percent. but also the decreased processing time would aid in preserving the palatability of the product (Corlett and Brown. The use of the multiple-barrier concept results in lowering the concentrations of acidulants or inhibitors.61 4. it would be advantageous to use the acids near these values. and chemical analyses can be found in the references by Ash and Ash (1995) and Doores (2002. physical parameters. Therefore. gaseous atmosphere.11 5. EFFECTIVE USE Various approaches have been used to study the efficiency of antimicrobial effects on cells.34 6.89 3.03 3. concentration. and acids with 8 to 12 carbons were more effective at neutrality and above. and other inhibitory compounds. They can control pectin gel formation.1). 1980).16 2. Some can be used . and degree of branching to inhibit or kill a wide variety of microorganisms.44 5. the choice of an acidulant may depend on secondary effects.

These organisms are found naturally associated with fermented products. are more tolerant to this acid than homofermentative lactic acid bacteria (homolactics). In addition.8. Numerous studies included in this chapter compare several organic acids for their antimicrobial effects. Salmonella Aertrycke (pH 4.0 than lactic acid bacteria. L. a more acid-tolerant organism.7. all of the groups were similarly affected (Woolford. many of these studies used high levels of the concentrated acids at low pH values. plantarum at pH 4. although they could also serve other functions in foods. application. Sauerkraut manufacture is a fermentation process brought about by a natural succession of microbes that inhabit the surface of cabbage leaves. as the name implies. Although these acid mixtures might be expected to occur naturally during fermentations by heterolactic bacteria. Concentrations of acid lower than those needed to inhibit Saccharomyces cerevisiae (pH 3. antimicrobial properties. The results of these studies are usually presented in the section describing the acid that gave the most antimicrobial effects. 1939) inhibited Bacillus cereus (pH 4.9) and Aspergillus niger (pH 4. In some instances. which exist naturally in many foods. this apparent synergy has not been demonstrated in other foods. 1966) The toxicology and safety of the acids must be considered when selecting an acid for use. are relatively nonpolar compounds. and molds. all these acids can be metabolized by the body primarily through lipid oxidation and via the tricarboxylic acid cycle. The succession typically begins with growth of coliforms and natural microflora followed by Leuconostoc mesenteroides (heterolactic). propionic acid. McDonald et al. and regulatory status is presented here for each acid.1) (Levine and Fellers. as such. At the levels and pH values in which these acids are normally used in foods. 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. Gram-positive bacteria. Owen (1946) suggested that acetic acid inhibited Gram-positive organisms. Most of the acids appear safe for use in food products. .6 to 4. yeasts. (1990) found that growth of L. These acids. Adams and Hall (1988) showed a weakly synergistic inhibitory effect between acetic and lactic acids with Salmonella enteritidis and Escherichia coli. The lipophilic acids include acetic acid and acetic acid derivatives. sauerkraut.4 to 5.0). They are usually added to foods for their antimicrobial properties. The salts of the acids are important in regulating the acidity of the foods (Gardner. then by Lactobacillus plantarum (homolactic). as the pH decreased to 4. however.0. and caprylic acid. ACETIC ACID Antimicrobial Properties Acetobacter and heterofermentative lactic acid bacteria (heterolactics) produce acetic acid as a byproduct of their metabolism and. the same hazards would not be expected. 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. Microorganisms vary in their susceptibility to acetic acid. This association could explain the apparent stability that occurs in natural fermentations. Several animal studies have indicated possible safety hazards. Bacillus and Clostridium species and Gram-negative bacteria were more inhibited at pH 6. such as pickles.94 Antimicrobials in Food as chemical leavening agents. would further indicate that hazards are unlikely.0.9). however. such as sauerkraut. and vinegar. and Staphylococcus aureus (pH 5. THE ACIDS Information on the toxicology. 1975b). whereas growth stopped in L. the information appears separately for each acid. which do not.9). mesenteroides ceased when the internal pH of the cells reached 5. The long-term consumption of these acids.

0% acetic acid and 1. coli. aureus preconditioned in acetate buffer (pH 4. E. A 0. 1984). especially with the use of pasteurized egg products. monocytogenes than lactic and citric acids and increased inhibition as the temperature of incubation .7% acetic acid in the aqueous phase and adjusted to pH 4. A concentration of 0. 1940). Malic acid allowed growth at pH 4.5. in the aqueous phase of CFM and RCM would still produce a microbiologically sound product. which has recognized antimicrobial properties.6) at 20°C shifted to pH 7.3.7% acetic acid with reduction to undetectable levels in 1 and 2 weeks with 0. monocytogenes was also reduced to undetectable levels in RCM within 10 days and in CFM within 14 days compared to 2 days for Salmonella. followed by citric. such as mayonnaise (Debevere.5 (Noda et al. 0.. Acetic acid exerted the most inhibition but had the lowest dissociation constant. Salmonella Blockley.15 M acetic acid and with 0. and hydrochloric acids (Nunheimer and Fabian. 1979).7 and 4. coli O157:H7 was inoculated into tryptic soy broth (TSB) containing acetic. 1988). pH 4.0. monocytogenes. B. 22°C) containing 1.0 with 0. lactic. malic.5%. pH 4. coli O157:H7 grew in all media at pH 5. Both studies concluded that the antimicrobial effect was the result of the undissociated molecule. and tartaric acid permitted growth at pH 4.7% acetic acid (Glass and Doyle.. 1982). 0. lactic. 1991).0 (Conner and Kotrola. respectively. respectively.1-M concentration of acetate and lactate completely inhibited multiplication at pH 6.3% acetic acid formulations. and exposed to 40°C became injured.33 M lactic acid (Wong and Chen. and tartaric acids. most likely because of the absence of egg yolks in CFM and the presence of egg white. pH 4. aureus.0% lactic.9 and 4.4 to 4.0. In addition. 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. tartaric.5. or 0. aureus. L. aureus was most sensitive to acetic. Traditionally acidic foods contain a single or multiple acids.6 and 4. citric.2 and 5. followed by lactic. respectively. Reduced-calorie mayonnaise (RCM) manufactured with 0. monocytogenes and Yersinia enterocolitica. some strains of S.1%. 1997).0 buffer. Buffered acidulant systems (brain heart infusion [BHI] broth. and Enterobacteriaceae in Japanese fermented soy sauce (Hayashi et al. At 25°C and 37°C. added or developed through fermentation.7% acetic acid. although more resistant than Salmonella. malic.0. L.0 with scant growth in TSB containing acetic acid. and HCl. cereus was inhibited at pH 5. respectively. growth occurred at pH 5.3%..5% and 0. pH 4. was reduced by 4 logs in both mayonnaises within 72 hours in formulations containing 0. When E. Similar results were noted when various acids were compared at the pH level required for a 90% and 99% reduction in numbers of S. E.6. therefore the standard use of 0. aureus in 12 hours (Minor and Marth. coli O157:H7 is considered more acid tolerant than strains of E. Gram-negative aerobic bacteria. or citric acid were far more effective in reducing numbers of S.6. Destruction of Salmonella occurred more quickly in CFM than RCM.4% to 0. 1970). 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.Organic Acids 95 S.2 and 4. This finding is important in the manufacture of medium acid foods containing more than one organic acid. 1995. tartaric.7% acetic acid. 1988). L. other ingredients can interact with acid to influence the degree of inhibitory action.. S. citric. malic. Conner et al.3% and 0. and E. but no injury occurred in cells not exposed to acidic conditions before heating (Smith et al. pH 4.1 and 5. whereas tartaric acid had one of the highest dissociation constants yet exerted the weakest inhibitory action.5 to 3. coli than unbuffered systems. It is unlikely that these products would contain such high initial levels of either of these organisms.9°C. Salmonella were inactivated within 48 hours in both RCM and CFM formulated with 0. The effect of temperature-acid interactions is noteworthy for the psychrotrophic pathogens.0 with hydrochloric acid was compared to cholesterol-free (egg yolk-free) reduced-calorie mayonnaise (CFM) manufactured with 0.5 for all acidified media at 10°C except for acetic acid.8. Acetic acid was effective at pH 5. Acetic acid caused greater inactivation of L. Streptococcus faecalis.8% reduced growth of Micrococcus and Bacillus species. and caused 50% inhibition of spore germination at pH 4.

and off-flavors. coli O157:H7 to nondetectable levels by enrichment methods. 25-g samples.3% inhibited L. containing from 0. Depending on the treatments. 1989). 1997. Treatments spanned varying amounts of time with concomitant decrease in microbial loads up to 3 logs. acidulants. Karapinar and Gönül. malic.5 to 3.9 log reduction for the nontreated seeds (Weissinger et al. citric acid caused greater injury than either lactic or acetic acids and injured organisms survived longer at low temperatures (Ahamad and Marth. 2%.96 Antimicrobials in Food decreased from 35°C to 13°C.. 1992). Exposure at 50°C for 12 hours with lower levels (100 and 300 mg/L of air) reduced the population >1. 1990). citric acid was more bactericidal than other acids at 25°C and 35°C. there can be adverse sensory effects including discoloration of meat. coli O157:H7.. 1992.0 for acetic acid. In addition. enterocolitica. seed germination was effected (Weissinger and Beuchat. and pH 4. concentration ≥0. This treatment reduced S. Immersion of alfalfa seeds in mixtures containing 5% lactic or citric acid for 10 minutes resulted in several log reductions.7 log without affecting germination of seeds. Acetic acid was more effective than lactic or citric acids in producing this effect (Adams et al. Similar growth inhibition was demonstrated for Y. respectively. monocytogenes irrespective of temperature.0. but L. 1973).5. but recovery required the presence of amino acids or peptones (Blankenship. Numerous organic acid combinations. however. 1981).6% to 3. injury and death rates were not affected by refrigeration temperatures for Salmonella Bareilly. 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.2 N for 4 min. and 0.0 have been used to rinse pork. and beef carcasses or meat tissue. 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. monocytogenes when compared to citric. Mung bean seeds inoculated with 3 to 5 logs of S. Parsley.01 N for 75 min. enterocolitica was not detectable after exposure to 2% and 5% acetic acid or 40% and 50% vinegar for 30 minutes (Karapinar and Gönül. lamb. a twofold reduction at pH 2. but resulted in changes in sensory characteristics. 1991. these foods can be the carriers of psychrotrophic pathogens. a mesophilic organism.05 N for 8. The minimum inhibitory pH level was pH 4.0 for 30 or 60 seconds (Biemuller et al. and L. off-odors.33 and 5.. and no reduction in microbial . lactic. Conversely. vapor treatment also reduced levels of resident microflora on seed but did not affect seed germination rates (Delaquis et al. 1992b).. enterocolitica. or 50%) for 15 or 30 minutes. and hydrochloric acids. 1 log unit at pH 2. 1990. Y. but acetic acid was less effective. 0. Acid injury did not appear to involve damage to ribosomes or other nucleic acid material.72 logs. When comparisons were based on equal molar concentrations.5. 1999). Typhimurium and E.10 N for 5 min. Dorsa et al.6 for lactic acid. Smulders and Greer. monocytogenes was isolated from 2 of 10. and temperature. 1989. Treatment at low temperature (4 days at 10°C) with 200 and 500 mg/L of air was effective in reducing Salmonella by 2. and hydrochloric acids at equal pH values and at all incubation times and temperatures (Sorrells et al.4 for malic.4 to 4.5 or 2. A 90% reduction of the population required exposure to 0. 2000). Dorsa. 1997. Total plate counts were reduced by 4 logs at pH 1. However. Sorrells and Enigl (1990) also demonstrated interactive effects between sodium chloride. Little et al.0% acetic acid at pH levels from 1. Acetic acid displayed the greatest antimicrobial action against L. such as Y.8 to 5. 1998). 40%. In addition.. E. Enteritidis were sprayed with acetic acid at pH 1. Because produce is typically refrigerated with only minimal processing. 1998.5 min.. or 5%) or vinegar (30%. citric.. enterocolitica at lower pH levels as the temperature increased. The greatest antimicrobial effect occurred at 35°C with the greatest survival at 10°C. A variety of organic acid treatments has been used to control microbial loads associated with meat carcasses and products (Dickson and Anderson. pH 4. inoculated with approximately 107 CFU/g Y. 0. monocytogenes were exposed to acetic acid at a concentration of 242 µL/L of air for 12 hours at 45°C. 1992a. Pork carcasses inoculated with 106 CFU/ml S. but malic acid was more bacteriostatic than other acids at 10°C. Brocklehurst and Lund. was dipped in acetic acid solutions (1%. Typhimurium. 2001).

and lactic acid bacteria counts.. An increase in organic material.. and stored for 28 days at 2°C in a high-oxygen barrier film. Lactobacillus species predominated as storage progressed. 1994. 1986). Hamby et al. 1995)..5% acetic acid were reduced more when treatments were conducted at 52°C than at 14. 1994). The treatment effectively reduced recovery of S. 1997). Enteritidis to 1 of 6 samples at pH 1. type of chemicals. 1987). Typhimurium on beefsteaks (Tinney et al. 1997. this was not significantly different from the controls. possibly because this tissue retained less moisture (20%) than lean tissue (75%) and the reduced water activity could have enhanced the antimicrobial effects. Unda et al. coli O157:H7 and S. Typhimurium reductions were greater when the bacteria were attached to fatty tissue. dirt. Meat was sprayed. 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.. 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. 1994). respectively. Anderson et al.. They found that acetic acid was a superior sanitizer compared to hypochlorite and had a greater residual effect. Typhimurium was reduced by 0. Microbial loads on beef carcasses subjected to machine washing and sanitization with 1.0.046% formic acids for 10 seconds caused discoloration from both treatments. some discoloration occurred (Cacciarelli et al. and application rate and sequence in the process (Gorman et al. monocytogenes was detected in 69% of loins and 33% of chops. 1989b). 1995).8 log CFU/cm2. Cutter et al. Cutter and Siragusa..4°C (Anderson et al. Application of a 2% lactic or 2% acetic acid spray to beef strip loins and stored at 1°C resulted in significantly lower aerobic and lactic acid bacteria counts over 112 days of storage (Goddard et al. 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. anaerobic. Incorporation of pulsed power electricity and a 2% acetic acid spray led to improved reduction of inoculated E..0.. 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.. Temperature played a greater role in reducing numbers than did the acid concentration. however.6% on meat using a 3% concentration of acetic acid before washing. Typhimurium followed by treatment with 2% acetic acid. 1994.S.5% acetic. 1997. resulted in less effective reduction of S. however. 1996). (1987) used intermittent sprays of 1% acetic acid or lactic acids. but beef treated with the mixture of acids did not differ in flavor from untreated beef (Bell et al..5 and 11 of 72 for pH 2.8 to 4. citric. It was noted that the use of acetic acid as a rinse for beef tissue led to sublethal injury of bacterial cells. (1980). Typhimurium. At this concentration.5. or manure. Dickson (1992) contaminated lean and beef tissue surfaces with S.. An 18% acetic acid or 12% lactic acid spray significantly reduced bacterial counts on lamb carcasses. Quartey-Papafio et al. coli. or manure slurry. 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.Organic Acids 97 numbers at pH 3.1 and 1. (1977) reduced bacterial counts by 99.5 to 0. the pH level decreased to 2. Dorsa et al. found that a 1% formic and 1% acetic acid combination exposed to beef for up to 20 minutes was effective in reducing a variety of bacterial species. which resulted in significant reductions in aerobic plate count (1.. Beef cubes treated with 1. and bleaching of the carcasses occurred (Ockerman et al. Hardin et al. Reductions in microbial counts depend on the water temperature.. vacuum packaged. Although washing of pork carcasses with 1. S. 1983).3 log/cm2). suggesting that acids had limited effectiveness (Fu et al. organic acids can be used in preevisceration rinse systems consisting of a water rinse followed by an organic acid rinse. Typhimurium.2% acetic acid or 0. a final application of acid washes could provide some residual effect during storage (Brackett et al.. As part of recently established guidelines by the U. Department of Agriculture Food Safety Inspection Service (FSIS). S. however. (1991) inoculated beef roasts by injection and on the . coli O157:H7. 1974). 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. S.6% acetic and 0. L. such as rumen fluid.

and 30°C (Oscroft et al. 1 mM perillaldehyde—or 0.86 log MPN/ml for the 0. Acidification of the chill water at the end of processing delivers a second intervention step to reduce microbial loads and to increase the shelf life of poultry.5 to 1. Poultry scald water containing 0. There was no significant difference in texture or sensory characteristics between the treatments.5% acetic acid reduced Enterobacteriaceae counts by 2. 1976). For pearl onions. aerobic plate counts were unaffected by the treatments. see Chapter 7) coupled with 0. Air injection did not affect reduction of these counts.05%.6% acetic acid under the same conditions.05% acetic acid. a 0. Adjustment of the chill water to pH 2. Stroup et al.35% solution required 7 days for equilibration for acetic and citric acid but 1 day for 0. Because of the possible attachment of Salmonella to broiler chicken skin. 1986). 1994).0 on inhibition of L.5 logs (Okrend et al. 1965).24 log (Lillard et al.3% acid and 2. Immersion of broilers for 10 minutes in 0. respectively.6% acetic acid-treated carcasses was darkened or yellowed (Dickens et al. Acetic and tartaric acids were more inhibitory than lactic and citric acids.6% acetic solution acid (pH 3. lactic. but Enterobacteriaceae counts were significantly reduced by 0. The acetic acid in the brine inhibited anaerobic and aerobic bacteria. As the incubation temperature to recover spores was lowered. and 0.2% to 0. and tartaric acid adjusted to pH 5. 0. Typhimurium using a model system.. 20°C. citric..5 mM. Despite these treatments. acetic acid caused the skin of the poultry to be hard and leathery. 1997). 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. greater than a 4% concentration of acid was needed to reduce levels by 2 logs (Tamblyn and Conner..7% acetic and 4 days for the same concentration of citric acid.0% acetic acid compared to 2 days for 0. Other studies continued to explore the use of acetic acid in chill water.35 log MPN/ml for the 0. of the same components—with 2% salt to inhibit growth of contaminating microorganisms for more than 20 days at 27°C. acetic acid was the most destructive acid at all combinations of parameters. Rubin (1978) found that acetic and lactic acid acted synergistically to retard growth of S.0% acetic acid caused instantaneous death.5%. further poultry processing can reintroduce microorganisms onto carcasses. with and without the use of air injection to agitate the chill water. Dickens and Whittemore (1994) exposed broilers to 0. Water pockets occurred under the skin of chicken carcasses with air-agitated samples (Dickens and Whittemore. fumaric.1% acetic acid at 52°C decreased levels of S.71 log most probable number (MPN)/ml (Dickens et al. The acid level could be reduced when used in combination with brines and sugar. Products are now being preserved by a number of antimicrobial compounds using a multiplebarrier approach to preservation..0) did not result in a significant reduction of aerobic plate counts but did significantly reduce levels of Enterobacteriaceae by 0. Whole pickles preserved with brine at 12° Brix and 0. Addition of 1. Levels of 0. 1987). citric. El-Shenawy and Marth (1989b) investigated the effect of acetic. Kurita and Koike (1983) combined 3% ethanol. although the skin of the 0. Expanding on their work.5% concentrations of acetic.9% acetic acid did not support growth or toxin formation of Clostridium botulinum (Ito et al. succinic. monocytogenes.5 with acetic acid was the most effective antimicrobial treatment followed by other acids in descending order of effectiveness: adipic.98 Antimicrobials in Food surface with approximately 103 CFU of Clostridium sporogenes and L. lactic. 1994). Intervention steps incorporated into poultry processing have been effective in reducing microbial contamination. but changes in pH and concentration of acid were more successful. Again.7% citric acid. 1994). 0. Although each individual acid influenced the heat resistance of Bacillus spores differently. and lactic (Mountney and O’Malley. monocytogenes.6 or 5.05% and 0. Acidified media showed greater inhibitory activity at 35°C than at 13°C. Typhimurium and Campylobacter jejuni by 0.6 depending on the concentration of acid in can brine within 1 day with 1. 1990)...6% acid solutions. germination and outgrowth of Bacillus . (1985) found that mushrooms reached an equilibrium pH of 4. Nisin (an approved bacteriocin for some foods.3% and 0. Despite the reduction in microbial numbers at this pH.

At the 0.2). It was found that acetic acid was the most effective acid. When used as a surface application. Studies on nonmammalian species showed that acetic acid at a concentration of 0. Grasshoppers treated with 0. it decomposes to yield oxygen and acetic acid. lactic. 1937). are especially susceptible to spoilage because of indigenous microbial populations. rats decreased their food intake level and lost 2.5 completely inhibited growth and aflatoxin production by Aspergillus parasiticus. 1949). Aspergillus fumigatus required 0. 1968).0. This treatment could also be used in recycling of water to reduce levels of yeasts and molds. it was not possible to determine the actual quantity of acid consumed.48 mg/m3 altered sensitivity to light. Parmeggiani and Sassi (1954) reported that workers . Cruess and Irish (1932) showed that apple juice containing 0. 2000). pneumonia (Gerhartz. however. 1921). it is effective against the black bread molds. niger. Nisin. 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. Ingestion of acetic acid by humans has caused burned lips (Palmer.0% concentration of acetic acid.5.8% to 1.0% acetic acid adjusted to pH 3.0 and below to inhibit growth (Kirby et al.29 mg/m3 caused changes in electroencephalograms.2 or 5 mg/m3 but not at 0. Levels of 0. Other forms of acetic acid have antimicrobial uses. Peracetic acid is formed by an oxygen molecule bound to the carboxyl carbon atom of acetic acid..8 and 0. Mori (1952) fed rats acetic acid ranging from 10 to 50 cm3 acid/kg of rice. 1958). Foods.04 M acetic acid at pH 3 to 4 showed chromosomal and chromatid breaks. 1971). glucono-delta lactone.001 M accelerated spindle activity and retarded anaphase in the mitotic cycle of the root tips of the onion. Rats consuming acetic acid in drinking water in concentrations of 0. lactic. followed by glucono-delta lactone. 1968). ready-to-eat products. On contact with organic substrates. suggesting its use as a decontamination agent for equipment (Hedberg and Miller. aerobic bacteria. however. a 1% concentration of acetic acid at pH 4. 1966). Damage from acetic acid resembled that from ionizing radiation or radiometric chemical damage (Manna and Mukherjee. but a concentration greater than 4% was needed when the pH was at 7. Rats fed 10% acetic acid and cats fed 20% acid developed ulcers in the gastric mucosa (Okabe et al. Populations of Pseudomonas aeruginosa were reduced by 99. and potatoes than peroxyacetic (80 ppm) acid alone (Hilgren and Salverda. Spore-forming bacteria are killed on surfaces at a concentration of 0. gastric erosion (O’Keane et al.2% (0.5 inhibited Saccharomyces ellipsoideus and Penicillium glaucum.5% level.6% or 0. A. 1932). interference with blood coagulation (Fin'ko. Rats exposed to vapor levels of acetic acid showed muscle imbalance at 0. Allium cepa (Makinen. A combination of these parameters could be used to extend the shelf life of these products.06 mg/m3.01% to 0. and citric acid when spores were heated at 95°C for 15 minutes and acetic. All rats showed mucosa damage owing to the feeding method. particularly precooked. cabbage. 1976).8% partially inhibited growth and decreased toxin formation by 70% and 90%.2 g/kg body weight) did not show any adverse effects. and 0.4% acetic acid at pH 5.01 mg/m3. in combination with organic acids. Human olfactory thresholds to acetic acid vapor were 0.. renal failure (Paar et al. 1969). Toxicology Acute toxicity data for acetic acid in mice and rats indicate varied tolerance levels by different routes of administration (Table 4. Takhirov (1969) suggested limits of 0. and death.0001 and 40% relative humidity within 10 minutes (Portner and Hoffman..60 mg/m3.999% after exposure to a 1. 1969).Organic Acids 99 spores were more restricted. displayed synergistic effects in increasing the destruction of the organism. Lower concentrations of 0. and citric acids when temperatures were reduced to 65°C for 60 minutes. and Rhizopus nigrificans at pH 3. and coliforms. Although acetic acid is generally used as an inhibitor of bacteria and yeasts. 1971). Johnson (1968) noted that rat gastric mucosa responded to a 100-mM concentration of acetic acid by releasing histamine into the interstitial fluid.0001 and 0. respectively (Buchanan and Ayres..2% acid at pH 5. chilled.

and tartaric acids.210 338 3. lactic.000 1. sodium Dehydroacetic Adipic Rabbit Caprylic Citric Mouse Mouse Enders (1941) Orö and Wretland (1961) Yokotani et al.960 525 3.810 625 3. (1950) Horn et al. and lungs with an air concentration of 0.530 380 1. calcium Propionate.310–3. No toxicologic data were available for peracetic acid. 1956).500 330 44 1.2 Available Acute Toxicity Data (LD50) for Various Acids Acid Acetic Animal Mouse Rat Mouse Rat Mouse Route of Administration Oral Intravenous Oral Intravenous Oral Oral Intravenous Intraperitoneal Oral Intravenous Intravenous Oral Intravenous Intravenous Intravenous Intraperitoneal Subcutaneous Oral Intraperitoneal Intraperitoneal Subcutaneous Intravenous Intravenous Intraperitoneal Intraperitoneal Intravenous Oral Oral Intravenous Oral Intravenous Oral Intravenous Intravenous LD50 (mg/kg body weight) 4. caused allergic reactions by producing canker sores (Tuft and Ettelson.700 725 884 5. In rare instances acetic acid. teeth.380–3.700 11. (1971) Rat Citrate.020 5. (1957) Acetate. (1971) Gruber and Halbeisen (1948) Horn et al. 1951).65 mg/L. (1957) exposed for 7 to 12 years to acetic acid vapors in the production of acetyl cellulose displayed corrosion of the hands. as well as citric. 1955).430 600 5.200 485 Reference Spector (1956) Orö and Wretland (1961) Spector (1956) Spector (1956) Spencer et al. Men having 7 to 25 years of exposure displayed blood abnormalities (Cesaro and Granata. (1957) Gruber and Halbeisen (1948) Yokotani et al. cold sensitivities (Wiseman and Adler. (1963) Horn et al. (1963) Hara et al. pharynx.100 1. eyes.100 Antimicrobials in Food TABLE 4. .340 580–1.900 680 275 2.790 940–960 42 203 961 2. sodium Rabbit Mouse Rat Rabbit Rat Guinea pig Mouse Rat Rat Mouse Gruber and Halbeisen (1948) Yokotani et al.430–4.040–5. and epidermal reactions (Weil and Rogers. although it is assumed that this derivative would be assimilated in a manner similar to that for acetic acid. 1956). (1971) Gruber and Halbeisen (1948) Gruber and Halbeisen (1948) Lactic Propionic Propionate.2 to 0.730 1. sodium Tartaric Spector (1956) Orö and Wretland (1961) Hara et al.460 1.860 2.

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

over 6 days.0% concentration of SA at pH 4. breve. SA alone or SA-MKP-treated fillets were not significantly different in odor from fresh controls up to 9 days. although it is assumed that this derivative would be assimilated in a manner similar to that for acetic acid. A combination of B. the indicator of spoilage. Histologic findings showed hypertrophy of the kidneys and ureters. monocytogenes in vacuum-packaged turkey bologna during storage at 4°C and was similar to 0.26% potassium sorbate or 2% sodium citrate in its efficacy (Wederquist et al. breve culture and stored at 4°C for 9 days (Al-Dagal and Bazaraa. SA at concentrations of 0. and held at 4°C for 12 days contained lower population levels of psychrotrophic and anaerobes than untreated samples (Zhuang et al. 0.2. 1976). SA extended shelf life to 12 days at 4°C. 1995b). 1994). B. 1. The combination of 0. vacuum packaged. . SA in combination with potassium sorbate and Lactococcus lactis ssp. and 1. but SA provided better sensory characteristics and more controlled growth of psychrotrophs than potassium sorbate with bifidobacteria. At lower acid concentrations of 0.7 logs when stored at 4°C compared with the nontreated fillets and increased shelf life by 6 days.25% potassium sorbate. cremoris culture added to catfish fillets was an effective treatment against growth of Gram-negative spoilage bacteria at 4°C (Kim and Hearnsberger. SA (0. sodium lactate (3%). sodium lactate. A combination of 0.5. Toxicology The LD50 (mean lethal dose) for SA in mice is presented in Table 4. Treatment with SA alone or in combination with bifidobacteria extended shelf life at 4°C by 3 days (Kim et al.. breve and SA exhibited an additive effect that extended shelf life still further.0% SA alone inhibited growth for more than 6 days at 4°C and counts increased only by 1.75% and 1% significantly reduced the initial aerobic plate counts of catfish fillets by 0.5% lactate was effective as an antimicrobial treatment in sliced servelat sausage over a 6-week storage life (Blom et al.5% SA.. No toxicologic data were available for calcium acetate (Food and Agricultural Organization. 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. and trisodium citrate extended shelf life to 6 days. 1996). Chickens fed diets supplemented with 5. Whole and peeled shrimp were immersed for 2 minutes in solutions containing 10% SA.7% SA and 0. 1995a)..3 or 1. The combination of 0.102 Antimicrobials in Food SA (10%). parasiticus was inhibited by a 1.25% acetate and 2.5% potassium sorbate. but they did appear brownish and watery (Kim et al.2 logs by day 12.. Psychrotrophic counts from untreated whole shrimp increased to >107/g. although a mild acetic acid odor was detected.. and increased mortality.6 to 0.6% or 0. 1994). Catfish fillets treated with 2% SA.44% SA showed decreased appetite.3% to 0.4% monopotassium phosphate (MKP) more effectively prolonged shelf life to 12 days compared with SA alone.8%.5%) was also effective in reducing populations of L. respectively (Buchanan and Ayres. untreated samples reached the spoilage mark within 10 days.5%). but bifidobacteria did not grow within 9 days. Color remained acceptable. 1999). 5% sodium lactate. 1997).5% trisodium citrate with or without B. Waterhouse and Scott (1962) attributed the toxic effect to the sodium ion rather than the acid moiety. 1951).5% lactic cultures or 0. depressed growth rate. growth and toxin formation were decreased 70% and 90%. Growth and aflatoxin production of A. A 22-year-old woman with sensitivity to acetic anhydride was not allergic to SA (Weil and Rogers.5 to 1. 1963). although MKP alone had no effect. and 2. potassium sorbate (1. For peeled shrimp.. 1999). or trisodium citrate were used alone or in combination with Bifidobacterium breve (5%) as a dip for fresh camel meat (Al-Sheddy et al. whereas potassium sorbate increased shelf life to 9 days.

2% sodium diacetate or 1% buffered sodium citrate and within 3 and 5 weeks for products containing 2.15% to 0. and 35°C (Shelef and Addala. a concentration of 0. and B.2% sodium diacetate. Enterococcus faecalis. monocytogenes and S. and Penicillium expansum. or 1. The addition of sodium diacetate affected the sensory qualities of the product precluding its use. it prevented mold growth (Chichester and Tanner. 1972).5% or 3. monocytogenes increased to approximately 106/g within 3 weeks. and stored for up to 40 days at 4°C. SODIUM DIACETATE Antimicrobial Properties Sodium diacetate was effective at a 0.29).55. L.4. monocytogenes and bactericidal . Lactobacillus curvatus and L. curvatus reached an end point of 107/g within 2 to 2. and when it was incorporated into butter wrappers. Shewanella putrefaciens.3 and incubated at 5°C.1% and 0. The minimum inhibitory concentration (MIC) of sodium diacetate was determined for L. and 6.2% sodium diacetate. However. or S. L. coli.2% sodium diacetate.5% at pH 4. Aspergillus glaucus. 5. the combination 0. Cooked-in-bag ham was formulated with additions of 0. sodium diacetate was also effective against hemorrhagic E.1721) and calcium (21 CFR 184. The MIC was determined to be 35 mM (pH 5. fumigatus. 6.1% to 0. monocytogenes was inhibited by sodium lactate and 0.3% sodium lactate.25) at 35°C.3% sodium lactate. and 28 mM (pH 5. respectively.3% sodium diacetate was effective in suppressing total aerobic counts. Lactobacillus fermentans. Sodium diacetate was more effective than acetic acid.1% and 0. 1981).4) at 20°C. niger. Sodium diacetate at concentrations of 0.2% SA. L. enterocolitica.5% sodium lactate was bacteriostatic to L. Sodium diacetate is equally effective in the baking industry where its inhibitory powers prevent the growth of bread mold and rope-forming bacteria.5% and 3. 0.3% and sodium lactate at concentrations of 2% to 3% were equally effective as antilisterial compounds in meat with little effect on pH and sensory characteristics (Mbandi and Shelef. Enteritidis were inoculated into a comminuted beef emulsion formulated with 0.1% to 2.05% to 0. aureus (Shelef and Addala. A. A concentration of 0. monocytogenes were inoculated into the cooked ham products at an initial level of 104 and 102/g of product. A. S. the addition of 0.5 inhibited these same strains and Mucor pusillus in feeds and silage. and the inhibitory effect was attributed to the diacetate moiety rather than pH alone. Enteritidis.25.1185) acetates are approved as GRAS substances for miscellaneous and general-purpose usage. Pseudomonas fluorescens.0.1% or 0. 32 mM (pH 5. cereus with no effect against Pseudomonas fragi. Saccharomyces species (Glabe and Maryanski. Y. L. In addition to L. However.5 inhibited Aspergillus flavus.2% sodium diacetate and 2.4% at pH 3. Calcium acetate is classified as a stabilizer when migrating from food packaging materials (21 CFR 181.Organic Acids 103 Application and Regulatory Status Sodium (21 CFR 184. 2. 1994). such as Bacillus mesentericus (subtilis).55) at 5°C. 20°C. SA can be used as a boiler additive for food-grade steam (Federal Register. In ground beef. 1962).5% level in malt syrups. monocytogenes was not significantly different in the presence of the individual inhibitors compared with control samples after 20 days at 10°C. The growth of L. or 1% buffered sodium citrate (Stekelenburg and Kant-Muermans 2001).5% sodium lactate or combinations of the acids and held at 5°C or 10°C. vacuum-packaged. An interactive effect was noted between temperature and concentration of acid in that growth was inhibited with decreasing concentrations of acid and temperatures. in products containing buffered sodium citrate. while having little effect on baker’s yeast. monocytogenes. 1994).5 weeks in products containing 0. Levels of 0. monocytogenes grown in BHI broth adjusted to pH 5.8% and 2.0% delayed mold growth in cheese spreads. 5. 2001).

monocytogenes by 0. Toxicology No toxicologic data were available for sodium diacetate (Food and Agriculture Organization. malt syrups. populations increased approximately 0. 1994). a more complex product. Lactate and pediocin enhanced the listericidal properties of diacetate suggesting some type of synergistic activity. . use levels range from 0. It is used in cheese spread. Sodium diacetate (0. plantarum at levels of 0. cerevisiae and 25 times more effective against P.4% (Glabe and Maryanski.3% and 0. The growth of L. Schlyter et al. 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. 1962). 2002). Sodium lactate did not affect the pH of the meat. although it is fungistatic at concentrations of 0. monocytogenes was reduced by 2. (1993b) used the multiple-barrier concept to limit growth of L.1754).2% sodium diacetate alone or in combination in beef bologna. 1993a). respectively. respectively. The powder dissolved in the surface moisture of the carcass to form acetic acid and SA and produced a surface pH of about 4. Slurries of uncured turkey breast. and pediocin (5000 U/ml).5%).3%. monocytogenes in mixtures held at 4°C for 42 days was inhibited at 0.1% (Wolf. Application and Regulatory Status Sodium diacetate is approved as a GRAS substance for miscellaneous and general-purpose usage (21 CFR 184. glaucum and A. 1981). DEHYDROACETIC ACID Antimicrobial Properties Dehydroacetic acid (DHA) has one of the highest dissociation constants (Table 4. Enterobacteriaceae counts dropped 1000-fold over 6 days at 2°C. sodium lactate (2. prepared with sodium diacetate (0. Although 1 M sodium lactate solution reduced populations within 2 days. As expected.. a fermentation by-product of lactic acid bacteria.1%.5 logs CFU/ml slurry. 0. Mixtures were incubated at 25°C or 4°C and sampled up to 7 and 42 days. lower concentrations of the antimicrobials were equally as effective. In bread and cake products..54 log reduction). Sodium dehydroacetate was twice as effective as sodium benzoate at pH 5.1%. Application of 4 M SA reduced the levels of L.5 logs within 6 days. In subsequent work (Mbandi and Shelef. but sodium diacetate reduced the pH slightly. 0. were pasteurized for 10 minutes at 68°C after which they were inoculated with L. Sensory characteristics of roasted chickens were not affected even at levels up to 8 g per chicken (Moye and Chambers. the combination of acids achieved greater reductions of both organisms at both temperatures compared to individual acids and greatly reduced background spoilage microflora. This acid is effective against Enterobacter aerogenes and L.104 Antimicrobials in Food to S. monocytogenes at a level of approximately 4. monocytogenes and Salmonella species was demonstrated using 2. and wrapping material. monocytogenes in turkey mixtures. Sodium diacetate was determined to be listericidal over the 7-day period at 25°C only at the 0. 1991). Enteritidis.1) of the acidulants and remains effective at higher pH ranges. although it is assumed that this derivative would be assimilated in a manner similar to that for acetic acid.59 log reduction).5% concentration (0. 1989). butter. producing a 2-log reduction after 7 days at 25°C (Schlyter et al. niger (Banwart.5% concentrations (0. 1950).5%) was listericidal in combination with ALTA™.25% to 0. L.3% and 0. stored aerobically at 5 and 10°C.6 logs in crabmeat stored at 4°C for 6 days when treated with 2 M sodium diacetate. the reduction of multiple strains of L.8.8 logs (Degnan et al.005% to 0.0 against S.5% sodium lactate and 0.5%). At lower temperatures.

appearance. 30..2). Enders (1941) showed similar symptoms in rabbits. even with prolonged contact (Spencer et al. although a dose of 200 mg/kg led to growth and organ changes.25% or 5% sodium dehydroacetate for 60 seconds were considered more marketable after 3 days of storage at 20°C than water-dipped berries. The effects of potassium sorbate. mortality. However. hematology. in addition to acidosis by the former route and intestinal adhesions by the latter route (Horn et al.1%. and caused slight histologic aberrations.0%. sodium p-hydroxybenzoate. but significant growth depression . The MIC in broth at pH 6. resulting in the loss of up to 20% of body weight. Toxicology The LD50 for DHA in rats is given in Table 4. 2000).2. thus extending marketability (Watada.0% adipic acid showed no effect at the two lowest levels. coli O157:H7 in broth and model meat products. Furthermore.05%. However. ADIPIC ACID Antimicrobial Properties Adipic acid has no antimicrobial properties other than those ascribed to its ability to reduce pH. 1957). benzoic acid. However. 1.130). weight gains were lower and mortality was higher than in the controls (Lang and Bartsch. This acid at the appropriate concentration level would inhibit the growth of any microorganisms susceptible to low pH values..Organic Acids 105 Strawberries dipped in or sprayed with 0. benzoic acid. 300 mg/kg led to severe weight loss and damage to internal organs. produced diarrhea. Application and Regulatory Status DHA or its sodium salt is GRAS when used in accordance with good manufacturing practices. Spencer et al. and sodium dehydroacetate and 16 mg/ml for sodium p-hydroxybenzoate. the 800 mg/day dosage depressed growth. 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. A 2-year feeding study with male and female rats fed 0.. 1950).1% of the substance showed no changes in growth. 1953). 1953). Sorbate was bacteriostatic at half the MIC for E. or histopathology. altered behavior. which included marked obstruction in the liver and kidneys by the oral route and polyuria by intravenous administration. and 0. 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. coli in homogenized beef but not in poultry (Yamamura et al.02%. at the higher dosage levels. and sodium dehydroacetate acid were investigated for three strains of E. It can also be used as a fungistat in cheese wrappers. Intravenous and intraperitoneal injection produced hemorrhaging in the lungs. The sodium salt of DHA was nonirritating to rabbit’s skin. Longer studies using male and female rats given 400 or 800 mg/day did not affect offspring. 0. Toxicology Acute toxicity data for adipic acid in mice and rabbits indicated varied tolerance levels by different routes of administration (Table 4. treatment with sodium dehydroacetate was effective in reducing respiration rate and delaying ripening. 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. or 100 mg/kg of DHA per day for 34 days. or 5.5 was 4 mg/ml for potassium sorbate. 1971). (1950) did not show any unusual effects in rats receiving 10. Oral administration of the acid to mice caused hemorrhaging in the small intestine with distension of the stomach. The 90-day feeding trials conducted on rats fed 0.

and biscuits because it imparts a smooth. 0. 3..04% for cheese. Toxicology Orö and Wretland (1961) determined the LD50 of caprylic acid for mice by intravenous administration (Table 4. Teratologic studies using mice. It is used as a flavoring adjuvant in levels not to exceed 0.1%.. meat products. and 0.8 µmol/ml (Kabara et al.3. Inhalation of adipic acid for 6 hours at a rate of 126 µg/l produced no unusual findings (Gage.0. weight gains for rats fed the two highest levels were significantly lower than those for control rats (Horn et al. The 2-year feeding trials using male rats fed 0. 1968). Sodium adipate at the 5% level decreased the rate of weight gain (Informatics. It is slightly soluble in water. 0.0%. frozen dairy desserts. and soft candy. 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.. 1943). and 205 mg/kg body weight.013% for baked goods. 1972). 1. 1970).005% for fats and oils. the inhibitory concentrations were not dramatically lower (Woolford. CAPRYLIC ACID Antimicrobial Properties When tested against a variety of Gram-positive and Gram-negative organisms and yeasts.1025).8% propionic acid. caprylic acid was ineffective at concentrations of up to 7.001% or less for other food categories.16% for snack foods. 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. not to be used in amounts exceeding good manufacturing practice. It also serves a function in the canning of vegetables. 288.0% level although the food intake level was the same. rancid taste. 0. No limit has been set on the acceptable daily intake for humans. 1980).1009). powdered fruit beverages. Caprylic acid at a 0. and in improving the melting characteristics of processed cheese (Gardner. baking powders. oily liquid that has a slightly unpleasant odor and a burning.106 Antimicrobials in Food occurred at the 5. Caprylic acid is also used as an antimicrobial agent for indirect use in cheese wraps. rats. coli when grown in a chemically defined medium and was more effective than 1. respectively (Food and Drug Research Laboratory. mildly acid flavor. 1975b). Application and Regulatory Status Adipic acid is a nonhygroscopic. refrigerated rolls. however. saturated dicarboxylic acid having low water solubility. . gelatins and puddings. Application and Regulatory Status Caprylic acid is a colorless. It is used in gelatin desserts. The MIC of caprylic acid was 100 µg/ml in TSB for Vibrio parahaemolyticus (Beuchat. 1957).0. 1972).0%. candies. Caprylic acid was also more inhibitory to Shigella species than either acetic or propionic acids (Nakamura and Zangar. however.0% acid did not show any abnormalities. and hamsters showed no significant difference from controls at levels of 263. Caprylic acid is approved as a GRAS substance for miscellaneous and general-purpose usage (21 CFR 184. Inc. At pH 6. caprylic acid inhibited bacteria and fungi at a lower concentration than either acetic or propionic acid.5% acetic acid and 1. At pH 5. as a sequestrant in oils.3% concentration inhibited E. and 5% adipic acid and female rats fed 1. 1973b). The acceptable daily intake for humans is listed in Table 4.2).

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. and 4. but lower pH levels were toxic.5% (pH 4.0.5) was more effective than lactic acid (pH 3. then lactic. 1973. 5. 1994).83 at 10°C and 7. propionic. respectively. canned in a brine containing acetic. aerobic counts at 20°C for pH 3. lactic.09. such as texture or appearance. and propionic acids and for 6 weeks in media containing HCl or lactic acid. Similar pH-temperature relationships were noted by Cole et al. 4.64 log/ml.8 and 2. pH was not considered a reliable indicator of preservation effectiveness (Fabian and Wadsworth. lower aerobic plate counts of 3. followed by acetic acid. 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. Okra. enterocolitica based on concentration of acid.. and 7 (Buchanan and Golden. citric. citric.5% sodium lactate was equally inhibitory as sodium citrate alone. (1989) reported that on an equal molar basis.0. Additional interactive effects were also noted in the presence of salt.7 juice were 4. respectively. Citric acid at a concentration of 0.12 log/ml.42 log/ml (Bizri and Wahem. but inhibition increased with decreasing pH (Murdock. 1997). Citric acid was more inhibitory to thermophilic bacteria than acetic or lactic acids. citric acid was more inhibitory than lactic acid.3. 1950). and 4. monocytogenes survived after 11 to 12 weeks in TSBYE adjusted with acetic. (1990). acetic. Yeast and mold counts were the reverse with counts of 2. 1990). Sodium citrate was also more effective than 2% sodium lactate. the highest inhibitory activity was associated with propionic.13.7 juice incubated at 20°C were 6. Based on pH. 4. monocytogenes after 4 weeks were pH 4. monocytogenes decreased to undetectable levels within 1 to 3 weeks at 30°C. and hydrochloric.0 with citric and hydrochloric acids (Conner et al. Fabian and Graham. A comparison of equimolar concentrations identified citric acid as the most antimicrobial.36 at 10°C. The activity of the undissociated portion of the acid was hydrochloric followed by lactic propionic.7 and stored for 7 days at 5°C.19 at 5°C.7% at pH 6. acetic. lactic. citric.1% at pH 4. or tartaric acid to achieve an equilibrium pH of 4. and 1. who determined that the minimum pH values that allowed survival of L.. 6. 1953). 1994). monocytogenes was achieved in trypticase soy yeast extract broth (TSBYE) adjusted to pH 5.5% sodium citrate and 1. whereas at 10°C. citric.4) in inhibiting Arcobacter butzleri in a broth system (Phillips. followed by hydrochloric.Organic Acids 107 CITRIC ACID Antimicrobial Properties Inhibition of L. 1987). Schoenemann et al. The inhibitory capacity of hydrochloric. was processed for 30 minutes in boiling water. L. Rate of inactivation of L. (1974).0 at 5°C. and phosphoric acid was compared in TSB for Y.0 with propionic acid. or malic acids did not change in physical or chemical attributes during processing compared with unacidified tomatoes (Schoenemann and Lopez. 1939. malic. phosphoric.4 and 0. propionic. and acetic. and degree of dissociation. aerobic counts for pH 3. 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. 2. All acids would be effective antibotulinal agents at that pH level (Nogueira et al.0 and 3.66 at 30°C. When tomato juice was adjusted with citric acid to pH 4. Sorrells et al. pH. The effect was temperature dependent in that survival of L. and the combination of 1. 1999). compared to counts in unacidified juice (6.5 with acetic and lactic acids. Citric acid was particularly inhibitory to flat-sour organisms isolated from tomato juice. phosphoric. indicating that citrate was the more effective compound. however. lactic. These experiments used citrate phosphate buffer in which the citric acid content was calculated at 1. fumaric. The authors suggested the inhibitory activity might be the result of the chelating ability of citric acid.6 log/ml were seen. Acidification impaired the color but enhanced the flavor of canned okra. Interaction was demonstrated between citric acid and sodium citrate added to BHI broth adjusted to pH 4.22 log/ml). Canned tomatoes acidified with citric. and acetic (Brackett. . Little bacteriostatic activity occurred at pH 5.

and 16°C (Valero et al.05 led to more inactivation of S. 1989. the product should . This process inhibited C.5) at 12°C but did not grow in broth acidified to pH 5. 5. 118°C.3. 12°C. Citric acid dips for foods not only retard some spoilage but also act as a chelator of metal ions responsible for enzymatic browning reactions. The preparation of these products included adjustment of the pH with acetic acid to 3. 2003). sporogenes spores per g were heated to an F0 of 0. thereby leading to its survival regardless of the change in pH (Ocio et al.5 were inoculated with C. The authors recommended that the final pH should be <3. 0. 4. Samples pretreated with 1% citric acid displayed significantly lower total numbers. 1991).0% citric acid for 5 or 30 minutes. 8°C.1 with holding temperatures between 18°C and 20°C for up to 3 days before consumption.2. 0. Mayonnaise made with >5% citric acid solution adjusted to pH below 4.. Growth in carrot puree was temperature dependent because B. The products were packed with or without modified atmosphere and stored at 4°C for 10 to 21 days. and 4.85.. By adding citric acid to can brine. If the amount of lemon juice was increased to 20 to 35 ml per egg yolk. cereus strains grown in model vegetable broths were conducted at typical refrigeration and abuse temperatures of 5°C.3 g/kg) for crawfish (Procambarus clarkii) stored at 4°C were not effective in retarding growth of L.5 followed by canning in a solution containing 200 ppm ethylenediamine tetraacetic acid (EDTA) controlled the outgrowth of spores of C. 1990). 2000. cereus could grow at pH 5. Enteritidis PT4 at 22 than 5°C. Both vegetable broths stimulated germination and growth of spores compared to nutrient broth. B. Cabbage and carrots were pretreated with the addition of a reduced calorie dressing or with a dip of 0. 1997. Only 2. Homemade mayonnaise has been a significant health hazard as a result of contamination by Salmonella from raw eggs (Membre et al.3 when at least 20 ml of lemon juice (citric acid) per fresh egg yolk was used. 1992). sporogenes decreased with decreasing pH levels.4 to 4.8% with the addition of EDTA. Samples containing the reduced calorie dressing were even lower in microbial numbers than aciddipped vegetables (Eytan et al. Although the heat resistance of C. but the shelf life was shortened at this higher temperature. 5. leading the authors to conclude that the extracts have a protective effect on C. sporogenes PA3679 after a sublethal heating process (Okereke et al.05 M) solution buffered to pH 3. and 121°C. 115°C. Citric acid dips (0.5).. 1992). botulinum type B when held for 70 days at 15°C or 14 days at 20°C.31% CA–2.108 Antimicrobials in Food Beelman et al. heat resistance was not affected by higher processing temperatures.9% and spoilage was further reduced to 16. (1989) developed an acid-blanch-chelate (ABC) process designed to reduce mesophilic and thermophilic bacterial spoilage in canned mushrooms.8.7 (control). Okereke et al.4. Silla Santos et al. 5. This process improved quality and increased the microbial stability of canned mushrooms (Okereke and Beelman. indicating a synergistic effect between pH and temperature.9.1% potassium sorbate (Houben and Krol. monocytogenes (Dorsa et al.05.34. 6. or 0. sporogenes. Notermans et al. sporogenes PA 3679 spores. The addition of potassium sorbate did not enhance stability. Growth curves developed for B. Extracts were then heated at 110°C. Microbial stability was achieved in liver paste formulated with 0. and 4.. Reduction in the thermal process time of canned liver paste was achieved through the addition of 0..1.65 and asparagus with added citric acid or glucono-delta-lactone to pH levels of 5.4% of cans containing mushrooms vacuum hydrated by the ABC process spoiled (Beelman et al. coliforms. and 0.14% CA (pH 5. (1985) dipped potatoes in a solution of 1% citric acid and 2% ascorbic acid for 2 minutes before vacuum packing and cooking. but could grow at 8°C only when the pH was higher (5. 1999). cereus grew in zucchini broth (pH 6.. Xiong et al.69) and processed with an F0 of 0. 0.0% TCA-0.. They found that acid blanching in a citric acid (0. 1994. 1990b).0 at temperatures below 16°C. Liver pastes inoculated with 106 to 107 Bacillus spores per g and 104 C.22.14% citric acid (CA).3.4 and 5. 1993)..29% CA. 1990a).2% or 1. and lactic acid bacteria counts than samples dipped with the lower concentration of citric acid.1 and 5.31% CA–2. provided temperatures were at 16°C. Mushroom extract acidified with citric acid to pH 6..0% trisodium citrate (TCA). this process reduced thermophilic spoilage from 68% to 23..

1967). A 0. fluorescens on beef carcasses (Thomson et al. casei.0% to 12. When acetic acid was used. with Arthrobacter simplex. 85°C. and 4%.. Similar destruction values were seen with potassium sorbate and sodium benzoate. lactic.08 to 4.5%.5% citric or 0.5 with citric. 1998). 0. or tartaric acid. malic. Sabouraud medium was adjusted with 0. leading to enhanced growth. and 3 to 4 hours at 85°C.25% concentration greatly reduced toxin production.75% for both citric and lactic acids had a slight growth-retarding effect on A. Healthy tissue had a pH of 5.. Using the multiple-barrier concept. 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.Organic Acids 109 be held at 22°C for longer than 72 hours. Sodium citrate can also exhibit bacteriostatic activity.75% lactic acid inhibited growth of Aspergillus versicolor. 1968).8. sodium citrate inhibited growth. 80°C. or shrimp and tomato sauce acidified with citric or acetic acids to pH 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. expansum (Reiss. Citric acid provided the maximal destruction of ascospores at 85°C. aureus showed inhibition both with increasing concentrations of citric acid and decreasing pH (Minor and Marth. The addition of garlic or mustard increased the death of S. or 1. when 35 ml lemon juice per egg yolk was added. Mangoes are subject to spoilage through infection with Penicillium cyclopium (Palejwala et al. the level of acidity decreased and sugars and nitrogenous compounds increased. 1970). Sodium citrate in concentrations of 0. it did not appear to affect attachment (Appl and Marshall. whereas infected tissue was around 4.2 or 4. Citrate inhibition of S. As little as 0. The amount of sodium citrate had a dual effect on Lactobacillus casei. Thermal resistance studies were conducted in mango juice adjusted to pH 3. no growth occurred at pH 5.0% was not as inhibitory to Streptococcus agalactiae when added to skim milk or fresh milk as citric acid at concentrations of 1%. Skim milk acidified with citric acid was more inhibitory to S. In concentrations of 12 to 18 µM/ml.. The metabolic effects of citrate on Arthrobacter species were twofold. 1984).5% sodium citrate were inhibitory to Salmonella anatum and Salmonella oranienburg (Davis and Barnes. 1968). aureus was overcome by adding calcium and magnesium ions.1 or less.8% gave a pH of 4. (1985) found no outgrowth or toxin production from spores of types A and B in TPGY medium acidified to pH 4. Enteritidis. 5 hours at 80°C.8.75% concentration of either acid did not affect the growth of P. and rinsing with a citric acid solution adjusted to pH 4. but salt had a protective effect on this organism.6 and incubated at 35°C. With Arthrobacter pascens.1% to 4.0% concentrations of citric acid and 0. Typhimurium than lactic and hydrochloric acids (Subramanian and Marth. During ripening. and in concentrations greater than 40 µM/ml. even in media containing up to 1% citrate.. however. suggesting that citrate was a chelator of ions essential for growth (Rammell. Branen and Keenan (1970) also suggested that citrate chelation of metal ions inhibited L. hard-cooked eggs allowed to equilibrate in 0.3% citric acid lowered the level of Salmonellae on poultry carcasses.016% to 1% malic acid as the sole carbon source because these acids predominate at these concentrations in a ripening mango.0. However. botulinum showed no change in numbers when inoculated in tomato puree. The MIC of 0. S. 1984). No toxin was detected in any of these foods over an 8-week shelf life (Post et al. 2%. 1976). A level of 0.5% lactic acid prevented toxin production.0 reduced the numbers of P. 1962).. citrate appeared to inhibit glucose utilization (Imai et al.75%. A level of 0. shrimp puree. the function of citrate appeared to be one of chelation of metal ions.. C.125% to 4% citric acid or 0.25% citric acid and 0.6.2% sodium benzoate were held for 30 days at 4°C or . Tsang et al.12 (Sinha et al. however. sodium citrate stimulated growth. 1970). 1952). Ascospores were able to withstand more than 6 hours of heating at 75°C. type E grew and produced toxin at 26°C in media containing citric acid at pH 4. the time decreased to 48 hours before use. Concentrations of 12. but 0. Survival studies for Neosartorya fischeri ascospores were conducted in mango and grape juices and heated at 75°C.2. a 0. and 90°C. parasiticus. 1985).

in the acid form (21 CFR 184. (1957) evaluated dosage levels in mice given the acid intravenously. 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. rats. Short-term studies on rats fed 0. Citric acid is approved as a GRAS substance for miscellaneous and general-purpose usage when used in accordance with good manufacturing practices. Gruber and Halbeisen (1948) used comparable levels of acid but suggested that many of the symptoms produced by high levels of citric acid resembled those of calcium deficiency.1195). Cramer et al. The acceptable daily intake for humans is listed in Table 4. Although there was no effect on weight gain. 1972).1625). 1981). E. 1971).. 7. aureus early in the fermentation and the increased acidity of the meat mixture allowed the fermentation to proceed more quickly. enterocolitica. This combination inhibited the growth of S. fruit preserves. It is approved for use in ice cream. Potassium sorbate at 0. and jams and jellies.2%. slight atrophy occurred in regions of the thymus and spleen (Yokotani et al.. (1982) used a combination of 0.75% glucono-deltalactone incorporated into sausage before fermentation. Acute toxicity data of sodium citrate are shown in Table 4. and rabbits indicated varied tolerance levels by different routes of administration (Table 4. A concentration of 0.05% potassium sorbate and citric acid at pH 5. Long-term studies involving rats fed quantities up to 1. the highest dosage contributed to enamel erosion.1033) or as the calcium (21 CFR 184. 1957) showed no abnormalities other than lowered weight gains and feed consumption in the 5% group. and 4. the urine contained calcium citrate. or sodium salt (21 CFR 184. Daly et al. Dalderup (1960) fed rats 1.0 to reduce the growth rate of S. Citric acid also acts synergistically with antioxidants to prevent rancidity by chelating metal ions (Gardner.75% citric acid alone for 21 days at 4°C.8% level. 2. and S. 3%.75% citric acid was sufficient to reduce inoculated populations of S.2%.02 mol sodium citrate and citric acid.2). or 5% citric acid (Bonting and Jansen. Death by all routes and in all animals resulted from respiratory or cardiac failure and hemorrhaging of the gastric mucosa. 4.5 depressed growth of L. Horn et al. Deaths resulted from acute acidosis and lung hemorrhages. It is highly water soluble and enhances the flavor of citrus-based foods. a by-product of yeast fermentation. coli. 1956.5.1% citric acid and 0. The authors concluded that citrate had a rachitogenic effect on the test animals.5. sherbets and ices. salad dressings. It is a precursor of diacetyl and therefore indirectly improves the flavor and aroma of a variety of cultured dairy products.. Horn et al.3.2% concentration in combination with citric or lactic acid at pH 5. potassium (21 CFR 184.7% sodium citrate (5% citric acid) fed to rabbits for 60 days produced no unusual effects (Packman et al. .110 Antimicrobials in Food in 0. (1956) fed vitamin D-free diets containing low calcium levels but adequate levels of phosphorus supplemented with 0. however. 1944). Studies by Yokotani and coworkers (1971) compared toxicities of commercial citric acid and Takeda citric acid. aureus (Fischer et al.. Y.. (1973) used a combination of 0. Restaino et al. It can control the pH for optimum gel formation. Toxicology Acute toxicity data for citric acid in mice. Application and Regulatory Status Citric acid is a tricarboxylic acid having a pleasant sour taste. Typhimurium. rouxii and Saccharomyces bisporus. beverages. 1985).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. plantarum but did not affect P. The number of cavities formed did not differ between the control and experimental groups. In another study.1751). and it is used as an acidulant in canned vegetables and dairy products. or 12 g citric acid per kg of a noncariogenic diet to show the possible effect of citric acid on the development of teeth. 1963).2.4%. aeruginosa (Restaino et al.

Cans packed with bacon and inoculated with C. citric. or tartaric acid based on weight when added to the heating medium. citric. When used in vacuum-packaged ground beef patties. and tartaric acids increased heat inactivation. 1947). coli O157:H7 reduced levels by 1 and 1. but the dimethyl and diethyl esters at a concentration of 20 mg per 2. Islam. 0. At the 1. 1996a. Fumaric acid was more fungicidal against Talaromyces flavus ascospores than acetic. fischeri (Conner and Beuchat. malic. botulinum swelled in 4 to 6 days at 30°C.0% and 1.15%) served as an acidulant (Ough and Kunkee.125%. Similar effects were noted with N. It was speculated that the antimicrobial properties of fumaric acid were related to a lower pKa than lactic or acetic acids. respectively. Fumaric acid by itself was only slightly inhibitory.1%) controlled fungal growth in tomato juice. These same preservatives provided a similar reduction in less than 5 hours and 2 hours when held at 25°C and 35°C.6 L in the vapor phase were also effective as a mold preventive (Huhtanen and Guy.5% concentration of fumaric acid inactivated ascospores after 20 minutes of heating at 80°C. Fumaric. Monomethyl and monoethyl (0.0% fumaric acid did not show any abnormalities. psychrotrophic. total aerobic counts were reduced to levels below those achieved through pasteurization. 1982). malic.. rats showed an increased mortality rate and more atrophy of the liver (Fitzhugh and Nelson.Organic Acids 111 FUMARIC ACID Antimicrobial Properties Fumaric acid (0.5.0% acid concentration at 82°C..1%. The use of fumaric acid to effect a 5-log reduction of E. and tartaric acids at a concentration of 2.2% fumaric acid or rabbits fed sodium fumarate at a concentration of 6.5% level. coli O157:H7 inoculated into unpreserved apple cider was investigated by Comes and Beelman (2002).0) were not lethal to T. flavus ascospores (Beuchat.9% (5% fumaric acid) showed no toxic effects (Packman et al.38% sodium fumarate or guinea pigs fed 1. A 0.8%. and di-n-alkyl fumarates were almost inactive (Dymicky et al. Methyl n-alkyl fumarates demonstrated lower activity.5%.05%) or sodium propionate (0. Fumaric acid was also more effective than lactic or acetic acid when used in meat products (Podolak et al. Monomethyl..2% showed considerable promise as a substitute for sodium nitrite in bacon.5 to 5. 1987). 1987). whereas the presence of fumaric acid esters in the bacon prevented swelling for up to 8 weeks.5% applied for 5 seconds at 55°C to the surface of lean beef inoculated with L.b).0 to 2. The fumarate esters not only prevented mold growth on bread (Huhtanen et al. dimethyl. and lethality increased as the pH decreased from 5. The addition of 0.1% and 1. 1963)..15%. The n-monoalkyl fumarates and maleates esterified with C13–C18 alcohols also possessed significant antibotulinal activity (Dymicky et al. respectively. 1988). monocytogenes and E. but malic acid did not. 1975). 0. Human feeding .2%) and dimethyl and diethyl fumarates (0. The 2-year studies using rats fed 0. 1987) heated in the presence of 2.3 logs.15% fumaric acid coupled with 0. Huhtanen (1983) and coworkers (1985) investigated the effect of various esters of fumaric acid on growth of C. 1982). 1984). However. 1974) and inhibitor of malolactic fermentation in wine (Pilone. Medium chain-length alkyl esters had more activity than aliphatic acids (Dymicky and Trenchard. Toxicology Weanling rats fed 0. coli O157:H7. Fumaric acid at concentrations of 1. 0.05% sodium benzoate followed by holding at 25°C for 6 hours before refrigeration at 4°C for 24 hours achieved reduction. botulinum types A and B in canned bacon. In addition to reduction of E. fumaric acid achieved the greatest reduction of aerobic. and coliform bacteria. 1981.. and ethyl esters of fumaric acid in concentrations of 0. citric.5% (pH 2. acetic. or 0.0% fumaric acid and 1. and 1.

an LD50 of 8. 1972). 1983. Lactic acid sprays have been effective in limiting microbial growth on meat carcasses under a variety of storage conditions.000 mg/kg was proposed (Levey et al. 1970).112 Antimicrobials in Food trials using a 500 mg/day oral dose for 1 year were also negative. 1988). 1946). 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. followed by vacuum packaging.3. lowered microbial counts after storage for 14 days at 2°C. Fumaric acid is used in fruit drinks. Although this additive has a strong acid taste. fumaric acid does display some antioxidant properties in fat-containing foods (Gardner. Because most other studies chose refrigeration storage temperatures. unsaturated.7 log CFU/cm2 (Visser et al. biscuit doughs. 1991). Lactic acid was an excellent inhibitor of spore-forming bacteria at pH 5. pork.350).3 of veal tongues. 1985). but a 2% concentration led to discoloration of the carcass surface (Smulders and Woolthuis. Enterobacteriaceae contaminated 50% of the untreated samples. combined with vacuum packaging and storage at 3°C. Fumaric acid and its salts are used for direct addition to food for human consumption (21 CFR 172. Lactic acid was more inhibitory to Mycobacterium tuberculosis as the pH decreased (Dubos. although more recently this acid has been used as a rinse for beef. 0. LACTIC ACID Antimicrobial Properties Lactic acid has been used more extensively for its sensory qualities than its antimicrobial properties in the past. and wines. Destruction rates for S. reduced aerobic mesophilic plate counts from 5..25% lactic acid spray of beef carcasses followed by a treatment of hot-boned cuts with 2% acid. . 1985). propionic.1% ascorbic acids selectively inhibited Enterobacteriaceae at 10°C. Lactic acid was about four times more effective than malic. the organism responsible for flat-sour spoilage in tomato juice (Rice and Pederson.. this study showed that lactic acid was effective at higher temperatures as well. The inhibitory capacity of this acid lies in its reduction of pH to levels below which bacteria cannot initiate growth. Application and Regulatory Status Fumaric acid is a nonhygroscopic. 1988).25% citric. Cold-pack cheese normally prepared with lactic acid was reformulated with acetic acid and inoculated with S.0% to 1. Woolthuis and Smulders. and acetic acid in limiting growth of Bacillus coagulans. Gill and Newton (1982) suggested that the inhibitory effect for psychrotrophic organisms was related to a decrease in pH rather than degree of dissociation. gelatin desserts. The acceptable daily intake for humans is listed in Table 4.6 to 2. 2% acetic. it blends with certain flavoring compounds to intensify the aftertaste of a flavor. In fermented foods.. and chicken carcasses. In addition to supplying acidity to a product. Osthold et al. 1950). Because of these dosage levels. A 1. Typhimurium. and storage for 10 days. (1984) found that spraying beef and sheep carcasses with a combination of 1% lactic. It increases the strength of gelatin gels.. which was reduced to 10% after treatment. Similar reductions in microbial counts occurred with a 1. 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. 1954). citric.25% concentration of acid sprayed on veal carcasses. Acid treatment when combined with vacuum packaging was more effective in prolonging shelf life than vacuum packaging alone (Smulders and Woolthuis. vacuum packaging. Typhimurium were similar for both acids (Park et al. 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. 1975a). pie fillings. A 2% L-lactic acid treatment at pH 2. dicarboxylic acid having low water solubility. and 0.

71°C. COM improved tenderness in muscle cuts. 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. coli O157:H7 and S. (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. coli was reduced by 6. coli counts was detected for beef dipped in acetic or lactic acid solutions compared with a water-dipped control at day 1. combinations of the treatments were examined. but odor was not affected (Ellebracht et al. Typhimurium levels decreased approximately 1 log. coli (Anderson and Marshall.3.3 M lactic acid solution (LAC. coli.b).5 logs (Anderson et al. or a combination (1:1) CAL/LAC (COM. total fecal coliforms. and 0. or 510°C.1% ascorbic acids adjusted to pH 2.25% citric.2%. The inoculated meat was subjected to sprays of tap water (15°C to 17°C. sub-primal meat was injected with one of three treatments: 0. the FPT supported lower microbial numbers than LPT.. 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. (1987) compared a combination of 1% lactic. Typhimurium-inoculated beef trimmings. 1999).. Dixon et al. A residual effect was noted for the lactic acid-treated tissue in that microbial counts were significantly decreased compared to the control steaks after 9 days of storage. 2001a. Postchill beef rounds inoculated with E. Acuff et al.6% or 1.. All products were stored at 4°C and examined for aerobic. When sequential treatments were employed using pre-chill and postchill lactic acid sprays. 30 psi). pH 5.3 M calcium chloride solution (CAL. When temperature changed from 20°C to 70°C or lactic and acetic acid concentrations increased individually to 3%. Meat color was affected after application and through storage at 4°C. reducing numbers by 2 logs and Enterobacteriaceae by 1. pH 3. Aerobic plate counts of meats injected with CAL were significantly higher than with LAC or COM. and sensory analysis were not affected by acid treatment. Similar work was conducted using lean pork trim (LPT) and fat-covered pork trim (FPT) (Castelo et al. 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. only suggesting that lactic acid can exert a continued antibacterial effect during the shelf life of the product. 2% acetic. and presumptive lactic acid bacteria. The combination of acids performed as well as 1% lactic or acetic acids alone.. The system alone produced a 3log reduction. E. Treatments containing lactic acid resulted in the lowest counts without affecting sensory qualities. 2001). In addition. 454°C. 1994). at 1°C to 2°C as a dip for rib eye steaks. aerobic bacterial counts and S.8 to 7. or 82°C. As in beef trim.. 1992). moisture content. 2% lactic acid (12°C to 15°C. 1998. but control cuts retained more desirable flavor profile (Eilers et al. and at two time intervals. 0. Furthermore. b). 76°C. coli O157:H7 or S. 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. but this effect was not seen with acetic acidtreated tissue.9 or 1% acetic acid at pH 3. 1990a. 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. psychrotrophic. or hot air at 371°C. Acid-treated samples were lighter in color as a result of leaching of the pigment during immersion.b).5 logs for E. Kotula and Thelappurate (1994) compared acetic acid and lactic acid solutions applied at two concentrations. Application of 95°C water alone or with 2% L-lactic acid was effective in reducing E.. <1 log for Enterobacteriaceae and 0. Typhimurium at 70°C. coli biotype 1 counts. pH 2. but shear values. Minimal reduction in total plate and E. 426°C.Organic Acids 113 Hot-boned. Lactic acid was most effective against S. 482°C. 20 or 120 seconds. .5) at a volume equal to 10% of the initial sub-primal weight.0). 0. 0. Beef trim meat used to make ground beef is of particular concern because of its higher microbial load.2 logs. 65 psi). Greater reductions on all microbial loads were seen in BTF compared to BTL.1).6 with either 1% lactic acid at pH 2. There was little difference in bactericidal activity between the combination and either acid alone on sub-primal cuts of beef. 2001a. and E.

Therefore. Woolthuis et al.2 or 4. whereas acetic acid was more effective than the acetic acid/gel treatment. monocytogenes by 1. vacuum packaging. 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.. coli O157:H7 was subjected to treatments of lowmolecular-weight polylactic acid. The combination of acids and heat treatment led to reduction of spore-forming organisms in frankfurters. or 360 seconds to 2% lactic acid. significant reductions were seen with all treatments. psychrotrophic Gram-positive bacteria. acetic acid (0. Elimination of these groups shifted the population to Gram-positive bacteria and yeasts. and 0. Lactic acid treatments alone were only 50% as effective as acid/alginate treatments. however. but no differences were seen between polylactic acid and lactic acid. or hydrochloric acid (Lynch and Potter. Beef was inoculated with L.5%). and polylactic acid. 1992. 1992).5 and 4. Y. 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. by immersing porcine livers in 0. 55°C) had little effect on the aerobic plate counts taken from the surface of pork carcasses.000 IU/ml nisin. malic. and 3200 units/ml pediocin. coli O157:H7 in beef burgers. or sodium lactate (4%) alone or in combination with pulse electric field or freezing was ineffective in reducing numbers of E. Lactic acid was effective in decreasing the number of Enterobacteriaceae as well as other Gram-negative mesophilic and psychrotrophic spoilage organisms. 1999). Beef inoculated with E. After 42 days of storage the initial inoculum of 5 logs was reduced to <1 log after using lactic acid. coli O157:H7. Beef cubes were inoculated with L.114 Antimicrobials in Food Lactic acid (2%). followed by mesophilic Enterobacteriaceae.. Gram-negative bacteria were the most sensitive to lactic acid. Salmonella species or Listeria species were not recovered nor were sensory characteristics affected (Prasai et al. nisin. 2002). monocytogenes. noncoated tissue became dehydrated during the 7-day study. Numbers of S. 1. Enterobacteriaceae.2 for B. Greater inactivation was noted for both organisms at the lower pH level when acetic or lactic acids were used but not with citric. A 1% concentration of lactic acid (pH 2.2% lactic acid for 5 minutes. monocytogenes and treated with 2% lactic acid. 1993). Frankfurter emulsions incorporated with acids to adjust pH to 5. (1984) effectively reduced total plate counts. Although the gels reduced populations of L. Psychrotrophic.6 for Bacillus stearothermophilus and pH 4. The use of these compounds was effective in reducing psychrotrophs and Enterobacteriaceae (Ariyapitipun et al. A novel approach to shelf life extension was the use of lactic or acetic acid-impregnated calcium alginate gels applied to the surface of beef tissue (Siragusa and Dickson. and nisin did not enhance the effects of lactic acid against E. . enterocolitica. lactic acid. 1993).. respectively (El-Khateib et al.7. lactobacilli and yeasts were the least sensitive. and nisin (400 IU/ml) were used alone or in combination in beef to inhibit L.8. monocytogenes (Ariyapitipun et al. low-molecular-weight polylactic acid (2% PLA). Lactic acid (2%). and Lactobacillaceae counts by 2 to 3 logs. but the population of microorganisms shifted. Five treatments were delivered to the pork skins: water-treated (control) or exposure at 21°C for 120. coagulans were heated to 121°C and 105°C or 110°C. these treatments were not deemed usable in the control of E. 240. 2002). and low-molecular-weight polylactic acid. Aeromonas hydrophila. there was no significant difference between nisin. or nisin and lactic acid.1. numbers of Salmonella species and Campylobacter species were reduced immediately and remained lower 24 hours after slaughter (Epling et al. 2000). The use of lactic acid for reduction of microbial numbers or pathogens has been similarly applied to pork products. After 28 days of storage. 1993). Typhimurium were reduced more by exposure to acetic acid/gel treatment than acetic acid alone.. monocytogenes before vacuum packaging and storage at 4°C.. All treatments significantly reduced the number of L. By increasing the concentration of lactic acid to 2%. monocytogenes. 40. These compounds reduced the level of L. lactic acid.6 log/6 cm2. coli O157:H7 inoculated into beef trimmings (Bolton et al. and storing for 5 days at 3°C. 1988). Not only did the microbial counts decrease as the time of decontamination increased... monocytogenes on beef tissue. 180. and 200 IU/ml of nisin (Mustapha et al.

None of the pathogens grew on lean tissue. The interaction of sodium chloride (0. coli O157:H7 rapidly died at pH 4. Acid treatments of fat tissues led to the reduction of pathogen numbers to nondetectable numbers within 4 days. thermosphacta. and a 2% lactic acid dip at pH 2. fragi and B. reduced levels of lactic acid (0.. E.2 decreased from 5. tissue was dipped for 15 seconds in water or lactic acid and held for 15 days at 4°C. 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.7 log CFU/g and Enterobacteriaceae from 3.6 log CFU/g.88 or 0.. coli O157:H7 grown in TSB at 37°C (Casey and Condon. Levels of psychrotrophs decreased from 3. sodium tripolyphosphate. Total microbial numbers from skin of birds immersed for 15 seconds at 19°C in 1% or 2% lactic acid at pH 2. This protective effect was even more pronounced with acetic acid (100. 1990). however.2 logs.5% sodium benzoate dip concluded that sensory qualities were acceptable (Hathcox et al. 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. pH 2. E. After inoculation. monocytogenes compared with monosodium phosphate. and A. and Beuchat. acidified with lactic acid..2 in media containing lactic. Although L. Salmonellae were eliminated from broiler carcasses after a 1-hour exposure. however. S..3 prolonged shelf life to 8 days (Zeitoun and Debevere. The treatments did not affect sensory quality. 2002).5% to 4%) and pH (4.0 than cells grown at 37°C. Lactic acid added to scald water alone had minimal effect on reducing the numbers of contaminated birds. or Brochothrix thermosphacta (Greer and Dilts.1°C. in sodium chloridefree. although tissue treated with acid affected the growth rate. discoloration still occurred and propylene glycol contributed an objectionable flavor (Izat et al. When treated broilers were packaged using modified atmospheres. grew on both tissue types after acid treatment. 1995). Lactic acid dips have also been used successfully in the poultry industry. 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. 1990a). P. such as Pseudomonas species. hydrophila numbers declined by 5 logs within 11 days of storage. The number of Salmonella-positive birds was also reduced as a function of time of the dip (Izat et al.9 to 2.2) was examined for E. Consumer evaluation of chicken treated with a 0. other compounds. respectively (Sawaya et al. or formic acids. pH 2. coli decreased 10. fragi. Hwang. Lactic acid added to broiler chill water resulted in the development of a brown coloration. The spoilage organisms. sodium acid pyrophosphate. that contributed to spoilage.7 log CFU/g (van der Marel et al. acetic. monocytogenes counts were reduced by 0. coli demonstrated a 10-fold decrease in numbers when grown in media containing sodium chloride.5% lactic acid/0. 1995).25%.23 in cells exposed to acid to 5. or sodium hexametaphosphate. shelf life was lengthened to >36 and 35 days at 4°C and 7°C.5 and 0.000-fold).. pH 2.79 in acid-free media. respectively. .3 to 2. 1988).62) were combined with propylene glycol (20%) in chill water.5%. 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. Chicken skin subjected to washing treatments using 1% lactic acid led to significant reductions of Salmonella species and L. Lactic acid was more effective than acetic acid when used as a 10-minute rinse for fresh-cut vegetables.Organic Acids 115 P. such as chlorine or chlorine dioxide. 1983). Typhimurium to almost nondetectable numbers. acidified media.8) and scald water (54°C/2 minutes) reduced the bacterial level of broilers inoculated with S. E. A 10% lactic acid and sodium lactate-buffered acid spray (pH 3. These treatments inhibited hydrogen sulfide-producing bacteria.2 to 3. Higher concentrations of the acid did not ensure greater decontamination nor did repeated treatments. 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. 1995).000-fold. 1995. produced similar reductions of approximately 1 log (Zhang and Farber.0) for chicken legs increased the shelf life from 6 to 12 days at 6°C. 1996). 1990b). but no protective effect was observed if the sodium chloride was added to the medium 45 minutes after the inoculation. Lactic acid (1%) added to both chill water (0°C to 1. In an effort to eliminate carcass discoloration.

1998.8. cultures decreased production of aflatoxin G1. Brochothrix. Yeasts were very broad in . parasiticus in association with L. Gram-positive bacteria including lactic acid bacteria. Ross et al. innocua from 804 to 982 mM. D (-) or DL. and lactic acid concentration has been developed (Mellefont et al. A predictive model for growth of E. appears naturally in meat products. The MIC of sodium lactate was investigated under optimum growth conditions (pH 6. With increasing levels of lactic acid. Bacillus. and in baking powders.2). increases meat yields. 1973) supported this view.. The Food and Agriculture Organization of the United Nations (Food and Agriculture Organization.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. Lactococcus. 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. lactis. (1970) recommended that the L (+) form should be used in feeding premature infants. sherbets. Gram-negative bacteria including Pseudomonas and Yersinia had a comparable range to Salmonella. improves juiciness. Carnobacterium.5 or 4. Presser et al.. and Staphylococcus ranged from 268 for S. Aflatoxin production also increased during growth of A. water activity. Premature infants fed acidified milk containing the isomeric forms of lactic acid.5% lactic acid (Trainer et al.116 Antimicrobials in Food El-Gazzar et al. 2003).75% lactic acid adjusted to pH levels of 3. pH. Lactic acid is approved as a GRAS substance for miscellaneous or general-purpose usage (21 CFR 184. confectionery products.. 1944). dehydration. Hamsters fed a cariogenic diet incorporating lactic acid in the drinking water (40 mg/100 ml) or in the feed (45. Toxicology The LD50 of lactic acid for test animals varied according to animal species (Table 4. coli in response to temperature. 1945). (1987) found that A. MIC values for Salmonella species ranged from 714 to 982 mM and a narrower range for L. Ballabriga et al. jellies. although Campylobacter was particularly sensitive with an MIC of 179 mM.3. 2003. The acceptable daily intake for humans is listed in Table 4.5% and 0. and vomiting.. parasiticus NRRL 2999 grown in a laboratory medium containing 0. Therefore. infants suffered stricture of the esophagus following ingestion of milk acidified with 1 teaspoon of 85% (Pitkin. see Shelef (1994) and De Wit and Rombouts (1990). For an excellent review of the antimicrobial nature of sodium lactate.1061). developed acidosis. monocytogenes and L.. It is used to adjust acidity in brines for pickles and olives. 1949). 1935) or 87. Luchese and Harrigan (1990) also noted an increase in aflatoxin production adjusted to pH 4. Application and Regulatory Status Lactic acid is a hygroscopic. In two other instances.5. to prevent discoloration in fruit.. syrupy liquid having a moderately strong acid taste. contributes to water-holding capacity. Lactic acid is used in the manufacture of jams. weight loss. aureus and Lactobacillus coryniformis to 804 mM for Brochothrix. and beverages.2 using HCl or lactic acid stimulated by substituting half of the carbon content by lactate and a reduction in production at pH 6. retains color.6 mg/100 g) did not differ in growth rate compared with control groups (Granados et al. 20°C) for a number of bacteria and yeasts isolated from spoiled and unspoiled meat products (Houtsma et al. Some enamel decalcification was apparent in the experimental groups. 1993). The autopsy revealed hemorrhaging and gangrenous gastritis (Young and Smith. Infants died after consuming milk acidified with an unknown quantity of lactic acid. Calcium lactate is used as a firming agent for apple slices. and extends the shelf life of products.

monocytogenes as the target organisms.0% sodium lactate. monocytogenes per gram of sausage..5%. with lower amounts of sodium lactate needed as pH and temperature dropped. potassium. with Debaryomyces and Candida being the most resistant at 1161 and 1339 mM. 1% sodium lactate delayed growth for 10 days. 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. spore germination. 6.1. 20°C. respectively. Pork liver sausage was formulated with sodium. Sodium. 1995) examined the effect of 3% sodium or calcium lactate added to pork liver sausage and various heat treatments.0.Organic Acids 117 their sensitivity. 10°C. and 2 M concentrations (Buchanan et al. At the abuse temperature of 20°C. At 30°C. and 4 using 0. L. Fresh pork sausage formulated with 0% to 3% sodium lactate was vacuum packaged and stored for up to 28 days at 4°C (Brewer et al.5. calcium. 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. Survivor curves were developed using L. indicating that inhibition was not the result of a lowering of water activity. Sodium chloride at the same water activity as sodium lactate (0. Addition of 2% sodium chloride enhanced the listericidal activity of the lactates (Weaver and Shelef.5. 1994a). Typhimurium when stored for 4 days at 20°C. The pH level was influential in inhibition by sodium lactate but not by sodium chloride. The effect of sodium lactate on toxin production. Calcium lactate was also more effective than sodium lactate in retarding growth of E. At a higher temperature of 20°C. 6. 1. 1993).0 at 4°C. 1995).. 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. Using 108 CFU/g as a microbial end point. innocua was modeled in broth adjusted to pH 5. toxin production was detected in media containing 4% sodium lactate within 11 days. As expected L. inhibition by sodium lactate was directly related to pH and temperature. 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 21.982) did not inhibit toxin production. Lactic acid/sodium lactate and acetic acid/SA were studied in a BHI broth at pH 7. 0.5. Samples were stored up to 50 days at 5°C and up to 10 days at 20°C.0. and 30°C (Houtsma et al. 3%. The calcium form was found to be more inhibitory than the sodium form.. its concentration. This level was listeriostatic at a moisture of >55%.. toxin formation was noted within 5 days for 0%. 5. 1991. botulinum strains in peptone yeast extract medium (Houtsma et al. 4% sodium or potassium lactate achieved reductions in L. monocytogenes was studied in a cooked meat model system containing varying moisture levels and 4% lactate.5%. the rate of inactivation was dependent on the acid. A subsequent study (Shelef and Potluri. 1994b). 6. 11 days for 1. coli O157:H7 and S. Potassium lactate (4%) and sodium chloride (3%) reduced growth somewhat at 5°C. Salts of lactic acid are equally as effective in muscle food systems. and heat resistance was tested on proteolytic C. and potassium lactate were equally effective. and 7. 1993). and the resultant pH. The effect of sodium lactate and sodium chloride on the growth of L.. comparable to a 3% concentration of calcium lactate. or calcium lactate at concentrations of 2%. This model was then evaluated for use in a bologna-type sausage system (Houtsma et al. lactates also contributed to lowering the aw of a food. no growth or toxin formation was detected after 32 days of incubation for media containing 4. and 15 days for 2. As expected. Chen and Shelef (1992) suggested that in addition to pH change. There did not appear to be a synergistic effect between a 4% concentration of lactate combined with 140 ppm . however. but the combination of 2% or 3% lactate and 2% sodium chloride at the same moisture level was inhibitory. Calcium lactate (2%) reduced populations below the inoculum level. or 4% and inoculated with 104 to 105 L. no toxin formation was detected after 49 days of incubation in media containing 3% sodium lactate. As expected. whereas a 2% level extended the time to 24 days. 1996). which might explain their prevalence in spoiled meat products. monocytogenes. spore germination did not lead to growth after 7 days.

Sodium lactate at concentrations of 3% and 4% were generally effective in limiting growth of L. Typhimurium. botulinum.0% or 3.c) injected beef top rounds with 0% to 4% sodium lactate.5 log CFU/cm2 in the same period. 2. S. Pork sausages manufactured with 25% fat or 8% fat with and without 3. The microflora changed from predominantly staphylococci and micrococci at 0 days to coryneform. and A.b. 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. or 4. Lactate increased cooking yields.5% SA or sodium diacetate as an antilisterial process for frankfurters. enterocolitica or L. At pH 6. then cooked and vacuum-packaged the roasts. micrococci. Red color was preserved by using 2% to 3% sodium lactate. Retention of nitrite was linked to higher pH levels and was unaffected by the incorporation of 1. monocytogenes was delayed for 4 and 12 weeks at 7°C and 3°C in unsmoked. Frankfurters containing 2.0% potassium lactate showed differences in spoilage rates. 1993). enterocolitica. monocytogenes and heated to 55°C to simulate a sous vide process (McMahon et al. coli O157:H7 and stored at 10°C. thermosphacta down to pH 5. the plate counts of treated roasts increased to 6. hydrophila and aerobic growth of the aeromonads (Grau. Another study (Bedie et al.. uncured bratwurst manufactured with 3. respectively (Porto et al. 0. aureus. Enterobacter cloacae. Although the aerobic plate counts for control meat increased from 2. such as wieners.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. The heat resistance of the two organisms was significantly lowered in beef containing either of the two concentrations of sodium lactate. or 4% sodium lactate were inoculated with L. enhanced flavor at the 1% level by adding a salty taste. Sodium lactate at a 6% concentration or 0.5 in a meat system prevented anaerobic growth of Serratia liquefaciens. 1994).25% sodium diacetate were the most effective treatments throughout the 120-day shelf life conducted at 4°C with minimal change in pH.7 to 8. C. monocytogenes during storage at refrigerator and abuse temperatures. Y. lactobacilli. the amount of L-lactate ordinarily found in muscle was not inhibitory to any of the strains. Levels of >3% sodium lactate or 1% each of lactate and diacetate were needed to prevented growth of Listeriae for 60 days at 4.05 and 6. A 100-mM concentration of sodium lactate buffered to pH 5.4%. but produced a mild throat irritation at a 4% concentration.4%. 1980)... 1991). 2. S. monocytogenes. A 3% concentration of sodium lactate at pH 7. or E. nitrite levels are reduced. (1991a. The growth of L. Both concentrations of potassium lactate significantly inhibited or delayed the growth of L. rely on sodium nitrite to inhibit growth of C. monocytogenes. Papadopoulos et al.. Cured meats. perfringens.2%. Sodium lactate was less effective against microorganisms when sausage contained soy concentrate.6% sodium lactate into the product (Kilic et al. Typhimurium. Papadopoulos et al. Over the storage life of the product. (1991b) did not find this effect in their studies. 1999).. or 3. 3%. .8% sodium lactate was inoculated with Y.5 reduced surface discoloration in chubs of fresh sausage and extended shelf life from 10 to 20 days.. Although Shelef and Yang (1991) suggested a possible aw effect for sodium lactate. Minced beef containing 0%. coli O157:H7 (Miller and Acuff.4% sodium lactate/0. 1981). monocytogenes and stored at 4°C and 10°C for up to 90 and 60 days. and yeasts at 42 days to homofermentative and heterofermentative lactobacilli and streptococci at 84 days.25% or 0. 2002).1. Lactates inhibited the growth of psychrotrophic organisms and delayed the pH decline (Bradford et al. 2%. 2002). 2002). sodium lactate permitted aerobic growth of B. The pH of the product was unaffected by incorporation of sodium lactate. S. 2001) compared the use of 3% or 6% sodium lactate.5.0% potassium lactate were inoculated with a five-strain cocktail of L. In retail products. 1997). Cooked beef top rounds containing 1%.118 Antimicrobials in Food potassium nitrite. sodium lactate caused more rapid surface discoloration (Lamkey et al. When raised to 210 mM.. and E..1% sodium diacetate (Glass et al.0 log CFU/cm2 after 84 days of storage.

Ground beef containing sodium alginate at concentrations up to 0. or 5°C. (1991) also found that 2% lactate inhibited the growth of C.2. Maas et al. whereas off-flavors developed in meat containing the 3.5% concentration. Unda et al. sporogenes. thermosphacta and E. Typhimurium but did inhibit P. or glycerol monolaurate enhanced inhibition of S.3%) was very effective in reducing numbers of B. and E only when lactic acid was used to reduce pH below 5... B. sodium lactate was the most effective during an 8-week storage period at 3°C. 5%. aureus grown anaerobically (Smith and Palumbo.3%. Lactates have been combined with other compounds in multiple-barrier studies.0) alone or combined with modified atmosphere packaging was examined on the survival of L. 2002). They also noted that the inhibitory component was the lactate moiety. Organoleptically. Maas et al. monocytogenes increased by only 0. 1991). Of all the treatments. Product was considered spoiled when microbial numbers reached 7 to 8 log CFU/g. the 2. Reduction in production of hydrogen sulfide and other sulfur-containing compounds resulted from the decreased populations of psychrotrophs. The influence of 2%. 7. or a combination of the two was inoculated with the two organisms and held at 5°C for 5 days. and 13 days for the 2%. fragi and S. but other samples were placed into packages that were gas flushed with a 90% carbon dioxide/10% oxygen mixture. 20°C. None of the treatments affected the growth of either organism. (1989) suggested that a possible mechanism for the action of lactate was the inhibition of a major anaerobic energy metabolism pathway necessary for growth. monocytogenes and the shelf life of chicken legs (Zeitoun and Debevere. Odor concomitant with growth of psychrotrophic spoilage organisms occurred in untreated samples within 8 days and within 9. They concluded that the two salts gave comparable results and could be used interchangeably. fragi at even at the 4% level. Some treated chicken was packaged without further modification. respectively. A 3% concentration of sodium lactate only minimally affected the growth of S. however. 7°C.Organic Acids 119 Shelef and Yang (1991) found that a 5. 5%. and 10% acid spray. suggesting that the lactate moiety was the effective component. Beef roasts containing sodium lactate. potassium sorbate.56 log during 17 days of storage for the legs treated with 10% buffer combined with MAP.. 10°C. Modified atmosphere packaging (MAP) extended storage life for products without acid sprays to 17 days and even longer with sprays. Sodium lactate at the same concentration had no effect. 1993). coli in a chicken model system after 14 days at 22°C but only delayed the growth of Lactobacillus alimentarius by about 2 days (Lemay et al. monocytogenes and C. Vacuumpackaged beef was treated with 2% sodium lactate. glycerol monolaurin. and 25°C. Toxicity occurred within 3 days in turkey without lactate and within 4 to 5. 10. Sodium lactate was more effective at reducing pathogen numbers at levels up to 3. monocytogenes in TSB and 4% limited growth of L. Typhimurium. or 3.5% than sodium gluconate. Lactate contributed to water-holding capacity and increased cooking yields.1°C. sporogenes were stored at 2°C. pediocin (1400 U/g). 2. or Microgard™ (2%) (Rozbeh et al. Levels of L. botulinum in comminuted raw turkey using levels of 2%. all were stored at 6°C. The meat was cooked to an internal temperature of 71. Excised skin tissue was analyzed for Listeriae for up to 17 days posttreatment.8%. which did not provide significant control (Stillmunkes et al.5%. Nisaplin (500 IU/g). 4 to 6. . vacuum packaged. or 7 to 8 days for each concentration. Lactic acid at 25 to 30 mEq in combination with sodium nitrite. (1989) demonstrated that sodium lactate was effective in retarding toxin formation by C. Notermans and Dufrenne (1981). found that glyceryl monolaurate at a concentration of 5 g/kg of a meat slurry inhibited C. or sodium gluconate and inoculated with L. 3%.0% concentration of sodium or potassium lactate delayed the growth of L. Harmayani et al.5%. nisin (1400 U/g. monocytogenes in comminuted chicken or beef when held at 35°C. botulinum types A. Sodium lactate (3.5% concentration was acceptable. 1980). or 10% lactic acid/sodium lactate buffer rinses (pH 3. (1991) looked at the interactive effects of sodium alginate and calcium or sodium lactate used in restructured meats on P. not sodium. and stored at 27°C. respectively. 1993). calcium lactate up to 0.

potassium (21 CFR 184.000 ppm malic acid before mating showed no significant differences from the controls (Hazelton Laboratories. Similar studies used low-fat ground beef patties formulated with carrageenan.0. In general. The acceptable daily intake for humans is listed in Table 4.98. Malic acid is approved as a GRAS substance for miscellaneous and general-purpose usage (21 CFR 184. and sodium lactates (21 CFR 184. dicarboxylic acid with high water solubility. monocytogenes for up to 50 days at 5°C. at pH 6. 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. Salmonella Senftenberg. MALIC ACID Antimicrobial Properties Malic acid is inhibitory to fungi and bacteria. jellies. Carrageenan had no effect on bacterial loads. 1975b). subtilis). 1992). 1970). however. a 2% lactate and 3% sodium chloride inhibited L. 1993). sodium lactate was more effective than the other additives in inhibiting growth of L. jams. fruit preserves. As the pH decreased to 5. Application and Regulatory Status Calcium (21 CFR 184. primarily for its flavoring and acidification characteristics (Gardner. and hydrolyzed vegetable protein and containing 1%. 1% kappa carrageenan. PROPIONIC ACID Antimicrobial Properties Propionic acid inhibited spore-forming bacteria. Some discoloration and lipid oxidation occurred. and sodium nitrite. and an alginate binder. The products were inoculated with L. It has a strong acid flavor but does not have the same buildup of acid taste as other acids. monocytogenes. especially rope bacteria (B. but the addition of 2% or 3% potassium lactate reduced microbial numbers..000 ppm. 1994). 1971a). sodium chloride.3. Nunheimer and Fabian (1940) found that malic acid was inhibitory against S.8% sodium lactate.1639)..1% sodium erythorbate. The acceptable daily intake for humans is listed in Table 4. It is used in sherbets and ices. 1972).0 or 4. the acid inhibited yeasts and molds but not to the same extent as bacteria (Woolford. food consumption decreased and growth rate declined (Hazelton Laboratories. inoculated with L. or 3% potassium lactate.1768) are also approved. Dogs fed the same three levels of malic acid showed no effects using the same parameters (Hazelton Laboratories. Reproductive experiments using rats fed 1000 and 10. Comminuted salmon was mixed with sodium lactate. monocytogenes and stored aerobically at 4°C.3. anatum. encapsulated salt.1069). and . and stored at 5°C or 10°C. Toxicology Malic acid fed to rats at levels of 500 and 5000 ppm in the basal diet had no effect.. 2%. at 50. monocytogenes and total aerobic plate counts (Harmayani et al. Application and Regulatory Status Malic acid is a nonhygroscopic. Sodium lactate displayed synergistic activity with nitrite and increasing concentrations of sodium chloride. vacuum packaged.1207).120 Antimicrobials in Food Raw and cooked ground beef were prepared with 1.0. 0. and beverages. thus affecting the sensory qualities of the meat (Egbert et al. 1971b). probably as a direct effect of pH manipulation. Chung and Goepfert (1970) compared various organic acids to determine the highest pH level that inhibited growth of S. aureus at pH 3.

and 35°C and prevented growth at 4°C (El-Shenawy and Marth. At a 6% concentration.5. L. and greater than 18 days for propionate. and propionate to a target level of pH 6. S.0. aeruginosa. Eklund (1985) concluded that propionic acid was not as inhibitory as benzoic acid for P.7 M propionic acid at pH 5. pH 4.. 1990). . monocytogenes grown at 13°C was extended by 7.6. and 5 with a 2% concentration. citric.. monocytogenes were undetectable. and S. citrate. tartaric.. The salts of propionic acid are also effective antimicrobial agents. Proteus vulgaris. pH 5.6 and incubated at 4°C.1. ZPNCP. 7.Organic Acids 121 Salmonella Tennessee when all other parameters were at their optimum. applesauce. Sodium propionate at a 0. and 4 days. DNA (deoxyribonucleic acid). the lag phase of L. Incorporation of calcium propionate into coatings for ready-to-eat products was effective in reducing problems with L. 4 for lactate and acetate.02% ascorbic acid.1. Sodium propionate at levels of 0. Products were stored for up to 24 days at 4°C or 8°C. acetic. A three-strain mixture of L. monocytogenes. Calcium propionate prevented rope formation caused by B. In laboratory studies examining the effect of sodium propionate on L. cherries. coli K12 and Salmonella. and Candida albicans. This substance not only postponed spoilage in fresh figs.0. or ZENCP. coli. 4. Nondetectable levels of the pathogen were found in products ZEN. and temperatures. At periodic intervals. Cherrington et al. and pH. particularly at the higher pH level.5. protein. or 4°C. 21°C. Sarcina lutea. and held at 28°C for 0 to 18 days.5 to 0.3% concentration in tryptose broth adjusted to pH 5.1% to 5% delayed growth of S.8. monocytogenes was inoculated into BHI broth containing 0.188%. ellipsoideus by as much as 5 days. acetate. degree of dissociation of the acid. pH 4. Citrate was most effective based on molarity (Miller et al.6 (O’Leary and Kralovec. lipid. Zein (Z) film coatings were dissolved in propylene glycol (ZP) or ethanol (ZE) with and without nisin (N. adjusted to pH 4. (1991) determined that 0. 13°C. pH 4. Golden et al. Exposure of L.0 and 13°C. or at a level of 0. 19°C. concentration of the organic acid. At a 5-mM concentration. monocytogenes (Janes et al. monocytogenes.08% EDTA.05.000 IU/g) or calcium propionate (CP. 5. Propionic acid was more effective than acetic acid as an antimicrobial. toxicity occurred after 7 days for pyruvate. tartaric. 1992). samples were removed from storage and tested for production of neurotoxin.4. and 35°C. Propionic acid at pH 5. 3 for citrate. The acids contributed to approximately 85% of the antimicrobial activity with 14% contributed by ascorbic acid. pyruvate. aureus. monocytogenes when used in conjunction with acetic acid in cold-pack cheese food (Ryser and Marth. 3. subtilis. 1988).0 with hydrochloric acid. or 3. fumaric and malic. pH 4. which led to an increase in cell mass without cell division and a decrease in viability (Cherrington et al. Torula species. syrup.6 with acetic. 21°C.0 was more inhibitory in 60 minutes compared to formic acid in 3 hours for E. Inactivation of L. 1992).0 minimized growth at 13°C. 1945). pH 5.9% sodium propionate or SA. pH 4. monocytogenes was the result of incubation temperature. E. 1993). 1989a). and lactate. little activity was ascribed to EDTA in combination with the acids. adipic. Much higher concentrations of propionic acid were bacteriostatic to B. When media containing 0.5 inhibited the growth of Salmonellae at a higher pH level than other acids (pH 5. lactic. 1. pH levels. respectively. Emphasizing the multiple-barrier concept in retarding growth. 1941). aureus.3% concentration was also effective at pH 5. (1995) examined the combination of sodium propionate and SA at a number of concentrations. the rate of RNA (ribonucleic acid). Toxin production occurred after 2 days for pyruvate. and stored at 28°C. 2002). and 0. Turkey breast meat was formulated with sodium salts of lactate.156%. acetate. monocytogenes to 8% sodium propionate for 60 minutes caused injury (Buazzi and Marth. Reduction of pH to 5. and cell-wall synthesis decreased. citric). 0. succinic.3. such as lima beans and peas (Wolford and Anderson. plantarum. and berries but also prevented spoilage in vegetables having a more neutral pH. the levels of L. 1%) and coated onto ready-toeat chicken samples dipped in L. or lactic acids in media (El-Shenawy and Marth. mesentericus (subtilis) in bread dough at a level of 0. 18 days for citrate.4. At pH 5. botulinum.0 in reducing numbers of L. inoculated with C. the organism was able to grow at up to a 0.3% sodium propionate was adjusted to pH 5. pH 5.

Hara. A. 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.85 (Guynot et al.93 in a model agar system on the retardation of growth of E. Not only was the final pH of the substrate critical. Ingle. Molds were inhibited by less calcium propionate by weight than sodium propionate.85.8 log reduction at 67% fat. no growth occurred. monocytogenes at 22% fat compared to 2. 1945). 1951. Suboptimal doses (0.03%.2). potassium sorbate. Additional studies using calcium and sodium propionate fed to mice. rubrum. sodium benzoate. Small amounts of β-alanine could overcome bacteriostatic action of sodium propionate for E. pH 4. β-alanine could not reverse the inhibitory effect of propionic acid in Aspergillus clavatus. and potassium sorbate at a level of 0. and rubrum. and humans showed no toxic effects (Graham et al. cooling surfaces. amstelodami.5 and 1. Sausage batter was mixed with 0. Graham and Grice.. 1965. flavus.5 logs. rather than later (Ghosh and Häggblom. with sodium lactate.b) examined several levels of calcium propionate. flavus and niger. However. Although mold spores are destroyed during baking.5 and water activity of 0. monocytogenes was studied in pork liver beaker sausage (Hu and Shelef. respectively.5. however. 1985). rats. monocytogenes by only 1. Eurotium amstelodami.5. 1954. B. coli.8 log reductions were observed. Adding yeast extracts previously fermented by Propionibacterium freudenreichii provided a source of propionic acid in bread formulation. 6. and sodium benzoate (0. and repens were inoculated by needle into cake analogues prepared with calcium propionate. and Penicillium corylophilum. There is evidence that the sodium salt has some local antihistaminic activity (Heseltine. reduced growth and aflatoxin formation of A. postprocess contamination from the atmosphere. 1953). (2001). Propionic acid at a concentration of 0. The inhibitory action could be the result of an interference with β-alanine synthesis (Wright and Skeggs. Spore germination was inhibited by 1402 ppm (Tzatzarakis et al. 1946). Toxicology Orö and Wretland (1961) determined the LD50 of propionic acid for mice. A 5% calcium propionate solution acidified to pH 5. Propionic acids and their salts are primarily inhibitory to molds. Bread produced with the extracts contained less ethanol and supported less spoilage by molds.8. Increasing the fat content from 22% to 67% decreased the growth of L.8% sodium lactate. and 0. 2002).3% by weight. Pseudomonas species.5 with lactic acid was as effective as a 10% unacidified solution in preventing surface mold growth on butter (Olson and Macy. 2002a. herbariorum. The antimicrobial activity of sodium propionate was most affected at 4°C by fat concentration with a 1.1%.1% sorbic acid or potassium sorbate.2%. All preservatives were effective at pH 6. and 7. 1952b). 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.03%) led to enhanced growth of Aspergillus and Penicillium isolates. (1963) determined the LD50 of calcium and sodium propionate for rats (Table 4. subtilis..8 to 0. Subsequent studies (Marin et al. and 0. A novel use of propionic acid to retard spoilage of bread was investigated by Gardner et al. Harshbarger. but also various organisms displayed different tolerances to the compounds (Olson and Macy. at 0. . 1996). Mold spoilage is a significant economic problem in bakery products.. 0.122 Antimicrobials in Food The effect of fat concentration on the efficacy of antimicrobial agents against L. 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.3%) at pH 4. 1955. 1940). 0. and wrapping materials reintroduce molds.0. 1940). herbariorum. and Hara et al. This inhibitory effect was more pronounced with the addition of acid at the time of inoculation. 1952a). 2000). 1942).5 log reduction of L. 1.003%. or Trichophyton mentagrophytes. and 0.0 and a water activity of 0. some species of Penicillium grew on media containing 5% propionic acid (Heseltine.2% sodium propionate.. None of the preservatives were effective at neutral pH. 0.

The acid form is readily miscible with water.38% in whole-wheat products.1221) and sodium propionate (21 CFR 184.0% succinic acid. but surface recontamination can occur during packaging. 1972). It has a slightly bitter taste and serves as a flavor enhancer. however. (1967) reduced the population of inoculated S. calcium (21 CFR 184.. No upper limits are prescribed for use of this additive. and 0. Calcium propionate is preferred. calcium and sodium propionate are listed as antimycotics when migrating from food-packaging material (21 CFR 181. dicarboxylic acid having low water solubility.1081) and its salts. and the sodium salt is more soluble than the calcium form. In addition. Thomson et al. A limit of 0.3. Propionic acid (21 CFR 184. 0. gradually increasing to 2. No limit has been set on the acceptable daily intake for humans. Propionates can be added to bread dough without interfering with leavening because there is little or no effect on yeast. Sodium propionate is recommended for use in chemically leavened products because the calcium ion interferes with the leavening action. It is normally found in cheese and as a metabolite in the ruminant gastrointestinal tract. 1965).1784). Succinic acid is approved as a GRAS substance for miscellaneous and general-purpose usage (21 CFR 184. for use in bread and rolls because the calcium contributes to the enrichment of the product (Chichester and Tanner.0 mg/day at 4 weeks and continuing at this level for 100 days. Its antimicrobial effect is limited to most yeast and bacteria. Toxicology In short-term studies. 1974). Salts of the acid have a slight cheeselike flavor. Application and Regulatory Status Succinic acid is a nonhygroscopic. Swiss cheese contains up to 1% propionic acid because of the growth and metabolism of propionibacteria.23). are approved as GRAS substances for miscellaneous and generalpurpose usage. except bread and rolls. . Succinic acid is used to modify the plasticity of bread doughs and in the production of edible fats. which conform to standards of identity.. and growth can be seen under the wrapper during storage. however a 3% or 5% concentration used at 60°C impaired the appearance of the product (Cox et al. SUCCINIC ACID Antimicrobial Properties Succinic acid successfully lowered microbial loads on poultry carcasses (Mountney and O’Malley. The test animals did not differ significantly in reproduction rates. 1944). Typhimurium by 60% on chicken carcasses after spraying with 1. Chick embryos developed normally when comparable dosages were injected into the air sac (Dye et al. This additive is also used as a mold inhibitor in cheese foods and spreads. hair appearance. which are associated with its manufacture and the characteristic Swiss cheese flavor. rats received subcutaneous injections of 0.5 mg succinic acid daily. 1972). or eye opening from the control group. The acceptable daily intake for humans is listed in Table 4. It can be incorporated into gelatin deserts and cake flavorings (Gardner. Propionic acid and its salts are used primarily as mold and rope inhibitors in bread.1091). tooth eruption. Baking destroys most molds.Organic Acids 123 Application and Regulatory Status Propionic acid is a monocarboxylic acid with a slightly pungent. 1980).32% can be used in flour and in white bread and rolls. disagreeable odor.3% in cheese products (Robach. This naturally formed additive becomes a developed preservative that limits mold growth on Swiss cheese.

and the production of energy by the cells. and cholesterol levels. The microbial cell normally reflects this equilibrium by attempting to maintain an internal pH near neutrality (Baird-Parker. It is thus essential to understand each of these concepts before consideration of the actual mode of action of the organic acids.1099). jellies and preserves.2% of the diet. molds. Fitzhugh and Nelson (1947) found similar results in long-term studies using 21-day-old weanling rats fed tartaric acid as 0. 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. this delicate balance is maintained and alteration of this balance causes destruction of the microbial cell. 1954). sugar. the most soluble of the solid acidulants. The acceptable daily intake for humans is listed in Table 4. 1980). mortality. and 215 mg/kg body weight. growth. 181. rats. and yeasts.. including increased nonprotein nitrogen. and phospholipids can be structurally altered by pH changes. 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.. Studies measuring teratogenic activity in pregnant mice.124 Antimicrobials in Food TARTARIC ACID Antimicrobial Properties Any antimicrobial property of tartaric acid is because of its ability to lower the pH. male rabbits consuming a diet containing 7. However. Inorganic acids. Tartaric acid is a GRAS substance for miscellaneous and general-purpose usage in accordance with good manufacturing practices (21 CFR 184. MODE OF ACTION The mode of action of organic acids in inhibiting microbial growth appears to be related to maintenance of acid–base equilibrium. nucleic acids. 0. which disappeared after they left the work site (Barsotti et al. . Workers exposed to tartaric acid dust measuring up to 32 mg/m3 in a manufacturing plant that produced the acid experienced tooth erosion.1%. Homeostasis is the tendency of a cell to sustain chemical equilibrium despite a fluctuation in the acid–base environment. Changes resulting from pH can destroy bacteria. 1973a).2).7% sodium tartrate (5% tartaric acid) did not show any changes in food consumption. and it prevents discoloration in cheese (Gardner. Biological and chemical systems depend on an interaction between acid and base systems.25 to 1 g to rabbits produced abnormalities in blood chemistry. The monopotassium salt (cream of tartar) is commonly found in baking powders.3. The workers also displayed skin eruptions. and 1. 225. 0. or gross histology compared with controls (Packman et al. has a strong tart taste that enhances grapelike flavors. respectively (Food and Drug Research Laboratory. hamsters. Proteins. It is used in fruit jams. 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. and rabbits demonstrated normal offspring at levels of 274.5%. such as hydrochloric. 1963). 1978). Toxicology Acute toxicity data for tartaric acid by the intravenous route for mice indicates an LD50 of 485 mg/kg body weight (Table 4. 1972). although some microorganisms such as Acetobacter can exist at and even require extremes of acidity (Langworthy. Rose’s (1924) work on subcutaneous administration of 0. and grape-flavored beverages. Application and Regulatory Status Tartaric acid.8%. sherbets. proton donation. Tartaric acid acts synergistically with antioxidants to prevent rancidity. Through the interaction of a series of chemical mechanisms.

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

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

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

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

A plot of the distribution of the three ionic forms can be calculated and is shown in Figure 5. The metabisulfite is the anhydride of the acid sulfite: – → 2– 2 HSO3 ← S2O5 + H2O When these salts are exposed to air. Table 5. It is useful to have the sulfur dioxide in a salt form.1. . 1968). and solubilities of each in water. sulfur dioxide can be written to show the equilibrium: → SO2 + H2 ← [H2SO3] – → [H2SO3] ← HSO3 + H+ – → HSO3 ← SO2– + H+ 3 The bracketed form indicates the sulfur dioxide associated with water. The pKa values for sulfur dioxide are 1. FORM AND SOLUBILITY In water solutions. The dry salts are easier to store and are less of a problem to handle than gaseous or liquid sulfur dioxide. as well as by other methods. their theoretical yields.20. Phillips. 1928.76 and 7. Most of these salts are hygroscopic and easily hydrolyzed (Schroeter.144 Antimicrobials in Food Sulfur dioxide can be prepared commercially from burning sulfur (oxidation). The anhydrous sodium and potassium salts are prepared by precipitation and then dehydrated with the appropriate hydroxide.2 lists the main forms of sulfur dioxide. Specific gravity for various water solutions of sulfur dioxide is shown in Table 5.1 Distribution of the ionized forms of sulfurous acid at various pHs. and from reducing gypsum. 1966). indicating a rather weak dibasic acid (Segal.1. 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. they show increasing stability in the order sulfite > bisulfite > metabisulfite (Mason. 1928). heating pyrites.

0292 1.Sulfur Dioxide and Sulfites 145 TABLE 5. After the oxygen disappeared. This reaction is extremely rapid.6 67.0001°F–1 TABLE 5.0393 1.008 1.003 1.018 1. The sulfur dioxide stabilizes the dehydroascorbic acid by reacting with the ketone bonds (Wisser et al.028 1.5 61.0493 20°C (68°F) 1.2 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. the second equation listed previously is a key reaction (Heinmann et al.0091 1.6 57. 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.4 s-1 for H2O2 with SO32. Holt and Kumar (1986) found an observed k = 41.4 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 pH 3.037 — Corrections are approximately 0..1 Specific Gravity of Various Sulfur Dioxide Water Solutions at Two Temperatures SO2 (g per 100 g) 1 2 3 6 8 10 a Specific Gravitya 15. much slower changes occurred in the sulfur dioxide content. 1970).4 53.0040 1. If ascorbic acid is being used in combination with sulfur dioxide.0191 1.56°C (60°F) 1. 1970).7 ± 3. . The sulfur dioxide scavenges the hydrogen peroxide formed and keeps further oxidation of the dehydroascorbic acid and other products to a minimum.40 and 15°C.8 25.

2 = Aerny (1986a. Glucose is by far the most abundant of the reactive aldehydes and ketones in most fruit juices. and fructose and sucrose did not react at all.12 — 2 99. and glucose reacted less rapidly. 1963).5 — 2. He also noted that phosphoglyceraldehyde would likely react. In botrytized grapes made into wine. Joslyn and Braverman. 2.7 × 102 — — — — 15. L-xylosone. raffinose reacted very slowly.0 0. Rhem (1964) noted that the rate of formation and the amount of sulfonate formed depended on the concentration of the reactive seven-member carbon ring system will react.1 0. and galacturonic acid were significant bisulfite binding forces (Burroughs and Sparks. 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. 1985).5 72 44 98 — 66 1 2 — — — — 0. 3 = Navara (1985). maltose. 1954). In moldy apples. and wines. form rapidly with aldehydes: – – → HSO3 + R-COH ← R-CHOH-SO3 All aldehydes form the hydroxysulfonates. the pH. 1964a.9 6 × 10–5 Note: 1 = Burroughs and Whiting (1960). 1954). and the temperature. 2-oxogluconic acid..b. 1986 a. d-threo-2. fermented ciders. Lea et al.6 7. Bisulfite addition products. 1954.1 87 3 50 32 18 — — — — — 26 — 45 32 0. Otherwise.146 Antimicrobials in Food TABLE 5.b). Relative percentages of aldehydes reacting with sulfur dioxide and their equilibrium constants are shown in Table 5. 1944). 1960. as much as 80% of the total sulfur dioxide may be bound by these types of carbonyls (Blouin. and arabinose reacted rapidly with bisulfite.3 (Burroughs and Whiting. Diethyl ketone reacts slowly and to a limited extent. pyruvate.5-hexodiulose. 1973.1 — 1. . 5-oxofructose. Ingram and Vas (1950) found that galactose.5-diketogluconic acid. but not all ketones react.5 3 5 8 Ka × 10–6 × 10–4 × 10–4 × 10-6 — 2 × 10–4 5 × 10–2 1. mannose. Those reacting the most rapidly formed complexes that dissociated the least. Aerny.7 90 1. α-ketoglutarate.b). acetaldehyde. Compared with model solutions. 2000).1 — 0. lactose. Navara. only ketones with a methyl group adjacent to the carbonyl or carbonyls that are part of a four. a Aerny (1986 a.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. 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.

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

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

1982. is very heat resistant and alcohol tolerant. and ethanol. Cruess (1912) found that 5. The juices high in insoluble solids ultimately had lower residual total sulfites. They indicated A.. plantarum. Pediococcus. Lactobacillus hilgardii. Fornachon (1957) noted that sulfur dioxide and pH were important factors in controlling lactic bacteria in wines. LafonLafourcade and Joyeux (1981) and Joyeux et al.. 1983).4 (Holt et al. Oenococcus oeni grows preferentially at lower pH but seems less tolerant to sulfur dioxide (Mayer.49 log Acetobacter per ml exposed to 100 and 200 mg/L of total sulfur dioxide in grape juice were reduced to 2. Lactobacillus plantarum. Leuconostoc mesenteroides subspecies dextranicum can also cause ropiness in beverages through the formation of dextrans. casei. Lafon-Lafourcade (1975) demonstrated the effectiveness of both free sulfur dioxide and bound sulfur dioxide in inhibiting Leuconostoc gracile in wine at pH 3. Lactobacillus buchneri. Additions of 100 mg/L bleached the color of the vinegar. red wine. This organism is particularly intolerant to sulfur dioxide. but much larger doses were required for bactericidal action. 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. The bacterium.0 in a buffered solution. Cell growth was completely prevented. Carr and Davies (1971) noted that relatively high amounts of free sulfurous acid were present in ciders that contained viable lactobacilli. Liu and Gallander (1983) demonstrated that lower pH wines underwent . and soft drinks were required to control acetic acid bacteria. 1983) indicated that around 50 mg/L of free sulfite could preserve wine vinegar for about half a year.5 mg SO2 per liter in the undissociated form was bacteriostatic to L. Addition of 30 mg/L of free sulfur dioxide killed the bacteria completely in 15 days. They found that up to 1.5 mg/L were bactericidal.. Watanabe and Ino (1984) and Juven and Shomen (1985) reported that up to 100 mg/L of total sulfites for grape juice. however. oeni was the most sensitive to SO2. Levels above 120 mg/L of total sulfur dioxide (free and bound) decreased the incidence of malolactic fermentation. He found that this genus also fixed a certain amount of the sulfur dioxide. to carbon dioxide and water. with lower pH increasing the effectiveness of the sulfur dioxide.47 log and 0.0 (Rhem and Wittmann. The homolactic species found in wines are Lactobacillus acidophilus. Leuconostoc. respectively. hilgardii. and Oenococcus. O. and P. (1980) reported that 30 mg/L of free sulfur dioxide is sufficient to inhibit malolactic fermentation in wine. Lactobacillus trichodes. 1949) is described as causing a hairlike growth in fortified wines. respectively. after 36 hours. Amerine et al. As little as 30 mg/L of added sulfur dioxide may be lethal to the species. Lactobacillus trichodes (Fornachon et al.5. pentosaceus to SO2.Sulfur Dioxide and Sulfites 149 oxidize ethanol in fermented beverages to acetic acid and. oeni. They are aerophilic and have pH optimum for growth of around 5. Pediococcus cerevisiae. and Oenococcus oeni (Amerine et al. As low as 75 to 80 mg/L of total sulfur dioxide prevents growth and 100 mg/L kills this species. Bacteria common in acid fruits and beverages are the lactic acid-producing genera Lactobacillus. Lactobacillus brevis. 1982. In model solutions containing Lactobacillus arabinosus and L. 320 and 651 mg/L of sulfurous acid were required. (1984) stated that the amounts of SO2 used in normal wine making are insufficient for acetic acid bacteria control. Concentrations above 1. Lactobacillus casei. aceti can grow in red wine with 25 mg/L of unbound sulfur dioxide present. 1962). further. pH. 1980). 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. and Pediococcus pentosaceus. 1994). Lactobacillus leichmannii. for antimicrobial action at pH 6. and the heterolactic species are Lactobacillus fermentum. 1979). 20 days was required to kill the bacteria when the same amount of aldehyde–bisulfite complex was used. Rhem and Wittmann (1962) reported that 200 mg/L sulfur dioxide killed Acetobacter at pH 6. Manca de Narda and Strosser de Saad (1987) investigated the tolerance of O. L.3 log CFU/ml. Dupuy and Maugenet (1963) noted that even small doses of sulfur dioxide inhibited the activity of the cells. Several reports (Karova and Kircheva.. Lactobacillus delbrueckii. Spirov et al.

. Davis et al. They suggested that these microorganisms could cause spoilage in wines that were considered sterile using conventional counting techniques. (1985) reviewed the use of sulfite as an additive to control microbiological changes occurring in meat products. In addition. oeni strains with unfavorable sensory results. 15–109. 190–241. (1988) demonstrated that without sufficient SO2 and adjusted pH.0 were as follows: Salmonella. Wibowo et al. Tompkin et al. the growth of O. Salmonella and E. (1988) tested 146 different strains of wild malolactic bacteria. such as Escherichia coli and Pseudomonas. and Hafnia alvei. O. The shelf life of ground beef was effectively increased from 1. The microorganisms tested and the concentration of free sulfite (µg/ml) necessary to inhibit their growth at pH 7. Lafon-Lafourcade et al. Ough et al. 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. The Gram-positive bacteria remaining grew more slowly than the Gram-negative bacteria.. Piracci (1984) found 0. Serratia marcescens. This is demonstrated in the use of sulfites in meats. than in inhibiting Gram-positive bacteria. state that sulfur dioxide is more effective against the growth of Gram-negative rods. oeni strains were less tolerant to the sulfur dioxide than Pediococcus paroulus strains or the Lactobacillus species. All strains grew at pH 4. oeni in red table wine occurs readily. Banks and Board (1982) tested several genera of Enterobacteriaceae isolated from sausage for their metabisulfite sensitivity. coli were inhibited to a greater extent by sulfites than other bacteria. 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.5 in beef broth and 20% tomato juice serum medium at 64 mg/L of total sulfur dioxide. (1983) determined that. oeni when SO2 was added and quite a variable response between strains. 50–195. It was found to tolerate up to 100 mg/L of SO2.5 mg/L of molecular sulfite was sufficient to control malolactic bacteria growth. Banks et al. The preservation of the color and odor of meats is improved by sulfite treatment and. 1988). The effect was inhibition of growth of the flora. oeni was the primary malolactic bacterium associated with wine. 1972). The delay was proportional to the concentration of the bisulfite addition. or in paired combinations. 65–136. 200–241.150 Antimicrobials in Food malolactic fermentation more slowly and that lower sulfur dioxide levels increased the fermentation rate of the O. Enterobacter agglomerans. There was some additive effect when used with dimethyl dicarbonate but not enough to warrant the use with SO2 for this purpose. 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. although after sulfite addition. (1987) found that vacuum packaging and a good oxygen barrier film decreased the . were completely effective. in the Bordeaux area of France. 83–142. coli. although slowing or prevention of growth of surface bacteria is probably important. 67–98. oeni PSU-1 used. 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. E. (1980) found the addition of 100 mg/kg of SO2 as sodium metabisulfite to canned pork inoculated with Clostridium botulinum spores delayed cell growth. They also noted that the interaction of sulfites with nitrites caused a lowering of the nitrites available for nitrosamine formation. Adams et al. None of the compounds alone. (1988) found significant delays in growth of O.8 days at 7°C storage with no treatment to 12. It survived alcoholic fermentation when others did not. This work confirms that wines with high total sulfur dioxide concentrations are more likely to undergo malolactic fermentation with other than O. Roberts and McWeeny (1972). Citrobacter freundii. a rapid decline in cell numbers occurred. They noted that sulfites shifted the microflora of the meat to Gram-positive bacteria from the normal Gramnegative flora. Yersinia enterocolitica. Sensory tests showed no adverse results. the main effect in meat appears to be the antioxidant properties (Roberts and McWeeney. in their review. The packaging used was a gas-permeable wrapping that allowed oxidative conditions (von Holy et al.6 days at 0°C with the addition of 250 mg/kg of sulfur dioxide. The remaining cells later multiplied to significant numbers. O.

between 2.81). such as “maintain the free SO2 at 20 mg/L. its use to prevent the growth of yeast in sweet table wines is seldom ever contemplated.20 7. was maintained for a longer period. In contrast.2–8. but the level of cadaverine was reduced (Bover-Cid et al.7 0.20 0. bailii at 25°C under aerobic conditions at pH 3. spoilage in sulfite-treated sausage. There was no effect on histamine.4) at 25 mg/L was sufficient to kill a culture of S. and several other genera were very susceptible.60 Rhem and Wittmann (1962). respectively.Sulfur Dioxide and Sulfites 151 TABLE 5. (1988) found that with yeast acclimatized to sulfur dioxide.0 and 3. Yeast The use of sulfur dioxide to deplete the wild yeast in grape juice is a standard practice dating back many years (Cruess. This was because of the lack of oxygen delaying yeast growth and the production of sulfite-binding substances.” for biological stability can be disastrous. Any rule of thumb addition.2 mg/L of molecular sulfur dioxide were necessary at the finish of fermentation and during aging. 2001). 1. 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. whereas Kloeckera.05 M free sulfur dioxide for Kloeckera apiculata to 2. resistance of yeast to sulfur dioxide ranges from 0. which inhibited growth. bailii. Delfini (1989) . cerevisiae that had been characterized as “SO2 resistant” for tolerance to high concentrations of sulfur dioxide.0.4). According to Warth (1985).1. with the pressure to reduce sulfur dioxide content. A 10-fold increase in molecular sulfur dioxide occurs between pH 4. and five others were able to produce adequate amounts of ethanol. Sudraud and Chauvet (1985) found that to maintain yeast stability. The yeast also showed increased resistance to the fungicide dimethyl dicarbonate.. the growth rates and cell yield were similar. Rhem and Wittmann (1962) determined the inactivation levels of sulfurous acid for a variety of yeast genera (Table 5.10–20. In fact.20 0.5 in tryptone yeast extract medium.0 mg/L were required. Rhodotorula. Haznedari (1979) tested 30 strains of S. Saccharomycodes ludwigii was nearly as tolerant as Z. Sodium sulfite addition in sausage was shown to affect biogenic amines. 1912). phenylethylamine.60 0.4 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. pH is extremely important in the effective use of sulfur dioxide. cerevisiae (105 CFU/ml) after 8 hours at 25°C. 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. or tryptamine. Molecular sulfur dioxide is the most effective form for inhibiting yeast. Pichia. Six strains could grow well at 1000 mg/L. Carr and Davies (1971) found sulfur dioxide incorporated into growth medium (pH 3.5 and 1. Thus the free sulfite. Ough et al. The amount of molecular SO2 (mg/L) in wine can be calculated using free SO2 mg/L/(1 + 10pH .8 mM for Z.40–0. Once the inhibition was overcome. The concentration of tyramine and putrescine increased in the presence of sulfite.0 and 3.

none seem to be capable of replacing the antioxidant property of SO2. found no correlation with dissolved oxygen levels in wort and no correlation with ATP sulfurase or sulfite reductase. With this genus. Ranote and Bains (1982) used 350 mg/L of sulfur dioxide to preserve Kinnow. Proper selection of the yeast strain to avoid high levels of sulfite is an obvious choice. Recommendations include cooling the fruit before crushing. Sethi and Anand (1984) had to use 692 mg/L in carrot preserves to inhibit Bacillus cereus. 1978). soft drinks. (1983). Chang et al. 1985.1 but not in the base material.152 Antimicrobials in Food reported the existence of very sulfite-resistant S. the sulfate permease was not repressed by methionine. Growth was found at the highest concentration of free sulfur dioxide used. Valouyko et al.. Hydrogen sulfide (H2S) was used (Ubigli et al. a late-harvest orange juice. Vernerova et al. Angelino et al. and wines because of its tolerance to SO2.. the main spoilage yeast is Saccharomyces. bailii. Z. This particular species was found to be very resistant to sulfurous acid. Generally. investigating the sulfur dioxide content of green beer. Some yeast strains can form rather large amounts of sulfur dioxide during juice fermentation (Table 5. 1985. ludwigii. S. (1985) used an initial 1000 mg/L of sulfur dioxide in the last stages of the manufacture . Hernandez. reducing the amount of sulfur dioxide before or during fermentation. cell numbers are depleted to very low levels at bottling. 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. bailii in comminuted orange drink was controlled by 230 mg/L of SO2 at pH 3. Spoilage by Z. Strain differences caused variation in the sulfur dioxide produced from 11 to 26 mg/L.7 (Lloyd. 1986a. settling and racking the juices. Although other agents may act as antimicrobial agents. The treatment was reported to be successful for juice. cerevisiae despite a reduction in the bacterial counts in the system at the same concentration. stated that the use of sulfide salts failed to protect wine and left negative sensory characteristics in the wine. Sand (1980) suggested that Z. however. Minarik and Navara (1977) reported finding the spoilage yeast S. Pearlstein (1988) found that extended aeration decreased sulfite production. 1985.5). and there was no correlation with the number of cells in the initial inoculum. which was at pH 3. 1985. Suzzi et al. (1985) tested 1700 Saccharomyces. using sulfide salts before fermentation. if proper sulfur dioxide levels are not maintained. especially wines in barrels. Schmitt et al. bailii was becoming a problem in juices. and low pH. (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. (1997) showed that sulfite up to 400 mg/L had no effect on S. sulfur dioxide. Galassi and Mancini. Gomes and da Silva Babo. S. (1989).6). 1975). There have been numerous reports on techniques to minimize the amount of sulfur dioxide used in wines (Aerny. They found a majority of the wines in the test to contain about 10 mg/L of total sulfur dioxide (Table 5. even at the legal limits. Patel et al. cerevisiae. Asvany. substituting other preservatives at bottling. Very little of the sulfur dioxide formed by the yeast remains in the free state. Breweries have an interest in sulfur dioxide as an antioxidant. 1982) as an alternative for pretreatment of grape juice before fermentation. and Schizosaccharomyces japonicus in the re-fermentation of sweet champagnes. and eliminating oxygen from bottled wine. This shows that most wines will not be free from sulfite labeling requirements even if no sulfur dioxide is added. In high-sulfite-producing yeast. Yeast can form sulfite from sulfate via sulfate permease. Dott et al. adjusting the pH downward if necessary. In normal sweet table wines.6 and 3.b. 1985). Spoilage yeast in dry wines are fairly rare. using yeast that produces minimum amounts of sulfur dioxide-binding components. alcohol. (1977) determined the cause for sulfite production to be sulfate permease inhibition by methionine. minimizing the air around the wine. Even at low pH (2. Brettanomyces can contaminate a winery. does not always inhibit growth and fermentation.4). ludwigii in a low-alcohol wine. Fruit juices and preserves can be protected from microbial spoilage by sulfur dioxide addition. excess amounts are used. ludwigii is also a noted spoiler in sweet grape juice containing high amounts of sulfur dioxide (Jakob. In a cell recycled ethanol fermentation system. because the pH is high. With modern filtration and sanitation technology.

(1976) Delfini et al. 44. (1976) Poulard and Brelet (1978) Delfini et al. 100. (1976) Delfini et al.5. 150 0.5 14. (1976) Minarik (1975) Eschenbruch and Bonish (1976) Heinzel et al. africana TABLE 5. 5 18–23 85 0. 1. (1976) Delfini et al. (1976) Delfini et al. bayanus S. 160. bailii S. 21.5. magna K. (1976) Poulard and Brelet (1978) Minarik (1975) Poulard and Brelet (1978) Minarik (1975) Delfini et al. 1300 22. (1976) Poulard and Brelet (1978) S. 40 0 18. 3. 52. chevalieri S.5 12 0 9 Reference Minarik (1975) Eschenbruch and Bonish (1976) Heinzel et al. rosei S. 170. 5. (1976) Delfini et al.6 Amounts of Sulfur Dioxide Found in Test Wines Fermented by 1700 Saccharomyces Yeasts (Suzzi et al. 35 20. 21. 100 5.Sulfur Dioxide and Sulfites 153 TABLE 5. 18. italicus S. 56.5. cerevisiae S.5 Formation of Sulfur Dioxide by Various Yeasts during Grape Juice Fermentations Yeast Saccharomyces carlsbergensis Sulfur Dioxide (mg/L) 12 150 80. 500 300. acidifacien S. rouxii Schizosaccharomyces pombe Torulopsis stellata Kloeckera apiculata K.5 0 0 0 1. (1976) Poulard and Brelet (1978) Delfini et al.. 4. pastorianus S. 500 5. 35. (1976) Delfini et al. uvarum S. (1976) Minarik (1975) Minarik (1975) Heinzel et al. (1976) Delfini et al. 1985) Range of SO2 Found in Synthetic Medium (mg/L) <10 10–20 20–30 30–40 >40 Average Total SO2 in Wine (mg/L) 10 17 22 34 58 Number of Strains 1292 354 50 2 2 . 76 17. 18. 20 1. 20 26 27 0 0. 3. (1976) Delfini et al.

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

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

including one death. Howland and Simon (1989). unless damaging doses are given. found that normal nonasthmatics were not affected by doses in the airway of up to 0. The review by Nicklas (1989) particularly stressed that many pharmaceuticals are preserved with sulfur dioxide. in controlled amounts. sulfite-related. Gershwin et al. No in vivo carcinogenic or mutagenic effects could be demonstrated with mice or rats. The variation may in part be the result of differing amounts of thiamine administered in these studies. A death related to a red wine that contained 93 mg/L of total sulfur dioxide was reported by Tsevat et al. studies indicated that certain asthmatic individuals were at risk by consuming relatively small amounts of sulfites. Only four of the 24 patients had a significant reduction in forced expiratory volume. However. There are many more papers and reviews on sulfite-induced reactions in people with asthma. Mathison et al. The food situation in relation to sulfite reactions is pertinent. They concluded that some asthmatics could be at risk in consuming wine-containing sulfites. leave a 900 mg/kg residue of sulfite on the product. Additionally. The researchers suggested that the role of sulfites and/or wine triggering asthmatic responses may be overstated. using Wistar rats. The Select Committee on GRAS Substances (1976) concluded that the average per capita consumption of 0.156 Antimicrobials in Food species). (1988) challenged eight people with asthma. could not show any teratogenic effects with heavy doses of sulfite but did show evidence of fetal toxicity. Early on. Taylor et al. the joint FAO/WHO Committee (Joint FAO/WHO. 1974) established the acceptable daily intake level at 0. Martin et al. Vally and Thompson (2001) exposed 12 wine-sensitive and 6 control asthmatics to wine with increasing concentrations of sulfites. (1987). In a recent study. Food and Drug Administration (FDA) (1985) agreed with the toxicology findings. Itami et al. 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). 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. when used as recommended. In a reexamination of the findings of the Select Committee on GRAS Substances (1976). (1985) reported on the effect of wine (white) with and without sulfur dioxide. on volunteer asthmatics.. much higher levels would result. use of SO2 was considered GRAS. Wines with lower concentrations of sulfites (<150 mg/L) did not elicit any response. The drinker consumed only one glass but was known to have asthma and sulfite hypersensitivity. adverse reactions. It appears from all the published reports that normal humans are reasonably tolerant to sulfur dioxide and. An extensive review by Gunnison and Jacobsen (1987) discussed in detail the possible mechanisms of sulfite hypersensitivity from which most asthmatics suffer. Dahl et al. to a number of foods treated with sulfites. A joint Food and Agriculture Organization and World Health Organization (Joint FAO/WHO. challenged them with lettuce treated with commercial. In 1983. in a replicated dose-response study.6% of these cases were classified as severe (Warner et al. For humans. but the medical aspects are beyond the scope of this chapter.6 ppm. (1989). Those with asthma had restriction in the airways and in one case a life-threatening reaction. 2000). studying patients known to be sulfite sensitive. (1985) discussed the possible mechanism for sulfite action on asthmatics and concluded that little is completely understood.2 mg/kg body weight per day was not a hazard to health. Until the early 1990s. Since the monitoring of food reactivity to sulfites started in the 1980s. (1987). there have been 1132 consumer complaints describing adverse reactions.7 mg SO2 per kilogram body weight per day. (1986) tested red wine-sensitive people with asthma for reactions against amines and sulfites in wine.S. They found that sulfur dioxide was the more severe causative reagent in all cases. can recover unaffected. sulfite-containing fresheners. (1986) showed that salad fresheners containing sulfites could. They found mixed responses from . but people with asthma responded to this dose level. the FDA considered the use of sulfites on fresh fruit and vegetables in restaurant foods a major problem area. Their report stated that no teratogenic effects had been reported and noted that sulfur dioxide had variable effects on mutagenicity to bacteria. Linn et al. the U. previously selected as SO2 sensitive by sulfite capsule tests. and 48. If the practice is abused. No significant differences were found between the groups in any lung function parameter. the FDA (1983) noted that they had received 90 reports of food-caused.

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

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. or irreversibly bound (Beck et al. TABLE 5..17 987. Sulfites can be described as free.32 980. Those sulfites that bind food matrices and are not converted to sulfur dioxide with heating or acidic conditions are irreversibly bound.29 990.16 963.04 990.28a 990.. A sample is placed in a distilling flask. and concentration varies by country.. Free sulfites are readily converted to sulfur dioxide upon acidification and can be quantitatively analyzed following distillation. One official method for measuring total sulfurous acid is the modified Monier-Williams procedure. The official methods of analysis for agricultural and food products are specific in their recommendations for analysis of sulfur dioxide (Table 5. 2000). 2000) Method Number 892. 2000). reversibly bound.20 975. Reversibly bound sulfites are converted to sulfur dioxide only after heat and acid or alkali treatments. garlic.02 961.09 962.30 990. Thiosulfonates are irreversibly bound (Beck et al.158 Antimicrobials in Food TABLE 5. acidified. and .7 Application of Sulfites in Foods as Antimicrobials and the Concentrations Used (Gould.8 AOAC International Official Methods of Analysis for Sulfites (Warner et al. purees.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.8). horseradish) Fruit juices Fruit-based sauces and related products Fruit pulps. 2000) Food Beer Fresh fruits Fresh vegetables (onion.

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

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

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

000 µg/g cannot be relied on to inhibit putrefactive anaerobes if other conditions are favorable for growth. research had clearly demonstrated that nitrite has significant antimicrobial properties. Clever as the scientific community has been in more clearly defining the antimicrobial properties of nitrite. By the middle 1950s. At levels between 22. 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. putrificum. found that sodium nitrate up to 22. solving a problem of sour hams. C. 6 of the 7 cultures were inhibited. 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.0% sodium chloride (salt). He suggested that nitrite. the highest levels tested.6 and held at 37°C. It is still difficult to design experiments that yield predictable. or 12.000 µg/g of sodium nitrate. One of the messages from the 1920s is that nitrate serves as a potential reservoir for conversion to nitrite and thus to nitrous acid. nitrite was considered to have no value in meat other than for flavor and color development. Unlike the other antimicrobial agents discussed in this text. despite meat having a pH closer to 6. During the 1940s and early 1950s. This concept became widely accepted. 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 and 44. reproducible results in all instances. data appeared that began to prove the opposite. 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. Levels of 3100 µg/g in pork infusion and 3900 µg/g in egg-meat. 44. were not inhibitory to any of the strains.Nitrite 171 documentary of Cassens (1990).000 µg/g. only salt was used in sufficient concentration in commercial hams to prevent proteolytic activity at 37°C. 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. of the three. putrificum. RESEARCH HISTORY BEFORE 1950 Tucker (1930) studied the proteolytic activity of a Clostridium putrificum isolate from sour ham. The list of recognized factors influencing the antimicrobial efficacy of nitrite in cured meats continues to grow 60 years later. Considering that research continues to reveal new interactions among the chemical constituents of cured meat.000 µg/g of sodium nitrite in a pork infusion broth buffered to pH 7. Proteolysis was inhibited by 4. This basic flaw in experimental design led to results that misguided and hardened opinions in the industry for 20 years. not nitrate. Most of the research on sour hams involved test tube experiments with culture media above pH 7. He concluded that. the value of nitrate.000 µg/g. and the value of nitrate was relegated to serving merely as a reservoir for the generation of nitrite. using Clostridium botulinum. Tanner and Evans (1933). The same results were obtained with Clostridium putrefaciens at 23°C. Until the early 1940s. irregular inhibition occurred among seven botulinal cultures. controversial history over whether nitrate and nitrite have antimicrobial properties. is responsible for retarding proteolysis by C. Research of the 1920s and 1930s focused on using nitrite to cure meat.000 µg/g did not inhibit the 12 strains tested in either pork infusion or egg-meat media. and Clostridium sporogenes. there has been a long. By then it was also more firmly believed that nitrous acid derived from nitrite in the acid environment of meat is responsible for microbial inhibition. The authors concluded that nitrate at concentrations as low as 22. At 44. . We are still ignorant of all the significant factors that influence the efficacy of nitrite or. Nitrate was believed to provide the antimicrobial effect. By the time the nitrosamine controversy arose in 1969 and the early 1970s. for that matter. and dealing with perishable canned hams that were being temperature abused in the marketplace.0.0.

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

” They postulated the anticlostridial mechanisms to consist of partial conversion of nitrate to hydroxylamine. botulinum and C. nitrite alone inhibited anaerobic putrefaction during 30 days of abuse at 37. although salt was the stronger inhibitor. and salt caused destruction of anaerobic spores at much lower temperatures. (1949) also examined the combined effect of heating and curing salts. fewer cans became putrid in the product with both nitrate and nitrite than in the product with nitrite only. (1945a) reached the same conclusion from tests in meat processed at 116°C. but not 2000 µg/g. thus leaving the meat in much the same state as freshly cooked uncured meat. of sodium nitrate. Two tests were reported with perishable canned ham. Nitrate. 1942). Subsequent tests (Tarr. Rapid chilling of the meat is not detrimental.” Their tests with fish muscle showed that nitrite delayed spoilage. Later tests involving C. In one. In both tests some cans with nitrate swelled because of the growth of Bacillus species. prevented growth of C. increasing nitrate did not increase the inhibition of C. nitrite. In the other. little or no inhibition was observed. Stumbo et al. workers at the time would have had difficulty applying the information to commercial practice. sporogenes during 4 weeks of incubation at 37. Despite these results. or repetitive heating did not increase this effect.. 1955). The authors suggested that the combination of heat. alone or in combination with other ingredients. Tarr and coworkers published a series of reports on the possible use of nitrite for preservation of fish. 1941) demonstrated the importance of pH to the efficacy of nitrite.Nitrite 173 of nitrate to nitrite in commercial bacon curing brines is mainly the result of the growth of micrococci.2°C. However. . thus serving as a desirable indicator of temperature abuse. which destroys anaerobes. Jensen and Hess found that heat (93°C for 4 hours) combined with 5000 µg/g. Others have shared this concern (Scott. 1942). At pH 5. How could reliance be placed on an unstable preservative that disappears during processing and storing? This question is still valid. The effect of pH was more firmly documented in a series of tests in nutrient broth inoculated with a variety of aerobes. nitrate. At pH 7. little or nothing is known regarding the possible bacteriostatic (or bactericidal) action of nitrites in meat pickles or in cured meats themselves. sporogenes 3679. the authors concluded that nitrate is beneficial for preventing the growth of putrefactive anaerobes in abused perishable canned ham. Jensen and Hess stated that nitrite reacts with protein during heating and is destroyed. The presence of nitrate or microbial action during the curing process is not essential for bacon flavor. Using the spiced ham medium they reported earlier. increasing salt and nitrite caused increased inhibition. longer heating times. 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. 1941) and that the mode of action was still unknown (Tarr. 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. 1945b) that nitrite appreciably delayed germination. While Tarr was demonstrating that nitrite has strong antimicrobial properties. sporogenes yielded similar results (Tarr. Tarr and Sutherland (1940) concluded that “as far as can be ascertained from the literature.7 and 6. They also reported (Stumbo et al. Yesair and Cameron (1942) pursued this idea but concluded that curing salts do not assist in thermal destruction but inhibit outgrowth.0. Jensen et al. did not appreciably influence spoilage in tubes processed from F0 = 1 to 16 and held for a year at 28°C. Jensen and Hess (1941) advocated the virtues of nitrate and perpetuated the belief that the sole function of nitrite was for cure color development. complete or strong microbial inhibition occurred. Greatly increased inhibition occurred in tubes of pork heated in the range of 50°C to 65°C for 30 minutes. Higher temperatures. They stated that the literature shows “unmistakenly that nitrate in the cure exerts a definite inhibitory effect upon bacteria. which inactivates catalase and allows the accumulation of hydrogen peroxide.01.2°C. Within the levels normally added to canned ham. Even if nitrite had been uniformly accepted as a critical factor in microbial protection.

(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. The relative roles of nitrate and nitrite as preservatives in cured meats were unclear. Nitrate was without effect. 1950–1960 Research of the 1950s clarified the relative significance of salt. 6.000 µg/g were required to prevent spoilage from anaerobic spore formers in pork. Although thermal destruction was not shown to be enhanced by the presence of curing salts. Strong opinions were expressed that nitrite alone was ineffective at the levels used in commercial practice.0. Salt was found to be the major factor retarding botulinal outgrowth in temperature-abused products. 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.4% or sodium nitrate in excess of 40. Halvorson (1955) later discussed the widespread problem of temperature abuse at the retail level. a combination of both nitrite and nitrate was the most inhibitory. During the 1930s some processors incubated perishable canned cured meat at 37°C as an index of “keeping quality. 5. and nitrite as preservative agents and expands this information to other microorganisms. Nitrate at the level permitted in meat (1250 µg/g) was without effect. Steinke and Foster (1951) investigated the effect of packaging liver sausage in an oxygenimpermeable film (Saran™) that was gaining acceptance in the industry.5%) and nitrite (150 to 170 µg/g) was much more effective than either substance alone. pasteurized.” This experience may have led to improvements in meat handling and equipment sanitation. Adding 200 µg/g of sodium nitrite was considerably more inhibitory to toxin formation than the addition of 1000 µg/g of sodium nitrate. and nitrite). even in the presence of salt or nitrite. but the condition of the meat was a factor. . by nitrite. and high heat resistance. however. and sterilized meat were of low. nitrate. 4. increased inhibition of PA 3679 was obtained as sodium nitrite was increased from 400 to 800 µg/g. yielded the maximum inhibition. Salt at the levels used at that time was considered the cornerstone for the preservation of cured meats.174 Antimicrobials in Food Vinton et al. 3. A mixed cure of salt (3. nitrate. SUMMARY FOR BEFORE 1950 A summary of the state of knowledge by 1950 might include the following: 1. It is perhaps for this reason that Jensen believed so strongly about the need for nitrate in perishable canned cured meats. Spores produced in raw. however. Nitrite was clearly shown to be an effective antimicrobial agent. 2.1 to 6. The disappearance of nitrite during processing and storage was assumed to make it an unreliable preservative. especially if the product pH was below 7. 7.37% and a pH range of 6. to some degree. there was increased inhibition of outgrowth of surviving spores by salt and. The basic formulation resulted in a product having a moderately high brine of 5. An underlying factor in the research before 1940 was the unfavorable experience of 1928 and 1929 with temperature-abused perishable canned hams. Bulman and Ayres (1952) found that levels of salt in excess of 4. The question was raised whether the growth of spoilage organisms is influenced by the depletion of nitrite to noninhibitory levels. The concept of a combined effect of heating in the presence of curing salts was proposed. respectively. A mixed cure of all three (salt.5.05% to 5. intermediate. In the absence of added salt.

Germination was apparently prevented because viable spores could be recovered. the legal requirement for trichina destruction. aureus was killed in hams when heated to an internal temperature of 58. botulinum.0%.g. Tests in broth media and raw pork led them to conclude that S. the control of salt concentration and resultant water activity was the most reliable bacteriostatic system for cured meats. In the United Kingdom.3 or below. found in the interior of cured meats. Earlier. 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. Pasteur. Nitrite was more inhibitory in the presence of ascorbate. aureus. S. The debated issue was the time and temperature for preenrichment. The requirements imposed on new products included a brine level of 5. Department of Agriculture (USDA) issued a regulation to assure the safety of new perishable canned cured meat products (e.3°C. Jensen. nitrite rapidly disappeared and was ineffective. this cautious view of the inhibitory effect of nitrite was expressed by Eddy (1957): “Taken in their totality. The brine requirement of 5. Inst. A pH of 5. who concluded that 1 ounce nitrate was optimal for gas formation by Bacillus species in temperature-abused perishable canned luncheon meat.. and 1 ounce of sodium nitrate per 100 pounds of meat.27.55) was autoclaved with glucose.5%. 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.5%. toxin formation was inhibited with a heat treatment as low as F0 = 0. At 6. and in the presence of nitrate. As late as 1957. the spores could germinate. a similar debate would include the time elapsed since manufacturing. although effective.5% sugar.S. This combination of ingredients was derived from the literature and unpublished results from Jensen. During the mid-1950s the U. A typical formula being used for large canned hams at that time included 0. botulinum. In North America.6 to 5. and a brine level of 3. aureus growth can occur in any combination of salt. 1/4 ounce sodium nitrate (156 µg/g). the addition of 1.8 was optimal for antibacterial efficacy. aureus to die rather rapidly in ham-curing pickle. B. and toxin would be produced. 1/8 ounce sodium nitrite (78 µg/g). growth would occur. At pH 5. and nitrite that is palatable and permissible.75% sugar. The authors postulated that nitrous acid was responsible for the bacteriostatic action of nitrite. Jensen concluded that if the brine content were less than 5.0% brine or above. Dack (1949) reported toxin inhibition in meat with brine levels above 6. these existing products were not affected. nitrate. a very small amount of added nitrite prevented staphylococcal growth when incubated anaerobically. brine values traditionally have been calculated at % brine = (% salt/% salt + % water) × 100. Scott (1955) concluded from the literature that because nitrate exhibited relatively poor antimicrobial inhibition and nitrite.Nitrite 175 Henry et al. if ever. (1956) examined whether curing salts plus the anaerobiosis of a vacuum package would inhibit the growth of S. unless protected by meat juices.9 to 5.5% would have caused consumer . They investigated why Staphylococcus aureus is rarely. Additional tests showed that if the broth medium (pH 6. who performed a series of tests with spiced ham inoculated with different strains of C.5 or above. nitrite enhanced bacterial growth in curing brine. (1954) reported that at pH 7. these observations leave no doubt inhibition by nitrite is at least a possibility.” An international symposium (Ann. 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. causing the cans to swell and forewarn consumers of a potential hazard.4%. was unstable.05. On adding a filter-sterilized solution of nitrite to a broth medium. less nitrite was necessary for inhibition as the pH was decreased from 6. 1955) on semipreserved meats in hermetically sealed containers included a curious debate. The USDA concluded that the high brine would retard the growth of C. They found S. 1 1/2 lb canned ham). Today. Lechowich et al. the growth of Bacillus species would occur. as suggested by Castellani and Niven (1955). Also. This effect was reversed by adding sulfhydryl compounds. Halvorson (1955) cited a private communication from L.

This research and a subsequent review by Silliker (1959) established the future direction for research on nitrite. toxin was produced without concomitant organoleptic spoilage. per se. 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. At brine levels approaching the inhibitory level.0% to 7. 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. Nitrite was shown to play a significant role in the stability of shelf-stable canned meat.. The system providing stability consisted of the combined effect of nitrite. as postulated by test tube experiments. (1958) used this class of product to show that nitrate played no role in retarding putrid spoilage. toxin was not produced.95% brine.8°C for up to 90 days. and 7. the following became increasingly clear: 1. 7. S. Nitrate could serve as an electron acceptor. At 8. thermal injury to the spores. or 5. the reasons for stability had not been defined. As expected. the product was organoleptically unacceptable — as the cans swelled — and toxin assays were performed. 6.09%. salt. its value as a preservative in perishable cured meats was still in doubt. At 5. This brought nitrate under renewed attack. toxin was produced without obvious organoleptic spoilage. permitting the growth of aerobes. Silliker et al. and a low indigenous spore level. although there have been several studies under other conditions in which toxin has been detected without obvious organoleptic spoilage. SUMMARY FOR 1950–1960 As 1960 approached. the primary effect of nitrite was to prevent germination and/or outgrowth of heat-injured spores.5%. Eddy and Ingram. had no antimicrobial effect.14% brine or less.125% and abused at 29. Verhoeven. Nitrate.0%) alone was not responsible for stability. other than its possible influence on water activity. aureus was killed during the heating process given to hams. 1950. Brine content was further shown to influence botulinal outgrowth and toxin formation. salt. Although shelf-stable canned cured luncheon meat had been produced for years. nitrate actively stimulated spoilage by aerobic spore formers. . and thermal injury to the low indigenous level of anaerobic spores. A significant observation was that at brine levels of 6. Anaerobic intolerance to nitrite within the interior of ham. 3.12%.4°C to 37. Brine level (0%. 1950. Because viable spores could be recovered from stable commercial product. such as micrococci and bacilli. 3.176 Antimicrobials in Food complaints of excessive saltiness in the new products. 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. Greenberg et al. (1959) subsequently demonstrated botulinal spore outgrowth in perishable canned cured meat having brine levels from 3. 5. Although nitrite was recognized as an effective antimicrobial agent. Several new reports of unstable commercially canned cured meats containing nitrate appeared (Hankins et al. The suspected role of nitrite was to prevent germination or outgrowth of surviving heat-injured spores. 4. was not confirmed. There have been no reported attempts to duplicate this effect of brine at levels approaching total inhibition.25%. Sodium nitrite levels were 22 µg/g or less at the time the meat was inoculated. 2. 1956).

making them unavailable for subsequent metabolism. even if nitrate is present as an alternative electron acceptor. Shelf-Stable Cured Meats Riemann (1963) supplemented existing information on the incidence. (1964). development ceased before lysis or rupture of the spore walls. It was postulated that nitrous acid reacts either with the cell itself or with various constituents of the medium. of spores of Bacillus species in the presence of less than 300 µg/g of sodium nitrite at pH 6. If the surviving spore counts between the enzyme-inactivating (74°C internal temperature) and the shelf-stable process (F0 = 0. In the course of this research. The increased use of vacuum packaging raised new questions about the microbiological safety of cured meats. spoilage. One reason given was that spores indigenous to meat emulsions have lower heat resistance than spores produced in laboratory media. Shank et al.2. Another factor in spore destruction is that a thermal process of F0 = 0. but at a reduced rate. . The impaired synthesis of heme prevents the formation of the required cytochrome systems. heat resistance. 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. and toxin formation from this low level of survivors during storage at 30°C to 37. but not in others. The presence of 25 µg/g of residual nitrite after processing and a relatively low brine level of 3. and outgrowth of spores with unpublished data from the Danish Meat Research Institute. a 4.0.017 viable cells/g remaining after processing to F0 = 0.203) were compared. 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.7°C for 6 months. the observations of Perigo et al. (1967) opened new possibilities. Gould (1964) observed germination. They concluded that nitrite and nitrate have either neutral or stimulatory effects on bacteria and that nitric oxide has virtually no effect.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. The thermal process used during the early 1960s by seven U.2 (Greenberg et al.. Research was initiated on irradiation for shelf-stable canned cured meats. 1965).6 at the center of the can would yield 10-fold higher values near the surface of the can. Immediately after germination. He estimated the thermal process caused a 4-log reduction of the indigenous spores. Nitrous acid was considered primarily responsible for the quantitative as well as qualitative changes that occur in the bacterial flora when meat is cured. The authors concluded that when oxygen is absent during the growth of some bacteria. Germination was completely prevented by sodium nitrite levels of 750 µg/g or higher at pH 6. Outbreaks of botulism from temperature-abused vacuum-packed smoked fish during the early 1960s added impetus to this research. producers of shelf-stable canned cured meat was reported to range from F0 = 0. and nitrate is not reduced anaerobically.5. In a study comparing irradiation with conventional thermal processing.36% did not prevent outgrowth. Hemin had no effect on nitrate reduction or growth of Escherichia coli or Bacillus polymyxa. The inhibited spores did not appear to become fully phase dark. heme synthesis is impaired. canned chopped ham was inoculated with botulinal spores and given 2 thermal processes. the initial spore load in the product is too high.S. 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.4 to 0.1 to 0. If. Nitrate and oxygen were considered interchangeable as electron acceptors required for heme synthesis in some bacteria. (1962) explored whether nitrite or the oxides of nitrogen were involved in the inhibition of Gram-negative bacteria in cured 5-log reduction occurred. The toxicity of nitrite was 3 to 5 times greater at pH 6 than at pH 7. with a median of 0. Product inoculated with the lowest inoculum level (173 spores/g) had 0. inhibition.

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

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

34 µg/g of residual nitrite). botulinum. Schmidt and Segner (1964) observed increased delay in the growth of four type E strains in culture media (neutral pH) as the nitrite level increased from 0 to 200 µg/g.0 to 4. perfringens inoculated into raw hams could survive the thermal process. At the highest brine level (4.1. botulinum in vacuum-packaged perishable cured meats were reported during this period. and temperature on a Microbacterium species in all-purpose Tween (APT) broth. This suggested that salt becomes more inhibitory as storage temperatures are decreased.8%).6. Similar results were obtained with jellied corned beef stored at 15°C and 25°C. the presence of nitrite caused very little or no inhibition.0 and below. residual nitrite. The combination of brine (3.8°C or 10°C.4% brine at 30°C. The rate of toxin formation decreased as the brine level increased from 3. but not at 29. Failure to obtain toxin production at 5°C may have been influenced by pH because 13 of 15 samples had a pH of 5.7 or less after 4 weeks. 30°C. A decrease in pH likely occurred during storage and prevented toxin formation in the other variables. the authors concluded that the levels used in ham would permit growth. Several studies on the growth of C. On sliced bologna. aureus was inoculated onto sliced chopped ham. The effect of brine on type E toxin production was more . and 37°C). Brownlie (1966) demonstrated the combined inhibitory effect of sodium nitrite concentration.35% and 3. Cooked sliced ham from five producers inoculated with type E spores yielded variable results at 30°C. Toxin was produced within 7 days at 30°C in bologna (3. pH. 21 were able to grow under the same conditions. Their findings that the type of cured meat influenced toxin production were particularly significant. Of 25 lactobacilli from cured meat. but no attempt was made to determine whether they could multiply if the hams were subsequently temperature abused. 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. and nitrite.8%. toxin was produced at 20°C to 30°C but not at 15°C. In sliced ham. Schmidt and Segner (1964) concluded that factors or a combination of factors provided a much longer shelf life than might be expected from salt content alone. toxin was detected in 2 days at 2. but not at 25°C if the brine was 5.1% or higher.4°C. 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). Another 25 strains of microbacteria failed to grow at pH 5. lower maximum population levels occurred in vacuum packages than in nonvacuum packages at 15°C and 22°C. Christiansen and Foster (1965) found type A botulinal toxin production to occur at the same rate on bologna whether packaged with vacuum or without.0. which was thought to be the result of product differences in brine and residual nitrite levels. and decreasing pH during temperature abuse were the suggested inhibitory factors. Nitrite was more inhibitory at 0°C than at the other temperatures tested (10°C and 25°C). toxin was produced only at 30°C. When S. No mention was made of pH and none of the agents were tested in combination.75%). In jellied ox tongue. At pH 6.180 Antimicrobials in Food nitrogen or vacuum was organoleptically acceptable. The four strains failed to produce toxin during 5 weeks at 10°C in sliced bologna (2. At pH 7. nitrate. A similar effect occurred with increasing salt from 0% to 4% during incubation at 7. Within 1 week the product pH had decreased from 6.8% brine). Pivnick and Bird (1965) found the oxygen permeability of packaging films to influence spoilage but not toxigenesis of sliced cured meats inoculated with C. Bologna was relatively resistant to type A toxin production but not ham.7% brine.5% brine and in 4 days at 4. the authors recommended that vacuum-packaged nonsterile processed foods be stored in the frozen state.5 in the presence of 200 µg/g of sodium nitrite. A total of 90% of the samples held at 25°C remained above pH 6. Toxin was not produced at 10°C or lower. pH 6. type E toxin was produced at 10°C to 25°C but not at 5°C. Gough and Alford (1965) showed that spores of C.1% to 4.0. type E toxin was not produced. In jellied pork tongue. From studies in thioglycollate medium containing salt. even at 30°C. a fermentable carbohydrate. The time required for toxin production decreased as the abuse temperature was increased (22°C.

as the brine level increased in the bologna and ham. (1958). increasing the nitrite level increased the time for toxin production. 5.4% brine. C. an increase in the level of residual nitrite also occurred. 3. The authors concluded that heat injury of spores is not necessary for nitrite to prevent toxin production in cured meats. and 8.8. The growth of S. At each temperature (20°C. The system responsible for the shelf stability of canned cured meats given less than a botulinal cooking was studied by several research groups. ham. Kafel and Ayres (1969) demonstrated that the growth of enterococci in culture media and. 1967). The effect of nitrite levels on types A and B botulinal toxin production in vacuum-packaged sliced ham (4..5% brine at pH 5. and Lactobacillus viridescens.3. 9% brine at pH 5. 1968) emphasized maintaining adequate salt levels to assure the microbiological safety of perishable vacuum-packaged cured meats. C. botulinum.8% to 4.1. Warnecke et al.9. . (1967) reported type E botulinal toxin production in ground beef held at 22. 25°C. perfringens. Toxin was also detected in bacon held at 12.8°C and 22. but this is highly questionable. An additional factor influencing the apparent significance of nitrite is the growth of competitive flora.3. in perishable canned hams can inhibit the growth of C. A new explanation for the shelf stability of canned cured meat was proposed. and an input nitrite level of 500 µg/g or more. and more than 10% brine at pH 6. during which time naturally occurring lactics drop the pH to 5. 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. more importantly.. Although not mentioned.5 to 5.5% brine at pH 6. All three products (bologna. 2.35% and after 8 days with 3. toxin was detected after 4 days with 2. Toxin was not produced in either bologna or country cured ham. and 30°C). considering the bacon had a brine level of 5.2 and 3% brine at all pH values.Nitrite 181 dramatic. (1967) suggested an inhibitory substance was formed from nitrite. botulinum type A seemed to be completely inhibited by 4.5. which disappeared during the thermal process.5% or above. The process of outgrowth was more sensitive than spore germination to nitrite.2°C. Riemann (Anon. Evidence was presented that the antimicrobial effect of nitrite is related to the formation of nitrous acid.1% brine) and jellied pork tongue (3. Using juices expressed from the newer sausage product. a brine level of 4. SUMMARY FOR 1960–1970 Progress made during the 1960s toward defining the role of nitrite in cured meats included the following: 1. Type E botulinal growth is inhibited by 2% brine at pH 5. The concept of thermal injury to spores that survive the processing of shelf-stable canned cured meats was confirmed. Toxin was not produced in product with 4.28%. The raw product was then sold at ambient temperature for cooking at home. Perigo et al. He concluded that C. In jellied pork tongue. 5.8 or less. and jellied pork tongue) were formulated with commercial levels of nitrate and nitrite. 4.1.5% brine at 20°C. In both products a marked delay in toxin production occurred at 20°C.6% brine at pH 6.2°C for 3 to 18 days. they determined that type B botulinal outgrowth was inhibited during 4 weeks at 30°C if the juice had a combined pH of 5. The product had been changed to a shelf-stable fermented product precooked to 80°C in an oxygen-impermeable casing.3% brine) was studied next (Pivnick et al. sporogenes. aureus is inhibited by more than 4% brine at pH 5. All of them reached the same general conclusions as those set forth by Silliker et al.

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

Considerable differences were noted in their redox potentials. Omitting any one or all three of the additives did not affect thermal destruction.01% strongly influenced the results.966. The pork was autoclaved before or after adding nitrite. inoculated (105/ml). The inhibitory level of nitrite was not influenced by pH within the range 5. The cans were then filled with brine (3% salt).5°C on the outgrowth of surviving spores was also examined by placing the heated tubes at 23°C for up to 1 year. salt.8% salt.6% salt.7 to 6.4). sealed. cooked.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. Ashworth and Spencer (1972) reported the equivalent levels to be . residual nitrite levels remained above 125 µg/g throughout 80 days at 30°C. This emphasized the importance of medium selection when studying the antimicrobial activity of nitrite and relating the results to commercially produced meats.. normal. nitrite and salt levels had lesser effects. Ashworth and Spencer (1972) described a model system of ground pork (25 g) in 1-ounce bottles. the solids removed. 150. botulinum. and then inoculated with vegetative clostridial cells.3.. and. It was reported that less nitrite was required for inhibition when it was added before autoclaving. ascorbate and cysteine) enhanced the antibotulinal effect of the meat suspensions but did not induce Perigo factor formation. (1967) reported equivalent inhibition with 8 µg/g of nitrite added before heating the medium and 52 µg/g added after heating the medium. cooked sausages. but not at levels above 0. The extract had 2. Reducing agents (e. A similar response with strain 3679 would not be unexpected. 200 µg/g sodium nitrite. the liquid placed into tubes. The magnitude of the effect was much less than was previously reported in culture media. (1967) medium and reinforced clostridial medium but not in liver veal or Wynne fluid media. aw 0. sporogenes spores to provide a level of 104 spores per can. Outgrowth was determined by blackening of the tubes as a result of ferric citrate having been added to the meat. as opposed to perishable canned cured meats heated in hot water to a minimum internal temperature of 66°C. Johnston and Loynes (1971) obtained Perigo factor formation in the Perigo et al. Perigo et al. however. and 50 µg/g of sodium nitrite. sporogenes strain PA 6982 in perishable canned cured meat as occurs with C. and heated (F0 = 0. respectively.4. A significant observation was that residual nitrite did not undergo normal depletion in the extract. The residual nitrite values of the four media and three meat suspensions were about the same after preparation. The effect of heating spores of PA 3679 at 115.5°C in ground pork containing 2. and 0. for inhibition. Small differences in the amount of sodium nitrite below 0. A 1:1 extract was next prepared with 3% saline and ground. (1975).” The effect of all three factors (pH. and overheated (115°C for 90 minutes) canned luncheon meat required 500. Farkas et al.. nitrite. The effect of nitrite.. showing a similar iron–nitrite interaction with C. and salt) appeared to be additive. The major factor influencing outgrowth was pH. pH. although they were “clear and undisputable subsidiary effects. Preliminary data.5% sodium tripolyphosphate was studied by Nordin et al. 115°C). Aqueous meat suspensions prepared from raw. thioglycollate) enhanced the effect of nitrite.01%. overlaid with paraffin oil. and heating at 115. 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.g. and pH 6. personal communication). The sausages were prepared with nitrite-containing curing salt. One of the sausages in each can was inoculated with C. In ground pork.Nitrite 183 sodium nitrite caused the greatest destruction when heated. exist (Christiansen et al. sodium ascorbate. After a loss of 15% to 25% during processing and an initial decline.6. (1973) studied the effect of adding nitrite to the brine solution for canned Vienna sausages. Sausages packed in brine with 400 µg/g of sodium nitrite and processed to F0 = 0.35 were as stable as sausages in brine without nitrite and processed to F0 = 2. Reducing agents (e. held overnight for nitrite equilibration. to some degree. thereby confirming earlier research. These observations led to the conclusion that a Perigo-type effect was detected in the meat system. cysteine. and then packed into cans. and heated to varying degrees.g. as already discussed. Between 120 and 200 µg/g sodium nitrite provided stability through 5 weeks of storage.

Residual nitrite. another inhibitory factor may be formed when nitrite is heated with meat.. 100.4 The product was held until the highest level of nitrite declined to less than 2 µg/g.5 and 22.1 µg/g) were the residual nitrite levels after 0 and 2 weeks in product made with 200 µg/g of nitrite. which indicates that residual nitrite influenced the results. 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. Botulinal . In summary.4 in raw meat juice with 4. 150. and 200 µg/g of sodium nitrite and processed to F0 = 0. the Perigo effect was of low magnitude. on the basis of added nitrite. Pivnick and Chang (1974) considered. The additional nitrite provided comparable residual levels after the various heat treatments. Ashworth et al. If residual nitrite is a valid means for comparison. 1967) that the nitrite that disappears during heating may be involved in the formation of the unknown inhibitor. per se. (1967) based their conclusions on the added level of nitrite. They concluded that although a PTF was formed.4.g. thermal destruction of spores and inhibition of the survivors by the salt). deaerated in boiling water.1 µg/g.5 or 22. Johnston et al.. Chang and Akhtar (1974) prepared buffered saline suspensions of canned luncheon meat made with 0. Perigo et al. and inoculated.000/g) added after heating and spores (20/g) added before or after heating.5% salt and 150 µg/g of sodium nitrite.184 Antimicrobials in Food 60 to 70 and 90 to 100 µg/g. It was originally suggested (Perigo et al.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. The data obtained with the spore inoculum should be more relevant to shelf-stable canned cured meats. less residual nitrite was required for inhibition when nitrite was added before heating. results similar to those just mentioned were obtained during the early stages of temperature abuse. that although the Perigo inhibitor may not be present in meat. The term Perigo-type factor (or PTF) was selected to describe the effect observed in their test. The nitrite level of the suspensions was adjusted after heating to 53. which had survived heating to F0 = 0. When spores were used. 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. (1969) found the inhibitor of Perigo et al. Shelf-stable canned cured luncheon meat was prepared with 0 to 300 µg/g of sodium nitrite and processed to F0 = 0. higher initial residual nitrite levels were required for inhibition. however. autoclaving. (1973) extended this research to the milder heating given to large perishable canned hams and obtained similar results. Adding salt (3. such as perishable canned ham. and it may also play a role in the safety and stability of pasteurized cured meats. concluded that a PTF was produced. The meat was then inoculated with botulinal spores. (1967) to be inactivated when added to meat. In all cases. Inhibition by nitrite diminished gradually over a period of 8 weeks at 37°C. Ashworth et al. The levels selected (53. The depletion rate of the nitrite added to the suspensions was not influenced by the amount of nitrite originally added to the meat. The amount of PTF formed was related to the amount of original nitrite added to the meat before heating. strongly influenced clostridial inhibition. with prolonged storage (42 days at 37°C). In this case alone. The authors found that as the original input nitrite level increased. This was considered evidence for an inhibitory factor other than residual nitrite. However. Similar residual nitrite levels were required to inhibit vegetative cells (160. rather than after (150 µg/g). Ashworth and Spencer based their conclusions on the residual level of nitrite. the relative inhibitory effect was quantitatively small. more was required when added before heating the meat. the conclusion that a Perigo effect was demonstrated must be tempered with the observation that. 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. Ashworth and Spencer (1972) found no relation between the amount of nitrite lost during heating and the inhibition obtained. more added nitrite was required in the pork if the nitrite was added before (300 µg/g). Actually. higher levels of added nitrite were required for inhibition. outgrowth and toxin production decreased.

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

Christiansen et al.5.5°C and 25°C. one reason was found to be the addition of sodium isoascorbate to the canned meat but not to the meat slurries. This led to the pork slurry system of Rhodes and Jarvis (1976) and used by Roberts et al. It was suggested that Bacillus species are less sensitive to nitrite than clostridia. 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. even with 300 µg/g of sodium nitrite. Grever (1974) examined the stability of emulsions normally used for preparing cooked sausage and liver sausage. 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.5% salt. Sodium nitrite and L-ascorbic acid were added as filter-sterilized solutions. the greatest inhibition occurred with 1. increasing salt (1. Adding L-ascorbic acid (1. (1976) found that heating botulinal spores in an aqueous extract of lean pork at 85°C. 3.0. 1978b) involving product held at 4. 15°C). At 20°C. diverse collection of botulinal strains in a broth medium and a cooked pork medium. 90°C.5%. a preliminary test in bacon led to the conclusion that L-ascorbic acid does not alter the antibotulinal properties of nitrite in bacon.0%) markedly increased the effectiveness of nitrite with none of the 38 strains tested showing growth in broth containing 50 µg/g sodium nitrite. recommended that large-scale tests be used to quantify the many interacting factors influencing the safety of perishable cured meats.5% salt. 1978a). The number of strains showing growth in broth was found to decrease with increasing nitrite (50.5%. decreasing temperature (25°C.. Autoclaving nitrite in the Perigo medium resulted in significantly more inhibition than autoclaving the nitrite in the meat slurry. Jarvis et al. thermal processing within the range of 63°C to 74°C (internal temperature) influenced neither residual nitrite levels nor botulinal inhibition in perishable canned cured meat . 101). The longer the product was held at refrigeration temperatures. (1977). 6. 6. 5. Stability was influenced by nitrite content. and whether the products were treated by a perishable or shelf-stable thermal process.8% or 3. (1976) to study the interactions of temperature of abuse. 22. Isoascorbate had enhanced the antibotulinal efficacy of nitrite in the canned meat. less inhibition occurred. neither salt level provided much inhibition. (1976).4°C or 10°C for up to half a year before placing at 27°C. The emulsions were filled into cans and heated. At 17. The antibotulinal effect of nitrite was considerably greater than that reported for the meat slurries of Roberts et al. Although less effective.1%) also increased the efficacy of nitrite in broth but had no effect. a pronounced salt–nitrite interaction occurred. 20°C.0% w/v). and inoculum level on botulinal growth and toxin production at pH 6.0%. decreasing pH (7. In another study. At 15°C. a race occurs between death of germinated botulinal cells and depletion of residual nitrite.5% or above. On investigation (Tompkin et al. Temperature of abuse was the most significant factor influencing the rate of botulinal growth. The spores heated at 70°C and 95°C were equally sensitive to the autoclaved nitrite. Botulinal outgrowth was dependent on the relative levels of residual nitrite and surviving botulinal cells. the more the residual nitrite levels declined and the less inhibitory the product became when placed at 27°C.5% but not 3.5°C. pro or con. 100.5°C. level of clostridial spores.0. in the cooked pork medium. Spores heated at lower temperatures (70°C or 80°C) were inhibited by 4. nitrite. 6. 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. salt. 4. perishable canned cured meat is placed at 27°C. 200 µg/g). The rate at which inoculated cans swelled at 27°C was inversely related to the level of nitrite added to the product.5).186 Antimicrobials in Food Baird-Parker and Baillie (1974) studied the growth of a large. This agreed with an earlier observation (Tompkin et al. or 25°C.. 150. At 20°C.5% salt (on water phase). product pH. 20 µg/g of nitrite in the Perigo medium and 210 µg/g in the slurry were equally inhibitory.0. Furthermore. (1978) concluded that when inoculated. 103. At the lower temperatures. or 95°C decreased the number that could grow in a recovery medium containing 2. The effect of nitrite in perishable canned cured meat was also reported by Tompkin et al. Jarvis et al. a lower concentration of L-ascorbic acid (0. and decreasing inoculum level (106.

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

.83%. more importantly. The rate of nitrite depletion was influenced by the type of soy. It was concluded that ascorbate did not affect the antibotulinal efficacy of nitrite. 1973). and 655 µg/g). (1979e) found the rate of germination of C. 105.8% brine and nitrite levels of 50 to 100 µg/g delayed botulinal outgrowth until the lactic flora decreased the pH to an inhibitory level. 450 µg/g of nitrate gave a variable response. a fairly rapid decrease in product pH during abuse. or 150 µg/g of nitrite declined to 5. 20. In a meat–soy mixture. The second test. Under these circumstances. (1973) formulated franks with various levels of nitrite and nitrate. A significant delay in botulinal toxin production occurred in pork. examined whether an increase in ascorbate over normal levels would alter the antibotulinal effect of nitrite. Nitrite was ineffective in a formulation of soy isolate and pork fat.08 or lower within 1 week at 27°C. The franks were produced in the same facilities and formulated as before (Hustad et al. The strong inhibitory effect observed was likely the result of the combined effects of nitrite. and.0 or less within 21 days. . Vacuum-Packaged and Fermented Meats The effect of nitrite in vacuum-packaged franks was studied on two occasions. 400 µg/g of sodium ascorbate. 1973). The pH of product made with 50. reported by Bowen and Deibel (1974) and Bowen et al. 40. and 156 µg/g). Assuming a similar pH response in the first test (Hustad et al.3°C) sausages containing dextrose. input versus residual nitrite. (1979d) compared the efficacy of nitrite (0 and 80 µg/g) in chicken. In the first. and omission of nitrate. In that variable the pH was 5. only those variables without nitrite became toxic at 27°C. beef. 100. (1979c) evaluated various soy proteins in their system. A later review of the pH data showed that all variables had a pH of 5. The depletion of residual nitrite was faster as the amount of beef was increased in a formulation containing soy. Hustad et al. in both the presence and absence of nitrite. The type of soy protein influenced the results. and pork in their system. botulinum spores in a chicken frank formulation to be unaffected by the addition of sodium nitrite (0. the addition of 156 µg/g of sodium nitrite was very effective in delaying botulinal toxin production. The rate of botulinal toxin production in formulations with soy protein was equal to or slower than in an all-meat formulation. the exception being one variable with 150 µg/g of nitrite. Adding 50 µg/g of sodium nitrite delayed botulinal toxin formation for more than 4 weeks during abuse at 27°C. and then only 2 of the 55 franks became toxic. Toxin was produced in all variables formulated without dextrose and more quickly in product having 0 or 50 µg/g of nitrite. Although product made with 150 µg/g of sodium nitrite did not become toxic. the levels of nitrite (0 to 150 µg/g) and ascorbate (9. The addition of 80 µg/g of sodium nitrite was relatively ineffective in chicken and beef. Toxic product occurred with 50 µg/g of sodium nitrite at all levels of ascorbate. the relatively high brine. and 4.188 Antimicrobials in Food Sofos et al. They concluded that the effect of nitrite was on delaying the development of the cells after germination and before toxin was produced.52% brine. 1975). In mildly heated (58. The rate of botulinal toxin production was slower in all soy protein formulations than in all meat formulations. about 2. and fewer toxic samples occurred in product with 100 or 150 µg/g of nitrite.6. The decline in pH caused by a lactic fermentation was also the dominant factor preventing botulinal outgrowth in fermented sausage (Christiansen et al.0 or less within 14 days. The finished franks had a brine of 4. and the products remained nontoxic.5% to 4. and the effect of ascorbate in franks should be restated or reexamined. except for changes in the ratio of pork to beef.8% fermentable carbohydrate. the conclusions in both studies concerning the inhibitory levels of nitrite. The pH of the dextrosefree product remained above 5. One toxic sample occurred at 100 µg/g and none at 150 µg/g of sodium nitrite. Sofos et al. Sofos et al. (1974). the two studies indicate that the combination of 4.. Toxin production was slower..

Nitrite 189 A request was made by the poultry industry to the U. The nitrate likely served as an electron acceptor for the growth of micrococci in the vacuum package. Shaw and Harding (1978) studied the effect of nitrate and nitrite on the microbial flora of Wiltshire bacon. medium 4. and 25°C) on the rate of botulinal toxin formation.80 and 0. and mixed cure with 200 µg/g of nitrite were compared. 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. A summary of the data showing the antibotulinal effect of nitrite in the two poultry products appears in Hauschild (1982). Raevuori (1975) studied the combined effect of sodium nitrite (0. 500. 2. After cooking in Saran™ casings. Bacon that was sliced and vacuum packaged developed a flora mainly of micrococci and lactics. The naturally occurring B.g. 1000.8% and 21. cereus). . 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.. The addition of ascorbate enhanced the antibotulinal effect of 100 µg/g but not 200 µg/g of nitrite. The data included botulinal challenge tests in frankfurters made with chicken meat and ham made with turkey meat. higher populations of S.9% to 4. and Moraxella-like bacteria. Erythorbate alone was not inhibitory. Raevouri encouraged more widespread use of erythorbate for sausage production in Finland.7% became toxic.9% became toxic. respectively.9%. Moraxella species. botulinum.7% to 6. 67. In opened packages. There was a general trend toward reduced residual nitrite levels at the tune of inoculation when ascorbate was added. If all samples containing mixed cure with and without ascorbate were compared. 42. botulinum.6%) on botulinal toxin production in vacuum-packaged back bacon. 20°C. and brine level (mild.4%. lower populations resulted and enterotoxin was not detected. Inoculation studies examined the effect of nitrite (0 and 100 µg/g) and storage temperature (15°C.7% and 18.S. and 1000 µg/g) on the growth of Bacillus cereus in cooked sausage placed at 20°C for 48 hours. Omitting nitrate led to higher numbers of Moraxella species in the cured bacon. growth was faster at 25°C than at 15°C. the sausages had relatively high pH and water activity values of 6. 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. 1977). B. Product with 100 µg/g of sodium nitrite remained nontoxic through 14 days at all three temperatures. (1977) that demonstrated the efficacy of sodium nitrite in poultry meat. and 2000 µg/g).98. and 34. Noninoculated product containing nitrite developed a slight unusual taste at 20°C owing to the growth of spore-forming spoilage organisms (e. The request included results by Christiansen et al. respectively. the interaction of nitrite and ascorbate is difficult to interpret.65 to 6. mixed cure with 100 µg/g of nitrite. If all samples with no mixed cure. When all samples having the mild and medium brine were compared. 100. cereus tested.9% became toxic. Erythorbate caused reductions in the residual nitrite and the redox potential of the product. It also was reported that S. As expected.. 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. Bacon Crowther et al. (1976) in the United Kingdom studied the effects of a mixture of nitrite (100 or 200 µg/g) and nitrate (250 µg/g). respectively. aureus developed and enterotoxin A was produced. In vacuum packages. The predominant flora of the bacon after curing consisted of micrococci.3%. cereus was evidently more resistant to nitrite than C. Because brine levels could not be held constant. Including nitrate in the bacon enhanced the growth of micrococci. 35. and 200 µg/g) and sodium erythorbate (0. regardless of the level of nitrite or ascorbate. Erythorbate was found to enhance the inhibitory effect of nitrite. Food and Drug Administration (FDA) for approval of sodium nitrite as an additive in poultry products. aureus grew well in the medium-salted bacon. A higher percentage of samples were toxic with the addition of 200 µg/g of nitrite than with 100 µg/g of nitrite. 19. ascorbate (0.

The initial period of delay depends on the inoculum level. Bowen and Deibel. 1980). There was considerable variation in toxin production in the bacon from the four plants. . The bacon was shipped to a laboratory. Tanaka et al. As the brine level exceeds 4. 10 appear in the technical literature (Christiansen et al. 1978. sucrose) were left to the discretion of the producing plant. it was suggested that nitrite could be important in delaying the sour spoilage caused by the growth of lactics. Temperatures below 27°C or 30°C increase the delay in outgrowth and the inhibitory effects of the foregoing factors. Botulinal outgrowth is influenced by the temperature of abuse. The USDA conducted a test to verify the antibotulinal effectiveness of a combined nitrite–sorbate mixture in bacon (Johnston.. The results demonstrated that the combination of nitrite–sorbate was equal to or better than nitrite alone. 1980b. and bacon with 40 µg/g of sodium nitrite plus 0. aside from the fact that the amount of added nitrite partially determines the level of residual nitrite. outgrowth is increasingly delayed. 4. Of the 30 tests. Ivey et al. 5. If a lactic fermentation develops in the interim. The level of residual nitrite at the time the bacon is abused influences the extent of the delay in botulinal outgrowth. bacon with 120 µg/g of sodium nitrite. Although the studies were performed for a variety of reasons. A total of 30 botulinal studies were performed with vacuum-packaged bacon during the 1970s in the United States and Canada. 6. Too high a level of botulinal cells when the bacon is abused can overwhelm the inhibitory system. These factors interact to influence botulinal inhibition. inoculated with 1000 spores/g. phosphate. The pH of the bacon during abuse significantly influences the potential for botulinal outgrowth. 3. The bacon was then incubated at 27°C for up to 56 days. Collins-Thompson et al. 1979).26% potassium sorbate.190 Antimicrobials in Food Because higher numbers of lactics were present in bacon with the lowest initial nitrite concentration. The level of nitrite added to the product is not important. The data indicate the following: 1.. Vacuum-packaged bacon prepared with 0. At least on the basis of the botulinal results. They can be more readily compared because they were similar in processing and final product. Within the levels and types used.0%. it is possible to glean the relative effect of nitrite by examining the nitrite-containing control variables from each test. and growth of a spoilage flora). Three types of bacon were produced in four commercial plants: bacon with no preservative. phosphates do not appear to influence botulinal outgrowth in bacon. Brine levels below 4. All three bacons contained 550 µg/g of sodium ascorbate or sodium isoascorbate. The levels of sodium chloride.. However.. 2. 8. This was likely the result of the interaction of the other components of the product (brine level. Sofos et al. it is difficult to assess their effect. and fermentable carbohydrate (e. the combination of relatively higher brine and decreasing pH can prevent botulinal outgrowth. ascorbate and isoascorbate can also have a negative effect by causing more rapid loss of residual nitrite during processing and storage.0% are not inhibitory to botulinal outgrowth. 1974. the data supported the potential approved use of a nitrite–sorbate blend to produce bacon. 1974. fermentable carbohydrate. 7. and vacuum packaged (100 g per package).g.. The addition of ascorbate or isoascorbate can act in concert with residual nitrite to retard botulinal outgrowth in freshly produced bacon. Because phosphates were present in almost all the studies.. 1974.7% sugar (sucrose) or more provides sufficient fermentable carbohydrate that naturally occurring lactics cause a decline in pH to inhibitory levels.

15%. Nitrous acid levels were calculated from the residual nitrite and pH values as described by Castellani and Niven (1955). and 0% oxygen yielded lower S. At pH 7. Genigeorgis et al. aureus is less acid tolerant under anaerobic conditions. A third sample with a pH of 5. Buchanan and Solberg (1972) studied the effect of nitrite (0. 500.3. 7. anaerobic toxin production occurred at pH 5. but it is unclear whether nitrite level influenced anaerobic enterotoxin production..3. aureus in brain heart infusion broth (BHI). Understanding why this occurs would be very instructive. aureus by blocking the sulfhydryl sites of either coenzyme A or α-lipoic acid. 200. aureus under aerobic but not anaerobic conditions. This suggested either that nitrite was not being used as an electron acceptor or the nitrite reductase system was blocked anaerobically. (1976) indicate that the growth of S. aureus is reduced and enterotoxin production can be prevented by the environmental conditions existing within vacuum-packaged cured meats.3) on the growth of S. Inoculated raw sausage (2.9% salt and 100 µg/g of sodium nitrite) incubated at 37°C in environments of 20%. Heterofermentative lactobacilli were more tolerant of salt than the homofermentative strains. aureus. but it remains debatable how much each of the possible factors (e. However. At pH 6.g. aureus populations as .34. 5%.6 or less).3. or both prevented anaerobic toxin production.01% salt. Very high levels of nitrite (1000 and 5000 µg/g) either prevented or severely retarded the growth of all strains. reduced pH. no residual nitrite.58. Collectively. low oxygen level. aureus and Other Bacteria A cluster of reports appeared during the early 1970s that dealt with the effect of nitrite on S. 8 µg/g enhanced the growth of the lactobacilli and. the increase in the adjustment phase was 10 hours or less aerobically and about 30 hours anaerobically. to a slight extent. 10%. nitrite was without effect in an aerobic environment and 500 µg/g or less was without effect anaerobically. One reason given was that S.3 in two samples with negligible nitrite levels. and initial pH (6. Extensive tests were also reported for laboratory-cured ham. nitrite. micrococci and staphylococci (24 strains). The commercially canned ham used for the test had a brine level of 3. it is uncertain whether high nitrous acid content. brine. The probability of toxin production at 10°C reportedly decreased as the level of nitrous acid increased.3 and 200 µg/g. They also concluded that vacuum packaging should improve the safety of cured meats relative to S. Lactobacilli (78 strains). (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. After considering the effects of changing pH during the growth of S. and 2000 µg/g). Anaerobic toxin production was prevented with 0. future research might demonstrate the involvement of additional factors.159 µg/g of nitrous acid also became toxic. because high nitrous acid values (>0. and product pH) contributes to this effect. aureus was found to be restricted to the periphery of commercially fermented sausage (Barber and Deibel. The authors questioned the hypothesis that nitrite inhibition is dependent on the conversion of nitrite to nitrous acid. the micrococci.6 µg/g) were possible only at reduced pH levels (pH 5. 1972).Nitrite 191 Tests with S. At 200 µg/g growth was delayed or slower. however.6 µg/g or more nitrous acid and pH values below 5. aureus. At 40 µg/g growth was comparable to that in the control without nitrite. Nitrite was metabolized by S. Buchanan and Solberg suggested that nitrite inhibits the growth of S. the data along with the aforementioned results of Christiansen and Foster (1965) and Crowther et al. and 0.0). thus preventing the normal metabolism of pyruvate. Toxin was produced in certain variables having residual nitrite levels as high as 145 µg/g. oxygen pressure.6%. Reasons for the reduced growth and enterotoxin production are evident from the reports. aureus and a review of the literature. growth of competitive flora. Of significant importance was a report that the growth of S. and Pediococcus cerevisiae (1 strain) were examined for their tolerance to nitrite in the presence and absence of 4. 1000. and an abnormally low pH of 5. Nurmi and Turunen (1970) studied the effect of adding nitrite to a previously autoclaved broth medium (pH 6. At higher temperatures (22°C and 30°C). 22 µg/g of nitrite.

Contrasting results were subsequently reported that showed the production of enterotoxin A to decrease as pH decreased.0 but to a lesser degree than at pH 7. Markus and Silverman (1970) reported enterotoxin A production in broth media (pH 6.5. decreasing incubation temperature. aureus. oxygen uptake. The evidence strongly suggested that nitrite exerted its inhibitory effect by oxidizing ferrous ion of an electron carrier(s) (e. In another report on non-spore-forming bacteria. 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. and pH). Formation of some similar inhibitor(s) (PTF) during the thermal processing of shelfstable canned cured meat appears possible. and 7.0 but not at pH 5. 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. salt level. and nitrate on either growth or enterotoxin production.7 (Morse and Baldwin. The formation of the Perigo factor in commercially cured meat is highly debatable. presumably. the sum of which influences stability. residual nitrite.. 1975) demonstrated the combined effect of pH. 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. nitrite. There was a general pattern of increased heat resistance as salt level (0. Nitrite did not affect glucose transport by S. Morse and Mah (1973) studied the effect of glucose on enterotoxin B synthesis in a broth medium buffered to an alkaline pH (7. SUMMARY FOR 1970–1980 The Perigo Factor in Cured Meats 1. and. Glucose repression of enterotoxin B production was also reported to occur at pH 6. and oxidative phosphorylation in Pseudomonas aeruginosa. salt.7). enterotoxin was not detected through 5 days of storage in the absence of oxygen.5 or 5. 1973). and decreasing pH.. no synergistic effect occurred between salt.8) to be unaffected by sodium nitrite (200 µg/ml) or sodium nitrate (1000 µg/ml). 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 8%) increased at pH 6. If manipulations of thermally processed meat were attempted to extract such an inhibitor. 4. and nitrate was converted to nitrite. 1973). Adding nitrite as an electron acceptor at the time of anaerobic shock prevented increased toxin production. and nitrite on the heat resistance and recovery of S.6 to 6.192 Antimicrobials in Food the oxygen level decreased. Perhaps because of the atypically high pH. Bean and Roberts (1974. and nitrite increased (Tompkin et al. when added to the recovery medium. The inhibitory effect of nitrite in the recovery medium increased with increasing salt content. salt increased. 2. Although adequate growth occurred within 48 hours. Rowe et al. the question could be raised whether the inhibitor would be altered or destroyed. Adding glucose caused decreased toxin production as a result of the rapid oxidative decarboxylation of pyruvate. 6.g. . (1979) reported that nitrite inhibits active transport. However. more available energy. the percentage of lactic acid as an end product. The effect of nitrite on heat resistance was inconsistent.0. increasing the level of nitrite had the general effect of reducing the recovery of both heat-damaged and undamaged cells. faecalis or Streptococcus lactis. both of which lack cytochromes. cytochrome oxidase) to the ferric state.

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

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

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 data provided estimates of the relative degree of botulinal risk for each product. TABLE 6. where P is the probability of outgrowth and toxigenesis from one spore.3–0.5 Thermoprocess (F0 ) 1.3 0. and commercial practices to arrive at guidelines to assure the microbiological safety of these products.0–4. .2 1. Since that time.7 4 2. They summarized research. existing government regulations. whereas turkey ham was a high-risk product. there has been a very rapid growth in the volume and variety of cured poultry products in the United States. Another major trend developed during this time. carbohydrate. the per capita consumption of red meats decreased while the consumption of poultry products increased.0 5. The level of risk differed among the various products.3 3. the impact of further reductions in salt deserved close examination.1–0. Their review was to assist in the development of a document for these products.5–5. He concluded that significant reductions in nitrite should not be made for all cured meats.1 Shelf Stable Canned Cured Meats: Minimal Brine and Thermoprocess with 150 mg/g Sodium Nitrite Product Luncheon meat % Brine 3–4 4.0 0.5 4.5 3. However.Nitrite 195 for sodium intake in the diet brought new pressure to reduce the salt content of cured meats. 1990). He proposed expressing botulinal inhibition as log I/P. Bacon was considered a low-risk product.0–5. the FDA acknowledged that prior sanction existed for the use of nitrite as an additive to poultry meat (Cassens.5 Ham and shoulder Sausages Source: Hauschild and Simonsen (1985).0–1. This was used to quantitatively compare the data from the various challenge tests.2–0. Considering the recent reductions in the amount of nitrite used for curing meats and the voluntary elimination of nitrate from most cured meats in the United States.5–1.5 0. Hauschild and Simonsen (1985) conducted an extensive review of the technology for producing shelf-stable canned cured meats (Table 6. selective reductions in nitrite could safely be made provided the levels of brine. This information was reiterated by Hauschild and Simonsen (1986). Throughout the decade. The level of risk could also differ among products of the same type as a result of differences in their brine concentrations.1).5 0. In 1983. Reductions in the recommended levels should be based on extensive plant experience and/or experimental work and controls to assure minimal spore levels. Reductions in the level of nitrite can be compensated for by moderate increases in the brine level and/or thermoprocess.0 0. further accelerating the trend of the past 40 years. and erythorbate are controlled.5 1. 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.

Some consumers find this amount of salt unacceptable. (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. botulinum was estimated to be about 0. By increasing the thermal process. and potassium sorbate (0% and . thereby permitting the more heat resistant C. Wagner and Busta (1983) compared the antibotulinal effect of combinations of sodium nitrite (40 and 120 µg/g). Additional data on the ham experiments were presented by Lücke et al. By increasing the processing temperature and/or prolonging the time of processing. The presence of sorbic acid slowed the rate of nitrite depletion. Sofos et al. Liver sausage was prepared with 83 µg/g of added sodium nitrite. 10°C.15 spores/kg in the product. and 6. Greater protection was obtained with 150 µg/g. Methods and media were recommended to facilitate the recovery of both the proteolytic and the psychrotrophic nonproteolytic strains of C. 1982). (1982). The use of nitrite in smoked fish in the United States has been restricted. cooled.4%. Toxin was detected after 9 to 11 days. Hauschild and Hilsheimer (1983) examined 138 samples of liver sausage from retail outlets in Canada.44%. After curing. Hauschild et al. botulinum. nitrite in the range of 0 to 100 µg/g had little or no effect on toxigenesis. heated. The antibotulinal efficacy of sorbic acid was pH dependent.24/kg. Perhaps the milder thermal process permitted the survival of a competitive flora that prevented toxigenesis. The total C. the inhibitory effect of the nitrite was reduced. As temperature was increased to 8°C. nitrite. toxin was detectable 10 cm from the injection site.8% to 4.2% brine.2%. and aw 0. The rate of pH decrease was slower as the brine level increased. Storage at 8°C until a nearly complete loss of nitrite occurred did not diminish the effect of the nitrite. sodium acid pyrophosphate (0% and 0. and incubated at 27°C.2 without loss of the benefit of sorbic acid. the pH of the formulation could be increased to pH 6. the inhibitory effect of the lactic fermentation and decreasing water activity values during fermentation and subsequent drying prevented botulinal toxicity. a high level of salt (5% brine) has been required.0 being required. They concluded that nonproteolytic. (1982) assessed the relative risk of botulism from temperature-abused liver sausage. and hams was investigated by Liicke et al.2. nitrite was more effective and an appreciable delay in toxicity was obtained with 50 and 100 µg/g of nitrite. As the hams continued through the completion of curing. The pH of the fillets decreased during 27°C incubation from about pH 6. The hams had a slight off-odor but no apparent putrefaction.4%). Cuppett et al. At brine levels of 3. but the recovery of viable botulinal cells became increasingly difficult. (1980a) studied the significance of pH on the efficacy of nitrite and sorbic acid mixtures in a chicken frank emulsion. The nitrite (40 µg/g) also significantly increased the inhibitory effect of the sorbic acid. The latter were suspected to be of particular importance as a cause of botulism from raw cured meat products in Europe (Lücke. overlaid with vaspar. psychrotrophic strains can grow and produce toxin during the early stages of curing before the salt and nitrite levels penetrate and reach inhibitory levels. Product with low levels of toxin often appeared acceptable. Similar results were obtained during the dry curing of hams at 8°C or 10°C.196 Antimicrobials in Food Botulinal Challenge Tests Tests in meat and fish products. The number of botulinal spores was estimated to be about 0. with levels of at least below pH 6. 4.98. competitive flora could have been destroyed. If a low level of nitrite (40 µg/g) was added. toxin production was increasingly more rapid. Tests in model systems. To prevent the growth of C. (1980). and 15°C. The tests involved vacuum-packaged smoked whitefish fillets prepared with about 2. toxin levels remained high. pH 6. fermented sausage.2. The test system consisted of a frank emulsion prepared from mechanically deboned chicken meat that was extruded into test tubes. and nitrate at 8°C. Toxin was not produced at 5°C. botulinum to dominate and develop toxin. Large bone-in hams were inoculated with nonproteolytic botulinal spores before curing in a solution of salt. Another possibility could be offered for this observation.3%. In fermented sausage. At a higher brine level of 5. Toxin production by a mixture of type E and nonproteolytic type B botulinal strains in liver sausage.4 to as low as 5.

Roberts et al.27 to 6. The frankfurters were made from chicken instead of beef and pork. sodium acid pyrophosphate was more effective than sodium hexametaphosphate or sodium tripolyphosphate. The combination of nitrite (40 µg/g). Considerable variation occurred between the meat from different animals within each breed.26%) in frankfurters. (1979b–e.6 to 6.3) and high-pH pork (pH 6. (1981a.65.27 to 6.0. A separate formula was developed for slurries prepared with low-pH pork (pH 5. Gibson et al.72). (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. 0. except for two outliers of 5. The test system avoided the problem of recontamination that occurs when peeling and packaging franks. The effect of sorbate was greater as salt levels . (1982) examined the interaction of potassium sorbate in their cured pork slurry system. and 4. with certain phosphates being more effective than others. but the reason for the increased inhibition when adding polyphosphate to the higher pH meat slurries is less evident.3 to 6. potassium sorbate significantly decreased toxin production. isoascorbate. sodium acid pyrophosphate (0.54. They also found that the antibotulinal efficacy of nitrite could be increased by the addition of phosphate. Tubes showing gas or other spoilage were preferentially selected for toxin assay.78 to 6. nitrate.b) evaluated the interactions of salt. 3. Nelson et al. No systematic difference in inhibition was observed with the meat from three breeds of pig.56. The pH of the four product variables was between 5. The levels of salt found in the slurries were often as much as 0. Of the polyphosphates tested. adding isoascorbate or nitrate. Roberts et al. At a given spore inoculum.Nitrite 197 0. It is unclear whether the target or analytical values for salt were used for statistical analysis. Jarvis et al. more like that of a reformed ham after heating.19. although the conclusions were stated on the basis of the target values of 2. This increased inhibition was not observed with nitrite levels of 75 to 125 µg/g. The pH of the pork used for the lower pH slurries ranged from 5.5%. The initial pH for the various products was 5. There appeared to be more botulinal inhibition with the addition of sodium acid pyrophosphate. nitrite. and thermal process on botulinal outgrowth in pork slurries of two different pH ranges (pH 5. The pH of the pork used for the higher pH slurries ranged from 6. 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. potassium sorbate (0. and 0. or decreasing the abuse temperature.65 to P80°C = 6.7 and 6. Statistically significant reductions in toxin production occurred with increasing salt level. They found the nitrite to be less effective in slurries prepared with meat from the shoulder than meat from the leg. Polyphosphate increased botulinal inhibition when added to combinations of sorbic acid or potassium sorbate and a low level of nitrite (40 µg/g).26%).2 units in the higher pH slurries.4%). (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. (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.7). The pH was important in all treatments tested.5% (wt/vol water) below the targeted values. or thermal process. This may alter the quantitative assessment of the individual and combined effects of the additives. although the trends in the data may not change. They used the same test system as in Sofos et al. Roberts et al.26% potassium sorbate. They found that increasing the heating from P80°C = 0. The polyphosphates did not influence botulinal inhibition in the absence of nitrite.4 and 5.1 to 0. The addition of polyphosphate caused an increase of about 0. The combination of 40 µg/g of sodium nitrite.36 and 6. One nitrite level (40 µg/g) was used throughout. polyphosphate. and pH control of the emulsion was the most effective to achieve reduced nitrite levels and provide enhanced botulinal inhibition.5% salt (wt/vol water). nitrite level.4% sodium acid pyrophosphate caused the greatest delay in toxin production. 1980a). The increase in pH could explain the decreased inhibition in the lower pH slurries.03.4 units in the lower pH slurries and about 0.5%.4 to 6.5 to 6. 1983).

Bactin was not produced when heating cysteine. 20. 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 stored at 30°C for 28 days. sealed with vaspar. unless ferric chloride was present and the pH was 6. and 200 µg/g of sodium nitrite. U. In another study of Perigo factor formation. which resulted in rapid botulinal outgrowth. 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. They found that the rate of depletion in their pork slurry system was influenced by many intrinsic and extrinsic factors. 50. and as the storage temperature decreased. No suggestion was offered for the increased botulinal inhibition when nitrate was added. (1989) prepared ground pork slurries with 0. The results are intriguing and leave relatively unexplored the underlying mechanisms influencing the results. the rate of nitrite depletion was influenced by storage temperature. forms an inhibitor when autoclaved in the presence of nitrite.5% to 3. botulinum. The significance of microbial growth on nitrite depletion in the slurries during storage was not addressed. the addition of nitrate. For example. This led Gibson et al.0. Residual nitrite levels after irradiation were inversely related to the irradiation dose. However. The probability of botulinal outgrowth decreased as the level of added nitrite increased above 50 µg/g. The amount of iron required for inhibitor formation was reportedly similar to the level found in muscle tissue. responded similarly. 40. and 50 kGy). and the addition of isoascorbate. 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. thioglycollate. The addition of nitrate led to higher residual nitrite levels for a longer period. 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). an iron-binding protein in casein. 30. They suggested that the bacteriostatic effect is the result of facilitated conversion of cysteine to Bactin and a concomitant removal of thio compounds. there was a marked decrease in the residual nitrite. Higher or lower levels prevented the inhibitory effect. Custer and Hansen (1980) reported that lactoferrin. At lower dose levels of 0 to 9 kGy. Pretreatment of the lactoferrin and transferrin with a protease was necessary for inhibitor formation.5%).198 Antimicrobials in Food increased (2. 100. a similar protein in blood serum.K. A rather high level of isoascorbate (1000 µg/g) caused nitrite to disappear rapidly and delayed the outgrowth of C. Their approach was to conduct large multifactorial experiments using pork slurries and then to analyze the data statistically and develop equations that could be used for predictive modeling. as pH levels decreased to below 6. As expected. They concluded that the level of residual nitrite did not influence botulinal outgrowth in their experiments. Transferrin. and iron in culture media. The slurries were irradiated. (1984) to examine the effect of various factors on the rate of nitrite depletion and botulinal outgrowth. The authors suggested that Bactin formation may be responsible for the inhibition observed in Perigo-type media. After higher doses of irradiation (10. which otherwise prevent bacteriostatic activity. the . The bacteriostatic action was reversed by the addition of cysteine. legislation was proposed to control the use of nitrite by monitoring the residual nitrite in cured meat products. 156. 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. 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. then inoculated with botulinal spores.

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

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

Nitrite 201 pH of the culture medium was lowered from 6.4 to 5. suggesting that nitrite inhibits the phosphoroclastic system. and a mixture of polymyxin and neomycin after heat injury at 70°C to 100°C. The enzyme system consists of two proteins. sporogenes in a glucose medium caused a rapid decrease in intracellular ATP concentration and an accumulation of pyruvate in the medium. botulinum (as opposed to reducing the antibotulinal efficacy of nitrite) in canned cured meat.6. 1980). They considered that nitrite inhibits C. Woods and Wood (1982) next reported that adding sodium nitrite to cell suspensions of C. RSNO (540 µg/g) was considerably less inhibitory than sodium nitrite (200 µg/g) when tested in inoculated perishable canned turkey at 27°C. Compared with earlier research. Nitric oxide was inhibitory.g. (1981) reported that ferric chloride or hemoglobin stimulates the growth of C. Recovery of heat-injured spores in a basal medium containing 3% salt or 1% salt with 200 µg/g of nitrite yielded comparable recovery rates. one of which is an iron protein containing nonheme iron and sulfide as a cubic Fe4S4 cluster.. The authors concluded that. but there was decreased activity in the ferredoxin from cells preincubated with nitrite. nitrite. The accumulation of pyruvate was the result of inhibition of the phosphoroclastic oxidoreductase. botulinum in pork. botulinum by making the iron of the heme groups in meat unavailable for growth. with resultant destruction of the iron–sulfur cluster.. Nitric oxide was found to rapidly and irreversibly inactivate the iron protein. Adding ferric chloride or myoglobin decreased the antibotulinal efficacy of nitrite. Chumney and Adams (1980) found C. thereby verifying observations that ascorbate increases the antibotulinal efficacy of nitrite in meat products. Adding nitrosylated myoglobin to the meat system resulted in a rather inhibitory system compared with adding untreated myoglobin. Their results supported the theory that nitrite is inhibitory to C. Nitrite did not react directly with ferredoxin. With addition of sodium ascorbate the intensity of the reaction was stronger. although nitric oxide can react with ferredoxin in vitro. Meyer (1981) investigated the effect of nitrite and nitric oxide as inhibitors of the nitrogenase enzyme system of Clostridium pasteurianum. . On an equimolar basis. The results suggested that nitrite is converted to nitric oxide. which then disrupts the iron–sulfur cluster of the protein and thereby inhibits nitrogenase activity. botulinum led to an accumulation of pyruvic acid. but this could be attributed to the formation of analytically detectable nitrite in the meat. the main site of inhibition within intact cells is the reaction of nitric oxide with the enzyme pyruvate-ferredoxin oxidoreductase. The decreased inhibition by nitrite when iron compounds were added appeared to be the result of a reduction in residual nitrite levels in the product rather than to the iron serving as nutrient to stimulate botulinal growth. a nutritional requirement for growth. Adding EDTA or denatured nitrosylated myoglobin increased the efficacy of nitrite. (1981) found that the addition of nitrite to a suspension of C. ferredoxin) that are essential for growth. They concluded that botulinal cells contain iron–sulfur proteins that react with nitrite to form iron–nitric oxide complexes. there was no noticeable relation between botulinal growth rate and residual nitrite level in the meat to which nitrosylmyoglobin had been added. the nitrite was relatively ineffective as an inhibitor. botulinum by inactivating iron–sulfur enzymes (e. (1983) used electron spin resonance to examine the spectra of botulinal cells treated with nitrite and ascorbate. They suggested the increased sensitivity to each agent was caused by the same cellular damage. This may have been the result of sulfite in the basal medium (tryptone sulfite neomycin agar) because sulfite destroys nitrite (Tompkin et al. Reddy et al. perfringens spores to be sensitive to inhibition by salt. Woods et al. This enzyme consists of a single protein molecule containing thiamine pyrophosphate and a single nonheme chromophore. Vahabzadeh et al. Collinge et al. 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. (1983) found carbon monoxide to be ineffective as a replacement of nitrite for inhibition of C. Also.

Research by Payne et al. decreasing pH and aw) influenced the rate of death. The relative sensitivity of C.5. sporogenes cells and isolated proteins. monocytogenes is quite resistant to the inhibitory effect of nitrite. In a broth medium at pH 6. The inhibitory effect of nitrite against C. more generally. In addition. In the research of Duncan and Foster (1968b) such blister formation was not reported following exposure to sodium nitrite. perfringens and fecal streptococci in broth media. (1991) and Roberts and Dainty (1991). which is more typical of cured meat and . At the pH values (6. the data more strongly demonstrate that L. At pH 6. the efficacy of sodium nitroprusside was explored (Joannou et al.5. One aspect that the more recent data suggests is that the interaction of nitric oxide with iron–sulfur complexes is irreversible. it is fairly certain that the mechanism of nitrite involves the inactivation of iron–sulfur proteins. such as ferredoxin and pyruvate oxidoreductase. (1990a. leading them to doubt that the inhibitory effect of nitrite is on preformed iron–sulfur proteins of the bacterial cell. The cell-wall surface was the primary point of attack for the nitrosyl complex associated with nitroprusside. However. and 37°C) had the strongest effect on growth rate and lag-phase duration. On the basis of the research conducted by these researchers and others.5.0) found in cured meats. botulinum was again addressed by Carpenter et al. Most cured meats have traditionally had brine levels of 3% or greater. It appears that the hypothesis proposed earlier is fairly close (Tompkin et al. sporogenes.5%) and sodium nitrite (50. the toxicity of nitrosyl compounds to bacteria. 19°C.0.0. and 200 µg/g). Gibson and Roberts (1986b) demonstrated the interactions of pH. Nitroprusside was used as a model to investigate the inhibitory mechanism of sodium nitroprusside and. perfringens foodborne illness involving cured meats. They were unable to demonstrate a direct action of the compounds on the iron–sulfur proteins. (1987). 1978). 5. perfringens was enhanced with a decrease in pH. The incubation temperature (5°C. Juntilla et al. nitrite was not an effective inhibitor at pH 7.. 120. It can be concluded that factors (e. (1989) found the rate of death of L. Miscellaneous Studies in Laboratory Media Botha and Holzapfel (1987) tested the effect of nitrite on Sporolactobacillus isolates from a variety of sources. a decrease in temperature of incubation. 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.5 and 6. although the nitrite had been autoclaved with the broth. and/or an increase in the salt concentration.6. The effect of nitrite on the growth of Listeria monocytogenes was examined in trypticase soy broth by Shahamat et al. Additional information and views on the mechanism of nitrite are available in Roberts et al.g. After continued research on the mechanism of compounds associated with the Perigo factor.b) examined the effect of nitrite and iron–sulfur–nitrosyl complexes on iron–sulfur clusters in C. The fecal streptococci proved to be quite resistant to nitrite.202 Antimicrobials in Food The debate over whether clostridial ferredoxin or pyruvate-ferredoxin oxidoreductase is the site of nitrite inhibition for C. and sodium nitrite on the inhibition of C. Their results led to the conclusion that both are inactivated. Consistent with previous reports over the past 60 years. perfringens to 3% to 4% salt could explain why there have been few outbreaks in C. Buchanan et al. A high salt concentration and low incubation temperatures were the more important factors influencing the inhibition of these bacteria. Sodium nitroprusside was found to be a very effective inhibitor of C. and 5. blisters at the surface of the cells were observed when viewed under the electron microscope. 28°C. (1989) tested the effect of nitrite on L. (1980).0% and 3. The cell walls showed a 90% decrease in thiol content when compared with untreated cells.. sodium chloride. 1998). some reduction in growth was observed with the addition of 200 µg/g of sodium nitrite. comparable to the inhibitory properties of the anion of Roussin’s black salt. monocytogenes in a broth medium at pH 7.0. The final pH of the products was about pH 4. monocytogenes to be similar in fermented sausage manufactured with different levels of salt (3. incubation temperature.. Sodium nitrite was added to the broth before autoclaving.

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

being insensitive to the presence of nitrite. B. The formulated raw meat was stuffed into a casing. Protoporphyrin. 100. and 200 µg/g) on the spoilage flora of a sliced vacuum-packed bologna-type sausage.. thermosphacta and Enterobacteriaceae at all temperatures of storage (2°C. B. Polish regulations require the heating of perishable canned . Nielsen (1983a) studied the effect of sodium nitrite (0..7 to 5.8. all the strains of L. and Moraxella and Moraxella-like organisms were increasingly inhibited by increasing the nitrite concentration and/or decreasing the temperature of storage (2°C. cereus in liver sausage has been a problem in Finland. and 200 µg/g) on the spoilage flora of cooked pork loin. 156 µg/g of sodium nitrite.204 Antimicrobials in Food were considered incapable of synthesizing heme compounds. sake tested were able to produce catalase when provided with hematin. Wolf and Hammes (1988) found all the strains of L. and processing to 68°C resulted in a shelf life of more than 16 weeks at 5°C. and lactic acid bacteria were inhibited to a limited extent. 100. The growth of B. particularly in the summer. whereas other iron-containing porphyrins (i. Nitrite inhibited the growth of B. 100. The spoilage of liver sausage was shown to be the result of S. the lactic acid bacteria. 5°C. Again. Silla and Simonsen (1985) studied the rate and type of spoilage of four cured meat products packaged under vacuum and in modified atmospheres. An industry survey found that liver sausage was commonly cooked to an internal temperature of 65°C. did not activate nitrite reductase or catalase activity. Increasing the level of sodium nitrite (0. The level of surviving enterococci decreased as the processing temperature increased (63°C. hemoglobin. Also.e. faecalis (Chyr et al. 63°C to 68°C) led to the growth of enterococci during storage at 7°C. and 20°C). This led to the lactic acid bacteria becoming the dominant spoilage flora in the vacuum-packed product. cereus can grow in the product even with the normal curing ingredients of salt. 68°C. Following the clue provided by Whittenbury (1964) that some strains of lactobacilli produce heme-containing catalase when grown in the presence of blood or hematin. (1988) reported that the growth of B. The Pediococcus isolate was not as heat resistant as the enterococcus. and 74°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. hematin) did activate the enzymes.3 units lower than with the other variables tested. Using good quality raw materials. as the storage temperature increased. myoglobin. gluconolactone (glucono delta lactone.. and citric acid in the product. chilled. 10°C. The enterococcus survived 65°C for 5 minutes or 60°C for 60 minutes with only 103 cells/ml broth. A marginal thermal process (i. Adding GDL was considered the most important. Additional additives were tested to reduce this risk. Gramnegative bacteria often constituted the major flora of the product without nitrite. thermosphacta. an iron-free compound. Lactobacilli were the dominant flora in all four products at the time of spoilage. Chyr et al. High levels of B. A correlation was found between the nitrite concentration at the time of slicing and packaging and the time for spoilage to occur. phosphate. thermosphacta may also have significantly influenced spoilage of two of the products. about 0. GDL). cooked. The spoilage flora of the liver sausage prepared without nitrite also included Pediococcus pentosaceus. and sodium nitrite (120 µg/g). sliced. and 10°C). and then vacuum packed. cereus was delayed or prevented by including a combination of erythorbate.2 to 0. yeasts. Enterobacteriaceae. and 150 µg/g) retarded microbial growth. Product with GDL had a pH value of 5. Grampositive cocci. plantarum tested were able to reduce nitrite when hematin was provided to cell suspensions. the Gram-negative flora proliferated in product with nitrite.e. In addition. dominated the spoilage flora in product with nitrite. 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. Kafel and Jozwik (1987) investigated the spoilage rates of commercially produced products from 6 meat processing plants in Poland. Omitting nitrite resulted in a perfume-like odor that was associated with the growth of enterococci. 50. Asplund et al. 5°C. 1981). plantarum and L. Nielsen (1983b) subsequently studied the effect of sodium nitrite (0. If the product is temperature abused.

high levels of iron (40 to 160 µg/g) increased the efficacy of nitrite. The ground meat was aerobically packaged and stored at 4°C to 5°C. These data reflect the influence of a mixture. When spoilage first occurred in each lot. Tests demonstrated that nonproteolytic. Extensive studies in meat and meat slurries demonstrated the interactions of nitrite. The spoilage rate of this second group of cans was 4%. From among 4322 cans of 8 different classes of perishable canned cured meats. respectively. This could explain the high number of cases that have occurred during certain periods in Europe (Tompkin. Low levels of iron (5 to 15 µg/g) reduced the efficacy of nitrite. 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. This led to the development of a model system for predicting botulinal outgrowth in food. of naturally occurring spores in fairly large cans of commercial product. and thermal process on botulinal outgrowth. 980 cans. 2. the NAS was not able to conclude that nitrate can act as either a carcinogen or mutagen. 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. 4. whether cooked or raw. 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. The NAS concluded that the evidence available about nitrite does not indicate that nitrite can act directly as a carcinogen in animals. or 23%. Nitrite was considerably more effective in white meat than in dark meat.8°C. The effect of nitrite on the aerobic spoilage flora of raw chicken patties was studied by Bushway et al. Bushway and Serreze (1984) found that adding iron to raw white meat caused an increase in the rate of aerobic microbial growth. The authors also investigated the effect of the inherent pH of the meat on the efficacy of nitrite. (1982). With the addition of sodium chloride (2. Adding EDTA enhanced the efficacy of nitrite and caused an increase in the lag phase for aerobic microbial growth. a concept .4.Nitrite 205 meats to a minimum internal temperature of 68. These conclusions meant that neither nitrite nor nitrate would be banned as food additives. psychrotrophic strains of C. isoascorbate. This could be influenced by the fact that white meat is lower in pH and iron content than dark meat. additional cans (8290) were removed from storage at around 8°C and incubated at 37°C for 3 days. even a low level of nitrite (40 µg/g) caused some inhibition in white meat but not in dark meat.5%). Perhaps some adjustment occurred in the microbial population. Adding EDTA alone had no significant impact on the rate of aerobic microbial growth. 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. The pH of the white and dark meat was 5.6. swelled. The reason for this effect on the aerobic spoilage flora was not known. pH. However. This led to the conclusion that the inherent pH of the meat is the major factor determining the effectiveness of nitrite on the rate of aerobic spoilage in chicken meat.6 and 6.4. A means to quantitatively compare the data from various challenge tests based on the probability of outgrowth and toxigenesis from one spore was proposed. 1980). This provided a basis for assessing the risk associated with different cured meats. Bushway and Jen (1984) found lower residual nitrite levels in chicken white meat than in dark meat. Likewise. If nitrite (130 µg/g) was also added. the efficacy of nitrite increased when the pH of dark meat was adjusted to 5. Furthermore. Bushway and Serreze (1984) also studied the effect of adding EDTA to raw dark chicken meat. the effect of iron on aerobic microbial growth depended on the amount of iron added. 3. The time elapsed was normally 7 to 10 days after production. phosphate. 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. SUMMARY FOR 1980–1990 1.

At pH 6. In subsequent tests the effect of various decolorized blood fractions was evaluated. delayed the time for toxin to be detected. but none have been adopted commercially. Some research continued on the Perigo factor. but no evidence was generated to link the Perigo factor to the antimicrobial effect that has been demonstrated in perishable. and the addition of plasma delayed the time for toxigenesis compared to the control sausage (Miller et al.0 and 5°C no growth was observed with any of the levels of sodium nitrite evaluated (50. pH. Adding hemoglobin. the rate of toxin formation was inversely related to the level of iron. is not more inoculated pack experiments but a rationale for interpreting them” (Ingram. Since 1990 there has been increased interest in control of L. Other available options have not received commercial acceptance. cured products that receive no heat treatment or are cooked to final internal temperatures in the range of 60°C to 75°C. or whole blood reduced the time for botulinal outgrowth and toxin formation compared with the control with nitrite. The debate over the risk of C. 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. (1991).3. 1974). monocytogenes in ready-to-eat foods. in my opinion. SINCE 1990 C. the USDA in 1988 initiated a series of increasingly restrictive policies on the rate of chilling for perishable cured meat and poultry products manufactured under USDA inspection. 200. Again.. In the absence of nitrite. Adding dried plasma. perfringens has not been a concern in cured meat and poultry products as evidenced by the relative lack of research on this pathogen during the past 40 years. The potential impact of various blood fractions as ingredients in sausage was investigated (Miller and Menichello. that has been expanded to include other pathogens and has gained acceptance throughout the world.0 sodium nitrite had little effect in delaying the time to detect visible growth except at the highest level tested (200 ppm) and a temperature of 15°C or below. Alternatives to nitrite continued to be investigated. monocytogenes in tryptose phosphate buffer under a wide variety of conditions (salt. Despite this evidence. At pH values of 6. 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. perfringens in commercially manufactured cured meat and poultry products has continued into the new millennium and serves as an example for the need of a risk assessment to clarify appropriate risk management policies. temperature. 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. red blood cells. This probably reflects the effect of the higher salt levels and nitrite found in cured products compared with noncured products. very slow growth did occur. aerobic/anaerobic conditions) and found sodium nitrite to interact with combinations of the factors tested. 6. 1991). 7.206 Antimicrobials in Food 5. however. Buchanan and Phillips (1990) studied the growth of L. using an automated tubidimetric system with multiwelled plates containing trypticase soy broth. 8. At the time he stated. Sausage formulated without sodium nitrite became toxic within 1 week compared with 3 weeks when nitrite was present. The mechanism of nitrite inhibition differs in different bacterial species. 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. 1993). The database was incorporated into the USDA Pathogen Modeling Program. found the efficacy of sodium nitrite was temperature and pH dependent. 400 µg/g). monocytogenes to the degree that . “What we need at the present time. Autoclaving the sodium nitrite in the medium did not increase the degree of inhibition against L. 100. At 20°C and below no visible growth occurred even with 50 µg/g sodium nitrite and a pH of 5. McClure et al.

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.Nitrite 207 had been observed with clostridia (see Perigo Factor). Applying the procedures described by Hauschild (1982). a combination of heat and sodium nitrite was necessary to prevent survival and growth (F0 = 0. 10°C) on the rate of growth of L. (1992). (1994) inoculated a variety of vacuum-packaged cooked sliced meats with L. 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). 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. and 15 days for L. and additives to achieve shelf stability of canned luncheon meat (Farkas and Andrássy. Adding 200 µg/g sodium nitrite to the sausage increased the time required to multiply from 10 cfu/g to 105 cfu/g by 9.7. (1997) examined the effect of nitrite on three lactics in vacuum-packed cooked sausage made with and without nitrite. inoculum level. (1995) examined the effect of sodium nitrite (0 and 200 ppm). Duffy et al. Perhaps the high iron content of liver pâté negated the effect of sodium nitrite and sodium erythorbate in this product. 4. McClure et al. Farber et al. The procedures described show promise for estimating and comparing the combined effects of multiple treatments on pathogen survival and growth. L. If the broth had been adjusted to pH 6. respectively. sporogenes. although sodium nitrite concentration was not varied. monocytogenes. heat. Liver paté has been implicated in outbreaks of listeriosis in the United Kingdom. botulinum. 3%). Buchanan et al. botulinum. mesenteroides. 1992b). 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. 1997).5% sodium chloride and 50 or 200 µg/g sodium nitrite inhibited growth during 125 days storage at 37°C. F0 = 0. Australia. irradiation. monocytogenes in the sliced vacuumpackaged product when stored at 4°C or 8°C. As in the case of earlier research with C. pH. 550 µg/g). The model test system was then used to evaluate various combinations of heat. Sameshima et al.7 with 50 µg/g). An extensive series of experiments led to the conclusion that nonthermal inactivation of L. and temperature of storage (4°C. Low levels of salt (1% to 2%) enhanced growth.. temperature. and Enterococcus faecalis. Using C. and sodium nitrite in laboratory medium and a predictive model for the growth of L. Fermented Meats and Dry Cured Hams Although removed from most cured meat products in the United States during the 1970s. and the United States. as had been reported earlier for C. monocytogenes by sodium nitrite is pH dependent and related to the concentration of undissociated nitrous acid (Buchanan and Golden. but levels above 3% were inhibitory. 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. smoked flavor can be found. they estimated the various formulations and thermal processes provided the equivalent of 4 to 7 log10 destruction of C. 1995.8 and containing 3. In regions of Northern Europe. sporogenes (9 × 105/ml) in reinforced clostridial medium. some . Only temperature proved to be a significant factor.0 and a sour. and other factors for inactivating spores and preventing the outgrowth of survivors. The effect of salt and nitrite on the growth of 24 lactics isolated from vacuum-packed cooked sausage was evaluated by Korkeala et al. Since about 1990. Farkas and Andrássy (1992a) described a test system to evaluate the combined effects of formulation. (1996) subsequently investigated the effects of salt. they found that broth adjusted to pH 5. viridescens.2 with 200 µg/g. sausages made with nitrite and having pH 5. sodium erythorbate (0. salt (1%. Leuconostoc isolates seemed more sensitive to the effects of sodium nitrite and salt in comparison to the homofermentative lactobacilli. monocytogenes in liver pâté.

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

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

As the amount of sodium nitrate increased. 1991. 1995). fruity. Cuppett et al. 2001a. 1983. late fermentation and gassiness resulting from Clostridium tyrobutyricum or C. 1984. poultry. botulinum type E (103 spores/g) at 27°C was inhibited for 35 days in smoked whitefish containing 4. nitrous acid) because a high percentage of the isolates from all three variables were able to reduce nitrate. the allowable level has been 500 mg potassium nitrate per kg fish to provide about 300 µg/g nitrate. Among the options considered was the use of a red pigment producing mold used to ferment rice. Monascus purpureus (Leistner et al. 25 g. (1987) found that toxin production by C. and 2 barrels. 1981). This chapter will not review the extensive literature on the use of nitrate to prevent cheese defects (e. For more than 1500 years the rice has been dried. In a second . Ariga et al.4% salt in the water phase. cold-smoked trout having 3. (1997) found the addition of sodium nitrite delayed or prevented toxin production by C. tofu. Fink-Gremmels et al. or 50 g of potassium nitrate was added to each of 200 barrels containing 90 to 100 kg fish.. flavor. toxin production was inhibited for 56 days.. The incidence of spoilage was 13.210 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. rice is fermented with molds of the genus Monascus to form a pleasant red color. Galesloot and Hassing.. 25 g. Assembly of Life Sciences (1981).. Canadian regulations permit this practice (Assembly of Life Sciences. 1983. 16 kg salt. 1988. The reader may recall that during the 1950s nitrate was found to be an electron acceptor in certain cured meat products. 8. Hyytiä et al.4% salt in the water phase.000 cfu/kg) and after 4 weeks with a low inoculum (800 cfu/kg). Two microbial hazards of concern in ready-to-eat fish products are C. Gouda and El-Zayat. When sodium nitrite (156 µg/g) and sodium ascorbate (550 µg/g) were added. rice wine) to improve color.. botulinum type E in vacuum-packed. for example. The reduction in spoilage was not considered to be the result of microbial inhibition by nitrate or its reaction products (nitrite. Adding nitrate caused the herring to be redder in color. 1984. saltpeter) in the curing of fishery products prior to 1950 throughout the world. butyricum). An opening to the literature on this subject can be obtained through references cited in Langsrud and Reinbold (1974). 1991. 1980). 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.b).b).. Tseng et al. particularly in China. respectively.. Nitrate was found to delay the depletion of trimethylamineoxide (TMAO). or putrid off-odors and offflavors. added to a variety of foods (e.g. sweet/sour. toxin was detected at 4 weeks at 4°C with a high inoculum level (20. in this form (called Angkak). Klijn et al.g. 1984a. 1999. They conducted an extensive study in which 0. Seafood The comprehensive review by Jarvis (1950) provides very few examples for the use of nitrate (i.3%. and 6 kg sucrose (Knøchel and Huss. and 50 g of sodium nitrate. with 0. 1991).e. monocytogenes (Gram. Cheese Nitrate has been added during the manufacture of certain European cheeses for more than 150 years (Pivnick.. and virtually no mention is made of its value as a preservative. and keeping quality (Leistner et al. In the Far East. fewer barrels were judged to be spoiled with sour. but no differences were noted for flavor or texture. fish. Gilles and Fryer. 2000). and. The additives provided no additional protection when the salt level in the fish was reduced to about 2. The 600 barrels were topped off with brine solution. pork. ground. 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. and evaluated over 18 months storage at 4°C to 6°C... In one study with 166 µg/g sodium nitrite. botulinum and L. sealed. and others (Mayenobe et al. Although not permitted in the United States. nitric oxide. In Denmark..

will prevent toxin production in cold smoked fish for several weeks beyond its sensory shelf life when packaged under reduced oxygen conditions. Two options are available in the event commercially manufactured product is implicated in botulism. that combination of salt and temperature. 1987). This may reflect survival or contamination. 1997). monocytogenes (Gram. Considering the lack of regulations and apparent variability in salt concentration in large quantities of cold smoked salmon distributed domestically and internationally. depending on the conditions of processing..5% salt in the water phase or 3. 2001a). 1987..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.. The panel expressed concern for the potential carcinogenic effects of nitrosamines.e. questions still remain about “aerobic packaging” and its value for controlling botulinal outgrowth.000 tons shipped internationally from 28 exporting countries to 52 importing countries (Duffes. if necessary. 1999). Cuppett et al. NOCs were not detected in two studies in which nitrite had been added to smoked fish (Pelroy et al. The addition of nitrite or nitrate increased the time the fish was considered acceptable by 3 or 4 weeks at 4°C. monocytogenes is common in smoked fish. Surveys conducted before the year 2000 indicate L. The panel was reluctant to recommend expanded use of sodium nitrite despite evidence showing that psychrotrophic C. sodium nitrite could be used as an additional control measure. although the numbers. 1982. Toxin production can be controlled with a combination of a moderate level of salt (3. and chub. whereas with salt alone. with a production of 12. 2001a): • • Psychrotrophic C. .0% salt if combined with 200 µg/g sodium nitrite (Gram. allowing for short periods of elevated temperatures up to 10°C. 1999).. In many countries there is no legal requirement for the minimum amount of salt. toxin was detected in 3 to 4 weeks. 1995).. 1997). federal regulations were adopted requiring vacuumpacked. 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. Adding potassium nitrate at 347 or 686 µg/g was equal to or more effective than sodium nitrite. botulinum can be expected to occur in smoked fish.5% in the water phase) and cold storage (<4.4°C) for at least 4 weeks. shad. 1982. although a minimum of 3% salt in the water phase is strongly recommended (Huss et al. Controlling the salt in the water phase and temperature should be adequate. a minimum concentration for salt in the water phase can be established. In 1992 more than 32. Following a review of the literature. Until there is evidence of a need. With the availability of lower cost farm-raised salmon the international market for cold smoked salmon has continued to increase. The addition of sodium nitrite is limited to smoked salmon. the salt in the water phase ranged from 2% to 4. it would appear the risk of botulism is lower than the research has suggested. 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. particularly in combination with controlled levels of salt (Pelroy et al. A level of 3% salt in the water phase will yield perfectly acceptable. but. brined shrimp). however. gravad fish. 2001b). a lower salt content is permitted (i. sablefish. a panel of experts reached the following consensus for its report to the FDA (Gram. In France. It is possible.. however. if present. it is highly unlikely that sodium nitrite will be adopted as an additive in smoked fish in Europe. and with refrigeration this combination is effective in inhibiting growth of C. botulinum (Huss.5% salt in the water phase). Analysis demonstrated that nitrite was produced during storage. First. Hyytiä et al. If packaged aerobically.. Cuppett et al. are low. botulinum would be further inhibited.6% (Duffes. 2.380 tons of cold smoked salmon in 1997. lightly preserved fish products (cold smoked fish. smoked fish to contain at least 3. but the panel did not consider the more recent risk assessments demonstrating the safety of dietary nitrite (see section on toxicology). Furthermore.

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 5.13% brine. perfringens swelled within 2 days at 37°C. and 720 µg/g of sodium nitrate at the time of opening and inoculation. S. the imported product had 4. including an isolate from the 1964 Aberdeen outbreak involving canned corned beef.. Typhimurium declined during the first 2 weeks at 5°C and then remained stable through 6 weeks. to produce cold smoked salmon with low levels. nor were analytical data presented for the product tested. In both products. Sodium lactate (2%. Survival of the inoculum in product held at 7°C varied with the product and inoculum level. Previous studies had found levels of 0. At the end of storage. The growth of S. Such levels would ensure the number does not increase above 100 cfu/g when the product is held at 5°C and eaten within 3 to 4 weeks. The authors implied that. 1994a). although not before some initial growth at 24°C. there was a gradual decline. Pelroy et al. Typhi has a nitrate reductase. (1994b) found the addition of sodium nitrite (190 to 200 µg/g) to be very effective in slowing the growth of L. Davidson and Webb (1973) also studied the growth of S. because S. A lower maximum population (106 versus 108/g) and slower die-off occurred in the imported product. increasing the salt concentration above the normal range for smoked fish is an unacceptable option for control. 1994. and held at room temperature. monocytogenes to salt and the adverse effect on flavor. The inocula died in wieners at both 18°C and 24°C. Because of the relatively high tolerance of L. w/w) was much more effective and prevented growth through 50 days at 5°C when used in combination with 3% salt. rewrapped in Saran™.26% brine.5 to 11.. were used for testing. Typhi in inoculated canned corned beef stored at room temperature and at 37°C. viable cells remained even after 6 months of storage. monocytogenes in salmon containing 3% salt in the water phase. Growth occurred in both products. Typhi remained after 20 months at 27°C to 38°C. Goepfert and Chung (1970) inoculated the surface of sliced bologna and liver cheese with S. often less than 1 cfu/g and at a low prevalence. however. 1991. The initial level of contamination is an important factor influencing growth (Guyer and Jemmi. 2000). one domestic and one imported.212 Antimicrobials in Food with strict adherence to good manufacturing practices. Cans inoculated with E. but a steady increase in numbers occurred at 24°C. At 5°C. Product from two sources. Pelroy et al. tests were not performed to show whether the presence of nitrate influenced growth. Typhimurium occurred more rapidly and to higher levels when the packages were opened. Typhimurium in vacuum-packaged luncheon meats. 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. Mock chicken supported the best growth at both temperatures tested (24°C and 37°C). Typhimurium grew in both vacuumpackaged products when held at room temperature.7 cfu/g in positive samples of cold smoked fish from five plants. it uses the nitrate in the product in place of oxygen as an alternative hydrogen acceptor. S. 1993. cans inoculated with C. Kittaka and Greenberg (personal communication) obtained similar results in corned beef inoculated with three different S. nitrite accelerated the death of all four strains. Peterson et al. Dehydration prevented growth if the packages were opened and left exposed at room temperature. Typhimurium or Salmonella anatum. however.6% salt. Viable S. Chemical data were not provided for either luncheon meat. This combination was more effective when a low inoculum level (10 cfu/g) was used as a challenge. 15 µg/g of sodium nitrite. coli remained flat. Very erratic growth occurred on bologna at 18°C. Similar results were obtained on ham. 14 µg/g of sodium nitrite.. (1993) found the rate of growth to be similar at 5°C for salt concentrations of 3% to 5% in the water phase. Typhi strains. It is reasonable to assume the products were recently produced. Rørvik. Peterson et al. and 45 µg/g of sodium nitrate. In subsequent research. The domestic product contained 4. Meers and Goode (1965) reported the rapid growth of S. the pH of mock chicken . After reaching 107/g. S. Anatum grew in bologna but not in liver cheese.

Growth occurred in the sausage containing 0 and 65 µg/g of nitrite. Under anaerobic conditions. The resistance of Salmonella to nitrite in culture media was demonstrated by Roberts and Garcia (1973). given the proper circumstances. All the enteropathogens grew in one or more of the products within 24 hours at 30°C.8%. Klebsiella. perfringens did not occur under any condition. respectively. no data were provided for brine or nitrite levels. nitrite was without effect. It is not possible to assess the influence of nitrite from the data.7.2% salt and 60 µg/g of potassium nitrite and a finished brine level of about 2.7 to 4. The rate of growth or death was influenced by the sodium nitrite content (0. The growth of Klebsiella and Enterobacter species on vacuum-packaged luncheon meats at 3°C was reported earlier by Hechelmann et al. 75.000/g). coli. Stiles et al.0. The product from one supplier remained above pH 6. (1979) tested the same bacteria on sliced bologna from two processors. the fastest generation times occurred in . (1967). At a high pH of 7. Leistner et al. This probably reflects the quantity of fermentable carbohydrate added to the product and/or the nature of the competitive flora.47 and 5. whether the nitrite was added before or after autoclaving the medium of Perigo et al. Typhimurium to utilize nitrate. The trend in the United Kingdom toward using less salt for processing ham was studied by Akman and Park (1974). but a mixture of Enterobacter. Typhimurium. Enteritidis were then used to study the effect of nitrite and salt on growth in nutrient broth at 22°C. Fermented sausage (teewurst) was prepared with four sodium nitrite levels (0. (1974).0. and the pH for the wieners was 4. and 100 µg/g) and the level of inoculum (100 or 100. and ammonia as the sole nitrogen source in a chemically defined minimal medium. viable counts of the 5 strains remained near the inoculum level. Page and Solberg (1980) found S. Aside from temperature of storage. In the low-brine ham at 22°C. that of the chopped hams was 70% and 59%. whereas the other frequently decreased to below pH 5. 50.58. and Hafnia strains readily multiplied at all nitrite levels. Although salt and nitrite values were not reported. The pH of the chopped ham with 70% and 59% moisture decreased to 5. Two strains of S. Again. The mean pH for the ham during storage decreased to no lower than 5.0. Both genera were prevalent in commercially produced luncheon meats and fermented sausages. The authors did not state whether the nitrite was added before or after autoclaving the medium. A high-brine ham had a brine level of about 6%.8% to 2.78. and 100 µg/g of sodium nitrite.8. 65.0 and 5% salt. No other chemical data were provided. growth was reduced by only about 30%. pH values were followed through storage. 4 of 5 strains of Salmonella grew readily. coli. 98. In the high-brine ham held at 22°C.5. Stiles and Ng (1979) studied ham and chopped ham obtained fresh from two processors. and B. S. aureus. It was concluded that cooked ham is a more likely source of salmonellosis if prepared with a low salt content.0.Nitrite 213 and bologna was about 6. S. Neither of two strains of S. Castellani and Niven (1955) reported S. Growth of C.0 by 160 µg/g of sodium nitrite.000). but pH changes were noted. A similar response occurred with enteropathogenic E. The moisture content of the hams was 70%. A low-brine ham was prepared with 1. At a low pH of 5. cereus) and then vacuum packaged. the primary factor influencing growth was product pH. and 130 µg/g) and two Salmonella inoculum levels (100 and 100. Nitrite and ammonia were assimilated at comparable rates. nitrite values were not given. commercially produced sliced ham and chopped ham did support the growth of the pathogens studied. 5% salt. including Salmonella.6 to 6. (1973) inoculated vacuum-packaged luncheon meat with a mixture of Salmonella strains and held the product at 8°C for 22 days. nitrite. perfringens. other than to say that fresh. The products were inoculated with five “enteropathogens” (C. Death occurred after 3 days in the sausage with the lower inoculum and 130 µg/g of nitrite. E. Salmonella Enteritidis was able to grow at 17°C but not at 10°C. The primary factors influencing growth were the temperature of storage and growth of competitive flora. The other four species were able to grow under certain conditions. 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. Typhimurium was inhibited at pH 6. that of the ham was near 7.

numbers increased during 72 hours in the presence of 0. Typhimurium used nitrite as the sole nitrogen source at concentrations as high as 400 µg/g. Typhimurium when grown anaerobically with nitrite as the sole nitrogen source. He recommended that a starter culture and/or gluconolactone be added to provide optimum safety. The presence of nitrite had no effect on aerobically grown cultures. 50. (1991). None of the variables tested would likely assure the destruction of Salmonella in this product category. (1984) prepared fermented sausages with sodium nitrite (0. and 100 µg/g sodium nitrite. found S. using a gradient gel plate system. vacuum packaged. Growth did not occur during fermentation or drying. being able to use nitrite in broth culture under anaerobic conditions at 5°C. This research was primarily concerned with preventing the growth of Salmonella. and incubated at 15°C or 27°C for 21 days. In a traditional Turkish dry sausage that is fermented and dried at temperatures . aureus and C. Nitrite was only inhibitory with the highest level of nitrite and when the franks were incubated at 15°C. 1991).0 to 6.6. coli. At concentrations of 500 µg/g and higher. pH (5. the effect of added phosphate was relatively minor. and sodium nitrite (0 to 400 µg/g) in BHI and incubation at 10°C to 35°C on the growth of Salmonella and E. 1977).2. Schillinger and Lücke (1989) measured Salmonella growth and/or survival in mettwurst. Collins-Thompson et al. Page et al. The rate of death of Salmonella Senftenberg in fermented sausage was fastest with 200 µg/g of sodium nitrite and next fastest with 100 µg/g of sodium nitrite (Puolanne. The presence of nitrite did not influence the survival of salmonellae. the inherent pH of the meat (normal pH 5. Similar results were observed with S. Hughes and McDermott (1989) used a procedure similar to that of Gibson and Roberts (1986a. and 156 µg/g) in frankfurters on a mixture of two strains of Salmonella. Isolates of Enterobacter and Serratia species were tolerant to nitrite. The death rate was slower with potassium nitrate (150 or 300 µg/g) or a combination of nitrate and a low level of nitrite (75 µg/g of potassium nitrate and 50 µg/g of sodium nitrite). coli was also apparent in tests involving two-dimensional gradient plates incubated at 20°C. coli.2).6 to 5. Typhimurium had shorter generation times and increased cell yields compared with nitrite-free cultures.8 versus 6. coli.8).214 Antimicrobials in Food media containing both nitrate and ammonia. The franks were inoculated on the surface. 6. 27°C. When grown anaerobically in a glucose-limited minimal medium with peptone and nitrite. except when the cultures were incubated at the lowest temperature tested (10°C). 50. and 200 µg/g) and three strains of Salmonella.8. S. At pH 5. S. and 37°C (McClure and Roberts. complete inhibition occurred. Gibson and Roberts (1986a) examined the interaction of salt (1% to 10% wt/vol). When examined after 10 weeks of incubation. 50. Factors influencing Salmonella growth in mettwurst that was cured and stored at 20°C include the amount of nitrite and salt. Typhimurium numbers decreased in BHI broth adjusted to pH 4. 150. but growth was delayed with 200 µg/g (Turanta and Ünlütürk. Thomas et al. sporogenes. a cured raw spreadable sausage. and 6. (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. (1985) identified a nitrite reductase in the cytoplasmic fraction of S. 100. S.8 and held at 24°C independent of sodium nitrite concentration. The reductase required NADH and was most active at pH 8.0. and the presence of lactic acid-producing bacteria.b) to determine the additional effect of eight different phosphates of E. The nitrite resistance of E. 1987). the addition of sugar. Collins-Thompson et al. The phosphates generally delayed the outgrowth of E. 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. Typhimurium to be relatively resistant to sodium nitrite across the ranges of salt and pH values found in nonfermented and acidulated foods. Rice and Pierson (1982) tested the effect of sodium nitrite (0.

coli O157:H7 (Chikthimmah et al. where . 1996). 1996).5. In ground pork held at 5ºC or 25ºC. coli O157:H7 at 37ºC were inhibited by 200 µg/g sodium nitrite at pH 5. particularly because salmonellae are destroyed during the manufacturing process for most cured meats. the death rate of S. 1997). coli O157:H7 in a broth system containing sodium nitrite was strongly pH dependent (i. nitrite... The inhibitory effect of nitrite was much greater at pH 5.. The highest survival involved a starter culture that produced the least amount of acid and yielded a relatively high final pH of 5. The effect of sodium nitrite (0. the inhibitory effect of smoke. the lowest pH tested and with increasing salt levels. sodium nitrite at 70 to 150 µg/g did not affect the growth or survival of E.7 × 105/g to nondetectable levels by 35 days in the sausages formulated with 80 µg/g nitrite. Riordan et al. 55ºC for 4 hours) were used. 1996). Effect of Nitrite on Yeasts and Molds A wide variety of yeasts and molds have been isolated from commercially cured meats.5. Inhibition of Aeromonas in broth has been found to be highly dependent on nitrite concentration. fermented to pH 4. salt level. The data indicate that the nitrite content of cured meats can influence the growth and survival of Salmonella and E. In Lebanon bologna. storage temperature. salt level. 1996. coli O157:H7 when lower temperature and/or shorter time combinations (e. 2001). and incubation temperature (Chou and Tsai. Inhibition was much greater at 28ºC than at 37ºC (Zaika et al. coli. pH. enterocolitica 0:3 to grow and survive during the manufacture of raw dry fermented sausage was examined by Asplund et al. The inoculum decreased from 1. (1998) found E. 1994). Adding sodium nitrite did not increase death of E. Inhibition of E. (1993). pH. The data for Salmonella can be summarized as follows. Inhibition of Yersinia enterocolitica in broth incubated aerobically was significantly influenced by the combined effect of nitrite concentration.. and 156 µg/g) had no effect on the death rate of E.e.5) and enhanced by lowering the incubation temperature (Buchanan and Bagi. 1994 and 1995). 1991). and temperature (Bhaduri et al.0. a fermented noncooked beef sausage. Typhimurium was not affected by the addition of 200 µg/g sodium nitrite (Turanta and Ünlütürk.8. 1993). 100.5% salt and 100 µg/g sodium nitrite. but even 1 g/L sodium nitrite was ineffective at higher pH values (Tsai and Chou. and 120 µg/g) on Y. S. pH. Pin et al. This sausage is not fermented or cooked and relies on relatively rapid dehydration to achieve microbial stability. coli O157:H7 numbers decreased less than 1 log10 in pepperoni formulated with 2. Another study involving anaerobic conditions yielded similar trends (Zaika et al. the addition of sodium nitrite (0.. and temperature. Preventing salmonellosis from cured meats is best accomplished by means other than relying on the level of nitrite in the product.. or ammonia under anaerobic conditions. They found some increased death of E.. coli O157:H7 with the addition of curing agents and drying at time–temperature combinations that appear to cause stress of the inoculum. In tryptic soy broth 4 strains of E. 80. coli O157:H7 (Yu and Chou. enterocolitica when tested in a meat system.g. 1994). but there is no published information that nitrite or nitrate inhibits their growth. This is not true for the clostridia. and. Raccach and Henningsen (1997) reported sodium nitrite and salt to act synergistically in the reduction of Y.5 to 6 hours) was next studied (Yu and Chou. 50. 78. Inhibition of Shigella flexneri in broth incubated aerobically was significantly influenced by the combined effect of nitrite concentration. The lack of information on the significance of nitrite and nitrate stems from the general acceptance that other factors largely determine the potential for yeast and mold spoilage of cured meats. and then dried for 7 days. pH 5. in a variety of commercially cured products. The effect of nitrite in Chinese-style sausage during drying at 50ºC to 60ºC for different time periods (2. That cured meats have been implicated in outbreaks of salmonellosis supports the available research that Salmonella can survive. Typhimurium has a nitrate reductase to reduce nitrate to nitrite and can assimilate inorganic nitrogen from nitrate. These factors include water activity. if not multiply. the availability of oxygen.Nitrite 215 not exceeding 24°C.

1957).5 to 15 minutes. During storage at 5°C for 28 days. Gardner. and other factors for controlling mesophilic clostridia.. The more rapidly produced products closely resemble those that have been produced in the United States over the past 30 to 40 years. 4. Control of these bacteria lies in proper handling of cooked product to be reworked into succeeding lots of meat. The effect of nitrite on aflatoxin production by Aspergillus parasiticus in fresh pork sausage was examined by Obioha et al. viridescens and other lactics has been developed by Grant and McCurdy (1986) and Grant et al.g.5 minutes. A system was developed to evaluate the combined effects of formulation.. SUMMARY FOR 1990–2002 1. (1949) described the characteristics of Lactobacillus and Leuconostoc strains isolated from commercially cured meats with greenish discoloration. The heat resistance of these bacteria has been reported (Niven et al. Except for a limited number of products to which nitrite and nitrate might be added. Increased heat resistance could be acquired by repetitive exposure to thermal processing. 6. circumstances. Nitrite can contribute to the inhibition of L. the product spoiled before aflatoxin reached detectable levels. 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. Green Discoloration in Cured Meats Niven et al. sorbate). 1972). 2.5 to 6. The name L. Iwata et al. 1967. inoculum level. yeasts and molds are of little or no public health importance. The mechanism of green discoloration was further elucidated by Shank and Lundquist (1963). At 26°C and 37°C.. 5. no aflatoxin production or mold growth occurred. but not all. and D71 = 1 to 2 minutes (Tompkin. D68 = 5. 1954. Additional information on factors affecting green discoloration by L. Despite this observation. the remaining nitrite enhanced growth and aflatoxin production. Endogenously formed nitric oxide and oxygen radicals can play an important role in human health. (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. All were salt tolerant and capable of growth at low temperature (5°C). 1957). 1986).5 minutes. (1988). Unpublished research by Shaparis and Christiansen has shown D values in minced ham to be D63 = 30. (1983). Niven et al. 3.216 Antimicrobials in Food permitted. Vrchlabsky and Leistner.g. The role of nitrate and nitrite in the manufacture of traditional and more rapidly produced fermented meats was clarified. In addition. 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.. 1965. Naturally occurring nitrate in vegetables accounts for more than 85% of the dietary intake of nitrate.5 = 12. The most significant development during this period was the growing body of data that dietary nitrate and nitrite are not carcinogenic (see Toxicology). monocytogenes under certain. These bacteria must not be given the opportunity to develop a population of increased heat resistance whereby the process will no longer be adequate to assure their destruction. 156 to 200 µg/g) initially restricted the profuse growth of mold. 1971). As residual nitrite decreased to less inhibitory levels. D65. . Evidence was presented indicating that the discoloration results from a reaction of hydrogen peroxide with the cured meat pigment. the results in pork sausage plus a test with media led to the conclusion that high levels of nitrite (e. the use of preservatives (e. heat. they are not major causes of spoilage. Conversion of nitrate in the saliva is the major source of nitrite in humans. viridescens was given to these bacteria (Niven and Evans.

3. regulations do not specify minimum nitrite levels for cured products. REGULATIONS GOVERNING THE USE OF NITRITE AND NITRATE The regulations limiting the use of nitrite and nitrate in cured meats vary widely among countries and have continued to change. Gerhardt. Nitrite is of relatively little value for controlling Gram-negative enteric pathogens in commercially prepared foods.17 and 424.177 (Code of Federal Regulations. 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 regulations were summarized in two comprehensive surveys (Meester.S. The current USDA regulations controlling the use of nitrite and nitrate in meat and poultry products are specified in 9 CFR 317. 1978). the method of assay applicable to each country should be used.Nitrite 217 7. The status of nitrite and nitrate in cured meats and cheese in the United Kingdom was reviewed in 1978 (Ministry of Agriculture. This further reduces the nitrite level in the product formulation and. meat and poultry regulations is that nitrite and nitrate are added on the basis of the uncured meat in the product formulation. ASSAY METHOD Because the levels of nitrite and nitrate permitted in foods are established by regulation. 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. Fisheries. Germany reduced the permitted level of nitrite in nitrite-containing curing salt by 20% (Leistner. The use of nitrite and nitrate in the processing of fish products is specified in 21 CFR 172. depending on the circumstances. U. 9. 1978).21-23 of the federal regulations (Code of Federal Regulations. Other countries limit the amount of nitrite and nitrate on the basis of the total weight of the product formulation.175. leads to lower residual nitrite levels after processing. 1981. With the exception of bacon and smoked chubs. the weight of this meat is subtracted from the fresh meat portion of the formulation and the amount of nitrite added is reduced accordingly. 2002b). This material is normally used as an ingredient at a level of about 5% in the manufacture of succeeding lots of the same product. 1986). Within the United States the methods of assay appear in the Official Methods of Analysis of the Association of Official Analytical Chemists . 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. The net effect of the U. 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. 172. As the percentage of extenders and other nonmeat ingredients is increased in the products. 1978). 1995). as proposed by the Expert Panel on Nitrites and Nitrosamines (Food Safety and Quality Service.S. 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.S. 172. the level of added nitrite is decreased.160. An example of rework is the round ends of sausages that are trimmed off before slicing the product. 1974. 2002a). Braathen.170. 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. Because it has already been cured. A unique feature of the U. It is also common to generate a certain amount of rework during the manufacture of sausage products. The most recent summary of the regulations in different countries appears in Cassens (1990). 1979). The use of nitrate or nitrite as additives in cheese and seafood products was clarified. 8. and 172. and Food.

estimates for the lethal dose have ranged from 2 to 9 g (about 33 to 250 mg/kg body weight). 1995). estimates for the toxic dose range from 1 to 8. estimates of the lethal dose have ranged from 4 to 30 g (about 70 to 500 mg/kg body weight). respectively.0%. 1994). 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. The normal range of methemoglobin levels is 0.3 mg sodium nitrite/kg body weight (Gangolli et al. Data on reproductive toxicity for nitrate were limited. TOXICOLOGY The acceptable daily intake for potassium nitrate and sodium nitrite have been reported to be 0 to 3. the uppermost level being found in infants and pregnant women. whereas 30 to 60 g of sodium nitrate have been given for 2 months as an acidifying diuretic without manifestation of adverse effects. International agreement has been reached on the specifications and analytical methods for potassium nitrate and sodium nitrite (JECFA. The no-adverseeffect dose level was estimated to be 2500 mg sodium nitrate/kg body weight/day (Gangolli et al.218 Antimicrobials in Food TABLE 6. the criterion most commonly used for nitrite toxicity.5% to 2. occurs at methemoglobin levels in excess of 10%. with the lower doses applying to infants who are more sensitive to methemoglobinemia. 2002). 1994). and it appears that nitrate per se is not genotoxic... 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 . For potassium nitrate.7 and 0 to 0. 1994). Cyanosis.06 mg/kg body weight. respectively (JECFA. (AOAC international. 1995). as evidence of toxicity. Source: Based on formulations in Pearson and Tauber (1974). Using this criterion.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. nonfat dry milk. The values are expressed as nitrate and nitrite ions. For sodium nitrite. and apply to all sources of intake but do not apply to infants younger than age 3 months. Two years of rat studies on nitrate showed no evidence of carcinogenicity..

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

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

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

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

Ray (1992). there were attempts to use nisin as a medical drug. Hara et al. Nisin is not used therapeutically in human or veterinary medicine nor is it used as an animal feed additive or for growth promotion. de Vuyst and Vandamme (1994). Nisin represents a safe. . 2001).1983).. Consumers do not want high levels of chemical food preservatives in their food and yet they increasingly look for convenient. 1952). 1992). 1993. both academic and industrial. 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. long history of successful usage as a food preservative. 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. (2000). 1962). We recommend that nisin (and other bacteriocins) should be considered as antimicrobial peptides and not referred to as antibiotics. nisin is not one of these agents. Nisin is a primary metabolite produced by a process involving ribosomal transcription and translation. long-life food that is of high quality but that is not severely processed (Gould. Hurst. natural method of preservation that can be used as part of a combined preservative system to increase the safety and shelf life of food. 1957). Delves-Broughton et al. Therefore. Before its development as a food preservative. 1951). 1962. Although there is no doubt that therapeutic drugs associated with medical usage should not be used in food applications. consequently nisin can be present naturally at low levels in both soured milk and cheese. most notably those by Hurst (1981.. There has been misplaced concern expressed in some quarters resulting from a confusion arising from references to nisin as an antibiotic (Hansen. Delves-Broughton (1990). Generally antibiotics are secondary metabolites. Nisin is a low-molecular-weight polypeptide produced by the bacterial dairy starter culture Lactococcus lactis subspecies lactis. the producer organism was classified as Lancefield serologic group N Streptococcus.” At that time. (now part of Danisco) in 1957. Increasing mass production and the longer distribution chains operating in many countries have led to an increase in foodborne illnesses with associated fatalities. 1988).238 Antimicrobials in Food Following its discovery in the late 1920s. Various studies have shown that nisin will not contribute to antibiotic drug resistance.. One of the earliest reports about “inhibitory streptococci” concerned milk in which cheese starter organisms failed to develop (Whitehead and Riddet. For instance. being active against a wide range of Grampositive bacteria. It remains the only bacteriocin allowed in food as an added preservative. and Thomas and Delves-Broughton (2001). (1996). 1933). Thomas et al. Delves-Broughton and Gasson (1994). who coined its name from the term “N inhibitory substance. It has a much broader spectrum than most other bacteriocins.. Since this group of organisms includes heat-resistant bacteria. developed between 1962 and 1965. Nisin has been studied by many research groups. 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. because of its many positive attributes — broad-spectrum antimicrobial activity. the first commercial extract of nisin was developed by Aplin & Barrett Ltd.. Hurst and Hoover (1993). several reviews have been published. 1981). 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 was also shown to be present in farmhouse cheese (Chevalier et al. Nisin-producing lactococci occur naturally in milk. Apart from numerous research papers on nisin. excellent safety record — and its designation as a natural food additive. the bacteriocin nisin has now been in use as a food preservative for almost 50 years (McLintock et al. The characterization of the molecule was first conducted by Mattick and Hirsch (1947). Food safety is a major concern internationally. 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. 1983). There are clear differences between this bacteriocin and pharmaceutical antibiotics (Cleveland and Tchikindas. Toxicity testing results demonstrating its safety for human consumption were published in 1962 (Frazer et al.

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

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. otherwise a loss of activity may occur as a result of salt precipitation or filtering. is the most widely used nisin assay.3 to 4. It is recommended that such solutions should not exceed a maximum concentration of 250 µg/ml. heating for 3 minutes at 250°F (121°C) destroyed 25% to 50% of the added nisin. 1992). (1965). 1 µg/g pure nisin is equivalent to 40 IU/g or 40 mg Nisaplin®/kg. The nisin preparation Nisaplin® was developed by Aplin & Barrett (now Danisco) between 1962 and 1965.2 as approximately 56 mg/ml. research in Danisco laboratories has shown that care must be taken when preparing high-concentration nisin solutions from commercial preparations such as Nisaplin®. In triangle taste trials conducted by Danisco. Delves-Broughton et al.5. To convert these units to equivalent Nisaplin® levels (mg/L. producing clearly distinguished and measurable zones of inhibition in the presence of nisin. mg/kg) or to International (Reading) Units (IU/ml.5 mg/ml at pH 6 and 0. 1970). and this bioassay method contains .1 to 6. These may be intended as stock solutions for the preparation of test dilutions in smallscale laboratory experiments. and contains 2. with levels of usage in food rarely above 0. Nisin has no significant taste and imparts no negative taste to foods in which it is used..025 mg/ml (equivalent to 1000 mg/kg Nisaplin®).5% nisin A with the remainder made up of added salts and milk solids derived from the fermentation of a modified milk medium by L lactis subspecies lactis. At high pH. Liu and Hansen (1990) suggested that the Dha and Dhb residues become more susceptible to modification as a result of the presence of nucleophiles. In this chapter nisin is expressed as levels of pure nisin (µg/ml or µg/g). The specifications for the purity and identity of nisin were set in 1969 by the FAO/WHO Joint Expert Committee on Food Additives (Anonymous. The effect of processing temperature and pH on the stability of nisin in different foods was studied by Heinemann et al. A similar degree of destruction was reported for highly acid foods having pH values of 3. 1966). Nisin is extracted from the test food by an acid-heat treatment. the levels of pure nisin should be multiplied by 40. Solubility in foods is never a problem because nisin levels are always very low.25 mg/ml at pH 8. Optimum heat stability occurs at pH 3 to 3.240 Antimicrobials in Food Nisin as a powder is extremely stable if stored at temperatures not exceeding 25°C away from direct sunlight in a sealed container allowing no moisture ingress. Pure nisin was deemed too potent for use in food. is the same value as the Reading Unit. dropping to 1. The horizontal agar diffusion test. Nisin is. International Unit. 1990. IU/g). however. 1969). The current term. This was contrary to previous work by Tramer (1964. This has a standard potency of 1 million International Units per g. equivalent to 1 µg of this preparation. volunteers could not detect 200 mg/L Nisaplin® in mineral water (equivalent to 5 µg pure nisin/g).5 (Davies et al. Both the solubility and stability of nisin in solution are affected by pH. and nisin activity was then defined in International Units. first developed by Tramer and Fowler (1964) and Fowler et al. suggesting that the molecule is protected to some extent by food components (Delves-Broughton. 1964). For example. 1998). (1975). However. Liu and Hansen (1990) reported nisin solubility at pH 2.25% biologically active nisin. Their results show that in low-acid foods at pH 6.5.9. and a specification was set for commercial nisin concentrates containing at least 2. successfully used in high-pH/heat-treated food products such as canned vegetables and pasteurized liquid egg. An international nisin reference preparation was later established by the WHO Committee on Biological Standardisation (Anonymous. 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.

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

damnosus. Can grow at low pH. Aerobes/anaerobes Aerobes/anaerobes. curvatus. Growth at low pH. beer.. Very nisin sensitive. Cause spoilage. luteus is indicator strain for the bioassay. buchneri. varians Spp. Varied nisin sensitivity. poor nutrient availability. Aerobes/anaerobes. pentosaceus inulinus aureus Source: From Thomas et al. 25°C–60°C.242 Antimicrobials in Food TABLE 7. tertium. butyricum. Cause spoilage (blowing. Cause blackening of canned food. Growth at pH 2. bulgaricus. and food-poisoning pathogens. Heat-resistant spore-forming thermophiles. licheniformis. Spore forming. monocytogenes Leuconostoc Listeria Micrococcus Pediococcus Sporolactobacillus Staphylococcus luteus. cells will usually be nonenergized in foods because they may well be experiencing adverse conditions caused by low temperature. thermophiles. M. brevis. Obligate anaerobes. producing slime. Heat-resistant aerobic and facultative anaerobic spore formers. salad dressings. quiescent cells may survive but their growth will be inhibited (Sahl.5–6. cured meat products. acidresistant. botulinum. cereus. putrificum. 20