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

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

sugars. A few. Surprisingly. It is important to note that. To accomplish the long-term shelf life necessary for such a marketing system. consumers expect foods to be readily available yearround. and lactate and diacetate to inactivate Listeria monocytogenes in processed meats (65FR17128. mold and rope inhibitors. plant. with rare exceptions. most others have seen extensive use only recently. Those preservatives used to prevent chemical deterioration include antioxidants. In addition. or chemicals applied for their insecticidal or herbicidal properties. to be “free” of foodborne pathogens.3(o)(2)) as “substances used to preserve food by preventing growth of microorganisms and subsequent spoilage. they will not preserve a food indefinitely. only proposed for use in foods. antibrowning compounds. including fungistats. Although some improvements have been made using packaging and processing systems to preserve foods without chemicals. It is essential that all involved in the decision process be acutely aware of the risks and benefits of all additives. Examples include nitrite inhibition of Clostridium botulinum in cured meats and. and animal sources. antimicrobials have been used increasingly as a primary intervention for inhibition or inactivation of pathogenic microorganisms in foods (Davidson and Zivanovic. few food antimicrobials have been used exclusively to control the growth of specific foodborne pathogens. Food and Drug Administration (FDA. Food antimicrobials are classified as “preservatives. However. to prevent enzymatic and nonenzymatic browning. lactoferrin. regulatory personnel. such as nisin. Today. DEFINITION AND FUNCTION OF CHEMICAL FOOD PRESERVATIVES Since prehistoric times. 2001). substances added to food by direct exposure thereof to wood smoke. Several months or years may elapse from the time food is produced until it is consumed. lipids. tends to prevent or retard deterioration thereof. nitrites.2). multiple effective means of preservation are often required. and heating have always been the primary methods used to prolong the shelf life of food products. antimicrobial preservatives play a significant role in protecting the food supply (Davidson et al. vinegars. fermenting.1 and 1.22(a)(5)) as “any chemical that. natamycin.” The traditional function of food antimicrobials is to prolong shelf life and preserve quality through inhibition of spoilage microorganisms. to prevent texture changes. because of changes in the marketing for foods to a more global system. Naturally occurring antimicrobials include compounds from microbial. and vitamins. because food antimicrobials are generally bacteriostatic or fungistatic. preservatives are used to prevent or retard both chemical and biological deterioration of foods. food antimicrobials are not able to conceal spoilage of a food product (i. and consumers. the food remains wholesome during its extended shelf life).” The FDA defines antimicrobial agents (21CFR 170. March 31. more recently.2 Antimicrobials in Food with scientists. for the most part. to prevent autoxidation of pigments. Those additives used to prevent biological deterioration are termed “antimicrobials. and sulfites. Drying. or oils extracted from spices.” Chemical preservatives are defined by the U. are approved by regulatory agencies in some countries for application .S. but does not include common salt.e. Today. products seldom are grown and sold locally as in the past. legislators. 2003). and to have a reasonably long shelf life.. and antistaling compounds. and lysozyme. foods produced in one area are often shipped to another area for processing and to several other areas for distribution. 21CFR 101.” Therefore. food processors. 2000). They are. The former are approved for use in foods by many international regulatory agencies (Tables 1.. Whereas some chemical food preservatives. chemicals have been added to preserve freshly harvested foods for later use. 2002). In addition. flavors. when added to food. selected organic acids as spray sanitizers against pathogens on beef carcasses. spices. such as salt. cooling. Antimicrobials may be classified as traditional or naturally occurring (Davidson. One of the reasons for increased use of chemical preservatives has been the change in the ways foods are produced and marketed. have been in use for many years. nisin and lysozyme against Clostridium botulinum in pasteurized processed cheese.

dehydroacetic acid Benzoic acid.170. Food and Drug Administration) Food Antimicrobialsa (Title 21 of the Code of Federal Regulations) Compound(s) Acetic acid.145 Molds Yeasts. lactates Lactoferrin Lysozyme Microbial Target Yeasts. and poultry products Cheese Cheese. margarine Beverages Meats. diacetates.177 184. dry sausage CFR Designation 184. 184.1768 GRAS Notice No.1005.3640. other bacteria Natamycin Nisin Molds Clostridium botulinum. bacteria (Gram positive) 172. cooked meat.6197.Food Antimicrobials — An Introduction 3 TABLE 1.S. dairy products. wines 184.1021. food antimicrobials permitted by USDA Food Safety and Inspection Service are listed in the Code of Federal Regulations. sauces Beverages. a combination of chemical preservatives and other preservation methods is needed (Leistner.2).133 184. 172.1207. .1550. syrups. 172. methyl. bacteria Yeasts. molds. and then the possible preservation systems must be evaluated via model studies and studies in the food product in question. 184.1 and 1. molds Yeasts Bacteria Bacteria Clostridium botulinum. 182. bacteria Primary Food Applications Baked goods.175.1784 182.1754. molds Bakery products. other bacteria 172. SELECTION OF ANTIMICROBIALS It is not an easy process to select the appropriate preservation system for a particular food product.1 Traditional or Regulatory-Approved (U.21 and 424. heptyl) of p-hydroxybenzoic acid) Propionic acid. meats. 184. Source: Davidson and Harrison. Naidu (2000). 184. 184. dairy products Most foods. beverages. Section 424. and poultry products Cured meats Beverages. 184. Sofos et al.1490.155 184. nitrate Parabens (alkyl esters (propyl. GRAS Notice No.1081.1185. 172.130 184. condiments. 182.1733 172.1221. fermented foods Meats Cheese. confections.1639. The target pathogen or spoilage microorganisms must be identified first. molds. propionates Sorbic acid.1538. baked goods. fats/oils. 184. 172. (1998).3795 Various For meat products. and Davidson and Zivanovic (2003).3089. GRAS Notice No.22. casings for frankfurters. 184. fruit products.160. fruit products. 184. benzoates Dimethyl dicarbonate Lactic acid. sorbates Sulfites a Clostridium botulinum Yeasts. 182. cooked meat. potato products. Extensive reviews on natural antimicrobials may be found in Dillon and Board (1994). casings for frankfurters. acetates. 2000). 172.1061. 2002 to foods (Tables 1.3225. Title 9. Generally. 182. GRN 000067 184. GRN 000065 Nitrite. 184. GRN 000064 Yeasts. wines Fruits.1721.1670.

For example. which have the ability to screen the antimicrobials because of the outer membrane lipopolysaccharide layer. bacteria. Each of these factors is discussed in detail. molds) and forms of those microorganisms (vegetative cells vs.4 Antimicrobials in Food TABLE 1.2 Regulatory-Approved Food Antimicrobial Designations in the European Union (E Numbers) and in Codex Alimentarius (INS Numbers) Compound Sorbic acid K sorbate Ca sorbate Benzoic acid Na benzoate K benzoate Ca benzoate Ethyl paraben Na ethyl paraben Propyl paraben Na propyl paraben Methyl paraben Na methyl paraben Sulfur dioxide Na sulfite Na hydrogen sulfite Na metabisulfite K metabisulfite Ca sulfite Ca hydrogen sulfite K hydrogen sulfite Nisin a E or INS Number 200 202 203 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 226 227 228 234 Compound Natamycin DMDCa K nitrite Na nitrite Na nitrate K nitrate Acetic acid K acetate Na acetate Na diacetate Ca acetate Lactic acid Propionic acid Na propionate Ca propionate K propionate Boric acid Na tetraborate Na lactate K lactate Ca lactate E or INS Number 235 242 249 250 251 252 260 261 262 262 263 270 280 281 282 283 284 285 325 326 327 Dimethyl dicarbonate. does growth in a synthetic .” Seldom. yeasts. 2002. the activity of very hydrophobic antimicrobials may be limited against Gram-negative bacteria. Source: Verbruggen. and the type of preservation or processing and storage systems used. negative) can have dramatic influences on apparent activity. Appropriate methods for in vitro evaluation of the activity of food antimicrobials are described in Chapter 21. Quite often. is not easy to achieve.. ANTIMICROBIAL SPECTRUM The initial selection of the antimicrobial is normally based on an assessment of the overall microbial spectrum of the chemical in question. or strains of microorganism. strain. including the spectrum of antimicrobial activity. however. “Methods for Activity of Assay and Evaluation of Results. species.g. the physicochemical properties and composition of the food product in question. the chemical properties of the antimicrobial. and Gram reaction (positive vs. 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. Even species. spores). a broad spectrum of activity. Selection of the proper antimicrobial depends on several primary factors. Few chemicals have the ability to inhibit several different types. although desired.

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

In the undissociated form.5 or greater. Eklund (1983) demonstrated that the anion does contribute slightly to antimicrobial activity. 1980.6). A lowering of the water activity can select for those organisms that have the ability to survive and/or grow at lower water activity. PROCESS FACTORS The type of preservation process used in conjunction with antimicrobials has a significant influence on the type and level of antimicrobial needed..87 4. and phospholipids. This is because the most effective antimicrobial form is the undissociated acid. there are very few compounds that are effective at low concentrations.g. allowing the antimicrobials access to the inner cell membrane. Certain preservation processes may result in the need to control sporeformers that have the ability to survive the heating process. which means their maximum activity will be in high-acid foods.79 4. Although the undissociated form of a weak acid has most of the antimicrobial activity. Vacuum or modified atmosphere packaging results in . Chelating compounds such as ethylenediamine tetraacetic acid (EDTA) or its salts have a potentiating effect on some antimicrobials.0 (Table 1. Branen and Davidson. organic acids can penetrate the cell membrane lipid bilayer more easily.75 4.. where microbial growth occurs (Branen et al. 1995. Packaging can directly alter the environment of the food and thus influence the overall growth pattern and type of organisms in the food. Once inside the cell. nucleic acids. thus requiring an antimicrobial capable of limiting the growth and activity of these organisms. For example. organic acids function at low concentrations only in high-acid foods (generally less than pH 4. 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.6 Antimicrobials in Food TABLE 1.5 to 4. All regulatory-approved organic acids used as antimicrobials have pKa values less than 5. They expand the activity of certain antimicrobials (e. It is theorized that chelators may destabilize the LPS layer of the outer cell membrane of the Gram-negative bacteria. Generally. which are not normally inhibited by the compounds alone (Cutter and Siragusa. lysozyme) to include Gram-negative bacteria. Bacteria maintain internal pH near neutrality to prevent conformational changes to the cell structural proteins.75 into the lipid areas of the food and away from the water phase. 2004). Protons generated from intracellular dissociation of the organic acid acidify the cytoplasm and must be extruded to the exterior. Rico-Munoz Davidson. pH of the food can result in ionization of an antimicrobial and a change in activity. some Gram-positive bacteria are more susceptible to certain antimicrobials in the presence of chelators including EDTA.3 pKa of Regulatory-Approved Organic Acids Compound or Group of Compounds Acetic acid Benzoic acid Lactic acid Propionic acid Sorbic acid pKa 4. Refrigeration generally selects for psychrotrophic Gram-negative microorganisms. thus indicating the need for a different antimicrobial. the acid dissociates because the cell interior has a higher pH than the exterior. For food products with a pH of 5. 1983). nisin. Because protons generated by the organic acid inside the cell must be extruded using energy in the form of adenosine triphosphate (ATP).19 3. In addition.3). enzymes. the constant influx of these protons will eventually deplete cellular energy.

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

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

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

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

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

were used to characterize commercial fruit juices (Fernandez de Simon et al.2). In four of the seven juices. Teuber. 1995).1).0. depending on the variety (Abdullah et al. p-hydroxybenzoic acid was detected at 0. 66. Yogurts have been found to contain natural levels of benzoic acid (Stijve and Hischenhuber.1% and in neutral media at 0. Orange. This may be of significance in countries like Switzerland. 1999). respectively).1) is a white granular or crystalline powder. apple. In an extensive review. Benzoic acid (molecular weight 122. Sodium benzoate (molecular weight 144. It accounted for approximately 16% of the growth inhibition of Saccharomyces bayanus and Pseudomonas fluorescens from ethanolic extracts of cranberries (Marwan and Nagel. O OH Benzoic Acid O O− Na+ Sodium Benzoate FIGURE 2. respectively). Its presence appears to occur as a by-product of the microbial degradation of either hippuric acid or phenylalanine in these products (Figure 2.1 Structures of benzoic acid and sodium benzoate. may also contribute to benzoic acid generation. Similar results were reported for fungi and yeasts (Cruess and Richert.. 1984. Sieber et al.2% but inactive in alkaline media. was found to have antimicrobial properties.2 g dissolves in 100 ml water at 0°C. 0. pear. 1929). occurs in pure form as colorless or white needles or leaflets. A third pathway. Benzoic acid has also been identified as a major constituent in extracts of blackberries (Humpf and Schreier.2 to 2. 18°C. It is soluble to a limited extent in water (0. 1989. oxidation of benzaldehyde. 20°C. along with benzoic acid. it is preferred for use in many cases. of mushrooms. Several new types of low-molecular-mass compounds were also identified. It is much more soluble in water than benzoic acid (62. Gabel (1921) was one of the first to demonstrate that benzoic acid was effective against bacteria in acid media at a level of 0.6 mg/l.3). MECHANISM OF ACTION It is the undissociated molecule of benzoic acid that is responsible for antimicrobial activity (Table 2.2). and 2. 1992). 1986a).1. apricot. peach. (1995) surveyed and analyzed many types of cultured dairy products and cheeses for the natural occurrence of benzoic acid (Table 2. and 74. For this reason.18. Potassium benzoate and calcium benzoate have also been approved for use. although their solubility in water is less than that of the sodium salt. and of fresh tomatoes (Marlatt et al. pineapple. 1991).. including derivatives of benzoic acid. .1). Benzoic acid occurs naturally in several foods and commodities (Table 2. also called phenylformic acid or benzenecarboxylic acid.8. 1992). and 100°C. where benzoic acid has not been approved as a food additive (Sieber et al.12 Antimicrobials in Food PHYSICAL AND CHEMICAL PROPERTIES AND NATURAL OCCURRENCES Benzoic acid (C6H5COOH) and sodium benzoate (C6H5COONa) have the structural formulas shown in Figure 2. Benzoic acid has also been identified as a natural by-product in culture filtrates of Lactobacillus plantarum (Niku-Paavola et al. and grape juices were analyzed to establish phenolic profiles unique to each type of juice... and a mixture of these. 1990).2 g dissolves in 100 ml water at 4°C.27. and 75°C. 1994).. Naturally occurring nonflavonoid phenolic compounds.

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

44 0. increasing the pH of the culture medium decreased the effectiveness of this preservative. Sorbate was more inhibitory than benzoate on the strains that were tested. NaCl.2 Biosynthesis of benzoic acid by lactic acid bacteria in fermented dairy products. Macris (1975) observed a rapid uptake of benzoic acid in Saccharomyces cerevisiae. 1997).144 4. The effect of temperature on the uptake was similar to that observed in enzymatic reactions.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. and sorbic and benzoic acids on the growth of 30 strains of food spoilage yeasts have been reported (Praphailong and Fleet. 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. Proteinaceous material was apparently involved in the uptake of this preservative. The effects of pH. The strong dependence of uptake on pH was because of the relative distribution of undissociated and dissociated forms in solution. The undissociated form was the only one taken up by cells. not to the pH itself. TABLE 2. Source: Adapted from Sieber et al.3 Effect of pH on the Dissociation of Benzoic Acid pH 3 4 5 6 7 pK Undissociated Acid (%) 93. The combined effects of pH and benzoate on the growth of these strains are presented in Table 2. Saturation was reached in about 2 minutes and remained constant thereafter.5 59.8 1. Zygosaccharomyces bailii and Yarrowia lipolytica were the most resistant to the two preservatives at pH 5.3 12.19 Source: From Baird-Parker (1980). . As expected.0. that only the undissociated acid was antimicrobial. (1995). sucrose. and that the toxicity of sodium benzoate in solution was a result of the undissociated benzoic acid molecule.4.

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

1966).. At pH 6. 50% of the trimethylamineN-oxide reductase activity was inhibited by 1. The production of lipase by P. 1980. In the citric acid cycle.5. enzymes involved in acetic acid metabolism and oxidative phosphorylation can be inhibited (Bosund. coli. 1962). Duration of the lag phase can be predicted from the model. Synthesis of messenger ribonucleic acid (mRNA) was critical for restoration of salt tolerance. yielding protons that acidify the alkaline interior of the cell. Hsiao and Siebert (1999) constructed a model based on physical and chemical properties of organic acids and principal components analysis.. 1977. such as benzoic. Increased permeability of cell membranes during the course of benzoate injury was not observed. Only those organic acids that are lipophilic. The authors suggested that the potency of weak acids. More than 99% of the viable cells of Listeria monocytogenes were injured after exposure to a solution of 8. causing uncoupling of both substrate transport and oxidative phosphorylation from the electron transport system (Freese et al.2 mM benzoic acid (Kruk and Lee. and Pseudomonas aeruginosa (Freese et al.5. Metabolic injury was evident by the inability of cells to tolerate 6% NaCl in tryptose agar and the ability to grow on this medium with no added salt.5% sodium benzoate (pH 7. Production of 12 of these was induced at pH 8. 1989). Gould. Uraih et al. One hypothesis is that they inhibit or kill microorganisms by interfering with the permeability of the microbial cell membrane.3). as food preservatives is related to their capacity specifically to reduce the intracellular pH. α-ketoglutarate and succinate dehydrogenases appear to be quite sensitive (Bosund. For example. 1989. resulting in nutritional starvation of cells (Gould et al. 1973). 1997). coli. 1975. 1956. benzoate addition resulted in an increased production of 33 proteins. Aflatoxin production by a toxigenic strain of Aspergillus flavus was greatly reduced by the presence of these compounds in synthetic media (Uraih and Chipley. A pH-related increase in protein production was observed when cultures of E. fluorescens was completely inhibited by sodium benzoate (Andersson et al... 1982).. in many bacteria and yeasts. 1978). and the ability of benzoic acid molecules to delocalize the negative charge of ions within the interior of cells and thus increase their membrane mobility. 1973. They also stated that although both the undissociated and dissociated forms of these acids cause the intracellular pH to fall. 1980).. benzoic acid or sodium benzoate can also inhibit specific enzyme systems within cells (Webb. Eklund. Davidson. show antimicrobial activity. Corlett and Brown.0) for 1 hour (Buazzi and Marth. The inhibitory potency of this acid can be determined by its lipid/water partition coefficient. 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. growth inhibition is the result predominantly of the undissociated acid. Inhibition of transport in turn results from the destruction of the proton-motive force caused by the continuous shuttle of protons into cells by benzoic acid (Davidson. Reduction . The effects of organic acid preservatives on the growth and intracellular pH of E. B. coli have been reported (Salmond et al. 1973). MICs were determined for each acid and used with principal components to produce models with good correlations. Acid-susceptible bacteria (Bacillus and Alicyclobacillus) and acid-resistant bacteria (Lactobacillus and Escherichia coli) were grown in media containing organic acids. Eklund. 1984). Theoretical ATP consumption for proton pumping can be directly correlated with reduction in cell yield. 4. 1980). In resting cells of E.0 as well as pH 6.16 Antimicrobials in Food 3.. Sheu et al. 2001). In addition to these mechanisms. In yeast cells. Similar effects have been found using a variety of microorganisms. 1997). its pK value (Table 2. 1980). coli were grown in liquid media with or without 20 mM benzoate (Lambert et al. 1976. membrane transport of amino acids is inhibited. 1992). In Bacillus subtilis (Freese. 1960. Benzoate also inhibits amino acid uptake in Penicillium chrysogenum (Hunter and Segel. E. and the effectiveness of undissociated acids as preservatives has been noted for many years (Ingram et al. such as benzoic acid. 1983. 1962). 1986). phosphofructokinase activity was inhibited (Francois et al.... subtilis. Chipley and Uraih.

coli and Salmonella Enteritidis (Chipley. flavus have been reported (Bauer et al.02% undissociated acid.5 and 2. Therefore. With the previously mentioned results in mind. In liquid media. 1972. and most yeasts and fungi are inhibited by 0. 1997).01% to 0.. The sensitivity of 42 yeast cultures to sorbic and benzoic acids. Increasing the concentration of sodium benzoate increased the percentage of inhibition at the end of incubation (10 days). MICs for some of the bacteria.. benzoic acid cannot be relied on to effectively preserve foods capable of supporting bacterial growth (Chichester and Tanner. In general. The scattering of MIC values was lower with potassium sorbate and benzoic acid and higher with sorbic acid and sodium benzoate.05% to 0. The authors concluded that subinhibitory concentrations of these compounds may stimulate toxin production in some cases.. as a weak inhibitor of poly(ADP ribose) polymerase (Oikawa et al. and environment from which the microorganism was originally isolated. aflatoxin B1 production was higher than in controls. 1980). 1979). including pH. The results suggested that these preservatives blocked an enzymatic step late in the biosynthetic pathway of aflatoxin B1. as an inhibitor of passive anion transport (Lucas-Heron and Fontenaille. composition of the growth medium. 1988). temperature. yeasts. Yagi et al. 1992)... and fungi involved in food poisoning and food spoilage are given in Tables 2. 1980). Sodium benzoate was found to control both growth and aflatoxin production by Aspergillus parasiticus in liquid media (El-Gazzar and Marth. 1981). 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. cerevisiae decreased the biomass and increased the specific oxygen uptake rate of cells (Verduyn et al. 4-dinitrophenol and cell envelope-associated enzymes of E. 1983).Sodium Benzoate and Benzoic Acid 17 was accompanied by the appearance of a yellow pigment that could be converted into aflatoxin B1 by cell-free extracts. The effects of several preservatives and antimicrobial agents on aflatoxin B1 production by A.1% of the undissociated acid. In benzoic acid-supplemented cultures. Benzoate may also serve as a scavenger for free radicals (Harvath. ANTIMICROBIAL ACTIVITY SPECTRUM OF ACTION Because the quantity of undissociated acid decreases with increasing pH (Table 2. 1980). all treated cultures produced measurable levels of toxin 3 to 7 days later than controls. as an inhibitor of Damino acid oxidases (Quay and Massay. with the greatest increase occurring when the medium contained 0. however. Addition of benzoate to chemostat cultures of S. and as an inhibitor of nitrosamine formation (Sung et al. . Food-poisoning and spore-forming bacteria are generally inhibited by 0.3% sodium benzoate. Baird-Parker. Fusarium species were more sensitive to benzoic acid (210 to 420 µg/ml) than were Penicillium species (250 to 3000 µg/ml). 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. potassium sorbate. 1977. these compounds are used primarily as antimycotic agents. prior exposure to the preservative. Currently. The average accumulation of mycelial dry weight was greater in the presence of benzoate than in its absence. 1987). 1974). Similar results were observed with 2. genus and species of the microorganism in question. This might result in inhibition of membrane-bound enzymes as a secondary effect.1979). the use of benzoic acid or sodium benzoate as a food preservative has been limited to those products that are acid in nature. Several factors interact to determine the MIC. It has been reported to have a flower-inducing effect in plants (Watanabe and Takimoto. Conversion could be prevented by addition of benzoic acid or sodium benzoate.6. 1979). and sodium benzoate was determined (Manganelli and Casolari. but many spoilage bacteria are much more resistant.3).

(1999).0 4.0 5.0 6. 1993).0 4. lentus Z.8 Fungi 5. Saccharomyces bayanus S.0 MICa Bacillus cereus Escherichia coli Klebsiella pneumoniae Lactobacillus sp.18 Antimicrobials in Food TABLE 2. Hansenula subpelliculosa Oospora lactis Pichia membranefaciens P.0 5.0 6. 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.5 Antimicrobial Spectrum of Benzoic Acid against Selected Bacteria.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.6–4.0 5.6–5.8 4.8 4.0 4.6 6. Yeasts. Listeria monocytogenes Micrococcus sp. Source: Adapted from Chipley (1983. Russell (1991).0 3.2–5.0 6. and Steels et al.2–5. Aspergillus parasiticus Aspergillus niger Byssochlamys nivea Chaetomonium globosum Cladosporium herbarum Mucor racemosus Penicillium sp.3 5. . rouxii 4.0–5.0 4.5–5.3 5. Pseudomonas sp.0 5.6 6.1 5.6 Yeasts 2.5 4. Pseudomonas aeruginosa Staphylococcus aureus Streptococcus sp.5 5.6 (4°C) 5.0 4.0 5.0 5.0 5. Zygosaccharomyces bailii Z.0–5. Penicillium citrinum Penicillium glaucum Rhizopus nigricans a 1500 20–300 >4000 2000 500 1000 100 30–120 30–280 2000 400–500 30–120 Minimum inhibitory concentration in µg/ml (ppm).0 3.0 4. Davidson and Juneja (1990).6 (21°C) 5. and Fungi Microorganisms pH Bacteria 6. pastori Rhodotorula sp. cerevisiae Torulopsis sp.0 2.3–6.

Yousef et al.5.6 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. Most were isolated from spoiled foods that had contained preservative. were tested against both the opaque and translucent morphotypes of Vibrio vulnificus (Sun and Oliver. Under appropriate conditions. 1979). including benzoic acid. 1989). Source: Adapted from Warth (1989c). Growth of this fungus was accompanied by production of fumitremorgin mycotoxins.. In a series of studies involving L. Isolates were grown at 25°C in yeast extract medium containing 5% glucose (pH 3. it was found that benzoic acid at concentrations of approximately 1000 to 3000 ppm had strong bacteriostatic. Salts. Fungal growth was reduced by lowering the pH of laboratory media from 7. but relatively modest bactericidal. Eight of these had a lethal effect on both morphotypes of this bacterium. enhanced aflatoxin production when present at low levels but became inhibitory at higher levels.5) without addition of benzoic acid. Both fungistatic and fungicidal properties have been attributed to benzoic acid.0 to 2. activities against cells in a liquid minimal medium. Ten generally regarded as safe (GRAS) substances. bacteriostatic and bactericidal properties of benzoic acid can also be demonstrated. monocytogenes (El-Shenawy and Marth. 1977). selected organic acids promoted growth and toxin production when added to the media. Small amounts (75 mg/L) of potassium sorbate or sodium benzoate completely inhibited germination of ascospores and subsequent outgrowth. Growth and aflatoxin production by toxigenic strains of Aspergillus were partially or completely inhibited by the undissociated form of six organic acid preservatives. The authors questioned the suitability of benzoic acid alone to control this pathogen in foods. according to the results of a study involving several strains of Trichophyton and Microsporum (Pelayo. 1989). such as sodium and potassium chlorides and sodium nitrate. including benzoic (Rusul and Marth. 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. Incubation of cells in minimal media caused injury that depended on the temperature of incubation but not on the presence of benzoic acid. 1988).. This organism was isolated from milk. 1988. Sodium benzoate has been suggested as an inhibitor of cellulose-decomposing bacteria and fungi (Sauer. 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.Sodium Benzoate and Benzoic Acid 19 TABLE 2. 1995). and its survival .

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

respectively. 1975). Antagonistic as well as synergistic effects were observed when a wine yeast strain of S.Sodium Benzoate and Benzoic Acid 21 1. irradiation. 1988). 1958). benzoic acid can be solubilized with a surfactant to hold it in solution in its undissociated form (Lewis and Payne. and 500 ppm sodium benzoate. However. 1984). in hours. RE. 1956).9) (Okoli and Ezenweke. 1976). 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.3 Effect of the medium on the inhibition of Saccharomyces bayanus by benzoic acid at 30°C and pH 4.0 50 100 150 ppm 200 250 300 350 FIGURE 2. followed by potassium sorbate and sodium benzoate. 1990).. The effects of pH and benzoate on the growth of yeasts in an experimental grape drink containing 1. Open circles: Trypticase® soy broth. refrigeration. Sodium benzoate prevented the growth of Staphylococcus aureus in an intermediate-moisture food (Boylan et al.4 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. and 37°C over a 25-day incubation period was significantly retarded by 75 ppm sulfur dioxide.8 0. Likewise. or drying. . the antimicrobial activity of benzoic acid is reduced by nonionic surfactants (de Navarre and Bailey. For example. A combination of benzoic acid and low temperatures was used effectively to reduce the numbers of yeasts in grape juice (Pederson et al.b). it may also be advantageous to combine preservatives with physical methods of food preservation.. it took the treated populations to reach a density of 70 Klett units divided by the time it took the control populations to reach the same value).5 volumes of CO2 have been reported (Kelly. 1961). Sulfur dioxide had the most significant effect on the rate of biomass and patulin production by this organism. Closed circles: yeast nitrogen base.2 0.0 0. such as heating. 30°C. depending on the chemical in question. Growth at 21°C. 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. cerevisiae was inoculated in a laboratory medium supplemented with several combinations of preservatives (Parish and Carroll.6 1/RE 0. relative effectiveness (the time. 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. Source: Marwan and Nagel (1986b). 150 ppm potassium sorbate. The presence of other chemicals may be beneficial or detrimental to the action of the preservative.0.

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

Three strains of T. and lethality was enhanced as the pH of the heating medium was reduced. TABLE 2. A strain of S. Warth (1977) reported that cells of Z. 1988) it was determined that energy was required for the reduction in cytoplasmic benzoate concentration and for the maintenance of internal pH. flavus. 1990).0 and 4. resulting in reduced growth yields and rates of cell production by yeasts. bailii grew in the presence of high concentrations (600 mg/L) of benzoic acid at pH values below the pK of this acid (4.. cells were resistant only when a sufficient energy source. .7). Fumaric. Heat resistance increased with age. The effects of sorbic and benzoic acids on the viability of ascospores varied depending on the strain and were influenced by other constituents in the heating medium. In a later study (Warth. and benzoic acids were the most effective. 1987). In an inoculated peach-based system. Several species of yeasts grown in the presence of benzoic acid tolerated 40% to 100% higher benzoic acid concentrations than yeasts grown in its absence (Table 2. Resistance resulted primarily from an inducible energy-requiring system that transported benzoate from the cell. and in some cases. Thus. Because this system required energy. heat tolerance appeared to be related to the morphology of the ascospores. such as glucose. 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. 1988). this isolate was less resistant to potassium sorbate.4 to 3.19). sorbic. the energy source was unavailable for growth. However.7 Resistance of Yeast Species to Benzoic Acid MIC of Benzoic Acid (mg/L) Isolate Saccharomyces cerevisiae Kluveromyces fragilis Kloeckera apiculata Hansenula anomala Saccharomyces cerevisiae Candida krusei Saccharomycodes ludwigii Schizosaccharomyces pombe Zygosaccharomyces bailii a Cells Grown without Benzoic Acid 100 125 125 140 175 300 300 325 600 Cells Grown with Benzoic Acida 170 173 188 223 285 440 650 567 1250 Adapted to benzoic acid by overnight growth in the presence of 0.5. The heat-resistant yeast Talaromyces flavus was isolated from fruit juice concentrates involved in spoilage of packaged reconstituted fruit juice (King and Halbrook. was present. Potassium sorbate and sodium benzoate prevented outgrowth.5. respectively (Ethiraj and Suresh. cerevisiae was found to have multiple resistances to both physical treatment and chemical preservatives (Guerzoni et al. The authors observed that preservative resistance may be strain specific. were tested for tolerance to several organic acids during and after heating (Beuchat. Source: Adapted from Warth (1988). isolated from spoiled fruit products.25 mM benzoic acid (first five species) or 2 mM benzoic acid (last four species). with the required concentrations lower when the pH of the growth media was reduced from 5.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. 1988).

1996). Removal or metabolism. 300. bailii can grow in concentrations of food preservatives in great excess of those normally. Foods at particular risk are acidic and contain relatively high levels of sugar. 1989b). Mustard spoilage by Z. The minimum pH for growth was not related to this acquired resistance. bailii has been described (Buchta et al. 1985). or an intrinsic sensitivity to benzoic acid or its anion (Warth. . Spoilage resulted from a Z.. 1989a).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. Some yeasts are intrinsically resistant to relatively high concentrations of preservatives (Stratford and Lambert. Zygosaccharomyces lentus. Growth of yeasts in the presence of benzoic acid reduced their subsequent permeability to this acid. The principal mechanism of uptake appeared to be diffusion of undissociated acid. 1989c). in which benzoic acid is continuously removed from the cell. lentus was likely to be more significant than Z. A new osmophilic. bailii strain that was resistant to both benzoic and sorbic acids. the capacity of the cell to remove it. The nonselective medium supported higher recovery of all nine strains from the six food products compared to the three selective media. 1999). Differences in resistance among yeast species could be the result of differences in the rate of penetration of the cell by benzoic acid. has been described (Steels et al. The Zygosaccharomyces genus also contains some of the most osmotolerant species known. Growth in the presence of weak acid preservatives appeared to involve a common resistance mechanism. Zygosaccharomyces and Saccharomyces are able to adapt to changing levels of preservatives such as benzoate. 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. 1996). The authors stated that microorganisms resist or adapt to weak acid preservatives (e. Adaptation can result in rapid growth in moderate levels or slow growth in previously inhibitory levels. Z. It was preserved using benzoic acid (1. Uptake rates of benzoic and propanoic acids in cells of Z. bailii grown in six commercial food products (Makdesi and Beuchat. and all strains were osmotolerant. and most can grow at very low pH. or legally. requiring only 5 to 15 minutes to reach equilibrium. bailii and S. They were resistant to sorbic acid (≤400 mg/l) and benzoic acid (≤900 mg/l) at pH 4. Resistant species may be less permeable than sensitive ones to undissociated benzoic acid (Warth. and 600 µg/ml).6) (Warth.0. benzoate) by one of the following three methods: 1. These media were also evaluated for enumerating two of these strains grown in blueberry syrup with or without sodium benzoate (0.0). 1989c).5 g/kg) and packaged in glass jars. results also suggested that the resistance mechanism. Eight tons of table mustard were exported to the United States. Benzoic acid was taken up 27 times faster by cells than propanoic acid. preservative-resistant spoilage yeast. The authors concluded that Z. Adaptation to higher preservative concentrations was demonstrated with benzoic acid. Growth at 4°C was significant. Three selective media were compared for detecting and enumerating nine strains of Z. and acetic acids and ethanol (Thomas and Davenport. 1988). Resistance to dimethyldicarbonate was greater than that of Z. benzoic. Cells grown in syrup without sodium benzoate were more sensitive to selective media than were cells grown in syrup containing the preservative. allowed in beverages and can adapt to concentrations even higher.. In another study (Warth. cerevisiae. 1999). 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.. is a common and major determinant of the preservative tolerance of yeast species (Table 2.2 to 7. Five strains of this yeast grew over a wide range of temperature (4°C to 25°C) and pH (2. bailii in the spoilage of refrigerated foods. Uptake of weak acid preservatives by passive diffusion is quite rapid. However.g. bailii were proportional to the concentration of undissociated acid (Warth. Glucose stimulated the rates of uptake and was also required for continuous elimination of benzoate from cells.

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

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

6. 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. (4) ring opening. one of the causal agents of wilt in banana plants. 1988). In the presence of benzoate. protocatechuate 3. and β-ketoadipate were identified as intermediates of benzoate metabolism (Figure 2. This is followed by conversion of the remainder of the molecule to acetyl-CoA. Niemetz et al. maleylpyruvate. and with Micrococcus (Haribabu et al. foods. 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. (3) introduction of a carbonyl group.. Benzoate was metabolized by 16 of these strains when added as the sole carbon source to liquid mineral media. including Pseudomonas. The proposed intermediates included benzoyl-CoA. was also capable of metabolizing benzoate (Kumari and Mahadevan. 1997). Harwood and Gibson (1997) stated that this pathway could be divided into several phases: (1) formation of CoA thioesters. iron. and cosmetics. benzoate and salicylate are metabolized via the protocatechuate and β-ketoadipate pathways. respectively (Shailubhai et al. both aerobically and anaerobically. (2) ring reduction. 1997). Nitrate. fumarylpyruvate. 1995).. Similar results have been reported with Bacillus stearothermophilus (Adams and Ribbons. which are central intermediates in the anaerobic metabolism of many aromatic compounds (Elder and Kelly. 4-dioxygenase was induced. and (5) a β-oxidation sequence. 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. 1995). Further metabolism proceeded via the catechol branch of the β-ketoadipate pathway with the occurrence of high specific activities of catechol 1. Seventeen strains of aerobic bacteria were tested for their ability to degrade 15 aromatic substrates (Lang. Kinetic models have been derived for the metabolism of [14C]-benzoate by Pseudomonas species (Simkins and Alexander. Pseudomonas solanacearum.. A mutant incapable of growth on benzoate was isolated and characterized.. Outlines of the new aerobic pathway were deduced. cis-muconate. 1996). and sulfate can be used as electron acceptors during metabolism (Colberg and Young. In Aspergillus niger. These results were confirmed by assaying key enzymes from benzoate-grown cells. The enzymes responsible for ring cleavage were induced depending on the substrate used for growth. 1982).. 1992). In Alcaligenes eutrophus. and fumarate + pyruvate. 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.Sodium Benzoate and Benzoic Acid 27 β-ketoadipate pathway. Hugo (1991) described the ability of nine species of Pseudomonas and Vibrio to degrade 18 aromatic compounds used as preservatives in medicines. Active transport of benzoate may occur in this organism (Thayer and Wheelis. 1982). gentisate. Both chromosomal and plasmid-encoded pathways have been described for the catabolism of benzoate in P. with Rhodococcus (Janke et al. Harwood and Gibson (1986) stated that aerobic degradation of benzoate by various Gramnegative bacteria. putida (Jeffrey et al. Catechol.5). 1994). such as glucose. Compared to relatively well-characterized aerobic mechanisms for metabolism of benzoate and other aromatic compounds. A permease-like protein involved in benzoate transport and degradation has now been isolated from cells of Acinetobacter (Collier et al. He reported that benzoic acid was easily metabolized by all nine bacterial species tested. 1984).5) (Ampe et al. cis. 3-hydroxybenzoyl-CoA. 1984). 1988). a commonly occurring soil bacterium... 1984).2-dioxygenase. anaerobic pathways have been far less studied and reported. 1993.2-dioxygenase for ring fission. One pathway proposed for the anaerobic microbial metabolism of benzoate is shown in Figure 2. The enzymes of these pathways appeared to be under coordinate control and were repressed in the presence of a primary substrate. ... end product of the β-ketoadipate pathway (Figure 2. 1990). 1997). whereas salicylate induced catechol 1. Anaerobic metabolism can also occur but involves reductive saturation of the nucleus. the rate of benzoate metabolism may be regulated by the presence of succinate.

Based on these results. monocytogenes by benzoic acid and other compounds (Ramos-Nino et al. 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. Nocardia asteroides and several fungi. Ganzfried and McFeeters (1976) developed a model system to evaluate binding of benzoic acid by lipids and proteins. the authors concluded that it may not be limited to a small number of microorganisms.6 Proposed pathway for the anaerobic microbial metabolism of benzoic acid. Dashed arrows indicate proposed intermediates. jejuni and demonstrated that this gene was not present in 17 isolates of C. including species of Rhizopus and Mucor. C. Both bound benzoic acid to approximately the same extent.. jejuni could be developed. the hippuricase assay is the only reliable biochemical assay that can distinguish C. 4-diene carboxyl-CoA O OH 2-Hydroxycyclohexane carboxyl-CoA Cyclohex-1.28 Antimicrobials in Food O OH CoA-SH Benzoic Acid O S-CoA 2H Benzoyl-CoA O S-CoA 2H O S-CoA H2O Cyclohex-1-ene carboxyl-CoA 2H OH O S-CoA H2O S-CoA 6-Hydroxycyclohex-1-ene carboxyl-CoA O S-CoA −2H Cyclohex-1. jejuni. 1996). .. antilisterial activity was much lower than predicted. coli. Currently. 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. 1988). jejuni from C. Harwood and Gibson (1997). making the models unacceptable under these conditions. coli. This enzyme cleaves hippuric acid into the constituent products glycine and benzoic acid. (1993). An equation was obtained to estimate the ratio of bound to unbound acid in food products based on their lipid and protein content. the authors proposed that a species-specific diagnostic DNA probe for human gastrointestinal infection caused by C. They also showed that the gene was absent in other Campylobacter species. Note: This sequence of reactions is compiled from several studies. were found to have this capability. Source: Adapted from Koch et al. In foods with high protein or lipid content. However. 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. one species. Microbial reduction of benzoate to benzyl alcohol under aerobic conditions has been reported (Kato et al. The authors isolated the gene responsible for the expression of hippuricase from 12 strains of C. Solid arrows indicate intermediates detected in benzoate-grown cultures. although stability was not adversely affected with time. 1979). Heintze (1978) reported that benzoic acid accumulated in the lipid phase of herring salad. In the genus Campylobacter.. has become recognized as a leading cause of human gastroenteritis (Hani and Chan. most of which were fungi. Models were tested in several food systems to determine the inhibition of 18 strains of L. Although this appears to be a unique reaction occurring in microbial cells under aerobic conditions. The N-benzoyl-glycine amidohydrolase (hippuricase) test is of paramount importance to differentiate C. 1995). jejuni (hippuricase positive) from other species. 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. For example.

184. Lueck (1980). the maximum permissible quantities generally range between 0. .4 g per 100 ml solution) than the sodium salt. Potassium benzoate is now commercially available and apparently evolved in response to consumers’ interest in reduced sodium intake.25%. 1977/1988. Inc. fruit products. REGULATORY STATUS In the United States. generate detectable levels of benzene. although its solubility in water is much less than the sodium salt (Pollard. 1994). and limitations of preservatives and antimicrobial agents. 1990). applications. including benzoic acid. In one case (a fruit juice–mineral water combination).9). Approximately 1. Similar results were reported by McNeal et al. under certain conditions. A Food Technology special report (1986) reviewed the characteristics. and lack of color. and other foods in several countries (Tables 2. the narrow pH range in which they are effective.1733) up to a maximum permitted level of 0. (1993) when they analyzed several foods and fruit-flavored soft drinks for benzene. Kimble (1977). Although the detectable yield was extremely low (<1 ppb). Secs. In addition. Calcium benzoate has also been approved for use. As food preservatives. the authors recommended that the combination of ascorbic acid and sodium benzoate in foods and beverages be evaluated more carefully. APPLICATIONS Benzoic acid and sodium benzoate are most suitable for foods and beverages that naturally are in a pH range below 4.1%. 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). In most other countries. USE IN VARIOUS FOOD SYSTEMS Benzoic acid and sodium benzoate have been widely used to preserve beverages. An ADI (average daily intake) value of 0 to 5 mg/kg of body weight has been specified for benzoic acid (Pollard. Title 21. and their toxicologic properties compared to some other preservatives have all contributed to recent efforts by food processors to replace benzoic acid and sodium benzoate with other preservatives with better characteristics. Regulatory agencies consider it to be GRAS to the same extent that sodium benzoate is GRAS as a preservative with the limitation of 0. 1990). Subsequent studies indicated that soft drinks containing both ascorbic acid and sodium benzoate could produce very low levels of detectable benzene. Product information for both salts is available (Miles Laboratories. Benzoate does not control the growth of high levels of microorganisms and therefore cannot be used to compensate for poor ingredients or processing. the use of benzoic acid and other chemicals in the processing and freezing of shrimp has been reviewed (Chandrasekaran.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.15% and 0. benzene levels higher than the permissible levels for mineral waters prompted a recall in one state. They may also include foods from which benzoate is excluded in the United States. They may also be used in certain standardized foods. their main advantages include low price. ease of incorporation into products. Applications of these preservatives to foods have been reviewed by Chichester and Tanner (1972). benzoic acid and sodium benzoate are GRAS preservatives (Code of Federal Regulations. The potassium salt is less water soluble (42. However.1%. the off-flavor they may impart to the foods they are to preserve. and Chipley (1983.5 or that can be brought into this range by acidification.8 and 2.1 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. bakery products. 1993).).1021 and 184.

salted Mayonnaise Mustard Pickles. yolk or whole Margarine. bailii in an inoculated salsa mayonnaise stored at room temperature (Wind and Restaino. Fresh catfish were dipped in various concentrations of sodium benzoate or potassium sorbate and smoked. The effectiveness of sodium benzoate and potassium sorbate. 2001). flavus was most sensitive to potassium sorbate. However. Several additives and preservatives were evaluated as inhibitors of melanosis (black spot) and microbial spoilage in prawns in chilled storage (Montero et al. Aspergillus flavus.a 1000 200 500 500 5000 1500 1500 1500 5000 2000 1000 1000 1000 10. 2000). . 4-hexylresorcinol was the most effective inhibitor of both melanosis and microbial spoilage in prawns. then evaluated during shelf life (Efiuvwevwere and Ajiboye.K. Penicillium sp. bailii. U. was evaluated against the growth of Z.S.3% sodium benzoate was three times longer than that of the controls (Ponce de Leon et al.. Source: Adapted from Chipley (1993).30 Antimicrobials in Food TABLE 2.02%) or sodium benzoate (0. b Soft drinks for consumption after dilution. separately or combined. 1995). and Penicillium sp. Orange juice not for manufacturing may not contain a preservative (federal standards of identity). c Soft drinks for consumption without dilution. A. d Good manufacturing practice. The shelf life of sardines stored in an acid brine containing 0. However. relishes Salads. ketchup Soft drinks containing fruit juice Soft drinks. 1996). Halotolerant fungi were isolated from salted dried fish (Chakrabarti and Varma. semipreserved Fruit juice Fruit pulp Jam.06%) or potassium sorbate (0. A maximum level of 2000 ppm may be used in orange juice for manufacturing. a maximum level of 1000 ppm benzoic acid or its salts may be used for all products listed. niger. prevented spoilage of the product by Z. separately or combined. Potassium sorbate was more effective than sodium benzoate. Propionic acid (0. Sorbate treatment was more effective than benzoate and increased the shelf life by 8 days. A combination of sodium benzoate and kojic acid was effective in inhibiting melanosis. was most sensitive to propionic acid and sodium benzoate. neither preservative. carbonated a Canada 1000 1000 1000 1000 1000 Denmark Norway Sweden U.8 Maximum Permitted Levels (ppm) for Benzoic Acid and Its Salts in Foods in Selected Countries Food Fish products Fish. Recent reports indicate that these preservatives continue to be of benefit in a variety of products. jellies Liquid egg white.04%) inhibited growth of all three fungi.. 1994).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. were dominant. but A. salad dressings Sauces.

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

A study was conducted to evaluate aqueous dipping solutions of organic acids or salts to control L. The antimicrobial can be applied directly onto the product surface where cells of L. but benzoate was about two to eight times more effective than sorbate (Splittstoesser et al. At 8°C.32 Antimicrobials in Food temperature. Survival of E. respectively. 1995). Chicken skin was inoculated with Salmonella spp. Washing the skin with solutions of either 0. Application of antimicrobials as sprays or mists to meats following processing may be more effective than their addition in the meat formulation.15% fumaric acid and 0. 2001). monocytogenes.0) made from milk acidified to pH 5. However. The treatment that was successful included addition of 0. Dock et al.6) or adding potassium sorbate (0. Tompkin et al. Large outbreaks of infection have been linked to consumption of contaminated coleslaw.. monocytogenes (Barker and Park.05% sodium benzoate followed by holding at 25°C for 6 hours and at 4°C for 24 hours. Both preservatives reduced the heat resistance of E. 5% sodium diacetate.9 with propionic acid. and wash solutions were evaluated for effectiveness in decontaminating the skin (Hwang and Beuchat. 1996. aureus. this antimicrobial was evaluated for control of postharvest diseases of various fruits and vegetables. jejuni. These strains grew well in unpasteurized and pasteurized apple juice. 1989).. 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. A preservative treatment was developed that was capable of achieving the FDA mandate for a 5-log reduction of E. cheeses. These solutions could be used for dips to sanitize chickens intended for frying before presentation to consumers (Hathcox et al. C. vacuum-packaged bologna stored at 4°C for up to 120 days. monocytogenes usually attach following cooking and during slicing and packaging (Farber and Peterkin. Listeria monocytogenes is recognized as a foodborne pathogen of major concern to humans. Resistance to these preservatives was greater at 4°C than at 25°C (Fisher and Golden. 1998). hot dogs. milk. Sorbates and benzoates are currently approved in various countries for use as dipping solutions to prevent fungal growth in dry sausages (Sofos. the combination of sorbate and benzoate was more effective than either preservative alone (Zhao et al. 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.05% sodium benzoate reduced the numbers of these pathogens compared to washing with water. Control strains of E. coli O157:H7 in apple juice and apple cider with or without potassium sorbate or sodium benzoate has been investigated. coli failed to grow in either type of apple juice. Ethanol-enhanced killing correlated with damage to the bacterial cytoplasmic membrane. Benzoate was one of the most effective compounds tested in the presence or absence of ethanol. or 5% potassium benzoate for 120 days (Samelis et al. 2000).3%) to cheese (pH 6. 1999).. monocytogenes on sliced. However. 1995). 1994). 1993). or S. L. USE AS A POSTHARVEST FUNGICIDE Because of the long and successful use of benzoic acid in the processed food industry. No significant increase in the numbers of this pathogen occurred on bologna slices treated with 2. 2002). Most research efforts have been directed toward E. the growth of some O157:H7 strains in apple cider was not affected by the presence of either preservative (Miller and Kaspar. 2001).. Combinations of organic acids. 1999.5% or 5% acetic acid. Unpasteurized apple cider has been implicated in several foodborne disease outbreaks caused by pathogenic microorganisms.3% or 0.. coli O157:H7 in cider because of the severity of illness this pathogen causes.. low pH.3%) to cheese (pH 6.. 2001). coli O157:H7. especially in younger children and the elderly. and luncheon meats. Contamination of many of these foods frequently occurs after processing. Growth was either prevented or delayed by adding sodium benzoate (0. growth was inhibited by benzoate or sorbate (Koodie and Dhople. several factors may influence the effectiveness of benzoic acid in the treatment of fresh .5% lactic acid combined with 0. and ethanol were very effective bactericidal treatments for L. coli O157:H7 in apple cider (Comes and Beelman.

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

Excretion of benzoic TABLE 2. is then excreted in the urine (White et al. Benzoate does not appear to be accumulated in the body. 1964). 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. 1964).11).b rabbit. Its formation can be increased greatly by administration of benzoic acid.0 g/kg body weight Results 50% mortality 100% mortality Subchronic Chronic 80 mg/kg body weight 3% of diet 4% of diet (as sodium benzoate) 1 g/day 12 g per 14 days 0. The overall reaction sequence proceeds as shown in Figure 2. 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. Route of administration was per os. (1909) and Dakin (1909) to conclude that sodium benzoate was not deleterious to human health. There is little danger of its being a toxicant or fire hazard under normal conditions of use. intramuscular.7 (White et al.10 Toxicity of Benzoic Acid and Sodium Benzoate by Exposure Categorya Category Acute Species Tested Rat Guinea pig. Sax and Lewis (1989) reported data for several animal species based on the route of administration (Table 2.3–4. and intraperitoneal routes (Sax and Lewis.10.0 g/kg body weight 1. cat. However. . synthesized in the liver..34 Antimicrobials in Food STORAGE AND HANDLING Sodium benzoate should be stored in a cool. Hippuric acid. The apparent reason for this involves a detoxifying mechanism whereby benzoate is absorbed from the intestine and “activated” by linkage with CoA to yield benzoyl coenzyme A.. TOXICOLOGY Several toxicologic studies involving different animal species have been conducted using both benzoic acid and sodium benzoate. This product is not corrosive. Containers should be kept closed as much as possible.7–4. In addition.4–2. 1989).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. the second is catalyzed by an acyltransferase enzyme. Source: Adapted from Lueck (1980). Extensive human feeding trials conducted early in the twentieth century led Chittenden et al. both benzoic acid and sodium benzoate are moderately toxic by ingestive. These have been summarized by exposure category in Table 2. dry place.

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

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. (1964). These authors analyzed plasma concentrations of benzoic and hippuric acids and urinary levels of hippuric acid after oral doses of sodium benzoate (40. These results have been confirmed in several animal studies. 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. 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). Different animal species differ in their ability to metabolize benzoic acid.. 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. S. 1990). Source: White et al. This may be related to differences in the ability of liver and kidney cells to carry out glycine and glucuronic acid conjugation (for review. The food preservatives were shown to have a strong prooxidant effect on aerobically grown yeast cells. 1999). the route of administration significantly affected the efficiency of benzoic acid metabolism (Nathan et al. Since the late 1970s. 1993). Cell sensitivity to these acids was enhanced by aerobic rather than anaerobic growth conditions. acid by this particular pathway was first reported by Dakin (1909). A single oral dose of 250 mg of benzoic acid was quantitatively metabolized to hippuric acid and excreted in the urine within 4 hours. 1991). Inhibition was caused by accumulation of benzoyl CoA with a resultant depletion of free CoA and acetyl CoA. or 160 mg/kg) were given at least 1 week apart to 6 healthy male volunteers.. 80. Akira et al.7 Metabolism of benzoic acid by mammals. 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. (1994) used deuterated benzoic acid and nuclear magnetic resonance spectroscopy to monitor metabolism in a healthy male volunteer. 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. In guinea pigs. . When applied topically. However. cerevisiae was used as a model system for testing antioxidant or prooxidant properties of benzoic acid and other weak organic acid food preservatives (Piper.36 Antimicrobials in Food O OH + ATP + HS-CoA Benzoic Acid O S-CoA Benzoyl CoA + H2N COOH O S-CoA + AMP + PPi Benzoyl CoA O NHCH2COOH + HS-CoA Hippuric Acid Glycine FIGURE 2.9%) of the absorbed benzoic acid was conjugated with glycine to form hippuric acid. see Chipley. 1991). only a small portion (6. 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. it was excreted almost completely as hippuric acid after systemic administration.. 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. They were also mutagenic to the yeast mitochondrial genome but only in the presence of oxygen.

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

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

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

however. The solubility of sorbic acid (Table 3. Commercially available salts include calcium. sorbates are considered effective food preservatives when used under sanitary conditions and in products processed using good manufacturing practices. especially ethanol. Inhibition of bacterial spore germination (Sofos et al. Sofos et al. however. while the health effects of sorbate were reexamined (Sofos. 1989). especially potassium sorbate. 1989. changes in the genetic material. in inhibition of microorganisms by sorbates. and inhibition of enzymes or transport functions (Sofos. and in glacial acetic acid. In food systems the solubility of the compound is estimated as higher than 50%. The solubility of sorbic acid is also higher in alcohols. and in other industrial applications (Sofos. and it has a weak but characteristic acrid odor and acid taste. 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. and it constitutes the most soluble form of sorbate (Table 3. probably through action on spore membranes or protease enzymes involved in germination (Blocher and Busta. substrate properties. has an antimicrobial potency of 74%. 1989. 1981). 1979a. some microbial strains are resistant to inhibition by sorbate or even metabolize the compound. Currently. The salts (e. 1989. but it increases with temperature. species and strains. Sofos. Robach and Sofos. depending on differences in microbial types. 1981.. The mechanisms of antimicrobial activity of sorbates are not fully defined (Sofos and Busta. and environmental factors. During the 1970s extensive research was performed on the potential for using sorbic acid and its salts as antibotulinal agents in meat products. 1986). There is variation. alterations in cell membranes. CHEMISTRY Sorbic acid is a straight-chain. The carboxyl group of sorbic acid is highly reactive and results in formation of various salts and esters. potassium sorbate) of sorbic acid may find more frequent applications in foods because of their greater solubility in water. and bacteria. Robach and Sofos. as animal feeds. including yeasts. 1980).4-hexadienoic acid.13 (Table 3.2% w/v at 20°C). sorbic acid and its more water-soluble salts. Inhibition of microbial metabolic function is probably the result of morphologic alterations in the structure of the cells. Sorbic acid forms colorless flakes or needles when crystallized.2) in water at room temperature is only 0. Sofos. or both. as pharmaceuticals and cosmetics.. In general. 1982). 1985. are collectively known as sorbates and are used widely throughout the world as preservatives for various foods. molds. 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. Sofos et al. The extensive use of sorbates as preservatives is based on their ability to inhibit or delay growth of numerous microorganisms. with a molecular weight of 112.22.. Good solubility (58. Sofos. The volatility of the compound in steam is useful in its isolation for quantitative detection in foods or other materials. which makes it valuable as a delayed-release form of sorbic acid (Sofos. 1981. 1989). Calcium sorbate has a water solubility of 1. sodium.15 g per 100 ml.50 Antimicrobials in Food activity against microbial growth and the potential application of the compound in additional food products (Sofos and Busta.2). 1992). 1979d) by sorbate is believed to occur at the connecting reactions of the germination process.. and . 1989). 1982). CH3CH=CH-CH=CH-COOH). the pH of the solution. The calcium salt is formed as a white odorless and tasteless powder. 1989). 1981.1).2% and is insoluble in fats. The molecular weight of potassium sorbate is 150. it is available as a white freeflowing powder or as granules.g. stability. Potassium sorbate may be manufactured as a powder or granules and. Under certain conditions. especially in combination with reduced nitrite levels for a reduction in nitrosamine formation (Sofos et al.. and potassium sorbates. The sodium salt has a water solubility of 32% (wt/vol). on an equivalent weight basis with the acid. 1986. trans-trans unsaturated fatty acid (2.

Oxidation and losses of sorbic acid are inhibited by antioxidants. acrolein. and β-carboxylactolein (Arya and Thakur.5 10 3 0. 1989). malonaldehyde.13 126 1.2 ease of manufacture make potassium sorbate the most widely used form in food systems (Sofos. mm Hg. Sofos.1 N NaOH to 0. °C Heat of combustion at 25°C. The rate of oxidation in aqueous solutions is increased at lower pH values by the presence of light and acids or increased temperatures. Aqueous solutions of sorbates are unstable and degrade through oxidation. acetaldehyde. appropriate packaging materials. Sorbic Acid 2. maximum% Source: From Sofos (1989). such as crotonaldehyde. In general. malonic acid. °C (COC ASTM D-92) Ionization constant at 25°C Density at 20°C g/ml Melting range. increasing the production of carbonyls. 1989. and other ingredients present (Sofos.22 — — 1.1 Physical and Chemical Properties of Sorbic Acid and Potassium Sorbate Property Chemical name Molecular formula Molecular weight Flash point. Btu/Lb Alkalinity/acidity Vapor pressure. . 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. % Water content. 1992).Sorbic Acid and Sorbates 51 TABLE 3.0 to 7. It is proposed that EDTA-Fe2+ complexes catalyze sorbic acid autoxidation. at 20°C 120°C 140°C Boiling point. formic acid. maximum% Ash. the partitioning of sorbic acid between the water and fat phases of foods depends on the pH of the food. 1981).927 — Potassium Sorbate 2.4-Hexadienoic acid potassium salt CH3-CH=CH-CH=CH=COOK 150. 1995).8 ml 0.36 Decomposes higher than 270°C — ml 0. maximum% Heavy metal content.4-Hexadienoic acid CH3-CH=CH-CH=CH-COOH 112. the type and amount of fat. 1988.0 with increased levels (50%) of soluble food components. whereas in the pure dry form they are stable. 1992).1 g — — — — — — >98 1. which take part in nonenzymatic browning.. Oxidation of sorbic acid yields various carbonyl compounds. The partition quotient is increased from 3.. such as sugars and salts (Sofos and Busta.0 10 3 — <0. Ethylenediamine tetraacetic acid (EDTA) enhances sorbate degradation probably as a result of iron scavenging from the packaging material (glass or polypropylene) through EDTA complexation.001 10 43 Decomposes 143 119 >98 0. maximum ppm Arsenic content. 1996). 1989).73 ∞ 10-5 — 132–137 11. °C 760 mm Hg 50 mm Hg 10 mm Hg Purity. and anaerobic conditions (Sofos.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. Thus.

50°C Pentane.00 55.12 0. 20% Ethanol. 50°C Benzene.08 0. the nature and pH of the food.60 1. 20°C Acetone.00 34.20 — — 61. 5% Ethanol.00 20.25 0. 10% solution Sodium chloride.30 2.30 6.b.00 0.30 Potassium Sorbate 58.26 0. 20°C Cottonseed oil.01 — — 0. 100% Propylene glycol. 60% solution Sodium chloride.07 0. 10% solution Sucrose. moisture content. light. amino acids. 20°C Cyclohexane.14 24.00–15.10 16.50–12. 50°C Propylene glycol. 20°C Carbon tetrachloride.0% enhanced sorbic acid destruction.00 0. 100°C Corn oil. 1980.30 0.2 Solubility (%) of Sorbic Acid and Potassium Sorbate Solvent Water.00 28. 25°C Pentane.29 — 12.90–14. 20°C Methanol.80 0. processing conditions.15 0.00 48.00 0.20 12. 85% Ethanol.00 — — — — 57. glacial Lactic acid. 1990). 20°C (pH 3.00–5. In general.00 5. 1992. 5% solution Sodium chloride.00 64. 95% Ethanol. but at 13. 20°C Glycerol. 75°C Benzene. 50% Phosphoric acid.50 12.00 — <0.15 0.. 20°C (pH 5. Sorbic Acid 0. 1986a.60 45.34 8.00 0.01 Losses of sorbic acid during storage of foods depend on sorbate levels. metal ions. other additives present.1) Water.10 0. Vidyasagar and Arya.02 0.08 2.60–14.28 1.00 47.10 0.04 11.. 50% Ethanol.03 — 0. oxidative degradation of .22 1.15 0. 20°C Propylene glycol. 15% solution Acetic acid. Gerschenson et al. 1994).00 45. 100°C Pentane.0% it had a protective effect (Guerrero et al.20 0.90 0.11 0. 20°C Source: From Sofos (1989). 85. 75°C Ethyl ether. 25°C Benzene.55 4.9) Water.80 2.4) Water. 20°C Sucrose.40 54. 50°C Water.50 2.52 Antimicrobials in Food TABLE 3.00 12.01 58.. and storage temperature and time (Bolin et al.01 0.5% and 8.31 9. Sodium chloride at 3. Sofos. antioxidants.00 — — — <0.02 0.5% Citric acid.50 0. 1987. 50°C Soybean oil. 1984. types of food materials present.52 1. packaging material. 40% solution Sucrose. 20°C Corn oil.16 0. 1983.05 5. 1989. 20°C (pH 4.

and the acetic acid-soluble components (0. 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. they are shipped and stored in fiber drums with a moisture barrier in the carton wall. Vojdani and Torres. 1989). Because the compounds are sensitive to heat. with a dissociation constant (pKa value) of 8. Charvalos et al. 1963. Troller and Olsen. Sofos and Busta. and sorbic aldehydes were also effective antimicrobials (Troller and Olsen. strong offflavors and odors. Although the salts of sorbic acid have been commercially developed. 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. 1992). salt-. 1990). dry. 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. These cartons should be kept closed in cool. 1992). A proportion of the binding is with the low-molecular-weight components of these fractions (Jideani and Wedzicha. These include esters. broadly tested. alcohols.8.b. some of these compounds are sorboyl palmitate. Several procedures have been patented for the manufacture of sorbates (Sofos. structure. and potential health problems (Lück. the water-. and light.3%). and widely used as antimicrobial agents. granules. 1980. physical state. sorbohydroxamic acid. Fractionation of the lipids and proteins from fura showed the 14C to be associated with the petroleum spirit crude fat extract (28±5%). New controlled-release. The lipid and protein fractions that appear to bind sorbic acid or its degradation products were identified with the help of 14C-sorbic acid. salt-. calcium or sodium (salts). Specifically. and amide derivatives. 1989).5±0. fura. 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. such as composition. In general. 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. aldehydes. When wheat flour doughs are treated with sorbic acid and heated. 1976). was an effective mold inhibitor over a wide pH range (3. Dialysis of the ethanol-.6 to 9. and dark storage areas. and it is influenced by the nature of the food and its components and by processing. 1976). A mixed anhydride of sorbic and palmitic acids called sorboyl palmitate has found some application in yeast-leavened bakery products. sorbamide. such as low solubility in water. The results show that sorbic acid binds with the lipid components. to a lesser extent. other derivatives of sorbic acid have also been examined.7%).. methyl sorbate. 1963). 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.2 ± 0. In addition. Uptake and diffusion of sorbate into foods should be influenced by properties of the substrate. sorbohydroxamic acid. and storage conditions (Sofos. moisture.. amine salts. 1986a.5±0. moisture. or solutions. 1994).7%). 1989. and the acidlabile nature of this binding is the result of the instability of the thiol-sorbic acid monoadducts under acid conditions (Khandelwal et al. 2001). however. 1981). ethyl sorbate. and water activity (Giannakopoulos and Guilbert. but commercial production of sorbic acid has mostly involved the methods of oxidation of .Sorbic Acid and Sorbates 53 sorbic acid in foods is less extensive than in aqueous solutions. and a further loss is apparent after the dough is cooked to give the traditional West African food.. Commercial application of such derivatives. and ethanol-soluble components of fura from pearl millet. 2000.. suspensions. is hindered by disadvantages. Sorbamide was 1000 times more inhibitory than sorbic acid against yeast alcohol dehydrogenase (Martoadiprawito and Whitaker. 1995). This suggests that sorbic acid is indeed reacting with thiol groups in the flour protein.2) (Dudman. the water-soluble (6. handling. a significant amount of sorbic acid is not recovered by extraction with methanol. the salt-soluble (3.9±0. the ethanol-soluble (4. Sofos.6%). Commercial applications of sorbates include use of the free acid or its potassium and. and sorbic anhydride.

Deuel et al. Heterosporium. and other suitable salts in the presence of necessary additives.. Cladosporium.b. Sporobolomyces. jams. Chaetomium. Endomycopsis. Pepularia. and purification procedures follow the second method. Candida. 1961. Rhodotorula. Kivanç. 1952. Ulocladium. Huang and Armstrong. Mold species inhibited by sorbates belong to the genera Alternaria. 2000). 1985a). 1988).b. Curvularia. 1998. hydroxides. Skirdal and Eklund. Cunninghamella. and others. Kaul et al. Gliocladium. fruits and juices. the inhibitory amounts of sorbic acid were reduced by 40% to 50%. Sporotrichum. 1996).. applied at 10 mg/ml. Marshall and Bullerman. Rosellinia. salad dressings. 1993. Gourama and Bullerman. Sorbates inhibit the formation of mycotoxins by various molds in culture media and in foods (Bullerman. 1954a. 1998. Yeasts inhibited by sorbates include species of the genera Brettanomyces. Penicillium. sorbates inhibit yeasts in fruit juices. such as carbonated beverages. and sorbic acid. whereas no growth was observed when the preservative concentration was increased to 220 ppm (El Halouat and Debevere. Saccharomyces. Truncatella.02% to 0. Effective antimicrobial concentrations of sorbates in most foods are in the range of 0. 1996.90). Tong and Draughon. and meat and fish products. Zygosaccharomyces rouxii is considered resistant to sorbate treatments. reaction conditions. Fan and Chen. Melnick et al. Trichoderma. El Halouat et al. Kloeckera. Colletotrichum. Geotrichum. Humicola. 1970.. Smith and Rollin. cakes. as well as against many bacteria (Sofos. Piper et al. and chocolate syrup (Restaino et al. ANTIMICROBIAL ACTIVITY INHIBITION Sorbic acid is active against yeasts and molds. after 21 days. Pederson et al. 1996).. Ascosphaera. Bracey et al. 1959. Use of sorbates for inhibition of yeasts is especially important in low-pH and/or intermediate water activity (aw) products.b. 1954a. bread. Torulopsis. Cephalosporium.. CO2 atmospheres. Torulaspora. 1979. 1985.b. Pichia. 1998). dried fruits. 1957. Helminthosporium. use of a hurdle approach resulted in a synergistic effect (El Halouat and Debevere. Garza et al. 1989). Hansenula. In addition to their effectiveness in fermented vegetables. 1972). Botrytis. sausages.. 1993. wines. syrups. Under an 80% CO2 atmosphere. 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. neither sorbate nor propionate. Cryptococccus. jellies. 1985. lower than the inoculum level (103 CFU/g). which is most widely used. potassium sorbate used at 150 ppm resulted in final counts. 1970. and smoked fish (Liewen and Marth. Sofos. Although.. 1952. The effectiveness of sorbates against yeasts has been documented by numerous studies (Emard and Vaughn. Pestalotiopsis. Numerous patents describing catalysts. Melnick and Luckmann. 1979. Geminder. Rhizopus. Debaryomyces. However. were able to depress mycelial weight and aflatoxin production by Aspergillus flavus or Aspergillus parasiticus cultured in yeast extract-sucrose (YES) broth (Fan . Tsai et al. 1982. Lennox and McElory. Aly. Ascochyta. Alkaline salts of sorbic acid are formed by its neutralization with alkali metal carbonates. Ferguson and Powrie. 1984. A major application of sorbates in food products is their use for inhibition of molds in cheeses (Chichester and Tanner. tomato products. Rhizoctonia. Sorbates also inhibit molds in butter. 1999). Mucor. cottage cheese. Liewen and Marth. Baldock et al. 1983.4-hexadienal and the condensation reaction of ketene and crotonaldehyde. and Zygosaccharomyces (Sofos.30%. Phoma.. Fusarium.. 1989). 1984. 1984. candy. Pullularia.. 1984. Matamoras-León. 1984.. 1992. Hurdles used in combination included aw. 1985a. Liewen and Marth. 1986. 1954a. Bhattacharya and Majumdar. Monilia.80 to 0. 1999. The inhibitory action of sorbate against yeasts was first documented in the 1950s in fermented vegetable products.54 Antimicrobials in Food 2. 1954a. Roland and Beuchat. Huang and Armstrong. Under this atmosphere and at aw values in the studied range (0. grains. 1984. Aspergillus. Numerous studies have also documented the effectiveness of sorbates against molds (Emard and Vaughn. 1983.

aw. Blocher and Busta. Alcaligenes. Klebsiella. 1996).1% potassium sorbate and 0.b. gas atmosphere. Bacillus.... 1979a–d. and/or vegetative cell division (Sofos et al. monocytogenes presence in two commercial cheese brines and thus could be used as an antilisterial agent in commercial brines. Lactobacillus... monocytogenes and Zygosaccharomyces bailii to inactivation by high hydrostatic pressure (Mackey et al. 1987a. Escherichia. 1987. coli O157:H7 during storage of apple cider (Miller and Kaspar. Chung and Lee. Proteus. Overall. Sofos. outgrowth. Campylobacter. In fact. under certain conditions. 1985. 1985a. however. 1974.. Potassium sorbate sensitized cells of L. potassium sorbate slightly decreased L. Liewen and Marth. however. 2000). 2001). temperature of storage. Zamora and Zaritzky. Pediococcus. SELECTIVE ACTION Microbial inhibition by sorbate is variable and depends on species. 1986. Serratia. monocytogenes in broth. 1989). it suppressed cysteine activation of listeriolysin. 1988). 1981. and other factors (Sofos.05% sorbic acid was found to inhibit growth of E.b. Kouassi and Shelef. Pseudomonas. which inhibited growth. 1993. 1995a. At the relatively high level of 1%. catalase-positive and catalase-negative. and mesophilic and psychrotrophic microorganisms.. 1989).2% to 0. Inhibition of bacteria by sorbate appears to cause an extension of the lag phase. Arthrobacter. The effect of sorbate on spore-forming bacteria may be exerted on spore germination. Salmonella. 1995a. it has also been shown that potassium sorbate or sodium benzoate did not affect survival of E. Certain bacterial strains are not inhibited by sorbate.. Alteromonas. Vibrio. 1984.. as well as spoilage and pathogenic bacteria. and their long-term stability in the high-salt and low pH environment of brines is not well documented (Larson et al. Seward et al. sorbates can inhibit Gram-positive and Gramnegative.b). Zhao et al. 2001). Moraxella. which could have a protective effect on E. Mycobacterium. 1994). 1999). the addition of sorbic acid (0.04% to 0. Aeromonas. Greer.. Tsay and Chou. Addition of 0. Staphylococcus. although sorbate did not affect growth of L. and some may even metabolize the compound (Sofos. 1988). A hydroxypropyl methylcellulose (HPMC) coating significantly reduced the number of viable Salmonella Montevideo cells on the surface of tomatoes. 1980a. 1981. increasing its survival. In other studies (Kouassi and Shelef.. Yersinia. Enterobacter. Koodie and Dhople. Palou et al. Monnet et al. It was concluded that combinations of sorbate with propionate or lactate. however. food-processing treatments.5% (Kumar et al. and others (El-Shenawy and Marth. sorbic acid is considered as a more effective inhibitor of yeasts and molds than bacteria (Skirdal and Eklund. Rusul and Marth. 1982. Bacterial species inhibited by sorbate belong to the genera Acetobacter. pH. 1997). could extend shelf life and increase safety (Kouassi and Shelef. with a lesser influence on rate and extent of growth (Larocco and Martin. however. 1988) and in a coldpack cheese food (Ryser and Marth. Depending on pH and concentration. 1995. but the cost effectiveness of adding high levels (1%) of substrate is questionable. 1988.. Sofos. 1982. type .Sorbic Acid and Sorbates 55 and Chen. aerobic and anaerobic. Acinetobacter.b. Smoot and Pierson. coli O157:H7. Achromobacter. Stimulation of mycotoxin formation by low levels of sorbate depends on species and strains of molds. 1999). 1989). 1993). 1988. Clostridium. and result in spoilage of the product (Zhao et al. 1982. 1981. subinhibitory levels of sorbate may simulate the production of mycotoxins (Bullerman and Olivigni. Micrococcus. Overall. Sofos. additives present. coli O157:H7 in apple cider (Koodie and Dhople. Gareis et al. Sanchis et al. 2000. 1989).1% sodium benzoate substantially decreased Escherichia coli O157:H7 contamination and suppressed the growth of yeasts and molds. strains. However.. 1987). 1993. 2000). composition of substrate. 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. Lund et al.4%) to HPMC did not substantially enhance bactericidal activity (Zhuang et al. 1995a. The combination of 0. storage temperature. sorbate inhibited or inactivated Listeria monocytogenes in a broth substrate (El-Shenawy and Marth.b.

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

substantial metabolism of sorbic acid. coli O157:H7 during storage . 1985a–c). When sorbate levels are high. aw. 1989). Mold strains of the genus Penicillium isolated from cheese treated with sorbate were able to grow and metabolize high (0. 1985a). plastic paint. 2000). It appears that selection may occur in sorbate-treated cheeses for certain molds tolerant to the compound (Schroeder and Bullerman. Products of sorbate metabolism by molds include 1. microbial flora. gas atmosphere.. Degradation of sorbate by lactic acid bacteria has been associated with geranium-type off-odors in wines and fermented vegetables. which is a volatile compound formed through a decarboxylation reaction and has a kerosene-like.. sorbate is completely oxidized to carbon dioxide and water. Some mold strains can grow and metabolize sorbate under certain conditions as detected in cheeses and fruit products (Melnick et al. 1989. Bullerman. 2000). 1989). Sorbic acid (0. dehydration of cultures. and Geotrichum (Sofos. 1985a. Steels et al. about a 7-fold increase in lethality (Splittstoesser et al.20%) sorbate levels (Marth et al. Liewen and Marth. In general. however. Sofos. 1995).. inoculum size. bailii resistance to sorbic acid activity. amount of sorbate present. Lück. some bacterial strains may also degrade sorbate under appropriate conditions.3-pentadiene. Degradation of sorbate by molds depends on species and strains. 1982). Finol et al.6 kcal/g.b. using it as a carbon source. 1989). 1980). Sofos.1%) reduced the D50°C of E.5-diene (Edinger and Splittstoesser.1% sorbate is usually sufficient to inhibit sensitive molds (Liewen and Marth. coli O157:H7 from 36 to 5. INTERACTIONS The antimicrobial activity of sorbates is influenced by compositional. Liewen and Marth. 1954b. Horwood et al.Sorbic Acid and Sorbates 57 DEGRADATION Animals and certain microorganisms can metabolize sorbate. caused by ethyl sorbate. Sofos. which was not an artifact caused by insufficient growth time. 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. The study showed that the resistance of Z. 1966. although many molds are sensitive to inhibition by sorbate.. there is also evidence of some ω-oxidation (Deuel et al. 1981. large inocula at high cell concentrations therefore require considerably higher concentrations of the inhibitor to prevent growth than do dilute cell suspensions. 4hexenoic acid. under certain conditions. under normal conditions of alimentation.. or binding of sorbic acid to dead cells. 1988). processing. a geranium-like odor is usually associated with wines treated with sorbate and contaminated with high microbial loads.18% to 1. 1985). In general. 1954b. Increasing the concentration of sorbic acid from 200 to 1000 mg/L in apple cider decreased the D50°C value of E. sorbate yields 6.2 min. Like caproic and butyric acids.. as a fatty acid through β-oxidation. certain strains are resistant and can metabolize the compound. and environmental factors. prior exposure to subinhibitory levels of sorbate. packaging. temperature.4-diene. pH. level of inoculum. Fusarium. 1986a. that there is no apparent relationship between sorbate resistance and the toxigenic properties of molds (Tsai et al. and type of substrate (Sofos. 1977. A study found a pronounced positive inoculum effect of Z... It appears. and other additives (Sofos and Busta... Other strains of molds that may degrade sorbate belong to the genera Aspergillus. 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. This metabolism is mostly associated with lactic acid-producing bacterial strains present as high inocula in sublethal concentrations of sorbate (Crowell and Guymon. 1989). or hydrocarbon-like odor (Marth et al. 1989. other ingredients. It should be noted that 0. and 2-ethoxyhexa-3. Because it is metabolized like other fatty acids. 1975. In addition to certain molds. 1981.. such as concentration. 1-ethoxyhexa-2. The MIC of sorbic acid increases with the size of the inoculum. These factors can act synergistically or be antagonistic and either enhance or negate the antimicrobial activity of sorbate (Sofos. of which 50% is biologically usable. 1992). 1966. 2000). Mucor.b).

1985a.0 (Raevuori. 1976.64 min (Splittstoesser et al. where it is needed for microbial control (Oka. as well as dormancy and recovery of heated microorganisms (Sofos. 1956.5 and 4.25 and 2. but increased levels of microbial contamination reduce antimicrobial activity. Sofos and Busta. the maximum pH for antimicrobial activity by most other common food preservatives is lower — for example. however.. 1990).. 1979a. 1960). 1996). Interactions of sorbate with heat may affect the rate and extent of microbial destruction during heating. might not be effective owing to their increased solubility in fat. 1981c). Cerruti et al. Sofos and Busta. Sucrose.5 is advantageous because it allows for their use in foods of higher pH values in which preservatives. Lowering the aw enhanced the resistance of the same organism to increasing concentrations of sorbate (Restaino et al. Sugar and salt. 1981. 1982). not as a substitute for appropriate sanitation and hygienic practices.76) (Cowles. Not only are certain strains of microorganisms resistant to inhibition by sorbate. 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. 5.1% sorbate (Bills et al. Sorbic acid (0. Hoffman et al. 1981). however. 1959. 1988). Lück. 1955. Nevertheless. Although activity is greater at low pH values. sorbates have the advantage of being effective at pH values as high as 6. Skirdal and Eklund.. 1982. 1983). Studies have indicated that the dissociated sorbic acid also had antimicrobial activity. however. 1981. The antimicrobial action of sorbate is pH dependent and increases as the pH of the substrate decreases.. 1980.. 1989). In environments of pH higher than 6.. 1993). 1955.4 (Liu et al. 1993). Increased amounts of fat in a product reduce the concentration of sorbate in the water phase. Gooding et al. Cerruti et al. 1941. which exhibited favorable antimicrobial properties against Vibrio vulnificus. whereas benzoic acid reduced it to 0. respectively (Sofos and Busta. 1954a. solutes should increase the inhibitory activity of sorbate by reducing the aw of the substrate (Sofos. Sodium chloride (1.1%) enhanced destruction of E. 1944. 1944.. Cerruti et al. Acott et al. 1985a). 1981.g.2 minutes. Sofos and Busta. 1955. 1993). 1957. Robach and Stateler. which is believed to be the effective antimicrobial form (Lück.b).1% sorbate rendered the cells more sensitive to inhibition by sorbate than preconditioning in 0% sucrose + 0. 1981)..5 (Bell et al. 1982. 1990). such as parabens. Lück. 1980. 1994). but it was 10 to 600 times less inhibitory than the dissociated acid (Eklund. 1989). more than 50% of the inhibition was the result of dissociated sorbic acid (Eklund. 1980. salt and sugars) also reduce the concentration of sorbate in the aqueous phase (Gooding et al. In contrast.0 to 4. Liewen and Marth.. approaching its dissociation constant (pKa = 4. 1983). 1987. Other food ingredients (e. however. 1980. Lund et al. certain studies have indicated antimicrobial activity by sorbate at pH values as high as 7...4 but not at pH 6. Sofos et al. and it may inhibit the repair and growth of . Sofos and Busta..58 Antimicrobials in Food of apple juice from 18 to 5. Use of artificial saltwater prevented dissociation of the 1% sorbic acid. Statham and McMeekin. Thus... 1988). Preconditioning of Saccharomyces rouxii cells in 60% sucrose + 0. In general.0 to 5. The increased antimicrobial activity of sorbate at lower pH values has been attributed to the increased amount of undissociated acid present. 1976. This popular theory has been questioned.. Beuchat. act synergistically to enhance the antimicrobial activity of sorbate (Costilow et al. 1989. 1997).5%) reduced the inhibition of Clostridium botulinum by sorbate in a nutrient broth (Wagner and Busta. Statham and McMeekin. have reduced the synergistic effect of sorbate and heat on thermal inactivation of microorganisms (Beuchat. 1955. 1984. Sorbate may enhance heat activation and destruction of spores. 1983. In certain instances. 1981a–c.5 for propionate and benzoate. glucose.. and sodium chloride.. however (Sofos. Rahn and Conn. sorbates should be used to preserve foods processed using good manufacturing practices. Sofos. 1981. coli O157:H7 cells at pH 3. supporting previous evidence that sorbic acid is effective in its undissociated form (Sun and Oliver. Sheneman and Costilow. 1988). 1976. Chung and Lee. 1976. it is believed that both undissociated and dissociated sorbic acids have antimicrobial activity (Skirdal and Eklund. The increased activity of sorbates at pH values higher than 5. 1989.0.

1996). Oloyede et al.. cerevisiae depending on pH (Cheng and Piper. 1999). 1985). 1979. 1980.. and types of acids (Restaino et al. 1996.Sorbic Acid and Sorbates 59 thermally injured organisms (Beuchat.. Park et al. Sorbic and benzoic acids affected the thermotolerance and heat shock response of S. a true aerophilic mold involved in the spoilage of grape preserves (Splittstoesser et al. Another report also indicated that potassium sorbate inhibited the heat-resistant N. microorganisms. 1984.. including meat (Wagner et al. Cells grown in sucrose-supplemented media tolerated higher concentrations of sorbate than did those grown in glucose-supplemented media. 1973. Sorbic acid ( opposed to glucose-containing media provides evidence that cell morphology influences heat resistance (Golden and Beuchat. 1961. McMeekin et al. 1994. 1983). as indicated with several food items. 1982. they can enhance its antimicrobial activity by increasing the concentration of undissociated sorbic acid. 1989). Although food acids may reduce the water solubility of sorbate. At low pH. Davidson .. 1982. In addition.. Sofos. 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. 1984. Lusher et al. It is of particular interest that 0. Huhtanen et al.. Concentrations of sorbate as high as 0.. such as butylated hydroxyanisole (BHA). 1999). Reductions of 5 to 10 log-units of E.. 1984). 1989). 1981a–d. The antimicrobial activity of sorbate is usually enhanced under vacuum or modified gas atmosphere storage conditions.. cerevisiae (Cheng et al. 1981. Heat resistance increased substantially when cells were grown in media containing sorbate.5 it acted as a powerful inducer of thermotolerance in the absence of sublethal heat treatment. 1972.. tertiary butyl hydroquinone (TBHQ).. 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. Elliot et al. 1985)... Sofos. Roland et al. Combinations of sorbate with antioxidants. Huhtanen and Feinberg. Roland and Beuchat. rouxii to sorbate was enhanced in cells preconditioned to elevated sorbate concentrations. but growth of surviving spores that had been exposed to high temperatures was greatly inhibited by sorbate concentrations as low as 0. Lopez et al. had increased antimicrobial activity compared with individual components (Klindworth et al. The finding that plasmolysis increased in cells grown in sucrose. Elliot and Gray. 1989). especially in media containing 0. 1982. 1989).1% potassium sorbate did not have any significant effects on the heat resistance of Bacillus stearothermophilus spores in distilled water. 1985. 1992). 1996). Gray et al. 1983. Banks et al.1% sorbate.2% to 0. and propyl gallate (PG)... 1982. 1980. Specific effects.4%). did not enhance the bactericidal activity of cellulose-based edible films against Salmonella Montevideo (Zhuang et al. but at pH 5. Inhibition of microbial growth by sorbate is more effective as storage temperature decreases. Tuncan and Martin. 1981. Robach. McMeekin et al. 1995). but the effect of sorbate on thermal inactivation and recovery of injured microorganisms is variable among species and strains. Roberts et al. 1989). 1984. 1980. 1994. 1982.. Splittstoesser et al. 1981.1% had little effect on the thermal resistance of ascospores of Neosartorya fischeri. sorbate inhibited induction of thermotolerance by sublethal heat shock. the specific anion itself may contribute antimicrobial activity (Juven.. 1976. 2000).. Park and Marth. 1989). Myers et al.. 1984) and fish products (Bremmer and Statham. 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.007% (Splittstoesser and Churey. indicating that the compound should be more useful as a preservative in refrigerated foods (Pederson et al. Combinations of carbon dioxide and sorbate have also been reported as effective inhibitors of microbial growth (Danzinger et al. Sorbate may also eliminate the protective effect of sucrose against the thermal inactivation of yeasts and molds (Sofos.. Statham et al. butylated hydroxytoluene (BHT). fischeri (Nielsen et al. and particularly in the presence of sucrose as opposed to glucose. 1984.. 1983. Low concentrations of sorbic and fumaric acids in the heating medium had little effect on the heat resistance of Eurotium herbariorum. Tolerance of Z.. however. Sorbic acid was found to induce thermotolerance without inducing the heat shock response through accumulation of trehalose in S.. 1970. however.. vary with substrates. 1988.

sorbate was more effective than benzoate against S. Poerschke and Cunningham. 1983. ascorbate.. Combinations of sorbate with benzoate or propionate may be used to expand the range of microorganisms inhibited with reduced concentrations of each preservative. 1994). sodium chloride concentration. 1993). Increase in the acidity and/or the NaCl concentration improved the effect of all the preservatives.5. 1960.98. as well as multifactorial interactions (Sofos. A study found that growth was not inhibited at pH 3. 1994). equivalent to a total sorbic acid concentration of 1300 mg/L. and potassium sorbate was evaluated against growth of three foodborne bacterial pathogens (Bacillus cereus.. 1988).. Bacillus coagulans. 1987. TellezGiron et al. Sorbate was completely effective against E. 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. with types of microorganisms.. pH 3. 1968. The effect of temperature. . These combinations offer the advantage of simultaneous inhibition of microbial growth and development of rancidity. 1990).. mixtures of sorbate with various antibiotics have demonstrated increased antimicrobial activity (Schmidt. 1981. Gourama and Bullerman. and Clostridium butyricum by adjustment of aw to 0. 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. verocytotoxigenic Escherichia coli. Marshall and Bullerman.. fatty acids.95 (Deak and Beuchat.97 and pH to 3. Robach. After 14 days at 30°C in the presence of 280 mg undissociated sorbic acid/L. However. 1985. Deak and Beuchat. 1978. 1982. Kabara. sulfur dioxide. 1957. Guerrrero et al. Ough and Ingraham.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.. 1981. 1979b. 1986. and S. Sofos. The ability of C. and aw 0. cereus... propylene glycol.8. 1985). except nitrite when used against S. however. 1986a. 1990). aureus) using gradient gel plates (Thomas et al. Interactions and increased antimicrobial effects have also been observed in combinations of sorbates with propionate. 1993).4 and addition of 250 ppm ascorbic acid.. glucose oxidase.06%) combined with aw values lower than 0. including sorbate. At temperatures of 12°C and below. Thomas and Wagner. Mendonca et al. 400 ppm sodium bisulfite. 1987. Z.. Numerous publications and patents describe interactions of sorbate with many other factors. and other compounds (Ferguson and Powrie. Bell and De Lacy.60 Antimicrobials in Food et al. Miller and Brown. 1989. aureus. certain amino acids. Wagner and Busta. 1995). sorbic acid resulted in marked inhibition. 1984.. or at 10°C (Deak and Beuchat. Several studies have also indicated increased antimicrobial effects when sorbate was combined with various phosphates (Ivey and Robach.. Robach et al. these concentrations of chemicals represented a greater than 50% reduction of the amounts needed to suppress growth when used individually (Matamoras-León et al. the log probability of growth of a single vegetative cell is approximately –4. 1982. The microbial stability of shelf-stable banana puree was enhanced through inhibition of inoculated osmophilic and nonosmophilic yeasts. Nelson et al. and mild heat (Guerrero et al. Variations existed... 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. 1989). In addition. Kadam et al.. 1999).. Lahellec et al. pH. Amano et al. 1993. 1994). Seward et al. 1960. 1994. At <25°C. molds. As indicated. 100 ppm potassium sorbate. 1999). 1994). bailii is highly resistant to individual inhibitory factors. Clostridium pasteurianum. 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. the log probability of growth of a single bacterium at 12°C after 60 d at pH 5. aureus when used with higher concentrations of NaCl (Thomas et al. the probability of growth was approximately –6 (Lund et al. antioxidants.. 1984. and substrates.06% potassium sorbate. in a medium containing 0. 1983. Potassium sorbate was found more inhibitory against yeasts than hydroxycinnamic acid (Stead. at aw of 0. coli at all temperature/pH/NaCl combinations and was the most effective preservative tested against B. 1981. Morad et al. 1988).. Thomas et al.93. sucrose fatty acid esters.

. fruit drinks. mixes.30 0. semimoist pet foods. pharmaceuticals. pies.3%. 1981.F. cosmetic products.02% to 0. low-calorie drinks Food emulsions: mayonnaise. 1989. 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.1% may be detectable in some foods. carbonated and noncarbonated beverages. cheese dips. 1989). jams. Selection of the most appropriate method depends on processing procedures. juices. 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. These factors include product pH and composition (fat and moisture). Part 133 (2001)]. or addition in a coating or packaging material (Table 3. jellies. but levels as low as 0.05–0.3% are tolerated. edible fat emulsion products. cottage cheese. sanitation and contamination.02–0. In general. 1992). as is the case with all food preservatives. animal feeds. including dairy products. fresh salads Fruit products: dried fruit.R. concentrates Beverages: still wines. confectionary Source: From Sofos (1989). olives.02–0. As yeast and mold inhibitors. presence of other ingredients and preservatives. syrups. doughnuts Vegetable products: fermented.3 Common Applications of Sorbates as Antimicrobial Agents Products Dairy products: aged cheeses.10 0. certain meat and fish products. margarine.Sorbic Acid and Sorbates 61 TABLE 3. federal standards of identity permit the use of sorbates as preservatives in more than 40 types of cheese. pickles.3). cake mixes. processed cheeses. In the United States. the extent of the antimicrobial activity of sorbate under commercial conditions is difficult to predict because it is influenced by a variety of factors. and convenience (Sofos. salad dressings Meat and fish products: smoked and salted fish. equipment available.4). 1989). sour cream. packaging. Levels (%) 0.03–0. amounts of 0. packaging materials.1% to 0. product processing.20 0. However. and technical preparations that come in contact with foods or the human body. purees.05–0. fillings. The compounds find application in human foods of all types. relishes. fudges. higher levels may cause undesirable changes in the taste of most foods. and storage temperature (Sofos and Busta. dusting with a powder.05–0. type of food. dry sausages Miscellaneous: dry sausage casings. spraying or immersing the food material in a solution. yogurt Bakery products: cakes. Application methods for sorbates include direct addition in the formulation. toppings. and sugar and confectionery items (Sofos.30 0. APPLICATIONS Sorbates are widely used food preservatives throughout the world. and cheese food [21 C. The most commonly used forms include sorbic acid and the potassium salt. gas atmosphere. sorbates have found wide application in various foods. They should be considered as adjuncts to proper sanitation and hygiene. fruit and vegetable products. Sofos. objectives to be accomplished. 1989) (Table 3. Although these concentrations have no major impact on food quality. cheese spreads. cheese spreads.03 0. In the United States.05–0.30 In general.25 0. DAIRY PRODUCTS Cheese preservation is one of the most common applications of sorbates. Amounts of sorbate used in foods are in the range of 0. bakery items. icings. fruit salads.02–0.10 0.

Colby.04 0.05–0.05–0. Monterey Jack. however (e.075–0. that sorbates should not be used in mold-ripened cheeses.05–0.5–10 20–40 20–40 2. however.05–0.3 Spraying Immersion or spraying Dry mixing Direct addition Dry mixing or direct addition Source: From Sofos (1989).075 0.05–0. Gouda Dry sausage or casings Dairy: Swiss. pasta filata. in general.3 0.1–0.1 0. is limited to the surface of cheese.5–5 5 Application Level (%) 0.3 0. vegetable. In certain products.3 0. fudges Vegetables: fresh salads (potato. preservation objectives. and domestic and imported cheeses.05–0.25 0. mozzarella Dried fruits: Prunes Raisins Figs Smoked and salted fish Bakery: Cheesecake Angel food cake Fruitcake Cake mixes Doughnut mixes Pie crust dough Dairy: processed cheese. and regulations. relishes. Sofos and Busta. It is obvious. blue. 1996). Addition of sorbate does not significantly affect the organoleptic properties of mozzarella cheese. Kivanç (1992) found that Penicillium was the major genus found on cheddar cheese.06 0. 1976.15 0.1 0.02–0. depending on the technique used to treat the cheese (i. pickles. 1981). Muenster. spreads Cottage cheese Bakery: icings. the high activity of sorbates against molds. such as Roquefort or blue cheese and Camembert cheese. olives. however.e.15 0.62 Antimicrobials in Food TABLE 3.5–5 5 2. For application on . Gruyere. and distribution. Edam. but they also protect human health by preventing the formation of toxic mold metabolites (mycotoxins).05–0. available facilities and equipment. toppings.02–0. sauerkraut) Fruits: syrups.1–0. In these applications sorbates not only prevent cheese spoilage by mold.3 0..4 0. which may vary among countries (Lück. Sorbate application.. 1976.g. sorbic acid may be incorporated into the cheese curd.1 0.05–0.05–0. Caciocarallo.2 0.1 0. and the improved action of sorbates at high pH values compared to other common preservatives (Lück.1 0. especially surface mold growth during storage.05–0. in which they can interfere with the desirable molds involved in the manufacture of these cheeses. cottage cheese). 1980). where mold development usually occurs.3 0.1–0. 1980. dipping or in brine salting).05–0.1 0. slaw.3 0. Siciliano.03–0.025–0.4 Methods of Application of Sorbates Methods Immersion Products Dairy: provolone. The method of application varies with the type of cheese. there may develop a slight objectionable flavor (Aly. fillings.1–0.15 0.1–0. Swiss cheese.1 0.06 0. Sorbates are widely used in preserving cheese owing to the susceptibility of all types of cheese to microbial deterioration. drinks Still wines Other: Margarine Semimoist pet food Bakery: pound cake Devil’s food cake Chocolate cake Solution (%) 20–40 2. beverages.075 0. aging. Emmentaler Dairy: cheddar.1–0.1–0.3 0.01–0.

(2) a sorbic acid powder for dusting. 1992). such as cottage cheese and yogurt (Mihyar et al.05% to 0. 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. thermophilus and Lactobacillus delbrueckii var. bulgaricus. use of sorbates in aged cheeses also reduces labor and other costs through reduction of the need for cheese washing and trimming.9. 1981). respectively) to maintain stability when kept at 5°C for 7 or 14 days. Sofos and Busta.. respectively) extended the shelf life by 14 days. 1989. respectively (Aly. especially at lower (≤6.30% (Lück. In another study.1 to 0. Therefore. 1980. The compound is insoluble in fat and only slightly soluble in water. spray.0 with hydrochloric. monocytogenes (Piccinin and Shelef.g. 1996).3%) or sodium benzoate (0. Amounts of sorbic acid permitted and applied in cheese preservation range from 0.3 g/dm2. Milk acidification to pH 6. Labaneh with higher initial yeast counts needed higher concentrations of preservative (>300 and >400 mg/kg for K-sorbate and Na-benzoate. potassium sorbate was added to the cheese (pH 6. or calcium sorbate impregnated into the packaging material or other coatings (e. monocytogenes survival in two commercial cheese brines (Larson et al. Concentrations applied to cheese surfaces range from 0. Treatment of mozzarella cheese with potassium sorbate in the kneading water or brine may be more effective than dipping (Aly. Weng and Chen. 15°C. 1997). 1995). and it was initiated through extensive research performed during the early stages of sorbate testing. wax) used in cheese storage and marketing (Han et al. or wash. Growth of Salmonella in soft cheese was inhibited if the cheese was prepared from milk that was acidified with propionic acid to pH 5. coli O157:H7 in queso fresco. In labaneh (a yogurt product) produced under strict hygienic processing measures and effectively chilled. and (3) sorbic acid. 1989).0) at levels of ≥0... most of the preservative remains on the surface of the cheese without migrating inside the cheese or dissolving in the water on the shelves during the long maturation period of these cheeses (Lück. and was especially effective against mold and yeast contamination by Penicillium roqueforti and Mucor miehi. Inclusion of sorbic acid in cottage cheese was effective as a preservative but not against L.07% sorbic acid are generally used in direct addition to cheese.05% to 0. or propionic acid significantly increased the effectiveness of potassium sorbate. The appearance of Penicillium verrucosum var. amounts of 2 to 4 g/m2 are used (Lück. potassium sorbate inhibited L. 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. and the cheese was stored at ≤30°C (Kasrazadeh and Genigeorgis. Calcium sorbate may be preferred in cheese preservation over other forms of sorbate owing to its solubility properties. 1994). which can be used to dip. In addition to reducing cheese spoilage and the possibility of aflatoxin formation. potassium sorbate (0. Addition of potassium sorbate (≤6%) to mozzarella cheese inhibited growth of Streptococcus salivarius var. acetic.3%) showed a significant effect in delaying growth of E. one of the following procedures may be followed: (1) potassium sorbate in solution or in a brine.3%. Furthermore. addition of potassium sorbate may increase the meltability and improve the fat leakage of certain cheeses (Aly. soft cheese (Kasrazadeh and Genigeorgis. when applied to the surface of hard. 1994). These concentrations are in agreement with use of these preservatives in other dairy products. This research demonstrated the effectiveness of sorbates against undesirable organisms without interfering with the desirable . and 25°C after 30 days (Kivanç. Sofos. The use of sorbates in the processing of vegetables was one of the first applications. 1996)..6°C) temperatures (Kasrazadeh and Genigeorgis. ripened cheeses. 1997). at the relatively high level of 1%. VEGETABLE PRODUCTS The water-soluble salts of sorbic acid are used widely in the preservation of a variety of plant products. initial numbers of yeasts were low and relatively low concentrations of potassium sorbate and sodium benzoate (<150 and <250 mg/kg. 1995). including fresh. and pickled vegetables (Sofos.Sorbic Acid and Sorbates 63 the surface of the cheese. 1998. 1980). potassium. 1999). however. 1976). whereas when applied to packaging films. fermented. Levels of 0. 1996).

. however.g. smaller quantities of sorbic acid are adequate for preservation because of the synergistic action of sorbate with sugar (Lück. Other fruit products that may be preserved with sorbates include maraschino cherries and strawberry puree. In wine processing.g. sorbates are used to prevent refermentation (Parish and Carroll. 1989).1% sorbate and 0.05% to 0. and putrefactive bacteria.02% to 0. in high-sugar products (e.. 1955. In these products sorbic acid acts as the microbial inhibitor and sulfur dioxide prevents oxidation and enzymatic spoilage (Lück. prunes.02% to 0..02% to 0. In fruit juice and drink processing. Mold and yeast spoilage is also prevented by sorbates in pickled vegetable products (e. Selection of precise levels of sorbate. Sorbate concentrations as low as 0. depends on products and specific fermentation conditions (e..02% to 0.g. fruit juices and syrups. however. Also. Lück. Concentrations as low as 0. cucumbers and olives). Sorbate levels in the range of 0. beverages.g. 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. to inhibit chemical. Good wine preservation is achieved with concentrations of 0. but it makes them susceptible to mold and yeast spoilage.10% may be used to preserve refrigerated fresh salads. Costilow et al.05% and 0. Addition of 0. the lower is the sorbate level needed for preservation.. coli O157:H7 and result in spoilage of the product (Zhao et al. raisins. 2001). sorbate is either added directly into the product or applied to the surface of the product or packaging material. 0. coli O157:H7 in apple cider. molds. or microbiological deterioration (e.05% are adequate for preserving high-moisture dried fruits (e. Other vegetable products preserved with sorbates include tomato products and fermented Asian sauces (Chichester and Tanner. Combinations of sorbic acid and sulfur dioxide are also used in the preservation of high-pulp fruit juices. 1994). or bacteria. figs). fruit cocktails. sorbate acts as an inhibitor of yeasts. Potassium sorbate and sodium benzoate were highly effective in reducing yeast and mold populations in tomato juice (Bizri and Wahem.g.1% potassium sorbate substantially inhibited E. and wines.10% sorbate reduce the number of bloaters and poor seed cavities.02% to 0. 1980). The higher moisture content in these products is preferred by the consumers. FRUIT PRODUCTS Fruit products preserved with sorbates throughout the world include dried fruits.1% sodium benzoate and 0. sorbates are used mostly at the preprocessing stage.20% inhibits growth of organisms not desired in vegetable fermentations. 1988a. 1980). 1994). sorbate is more effective than benzoate owing to the pH of these products. In these products. Sofos. Also. Lück. 1993). In sweet cucumber packs. coli O157:H7 growth was inhibited in both tryptic soy broth and in apple cider by sorbic acid applied at 0. Because potassium may result in potassium bitartrate precipitation in some wines.10% are adequate in improving the preservation of soft drinks. while additionally suppressing the growth of yeasts and molds.05% and as such may be an option that processors have to reduce contamination in their products (Koodie and Dhople. Potassium sorbate was found more effective than chitosan in inhibiting growth of Aspergillus niger on lowsugar candied “kumquat” (Fang et al. 1980. Potassium sorbate is preferred because of its solubility. Depending on the particular product. Use of the combination of 0. Use of sorbates at levels between 0.g. dried fruits). preserves. The lower the moisture content of the product (e. together with sulfur dioxide and pasteurization. the pH values of fermented green olives favor the use of 0.02% sorbic acid in combination with .. Another study showed that E. jams and jellies). 1980).b). salt concentration).05% sorbates in the fermenting and holding tanks.05% sorbate to a 15% to 20% brine retards yeast scum formation. Levels of 0. jellies. jams.. enzymatic.. 1952. such as yeasts. sodium sorbate may be used to avoid the problem.. molds. 1972.64 Antimicrobials in Food fermentations. In sweet relishes.5% acetic acid completely inhibit microbial spoilage at sucrose concentrations ranging from 2% to 40%. which may influence the survival of E. fermentation). At these concentrations the selective action of sorbic acid allows growth of the desirable lactic acidproducing organisms.

and other beverages. which is especially important in preservation of juices. soft drinks. Concentrations of 0. However. in general. 1972. Sorbate acts as the inhibitor of yeast refermentation during storage. such as S.. 1988). and marmalades). brown-and-serve products).g. sorbates are more common meat preservatives in other countries. 1989). BAKERY PRODUCTS Although propionic acid-based preservatives are the most widely used compounds in preserving bakery goods. For this purpose a solution of up to 10% potassium sorbate may be used to protect unrefrigerated dry sausages.30% sorbic acid are adequate for preserving bakery products. Yeast-leavened products may be protected with sorbate spraying after baking. pizza shells and toppings. including cakes and confectionery. 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. Use of this compound has the advantage of preserving yeast-leavened bread without interfering with the fermentation process (Lück. 1980). York and Vaughn. sorbates are used in combination with benzoate in preserving fruit juices and drinks. cooked sausage product. 1989). 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. upon heating in the baking process. It has also been used in champagne preservation (Sofos. mold inhibition may be achieved by sprinkling sorbate on the surface of the product. Sorbates should be valuable in baking powder-raised products. Sorbates are also used in the preservation of pie crusts and fillings. A few sporadic studies in the 1950s and 1960s also indicated its potential as a preservative for meat products (Sofos. 1952. toppings. the compound is chemically degraded and releases sorbic acid after the yeast-leavened dough is raised. 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. 1999).10% sorbate inhibit the spoilage of cake icings. unless the amount of yeast and the leavening time are increased. In 1974. To avoid the effect of sorbates on desirable yeasts in yeast-leavened products. 2000). sorbates may also be useful in preventing aflatoxin formation (Lück. however. sorbates are also valuable because they are active at higher pH values and effective against molds (Chichester and Tanner. In fruit preserves (e.. the derivative sorboyl palmitate. aureus.004% sulfur dioxide.. may also be used. In addition to undesirable molds. In such applications sorbate may be used in the coating oil or the pan grease (e. 1989).Sorbic Acid and Sorbates 65 0. 1989).002% to 0. figs). and fillings. Sorbate levels above 0. 1989. refrigerated dough products. sorbate also inhibits rope-forming bacteria and pathogens. examined its activity against various pathogenic bacteria in an uncured.. Concentrations of 0. Tompkin et al. 1989). In addition to preventing mold spoilage. 1980). 1980). Sorbate can also be added before bottling the wine and is very valuable in the preservation of sweet wines. Lück.05% to 0. (1974) . Potassium sorbate increased the shelf life of acidified tortillas. jellies. which is a mixed anhydride of sorbic and palmitic acids that has no direct antimicrobial activity. Use of sorbates must be avoided in products raised by yeasts. and doughnut mixes (Sofos. Sorbates. 1969.03% to 0. Sofos.g. the dipping method of application is more effective (Sofos. Often.. Tompkin et al.03% are not recommended in wine preservation because they may impart an off-flavor to the delicate taste of wine.g. may be more suitable for use in fruit products than other common preservatives because of their milder organoleptic properties and their neutral taste. especially when used in combination with calcium propionate (Tellez-Giron et al. jams. 1976. and sulfur dioxide protects against enzymatic and oxidative changes and bacterial fermentation. In dried products with an irregular surface conformation (e. such as Japan and Korea (Sofos. 1954).. in cream pies (Schmidt et al. muffins..

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

it may be desirable to apply sorbic acid during malting (Ogundiwin et al.30% can be effective mold inhibitors. 1982. 1983. such as benzoates. Robach and Hickey. inhibiting their growth. Regenstein. This optimal concentration of sorbic acid coincided with the lethal dosage for most of the associated fungal microorganisms during malting. potato chips. depending on moisture content. Application of potassium sorbate with bifidobacteria extended the shelf life of whole and peeled shrimp by 3 days (Al-Dagal and Bazaraa. in which the emulsifiers may be inactivated by nonionic compounds (Chichester and Tanner. Shaw et al. 1983. 1972. Sorbic acid at a concentration of 0. tomato juice. 1983. 1959. Samples treated with sorbic acid lost biological activity within a few days of aerobic storage (confirmed by aerobic . however.. botulinum in shucked scallops developed slightly more rapidly at 27°C in vacuum packages when sorbate (0. mayonnaise.. They include high-moisture marshmallow candy. Debevere and Voets. 1989). Bremner and Statham. the esters of sorbic acid (e. 1981. sorbic acid may be mixed with its potassium salt or with other preservatives. 1972. 1976.. tangerine sherbet base. cut squash. Chung and Lee. strawberry puree. and similar products. 1991). are also effective in preventing mold growth on animal feeds during storage.. A sorbate (0. such as margarine. sour cream.10%. OTHER APPLICATIONS Other items that may be preserved by sorbic acid and its salts include pharmaceuticals. especially those of the emulsion type. 1989). pineapples. 1978. Statham and Bremner. 1988). 1980.5% did not adversely affect the germination energies and germination capacities of sorghum grain and did not significantly affect malting loss. 1989). Sofos and Busta. ethyl and propyl) may find applications in cosmetics and pharmaceuticals. In certain instances and for better preservation.. In Asian countries sorbates are commonly used in combination with other preservatives to preserve fish sausages and similarly processed fish and meat products. diastatic powers. 1972. 1980. and animal feeds (Sofos.1%)–benzoate (0. Fey and Regenstein. at which they prevent growth of molds and osmophilic yeasts in items such as fillings for chocolate and pralines (Chichester and Tanner. prepeeled carrots. as well as fermented plant foods (Chichester and Tanner.20%. salad dressings. Lück.03% to 0. 1999). 1982).. One study. 1981). in which levels of up to 0.g. delicatessen items. Other food-related applications of sorbates include sugar-based and confectionery products at concentrations of 0. Sorbates may be particularly important in intermediate-moisture (25% moisture content) pet foods. 1972. Sorbic acid levels in the range of 0. Sharp et al. Mayer and Hillebrandt (1997) established that chemical preservation with sorbic acid was a possible way of preventing potato pulp from spoiling for subsequent practical application (feed). Sorbates are also used to prevent mold growth on the surface of dried and smoked fish (e. dried cod). Mexican-type hot sauces. and cold and hot water extracts of the malt samples.Sorbic Acid and Sorbates 67 of bacon treated only with nitrite. 1976).1%) was used (Fletcher et al.. In addition. thus. Lück. MISCELLANEOUS FOOD PRODUCTS Sorbates are commonly used in the preservation of oil-in-water or water-in-oil emulsion-based products. 1995).05% to 0. 1983. 1982. 1989). garlic oil. Bremner et al. which is longer than the preservation (31 days) achieved by the most effective bacteriocin (nisin) tested (Einarsson and Lauzon. and pancake batter (Sofos. 1976. shredded cabbage. This should assure mycotoxin-free animal feeds and thus eliminate any hazards associated with mycotoxin-contaminated feed. Lynch and Potter. The literature contains reports on several other food products that may be preserved with sorbates (Sofos. Sofos. Several studies have reported improved preservation of various types of fish with sorbate (Geminder. cucumbers.1%) solution preserved brined shrimp for 59 days. 1986). indicated that toxin production by C.g. 1982. orange peels. Robach and Sofos. Lück. 1978. cosmetic products. the flavors of both types of bacon were equally desirable (National Academy of Sciences.

did not influence the susceptibility of the bees to the fungus Ascosphaera aggregate (Goettel and Duke. and vacuoles. and it may be possible that several of them may be functional under various conditions. Inhibition has involved various species of bacteria in laboratory culture media and in foods and has been influenced by species. Other proposed mechanisms of inhibition of microbial growth by sorbate include alterations in the morphology. environmental conditions. 1986. 1985). 1976. 1973. Under certain conditions sorbates have changed the morphology and appearance of microbial cells (Statham and McMeekin. Such changes have been observed in yeast cells as dense phosphoprotein granules. 1985a. MECHANISMS OF ACTION Sorbate concentrations used in foods (<0. the outer cell wall was absent in many areas (Ronning and Frank. 1986). 1986. whereas higher amounts may result in death. botulinum were long. Seward et al. adhesives. Blocher and Busta. 1979d. 1988.. and the type of food processing. 1989). Blanching (80°C) and the application of 0. but the exact mechanisms of antimicrobial activity are not well defined (Freese et al... the surviving microorganisms may resume growth and spoil the food. and not triggering of germination. 1978. Sofos. when present. Cells of C. and glues (Lück. Sofos. resulted in minicells. Although the significance of such alterations is unknown. Other reported uses of sorbates include preservation of tobacco products. 1985). type of substrate. Several studies have indicated that sorbate inhibits bacterial spore germination (Sofos et al. 1989). 1985. Another study reported that sorbate inhibited spores triggered to germinate or after germinant binding (Blocher and Busta. It was also postulated that sorbate probably inhibits a postgerminant binding step in the process of germination. when the sorbate hurdle is reduced or removed. with bulbous formation and defective division (Seward et al. when used individually or in combination as preservatives of an artificial diet for leafcutting bees. Published data have suggested that sorbate acts as a competitive and reversible inhibitor of amino acid-induced germination (Smoot and Pierson. strains.0 increased cell hydrophobicity and cell wall lysis on exposure to lysozyme. and the inner cell wall appeared to be thickened. 1989). Freese and Levin. pH. including types and species of microorganisms. and function of cell membranes and inhibition of transport functions and metabolic activity (Sofos. and clostridia common in the spoilage of silage and. Sofos. reduced ensiling losses and increased the feed value characteristics of citrus pulp silages (Filya et al. Septation...1% sorbic acid to citrus pulp were effective in inhibiting yeasts. 1982. 1981.. Sorbate inhibits cell growth and multiplication as well as germination and outgrowth of spore-forming bacteria. 1989. Wagner and Busta. 1996). Death of microorganisms exposed to high concentrations of preservatives. 1989). increased numbers and variable sizes of mitochondria. 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.68 Antimicrobials in Food degradation of starch and triglycerides). Although the presence of enzyme activities was shown. Treatment of Alteromonas putrefaciens with sorbate at pH 7.3%) usually inhibit microorganisms. Sofos et al. 2001). 1989). Sofos et al. 1989). There was also evidence of other membrane damage in sorbate-treated cells (Statham and McMeekin. molds.. 1988). sporogenes were usually filamentous and nonseptate but with distorted shapes characterized by numerous bends and bulges. irregular nuclei. Sorbate-treated cells of C. Several mechanisms of inhibition of metabolic function by sorbate have been proposed. such as sorbate. and sorbate concentration (Sofos. Sofos. enterobacteria. Smoot and Pierson. integrity.. which could be overcome to some extent by addition of magnesium ions. Ronning and Frank. 1981). Consequently. 1982. therefore. there was no detection of pectinolytic activities (Mayer and Hillebrandt.b). 1997). 1986. they may be the result of the incorporation of sorbate into specific cell structures and alteration of biosynthetic processes in the cell (Sofos et al. and it was thus concluded that it inhibits spore commitment to germination. has been attributed to generation of holes in the cell membrane . 1989). The preservatives sorbic acid and methyl paraben.

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

such as increased proton pumping by the membrane H+-ATPase. Specific inhibition of metabolism is also unlikely because inhibitory activity is shared by several small carbon compounds. Ronning and Frank. Evidence also suggests that this energy-demanding stress could also be the result of the induction of ATP-driven plasma membrane pumps for active extrusion of weak acids from the cell (Henriques et al. This could then discharge the pH gradient required for ATP formation according to the chemostatic theory of oxidative phosphorylation (Sofos. 1994). Thus. 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. 1987. A stringent response involves readjustments occurring in bacteria when amino acids become limiting or their specific ratios are disturbed (Sofos. 2000). cerevisiae (Piper et al. 1996). 1997). a shift in charge may potentially take place and lead to a decrease in the net negative intercellular charge. Hunter and Segal. 2001). 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. In fact. 1985. which ensured that pHi did not decline when cells were exposed to sorbic acid (Henriques et al.. weak-acid preservatives such as sorbic acid and lead to starvation of cells from compounds that are transported actively by the PMF (Eklund. Salmond et al.. a pump recently identified as the Pdr12 protein is induced by sorbic acid in S. which became . 2000). As the hydrogen influx exceeds the pumped efflux. 1989. it has been shown that sorbic acid acts at high pH where weak-acid preservatives are not expected to be active (Stratford and Anslow. cerevisiae yeast cells in the presence of benzoate or sorbate at an extracellular pH value of 6. Inhibition of microbial growth by sorbate may be based on neutralization of the PMF that exists across the cell membranes by lipophilic.8 elicited a set of metabolic effects on sugar metabolism. the difference across the cell membrane is large. 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. resulting in inhibition of growth but in maintenance of cell viability (Sofos... found no decreases in ATP in the presence of sorbic acid (Sofos. 1997). and the amount of acid entering and dissociating in the cytoplasm is higher. Similar degrees of inhibition by sorbic alcohol and sorbic aldehyde suggest that sorbic acid does not act as a weak-acid preservative. however. According to this theory. 2000). 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. 2000). Cells of Z. little correlation was found between reduced growth on exposure to sorbic acid and reduction of intracellular pH (pHi) (Bracey et al. Inhibition of uptake of such components is believed to induce a stringent-type regulatory response in the cells. Conversely. 1989). 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.. Sofos. 1973). which is one of the components of the PMF (Sofos. 2000). 1968. 1983. This was partly attributed to the activation of protective mechanisms. as indicated.. 1998).. 1998). 1980). In another study using a novel method to measure pH in growing cells.. 1980.70 Antimicrobials in Food which act as molecular chaperones (de Nobel et al. In lower pH environments. 1998). which decreases the intracellular pH and dissipates the PMF of the membrane that energizes transport of compounds such as amino and keto acids. growth inhibition correlated with an increase in the intracellular adenosine diphosphate (ADP)/ATP ratio as a result of increased ATP consumption by the cells. Preincubation of S. Previous studies with bacteria.. 1992. Sorbic acid in its MIC releases far fewer protons than other weak-acid preservatives. 1984. 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. Because inhibition of microbial growth by sorbate has been associated with decreased levels of ATP (Harada et al. 1988. 1998). Indeed. undissociated sorbic acid acts as a protonophore. Therefore.

TOXICOLOGY AND SAFETY In the United States. Thus. Sofos. in such situations. 1980.. 3.2 to 10. These effects can be summarized as follows: (1) reduced glucose consumption. 1993). However.Sorbic Acid and Sorbates 71 apparent after the subsequent addition of glucose. 1955).or longterm exposure. based on acute toxicity. 1989). 1998. 2000). Rat feeding studies have indicated that a dosage of 10% sorbic acid in the diet could be tolerated for 40 days (Lück. depending on the type of microorganism..5 g/kg body weight (Smyth and Carpenter. For comparison. the World Health Organization has set the acceptable daily intake for sorbate at 25 mg/kg body weight per day. 1948. Reasons for this conclusion include the following: 1. 1976).6-bisphosphatase and phosphoenolpyruvate carboxy-kinase. Lück. sorbate is considered one of the least harmful preservatives in use. and because it is a metabolizable fatty acid. the LD50 for common salt (NaCl) is 5 g/kg body weight. 1989. 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.. the mechanisms involved in inhibition of microbial metabolism and proliferation by sorbic and other similar lipophilic acid preservatives appear to be different. Overall. 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. 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. 1954a). 1954a. Lück. Sofos.6-bisphosphate. (2) inhibition of glucose. 1980). Sorbic acid has been shown to inhibit microbial growth. which is the difference in electrical potential.. sorbate is considered a GRAS compound. Demaree et al. Bracey et al.. and (5) block of catabolite inactivation of fructose-1. but 8% sorbic acid resulted in an increase in liver weight (Deuel et al. The compounds were fed to various animal species for determination of acute toxicity. uptake of the compound based on pH difference across the cell membrane would not be adequate to explain the observed antimicrobial activity. (4) substantial reduction of glucose-triggered peak of fructose 2. (3) suppression of glucose-triggered peak of hexose monophosphates. Thus. its effect on the other component of the PMF. 1993). although less efficiently.and fructose-phosphorylating activities. unlike former assumptions. 1954a. however. 1955. carcinogenicity. Food and Drug Research Laboratories. The relative nontoxicity of sorbates was established early in their testing as food preservatives. Deuel et al. 2. . Although dissociation of the acid inside the cell may eliminate the pH difference across the membrane. On extending the feeding period to 120 days. even at pH values near neutrality. but not of cytoplasmic malate dehydrogenase (Burlini et al. Thus. 2000). On the whole. this pattern resulted in prevention of glucose-induced switch of metabolism from a gluconeogenic to a glycolytic state. 1998. Acute toxicity studies with rats have determined LD50 (mean lethal dose) values for sorbates in the range of 4.. the growth rate and liver weight of the animals increased (Demaree et al.. substrates. it is believed to be adequate to energize the uptake of substances needed for cell maintenance and growth. these studies demonstrated the relative harmlessness of sorbates and their relative superiority in safety compared to other chemical additives. 1992). 1973. Overall. 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. as well as influence on metabolism. Additional studies with rats and dogs have shown no damage with feeding 5% sorbic acid for 90 days. and environmental conditions (Sofos. and teratogenicity after short. is smaller..

and Robach and Adam (1980) were able to induce such symptoms from panelists consuming commercial bacon manufactured without sorbate. 1981. (2) formation of the c-nitroso mutagens requires the presence of excess nitrite. 1989). Shtenberg and Ignat’ev. Litton Biometrics. forming high-molecular-weight products that do not seem to result in a nutritional loss. leading to decreased amino acid availability (Quattrucci and Masci. Department of Agriculture. 1976. 1989). 1970. cysteine. Patrizi et al.30% in food processing. apparently because of an increased caloric intake resulting from the metabolizable sorbic acid. Under conditions typical of . 1980) and because sorbic acid is an unsaturated fatty acid. 1999). sorbates have been extensively examined for skin tolerance (Lück. Kito et al. Osawa and Namiki. No carcinogenic or mutagenic effects have been observed with sorbate alone (Dickens et al. 1976). 1980a. probably because the acid environment of the stomach leads to the breakdown of the sorbic-protein adducts (Quattrucci and Masci. 1979. The products of such reactions have the potential of being mutagenic (Sofos. 1975. 1982). no such symptoms were observed by other individuals who tasted sorbate–nitrate experimental bacon from other studies... In addition. Sorbic acid has a conjugated system of double bonds. Sofos and Busta. Department of Agriculture. The literature is contradictory. it may react with nitrite in products such as cured meats. and vegetable juices and is inactivated by heat (Kada.. 2001). does not appear to create toxicologic problems. in general. 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. Thus.. however. 1975. 1981. and in some instances the growth rate of sorbate-fed animals increased significantly. 1979. 2000a. Some taste panelists reported certain “allergic-type” symptoms after tasting uninoculated experimental bacon (U. may undergo nonenzymatic browning reactions when stored with sorbic acid at a favorable range of aw and temperature (Quattrucci and Masci. and some very sensitive individuals may show irritations even at lower levels (Lück. As preservatives for cosmetics and pharmaceutical products.0.S. 1976. Average sorbic acid concentrations for skin irritations are in the range of 1%. 1989). Namiki. 1992). This. 1974. It has been regarded that amino-compounds. the potential for such irritations in commercial products is minor. but it indicates that most people are not affected by sorbic acid applied to the skin. Lück. no direct relation could be proved between symptoms and specific ingredients used in formulating the bacon (U. Sorbates were found to react easily with proteins. 1994). Although it was implied that sorbate might have been involved in producing those symptoms.. which makes it susceptible to nucleophilic attack. Considering the average use levels of 0. 1968. Khoudokormoff.S. of course. Some sensitive individuals. 1981.. 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. 1980.10% to 0.. 1979b). and (3) mutagen formation is inhibited by such ingredients as ascorbic acid. 1979b. This reaction was demonstrated in model systems but. 1983. 1982). Sofos. Hendy et al. 1980. 1992). 1981. Food and Drug Research Laboratories. does not rule out the possibility that high concentrations of sorbate may act as irritants to certain susceptible individuals (Sofos. 1982. National Academy of Sciences. 1986. which is the average pH of most meat products. 1977. 1981). Gaunt et al. 1978. 1980. No abnormalities have been observed. however. 1993. including lysine and glutamate. 1992).. There has been some concern about possible sorbate–nitrite reactions when added to the same substrate (Sofos. botulinum.b). 1982. 1981.5. various studies have indicated the following: (1) nitrite reacts with sorbic acid to form mutagens optimally at pH 3. not at 6. sometimes giving mutagenic products (Ferrand et al. show skin irritations when exposed to sorbic acid (National Academy of Sciences. Robach et al. Osawa 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. In general. 1974. to some extent. Dejobert et al. Berry and Blumer.. Walker (1990) concluded that sorbate use. Because there is evidence of reactions between nitrite and fatty acids (Benedict. it may apply also to more complex systems such as foods.

10) by the color reaction with αthiobarbituric acid (Roy et al. The spectrophotometric (ultraviolet/UV. at 20°C. 1962) and spectrophotometric (Melnick and Luckman. . 1955. The formation of new products by addition reactions using ethyl sorbate and various amines led.15 and 975. and solvent extension using ethyl or petroleum ether. cheese. methods 971. 1995.31). Extraction methods include acid-steam distillation. 1983. 1995. method 975.. 1980). it involves measurement of absorbance at 260 nm (250 to 290 nm).. 1978.b). showed that none of the products studied presented mutagenic or genotoxic activities (Ferrand et al. 1972..22). however. 1999. sodium hydroxide. Sorbate.. 1989). Potential injury in cell hepatocytes induced by potassium sorbate was prevented by antioxidants (Sugihara et al. sorbates appear to be one of the safest food preservatives available. and methylene chloride. or direct analysis has been used (Sofos. enzymatic. DETECTION AND ANALYSIS Analytic methods used or tested for qualitative and quantitative detection of sorbates in foods include acidimetry. 1989). 1976. filtration. Yamamoto et al. Mutagenesis studies. in cyclic interaction products. Noda et al. Detection methods require quantitative extraction and separation of sorbic acid from the food material without food ingredient interference (DeLuca et al. Sofos. to linear monoadducts and.. 1982. 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. although chromatographic methods have gained acceptance in recent years. Chung. Holley and Millard. polarography. 1981.. 1989). Such combinations have involved extraction under acid conditions from the steam distillate and reextraction with sodium hydroxide. bromometry. involving the Ames test and genotoxicity studies with HeLa cells and on plasmid DNA.. Extraction by steam distillation has been used extensively.b. Puttermans et al. 1981a. 1988). cheese (AOAC Int. 2000). Sofos. Mandrou et al. Sofos. the method was modified by Alderton and Lewis (1958). Sofos. Wilamowski. Massey et al. benzoate. dialysis. It should be noted. After development by Melnick and Luckman (1954a). The method is simple and usually very specific (Lück. however. Overall. Larsson. 1976. Sofos. successive extractions with ether. to cyclic derivatives resulting from double addition (Ferrand et al. bakery items.. 1989). dichloromethane. whereas combinations of various treatments have also been used to improve extraction and reduce interference (Sofos.. 1974. In some foods. 2000a... 1973. colorimetry. 1960. AOAC Int. Lathia and Schellhoeh. and propionate (Sofos. Special extraction and purification steps have been proposed in the literature to reduce interference problems (Sofos. method 975.. had no effect on nitrosamine formation from aminopyrine and sodium nitrite in animal stomachs (Kawanishi et al. No synergism in acute toxicity has been detected for combinations of sorbic acid with various other additives. 1983. however. 1980). absorption) procedure has also been used in many foods. did not protect against the toxic effects of other substances (Daoud and Griffin. Rao et al. The derivatives of sorbic acid are also relatively nontoxic.b). however. at 50°C. Several modifications have been proposed as useful in avoiding interference and in improving the accuracy of the steam distillation procedure (Luckmann and Melnick.. dialysis and solvent extraction. Sorbic acid. 1954a). and isooctane. 1981. 1962. and sausage products (Maxstadt and Karasz. 1972.. Stafford.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. that certain studies have reported inhibition by sorbate of carcinogenic nitrosamine formation in model systems (Tanaka et al. and wine (AOAC Int. wine. including parabens. 2000). Montano et al. selective gas diffusion. including fruit products. 1982. 1980). spectrophotometry.. 1997). and double distillation and ether extraction (Tjan and Konter. 1989). have been colorimetric (Schmidt. 2000a. The most widely used methods. 1989). 1989).. 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. and chromatography.

In the United States.. 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. 1978) by a select committee of the U. 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 974. 1998).10) (AOAC Int. 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. 1995). Solid extraction has been used as a cleanup procedure for the determination of sorbic acid by liquid chromatography in fruit products (Mandrou et al. 2001).16) for sorbic and benzoic acids in foods (AOAC Int.. considering their extensive testing. 2002). and micellar electrokinetic capillary chromatography (MECC) and ion chromatography. Castineira et al.. Moreover. Microdialysis was used to extract sorbic acid and benzoic acid from food to be separated and detected by HPLC (Mannino and Cosio. 1998. which is proportional to sorbic acid levels in the sample. Food and Drug Administration. that in addition to sorbate. It is obvious. They include gas chromatography. thin-layer chromatography. especially for high sample throughputs (Hofer and Jenewein.. preservatives.74 Antimicrobials in Food 1989).. 2000). A gas chromatographic method is a first action procedure (AOAC Int. In addition. 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. REGULATORY STATUS Several forms of sorbate are allowed for use in a variety of foods throughout the world (Sofos. and other substrates (Cancalon. 2001). The use of reversed-phase highperformance liquid chromatography (RP-HPLC) was found to be a simple. 2000). 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. ion chromatography was effective in simultaneously determining artificial sweeteners. and advantages over other preservatives. highperformance liquid chromatography (HPLC). Inhibition of microorganisms has also been evaluated as a procedure for the qualitative detection of sorbate (Ellerman. rapid method with high reproducibility for determining sorbic acid and other additives in fruit juices (Zou et al. 1989). precise.S. This major use may expand in the future. method 980. Others. sorbic acid and potassium sorbate are GRAS. The literature contains numerous reports of chromatographic methods used or evaluated for determination of sorbate in food products (Sofos. have reported that cleanup procedures did not improve determination by liquid chromatography (Benassi and Cecchi. such inhibition may be caused by a number of other inhibitors. 1999). 1999. Capillary electrophoresis was also applied for detection in citrus juices. 2000). low toxicity.. 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. caffeine. 2000). In addition.. sorbates in the United States . 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. The method is an official first action procedure for ground beef (AOAC Int. however. Dobiasova et al. where the absorbance is measured at 300 nm and is specific for sorbyl CoA. it can be used for the analysis of intensely colored juice samples. and theophylline and as such may be a beneficial alternative to conventional HPLC. method 983.17) and final action procedure for wines (AOAC Int. 2000. 2000). The reaction is quantified by irreversibly hydrolyzing pyrophosphate to phosphate in the presence of inorganic pyrophosphatase. method 974. Thus. wine. 1990). and this status has been reaffirmed (U. 1996). 1989).S. Mercier et al. however. paper chromatography. Department of Agriculture. 1977). HPLC was found to be adequate in obtaining accurate stability data for sorbic acid in creams (de Villiers and Bergh. theobromine. and reliable and is thus well suited for routine determinations. The method was found to be fast.08) and dairy products (AOAC Int. In addition.

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

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

41 4. The multiple-barrier concept (“hurdle” concept) has gained popularity for use in a variety of foods in recent years. gaseous atmosphere.75 5. The use of the multiple-barrier concept results in lowering the concentrations of acidulants or inhibitors.89 3. Comprehensive texts describing applications.75 4. The inhibitory effect of acids has been compared based on pH.Organic Acids 93 TABLE 4. normality. 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.87 4. and other inhibitory compounds. Hoffman et al.61 4. organic acids are usually more effective as antimycotic agents.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). water activity. EFFECTIVE USE Various approaches have been used to study the efficiency of antimicrobial effects on cells. and protect color.44 5. and acids with 8 to 12 carbons were more effective at neutrality and above. adjustments in pH levels can be coupled with changes in temperature. or molarity to achieve final pH.11 5. 1980).39 times. the choice of an acidulant may depend on secondary effects. and chemical analyses can be found in the references by Ash and Ash (1995) and Doores (2002. pK1 4.1). but also the decreased processing time would aid in preserving the palatability of the product (Corlett and Brown. and degree of branching to inhibit or kill a wide variety of microorganisms. type.40 4.98 pK2 pK3 5. Because some experiments express concentration in percent.43 4.16 2.27 4. Effective use of an acidulant depends on the dissociation constant (pKa) or the pH at which 50% of the total acid is dissociated. chain length. This primary concern limits the use of acidulants to foods with pH values of less than 5. In addition to any antimicrobial effects an acid possesses. Acids contribute to the taste and tartness of a product. aid in the inversion of sucrose. 2003). Given that this value lies at the lower limits of growth for many bacteria. it would be advantageous to use the acids near these values. Therefore.34 6. This concept is based on the premise that foods can be preserved using several inhibitors concurrently rather than relying on a single factor. Because the undissociated portion of the molecule is believed to be responsible for the antimicrobial effect. physical parameters. prevent browning. concentration. The pKa of most organic acids lies between pH 3 and 5 (Table 4.0. They can control pectin gel formation.03 3.08 3.77 4. They can create a synergistic relationship with antioxidants by chelating metal ions. Some can be used . Not only would this interaction ensure commercial sterility of the food. (1939) observed that acids with fewer than 7 carbons were more effective at lower pH.14 3.

The succession typically begins with growth of coliforms and natural microflora followed by Leuconostoc mesenteroides (heterolactic). sauerkraut. They are usually added to foods for their antimicrobial properties. Several animal studies have indicated possible safety hazards. are more tolerant to this acid than homofermentative lactic acid bacteria (homolactics). application. however. 1975b). many of these studies used high levels of the concentrated acids at low pH values. the same hazards would not be expected.9) and Aspergillus niger (pH 4. yeasts. Concentrations of acid lower than those needed to inhibit Saccharomyces cerevisiae (pH 3.0. Although these acid mixtures might be expected to occur naturally during fermentations by heterolactic bacteria. L. which exist naturally in many foods. Adams and Hall (1988) showed a weakly synergistic inhibitory effect between acetic and lactic acids with Salmonella enteritidis and Escherichia coli. all these acids can be metabolized by the body primarily through lipid oxidation and via the tricarboxylic acid cycle.8. as the name implies.9). however. plantarum at pH 4. In addition. such as pickles. These organisms are found naturally associated with fermented products. and Staphylococcus aureus (pH 5.0). and regulatory status is presented here for each acid. Numerous studies included in this chapter compare several organic acids for their antimicrobial effects. The lipophilic acids include acetic acid and acetic acid derivatives.94 Antimicrobials in Food as chemical leavening agents. antimicrobial properties. At the levels and pH values in which these acids are normally used in foods. 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.1) (Levine and Fellers. the information appears separately for each acid. such as sauerkraut. Sauerkraut manufacture is a fermentation process brought about by a natural succession of microbes that inhabit the surface of cabbage leaves.0. ACETIC ACID Antimicrobial Properties Acetobacter and heterofermentative lactic acid bacteria (heterolactics) produce acetic acid as a byproduct of their metabolism and. and molds. which do not.0 than lactic acid bacteria. McDonald et al. plantarum maintained its pH gradient in the presence of either 160 mM sodium acetate (SA) or sodium lactate down to an external pH of 3. whereas growth stopped in L. This association could explain the apparent stability that occurs in natural fermentations. 1966) The toxicology and safety of the acids must be considered when selecting an acid for use. a more acid-tolerant organism. Most of the acids appear safe for use in food products. Owen (1946) suggested that acetic acid inhibited Gram-positive organisms. and vinegar. mesenteroides ceased when the internal pH of the cells reached 5. The long-term consumption of these acids. . The salts of the acids are important in regulating the acidity of the foods (Gardner. In some instances.9).6 to 4. (1990) found that growth of L. Bacillus and Clostridium species and Gram-negative bacteria were more inhibited at pH 6. then by Lactobacillus plantarum (homolactic). as the pH decreased to 4. The results of these studies are usually presented in the section describing the acid that gave the most antimicrobial effects. are relatively nonpolar compounds. Microorganisms vary in their susceptibility to acetic acid. would further indicate that hazards are unlikely. although they could also serve other functions in foods. this apparent synergy has not been demonstrated in other foods. 1939) inhibited Bacillus cereus (pH 4. all of the groups were similarly affected (Woolford. and caprylic acid. Salmonella Aertrycke (pH 4. propionic acid.4 to 5. These acids.7. Gram-positive bacteria. THE ACIDS Information on the toxicology. as such.

Similar results were noted when various acids were compared at the pH level required for a 90% and 99% reduction in numbers of S.4 to 4.7% acetic acid with reduction to undetectable levels in 1 and 2 weeks with 0. Conner et al. Streptococcus faecalis. aureus preconditioned in acetate buffer (pH 4. Acetic acid caused greater inactivation of L. B.15 M acetic acid and with 0. and tartaric acid permitted growth at pH 4. although more resistant than Salmonella. especially with the use of pasteurized egg products. therefore the standard use of 0. and HCl. 1979).0 with scant growth in TSB containing acetic acid. 1997).0 buffer. respectively.1-M concentration of acetate and lactate completely inhibited multiplication at pH 6. L. E. monocytogenes than lactic and citric acids and increased inhibition as the temperature of incubation . 1991). coli than unbuffered systems.3% and 0. pH 4. which has recognized antimicrobial properties. aureus.0 with hydrochloric acid was compared to cholesterol-free (egg yolk-free) reduced-calorie mayonnaise (CFM) manufactured with 0.8. Both formulations were inoculated with approximately 108 CFU/ml of an 8-strain mixture of Salmonella or a 6-strain cocktail of Listeria monocytogenes and held at 23.0% lactic.3.6) at 20°C shifted to pH 7. Salmonella Blockley.9°C. aureus in 12 hours (Minor and Marth. The effect of temperature-acid interactions is noteworthy for the psychrotrophic pathogens..9 and 4. pH 4. monocytogenes and Yersinia enterocolitica. 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.7 and 4. L. 1995..0. When E.5% and 0. and E.5 for all acidified media at 10°C except for acetic acid. added or developed through fermentation. 1988).0 (Conner and Kotrola. coli O157:H7 is considered more acid tolerant than strains of E. Traditionally acidic foods contain a single or multiple acids.Organic Acids 95 S. Acetic acid exerted the most inhibition but had the lowest dissociation constant.1 and 5.0% acetic acid and 1.2 and 5. This finding is important in the manufacture of medium acid foods containing more than one organic acid.5.0 with 0.1%. 1970). but no injury occurred in cells not exposed to acidic conditions before heating (Smith et al. malic.7% acetic acid.6.0. and exposed to 40°C became injured. A 0. and caused 50% inhibition of spore germination at pH 4. Malic acid allowed growth at pH 4. respectively.. and Enterobacteriaceae in Japanese fermented soy sauce (Hayashi et al.6 and 4. and tartaric acids. A concentration of 0.8% reduced growth of Micrococcus and Bacillus species. coli O157:H7 was inoculated into tryptic soy broth (TSB) containing acetic. aureus was most sensitive to acetic. coli. 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 5.7% acetic acid (Glass and Doyle. S.3% acetic acid formulations. It is unlikely that these products would contain such high initial levels of either of these organisms. other ingredients can interact with acid to influence the degree of inhibitory action. Acetic acid was effective at pH 5.5 (Noda et al. was reduced by 4 logs in both mayonnaises within 72 hours in formulations containing 0. and hydrochloric acids (Nunheimer and Fabian. 1988). such as mayonnaise (Debevere. respectively. citric. 0.7% acetic acid.5 to 3. or 0. 22°C) containing 1. Salmonella were inactivated within 48 hours in both RCM and CFM formulated with 0. citric. followed by lactic. 1982). in the aqueous phase of CFM and RCM would still produce a microbiologically sound product.2 and 4. E. monocytogenes.7% acetic acid in the aqueous phase and adjusted to pH 4. growth occurred at pH 5. whereas tartaric acid had one of the highest dissociation constants yet exerted the weakest inhibitory action. malic. lactic. 1940). 0. some strains of S. pH 4. most likely because of the absence of egg yolks in CFM and the presence of egg white. coli O157:H7 grew in all media at pH 5. Both studies concluded that the antimicrobial effect was the result of the undissociated molecule.6.. tartaric. malic. In addition.5%.3%. Buffered acidulant systems (brain heart infusion [BHI] broth. L. aureus.33 M lactic acid (Wong and Chen. Destruction of Salmonella occurred more quickly in CFM than RCM. respectively. Reduced-calorie mayonnaise (RCM) manufactured with 0. or citric acid were far more effective in reducing numbers of S. lactic. pH 4. Gram-negative aerobic bacteria. pH 4.5.4% to 0. 1984). tartaric.0. At 25°C and 37°C. cereus was inhibited at pH 5.

When comparisons were based on equal molar concentrations. and hydrochloric acids. Numerous organic acid combinations. respectively. coli O157:H7. lamb. 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.33 and 5.5 to 3. enterocolitica at lower pH levels as the temperature increased.4 for malic. 2001). Because produce is typically refrigerated with only minimal processing..96 Antimicrobials in Food decreased from 35°C to 13°C. and off-flavors. monocytogenes were exposed to acetic acid at a concentration of 242 µL/L of air for 12 hours at 45°C. citric acid caused greater injury than either lactic or acetic acids and injured organisms survived longer at low temperatures (Ahamad and Marth.. E. Depending on the treatments. 1991. Treatment at low temperature (4 days at 10°C) with 200 and 500 mg/L of air was effective in reducing Salmonella by 2.7 log without affecting germination of seeds. but acetic acid was less effective. acidulants. 1999). or 50%) for 15 or 30 minutes. these foods can be the carriers of psychrotrophic pathogens.05 N for 8.. there can be adverse sensory effects including discoloration of meat. The greatest antimicrobial effect occurred at 35°C with the greatest survival at 10°C. malic. a twofold reduction at pH 2. monocytogenes when compared to citric. 1992. 1981). 2000). but recovery required the presence of amino acids or peptones (Blankenship.9 log reduction for the nontreated seeds (Weissinger et al. or 5%) or vinegar (30%. was dipped in acetic acid solutions (1%. 40%. 25-g samples. vapor treatment also reduced levels of resident microflora on seed but did not affect seed germination rates (Delaquis et al. Brocklehurst and Lund. but resulted in changes in sensory characteristics. 1990). 1997.0 for acetic acid. inoculated with approximately 107 CFU/g Y. enterocolitica.0 have been used to rinse pork. A 90% reduction of the population required exposure to 0. containing from 0. 1989). Treatments spanned varying amounts of time with concomitant decrease in microbial loads up to 3 logs. In addition. and beef carcasses or meat tissue. Exposure at 50°C for 12 hours with lower levels (100 and 300 mg/L of air) reduced the population >1. and pH 4. enterocolitica was not detectable after exposure to 2% and 5% acetic acid or 40% and 50% vinegar for 30 minutes (Karapinar and Gönül.5 min.3% inhibited L. In addition.. Acetic acid displayed the greatest antimicrobial action against L. pH 4.4 to 4.5 or 2. Mung bean seeds inoculated with 3 to 5 logs of S. 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 1.72 logs. A variety of organic acid treatments has been used to control microbial loads associated with meat carcasses and products (Dickson and Anderson. 1998). 1997.2 N for 4 min. Dorsa et al.6% to 3. 1 log unit at pH 2. 1992a. and hydrochloric acids at equal pH values and at all incubation times and temperatures (Sorrells et al. enterocolitica. and no reduction in microbial . 0. monocytogenes was isolated from 2 of 10. Sorrells and Enigl (1990) also demonstrated interactive effects between sodium chloride. Parsley. Enteritidis were sprayed with acetic acid at pH 1. seed germination was effected (Weissinger and Beuchat. Smulders and Greer. coli O157:H7 to nondetectable levels by enrichment methods. Immersion of alfalfa seeds in mixtures containing 5% lactic or citric acid for 10 minutes resulted in several log reductions. 1989.0 for 30 or 60 seconds (Biemuller et al. 1973). 0. The minimum inhibitory pH level was pH 4. and L. Typhimurium. injury and death rates were not affected by refrigeration temperatures for Salmonella Bareilly.. Acetic acid was more effective than lactic or citric acids in producing this effect (Adams et al. Conversely.5. citric acid was more bactericidal than other acids at 25°C and 35°C.. Typhimurium and E. 1992b). however. However. and temperature. Little et al. 2%. Y. Pork carcasses inoculated with 106 CFU/ml S. lactic.5. but L. concentration ≥0. monocytogenes irrespective of temperature. Karapinar and Gönül. 1990. Total plate counts were reduced by 4 logs at pH 1.. a mesophilic organism. but malic acid was more bacteriostatic than other acids at 10°C.10 N for 5 min. Similar growth inhibition was demonstrated for Y. Dorsa.8 to 5. such as Y.6 for lactic acid. 1992). 1998. citric.0. and 0. off-odors. Acid injury did not appear to involve damage to ribosomes or other nucleic acid material.0% acetic acid at pH levels from 1. This treatment reduced S.01 N for 75 min.

They found that acetic acid was a superior sanitizer compared to hypochlorite and had a greater residual effect. 1996). (1991) inoculated beef roasts by injection and on the . and stored for 28 days at 2°C in a high-oxygen barrier film. Enteritidis to 1 of 6 samples at pH 1. anaerobic.. The treatment effectively reduced recovery of S. L. Meat was sprayed. 1987). but beef treated with the mixture of acids did not differ in flavor from untreated beef (Bell et al. such as rumen fluid. As part of recently established guidelines by the U.1 and 1.. Dickson (1992) contaminated lean and beef tissue surfaces with S. 1983). 1995). Typhimurium was reduced by 0. S. Temperature played a greater role in reducing numbers than did the acid concentration. 1994).5 and 11 of 72 for pH 2. and application rate and sequence in the process (Gorman et al. Typhimurium. Quartey-Papafio et al. An increase in organic material.5% acetic. vacuum packaged.6% on meat using a 3% concentration of acetic acid before washing.4°C (Anderson et al. coli O157:H7. Anderson et al. however. Hardin et al... and lactic acid bacteria counts. citric. dirt.. Typhimurium.3 log/cm2).2% acetic acid or 0. 1994.046% formic acids for 10 seconds caused discoloration from both treatments.5% acetic acid were reduced more when treatments were conducted at 52°C than at 14. and bleaching of the carcasses occurred (Ockerman et al.. the pH level decreased to 2. Lactobacillus species predominated as storage progressed. 1997).. Microbial loads on beef carcasses subjected to machine washing and sanitization with 1. Typhimurium reductions were greater when the bacteria were attached to fatty tissue. Typhimurium followed by treatment with 2% acetic acid. 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. a final application of acid washes could provide some residual effect during storage (Brackett et al. this was not significantly different from the controls. coli O157:H7 and S..0. possibly because this tissue retained less moisture (20%) than lean tissue (75%) and the reduced water activity could have enhanced the antimicrobial effects. respectively.. however. Unda et al. or manure slurry.S.5 to 0.Organic Acids 97 numbers at pH 3.. or manure. 1994). resulted in less effective reduction of S.. 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. which resulted in significant reductions in aerobic plate count (1..0. Department of Agriculture Food Safety Inspection Service (FSIS). 1997.. 1974). (1977) reduced bacterial counts by 99.8 to 4. 1994. 1989b). Beef cubes treated with 1.6% acetic and 0. Further work by Anderson and Marshall (1989) demonstrated that application of acetic acid at 70°C was most effective in sanitizing beef semitendinous muscle inoculated with E. At this concentration. 1995). organic acids can be used in preevisceration rinse systems consisting of a water rinse followed by an organic acid rinse. suggesting that acids had limited effectiveness (Fu et al. Typhimurium on beefsteaks (Tinney et al. Cutter et al. 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. 1986). Dorsa et al. S. Although washing of pork carcasses with 1. Cutter and Siragusa. Hamby et al. however. monocytogenes was detected in 69% of loins and 33% of chops. 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. An 18% acetic acid or 12% lactic acid spray significantly reduced bacterial counts on lamb carcasses. (1980). type of chemicals. (1987) used intermittent sprays of 1% acetic acid or lactic acids. some discoloration occurred (Cacciarelli et al..8 log CFU/cm2. It was noted that the use of acetic acid as a rinse for beef tissue led to sublethal injury of bacterial cells. 1997. 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.5. Incorporation of pulsed power electricity and a 2% acetic acid spray led to improved reduction of inoculated E. 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. 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. Reductions in microbial counts depend on the water temperature. S. coli.

fumaric.9% acetic acid did not support growth or toxin formation of Clostridium botulinum (Ito et al. further poultry processing can reintroduce microorganisms onto carcasses. 1994). 20°C. Again.0 on inhibition of L.1% acetic acid at 52°C decreased levels of S. Kurita and Koike (1983) combined 3% ethanol. El-Shenawy and Marth (1989b) investigated the effect of acetic.5 mM. 1997).3% and 0..86 log MPN/ml for the 0. monocytogenes. For pearl onions. Despite the reduction in microbial numbers at this pH. Other studies continued to explore the use of acetic acid in chill water. 1990). 1 mM perillaldehyde—or 0. aerobic plate counts were unaffected by the treatments.5 with acetic acid was the most effective antimicrobial treatment followed by other acids in descending order of effectiveness: adipic.. 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 logs (Okrend et al. Typhimurium using a model system. Rubin (1978) found that acetic and lactic acid acted synergistically to retard growth of S. see Chapter 7) coupled with 0. Typhimurium and Campylobacter jejuni by 0. acetic acid was the most destructive acid at all combinations of parameters. a 0. and 30°C (Oscroft et al. greater than a 4% concentration of acid was needed to reduce levels by 2 logs (Tamblyn and Conner. Nisin (an approved bacteriocin for some foods. respectively. citric. 0.35 log MPN/ml for the 0. Dickens and Whittemore (1994) exposed broilers to 0.5%.98 Antimicrobials in Food surface with approximately 103 CFU of Clostridium sporogenes and L.05% and 0. Acidified media showed greater inhibitory activity at 35°C than at 13°C. succinic. germination and outgrowth of Bacillus .05% acetic acid. lactic.2% to 0.6% acetic acid under the same conditions. 1994).5 to 1.6 or 5. 1976). Adjustment of the chill water to pH 2.6% acetic solution acid (pH 3.0% acetic acid compared to 2 days for 0. There was no significant difference in texture or sensory characteristics between the treatments. The acid level could be reduced when used in combination with brines and sugar. Air injection did not affect reduction of these counts. Products are now being preserved by a number of antimicrobial compounds using a multiplebarrier approach to preservation. Intervention steps incorporated into poultry processing have been effective in reducing microbial contamination.7% citric acid. monocytogenes.0) did not result in a significant reduction of aerobic plate counts but did significantly reduce levels of Enterobacteriaceae by 0. Poultry scald water containing 0. Levels of 0. but Enterobacteriaceae counts were significantly reduced by 0. 1994).71 log most probable number (MPN)/ml (Dickens et al.05%. acetic acid caused the skin of the poultry to be hard and leathery. Because of the possible attachment of Salmonella to broiler chicken skin. Despite these treatments. of the same components—with 2% salt to inhibit growth of contaminating microorganisms for more than 20 days at 27°C.35% solution required 7 days for equilibration for acetic and citric acid but 1 day for 0... As the incubation temperature to recover spores was lowered. citric.6 depending on the concentration of acid in can brine within 1 day with 1. Although each individual acid influenced the heat resistance of Bacillus spores differently. Whole pickles preserved with brine at 12° Brix and 0. but changes in pH and concentration of acid were more successful. (1985) found that mushrooms reached an equilibrium pH of 4.24 log (Lillard et al. with and without the use of air injection to agitate the chill water.3% acid and 2. 1965). Stroup et al. and lactic (Mountney and O’Malley. lactic. 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. Water pockets occurred under the skin of chicken carcasses with air-agitated samples (Dickens and Whittemore. 0. 1986). Expanding on their work.7% acetic and 4 days for the same concentration of citric acid. Acetic and tartaric acids were more inhibitory than lactic and citric acids. Addition of 1.. The acetic acid in the brine inhibited anaerobic and aerobic bacteria.5% acetic acid reduced Enterobacteriaceae counts by 2.6% acetic acid-treated carcasses was darkened or yellowed (Dickens et al.5% concentrations of acetic. and 0. although the skin of the 0. Immersion of broilers for 10 minutes in 0..0% acetic acid caused instantaneous death. and tartaric acid adjusted to pH 5.6% acid solutions. 1987).

aerobic bacteria. cabbage.5 inhibited Saccharomyces ellipsoideus and Penicillium glaucum. 1969). It was found that acetic acid was the most effective acid.01% to 0.6% of their body weight (Sollman. but a concentration greater than 4% was needed when the pH was at 7. and 0. Foods. ready-to-eat products. Takhirov (1969) suggested limits of 0. lactic. Rats consuming acetic acid in drinking water in concentrations of 0.001 M accelerated spindle activity and retarded anaphase in the mitotic cycle of the root tips of the onion. lactic. Although acetic acid is generally used as an inhibitor of bacteria and yeasts. gastric erosion (O’Keane et al. respectively (Buchanan and Ayres. Cruess and Irish (1932) showed that apple juice containing 0.5 completely inhibited growth and aflatoxin production by Aspergillus parasiticus. displayed synergistic effects in increasing the destruction of the organism. Spore-forming bacteria are killed on surfaces at a concentration of 0.29 mg/m3 caused changes in electroencephalograms. Rats fed 10% acetic acid and cats fed 20% acid developed ulcers in the gastric mucosa (Okabe et al. 1968). are especially susceptible to spoilage because of indigenous microbial populations. 1932).. 1971). 1958). 1949).5% level. Damage from acetic acid resembled that from ionizing radiation or radiometric chemical damage (Manna and Mukherjee. interference with blood coagulation (Fin'ko. 1976).2 g/kg body weight) did not show any adverse effects. Lower concentrations of 0.5.0% acetic acid adjusted to pH 3.2% acid at pH 5. it was not possible to determine the actual quantity of acid consumed.8% partially inhibited growth and decreased toxin formation by 70% and 90%.0. Rats exposed to vapor levels of acetic acid showed muscle imbalance at 0. A. 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. At the 0.4% acetic acid at pH 5. particularly precooked. On contact with organic substrates. glucono-delta lactone. it is effective against the black bread molds.2 or 5 mg/m3 but not at 0. 1921). niger. and death.2). chilled. When used as a surface application. and citric acid when spores were heated at 95°C for 15 minutes and acetic.2% (0.01 mg/m3. A combination of these parameters could be used to extend the shelf life of these products.999% after exposure to a 1. Ingestion of acetic acid by humans has caused burned lips (Palmer. Levels of 0. Other forms of acetic acid have antimicrobial uses.0% concentration of acetic acid. Mori (1952) fed rats acetic acid ranging from 10 to 50 cm3 acid/kg of rice. and coliforms.48 mg/m3 altered sensitivity to light. in combination with organic acids. 1937). followed by glucono-delta lactone.04 M acetic acid at pH 3 to 4 showed chromosomal and chromatid breaks. Grasshoppers treated with 0. a 1% concentration of acetic acid at pH 4. however. Peracetic acid is formed by an oxygen molecule bound to the carboxyl carbon atom of acetic acid. and Rhizopus nigrificans at pH 3. Toxicology Acute toxicity data for acetic acid in mice and rats indicate varied tolerance levels by different routes of administration (Table 4. Aspergillus fumigatus required 0.8 and 0. Populations of Pseudomonas aeruginosa were reduced by 99. 1971). Parmeggiani and Sassi (1954) reported that workers . and potatoes than peroxyacetic (80 ppm) acid alone (Hilgren and Salverda. and citric acids when temperatures were reduced to 65°C for 60 minutes. 2000). 1966). however..Organic Acids 99 spores were more restricted.0001 and 40% relative humidity within 10 minutes (Portner and Hoffman.0 and below to inhibit growth (Kirby et al. Nisin. pneumonia (Gerhartz. Studies on nonmammalian species showed that acetic acid at a concentration of 0. Allium cepa (Makinen.. renal failure (Paar et al. 1969).. suggesting its use as a decontamination agent for equipment (Hedberg and Miller. This treatment could also be used in recycling of water to reduce levels of yeasts and molds.60 mg/m3.8% to 1.0001 and 0. rats decreased their food intake level and lost 2. it decomposes to yield oxygen and acetic acid. Johnson (1968) noted that rat gastric mucosa responded to a 100-mM concentration of acetic acid by releasing histamine into the interstitial fluid. 1968).06 mg/m3. All rats showed mucosa damage owing to the feeding method.6% or 0. Human olfactory thresholds to acetic acid vapor were 0.

100 Antimicrobials in Food TABLE 4. teeth. (1963) Horn et al. (1963) Hara et al.900 680 275 2.500 330 44 1. (1971) Gruber and Halbeisen (1948) Horn et al.430–4. calcium Propionate.200 485 Reference Spector (1956) Orö and Wretland (1961) Spector (1956) Spector (1956) Spencer et al. and tartaric acids. and lungs with an air concentration of 0. 1955). Men having 7 to 25 years of exposure displayed blood abnormalities (Cesaro and Granata.530 380 1. eyes.790 940–960 42 203 961 2. (1971) Rat Citrate. (1957) Gruber and Halbeisen (1948) Yokotani et al.860 2. as well as citric. sodium Tartaric Spector (1956) Orö and Wretland (1961) Hara et al.810 625 3. pharynx.040–5.730 1. caused allergic reactions by producing canker sores (Tuft and Ettelson.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) 4.100 1. sodium Dehydroacetic Adipic Rabbit Caprylic Citric Mouse Mouse Enders (1941) Orö and Wretland (1961) Yokotani et al. sodium Rabbit Mouse Rat Rabbit Rat Guinea pig Mouse Rat Rat Mouse Gruber and Halbeisen (1948) Yokotani et al.460 1.340 580–1. (1957) exposed for 7 to 12 years to acetic acid vapors in the production of acetyl cellulose displayed corrosion of the hands. and epidermal reactions (Weil and Rogers.020 5.310–3. (1971) Gruber and Halbeisen (1948) Gruber and Halbeisen (1948) Lactic Propionic Propionate.65 mg/L.430 600 5.700 11.700 725 884 5.000 1. 1951). (1950) Horn et al.2 to 0. No toxicologic data were available for peracetic acid. although it is assumed that this derivative would be assimilated in a manner similar to that for acetic acid. (1957) Acetate. lactic. 1956).210 338 3. . In rare instances acetic acid.960 525 3. 1956).380–3. cold sensitivities (Wiseman and Adler.

3 Acceptable Daily Intake for Humans Limitations (mg/kg body weight) Acid Acetic Acetate. Ca+. K+. Na+ Malic DL-malic Propionic Propionate. 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. salad dressings. the malic acid content of malic acid should not exceed 0. The data are scarce for the use of peracetic acid in food products. c Content of DL-malic acid. 1954). catsup. which limits its use. It is the principal component of vinegars and as such is primarily used for its flavoring abilities. it has been added to infant feeding formulas to replace lactic acid (Seymour et al. Ca+.. Application and Regulatory Status Acetic acid is a monocarboxylic acid with a pungent odor and taste. 1980).3. Mendonca et al. K+.. K+. Acetic acid is generally regarded as safe (GRAS) for miscellaneous and general-purpose usage (21 CFR 184.05%. 1939). . It is used in condiments such as mustard. 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. The salt form. It is highly soluble in water. Ca+. the estimated acceptable daily intakes listed here do not include amounts occurring naturally. Na+ Fumaric Lactic DL-lactic Lactate. NH4+.Organic Acids 101 TABLE 4. Because of cost and antimicrobial action. b Content of DL-lactic acid. requires different handling and use procedures than the acid. ACETATES Antimicrobial Properties Several derivatives of acetic acid are currently in use as antimicrobial agents. K+. however.. The acceptable daily intakes for humans for acetic acid and its derivatives are listed in Table 4. Na+ Sodium diacetate Adipic Citrica Citrate. Ca+. (1989a) used a dip of SA in combination with potassium sorbate (10%) and phosphates (10%) to extend the shelf life of pork chops. Na+ a Unconditional Not limited Not limited 0–15 Conditional Reference FAO FAO FAO FAO FAO FAO FAO FAO FAO FAO FAO FAO FAO FAO FAO FAO (1965 (1963. Na+ Tartaric Tartrate. and mayonnaise and is found in pickled products such as sausages and pigs’ feet. 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.1005). K+.

SA extended shelf life to 12 days at 4°C. Waterhouse and Scott (1962) attributed the toxic effect to the sodium ion rather than the acid moiety. or trisodium citrate were used alone or in combination with Bifidobacterium breve (5%) as a dip for fresh camel meat (Al-Sheddy et al. Catfish fillets treated with 2% SA. 1..7 logs when stored at 4°C compared with the nontreated fillets and increased shelf life by 6 days. Chickens fed diets supplemented with 5.2 logs by day 12.2. potassium sorbate (1. A combination of B. and trisodium citrate extended shelf life to 6 days. and 2. No toxicologic data were available for calcium acetate (Food and Agricultural Organization. although a mild acetic acid odor was detected. 1996).5%).5% lactic cultures or 0. 1976). 1995a). SA alone or SA-MKP-treated fillets were not significantly different in odor from fresh controls up to 9 days. 5% sodium lactate. and held at 4°C for 12 days contained lower population levels of psychrotrophic and anaerobes than untreated samples (Zhuang et al. 1994). 1994). Psychrotrophic counts from untreated whole shrimp increased to >107/g. Whole and peeled shrimp were immersed for 2 minutes in solutions containing 10% 6-week storage life (Blom et al. growth and toxin formation were decreased 70% and 90%. The combination of 0. B.0% concentration of SA at pH 4..3 or 1.5% potassium sorbate.5 to 1. .3% to 0.. but they did appear brownish and watery (Kim et al. breve.26% potassium sorbate or 2% sodium citrate in its efficacy (Wederquist et al. 1951).5% lactate was effective as an antimicrobial treatment in sliced servelat sausage over a 4. monocytogenes in vacuum-packaged turkey bologna during storage at 4°C and was similar to 0. For peeled shrimp. 1997). whereas potassium sorbate increased shelf life to 9 days.. but SA provided better sensory characteristics and more controlled growth of psychrotrophs than potassium sorbate with bifidobacteria.25% acetate and 2. SA in combination with potassium sorbate and Lactococcus lactis ssp. A 22-year-old woman with sensitivity to acetic anhydride was not allergic to SA (Weil and Rogers. 1963).25% potassium sorbate. depressed growth rate. Histologic findings showed hypertrophy of the kidneys and ureters. breve culture and stored at 4°C for 9 days (Al-Dagal and Bazaraa.4% monopotassium phosphate (MKP) more effectively prolonged shelf life to 12 days compared with SA alone.6 to 0. although it is assumed that this derivative would be assimilated in a manner similar to that for acetic acid.. parasiticus was inhibited by a 1.75% and 1% significantly reduced the initial aerobic plate counts of catfish fillets by 0.7% SA and 0. over 6 days.5% trisodium citrate with or without B..102 Antimicrobials in Food SA (10%).5%) was also effective in reducing populations of L. A combination of 0.5% SA.6% or 0. 0. 1999). 1999). At lower acid concentrations of 0. and 1. Growth and aflatoxin production of A.44% SA showed decreased appetite.5. 1995b). the indicator of spoilage. cremoris culture added to catfish fillets was an effective treatment against growth of Gram-negative spoilage bacteria at 4°C (Kim and Hearnsberger. but bifidobacteria did not grow within 9 days. Color remained acceptable. although MKP alone had no effect. The combination of 0. breve and SA exhibited an additive effect that extended shelf life still further. sodium lactate (3%). Toxicology The LD50 (mean lethal dose) for SA in mice is presented in Table 4. untreated samples reached the spoilage mark within 10 days. SA at concentrations of 0. 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. sodium lactate. SA (0. and increased mortality. respectively (Buchanan and Ayres.0% SA alone inhibited growth for more than 6 days at 4°C and counts increased only by 1. vacuum packaged. Treatment with SA alone or in combination with bifidobacteria extended shelf life at 4°C by 3 days (Kim et al.8%.

2% sodium diacetate. S. 2001). monocytogenes grown in BHI broth adjusted to pH 5. coli. 1981).55) at 5°C. Y.5 inhibited these same strains and Mucor pusillus in feeds and silage.5% and 3. 2. 1994). and stored for up to 40 days at 4°C.3 and incubated at 5°C. and B. monocytogenes and bactericidal . A concentration of 0.2% sodium diacetate.25.1% or 0. The minimum inhibitory concentration (MIC) of sodium diacetate was determined for L. or 1% buffered sodium citrate (Stekelenburg and Kant-Muermans 2001). cereus with no effect against Pseudomonas fragi. 20°C. L. Aspergillus glaucus.05% to 0. In ground beef.3% sodium lactate. The growth of L. L. respectively. and when it was incorporated into butter wrappers. 1972). and 28 mM (pH 5. Lactobacillus curvatus and L.4.5% at pH 4. In addition to L.1721) and calcium (21 CFR 184. while having little effect on baker’s yeast. it prevented mold growth (Chichester and Tanner. monocytogenes was not significantly different in the presence of the individual inhibitors compared with control samples after 20 days at 10°C.4% at pH 3. Sodium diacetate was more effective than acetic acid. Calcium acetate is classified as a stabilizer when migrating from food packaging materials (21 CFR 181. Enterococcus faecalis.2% sodium diacetate. A. and 6. Cooked-in-bag ham was formulated with additions of 0. vacuum-packaged. Enteritidis. 1994).3% sodium diacetate was effective in suppressing total aerobic counts. the combination 0. However.8% and 2. 6. 0. L.1185) acetates are approved as GRAS substances for miscellaneous and general-purpose usage.Organic Acids 103 Application and Regulatory Status Sodium (21 CFR 184. The MIC was determined to be 35 mM (pH 5.5% sodium lactate or combinations of the acids and held at 5°C or 10°C. SA can be used as a boiler additive for food-grade steam (Federal Register.1% and 0.5 weeks in products containing 0. Saccharomyces species (Glabe and Maryanski. or S. Sodium diacetate is equally effective in the baking industry where its inhibitory powers prevent the growth of bread mold and rope-forming bacteria. the addition of 0. and Penicillium expansum. An interactive effect was noted between temperature and concentration of acid in that growth was inhibited with decreasing concentrations of acid and temperatures. monocytogenes.25) at 35°C. 5. sodium diacetate was also effective against hemorrhagic E.5% level in malt syrups.5% sodium lactate was bacteriostatic to L.0% delayed mold growth in cheese spreads. fumigatus.2% sodium diacetate and 2. curvatus reached an end point of 107/g within 2 to 2. a concentration of 0. and the inhibitory effect was attributed to the diacetate moiety rather than pH alone. niger. SODIUM DIACETATE Antimicrobial Properties Sodium diacetate was effective at a 0. Enteritidis were inoculated into a comminuted beef emulsion formulated with 0.1% and 0. monocytogenes was inhibited by sodium lactate and 0. monocytogenes were inoculated into the cooked ham products at an initial level of 104 and 102/g of product. Sodium diacetate at concentrations of 0.5 inhibited Aspergillus flavus. and 35°C (Shelef and Addala. monocytogenes increased to approximately 106/g within 3 weeks. or 1. 1962). A. monocytogenes and S.2% SA. 5. The addition of sodium diacetate affected the sensory qualities of the product precluding its use. in products containing buffered sodium citrate.55.1% to 0.0. Pseudomonas fluorescens.3% sodium lactate.1% to 2.5% or 3. aureus (Shelef and Addala.15% to 0.29). Lactobacillus fermentans. such as Bacillus mesentericus (subtilis).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. enterocolitica.4) at 20°C. Levels of 0. L.2% sodium diacetate or 1% buffered sodium citrate and within 3 and 5 weeks for products containing 2. However. Shewanella putrefaciens. 32 mM (pH 5.

use levels range from 0.. although it is fungistatic at concentrations of 0. This acid is effective against Enterobacter aerogenes and L. the combination of acids achieved greater reductions of both organisms at both temperatures compared to individual acids and greatly reduced background spoilage microflora. As expected.2% sodium diacetate alone or in combination in beef bologna. glaucum and A. Toxicology No toxicologic data were available for sodium diacetate (Food and Agriculture Organization.4% (Glabe and Maryanski.8 logs (Degnan et al.3% and 0. plantarum at levels of 0.0 against S. 1993a). monocytogenes in mixtures held at 4°C for 42 days was inhibited at 0.1%. 1994). 0. monocytogenes and Salmonella species was demonstrated using 2. cerevisiae and 25 times more effective against P. monocytogenes in turkey mixtures. 2002).25% to 0. Application and Regulatory Status Sodium diacetate is approved as a GRAS substance for miscellaneous and general-purpose usage (21 CFR 184. 1950). Application of 4 M SA reduced the levels of L. Schlyter et al.005% to 0. respectively. In subsequent work (Mbandi and Shelef. In bread and cake products. although it is assumed that this derivative would be assimilated in a manner similar to that for acetic acid.5% concentration (0.5% sodium lactate and 0.1% (Wolf. Sensory characteristics of roasted chickens were not affected even at levels up to 8 g per chicken (Moye and Chambers.1754). 1989).5%).104 Antimicrobials in Food to S.. Sodium diacetate was determined to be listericidal over the 7-day period at 25°C only at the 0. monocytogenes was reduced by 2. a more complex product. Sodium diacetate (0. Mixtures were incubated at 25°C or 4°C and sampled up to 7 and 42 days. 1991). Slurries of uncured turkey breast. The growth of L.5% concentrations (0. . Sodium lactate did not affect the pH of the meat. Enteritidis.8. It is used in cheese spread. and pediocin (5000 U/ml). 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. the reduction of multiple strains of L.3% and 0. DEHYDROACETIC ACID Antimicrobial Properties Dehydroacetic acid (DHA) has one of the highest dissociation constants (Table 4. stored aerobically at 5 and 10°C. populations increased approximately 0. malt syrups.5%) was listericidal in combination with ALTA™. niger (Banwart. Although 1 M sodium lactate solution reduced populations within 2 days. Enterobacteriaceae counts dropped 1000-fold over 6 days at 2°C. a fermentation by-product of lactic acid bacteria. 0.1%. butter. (1993b) used the multiple-barrier concept to limit growth of L. Lactate and pediocin enhanced the listericidal properties of diacetate suggesting some type of synergistic activity.54 log reduction). Sodium dehydroacetate was twice as effective as sodium benzoate at pH 5. L.6 logs in crabmeat stored at 4°C for 6 days when treated with 2 M sodium diacetate. 1981).5%). monocytogenes at a level of approximately 4. lower concentrations of the antimicrobials were equally as effective. monocytogenes by 0. were pasteurized for 10 minutes at 68°C after which they were inoculated with L. 1962). respectively. but sodium diacetate reduced the pH slightly.1) of the acidulants and remains effective at higher pH ranges. producing a 2-log reduction after 7 days at 25°C (Schlyter et al. sodium lactate (2.5 logs CFU/ml slurry. The powder dissolved in the surface moisture of the carcass to form acetic acid and SA and produced a surface pH of about 4. prepared with sodium diacetate (0.59 log reduction). At lower temperatures. and wrapping material.3%.5 logs within 6 days.

1953). which included marked obstruction in the liver and kidneys by the oral route and polyuria by intravenous administration. but significant growth depression . mortality. and sodium dehydroacetate acid were investigated for three strains of E. at the higher dosage levels. 1957). coli O157:H7 in broth and model meat products. 30. However. Enders (1941) showed similar symptoms in rabbits. appearance. A 2-year feeding study with male and female rats fed 0. This acid at the appropriate concentration level would inhibit the growth of any microorganisms susceptible to low pH values. 2000). although a dose of 200 mg/kg led to growth and organ changes. 0. Longer studies using male and female rats given 400 or 800 mg/day did not affect offspring.0% adipic acid showed no effect at the two lowest levels. and caused slight histologic aberrations.25% or 5% sodium dehydroacetate for 60 seconds were considered more marketable after 3 days of storage at 20°C than water-dipped berries. weight gains were lower and mortality was higher than in the controls (Lang and Bartsch. 1950). sodium p-hydroxybenzoate. and sodium dehydroacetate and 16 mg/ml for sodium p-hydroxybenzoate. 1971).1%. Toxicology Acute toxicity data for adipic acid in mice and rabbits indicated varied tolerance levels by different routes of administration (Table 4. Sorbate was bacteriostatic at half the MIC for E.2.5 was 4 mg/ml for potassium sorbate. benzoic acid. 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. (1950) did not show any unusual effects in rats receiving 10. The 90-day feeding trials conducted on rats fed 0. or 5. the 800 mg/day dosage depressed growth. treatment with sodium dehydroacetate was effective in reducing respiration rate and delaying ripening. hematology. or 100 mg/kg of DHA per day for 34 days. altered behavior. Toxicology The LD50 for DHA in rats is given in Table 4.. It can also be used as a fungistat in cheese wrappers.130). thus extending marketability (Watada.05%. 300 mg/kg led to severe weight loss and damage to internal organs. resulting in the loss of up to 20% of body weight. 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. in addition to acidosis by the former route and intestinal adhesions by the latter route (Horn et al. However. The MIC in broth at pH 6. Furthermore.0%. produced diarrhea. or histopathology.1% of the substance showed no changes in growth.02%. and 0..2). 1953). Oral administration of the acid to mice caused hemorrhaging in the small intestine with distension of the stomach. benzoic acid. The effects of potassium sorbate. Application and Regulatory Status DHA or its sodium salt is GRAS when used in accordance with good manufacturing practices. The sodium salt of DHA was nonirritating to rabbit’s skin.. Spencer et al.Organic Acids 105 Strawberries dipped in or sprayed with 0. 1. Intravenous and intraperitoneal injection produced hemorrhaging in the lungs. even with prolonged contact (Spencer et al. coli in homogenized beef but not in poultry (Yamamura et al. However. ADIPIC ACID Antimicrobial Properties Adipic acid has no antimicrobial properties other than those ascribed to its ability to reduce pH. 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.

It is used in gelatin desserts.16% for snack foods. Application and Regulatory Status Adipic acid is a nonhygroscopic. No limit has been set on the acceptable daily intake for humans. coli when grown in a chemically defined medium and was more effective than 1.0.8 µmol/ml (Kabara et al. 1975b).106 Antimicrobials in Food occurred at the 5. however. Caprylic acid at a 0.3. caprylic acid inhibited bacteria and fungi at a lower concentration than either acetic or propionic acid. refrigerated rolls.8% propionic acid.0. Inhalation of adipic acid for 6 hours at a rate of 126 µg/l produced no unusual findings (Gage.013% for baked goods. frozen dairy desserts. and in improving the melting characteristics of processed cheese (Gardner.1025). At pH 5.5% acetic acid and 1. gelatins and puddings. the inhibitory concentrations were not dramatically lower (Woolford. 1973b). 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.005% for fats and oils.. not to be used in amounts exceeding good manufacturing practice. It is slightly soluble in water. Application and Regulatory Status Caprylic acid is a colorless. and soft candy. however. and biscuits because it imparts a smooth. It also serves a function in the canning of vegetables.0%. Sodium adipate at the 5% level decreased the rate of weight gain (Informatics. At pH 6. CAPRYLIC ACID Antimicrobial Properties When tested against a variety of Gram-positive and Gram-negative organisms and yeasts. It is used as a flavoring adjuvant in levels not to exceed 0. Inc.0% level although the food intake level was the same. The acceptable daily intake for humans is listed in Table 4. rancid taste. and 205 mg/kg body weight. 0. 1970). . 3.1%. 0. Toxicology Orö and Wretland (1961) determined the LD50 of caprylic acid for mice by intravenous administration (Table 4. 1943). 1. baking powders.1009). 1972).3% concentration inhibited E. and 5% adipic acid and female rats fed 1.. weight gains for rats fed the two highest levels were significantly lower than those for control rats (Horn et al. caprylic acid was ineffective at concentrations of up to 7. and 0. respectively (Food and Drug Research Laboratory. rats. Caprylic acid is approved as a GRAS substance for miscellaneous and general-purpose usage (21 CFR 184. powdered fruit beverages. meat products. saturated dicarboxylic acid having low water solubility. The MIC of caprylic acid was 100 µg/ml in TSB for Vibrio parahaemolyticus (Beuchat.04% for cheese. 1957).0%. 1968).0% acid did not show any abnormalities.001% or less for other food categories. 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. Caprylic acid was also more inhibitory to Shigella species than either acetic or propionic acids (Nakamura and Zangar. Caprylic acid is also used as an antimicrobial agent for indirect use in cheese wraps. and hamsters showed no significant difference from controls at levels of 263.. 0. as a sequestrant in oils. oily liquid that has a slightly unpleasant odor and a burning. 288. Teratologic studies using mice.2). mildly acid flavor. candies. 1972). The 2-year feeding trials using male rats fed 0. 1980).

Sorrells et al. 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. indicating that citrate was the more effective compound. Citric acid at a concentration of 0. A comparison of equimolar concentrations identified citric acid as the most antimicrobial. citric. then lactic. but inhibition increased with decreasing pH (Murdock. such as texture or appearance.7 juice incubated at 20°C were 6. pH was not considered a reliable indicator of preservation effectiveness (Fabian and Wadsworth. When tomato juice was adjusted with citric acid to pH 4. Based on pH. acetic. followed by acetic acid.0.Organic Acids 107 CITRIC ACID Antimicrobial Properties Inhibition of L. 1939. . lactic. 5.4 and 0. The authors suggested the inhibitory activity might be the result of the chelating ability of citric acid. 1973.66 at 30°C. Citric acid was particularly inhibitory to flat-sour organisms isolated from tomato juice.7% at pH 6.19 at 5°C. propionic. 1953). The inhibitory capacity of hydrochloric. 1950). Yeast and mold counts were the reverse with counts of 2. and propionic acids and for 6 weeks in media containing HCl or lactic acid.0 with citric and hydrochloric acids (Conner et al.09. 4. aerobic counts at 20°C for pH 3.13. Additional interactive effects were also noted in the presence of salt. monocytogenes after 4 weeks were pH 4.42 log/ml (Bizri and Wahem.8 and 2.4) in inhibiting Arcobacter butzleri in a broth system (Phillips. monocytogenes survived after 11 to 12 weeks in TSBYE adjusted with acetic. Sodium citrate was also more effective than 2% sodium lactate. followed by hydrochloric. 1987). These experiments used citrate phosphate buffer in which the citric acid content was calculated at 1. 1994).64 log/ml. and 7 (Buchanan and Golden. The effect was temperature dependent in that survival of L. canned in a brine containing acetic. lactic.0 at 5°C. Interaction was demonstrated between citric acid and sodium citrate added to BHI broth adjusted to pH 4. L. compared to counts in unacidified juice (6. the highest inhibitory activity was associated with propionic. citric. 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. Schoenemann et al.3. 1999). and the combination of 1.. Acidification impaired the color but enhanced the flavor of canned okra. citric. whereas at 10°C. Similar pH-temperature relationships were noted by Cole et al. Canned tomatoes acidified with citric. 1994). and acetic (Brackett.0 with propionic acid. and acetic. pH. Okra. and 4. All acids would be effective antibotulinal agents at that pH level (Nogueira et al. aerobic counts for pH 3. monocytogenes was achieved in trypticase soy yeast extract broth (TSBYE) adjusted to pH 5.83 at 10°C and 7. however. 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. or malic acids did not change in physical or chemical attributes during processing compared with unacidified tomatoes (Schoenemann and Lopez. phosphoric. citric acid was more inhibitory than lactic acid.. Rate of inactivation of L.5% sodium citrate and 1.5% sodium lactate was equally inhibitory as sodium citrate alone. 4.7 and stored for 7 days at 5°C. Little bacteriostatic activity occurred at pH 5. lower aerobic plate counts of 3. (1990).1% at pH 4. and 4. respectively.5% (pH 4. acetic. enterocolitica based on concentration of acid.22 log/ml). 1990). and 1. was processed for 30 minutes in boiling water. (1974). 1997). but lower pH levels were toxic. who determined that the minimum pH values that allowed survival of L.12 log/ml. The activity of the undissociated portion of the acid was hydrochloric followed by lactic propionic.5 with acetic and lactic acids. malic. 6.5) was more effective than lactic acid (pH 3.0.7 juice were 4. or tartaric acid to achieve an equilibrium pH of 4. monocytogenes decreased to undetectable levels within 1 to 3 weeks at 30°C. and hydrochloric. Fabian and Graham.0 and 3. citric. respectively. and degree of dissociation. 2. fumaric. and phosphoric acid was compared in TSB for Y. Citric acid was more inhibitory to thermophilic bacteria than acetic or lactic acids. lactic.36 at 10°C. phosphoric. (1989) reported that on an equal molar basis. propionic.6 log/ml were seen.

2.2% or 1. 1990). but the shelf life was shortened at this higher temperature. Enteritidis PT4 at 22 than 5°C. If the amount of lemon juice was increased to 20 to 35 ml per egg yolk.1 and 5. 2003). 0.29% CA. heat resistance was not affected by higher processing temperatures.. The preparation of these products included adjustment of the pH with acetic acid to 3. Both vegetable broths stimulated germination and growth of spores compared to nutrient broth. Notermans et al. thereby leading to its survival regardless of the change in pH (Ocio et al. Xiong et al.108 Antimicrobials in Food Beelman et al. sporogenes PA 3679 spores. this process reduced thermophilic spoilage from 68% to 23. 1992).5). Growth in carrot puree was temperature dependent because B.3 g/kg) for crawfish (Procambarus clarkii) stored at 4°C were not effective in retarding growth of L..9. Only 2.5 followed by canning in a solution containing 200 ppm ethylenediamine tetraacetic acid (EDTA) controlled the outgrowth of spores of C. 1990a).22.5 were inoculated with C.. B.. sporogenes. The authors recommended that the final pH should be <3. Liver pastes inoculated with 106 to 107 Bacillus spores per g and 104 C. botulinum type B when held for 70 days at 15°C or 14 days at 20°C. This process inhibited C. and 16°C (Valero et al. sporogenes spores per g were heated to an F0 of 0.05 M) solution buffered to pH 3.. 5.8.0% trisodium citrate (TCA).4 and 5. Samples pretreated with 1% citric acid displayed significantly lower total numbers.69) and processed with an F0 of 0.31% CA–2.0 at temperatures below 16°C. Although the heat resistance of C. or 0. 1992). 115°C. sporogenes decreased with decreasing pH levels. Mushroom extract acidified with citric acid to pH 6.1% potassium sorbate (Houben and Krol. This process improved quality and increased the microbial stability of canned mushrooms (Okereke and Beelman. Citric acid dips (0.. and 121°C. and 0. 0. sporogenes PA3679 after a sublethal heating process (Okereke et al. 1991). 5. 6. cereus strains grown in model vegetable broths were conducted at typical refrigeration and abuse temperatures of 5°C. The products were packed with or without modified atmosphere and stored at 4°C for 10 to 21 days. Cabbage and carrots were pretreated with the addition of a reduced calorie dressing or with a dip of 0. 1994. 1990b). 8°C. but could grow at 8°C only when the pH was higher (5.8% with the addition of EDTA. indicating a synergistic effect between pH and temperature.. 2000.85. cereus grew in zucchini broth (pH 6.05 led to more inactivation of S. 1999).0% TCA-0. leading the authors to conclude that the extracts have a protective effect on C.3 when at least 20 ml of lemon juice (citric acid) per fresh egg yolk was used.05. Samples containing the reduced calorie dressing were even lower in microbial numbers than aciddipped vegetables (Eytan et al.9% and spoilage was further reduced to 16. Extracts were then heated at 110°C.3. and 4. Okereke et al. provided temperatures were at 16°C.1 with holding temperatures between 18°C and 20°C for up to 3 days before consumption.5) at 12°C but did not grow in broth acidified to pH 5.4. 5. The addition of potassium sorbate did not enhance stability. By adding citric acid to can brine. Microbial stability was achieved in liver paste formulated with 0.0% citric acid for 5 or 30 minutes. 1989..3. 4.. 118°C.. the product should . Mayonnaise made with >5% citric acid solution adjusted to pH below 4. Citric acid dips for foods not only retard some spoilage but also act as a chelator of metal ions responsible for enzymatic browning reactions. coliforms.14% CA (pH 5.4% of cans containing mushrooms vacuum hydrated by the ABC process spoiled (Beelman et al. Growth curves developed for B. Homemade mayonnaise has been a significant health hazard as a result of contamination by Salmonella from raw eggs (Membre et al. and lactic acid bacteria counts than samples dipped with the lower concentration of citric acid.34. Reduction in the thermal process time of canned liver paste was achieved through the addition of 0. cereus could grow at pH 5.4 to 4. (1989) developed an acid-blanch-chelate (ABC) process designed to reduce mesophilic and thermophilic bacterial spoilage in canned mushrooms. 1997. (1985) dipped potatoes in a solution of 1% citric acid and 2% ascorbic acid for 2 minutes before vacuum packing and cooking. monocytogenes (Dorsa et al. They found that acid blanching in a citric acid (0.1. 12°C.14% citric acid (CA). Silla Santos et al.31% CA–2.65 and asparagus with added citric acid or glucono-delta-lactone to pH levels of 5. 1993).7 (control). and 4. 0.

25% citric acid and 0. Enteritidis.5% citric or 0. leading to enhanced growth. Mangoes are subject to spoilage through infection with Penicillium cyclopium (Palejwala et al. no growth occurred at pH 5. and 4%.016% to 1% malic acid as the sole carbon source because these acids predominate at these concentrations in a ripening mango. The metabolic effects of citrate on Arthrobacter species were twofold. 1962).5% lactic acid prevented toxin production. and 3 to 4 hours at 85°C. and in concentrations greater than 40 µM/ml. S. even in media containing up to 1% citrate. Citric acid provided the maximal destruction of ascospores at 85°C..0% was not as inhibitory to Streptococcus agalactiae when added to skim milk or fresh milk as citric acid at concentrations of 1%.75% lactic acid inhibited growth of Aspergillus versicolor. 1970). aureus was overcome by adding calcium and magnesium ions. A level of 0.0% to 12.. 0. However. fluorescens on beef carcasses (Thomson et al. The addition of garlic or mustard increased the death of S. when 35 ml lemon juice per egg yolk was added. Sabouraud medium was adjusted with 0. sodium citrate stimulated growth. Sodium citrate can also exhibit bacteriostatic activity. and rinsing with a citric acid solution adjusted to pH 4.6. however. 1970). 85°C. a 0. The MIC of 0. Thermal resistance studies were conducted in mango juice adjusted to pH 3. or shrimp and tomato sauce acidified with citric or acetic acids to pH 4. 80°C. 1968). The amount of sodium citrate had a dual effect on Lactobacillus casei. Typhimurium than lactic and hydrochloric acids (Subramanian and Marth.Organic Acids 109 be held at 22°C for longer than 72 hours.0% concentrations of citric acid and 0. With Arthrobacter pascens. with Arthrobacter simplex. Citrate inhibition of S. When acetic acid was used. Branen and Keenan (1970) also suggested that citrate chelation of metal ions inhibited L.75%. malic. but salt had a protective effect on this organism. A level of 0. Healthy tissue had a pH of 5..8% gave a pH of 4.75% for both citric and lactic acids had a slight growth-retarding effect on A. but 0. hard-cooked eggs allowed to equilibrate in 0. Skim milk acidified with citric acid was more inhibitory to S. citrate appeared to inhibit glucose utilization (Imai et al.0.8.0 reduced the numbers of P.75% concentration of either acid did not affect the growth of P. 5 hours at 80°C. 1985).. the function of citrate appeared to be one of chelation of metal ions. In concentrations of 12 to 18 µM/ml. 1976). the time decreased to 48 hours before use. 1968). lactic. Similar destruction values were seen with potassium sorbate and sodium benzoate. C. A 0. 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.25% concentration greatly reduced toxin production.2% sodium benzoate were held for 30 days at 4°C or .. 2%. or 1.1% to 4. casei. sodium citrate inhibited growth. No toxin was detected in any of these foods over an 8-week shelf life (Post et al. and 90°C. Survival studies for Neosartorya fischeri ascospores were conducted in mango and grape juices and heated at 75°C. Tsang et al. (1985) found no outgrowth or toxin production from spores of types A and B in TPGY medium acidified to pH 4. suggesting that citrate was a chelator of ions essential for growth (Rammell.12 (Sinha et al. parasiticus.3% citric acid lowered the level of Salmonellae on poultry carcasses. During ripening. 1998). botulinum showed no change in numbers when inoculated in tomato puree.5 with citric.5% sodium citrate were inhibitory to Salmonella anatum and Salmonella oranienburg (Davis and Barnes. 1952). Concentrations of 12.6 and incubated at 35°C.08 to 4.2 or 4.125% to 4% citric acid or 0.2.. 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. 1984). Sodium citrate in concentrations of 0. As little as 0. expansum (Reiss. 1984). however.8.1 or less. or tartaric acid. 1967). 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. it did not appear to affect attachment (Appl and Marshall. aureus showed inhibition both with increasing concentrations of citric acid and decreasing pH (Minor and Marth. whereas infected tissue was around 4. the level of acidity decreased and sugars and nitrogenous compounds increased. shrimp puree.5%. Using the multiple-barrier concept.

2%.4%. or 5% citric acid (Bonting and Jansen. and 4. Dalderup (1960) fed rats 1. however. or sodium salt (21 CFR 184.02 mol sodium citrate and citric acid. 1972). (1982) used a combination of 0. Application and Regulatory Status Citric acid is a tricarboxylic acid having a pleasant sour taste.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 4.75% citric acid alone for 21 days at 4°C. beverages.5.0 to reduce the growth rate of S. the urine contained calcium citrate.1751).7% sodium citrate (5% citric acid) fed to rabbits for 60 days produced no unusual effects (Packman et al. and rabbits indicated varied tolerance levels by different routes of administration (Table 4. (1957) evaluated dosage levels in mice given the acid intravenously.75% glucono-deltalactone incorporated into sausage before fermentation.110 Antimicrobials in Food in 0.2%. (1956) fed vitamin D-free diets containing low calcium levels but adequate levels of phosphorus supplemented with 0. Studies by Yokotani and coworkers (1971) compared toxicities of commercial citric acid and Takeda citric acid.1195). rats. aeruginosa (Restaino et al. and S. It can control the pH for optimum gel formation..5 depressed growth of L.8% level. 4. fruit preserves.. It is a precursor of diacetyl and therefore indirectly improves the flavor and aroma of a variety of cultured dairy products. . 1985).3. in the acid form (21 CFR 184.. Acute toxicity data of sodium citrate are shown in Table 4. Subcutaneous injections of 320 to 1200 mg/kg body weight of sodium citrate into dogs decreased blood calcium levels while increasing calcium levels in urine (Gomori and Gulyas. slight atrophy occurred in regions of the thymus and spleen (Yokotani et al. Typhimurium.75% citric acid was sufficient to reduce inoculated populations of S. Death by all routes and in all animals resulted from respiratory or cardiac failure and hemorrhaging of the gastric mucosa. Potassium sorbate at 0. sherbets and ices. aureus early in the fermentation and the increased acidity of the meat mixture allowed the fermentation to proceed more quickly. 1956. E. salad dressings. aureus (Fischer et al. potassium (21 CFR 184.. It is approved for use in ice cream. Toxicology Acute toxicity data for citric acid in mice.1625). The number of cavities formed did not differ between the control and experimental groups.2). the highest dosage contributed to enamel erosion. 3%. Long-term studies involving rats fed quantities up to 1. 1981). Restaino et al. 2. It is highly water soluble and enhances the flavor of citrus-based foods.5. Deaths resulted from acute acidosis and lung hemorrhages. The authors concluded that citrate had a rachitogenic effect on the test animals. plantarum but did not affect P. The acceptable daily intake for humans is listed in Table 4.1033) or as the calcium (21 CFR 184. or 12 g citric acid per kg of a noncariogenic diet to show the possible effect of citric acid on the development of teeth. 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. Cramer et al. and jams and jellies. Although there was no effect on weight gain. Short-term studies on rats fed 0. rouxii and Saccharomyces bisporus. This combination inhibited the growth of S. 1957) showed no abnormalities other than lowered weight gains and feed consumption in the 5% group. Y. 1944). Citric acid also acts synergistically with antioxidants to prevent rancidity by chelating metal ions (Gardner.2. A concentration of 0.. In another study. (1973) used a combination of 0. 7. Horn et al. enterocolitica. Horn et al.05% potassium sorbate and citric acid at pH 5. and it is used as an acidulant in canned vegetables and dairy products.2% concentration in combination with citric or lactic acid at pH 5. Daly et al. 1971).1% citric acid and 0. a by-product of yeast fermentation. coli. Citric acid is approved as a GRAS substance for miscellaneous and general-purpose usage when used in accordance with good manufacturing practices. 1963).

and lethality increased as the pH decreased from 5. 0. Medium chain-length alkyl esters had more activity than aliphatic acids (Dymicky and Trenchard.38% sodium fumarate or guinea pigs fed 1.. acetic. 1975). Islam..5. 1987). malic.. or tartaric acid based on weight when added to the heating medium. psychrotrophic.5% level. flavus ascospores (Beuchat. and 1.0% acid concentration at 82°C.5%. 1963).05%) or sodium propionate (0.125%. whereas the presence of fumaric acid esters in the bacon prevented swelling for up to 8 weeks.8%. monocytogenes and E. respectively. Monomethyl and monoethyl (0. Monomethyl. 0.6 L in the vapor phase were also effective as a mold preventive (Huhtanen and Guy.1%) controlled fungal growth in tomato juice.15%. 1996a. fumaric acid achieved the greatest reduction of aerobic.5% applied for 5 seconds at 55°C to the surface of lean beef inoculated with L. The use of fumaric acid to effect a 5-log reduction of E.Organic Acids 111 FUMARIC ACID Antimicrobial Properties Fumaric acid (0.5 to 5. respectively. and coliform bacteria. 1974) and inhibitor of malolactic fermentation in wine (Pilone.5% (pH 2. Toxicology Weanling rats fed 0.0% fumaric acid and 1.0% fumaric acid did not show any abnormalities.2% fumaric acid or rabbits fed sodium fumarate at a concentration of 6. These same preservatives provided a similar reduction in less than 5 hours and 2 hours when held at 25°C and 35°C. dimethyl. In addition to reduction of E. fischeri (Conner and Beuchat. and tartaric acids at a concentration of 2. citric.05% sodium benzoate followed by holding at 25°C for 6 hours before refrigeration at 4°C for 24 hours achieved reduction.15%) served as an acidulant (Ough and Kunkee. Fumaric acid by itself was only slightly inhibitory. Methyl n-alkyl fumarates demonstrated lower activity.1%. Fumaric acid was more fungicidal against Talaromyces flavus ascospores than acetic.5% concentration of fumaric acid inactivated ascospores after 20 minutes of heating at 80°C. malic. or 0. A 0. 1981.9% (5% fumaric acid) showed no toxic effects (Packman et al.0 to 2.. When used in vacuum-packaged ground beef patties. total aerobic counts were reduced to levels below those achieved through pasteurization. However. The fumarate esters not only prevented mold growth on bread (Huhtanen et al.1% and 1. It was speculated that the antimicrobial properties of fumaric acid were related to a lower pKa than lactic or acetic acids. Fumaric acid at concentrations of 1.0% and 1. and tartaric acids increased heat inactivation. 1947). Fumaric.2%) and dimethyl and diethyl fumarates (0. 1984). rats showed an increased mortality rate and more atrophy of the liver (Fitzhugh and Nelson. Human feeding . coli O157:H7. but malic acid did not. At the 1. The 2-year studies using rats fed 0. and di-n-alkyl fumarates were almost inactive (Dymicky et al.3 logs. 1987). coli O157:H7 reduced levels by 1 and 1. 1988). and ethyl esters of fumaric acid in concentrations of 0..15% fumaric acid coupled with 0. botulinum swelled in 4 to 6 days at 30°C. citric. 1982). 0. The addition of 0. 1982). botulinum types A and B in canned bacon. Similar effects were noted with N.b). Huhtanen (1983) and coworkers (1985) investigated the effect of various esters of fumaric acid on growth of C. Fumaric acid was also more effective than lactic or acetic acid when used in meat products (Podolak et al.0) were not lethal to T. The n-monoalkyl fumarates and maleates esterified with C13–C18 alcohols also possessed significant antibotulinal activity (Dymicky et al. 1987) heated in the presence of 2. but the dimethyl and diethyl esters at a concentration of 20 mg per 2. coli O157:H7 inoculated into unpreserved apple cider was investigated by Comes and Beelman (2002).2% showed considerable promise as a substitute for sodium nitrite in bacon. Cans packed with bacon and inoculated with C. citric.

25% concentration of acid sprayed on veal carcasses. A 2% L-lactic acid treatment at pH 2. reduced aerobic mesophilic plate counts from 5. this study showed that lactic acid was effective at higher temperatures as well. Because of these dosage levels. 1954). the organism responsible for flat-sour spoilage in tomato juice (Rice and Pederson. 1970).350).0% to 1. It increases the strength of gelatin gels. although more recently this acid has been used as a rinse for beef. vacuum packaging. and 0. 1985). pie fillings.1% ascorbic acids selectively inhibited Enterobacteriaceae at 10°C. 1975a). In addition to supplying acidity to a product.0 but was ineffective against yeasts and molds (Woolford. Fumaric acid and its salts are used for direct addition to food for human consumption (21 CFR 172. 1950). gelatin desserts.. The inhibitory capacity of this acid lies in its reduction of pH to levels below which bacteria cannot initiate growth. 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.3. A 1. 1983. dicarboxylic acid having low water solubility. Cold-pack cheese normally prepared with lactic acid was reformulated with acetic acid and inoculated with S. 1988).3 of veal tongues. and storage for 10 days. Acid treatment when combined with vacuum packaging was more effective in prolonging shelf life than vacuum packaging alone (Smulders and Woolthuis. . Osthold et al.6 to 2. The acceptable daily intake for humans is listed in Table 4. Fumaric acid is used in fruit drinks. Typhimurium. and wines.25% citric. Typhimurium were similar for both acids (Park et al. 1946). it blends with certain flavoring compounds to intensify the aftertaste of a flavor. Because most other studies chose refrigeration storage temperatures. fumaric acid does display some antioxidant properties in fat-containing foods (Gardner. combined with vacuum packaging and storage at 3°C. Application and Regulatory Status Fumaric acid is a nonhygroscopic. 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. 1972).. (1984) found that spraying beef and sheep carcasses with a combination of 1% lactic. In fermented foods. Enterobacteriaceae contaminated 50% of the untreated samples. 1991)..25% lactic acid spray of beef carcasses followed by a treatment of hot-boned cuts with 2% acid. 2% acetic. 1985). 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. lowered microbial counts after storage for 14 days at 2°C. biscuit doughs.112 Antimicrobials in Food trials using a 500 mg/day oral dose for 1 year were also negative.7 log CFU/cm2 (Visser et al. and chicken carcasses. Lactic acid was more inhibitory to Mycobacterium tuberculosis as the pH decreased (Dubos. 0. Destruction rates for S. 1988). Similar reductions in microbial counts occurred with a 1.000 mg/kg was proposed (Levey et al.. pork. citric. Woolthuis and Smulders. an LD50 of 8. but a 2% concentration led to discoloration of the carcass surface (Smulders and Woolthuis. Lactic acid was an excellent inhibitor of spore-forming bacteria at pH 5. followed by vacuum packaging. Although this additive has a strong acid taste. which was reduced to 10% after treatment. unsaturated. Lactic acid was about four times more effective than malic. LACTIC ACID Antimicrobial Properties Lactic acid has been used more extensively for its sensory qualities than its antimicrobial properties in the past. lactic acid coupled with other antigrowth factors excreted by lactic acid-producing microorganisms inhibits competing microorganisms. propionic. Lactic acid sprays have been effective in limiting microbial growth on meat carcasses under a variety of storage conditions.

. When sequential treatments were employed using pre-chill and postchill lactic acid sprays. 71°C. Minimal reduction in total plate and E. When temperature changed from 20°C to 70°C or lactic and acetic acid concentrations increased individually to 3%. Similar work was conducted using lean pork trim (LPT) and fat-covered pork trim (FPT) (Castelo et al. 0. coli O157:H7 and S. 2001a. As in beef trim.b). . 76°C. Application of 95°C water alone or with 2% L-lactic acid was effective in reducing E. or 510°C. pH 3. Typhimurium were subjected to an automated wash water system (Castillo 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. 2001a. 2001). but control cuts retained more desirable flavor profile (Eilers et al. The combination of acids performed as well as 1% lactic or acetic acids alone. Acid-treated samples were lighter in color as a result of leaching of the pigment during immersion. coli. aerobic bacterial counts and S. 0.5 logs (Anderson et al. 30 psi). 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. and at two time intervals.8 to 7. Significantly higher aerobic plate counts were obtained from hot-boned cuts over 17 days postmortem compared to cold-boned cuts. Lactic acid was most effective against S. The inoculated meat was subjected to sprays of tap water (15°C to 17°C. total fecal coliforms. but odor was not affected (Ellebracht et al. moisture content. reducing numbers by 2 logs and Enterobacteriaceae by 1.3 M lactic acid solution (LAC. 482°C. but shear values..6 with either 1% lactic acid at pH 2. b). 65 psi). and presumptive lactic acid bacteria. (1987) compared a combination of 1% lactic. Furthermore.1).. coli counts was detected for beef dipped in acetic or lactic acid solutions compared with a water-dipped control at day 1.2 logs.0). Meat color was affected after application and through storage at 4°C. coli biotype 1 counts. All products were stored at 4°C and examined for aerobic. psychrotrophic. 1994). 1998.. In addition. Acuff et al. 454°C. Kotula and Thelappurate (1994) compared acetic acid and lactic acid solutions applied at two concentrations.5) at a volume equal to 10% of the initial sub-primal weight. COM improved tenderness in muscle cuts.3 M calcium chloride solution (CAL. but this effect was not seen with acetic acidtreated tissue. 1990a. 1999).9 or 1% acetic acid at pH 3. sub-primal meat was injected with one of three treatments: 0. or hot air at 371°C. Microbial counts obtained from ground beef made from the treated beef rounds that were subjected to the multiple sprays were significantly lower than samples receiving a postchill rinse. coli was reduced by 6. Dixon et al.5 logs for E. hot water (30 psi) at 65°C. Typhimurium-inoculated beef trimmings. Typhimurium levels decreased approximately 1 log. 20 or 120 seconds. at 1°C to 2°C as a dip for rib eye steaks.. 1992).2%. and 0. and E. 0. 2% acetic. and sensory analysis were not affected by acid treatment.Organic Acids 113 Hot-boned. the FPT supported lower microbial numbers than LPT. only suggesting that lactic acid can exert a continued antibacterial effect during the shelf life of the product. (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.b). The system alone produced a 3log reduction. coli (Anderson and Marshall.6% or 1. 426°C. coli O157:H7 or S. pH 5. Postchill beef rounds inoculated with E. <1 log for Enterobacteriaceae and 0. 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.25% citric. or a combination (1:1) CAL/LAC (COM. E.1% ascorbic acids adjusted to pH 2. 2% lactic acid (12°C to 15°C. Beef trim meat used to make ground beef is of particular concern because of its higher microbial load. Treatments containing lactic acid resulted in the lowest counts without affecting sensory qualities. Typhimurium at 70°C. pH 2. Beef trim was fabricated to make predominantly beef trim lean (BTL) and beef trim fat (BTF) products that were inoculated with bovine fecal samples (Kang et al.3. combinations of the treatments were examined. or 82°C. Greater reductions on all microbial loads were seen in BTF compared to BTL.. Aerobic plate counts of meats injected with CAL were significantly higher than with LAC or COM. There was little difference in bactericidal activity between the combination and either acid alone on sub-primal cuts of beef.

and 200 IU/ml of nisin (Mustapha et al. Beef cubes were inoculated with L. 1992). 1993).5%).6 log/6 cm2. but the population of microorganisms shifted. and nisin did not enhance the effects of lactic acid against E. 1993). 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. lactobacilli and yeasts were the least sensitive.2 or 4. enterocolitica. by immersing porcine livers in 0. 2000). noncoated tissue became dehydrated during the 7-day study. The combination of acids and heat treatment led to reduction of spore-forming organisms in frankfurters. The use of these compounds was effective in reducing psychrotrophs and Enterobacteriaceae (Ariyapitipun et al. and polylactic acid. Not only did the microbial counts decrease as the time of decontamination increased. Elimination of these groups shifted the population to Gram-positive bacteria and yeasts. After 42 days of storage the initial inoculum of 5 logs was reduced to <1 log after using lactic acid. Frankfurter emulsions incorporated with acids to adjust pH to 5. and nisin (400 IU/ml) were used alone or in combination in beef to inhibit L. Psychrotrophic. coli O157:H7 in beef burgers. 55°C) had little effect on the aerobic plate counts taken from the surface of pork carcasses. but no differences were seen between polylactic acid and lactic acid. Salmonella species or Listeria species were not recovered nor were sensory characteristics affected (Prasai et al. 240. monocytogenes before vacuum packaging and storage at 4°C. Enterobacteriaceae. significant reductions were seen with all treatments. Beef was inoculated with L. coli O157:H7 was subjected to treatments of lowmolecular-weight polylactic acid..7. acetic acid (0. coli O157:H7 inoculated into beef trimmings (Bolton et al. Lactic acid treatments alone were only 50% as effective as acid/alginate treatments. 1993). . 180. and 0. and low-molecular-weight polylactic acid. (1984) effectively reduced total plate counts. Although the gels reduced populations of L. monocytogenes on beef tissue. monocytogenes. Woolthuis et al. and Lactobacillaceae counts by 2 to 3 logs. coagulans were heated to 121°C and 105°C or 110°C. these treatments were not deemed usable in the control of E. lactic acid. monocytogenes. These compounds reduced the level of L. Beef inoculated with E.. coli O157:H7. low-molecular-weight polylactic acid (2% PLA). 2002).6 for Bacillus stearothermophilus and pH 4.2 for B. 1. Gram-negative bacteria were the most sensitive to lactic acid. lactic acid.2% lactic acid for 5 minutes. After 28 days of storage. or hydrochloric acid (Lynch and Potter.. Numbers of S. 1992.. monocytogenes (Ariyapitipun et al. monocytogenes and treated with 2% lactic acid. 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. and 3200 units/ml pediocin.1.. nisin.. monocytogenes by 1. malic. Aeromonas hydrophila. A 1% concentration of lactic acid (pH 2.114 Antimicrobials in Food Lactic acid (2%). 40. or sodium lactate (4%) alone or in combination with pulse electric field or freezing was ineffective in reducing numbers of E. Therefore.000 IU/ml nisin. respectively (El-Khateib et al.. By increasing the concentration of lactic acid to 2%.8. whereas acetic acid was more effective than the acetic acid/gel treatment. vacuum packaging. Y. 2002). there was no significant difference between nisin. 1988). 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.5 and 4. Lactic acid was effective in decreasing the number of Enterobacteriaceae as well as other Gram-negative mesophilic and psychrotrophic spoilage organisms. followed by mesophilic Enterobacteriaceae. numbers of Salmonella species and Campylobacter species were reduced immediately and remained lower 24 hours after slaughter (Epling et al. Greater inactivation was noted for both organisms at the lower pH level when acetic or lactic acids were used but not with citric. psychrotrophic Gram-positive bacteria. The use of lactic acid for reduction of microbial numbers or pathogens has been similarly applied to pork products. Lactic acid (2%). or 360 seconds to 2% lactic acid. All treatments significantly reduced the number of L. Typhimurium were reduced more by exposure to acetic acid/gel treatment than acetic acid alone. and storing for 5 days at 3°C. or nisin and lactic acid. Five treatments were delivered to the pork skins: water-treated (control) or exposure at 21°C for 120. however. 1999).

.0) for chicken legs increased the shelf life from 6 to 12 days at 6°C. Chicken skin subjected to washing treatments using 1% lactic acid led to significant reductions of Salmonella species and L. S. tissue was dipped for 15 seconds in water or lactic acid and held for 15 days at 4°C.0 than cells grown at 37°C. Lactic acid (1%) added to both chill water (0°C to 1. acidified with lactic acid. pH 2. 2002).2) was examined for E.2 logs. This protective effect was even more pronounced with acetic acid (100. respectively. 1995. respectively (Sawaya et al. Although L.7 log CFU/g (van der Marel et al. E. Lactic acid dips have also been used successfully in the poultry industry. however. grew on both tissue types after acid treatment. E. Typhimurium to almost nondetectable numbers. although tissue treated with acid affected the growth rate. thermosphacta.1°C. reduced levels of lactic acid (0. Higher concentrations of the acid did not ensure greater decontamination nor did repeated treatments.7 log CFU/g and Enterobacteriaceae from 3.2 decreased from 5.. but no protective effect was observed if the sodium chloride was added to the medium 45 minutes after the inoculation.000-fold). When treated broilers were packaged using modified atmospheres.2 in media containing lactic. 1988). 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. sodium tripolyphosphate..8) and scald water (54°C/2 minutes) reduced the bacterial level of broilers inoculated with S.5 and 0. Salmonellae were eliminated from broiler carcasses after a 1-hour exposure. in sodium chloridefree. . or formic acids. sodium acid pyrophosphate. 1995). monocytogenes counts were reduced by 0. Lactic acid added to broiler chill water resulted in the development of a brown coloration. 1983). In an effort to eliminate carcass discoloration. hydrophila numbers declined by 5 logs within 11 days of storage. 1996).62) were combined with propylene glycol (20%) in chill water. 1990b). or Brochothrix thermosphacta (Greer and Dilts. Cells grown at 46°C were more heat resistant and better able to grow in media adjusted with acetic and lactic acids to pH 5.Organic Acids 115 P.5%.25%. Pretreatment of broiler carcasses with lactic acid buffer increased shelf life by 6 to 7 days at 4°C and 5 to 6 days at 7°C.2 to 3. 1995). discoloration still occurred and propylene glycol contributed an objectionable flavor (Izat et al. 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..000-fold. monocytogenes compared with monosodium phosphate. Lactic acid was more effective than acetic acid when used as a 10-minute rinse for fresh-cut vegetables. coli O157:H7 grown in TSB at 37°C (Casey and Condon.6 log CFU/g.3 prolonged shelf life to 8 days (Zeitoun and Debevere. or sodium hexametaphosphate.88 or 0. P. and a 2% lactic acid dip at pH 2.5% lactic acid/0. produced similar reductions of approximately 1 log (Zhang and Farber. Total microbial numbers from skin of birds immersed for 15 seconds at 19°C in 1% or 2% lactic acid at pH 2.. Consumer evaluation of chicken treated with a 0. and Beuchat. 1990). shelf life was lengthened to >36 and 35 days at 4°C and 7°C. Acid treatments of fat tissues led to the reduction of pathogen numbers to nondetectable numbers within 4 days. that contributed to spoilage. pH 2. 1990a). Levels of psychrotrophs decreased from 3. fragi.5% sodium benzoate dip concluded that sensory qualities were acceptable (Hathcox et al. None of the pathogens grew on lean tissue. acidified media. After inoculation.5% to 4%) and pH (4. E. coli demonstrated a 10-fold decrease in numbers when grown in media containing sodium chloride. A 10% lactic acid and sodium lactate-buffered acid spray (pH 3.3 to 2. acetic. Hwang.9 to 2. The interaction of sodium chloride (0. such as Pseudomonas species. however. and A. The number of Salmonella-positive birds was also reduced as a function of time of the dip (Izat et al. The spoilage organisms. These treatments inhibited hydrogen sulfide-producing bacteria. coli O157:H7 rapidly died at pH 4. fragi and B. Lactic acid added to scald water alone had minimal effect on reducing the numbers of contaminated birds. coli decreased 10. 1995). pH 2. The treatments did not affect sensory quality. other compounds.23 in cells exposed to acid to 5. such as chlorine or chlorine dioxide. 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 5.79 in acid-free media.

Lactic acid is used in the manufacture of jams. It is used to adjust acidity in brines for pickles and olives. Lactococcus. innocua from 804 to 982 mM. Toxicology The LD50 of lactic acid for test animals varied according to animal species (Table 4. D (-) or DL. confectionery products. Brochothrix.6 mg/100 g) did not differ in growth rate compared with control groups (Granados et al.2). MIC values for Salmonella species ranged from 714 to 982 mM and a narrower range for L. although Campylobacter was particularly sensitive with an MIC of 179 mM.116 Antimicrobials in Food El-Gazzar et al. and beverages. In two other instances. Infants died after consuming milk acidified with an unknown quantity of lactic acid. With increasing levels of lactic acid. (1987) found that A. 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. 1973) supported this view. developed acidosis.5 or 4. 1993). 1949). lactis.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... 20°C) for a number of bacteria and yeasts isolated from spoiled and unspoiled meat products (Houtsma et al. sherbets. appears naturally in meat products. improves juiciness.. and Staphylococcus ranged from 268 for S. contributes to water-holding capacity. Bacillus. Lactic acid is approved as a GRAS substance for miscellaneous or general-purpose usage (21 CFR 184. pH. (1970) recommended that the L (+) form should be used in feeding premature infants.8. weight loss.. jellies. Gram-positive bacteria including lactic acid bacteria.5% lactic acid (Trainer et al. A predictive model for growth of E. aureus and Lactobacillus coryniformis to 804 mM for Brochothrix. infants suffered stricture of the esophagus following ingestion of milk acidified with 1 teaspoon of 85% (Pitkin. monocytogenes and L. Ross et al. 2003. 1944). Some enamel decalcification was apparent in the experimental groups. cultures decreased production of aflatoxin G1. to prevent discoloration in fruit. The MIC of sodium lactate was investigated under optimum growth conditions (pH 6. Carnobacterium. increases meat yields. Ballabriga et al. coli in response to temperature. Presser et al.5% and 0. The autopsy revealed hemorrhaging and gangrenous gastritis (Young and Smith. The acceptable daily intake for humans is listed in Table 4. and extends the shelf life of products.2 using HCl or lactic acid stimulated by substituting half of the carbon content by lactate and a reduction in production at pH 6. Luchese and Harrigan (1990) also noted an increase in aflatoxin production adjusted to pH 4.3. Yeasts were very broad in . Therefore. parasiticus in association with L. Aflatoxin production also increased during growth of A.. Calcium lactate is used as a firming agent for apple slices. The Food and Agriculture Organization of the United Nations (Food and Agriculture Organization. 2003).1061).75% lactic acid adjusted to pH levels of 3. Premature infants fed acidified milk containing the isomeric forms of lactic acid. Application and Regulatory Status Lactic acid is a hygroscopic.5. and vomiting. see Shelef (1994) and De Wit and Rombouts (1990). 1998. syrupy liquid having a moderately strong acid taste. and in baking powders. dehydration. For an excellent review of the antimicrobial nature of sodium lactate. retains color. 1935) or 87. water activity. Gram-negative bacteria including Pseudomonas and Yersinia had a comparable range to Salmonella. 1945). parasiticus NRRL 2999 grown in a laboratory medium containing 0. and lactic acid concentration has been developed (Mellefont et al. Hamsters fed a cariogenic diet incorporating lactic acid in the drinking water (40 mg/100 ml) or in the feed (45..

. 6. 1995) examined the effect of 3% sodium or calcium lactate added to pork liver sausage and various heat treatments. Samples were stored up to 50 days at 5°C and up to 10 days at 20°C. 10°C. 5. spore germination.5. toxin production was detected in media containing 4% sodium lactate within 11 days. 1994a).0. At the abuse temperature of 20°C. lactates also contributed to lowering the aw of a food. monocytogenes was studied in a cooked meat model system containing varying moisture levels and 4% lactate.5%. Salts of lactic acid are equally as effective in muscle food systems. inhibition by sodium lactate was directly related to pH and temperature. respectively. 1996). Chen and Shelef (1992) suggested that in addition to pH change. 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. As expected. 1995). A subsequent study (Shelef and Potluri. Survivor curves were developed using L. The effect of sodium lactate on toxin production.982) did not inhibit toxin production. As expected L. Sodium. 1% sodium lactate delayed growth for 10 days. There did not appear to be a synergistic effect between a 4% concentration of lactate combined with 140 ppm . 1991. and 30°C (Houtsma et al.. 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. botulinum strains in peptone yeast extract medium (Houtsma et al. 6. 4% sodium or potassium lactate achieved reductions in L. calcium. potassium. and heat resistance was tested on proteolytic C. but the combination of 2% or 3% lactate and 2% sodium chloride at the same moisture level was inhibitory. Addition of 2% sodium chloride enhanced the listericidal activity of the lactates (Weaver and Shelef. with lower amounts of sodium lactate needed as pH and temperature dropped. This level was listeriostatic at a moisture of >55%.Organic Acids 117 their sensitivity.. Sodium chloride at the same water activity as sodium lactate (0.5. 3%. the rate of inactivation was dependent on the acid. 11 days for 1. As expected. At a higher temperature of 20°C. The calcium form was found to be more inhibitory than the sodium form. 6. which might explain their prevalence in spoiled meat products. innocua was modeled in broth adjusted to pH 5. comparable to a 3% concentration of calcium lactate.5. monocytogenes was inhibited by combination of 3% lactate and heat sterilization (121°C for 15 minutes) followed by a water bath heat process (70°C internal temperature) and least affected when lactate was added to sausage without heat treatment.0.0% sodium lactate. L. no growth or toxin formation was detected after 32 days of incubation for media containing 4. or 4% and inoculated with 104 to 105 L. monocytogenes. toxin formation was noted within 5 days for 0%. Using 108 CFU/g as a microbial end point.5%. monocytogenes as the target organisms. and 7. This model was then evaluated for use in a bologna-type sausage system (Houtsma et al. no toxin formation was detected after 49 days of incubation in media containing 3% sodium lactate. and the resultant pH.. The pH level was influential in inhibition by sodium lactate but not by sodium chloride. spore germination did not lead to growth after 7 days. however. monocytogenes per gram of sausage. and 4 using 0. or calcium lactate at concentrations of 2%. and potassium lactate were equally effective. indicating that inhibition was not the result of a lowering of water activity. At 30°C. Lactic acid/sodium lactate and acetic acid/SA were studied in a BHI broth at pH 7. Pork liver sausage was formulated with sodium. its concentration. 1994b).1. with Debaryomyces and Candida being the most resistant at 1161 and 1339 mM. Potassium lactate (4%) and sodium chloride (3%) reduced growth somewhat at 5°C. and 15 days for 2. 1993). 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. whereas a 2% level extended the time to 24 days. 1. The effect of sodium lactate and sodium chloride on the growth of L. Typhimurium when stored for 4 days at 20°C. 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. 1993).0 at 4°C. coli O157:H7 and S. Calcium lactate (2%) reduced populations below the inoculum level. 20°C. Calcium lactate was also more effective than sodium lactate in retarding growth of E.. 0. and 2 M concentrations (Buchanan et al.

Pork sausages manufactured with 25% fat or 8% fat with and without 3. 2.0% potassium lactate were inoculated with a five-strain cocktail of L.5 log CFU/cm2 in the same period. monocytogenes was delayed for 4 and 12 weeks at 7°C and 3°C in unsmoked. 2002). Lactate increased cooking yields. 2002). lactobacilli. enterocolitica. (1991b) did not find this effect in their studies. Levels of >3% sodium lactate or 1% each of lactate and diacetate were needed to prevented growth of Listeriae for 60 days at 4. The heat resistance of the two organisms was significantly lowered in beef containing either of the two concentrations of sodium lactate. Sodium lactate at concentrations of 3% and 4% were generally effective in limiting growth of L.5°C Lactate influenced the growth of lactic acid bacteria in meat based on the pH level and the atmospheric conditions under which postrigor meat was held (Grau.7 to 8. Although the aerobic plate counts for control meat increased from 2.6% sodium lactate into the product (Kilic et al. Cured meats. or E.25% or 0.0% potassium lactate showed differences in spoilage rates.. such as wieners. Minced beef containing 0%. uncured bratwurst manufactured with 3. Lactates inhibited the growth of psychrotrophic organisms and delayed the pH decline (Bradford et al. or 4. 1980). Although Shelef and Yang (1991) suggested a possible aw effect for sodium lactate. Frankfurters containing 2.4%. In retail products. or 3. coli O157:H7 (Miller and Acuff.5 reduced surface discoloration in chubs of fresh sausage and extended shelf life from 10 to 20 days. sodium lactate caused more rapid surface discoloration (Lamkey et al.5.b. monocytogenes. monocytogenes. Red color was preserved by using 2% to 3% sodium lactate.05 and 6. Y. but produced a mild throat irritation at a 4% concentration.4%. thermosphacta down to pH 5. monocytogenes and heated to 55°C to simulate a sous vide process (McMahon et al.. Papadopoulos et al. botulinum. Typhimurium. then cooked and vacuum-packaged the roasts. 1981). or 4% sodium lactate were inoculated with L.2%. Typhimurium. micrococci. 1991). enterocolitica or L. Sodium lactate was less effective against microorganisms when sausage contained soy concentrate.0 log CFU/cm2 after 84 days of storage. (1991a.8% sodium lactate was inoculated with Y. Both concentrations of potassium lactate significantly inhibited or delayed the growth of L. 2002). sodium lactate permitted aerobic growth of B.. coli O157:H7 and stored at 10°C. The pH of the product was unaffected by incorporation of sodium lactate. Papadopoulos et al. enhanced flavor at the 1% level by adding a salty taste.5 in a meat system prevented anaerobic growth of Serratia liquefaciens. Sodium lactate at a 6% concentration or 0. .. and yeasts at 42 days to homofermentative and heterofermentative lactobacilli and streptococci at 84 days. perfringens.1. Enterobacter cloacae. 0. C.1% sodium diacetate (Glass et al. Retention of nitrite was linked to higher pH levels and was unaffected by the incorporation of 1. aureus. and A. monocytogenes during storage at refrigerator and abuse temperatures. 1999). When raised to 210 mM. the amount of L-lactate ordinarily found in muscle was not inhibitory to any of the strains. 2%..25% sodium diacetate were the most effective treatments throughout the 120-day shelf life conducted at 4°C with minimal change in pH. the plate counts of treated roasts increased to 6.. 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. Over the storage life of the product. S. 2. S. 1997). The microflora changed from predominantly staphylococci and micrococci at 0 days to coryneform.4% sodium lactate/0. monocytogenes and stored at 4°C and 10°C for up to 90 and 60 days. 1993). A 100-mM concentration of sodium lactate buffered to pH 5. At pH 6. hydrophila and aerobic growth of the aeromonads (Grau. The growth of L. nitrite levels are reduced.0% or 3. 2001) compared the use of 3% or 6% sodium lactate... and E. respectively (Porto et al. 3%. 1994).5% SA or sodium diacetate as an antilisterial process for frankfurters. Another study (Bedie et al.118 Antimicrobials in Food potassium nitrite.c) injected beef top rounds with 0% to 4% sodium lactate. S. rely on sodium nitrite to inhibit growth of C. A 3% concentration of sodium lactate at pH 7. Cooked beef top rounds containing 1%.

Sodium lactate (3.. respectively. None of the treatments affected the growth of either organism. calcium lactate up to 0. Typhimurium. A 3% concentration of sodium lactate only minimally affected the growth of S. and stored at 27°C. or 3. or 10% lactic acid/sodium lactate buffer rinses (pH 3. whereas off-flavors developed in meat containing the 3. monocytogenes in TSB and 4% limited growth of L. Typhimurium but did inhibit P.8%. 2.5% concentration. or glycerol monolaurate enhanced inhibition of S. Sodium lactate was more effective at reducing pathogen numbers at levels up to 3. Of all the treatments. Modified atmosphere packaging (MAP) extended storage life for products without acid sprays to 17 days and even longer with sprays. or a combination of the two was inoculated with the two organisms and held at 5°C for 5 days. Nisaplin (500 IU/g).5% concentration was acceptable. which did not provide significant control (Stillmunkes et al. . Beef roasts containing sodium lactate. Organoleptically.2. Some treated chicken was packaged without further modification. monocytogenes and the shelf life of chicken legs (Zeitoun and Debevere. 2002).5% than sodium gluconate. 7. botulinum types A. Vacuumpackaged beef was treated with 2% sodium lactate. The meat was cooked to an internal temperature of 71. or sodium gluconate and inoculated with L. glycerol monolaurin. and E only when lactic acid was used to reduce pH below 5. 4 to 6. 3%. 1991). or 5°C. however. Odor concomitant with growth of psychrotrophic spoilage organisms occurred in untreated samples within 8 days and within 9. and 13 days for the 2%. all were stored at 6°C.5%. or Microgard™ (2%) (Rozbeh et al. thermosphacta and E.56 log during 17 days of storage for the legs treated with 10% buffer combined with MAP. not sodium. (1989) suggested that a possible mechanism for the action of lactate was the inhibition of a major anaerobic energy metabolism pathway necessary for growth. 10°C. Levels of L. 1993). They also noted that the inhibitory component was the lactate moiety. and 25°C. Harmayani et al. fragi at even at the 4% level. 5%. aureus grown anaerobically (Smith and Palumbo.3%. monocytogenes in comminuted chicken or beef when held at 35°C. pediocin (1400 U/g). 7°C.5%. They concluded that the two salts gave comparable results and could be used interchangeably. sporogenes were stored at 2°C. Notermans and Dufrenne (1981). suggesting that the lactate moiety was the effective component.. Maas et al. and 10% acid spray.0) alone or combined with modified atmosphere packaging was examined on the survival of L. Lactic acid at 25 to 30 mEq in combination with sodium nitrite. 5%. (1991) also found that 2% lactate inhibited the growth of C. fragi and S. or 7 to 8 days for each concentration.Organic Acids 119 Shelef and Yang (1991) found that a 5. nisin (1400 U/g. but other samples were placed into packages that were gas flushed with a 90% carbon dioxide/10% oxygen mixture. Sodium lactate at the same concentration had no effect. the 2. Excised skin tissue was analyzed for Listeriae for up to 17 days posttreatment.1°C. Lactates have been combined with other compounds in multiple-barrier studies.. Toxicity occurred within 3 days in turkey without lactate and within 4 to 5. Lactate contributed to water-holding capacity and increased cooking yields. The influence of 2%. respectively. monocytogenes increased by only 0. botulinum in comminuted raw turkey using levels of 2%. 1993). 1980). found that glyceryl monolaurate at a concentration of 5 g/kg of a meat slurry inhibited C. vacuum packaged. (1991) looked at the interactive effects of sodium alginate and calcium or sodium lactate used in restructured meats on P.0% concentration of sodium or potassium lactate delayed the growth of L. B. Product was considered spoiled when microbial numbers reached 7 to 8 log CFU/g. sodium lactate was the most effective during an 8-week storage period at 3°C. Reduction in production of hydrogen sulfide and other sulfur-containing compounds resulted from the decreased populations of psychrotrophs. 20°C. Unda et al. monocytogenes and C. Ground beef containing sodium alginate at concentrations up to 0.3%) was very effective in reducing numbers of B. 10. (1989) demonstrated that sodium lactate was effective in retarding toxin formation by C. potassium sorbate. 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. sporogenes. Maas et al.

PROPIONIC ACID Antimicrobial Properties Propionic acid inhibited spore-forming bacteria. jellies.. Comminuted salmon was mixed with sodium lactate. and stored at 5°C or 10°C.98.1% sodium erythorbate. thus affecting the sensory qualities of the meat (Egbert et al. 1972). The acceptable daily intake for humans is listed in Table 4. and an alginate binder. 1994). Chung and Goepfert (1970) compared various organic acids to determine the highest pH level that inhibited growth of S. Application and Regulatory Status Calcium (21 CFR 184. at 50. sodium chloride. encapsulated salt. Some discoloration and lipid oxidation occurred. 1993).000 ppm malic acid before mating showed no significant differences from the controls (Hazelton Laboratories. and hydrolyzed vegetable protein and containing 1%. probably as a direct effect of pH manipulation. Dogs fed the same three levels of malic acid showed no effects using the same parameters (Hazelton Laboratories.. inoculated with L. however. In general.0. 1970). and . Reproductive experiments using rats fed 1000 and 10. but the addition of 2% or 3% potassium lactate reduced microbial numbers. anatum.0 or 4. The products were inoculated with L. monocytogenes and total aerobic plate counts (Harmayani et al. Similar studies used low-fat ground beef patties formulated with carrageenan. especially rope bacteria (B. jams. It is used in sherbets and ices. 1992).8% sodium lactate. 0. The acceptable daily intake for humans is listed in Table 4.000 ppm.0. Toxicology Malic acid fed to rats at levels of 500 and 5000 ppm in the basal diet had no effect. or 3% potassium lactate. monocytogenes. 1971b). subtilis). and sodium nitrite.1207). vacuum packaged. and beverages.3. a 2% lactate and 3% sodium chloride inhibited L. monocytogenes for up to 50 days at 5°C. As the pH decreased to 5. Application and Regulatory Status Malic acid is a nonhygroscopic. Sodium lactate displayed synergistic activity with nitrite and increasing concentrations of sodium chloride. Carrageenan had no effect on bacterial loads.1768) are also approved. dicarboxylic acid with high water solubility. Malic acid is approved as a GRAS substance for miscellaneous and general-purpose usage (21 CFR 184. Nunheimer and Fabian (1940) found that malic acid was inhibitory against S. aureus at pH 3. primarily for its flavoring and acidification characteristics (Gardner.1069). Salmonella Senftenberg. fruit preserves. 2%.120 Antimicrobials in Food Raw and cooked ground beef were prepared with 1.3. It has a strong acid flavor but does not have the same buildup of acid taste as other acids. potassium (21 CFR 184. 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. sodium lactate was more effective than the other additives in inhibiting growth of L. and sodium lactates (21 CFR 184. the acid inhibited yeasts and molds but not to the same extent as bacteria (Woolford. monocytogenes and stored aerobically at 4°C. 1971a). food consumption decreased and growth rate declined (Hazelton Laboratories. MALIC ACID Antimicrobial Properties Malic acid is inhibitory to fungi and bacteria.1639). at pH 6. 1% kappa carrageenan. 1975b)..

1990). subtilis. or 3. acetate. Emphasizing the multiple-barrier concept in retarding growth. lipid. concentration of the organic acid. Products were stored for up to 24 days at 4°C or 8°C. At periodic intervals. pH 4. protein. 3. 1. 0. and S. lactic. aeruginosa. citrate. inoculated with C. and 35°C. cherries. DNA (deoxyribonucleic acid). monocytogenes was inoculated into BHI broth containing 0. 2002). 1989a). monocytogenes was the result of incubation temperature. Propionic acid at pH 5. 1%) and coated onto ready-toeat chicken samples dipped in L. 18 days for citrate. pH levels.156%.. and stored at 28°C.3% concentration in tryptose broth adjusted to pH 5. citric). pH 4. and lactate.1% to 5% delayed growth of S.9% sodium propionate or SA. particularly at the higher pH level.05.1. Reduction of pH to 5.0 with hydrochloric acid..000 IU/g) or calcium propionate (CP. Proteus vulgaris.3% concentration was also effective at pH 5. ellipsoideus by as much as 5 days. This substance not only postponed spoilage in fresh figs. 21°C. At a 6% concentration. Incorporation of calcium propionate into coatings for ready-to-eat products was effective in reducing problems with L.1. 13°C. E. pH 4. and pH. plantarum. samples were removed from storage and tested for production of neurotoxin. and 5 with a 2% concentration.4. A three-strain mixture of L.5. 1993). Nondetectable levels of the pathogen were found in products ZEN.4. coli K12 and Salmonella. (1991) determined that 0. Torula species. 1992). Propionic acid was more effective than acetic acid as an antimicrobial. aureus. ZPNCP. the levels of L. which led to an increase in cell mass without cell division and a decrease in viability (Cherrington et al. When media containing 0.6 (O’Leary and Kralovec. adipic. citric. S.5 inhibited the growth of Salmonellae at a higher pH level than other acids (pH 5.. 19°C. monocytogenes when used in conjunction with acetic acid in cold-pack cheese food (Ryser and Marth.188%. monocytogenes. aureus. such as lima beans and peas (Wolford and Anderson. the organism was able to grow at up to a 0. toxicity occurred after 7 days for pyruvate. 1988). 3 for citrate. 7. and propionate to a target level of pH 6.Organic Acids 121 Salmonella Tennessee when all other parameters were at their optimum.0 minimized growth at 13°C.08% EDTA.5. and 0. 4 for lactate and acetate. 1992).0 and 13°C.6 with acetic. and held at 28°C for 0 to 18 days. and temperatures. Golden et al.0 was more inhibitory in 60 minutes compared to formic acid in 3 hours for E. tartaric. Citrate was most effective based on molarity (Miller et al. monocytogenes were undetectable. Exposure of L. and Candida albicans. botulinum. At pH 5. coli. and greater than 18 days for propionate. L. adjusted to pH 4.5 to 0. 5. pH 5. the lag phase of L. respectively. pH 5.3.7 M propionic acid at pH 5. (1995) examined the combination of sodium propionate and SA at a number of concentrations. and berries but also prevented spoilage in vegetables having a more neutral pH.02% ascorbic acid. 4. mesentericus (subtilis) in bread dough at a level of 0.0 in reducing numbers of L. Toxin production occurred after 2 days for pyruvate.6. Sodium propionate at levels of 0. Much higher concentrations of propionic acid were bacteriostatic to B. Inactivation of L. Sarcina lutea. Cherrington et al. the rate of RNA (ribonucleic acid). At a 5-mM concentration. succinic. Zein (Z) film coatings were dissolved in propylene glycol (ZP) or ethanol (ZE) with and without nisin (N. acetate.8. 1941). or ZENCP. Eklund (1985) concluded that propionic acid was not as inhibitory as benzoic acid for P. 1945). syrup. Calcium propionate prevented rope formation caused by B. monocytogenes (Janes et al. degree of dissociation of the acid. or 4°C. The salts of propionic acid are also effective antimicrobial agents. little activity was ascribed to EDTA in combination with the acids. or lactic acids in media (El-Shenawy and Marth. 21°C. Sodium propionate at a 0. . monocytogenes to 8% sodium propionate for 60 minutes caused injury (Buazzi and Marth. and 4 days. applesauce. and cell-wall synthesis decreased. The acids contributed to approximately 85% of the antimicrobial activity with 14% contributed by ascorbic acid. monocytogenes. acetic. Turkey breast meat was formulated with sodium salts of lactate. tartaric.6 and incubated at 4°C. fumaric and malic. or at a level of 0. monocytogenes grown at 13°C was extended by 7. In laboratory studies examining the effect of sodium propionate on L.0.3% sodium propionate was adjusted to pH 5. and 35°C and prevented growth at 4°C (El-Shenawy and Marth. pH 4. pH 5.0. pyruvate. pH 4.

8 log reductions were observed. monocytogenes by only 1. Mold spoilage is a significant economic problem in bakery products. 1946). However. Spore germination was inhibited by 1402 ppm (Tzatzarakis et al. rather than later (Ghosh and Häggblom.8 log reduction at 67% fat.3% by weight. 1952b). Propionic acids and their salts are primarily inhibitory to molds. Pseudomonas species. at 0. Graham and Grice. or Trichophyton mentagrophytes.5 and 1. The inhibitory action could be the result of an interference with β-alanine synthesis (Wright and Skeggs. Bread produced with the extracts contained less ethanol and supported less spoilage by molds. subtilis.1%.8 to 0. and 0. None of the preservatives were effective at neutral pH.. some species of Penicillium grew on media containing 5% propionic acid (Heseltine. and humans showed no toxic effects (Graham et al. coli. Adding yeast extracts previously fermented by Propionibacterium freudenreichii provided a source of propionic acid in bread formulation.. Toxicology Orö and Wretland (1961) determined the LD50 of propionic acid for mice. Propionic acid at a concentration of 0. 1951. monocytogenes at 22% fat compared to 2. 1955.1% sorbic acid or potassium sorbate.03%. Harshbarger. (2001).0. 0.. 6. 1940).122 Antimicrobials in Food The effect of fat concentration on the efficacy of antimicrobial agents against L. The antimicrobial activity of sodium propionate was most affected at 4°C by fat concentration with a 1. rats. potassium sorbate. rubrum. 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. β-alanine could not reverse the inhibitory effect of propionic acid in Aspergillus clavatus. amstelodami. All preservatives were effective at pH 6. Small amounts of β-alanine could overcome bacteriostatic action of sodium propionate for E. There is evidence that the sodium salt has some local antihistaminic activity (Heseltine.85 (Guynot et al. monocytogenes was studied in pork liver beaker sausage (Hu and Shelef. and Penicillium corylophilum.8. Hara. 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. and repens were inoculated by needle into cake analogues prepared with calcium propionate. 1954. 1996). and Hara et al.b) examined several levels of calcium propionate. herbariorum. no growth occurred. but also various organisms displayed different tolerances to the compounds (Olson and Macy. and 0. 1940). postprocess contamination from the atmosphere..5 and water activity of 0. Sausage batter was mixed with 0. Additional studies using calcium and sodium propionate fed to mice.85. Not only was the final pH of the substrate critical.0 and a water activity of 0. and 0.2% sodium propionate. and wrapping materials reintroduce molds. 2002). with sodium lactate. and sodium benzoate (0. 0. A novel use of propionic acid to retard spoilage of bread was investigated by Gardner et al.003%. and rubrum. 1942). B.03%) led to enhanced growth of Aspergillus and Penicillium isolates. however.5. 2000).2). and potassium sorbate at a level of 0. Ingle.3%) at pH 4.2%. pH 4.93 in a model agar system on the retardation of growth of E. Suboptimal doses (0. sodium benzoate. (1963) determined the LD50 of calcium and sodium propionate for rats (Table 4. A 5% calcium propionate solution acidified to pH 5. This inhibitory effect was more pronounced with the addition of acid at the time of inoculation.5 log reduction of L. 2002a. Eurotium amstelodami. A. reduced growth and aflatoxin formation of A. Increasing the fat content from 22% to 67% decreased the growth of L. 1945).5 with lactic acid was as effective as a 10% unacidified solution in preventing surface mold growth on butter (Olson and Macy. 1952a). 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. 1. flavus and niger.5 logs. and 7. 1965. 0.5. cooling surfaces. flavus. Subsequent studies (Marin et al. . 1953). respectively. 1985). Molds were inhibited by less calcium propionate by weight than sodium propionate. herbariorum. Although mold spores are destroyed during baking.8% sodium lactate.

tooth eruption. 1974). 1944). The acceptable daily intake for humans is listed in Table 4. Succinic acid is used to modify the plasticity of bread doughs and in the production of edible fats.3. gradually increasing to 2. No upper limits are prescribed for use of this additive. but surface recontamination can occur during packaging. The acid form is readily miscible with water. and 0. Propionates can be added to bread dough without interfering with leavening because there is little or no effect on yeast. Its antimicrobial effect is limited to most yeast and bacteria. Application and Regulatory Status Succinic acid is a nonhygroscopic. 1972). calcium and sodium propionate are listed as antimycotics when migrating from food-packaging material (21 CFR 181. are approved as GRAS substances for miscellaneous and generalpurpose usage.23). 0.1221) and sodium propionate (21 CFR 184. Calcium propionate is preferred. Thomson et al. rats received subcutaneous injections of 0.Organic Acids 123 Application and Regulatory Status Propionic acid is a monocarboxylic acid with a slightly pungent. A limit of 0. and growth can be seen under the wrapper during storage.1784). Chick embryos developed normally when comparable dosages were injected into the air sac (Dye et al. Propionic acid and its salts are used primarily as mold and rope inhibitors in bread. (1967) reduced the population of inoculated S. which conform to standards of identity. 1980). Typhimurium by 60% on chicken carcasses after spraying with 1.1091). It can be incorporated into gelatin deserts and cake flavorings (Gardner. Toxicology In short-term studies. however a 3% or 5% concentration used at 60°C impaired the appearance of the product (Cox et al. disagreeable odor. The test animals did not differ significantly in reproduction rates. Propionic acid (21 CFR 184.32% can be used in flour and in white bread and rolls. No limit has been set on the acceptable daily intake for humans. This additive is also used as a mold inhibitor in cheese foods and spreads. This naturally formed additive becomes a developed preservative that limits mold growth on Swiss cheese.1081) and its salts. . Sodium propionate is recommended for use in chemically leavened products because the calcium ion interferes with the leavening action. except bread and rolls..5 mg succinic acid daily. however.38% in whole-wheat products. SUCCINIC ACID Antimicrobial Properties Succinic acid successfully lowered microbial loads on poultry carcasses (Mountney and O’Malley. or eye opening from the control group. which are associated with its manufacture and the characteristic Swiss cheese flavor. dicarboxylic acid having low water solubility.0 mg/day at 4 weeks and continuing at this level for 100 days. calcium (21 CFR 184. Baking destroys most molds. Succinic acid is approved as a GRAS substance for miscellaneous and general-purpose usage (21 CFR 184. hair appearance. 1972). It is normally found in cheese and as a metabolite in the ruminant gastrointestinal tract.3% in cheese products (Robach. Salts of the acid have a slight cheeselike flavor. It has a slightly bitter taste and serves as a flavor enhancer. In addition. 1965). Swiss cheese contains up to 1% propionic acid because of the growth and metabolism of propionibacteria.. and the sodium salt is more soluble than the calcium form.0% succinic acid. for use in bread and rolls because the calcium contributes to the enrichment of the product (Chichester and Tanner.

It is thus essential to understand each of these concepts before consideration of the actual mode of action of the organic acids. or gross histology compared with controls (Packman et al. mortality. and 1. Proteins. Biological and chemical systems depend on an interaction between acid and base systems. The workers also displayed skin eruptions. However.2). Changes resulting from pH can destroy bacteria. and yeasts. Studies measuring teratogenic activity in pregnant mice. rats.. has a strong tart taste that enhances grapelike flavors.8%. Toxicology Acute toxicity data for tartaric acid by the intravenous route for mice indicates an LD50 of 485 mg/kg body weight (Table 4. Inorganic acids. growth. and cholesterol levels. Application and Regulatory Status Tartaric acid. 1980). 0. respectively (Food and Drug Research Laboratory. .1%. 181. Through the interaction of a series of chemical mechanisms. and grape-flavored beverages. jellies and preserves. Homeostasis is the tendency of a cell to sustain chemical equilibrium despite a fluctuation in the acid–base environment.3. molds. 1978). sherbets. The acceptable daily intake for humans is listed in Table 4. nucleic acids. 0. 225.7% sodium tartrate (5% tartaric acid) did not show any changes in food consumption. 1954). such as hydrochloric. 1972). 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. Fitzhugh and Nelson (1947) found similar results in long-term studies using 21-day-old weanling rats fed tartaric acid as 0. Rose’s (1924) work on subcutaneous administration of 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.124 Antimicrobials in Food TARTARIC ACID Antimicrobial Properties Any antimicrobial property of tartaric acid is because of its ability to lower the pH. the most soluble of the solid acidulants. 1973a). It is used in fruit jams. including increased nonprotein nitrogen. male rabbits consuming a diet containing 7. this delicate balance is maintained and alteration of this balance causes destruction of the microbial cell. The availability of metallic ions to the organism is also altered and becomes a function of membrane permeability because membranes are less permeable to charged molecules than to uncharged molecules. proton donation. and rabbits demonstrated normal offspring at levels of 274. The microbial cell normally reflects this equilibrium by attempting to maintain an internal pH near neutrality (Baird-Parker.1099).2% of the diet.5%. MODE OF ACTION The mode of action of organic acids in inhibiting microbial growth appears to be related to maintenance of acid–base equilibrium. and it prevents discoloration in cheese (Gardner. sugar. Tartaric acid is a GRAS substance for miscellaneous and general-purpose usage in accordance with good manufacturing practices (21 CFR 184. The terms strong or weak are used to describe acids and reflect the degree with which acids readily donate a proton or dissociate in aqueous solutions. and the production of energy by the cells. hamsters. Tartaric acid acts synergistically with antioxidants to prevent rancidity. and 215 mg/kg body weight.. The monopotassium salt (cream of tartar) is commonly found in baking powders. and phospholipids can be structurally altered by pH changes.25 to 1 g to rabbits produced abnormalities in blood chemistry. 1963). although some microorganisms such as Acetobacter can exist at and even require extremes of acidity (Langworthy.

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

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

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

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

their theoretical yields.76 and 7. FORM AND SOLUBILITY In water solutions.20. 1966). A plot of the distribution of the three ionic forms can be calculated and is shown in Figure 5.1. The metabisulfite is the anhydride of the acid sulfite: – → 2– 2 HSO3 ← S2O5 + H2O When these salts are exposed to air.2 lists the main forms of sulfur dioxide. The dry salts are easier to store and are less of a problem to handle than gaseous or liquid sulfur dioxide. 1968). as well as by other methods. The anhydrous sodium and potassium salts are prepared by precipitation and then dehydrated with the appropriate hydroxide. Most of these salts are hygroscopic and easily hydrolyzed (Schroeter. It is useful to have the sulfur dioxide in a salt form.1 Distribution of the ionized forms of sulfurous acid at various pHs. and from reducing gypsum. 1928. and solubilities of each in water. Table 5. 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. .144 Antimicrobials in Food Sulfur dioxide can be prepared commercially from burning sulfur (oxidation). 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. indicating a rather weak dibasic acid (Segal. Specific gravity for various water solutions of sulfur dioxide is shown in Table 5. 1928).1. Phillips. The pKa values for sulfur dioxide are 1. heating pyrites. they show increasing stability in the order sulfite > bisulfite > metabisulfite (Mason.

0393 1.Sulfur Dioxide and Sulfites 145 TABLE 5.0040 1.037 — Corrections are approximately 0.8 25.40 and 15°C.4 53. 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. After the oxygen disappeared.003 1. 1970).56°C (60°F) 1. the second equation listed previously is a key reaction (Heinmann et al. 1970).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. .0001°F–1 TABLE 5.6 67. If ascorbic acid is being used in combination with sulfur dioxide. The sulfur dioxide scavenges the hydrogen peroxide formed and keeps further oxidation of the dehydroascorbic acid and other products to a minimum.4 s-1 for H2O2 with SO32.5 61.4 H2O Solubility (g/L) 110 (20°C) 250 (20°C) 280 (40°C) 240 (25°C) 1000 (20°C) 3000 (20°C) 250 (0°C) 540 (20°C) REACTIVITY Wedzicha (1984) briefly reviewed the chemical interactions of sulfur dioxide. much slower changes occurred in the sulfur dioxide content.008 1. This reaction is extremely rapid.028 1.018 1.0292 pH 3.6 57..0191 1. Holt and Kumar (1986) found an observed k = 41. The sulfur dioxide stabilizes the dehydroascorbic acid by reacting with the ketone bonds (Wisser et al.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 15.0493 20°C (68°F) 1.0091 1.7 ± 3.

and wines. Bisulfite addition products.7 90 1. lactose. L-xylosone. natural fruit juices always bind more sulfur dioxide than would be calculated from the glucose present in the juice (Joslyn and seven-member carbon ring system will react. In moldy apples. 2000).5 — 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. Joslyn and Braverman. mannose.b. maltose.6 7. 1954). Lea et al. 2-oxogluconic acid. Otherwise. form rapidly with aldehydes: – – → HSO3 + R-COH ← R-CHOH-SO3 All aldehydes form the hydroxysulfonates.b). Diethyl ketone reacts slowly and to a limited extent.5-diketogluconic acid.0 0. pyruvate.1 0.5 3 5 8 Ka × 10–6 × 10–4 × 10–4 × 10-6 — 2 × 10–4 5 × 10–2 1.3 Relative Percentage of Aldehydes that React with Sulfur Dioxide and Their Equilibrium Constants (K) Compound Acetaldehyde Pyruvic acid α-Ketoglutaric acid Glyoxylic acid L-Xylosone Oxaloacetic acid Glucuronic Acid Monogalacturonic Acid Rhamnose Trigalacturonic Acid Arabinose Xylose Fructose Glucose Malvidin-3-glucoside 1 100 66 47 — 27 — — 2. Ingram and Vas (1950) found that galactose. 5-oxofructose.5 72 44 98 — 66 1 2 — — — — 0. Aerny.5-hexodiulose. Rhem (1964) noted that the rate of formation and the amount of sulfonate formed depended on the concentration of the reactive substances. Those reacting the most rapidly formed complexes that dissociated the least. fermented ciders. 1954. . acetaldehyde. 1960. the hydroxysulfonic acids (Suter. 1954). 1973. as much as 80% of the total sulfur dioxide may be bound by these types of carbonyls (Blouin. the pH. but not all ketones react.7 × 102 — — — — 15.9 6 × 10–5 Note: 1 = Burroughs and Whiting (1960). raffinose reacted very slowly. and arabinose reacted rapidly with bisulfite. In botrytized grapes made into wine.. Compared with model solutions. Navara.12 — 2 99. 2. He also noted that phosphoglyceraldehyde would likely react. 3 = Navara (1985). and galacturonic acid were significant bisulfite binding forces (Burroughs and Sparks.146 Antimicrobials in Food TABLE 5. 1964a. and fructose and sucrose did not react at all.1 87 3 50 32 18 — — — — — 26 — 45 32 0. 2 = Aerny (1986a. α-ketoglutarate. 1944). and the temperature. d-threo-2. Glucose is by far the most abundant of the reactive aldehydes and ketones in most fruit juices. Relative percentages of aldehydes reacting with sulfur dioxide and their equilibrium constants are shown in Table 5. 1985).b).3 (Burroughs and Whiting.1 — 1. 1986 a. and glucose reacted less rapidly. only ketones with a methyl group adjacent to the carbonyl or carbonyls that are part of a four.1 — 0. 1963). a Aerny (1986 a.

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

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

Lafon-Lafourcade (1975) demonstrated the effectiveness of both free sulfur dioxide and bound sulfur dioxide in inhibiting Leuconostoc gracile in wine at pH 3. In model solutions containing Lactobacillus arabinosus and L. 1983) indicated that around 50 mg/L of free sulfite could preserve wine vinegar for about half a year. This organism is particularly intolerant to sulfur dioxide. and Oenococcus oeni (Amerine et al. casei. respectively. Several reports (Karova and Kircheva. but much larger doses were required for bactericidal action. Lactobacillus leichmannii. and ethanol.Sulfur Dioxide and Sulfites 149 oxidize ethanol in fermented beverages to acetic acid and. oeni. and soft drinks were required to control acetic acid bacteria.5 mg/L were bactericidal. Manca de Narda and Strosser de Saad (1987) investigated the tolerance of O. They indicated A. oeni was the most sensitive to SO2. and Pediococcus pentosaceus. Spirov et al. hilgardii. Watanabe and Ino (1984) and Juven and Shomen (1985) reported that up to 100 mg/L of total sulfites for grape juice. 1983). further. after 36 hours. Fornachon (1957) noted that sulfur dioxide and pH were important factors in controlling lactic bacteria in wines. pH. As little as 30 mg/L of added sulfur dioxide may be lethal to the species. 1980). He found that this genus also fixed a certain amount of the sulfur dioxide. (1984) stated that the amounts of SO2 used in normal wine making are insufficient for acetic acid bacteria control.. LafonLafourcade and Joyeux (1981) and Joyeux et al. O. Lactobacillus trichodes. 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. 1994). plantarum. Pediococcus. The homolactic species found in wines are Lactobacillus acidophilus.. Lactobacillus buchneri. Cruess (1912) found that 5. 20 days was required to kill the bacteria when the same amount of aldehyde–bisulfite complex was used. (1980) reported that 30 mg/L of free sulfur dioxide is sufficient to inhibit malolactic fermentation in wine. As low as 75 to 80 mg/L of total sulfur dioxide prevents growth and 100 mg/L kills this species. and P. 1982. 1979). Oenococcus oeni grows preferentially at lower pH but seems less tolerant to sulfur dioxide (Mayer. however. 1962). and the heterolactic species are Lactobacillus fermentum. Leuconostoc mesenteroides subspecies dextranicum can also cause ropiness in beverages through the formation of dextrans. and Oenococcus. to carbon dioxide and water.5 mg SO2 per liter in the undissociated form was bacteriostatic to L. 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. Lactobacillus hilgardii. Lactobacillus brevis. Bacteria common in acid fruits and beverages are the lactic acid-producing genera Lactobacillus.. The juices high in insoluble solids ultimately had lower residual total sulfites. is very heat resistant and alcohol tolerant. 1949) is described as causing a hairlike growth in fortified wines. 1982. Pediococcus cerevisiae. Liu and Gallander (1983) demonstrated that lower pH wines underwent . Additions of 100 mg/L bleached the color of the vinegar. Amerine et al. Leuconostoc. Cell growth was completely prevented. Lactobacillus casei. Lactobacillus plantarum. with lower pH increasing the effectiveness of the sulfur dioxide. Levels above 120 mg/L of total sulfur dioxide (free and bound) decreased the incidence of malolactic fermentation. Lactobacillus trichodes (Fornachon et al.3 log CFU/ml. The bacterium. They found that up to 1. L. for antimicrobial action at pH 6. 320 and 651 mg/L of sulfurous acid were required. Concentrations above 1.5. pentosaceus to SO2. Addition of 30 mg/L of free sulfur dioxide killed the bacteria completely in 15 days. Lactobacillus delbrueckii.47 log and 0.49 log Acetobacter per ml exposed to 100 and 200 mg/L of total sulfur dioxide in grape juice were reduced to 2.0 (Rhem and Wittmann. aceti can grow in red wine with 25 mg/L of unbound sulfur dioxide present.. Rhem and Wittmann (1962) reported that 200 mg/L sulfur dioxide killed Acetobacter at pH 6.4 (Holt et al.0 in a buffered solution. They are aerophilic and have pH optimum for growth of around 5. respectively. Carr and Davies (1971) noted that relatively high amounts of free sulfurous acid were present in ciders that contained viable lactobacilli. red wine. Dupuy and Maugenet (1963) noted that even small doses of sulfur dioxide inhibited the activity of the cells.

Wibowo et al. 65–136.150 Antimicrobials in Food malolactic fermentation more slowly and that lower sulfur dioxide levels increased the fermentation rate of the O. oeni strains were less tolerant to the sulfur dioxide than Pediococcus paroulus strains or the Lactobacillus species. 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. state that sulfur dioxide is more effective against the growth of Gram-negative rods. In addition. The effect was inhibition of growth of the flora. (1988) found significant delays in growth of O. oeni when SO2 was added and quite a variable response between strains. All strains grew at pH 4.5 in beef broth and 20% tomato juice serum medium at 64 mg/L of total sulfur dioxide.5 mg/L of molecular sulfite was sufficient to control malolactic bacteria growth. This is demonstrated in the use of sulfites in meats. 50–195. (1988) demonstrated that without sufficient SO2 and adjusted pH. They also noted that the interaction of sulfites with nitrites caused a lowering of the nitrites available for nitrosamine formation. The delay was proportional to the concentration of the bisulfite addition. Citrobacter freundii. Adams et al. Tompkin et al. The shelf life of ground beef was effectively increased from 1. in their review. The packaging used was a gas-permeable wrapping that allowed oxidative conditions (von Holy et al. although slowing or prevention of growth of surface bacteria is probably important. 200–241. Ough et al. There was some additive effect when used with dimethyl dicarbonate but not enough to warrant the use with SO2 for this purpose. 83–142. 15–109. O. than in inhibiting Gram-positive bacteria. They noted that sulfites shifted the microflora of the meat to Gram-positive bacteria from the normal Gramnegative flora. (1980) found the addition of 100 mg/kg of SO2 as sodium metabisulfite to canned pork inoculated with Clostridium botulinum spores delayed cell growth. 67–98. were completely effective. such as Escherichia coli and Pseudomonas. 190–241. The preservation of the color and odor of meats is improved by sulfite treatment and. 1972). This work confirms that wines with high total sulfur dioxide concentrations are more likely to undergo malolactic fermentation with other than O. coli. oeni strains with unfavorable sensory results. The microorganisms tested and the concentration of free sulfite (µg/ml) necessary to inhibit their growth at pH 7. Millet and Lonvaud-Funel (2000) reported that sulfites cause a portion of lactic acid and acetic acid bacterial populations to enter a viable but nonculturable (VBNC) state. the main effect in meat appears to be the antioxidant properties (Roberts and McWeeney. Sensory tests showed no adverse results. (1985) reviewed the use of sulfite as an additive to control microbiological changes occurring in meat products. They suggested that these microorganisms could cause spoilage in wines that were considered sterile using conventional counting techniques. a rapid decline in cell numbers occurred. Reddy and Mandokhot (1987) found that minced goat meat could be preserved up to 11 to 13 days if held at 7°C with 450 mg/L of sulfur dioxide added. Yersinia enterocolitica. It was found to tolerate up to 100 mg/L of SO2. oeni PSU-1 used. Davis et al. The remaining cells later multiplied to significant numbers. Enterobacter agglomerans.8 days at 7°C storage with no treatment to 12.. (1987) found that vacuum packaging and a good oxygen barrier film decreased the . Salmonella and E. 1988). (1983) determined that. oeni in red table wine occurs readily. Banks et al.6 days at 0°C with the addition of 250 mg/kg of sulfur dioxide. Lafon-Lafourcade et al. the growth of O.0 were as follows: Salmonella. E. Roberts and McWeeny (1972). although after sulfite addition. None of the compounds alone. Splittstoesser and Stoyla (1989) looked at five different regulatory-approved additives to determine if they could suppress malolactic bacterial growth in grape juice as a replacement for sulfites. coli were inhibited to a greater extent by sulfites than other bacteria. oeni was the primary malolactic bacterium associated with wine. Serratia marcescens. O. Piracci (1984) found 0. The Gram-positive bacteria remaining grew more slowly than the Gram-negative bacteria. and Hafnia alvei. Banks and Board (1982) tested several genera of Enterobacteriaceae isolated from sausage for their metabisulfite sensitivity. It survived alcoholic fermentation when others did not. or in paired combinations. (1988) tested 146 different strains of wild malolactic bacteria. in the Bordeaux area of France..

but the level of cadaverine was reduced (Bover-Cid et al. The concentration of tyramine and putrescine increased in the presence of sulfite. 1. Once the inhibition was overcome.5 in tryptone yeast extract medium.10–20. with the pressure to reduce sulfur dioxide content. whereas Kloeckera. Rhem and Wittmann (1962) determined the inactivation levels of sulfurous acid for a variety of yeast genera (Table 5.” for biological stability can be disastrous. which inhibited growth. such as “maintain the free SO2 at 20 mg/L. Pichia. was maintained for a longer period. According to Warth (1985). resistance of yeast to sulfur dioxide ranges from 0.20 7. In contrast. pH is extremely important in the effective use of sulfur dioxide. the growth rates and cell yield were similar.2 mg/L of molecular sulfur dioxide were necessary at the finish of fermentation and during aging.2–8. its use to prevent the growth of yeast in sweet table wines is seldom ever contemplated.4) at 25 mg/L was sufficient to kill a culture of S.60 Rhem and Wittmann (1962).Sulfur Dioxide and Sulfites 151 TABLE 5. Thus the free sulfite. (1988) found that with yeast acclimatized to sulfur dioxide.1. bailii at 25°C under aerobic conditions at pH 3.81). A 10-fold increase in molecular sulfur dioxide occurs between pH 4. cerevisiae that had been characterized as “SO2 resistant” for tolerance to high concentrations of sulfur dioxide.7 0.0 mg/L were required. Yeast The use of sulfur dioxide to deplete the wild yeast in grape juice is a standard practice dating back many years (Cruess. 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. Rhodotorula.. Haznedari (1979) tested 30 strains of S. Any rule of thumb addition.20 0. Carr and Davies (1971) found sulfur dioxide incorporated into growth medium (pH 3. spoilage in sulfite-treated sausage. This was because of the lack of oxygen delaying yeast growth and the production of sulfite-binding substances.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 0.0 and 3. phenylethylamine. and several other genera were very susceptible. cerevisiae (105 CFU/ml) after 8 hours at 25°C. or tryptamine. There was no effect on histamine.8 mM for Z. and five others were able to produce adequate amounts of ethanol.05 M free sulfur dioxide for Kloeckera apiculata to 2.60 0.5 and 1. between 2. The amount of molecular SO2 (mg/L) in wine can be calculated using free SO2 mg/L/(1 + 10pH . Sudraud and Chauvet (1985) found that to maintain yeast stability.4). 2001). Six strains could grow well at 1000 mg/L. Delfini (1989) .0. bailii. respectively. Sodium sulfite addition in sausage was shown to affect biogenic amines.40–0. Molecular sulfur dioxide is the most effective form for inhibiting yeast. The yeast also showed increased resistance to the fungicide dimethyl dicarbonate.0 and 3. 1912). Goto (1980) determined viable counts of various wild yeasts in grape juice in the presence of sulfur dioxide and found Torulopsis and Saccharomyces were the most tolerant. In fact. Saccharomycodes ludwigii was nearly as tolerant as Z. Ough et al.20 0.

With modern filtration and sanitation technology. Proper selection of the yeast strain to avoid high levels of sulfite is an obvious choice. Spoilage yeast in dry wines are fairly rare. and low pH. There have been numerous reports on techniques to minimize the amount of sulfur dioxide used in wines (Aerny. Breweries have an interest in sulfur dioxide as an antioxidant. Dott et al. (1997) showed that sulfite up to 400 mg/L had no effect on S. Although other agents may act as antimicrobial agents. Very little of the sulfur dioxide formed by the yeast remains in the free state.5). using sulfide salts before fermentation. and Schizosaccharomyces japonicus in the re-fermentation of sweet champagnes..b. 1975). Sethi and Anand (1984) had to use 692 mg/L in carrot preserves to inhibit Bacillus cereus. (1985) used an initial 1000 mg/L of sulfur dioxide in the last stages of the manufacture . 1978). stated that the use of sulfide salts failed to protect wine and left negative sensory characteristics in the wine. adjusting the pH downward if necessary. Yeast can form sulfite from sulfate via sulfate permease. 1985. minimizing the air around the wine. cerevisiae. ludwigii. Fruit juices and preserves can be protected from microbial spoilage by sulfur dioxide addition. bailii.6). however. even at the legal limits.7 (Lloyd. Generally. (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. 1986a. none seem to be capable of replacing the antioxidant property of SO2.4). excess amounts are used. Strain differences caused variation in the sulfur dioxide produced from 11 to 26 mg/L. Hernandez. ludwigii in a low-alcohol wine. Sand (1980) suggested that Z. Angelino et al. and there was no correlation with the number of cells in the initial inoculum. bailii in comminuted orange drink was controlled by 230 mg/L of SO2 at pH 3. Ranote and Bains (1982) used 350 mg/L of sulfur dioxide to preserve Kinnow. alcohol. 1985. Hydrogen sulfide (H2S) was used (Ubigli et al. Minarik and Navara (1977) reported finding the spoilage yeast S. 1985. Even at low pH (2.152 Antimicrobials in Food reported the existence of very sulfite-resistant S. Asvany. settling and racking the juices.1 but not in the base material. found no correlation with dissolved oxygen levels in wort and no correlation with ATP sulfurase or sulfite reductase. substituting other preservatives at bottling. which was at pH 3. Brettanomyces can contaminate a winery. using yeast that produces minimum amounts of sulfur dioxide-binding components. does not always inhibit growth and fermentation. Recommendations include cooling the fruit before crushing. Suzzi et al. In a cell recycled ethanol fermentation system. They found a majority of the wines in the test to contain about 10 mg/L of total sulfur dioxide (Table 5.6 and 3. Some yeast strains can form rather large amounts of sulfur dioxide during juice fermentation (Table 5. (1989). Z. 1985. ludwigii is also a noted spoiler in sweet grape juice containing high amounts of sulfur dioxide (Jakob. Growth was found at the highest concentration of free sulfur dioxide used. if proper sulfur dioxide levels are not maintained. The treatment was reported to be successful for juice. Patel et al. reducing the amount of sulfur dioxide before or during fermentation. soft drinks. Schmitt et al. (1977) determined the cause for sulfite production to be sulfate permease inhibition by methionine. especially wines in barrels. Pearlstein (1988) found that extended aeration decreased sulfite production. cell numbers are depleted to very low levels at bottling. This particular species was found to be very resistant to sulfurous acid. a late-harvest orange juice. and wines because of its tolerance to SO2. Vernerova et al.. and eliminating oxygen from bottled wine. Chang et al. 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. S. Valouyko et al. With this genus. (1985) tested 1700 Saccharomyces. because the pH is high. Gomes and da Silva Babo. investigating the sulfur dioxide content of green beer. sulfur dioxide. Spoilage by Z. the main spoilage yeast is Saccharomyces. 1982) as an alternative for pretreatment of grape juice before fermentation. 1985). This shows that most wines will not be free from sulfite labeling requirements even if no sulfur dioxide is added. the sulfate permease was not repressed by methionine. Galassi and Mancini. cerevisiae despite a reduction in the bacterial counts in the system at the same concentration. bailii was becoming a problem in juices. (1983). In normal sweet table wines. S. In high-sulfite-producing yeast.

35 20. cerevisiae S.5 14. (1976) Poulard and Brelet (1978) S.Sulfur Dioxide and Sulfites 153 TABLE 5. 170. (1976) Delfini et al.5. 100 5. 20 26 27 0 0. acidifacien S. (1976) Delfini et al. 76 17. 18. rosei S. magna K.5 12 0 9 Reference Minarik (1975) Eschenbruch and Bonish (1976) Heinzel et al. (1976) Delfini et al. 3. 3. (1976) Poulard and Brelet (1978) Delfini et al. (1976) Minarik (1975) Eschenbruch and Bonish (1976) Heinzel et al.6 Amounts of Sulfur Dioxide Found in Test Wines Fermented by 1700 Saccharomyces Yeasts (Suzzi et al. 52. bailii S. 4. africana TABLE 5. 21. 160. 20 1. uvarum S. (1976) Delfini et al. pastorianus S.5 0 0 0 1. 150 0.5. 21. 35. bayanus S. (1976) Poulard and Brelet (1978) Delfini et al. 56.5 Formation of Sulfur Dioxide by Various Yeasts during Grape Juice Fermentations Yeast Saccharomyces carlsbergensis Sulfur Dioxide (mg/L) 12 150 80.5. 1300 22. (1976) Delfini et al. (1976) Delfini et al. (1976) Poulard and Brelet (1978) Minarik (1975) Poulard and Brelet (1978) Minarik (1975) Delfini et al. 500 300. 40 0 18. 18. 100. italicus S. (1976) Minarik (1975) Minarik (1975) Heinzel et al. (1976) Delfini et al. chevalieri S. rouxii Schizosaccharomyces pombe Torulopsis stellata Kloeckera apiculata K. 5 18–23 85 0. 44. 500 5. 1. (1976) Delfini et al.. 5. 1985) Range of SO2 Found in Synthetic Medium (mg/L) <10 10–20 20–30 30–40 >40 Average Total SO2 in Wine (mg/L) 10 17 22 34 58 Number of Strains 1292 354 50 2 2 .

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

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

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

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

16 963.28a 990. The official methods of analysis for agricultural and food products are specific in their recommendations for analysis of sulfur dioxide (Table 5. Reversibly bound sulfites are converted to sulfur dioxide only after heat and acid or alkali treatments. Sulfites can be described as free. purees.. A sample is placed in a distilling flask.8 AOAC International Official Methods of Analysis for Sulfites (Warner et al. 2000). TABLE 5. Thiosulfonates are irreversibly bound (Beck et al.02 961. acidified.20 975.09 962.. Those sulfites that bind food matrices and are not converted to sulfur dioxide with heating or acidic conditions are irreversibly bound. 2000) Method Number 892. garlic. or irreversibly bound (Beck et al.32 980.29 990. 2000).. Free sulfites are readily converted to sulfur dioxide upon acidification and can be quantitatively analyzed following distillation. and .7 Application of Sulfites in Foods as Antimicrobials and the Concentrations Used (Gould.30 990.8).158 Antimicrobials in Food TABLE 5. horseradish) Fruit juices Fruit-based sauces and related products Fruit pulps.04 990. 2000) Food Beer Fresh fruits Fresh vegetables (onion. One official method for measuring total sulfurous acid is the modified Monier-Williams procedure. 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. reversibly bound. and concentration varies by country.17 987.31 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.

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

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

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

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

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

but not 2000 µg/g. Two tests were reported with perishable canned ham. increasing salt and nitrite caused increased inhibition.Nitrite 173 of nitrate to nitrite in commercial bacon curing brines is mainly the result of the growth of micrococci. In both tests some cans with nitrate swelled because of the growth of Bacillus species. botulinum and C.0.2°C.” Their tests with fish muscle showed that nitrite delayed spoilage. Subsequent tests (Tarr. although salt was the stronger inhibitor. In one. 1942). At pH 7.” They postulated the anticlostridial mechanisms to consist of partial conversion of nitrate to hydroxylamine. little or no inhibition was observed. Stumbo et al. (1945a) reached the same conclusion from tests in meat processed at 116°C. Nitrate. nitrite alone inhibited anaerobic putrefaction during 30 days of abuse at 37. prevented growth of C. increasing nitrate did not increase the inhibition of C. The presence of nitrate or microbial action during the curing process is not essential for bacon flavor.7 and 6. Later tests involving C. Tarr and coworkers published a series of reports on the possible use of nitrite for preservation of fish. complete or strong microbial inhibition occurred. They also reported (Stumbo et al. Rapid chilling of the meat is not detrimental. 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. sporogenes yielded similar results (Tarr. little or nothing is known regarding the possible bacteriostatic (or bactericidal) action of nitrites in meat pickles or in cured meats themselves. workers at the time would have had difficulty applying the information to commercial practice. Jensen and Hess stated that nitrite reacts with protein during heating and is destroyed. of sodium nitrate. sporogenes 3679. thus leaving the meat in much the same state as freshly cooked uncured meat. The effect of pH was more firmly documented in a series of tests in nutrient broth inoculated with a variety of aerobes. fewer cans became putrid in the product with both nitrate and nitrite than in the product with nitrite only. sporogenes during 4 weeks of incubation at 37. nitrite. 1955). Within the levels normally added to canned ham. The authors suggested that the combination of heat. Jensen and Hess found that heat (93°C for 4 hours) combined with 5000 µg/g. (1949) also examined the combined effect of heating and curing salts. thus serving as a desirable indicator of temperature abuse. 1942). Using the spiced ham medium they reported earlier. nitrate. They stated that the literature shows “unmistakenly that nitrate in the cure exerts a definite inhibitory effect upon bacteria. Yesair and Cameron (1942) pursued this idea but concluded that curing salts do not assist in thermal destruction but inhibit outgrowth. At pH 5. alone or in combination with other ingredients. Higher temperatures. Greatly increased inhibition occurred in tubes of pork heated in the range of 50°C to 65°C for 30 minutes. 1945b) that nitrite appreciably delayed germination. 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. longer heating times. the authors concluded that nitrate is beneficial for preventing the growth of putrefactive anaerobes in abused perishable canned ham. Even if nitrite had been uniformly accepted as a critical factor in microbial protection.01. which inactivates catalase and allows the accumulation of hydrogen peroxide. 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. or repetitive heating did not increase this effect. 1941) demonstrated the importance of pH to the efficacy of nitrite.2°C. Others have shared this concern (Scott. Despite these results. In the other. . which destroys anaerobes. 1941) and that the mode of action was still unknown (Tarr. While Tarr was demonstrating that nitrite has strong antimicrobial properties. and salt caused destruction of anaerobic spores at much lower temperatures. Jensen et al. Tarr and Sutherland (1940) concluded that “as far as can be ascertained from the literature. How could reliance be placed on an unstable preservative that disappears during processing and storing? This question is still valid. However..

Although thermal destruction was not shown to be enhanced by the presence of curing salts. The question was raised whether the growth of spoilage organisms is influenced by the depletion of nitrite to noninhibitory levels. 3. 2. and high heat resistance. The concept of a combined effect of heating in the presence of curing salts was proposed. Nitrate at the level permitted in meat (1250 µg/g) was without effect. Salt at the levels used at that time was considered the cornerstone for the preservation of cured meats. The basic formulation resulted in a product having a moderately high brine of 5. The disappearance of nitrite during processing and storage was assumed to make it an unreliable preservative.174 Antimicrobials in Food Vinton et al. SUMMARY FOR BEFORE 1950 A summary of the state of knowledge by 1950 might include the following: 1. yielded the maximum inhibition. especially if the product pH was below 7. A mixed cure of all three (salt.” This experience may have led to improvements in meat handling and equipment sanitation. A mixed cure of salt (3.0.5%) and nitrite (150 to 170 µg/g) was much more effective than either substance alone. there was increased inhibition of outgrowth of surviving spores by salt and. to some degree. a combination of both nitrite and nitrate was the most inhibitory.37% and a pH range of 6. intermediate. Halvorson (1955) later discussed the widespread problem of temperature abuse at the retail level. nitrate. Nitrate was promoted as an inhibitor of anaerobic spoilage and to enhance the swelling of perishable canned cured meat by Bacillus species when temperature abused. During the 1930s some processors incubated perishable canned cured meat at 37°C as an index of “keeping quality. however. Nitrite was clearly shown to be an effective antimicrobial agent.000 µg/g were required to prevent spoilage from anaerobic spore formers in pork. 1950–1960 Research of the 1950s clarified the relative significance of salt. Salt was found to be the major factor retarding botulinal outgrowth in temperature-abused products. An underlying factor in the research before 1940 was the unfavorable experience of 1928 and 1929 with temperature-abused perishable canned hams. Spores produced in raw. and nitrite as preservative agents and expands this information to other microorganisms. . but the condition of the meat was a factor. and sterilized meat were of low. Adding 200 µg/g of sodium nitrite was considerably more inhibitory to toxin formation than the addition of 1000 µg/g of sodium nitrate. even in the presence of salt or nitrite. Steinke and Foster (1951) investigated the effect of packaging liver sausage in an oxygenimpermeable film (Saran™) that was gaining acceptance in the industry. 4. and nitrite).4% or sodium nitrate in excess of 40. nitrate. by nitrite. (1947) found the heat resistance of spore crops was not altered by adding nitrite and nitrate to the meat on which the spores were grown. 5. 7. 6. increased inhibition of PA 3679 was obtained as sodium nitrite was increased from 400 to 800 µg/g. It is perhaps for this reason that Jensen believed so strongly about the need for nitrate in perishable canned cured meats.05% to 5. Strong opinions were expressed that nitrite alone was ineffective at the levels used in commercial practice. The relative roles of nitrate and nitrite as preservatives in cured meats were unclear.5. however. Nitrate was without effect. respectively. pasteurized. Bulman and Ayres (1952) found that levels of salt in excess of 4. In the absence of added salt.1 to 6.

nitrite enhanced bacterial growth in curing brine. this cautious view of the inhibitory effect of nitrite was expressed by Eddy (1957): “Taken in their totality. 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. nitrite rapidly disappeared and was ineffective. aureus to die rather rapidly in ham-curing pickle.5% would have caused consumer . The brine requirement of 5. Today. growth would occur. Germination was apparently prevented because viable spores could be recovered.8 was optimal for antibacterial efficacy. B.Nitrite 175 Henry et al. Nitrite was more inhibitory in the presence of ascorbate.0%. aureus was killed in hams when heated to an internal temperature of 58. 1955) on semipreserved meats in hermetically sealed containers included a curious debate. Also. The USDA concluded that the high brine would retard the growth of C. and a brine level of 3. the growth of Bacillus species would occur. these observations leave no doubt inhibition by nitrite is at least a possibility.3°C. A pH of 5. 1/8 ounce sodium nitrite (78 µg/g). They found S. toxin formation was inhibited with a heat treatment as low as F0 = 0. In the United Kingdom. less nitrite was necessary for inhibition as the pH was decreased from 6. Jensen. At 6. A typical formula being used for large canned hams at that time included 0. unless protected by meat juices. who concluded that 1 ounce nitrate was optimal for gas formation by Bacillus species in temperature-abused perishable canned luncheon meat. 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. (1954) reported that at pH 7. This combination of ingredients was derived from the literature and unpublished results from Jensen. the legal requirement for trichina destruction.5 or above. nitrate. botulinum. the addition of 1. Pasteur. The authors postulated that nitrous acid was responsible for the bacteriostatic action of nitrite. At that time European microbiologists commonly preincubated perishable canned cured meats before microbiological examination. a very small amount of added nitrite prevented staphylococcal growth when incubated anaerobically.” An international symposium (Ann.0% brine or above. The debated issue was the time and temperature for preenrichment. was unstable. Lechowich et al. Because the procedures traditionally used for large canned hams had a history of safety. Dack (1949) reported toxin inhibition in meat with brine levels above 6. Inst.5%. This effect was reversed by adding sulfhydryl compounds.5%. Scott (1955) concluded from the literature that because nitrate exhibited relatively poor antimicrobial inhibition and nitrite.5% sugar. the spores could germinate. They investigated why Staphylococcus aureus is rarely.27. a similar debate would include the time elapsed since manufacturing. and toxin would be produced. aureus growth can occur in any combination of salt.3 or below. 1 1/2 lb canned ham)..6 to 5. causing the cans to swell and forewarn consumers of a potential hazard. the control of salt concentration and resultant water activity was the most reliable bacteriostatic system for cured meats. As late as 1957. Halvorson (1955) cited a private communication from L. Tests in broth media and raw pork led them to conclude that S. aureus. On adding a filter-sterilized solution of nitrite to a broth medium. At pH 5. these existing products were not affected. and in the presence of nitrate.55) was autoclaved with glucose. Additional tests showed that if the broth medium (pH 6. The requirements imposed on new products included a brine level of 5. and nitrite that is palatable and permissible. Jensen concluded that if the brine content were less than 5. and 1 ounce of sodium nitrate per 100 pounds of meat. brine values traditionally have been calculated at % brine = (% salt/% salt + % water) × 100. Earlier. as suggested by Castellani and Niven (1955). botulinum. Department of Agriculture (USDA) issued a regulation to assure the safety of new perishable canned cured meat products (e.S.75% sugar. In North America. (1956) examined whether curing salts plus the anaerobiosis of a vacuum package would inhibit the growth of S. 1/4 ounce sodium nitrate (156 µg/g). During the mid-1950s the U.4%. found in the interior of cured meats. who performed a series of tests with spiced ham inoculated with different strains of C.05.9 to 5. although effective. S.g. if ever.

its value as a preservative in perishable cured meats was still in doubt. toxin was not produced. although there have been several studies under other conditions in which toxin has been detected without obvious organoleptic spoilage. the primary effect of nitrite was to prevent germination and/or outgrowth of heat-injured spores. Because viable spores could be recovered from stable commercial product. 6.5%.125% and abused at 29. and a low indigenous spore level. Brine level (0%.12%. At 5. 3. 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. Silliker et al. Several new reports of unstable commercially canned cured meats containing nitrate appeared (Hankins et al. (1959) subsequently demonstrated botulinal spore outgrowth in perishable canned cured meat having brine levels from 3. was not confirmed. Nitrate. A significant observation was that at brine levels of 6. other than its possible influence on water activity. and thermal injury to the low indigenous level of anaerobic spores. Nitrate could serve as an electron acceptor. As expected.09%. This research and a subsequent review by Silliker (1959) established the future direction for research on nitrite. had no antimicrobial effect. toxin was produced without concomitant organoleptic spoilage. Verhoeven. Sodium nitrite levels were 22 µg/g or less at the time the meat was inoculated. This brought nitrate under renewed attack. toxin was produced without obvious organoleptic spoilage. Brine content was further shown to influence botulinal outgrowth and toxin formation. 1950. Nitrite was shown to play a significant role in the stability of shelf-stable canned meat. the following became increasingly clear: 1. 2. 4. 1950. 5. Greenberg et al. nitrate actively stimulated spoilage by aerobic spore formers. S. 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. thermal injury to the spores. There have been no reported attempts to duplicate this effect of brine at levels approaching total inhibition. SUMMARY FOR 1950–1960 As 1960 approached. salt. or 5.95% brine. Although shelf-stable canned cured luncheon meat had been produced for years. aureus was killed during the heating process given to hams. as postulated by test tube experiments. At 8. Eddy and Ingram.14% brine or less. The suspected role of nitrite was to prevent germination or outgrowth of surviving heat-injured spores. salt. the product was organoleptically unacceptable — as the cans swelled — and toxin assays were performed. Anaerobic intolerance to nitrite within the interior of ham. per se.25%. 1956). such as micrococci and bacilli.. The system providing stability consisted of the combined effect of nitrite.8°C for up to 90 days. permitting the growth of aerobes. 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. At brine levels approaching the inhibitory level.176 Antimicrobials in Food complaints of excessive saltiness in the new products. Although nitrite was recognized as an effective antimicrobial agent. 7.0% to 7.0%) alone was not responsible for stability. . and 7. the reasons for stability had not been defined. (1958) used this class of product to show that nitrate played no role in retarding putrid spoilage. 3.4°C to 37.

even if nitrate is present as an alternative electron acceptor.0. but not in others. He concluded that most of the spores are killed during the thermal processing of shelf-stable canned cured meat. Germination was completely prevented by sodium nitrite levels of 750 µg/g or higher at pH 6. Shank et al. of spores of Bacillus species in the presence of less than 300 µg/g of sodium nitrite at pH 6. (1962) explored whether nitrite or the oxides of nitrogen were involved in the inhibition of Gram-negative bacteria in cured meats. They concluded that nitrite and nitrate have either neutral or stimulatory effects on bacteria and that nitric oxide has virtually no effect. 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. canned chopped ham was inoculated with botulinal spores and given 2 thermal processes.017 viable cells/g remaining after processing to F0 = 5-log reduction occurred. and nitrate is not reduced anaerobically.6 at the center of the can would yield 10-fold higher values near the surface of the can.. inhibition. Product inoculated with the lowest inoculum level (173 spores/g) had 0. but at a reduced rate. The inhibited spores did not appear to become fully phase dark. The surviving spores are subsequently inhibited by the salt and nitrite.2 (Greenberg et al. and outgrowth of spores with unpublished data from the Danish Meat Research Institute. The toxicity of nitrite was 3 to 5 times greater at pH 6 than at pH 7. and toxin formation from this low level of survivors during storage at 30°C to 37. The presence of 25 µg/g of residual nitrite after processing and a relatively low brine level of 3. If.203) were compared.Nitrite 177 1960–1970 After 1960 increased emphasis was directed toward the role of nitrite in the total inhibitory system in cured meats and its effect on thermally injured spores. The impaired synthesis of heme prevents the formation of the required cytochrome systems. The thermal process used during the early 1960s by seven U. Nitrate and oxygen were considered interchangeable as electron acceptors required for heme synthesis in some bacteria. One reason given was that spores indigenous to meat emulsions have lower heat resistance than spores produced in laboratory media. The increased use of vacuum packaging raised new questions about the microbiological safety of cured meats. Hemin had no effect on nitrate reduction or growth of Escherichia coli or Bacillus polymyxa. with a median of 0. however.5. making them unavailable for subsequent metabolism. heme synthesis is impaired.1 to 0. spoilage. He estimated the thermal process caused a 4-log reduction of the indigenous spores. If the surviving spore counts between the enzyme-inactivating (74°C internal temperature) and the shelf-stable process (F0 = 0. Research was initiated on irradiation for shelf-stable canned cured meats. the larger number of spores that survive the heat process increases the probability that at least one will be able to grow despite the preservative system. Nitrous acid was considered primarily responsible for the quantitative as well as qualitative changes that occur in the bacterial flora when meat is cured. heat resistance. In the course of this research. Gould (1964) observed germination. The authors concluded that when oxygen is absent during the growth of some bacteria. In a study comparing irradiation with conventional thermal processing.7°C for 6 months. Shelf-Stable Cured Meats Riemann (1963) supplemented existing information on the incidence. a 4. 1965). (1967) opened new possibilities. (1964). It was postulated that nitrous acid reacts either with the cell itself or with various constituents of the medium. producers of shelf-stable canned cured meat was reported to range from F0 = 0.2.S.36% did not prevent outgrowth. Immediately after germination. the observations of Perigo et al. development ceased before lysis or rupture of the spore walls.4 to 0. the initial spore load in the product is too high. Outbreaks of botulism from temperature-abused vacuum-packed smoked fish during the early 1960s added impetus to this research. . Another factor in spore destruction is that a thermal process of F0 = 0.

and (4) the germination and autolysis of surviving spores during refrigerated storage of the meat because of the adverse conditions for outgrowth (low temperature and high residual nitrite).. The process involved a unique sequence of adding the curing ingredients. and appeared to be the chief preservative against putrefactive anaerobic spoilage. including (1) the destruction of vegetative cells by heating to the initial internal temperature of 71°C. spores tended to be protected by salt and nitrate. The refrigerated storage period was necessary to assure stability. 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. but nitrite enhanced heat injury. Increased stability also resulted from improving the canning process to reduce or prevent voids.g.g. and the product was given a long thermal process at a low temperature (e. 30%) and poor meat texture. 75 to 150 µg/g of added sodium nitrite to provide about 20 µg/g after processing to F0 = 0.000 µg/g) increased the apparent heat resistance of the spores. Kueck et al. 1 psig). At 2% to 4%. and then thermally processed. Duncan and Foster (1968a) also reported that heated putrefactive anaerobe spores were less tolerant of salt. Ascorbic acid was then uniformly distributed throughout the meat. This process was used commercially with the approval of the USDA. the normal pH value of canned luncheon meat. salt and sodium nitrate at 0. sealed. (3) destruction of spores on the surface of the meat by the short but high thermal process.. The effect of nitrite was pH dependent and led the authors to support the position that undissociated nitrous acid is the inhibitory agent. The success of the process was believed to be the result of a combination of factors. The product was then heated for a short time at a high temperature (e. A nitric oxide-generating material. The process was considered to greatly increase the amount of nitric oxide available for use as an antibacterial agent. The meat mixture was then filled into cans.5 before placing them onto culture media containing salt. These data reemphasized the importance of spore contamination level for the stability of shelf-stable canned cured meats. The product was then cooled in tap water and placed into refrigerated storage (e. 2 to 2 1/2 hours in 74°C water) to achieve an internal temperature of at least 71°C. The process had to be properly controlled to be successful (Kueck. nitrite. Curing salts interfered with some stage of germination and development of the surviving heated spores at concentrations that were ineffective with unheated spores. Schack and Taylor (1966) described a procedure to produce shelf-stable canned cured meat with improved quality as a result of reduced thermal process. but the process was not dependable and was abandoned. The thermal process required to achieve shelf stability of canned hams larger than 1 1/2 pounds caused excessive purge (e. especially at pH 6. Roberts and Ingram (1966) heated spores in water from F0 = 0..0.1 to 0. When heated and cultured in the presence of the agents.3°C) before release as a shelf-stable product.5% to 4. The two Bacillus species were rendered more sensitive to salt by lower heat treatments than clostridia. nitrite. was then added and mixed into the meat while under a positive pressure (e. Nitrite was strongly inhibitory. such as sodium nitrite. 15 minutes at 113°C to 118°C). A 3-pound meat portion was filled into each can. and nitrate than unheated spores.g. Their results confirmed the concept of spore injury and increased sensitivity to salt and nitrite proposed by Silliker et al. and nitrate.g.4. (1965) developed a new process for the production of 3-pound shelf-stable canned hams with a purge of about 17% and acceptable texture and flavor. (1958). .178 Antimicrobials in Food Spencer (1966) concluded from the literature that 3.. This process caused the surface of the meat in the can to reach 96°C to 107°C. heat resistance was decreased.. First. 30 days at 6°C to 8. the meat was ground or chopped and then deaerated and backflushed with nitrogen to displace free oxygen. This process was tested extensively in at least one commercial plant. When heated in the presence of these agents. They demonstrated that increased stability could be achieved by omitting sodium nitrate and sucrose from the formulation. personal communication). Nitrate had no effect beyond that on water activity.g. and a low incidence of clostridial spores (less than 1/g) were necessary for the safety and stability of shelf-stable canned cured meats.0015 to 1. (2) partial destruction of spores by the initial long thermal process but at the same time retaining a high level of residual nitrite.5% to 1% (5000 to 10.0% brine.

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

In jellied pork tongue. A similar effect occurred with increasing salt from 0% to 4% during incubation at 7. a fermentable carbohydrate.8% brine). botulinum.6.0 to 4. When S. At the highest brine level (4. 21 were able to grow under the same conditions. Of 25 lactobacilli from cured meat.1% or higher. botulinum in vacuum-packaged perishable cured meats were reported during this period. and nitrite. the authors concluded that the levels used in ham would permit growth. The effect of brine on type E toxin production was more . The four strains failed to produce toxin during 5 weeks at 10°C in sliced bologna (2. Similar results were obtained with jellied corned beef stored at 15°C and 25°C.7% brine.1. On sliced bologna. Brownlie (1966) demonstrated the combined inhibitory effect of sodium nitrite concentration. Cooked sliced ham from five producers inoculated with type E spores yielded variable results at 30°C.8%. Another 25 strains of microbacteria failed to grow at pH 5. but no attempt was made to determine whether they could multiply if the hams were subsequently temperature abused. pH. increasing the amount of nitrite from 25 to 200 µg/g caused progressively greater inhibition. Toxin was produced within 7 days at 30°C in bologna (3. but not at 25°C if the brine was 5. residual nitrite. Nitrite was more inhibitory at 0°C than at the other temperatures tested (10°C and 25°C). type E toxin was not produced.75%). Failure to obtain toxin production at 5°C may have been influenced by pH because 13 of 15 samples had a pH of 5.35% and 3. A decrease in pH likely occurred during storage and prevented toxin formation in the other variables.8%). The time required for toxin production decreased as the abuse temperature was increased (22°C. From studies in thioglycollate medium containing salt. perfringens inoculated into raw hams could survive the thermal process. The rate of toxin formation decreased as the brine level increased from 3. the presence of nitrite caused very little or no inhibition. toxin was produced only at 30°C. and temperature on a Microbacterium species in all-purpose Tween (APT) broth. toxin was produced at 20°C to 30°C but not at 15°C. nitrate. Several studies on the growth of C. 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.5 in the presence of 200 µg/g of sodium nitrite.180 Antimicrobials in Food nitrogen or vacuum was organoleptically acceptable. Within 1 week the product pH had decreased from 6. and 37°C). even at 30°C. but not at 29.0. 30°C. No mention was made of pH and none of the agents were tested in combination.7 or less after 4 weeks. lower maximum population levels occurred in vacuum packages than in nonvacuum packages at 15°C and 22°C. which was thought to be the result of product differences in brine and residual nitrite levels. Gough and Alford (1965) showed that spores of C. Their findings that the type of cured meat influenced toxin production were particularly significant. and decreasing pH during temperature abuse were the suggested inhibitory factors.4% brine at 30°C. At pH 7. pH 6. 34 µg/g of residual nitrite). This would help to explain the dominance of the lactobacilli as spoilage organisms in many perishable cured meats. Bologna was relatively resistant to type A toxin production but not ham. toxin was detected in 2 days at 2. At pH 6.0 and below.8°C or 10°C. the authors recommended that vacuum-packaged nonsterile processed foods be stored in the frozen state. In jellied ox tongue.4°C. Pivnick and Bird (1965) found the oxygen permeability of packaging films to influence spoilage but not toxigenesis of sliced cured meats inoculated with C.5% brine and in 4 days at 4. The combination of brine (3. 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). Christiansen and Foster (1965) found type A botulinal toxin production to occur at the same rate on bologna whether packaged with vacuum or without.0.1% to 4. type E toxin was produced at 10°C to 25°C but not at 5°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. This suggested that salt becomes more inhibitory as storage temperatures are decreased. aureus was inoculated onto sliced chopped ham. A total of 90% of the samples held at 25°C remained above pH 6. Toxin was not produced at 10°C or lower.

Using juices expressed from the newer sausage product. An additional factor influencing the apparent significance of nitrite is the growth of competitive flora. Toxin was not produced in product with 4. At each temperature (20°C. All of them reached the same general conclusions as those set forth by Silliker et al. Type E botulinal growth is inhibited by 2% brine at pH 5. and jellied pork tongue) were formulated with commercial levels of nitrate and nitrite. Kafel and Ayres (1969) demonstrated that the growth of enterococci in culture media and. toxin was detected after 4 days with 2. but this is highly questionable. an increase in the level of residual nitrite also occurred.8.28%. 9% brine at pH 5... The concept of thermal injury to spores that survive the processing of shelf-stable canned cured meats was confirmed. botulinum.1% brine) and jellied pork tongue (3. and 30°C).6% brine at pH 6. The product had been changed to a shelf-stable fermented product precooked to 80°C in an oxygen-impermeable casing. Toxin was not produced in either bologna or country cured ham.8% to 4. Perigo et al.5 to 5.3. as the brine level increased in the bologna and ham.35% and after 8 days with 3. The growth of S. sporogenes. Warnecke et al. Evidence was presented that the antimicrobial effect of nitrite is related to the formation of nitrous acid. The process of outgrowth was more sensitive than spore germination to nitrite.3% brine) was studied next (Pivnick et al.1. The authors concluded that heat injury of spores is not necessary for nitrite to prevent toxin production in cured meats. a brine level of 4.9. aureus is inhibited by more than 4% brine at pH 5. and more than 10% brine at pH 6.2°C for 3 to 18 days. considering the bacon had a brine level of 5.5% or above. C.5% brine at pH 6.3. 5.8°C and 22. they determined that type B botulinal outgrowth was inhibited during 4 weeks at 30°C if the juice had a combined pH of 5. All three products (bologna.1.2°C. 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. (1958). Toxin was also detected in bacon held at 12. during which time naturally occurring lactics drop the pH to 5. in perishable canned hams can inhibit the growth of C. In jellied pork tongue. C. more importantly.8 or less. SUMMARY FOR 1960–1970 Progress made during the 1960s toward defining the role of nitrite in cured meats included the following: 1.5% brine at pH 5. The system responsible for the shelf stability of canned cured meats given less than a botulinal cooking was studied by several research groups. perfringens. 3. (1967) suggested an inhibitory substance was formed from nitrite.4% brine. Although not mentioned. 1968) emphasized maintaining adequate salt levels to assure the microbiological safety of perishable vacuum-packaged cured meats. 5. increasing the nitrite level increased the time for toxin production. The raw product was then sold at ambient temperature for cooking at home.2 and 3% brine at all pH values. He concluded that C. 4. Riemann (Anon.5. . and an input nitrite level of 500 µg/g or more. In both products a marked delay in toxin production occurred at 20°C. 1967). and Lactobacillus viridescens. botulinum type A seemed to be completely inhibited by 4.5% brine at 20°C.Nitrite 181 dramatic. 2. which disappeared during the thermal process. 25°C. (1967) reported type E botulinal toxin production in ground beef held at 22. ham. The effect of nitrite levels on types A and B botulinal toxin production in vacuum-packaged sliced ham (4. A new explanation for the shelf stability of canned cured meat was proposed. and 8.

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

thioglycollate) enhanced the effect of nitrite. A 1:1 extract was next prepared with 3% saline and ground. 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. and heating at 115. personal communication). cooked sausages. sporogenes strain PA 6982 in perishable canned cured meat as occurs with C. (1967) reported equivalent inhibition with 8 µg/g of nitrite added before heating the medium and 52 µg/g added after heating the medium. This emphasized the importance of medium selection when studying the antimicrobial activity of nitrite and relating the results to commercially produced meats. and then packed into cans.g. salt. aw 0.7 to 6. The effect of nitrite. The major factor influencing outgrowth was pH. Ashworth and Spencer (1972) reported the equivalent levels to be . and heated (F0 = 0. the solids removed. to some degree.. botulinum. nitrite and salt levels had lesser effects. The cans were then filled with brine (3% salt). respectively. Sausages packed in brine with 400 µg/g of sodium nitrite and processed to F0 = 0. Ashworth and Spencer (1972) described a model system of ground pork (25 g) in 1-ounce bottles. (1975). although they were “clear and undisputable subsidiary effects. nitrite. and 0.g.6.3. The inhibitory level of nitrite was not influenced by pH within the range 5. 115°C). The magnitude of the effect was much less than was previously reported in culture media. cooked. thereby confirming earlier research. 200 µg/g sodium nitrite.. and 50 µg/g of sodium nitrite. overlaid with paraffin oil. Perigo et al. exist (Christiansen et al.5°C in ground pork containing 2. and heated to varying degrees. ascorbate and cysteine) enhanced the antibotulinal effect of the meat suspensions but did not induce Perigo factor formation. held overnight for nitrite equilibration.6% salt. Between 120 and 200 µg/g sodium nitrite provided stability through 5 weeks of storage. as already discussed. Considerable differences were noted in their redox potentials. showing a similar iron–nitrite interaction with C.5°C on the outgrowth of surviving spores was also examined by placing the heated tubes at 23°C for up to 1 year. A similar response with strain 3679 would not be unexpected. as opposed to perishable canned cured meats heated in hot water to a minimum internal temperature of 66°C. cysteine. sporogenes spores to provide a level of 104 spores per can.4). 150. (1967) medium and reinforced clostridial medium but not in liver veal or Wynne fluid media. In ground pork.8% salt.01%. and pH 6. sodium ascorbate..Nitrite 183 sodium nitrite caused the greatest destruction when heated. pH. inoculated (105/ml). Preliminary data.5% sodium tripolyphosphate was studied by Nordin et al.” The effect of all three factors (pH. the liquid placed into tubes. residual nitrite levels remained above 125 µg/g throughout 80 days at 30°C. (1973) studied the effect of adding nitrite to the brine solution for canned Vienna sausages. normal. Small differences in the amount of sodium nitrite below 0. The pork was autoclaved before or after adding nitrite.g. The sausages were prepared with nitrite-containing curing salt. and overheated (115°C for 90 minutes) canned luncheon meat required 500. 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.01% strongly influenced the results. and salt) appeared to be additive.4. and then inoculated with vegetative clostridial cells. One of the sausages in each can was inoculated with C.966. sealed. Johnston and Loynes (1971) obtained Perigo factor formation in the Perigo et al. but not at levels above 0. After a loss of 15% to 25% during processing and an initial decline. The effect of heating spores of PA 3679 at 115. Aqueous meat suspensions prepared from raw. Reducing agents (e. Omitting any one or all three of the additives did not affect thermal destruction. The residual nitrite values of the four media and three meat suspensions were about the same after preparation. It was reported that less nitrite was required for inhibition when it was added before autoclaving. The extract had 2. Farkas et al. for inhibition. however. and. Outgrowth was determined by blackening of the tubes as a result of ferric citrate having been added to the meat. A significant observation was that residual nitrite did not undergo normal depletion in the extract. Reducing agents (e.. These observations led to the conclusion that a Perigo-type effect was detected in the meat system.35 were as stable as sausages in brine without nitrite and processed to F0 = 2.

g. The data obtained with the spore inoculum should be more relevant to shelf-stable canned cured meats. more added nitrite was required in the pork if the nitrite was added before (300 µg/g).4 The product was held until the highest level of nitrite declined to less than 2 µg/g. per se. They concluded that although a PTF was formed. Pivnick and Chang (1974) considered. (1969) found the inhibitor of Perigo et al. The additional nitrite provided comparable residual levels after the various heat treatments. results similar to those just mentioned were obtained during the early stages of temperature abuse. In all cases. 100. less residual nitrite was required for inhibition when nitrite was added before heating. 1967) that the nitrite that disappears during heating may be involved in the formation of the unknown inhibitor. the levels of nitrite required to obtain inhibition were about the same or lower if added before heating than if added after heating. (1973) extended this research to the milder heating given to large perishable canned hams and obtained similar results. thermal destruction of spores and inhibition of the survivors by the salt). another inhibitory factor may be formed when nitrite is heated with meat. 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. When spores were used. Actually. The levels selected (53. Residual nitrite. that although the Perigo inhibitor may not be present in meat. the conclusion that a Perigo effect was demonstrated must be tempered with the observation that. Similar residual nitrite levels were required to inhibit vegetative cells (160. In summary.5 and 22. outgrowth and toxin production decreased.1 µg/g) were the residual nitrite levels after 0 and 2 weeks in product made with 200 µg/g of nitrite. The amount of PTF formed was related to the amount of original nitrite added to the meat before heating. Ashworth et al.5% salt and 150 µg/g of sodium nitrite. strongly influenced clostridial inhibition. (1967) based their conclusions on the added level of nitrite. With excessive heating. Ashworth and Spencer (1972) found no relation between the amount of nitrite lost during heating and the inhibition obtained. The inhibitory levels of residual nitrite for two heating processes — heating from 80°C for 4 hours and heating from 20°C to 70°C over a 4-hour period — did not differ. with prolonged storage (42 days at 37°C). (1967) to be inactivated when added to meat. such as perishable canned ham. Inhibition by nitrite diminished gradually over a period of 8 weeks at 37°C. deaerated in boiling water. If residual nitrite is a valid means for comparison. and 200 µg/g of sodium nitrite and processed to F0 = 0.1 µg/g.184 Antimicrobials in Food 60 to 70 and 90 to 100 µg/g. 150. This was considered evidence for an inhibitory factor other than residual nitrite.5 or 22. Johnston et al. the Perigo effect was of low magnitude. Ashworth et al. and inoculated. Botulinal . In this case alone. The meat was then inoculated with botulinal spores. It was originally suggested (Perigo et al. which had survived heating to F0 = 0. Ashworth and Spencer based their conclusions on the residual level of nitrite. The nitrite level of the suspensions was adjusted after heating to 53. Chang and Akhtar (1974) prepared buffered saline suspensions of canned luncheon meat made with 0.4 in raw meat juice with 4. The authors found that as the original input nitrite level increased. Adding salt (3. the relative inhibitory effect was quantitatively small. which indicates that residual nitrite influenced the results. autoclaving. higher levels of added nitrite were required for inhibition. on the basis of added nitrite. however. However.4. 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. more was required when added before heating the meat.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. and it may also play a role in the safety and stability of pasteurized cured meats.000/g) added after heating and spores (20/g) added before or after heating. The term Perigo-type factor (or PTF) was selected to describe the effect observed in their test. rather than after (150 µg/g).. Perigo et al. The depletion rate of the nitrite added to the suspensions was not influenced by the amount of nitrite originally added to the meat.. concluded that a PTF was produced.

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

0%. product pH. 103. Stability was influenced by nitrite content. The longer the product was held at refrigeration temperatures. The antibotulinal effect of nitrite was considerably greater than that reported for the meat slurries of Roberts et al.5°C and 25°C. decreasing temperature (25°C. a race occurs between death of germinated botulinal cells and depletion of residual nitrite. Christiansen et al. 6. Temperature of abuse was the most significant factor influencing the rate of botulinal growth. (1976) found that heating botulinal spores in an aqueous extract of lean pork at 85°C. The effect of nitrite in perishable canned cured meat was also reported by Tompkin et al. (1978) concluded that when inoculated. even with 300 µg/g of sodium nitrite. 4.5% salt.8% or 3. level of clostridial spores. a preliminary test in bacon led to the conclusion that L-ascorbic acid does not alter the antibotulinal properties of nitrite in bacon. 90°C. neither salt level provided much inhibition. pro or con.. 6. At the lower temperatures. 100. 1978a). 200 µg/g). in the cooked pork medium. This agreed with an earlier observation (Tompkin et al. Furthermore. less inhibition occurred. 1978b) involving product held at 4.5°C. the impact of increasing the nitrite level from 40 to 300 µg/g was minor and became less so as the temperature of abuse increased to 22. At 17. 20°C. At 20°C. Jarvis et al. 3. perishable canned cured meat is placed at 27°C. the greatest inhibition occurred with 1. Spores heated at lower temperatures (70°C or 80°C) were inhibited by 4. nitrite. or 95°C decreased the number that could grow in a recovery medium containing 2. 6. This led to the pork slurry system of Rhodes and Jarvis (1976) and used by Roberts et al.5). a lower concentration of L-ascorbic acid (0. A statistically significant salt–nitrite interaction was observed after heating (70°C to 95°C) only if the recovery medium contained salt levels of 4. decreasing pH (7.0% w/v).5%. 20 µg/g of nitrite in the Perigo medium and 210 µg/g in the slurry were equally inhibitory.5% but not 3. and whether the products were treated by a perishable or shelf-stable thermal process.4°C or 10°C for up to half a year before placing at 27°C. and decreasing inoculum level (106.0. recommended that large-scale tests be used to quantify the many interacting factors influencing the safety of perishable cured meats. one reason was found to be the addition of sodium isoascorbate to the canned meat but not to the meat slurries.5%. Although less effective. (1976) to study the interactions of temperature of abuse. 150.0. Adding L-ascorbic acid (1. Botulinal outgrowth was dependent on the relative levels of residual nitrite and surviving botulinal cells.186 Antimicrobials in Food Baird-Parker and Baillie (1974) studied the growth of a large.5% salt (on water phase). Isoascorbate had enhanced the antibotulinal efficacy of nitrite in the canned meat. 15°C).1%) also increased the efficacy of nitrite in broth but had no effect. 5.. Sodium nitrite and L-ascorbic acid were added as filter-sterilized solutions. salt. At 20°C. increasing salt (1.5% salt. On investigation (Tompkin et al. It was suggested that Bacillus species are less sensitive to nitrite than clostridia. diverse collection of botulinal strains in a broth medium and a cooked pork medium.5. The rate at which inoculated cans swelled at 27°C was inversely related to the level of nitrite added to the product.0%) markedly increased the effectiveness of nitrite with none of the 38 strains tested showing growth in broth containing 50 µg/g sodium nitrite.0. (1976). Autoclaving nitrite in the Perigo medium resulted in significantly more inhibition than autoclaving the nitrite in the meat slurry. 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. or 25°C. In another study. The emulsions were filled into cans and heated. the more the residual nitrite levels declined and the less inhibitory the product became when placed at 27°C.5% or above. (1977). The spores heated at 70°C and 95°C were equally sensitive to the autoclaved nitrite. 101). At 15°C. 22. Grever (1974) examined the stability of emulsions normally used for preparing cooked sausage and liver sausage. Jarvis et al.5°C. The number of strains showing growth in broth was found to decrease with increasing nitrite (50. and inoculum level on botulinal growth and toxin production at pH 6. 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 . a pronounced salt–nitrite interaction occurred.

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

Under these circumstances. the relatively high brine. The second test. and. A later review of the pH data showed that all variables had a pH of 5. The finished franks had a brine of 4. about 2. the exception being one variable with 150 µg/g of nitrite. Toxic product occurred with 50 µg/g of sodium nitrite at all levels of ascorbate. (1979c) evaluated various soy proteins in their system. One toxic sample occurred at 100 µg/g and none at 150 µg/g of sodium nitrite. (1979e) found the rate of germination of C. and omission of nitrate. and 4. It was concluded that ascorbate did not affect the antibotulinal efficacy of nitrite.5% to 4. and 156 µg/g). 105. Toxin production was slower. and then only 2 of the 55 franks became toxic. The strong inhibitory effect observed was likely the result of the combined effects of nitrite.. a fairly rapid decrease in product pH during abuse. the conclusions in both studies concerning the inhibitory levels of nitrite. Sofos et al. 1973). the two studies indicate that the combination of 4. In a meat–soy mixture.52% brine. except for changes in the ratio of pork to beef. The depletion of residual nitrite was faster as the amount of beef was increased in a formulation containing soy. The rate of nitrite depletion was influenced by the type of soy. examined whether an increase in ascorbate over normal levels would alter the antibotulinal effect of nitrite. The pH of the dextrosefree product remained above 5. and the effect of ascorbate in franks should be restated or reexamined. Vacuum-Packaged and Fermented Meats The effect of nitrite in vacuum-packaged franks was studied on two occasions.0 or less within 14 days.. (1979d) compared the efficacy of nitrite (0 and 80 µg/g) in chicken. The addition of 80 µg/g of sodium nitrite was relatively ineffective in chicken and beef. 1973). The franks were produced in the same facilities and formulated as before (Hustad et al. 450 µg/g of nitrate gave a variable response. A significant delay in botulinal toxin production occurred in pork. Sofos et al. Toxin was produced in all variables formulated without dextrose and more quickly in product having 0 or 50 µg/g of nitrite. The type of soy protein influenced the results. Although product made with 150 µg/g of sodium nitrite did not become toxic. reported by Bowen and Deibel (1974) and Bowen et al.0 or less within 21 days. input versus residual nitrite. 40. 20..83%. The decline in pH caused by a lactic fermentation was also the dominant factor preventing botulinal outgrowth in fermented sausage (Christiansen et al. and pork in their system. more importantly. (1973) formulated franks with various levels of nitrite and nitrate.08 or lower within 1 week at 27°C. or 150 µg/g of nitrite declined to 5.6. Assuming a similar pH response in the first test (Hustad et al. only those variables without nitrite became toxic at 27°C. the levels of nitrite (0 to 150 µg/g) and ascorbate (9. In that variable the pH was 5.3°C) sausages containing dextrose. In mildly heated (58. The rate of botulinal toxin production in formulations with soy protein was equal to or slower than in an all-meat formulation. The pH of product made with 50. the addition of 156 µg/g of sodium nitrite was very effective in delaying botulinal toxin production. (1974). and 655 µg/g). in both the presence and absence of nitrite. . The rate of botulinal toxin production was slower in all soy protein formulations than in all meat formulations. Adding 50 µg/g of sodium nitrite delayed botulinal toxin formation for more than 4 weeks during abuse at 27°C. and the products remained nontoxic.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. In the first. 1975).188 Antimicrobials in Food Sofos et al. and fewer toxic samples occurred in product with 100 or 150 µg/g of nitrite. 400 µg/g of sodium ascorbate. Nitrite was ineffective in a formulation of soy isolate and pork fat. beef. They concluded that the effect of nitrite was on delaying the development of the cells after germination and before toxin was produced. 100.8% fermentable carbohydrate. botulinum spores in a chicken frank formulation to be unaffected by the addition of sodium nitrite (0. Hustad et al.

The addition of ascorbate enhanced the antibotulinal effect of 100 µg/g but not 200 µg/g of nitrite. cereus). 1977). 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.9% to 4. The nitrate likely served as an electron acceptor for the growth of micrococci in the vacuum package. cereus tested. If all samples with no mixed cure. Raevuori (1975) studied the combined effect of sodium nitrite (0.7% became toxic. The data included botulinal challenge tests in frankfurters made with chicken meat and ham made with turkey meat. After cooking in Saran™ casings. Erythorbate caused reductions in the residual nitrite and the redox potential of the product. Including nitrate in the bacon enhanced the growth of micrococci. lower populations resulted and enterotoxin was not detected. 42. and 1000 µg/g) on the growth of Bacillus cereus in cooked sausage placed at 20°C for 48 hours. growth was faster at 25°C than at 15°C.Nitrite 189 A request was made by the poultry industry to the U. Erythorbate alone was not inhibitory. 20°C. and 2000 µg/g). Product with 100 µg/g of sodium nitrite remained nontoxic through 14 days at all three temperatures. 100.. A higher percentage of samples were toxic with the addition of 200 µg/g of nitrite than with 100 µg/g of nitrite.9%. higher populations of S.65 to 6.4%.g.S. A summary of the data showing the antibotulinal effect of nitrite in the two poultry products appears in Hauschild (1982). and brine level (mild. mixed cure with 100 µg/g of nitrite. 2. 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. botulinum. and 34. and 25°C) on the rate of botulinal toxin formation.6%) on botulinal toxin production in vacuum-packaged back bacon. Moraxella species.98.9% became toxic. As expected. Inoculation studies examined the effect of nitrite (0 and 100 µg/g) and storage temperature (15°C. Erythorbate was found to enhance the inhibitory effect of nitrite. The request included results by Christiansen et al.7% and 18.3%. (1976) in the United Kingdom studied the effects of a mixture of nitrite (100 or 200 µg/g) and nitrate (250 µg/g). Raevouri encouraged more widespread use of erythorbate for sausage production in Finland.7% to 6. Because brine levels could not be held constant. Noninoculated product containing nitrite developed a slight unusual taste at 20°C owing to the growth of spore-forming spoilage organisms (e. 500. Omitting nitrate led to higher numbers of Moraxella species in the cured bacon. B. 19. respectively. Bacon Crowther et al. medium 4. regardless of the level of nitrite or ascorbate. 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. There was a general trend toward reduced residual nitrite levels at the tune of inoculation when ascorbate was added. Shaw and Harding (1978) studied the effect of nitrate and nitrite on the microbial flora of Wiltshire bacon. Bacon that was sliced and vacuum packaged developed a flora mainly of micrococci and lactics. botulinum. and Moraxella-like bacteria. The predominant flora of the bacon after curing consisted of micrococci. 1000. respectively. 35. aureus grew well in the medium-salted bacon. If all samples containing mixed cure with and without ascorbate were compared. 67. ascorbate (0. Food and Drug Administration (FDA) for approval of sodium nitrite as an additive in poultry products. the sausages had relatively high pH and water activity values of 6. In opened packages.9% became toxic. aureus developed and enterotoxin A was produced. cereus was evidently more resistant to nitrite than C. (1977) that demonstrated the efficacy of sodium nitrite in poultry meat. and 200 µg/g) and sodium erythorbate (0. When all samples having the mild and medium brine were compared. . and mixed cure with 200 µg/g of nitrite were compared.8% and 21. In vacuum packages. respectively. The naturally occurring B.. 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.80 and 0. It also was reported that S. the interaction of nitrite and ascorbate is difficult to interpret.

However. Sofos et al. If a lactic fermentation develops in the interim. All three bacons contained 550 µg/g of sodium ascorbate or sodium isoascorbate. and bacon with 40 µg/g of sodium nitrite plus 0. 6. the data supported the potential approved use of a nitrite–sorbate blend to produce bacon. Temperatures below 27°C or 30°C increase the delay in outgrowth and the inhibitory effects of the foregoing factors. At least on the basis of the botulinal results.. There was considerable variation in toxin production in the bacon from the four plants. The bacon was shipped to a laboratory. Too high a level of botulinal cells when the bacon is abused can overwhelm the inhibitory system. 7.0%.. Collins-Thompson et al. and fermentable carbohydrate (e..26% potassium sorbate. sucrose) were left to the discretion of the producing plant. outgrowth is increasingly delayed. A total of 30 botulinal studies were performed with vacuum-packaged bacon during the 1970s in the United States and Canada. Brine levels below 4. 1974. Because phosphates were present in almost all the studies. the combination of relatively higher brine and decreasing pH can prevent botulinal outgrowth.g. The level of nitrite added to the product is not important. Of the 30 tests. Ivey et al. The data indicate the following: 1. 1980). and growth of a spoilage flora). The bacon was then incubated at 27°C for up to 56 days. They can be more readily compared because they were similar in processing and final product. bacon with 120 µg/g of sodium nitrite. phosphate. The level of residual nitrite at the time the bacon is abused influences the extent of the delay in botulinal outgrowth. The results demonstrated that the combination of nitrite–sorbate was equal to or better than nitrite alone. Bowen and Deibel.0% are not inhibitory to botulinal outgrowth. The initial period of delay depends on the inoculum level. it was suggested that nitrite could be important in delaying the sour spoilage caused by the growth of lactics. 1974. 8. 5. Within the levels and types used. 3. This was likely the result of the interaction of the other components of the product (brine level. 1979).. Vacuum-packaged bacon prepared with 0. Although the studies were performed for a variety of reasons. The USDA conducted a test to verify the antibotulinal effectiveness of a combined nitrite–sorbate mixture in bacon (Johnston. These factors interact to influence botulinal inhibition. Three types of bacon were produced in four commercial plants: bacon with no preservative. 1974. ascorbate and isoascorbate can also have a negative effect by causing more rapid loss of residual nitrite during processing and storage. 1978. As the brine level exceeds 4. phosphates do not appear to influence botulinal outgrowth in bacon. and vacuum packaged (100 g per package).7% sugar (sucrose) or more provides sufficient fermentable carbohydrate that naturally occurring lactics cause a decline in pH to inhibitory levels. aside from the fact that the amount of added nitrite partially determines the level of residual nitrite. fermentable carbohydrate. Tanaka et al. Botulinal outgrowth is influenced by the temperature of abuse. 4. The levels of sodium chloride. it is possible to glean the relative effect of nitrite by examining the nitrite-containing control variables from each test. inoculated with 1000 spores/g.. 10 appear in the technical literature (Christiansen et al. 2. 1980b.. . The addition of ascorbate or isoascorbate can act in concert with residual nitrite to retard botulinal outgrowth in freshly produced bacon.190 Antimicrobials in Food Because higher numbers of lactics were present in bacon with the lowest initial nitrite concentration. it is difficult to assess their effect. The pH of the bacon during abuse significantly influences the potential for botulinal outgrowth.

15%. 1972). and 2000 µg/g). the increase in the adjustment phase was 10 hours or less aerobically and about 30 hours anaerobically. Understanding why this occurs would be very instructive. oxygen pressure. aureus is reduced and enterotoxin production can be prevented by the environmental conditions existing within vacuum-packaged cured meats. One reason given was that S. nitrite. Toxin was produced in certain variables having residual nitrite levels as high as 145 µg/g.34. Nurmi and Turunen (1970) studied the effect of adding nitrite to a previously autoclaved broth medium (pH 6.g.0). reduced pH.3 in two samples with negligible nitrite levels. 10%. 22 µg/g of nitrite. Reasons for the reduced growth and enterotoxin production are evident from the reports. After considering the effects of changing pH during the growth of S. aureus and a review of the literature.3 and 200 µg/g. This suggested either that nitrite was not being used as an electron acceptor or the nitrite reductase system was blocked anaerobically. At higher temperatures (22°C and 30°C). and product pH) contributes to this effect.3) on the growth of S. thus preventing the normal metabolism of pyruvate. aureus by blocking the sulfhydryl sites of either coenzyme A or α-lipoic acid. 200. however. Collectively. Very high levels of nitrite (1000 and 5000 µg/g) either prevented or severely retarded the growth of all strains. it is uncertain whether high nitrous acid content.6 or less). aureus under aerobic but not anaerobic conditions.01% salt. brine. At pH 7. The probability of toxin production at 10°C reportedly decreased as the level of nitrous acid increased. because high nitrous acid values (>0. Buchanan and Solberg (1972) studied the effect of nitrite (0. At 40 µg/g growth was comparable to that in the control without nitrite. However. Nitrous acid levels were calculated from the residual nitrite and pH values as described by Castellani and Niven (1955). They also concluded that vacuum packaging should improve the safety of cured meats relative to S. but it is unclear whether nitrite level influenced anaerobic enterotoxin production. At 200 µg/g growth was delayed or slower.Nitrite 191 Tests with S. and an abnormally low pH of 5. Heterofermentative lactobacilli were more tolerant of salt than the homofermentative strains. aureus is less acid tolerant under anaerobic conditions. low oxygen level. A third sample with a pH of 5. nitrite was without effect in an aerobic environment and 500 µg/g or less was without effect anaerobically. The commercially canned ham used for the test had a brine level of 3. Anaerobic toxin production was prevented with 0.6 µg/g or more nitrous acid and pH values below 5. to a slight extent. aureus was found to be restricted to the periphery of commercially fermented sausage (Barber and Deibel.6%. 7.58. aureus populations as . (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. Extensive tests were also reported for laboratory-cured ham.3. At pH 6. and initial pH (6. Buchanan and Solberg suggested that nitrite inhibits the growth of S. Of significant importance was a report that the growth of S. 1000. and 0% oxygen yielded lower S. 8 µg/g enhanced the growth of the lactobacilli and.6 µg/g) were possible only at reduced pH levels (pH 5. aureus. aureus. the data along with the aforementioned results of Christiansen and Foster (1965) and Crowther et al.9% salt and 100 µg/g of sodium nitrite) incubated at 37°C in environments of 20%. growth of competitive flora. and 0. micrococci and staphylococci (24 strains). 5%. aureus in brain heart infusion broth (BHI). no residual nitrite. but it remains debatable how much each of the possible factors (e. or both prevented anaerobic toxin production. Lactobacilli (78 strains).159 µg/g of nitrous acid also became toxic. anaerobic toxin production occurred at pH 5. future research might demonstrate the involvement of additional factors.3. Genigeorgis et al. the micrococci. Nitrite was metabolized by S. The authors questioned the hypothesis that nitrite inhibition is dependent on the conversion of nitrite to nitrous acid. and Pediococcus cerevisiae (1 strain) were examined for their tolerance to nitrite in the presence and absence of 4. (1976) indicate that the growth of S.. aureus and Other Bacteria A cluster of reports appeared during the early 1970s that dealt with the effect of nitrite on S. 500. Inoculated raw sausage (2.3.

no synergistic effect occurred between salt. and 8%) increased at pH 6. Contrasting results were subsequently reported that showed the production of enterotoxin A to decrease as pH decreased. 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.0.. aureus. decreasing incubation temperature. nitrite. 6. (1979) reported that nitrite inhibits active transport. faecalis or Streptococcus lactis. Morse and Mah (1973) studied the effect of glucose on enterotoxin B synthesis in a broth medium buffered to an alkaline pH (7.g.192 Antimicrobials in Food the oxygen level decreased. the question could be raised whether the inhibitor would be altered or destroyed.. However.6 to 6. Markus and Silverman (1970) reported enterotoxin A production in broth media (pH 6. and pH). salt. oxygen uptake.g. and nitrite on the heat resistance and recovery of S. salt level. Perhaps because of the atypically high pH. Nitrite did not affect glucose transport by S. 4. and decreasing pH. residual nitrite. They instead rely on glycolysis for generating adenosine triphosphate (ATP) when grown on glucose and transport glucose via phosphoenolpyruvate-phosphotransferase rather than by active transport. the sum of which influences stability. cytochrome oxidase) to the ferric state. and 7. more available energy. SUMMARY FOR 1970–1980 The Perigo Factor in Cured Meats 1. 1973). Formation of some similar inhibitor(s) (PTF) during the thermal processing of shelfstable canned cured meat appears possible.7). enterotoxin was not detected through 5 days of storage in the absence of oxygen. and nitrite increased (Tompkin et al. The effect of nitrite on heat resistance was inconsistent. presumably. Adding glucose caused decreased toxin production as a result of the rapid oxidative decarboxylation of pyruvate.8) to be unaffected by sodium nitrite (200 µg/ml) or sodium nitrate (1000 µg/ml). There was a general pattern of increased heat resistance as salt level (0. Adding nitrite as an electron acceptor at the time of anaerobic shock prevented increased toxin production. both of which lack cytochromes.5 or 5. Glucose repression of enterotoxin B production was also reported to occur at pH 6. .0 but to a lesser degree than at pH 7. The inhibitory effect of nitrite in the recovery medium increased with increasing salt content. Bean and Roberts (1974.. 1973). increasing the level of nitrite had the general effect of reducing the recovery of both heat-damaged and undamaged cells. salt increased. 2.5. The formation of the Perigo factor in commercially cured meat is highly debatable. Although adequate growth occurred within 48 hours. In another report on non-spore-forming bacteria. Rowe et al. the percentage of lactic acid as an end product. 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.7 (Morse and Baldwin. Subjecting the cells to anaerobic shock (sparging with 95% N2 and 5% CO2) to inhibit pyruvate decarboxylation caused an increase in the rate and quantity of toxin produced. and. and nitrate on either growth or enterotoxin production. 1975) demonstrated the combined effect of pH. The evidence strongly suggested that nitrite exerted its inhibitory effect by oxidizing ferrous ion of an electron carrier(s) (e. and nitrate was converted to nitrite. and oxidative phosphorylation in Pseudomonas aeruginosa. when added to the recovery medium. If manipulations of thermally processed meat were attempted to extract such an inhibitor.0 but not at pH 5.

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

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

Nitrite 195 for sodium intake in the diet brought new pressure to reduce the salt content of cured meats.7 4 2.2 1. This was used to quantitatively compare the data from the various challenge tests. the FDA acknowledged that prior sanction existed for the use of nitrite as an additive to poultry meat (Cassens. . This information was reiterated by Hauschild and Simonsen (1986). the per capita consumption of red meats decreased while the consumption of poultry products increased. Bacon was considered a low-risk product. whereas turkey ham was a high-risk product.3 3. Reductions in the level of nitrite can be compensated for by moderate increases in the brine level and/or thermoprocess. Their review was to assist in the development of a document for these products.3–0. 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.5–1. He proposed expressing botulinal inhibition as log I/P. TABLE 6. where P is the probability of outgrowth and toxigenesis from one spore.5 4.5 1.1). there has been a very rapid growth in the volume and variety of cured poultry products in the United States. The data provided estimates of the relative degree of botulinal risk for each product. and commercial practices to arrive at guidelines to assure the microbiological safety of these products.1 Shelf Stable Canned Cured Meats: Minimal Brine and Thermoprocess with 150 mg/g Sodium Nitrite Product Luncheon meat % Brine 3–4 4. carbohydrate.5 Ham and shoulder Sausages Source: Hauschild and Simonsen (1985). Since that time. The level of risk differed among the various products. They summarized research.3 0. Hauschild and Simonsen (1985) conducted an extensive review of the technology for producing shelf-stable canned cured meats (Table 6.1–0.5 Thermoprocess (F0 ) 1. Another major trend developed during this time.5 0. Reductions in the recommended levels should be based on extensive plant experience and/or experimental work and controls to assure minimal spore levels.0–4. In 1983.0 5. Considering the recent reductions in the amount of nitrite used for curing meats and the voluntary elimination of nitrate from most cured meats in the United States.5 0.0–5. 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 impact of further reductions in salt deserved close examination.2–0.0–1.5 3. The level of risk could also differ among products of the same type as a result of differences in their brine concentrations. and erythorbate are controlled. He concluded that significant reductions in nitrite should not be made for all cured meats.0 0. existing government regulations. However. 1990). Throughout the decade. further accelerating the trend of the past 40 years. selective reductions in nitrite could safely be made provided the levels of brine.5–5.0 0.

botulinum was estimated to be about 0. Storage at 8°C until a nearly complete loss of nitrite occurred did not diminish the effect of the nitrite. overlaid with vaspar. toxin levels remained high. At brine levels of 3.2.15 spores/kg in the product. Hauschild and Hilsheimer (1983) examined 138 samples of liver sausage from retail outlets in Canada.2. with levels of at least below pH 6. botulinum.24/kg. Perhaps the milder thermal process permitted the survival of a competitive flora that prevented toxigenesis. toxin was detectable 10 cm from the injection site. and 15°C. The rate of pH decrease was slower as the brine level increased. The latter were suspected to be of particular importance as a cause of botulism from raw cured meat products in Europe (Lücke. Another possibility could be offered for this observation. Toxin was not produced at 5°C.4%). In fermented sausage. Greater protection was obtained with 150 µg/g. pH 6. The total C. botulinum to dominate and develop toxin. toxin production was increasingly more rapid.4%. Liver sausage was prepared with 83 µg/g of added sodium nitrite. The test system consisted of a frank emulsion prepared from mechanically deboned chicken meat that was extruded into test tubes. Some consumers find this amount of salt unacceptable. Toxin was detected after 9 to 11 days.2% brine. The use of nitrite in smoked fish in the United States has been restricted. fermented sausage. To prevent the growth of C. Large bone-in hams were inoculated with nonproteolytic botulinal spores before curing in a solution of salt. The tests involved vacuum-packaged smoked whitefish fillets prepared with about 2. nitrite in the range of 0 to 100 µg/g had little or no effect on toxigenesis. At a higher brine level of 5. Sofos et al.4 to as low as 5. but the recovery of viable botulinal cells became increasingly difficult. Additional data on the ham experiments were presented by Lücke et al. Tests in model systems. and 6. the inhibitory effect of the lactic fermentation and decreasing water activity values during fermentation and subsequent drying prevented botulinal toxicity. (1982) assessed the relative risk of botulism from temperature-abused liver sausage. (1980). Hauschild et al. heated. and hams was investigated by Liicke et al. 1982). thereby permitting the more heat resistant C. psychrotrophic strains can grow and produce toxin during the early stages of curing before the salt and nitrite levels penetrate and reach inhibitory levels. By increasing the processing temperature and/or prolonging the time of processing. 10°C. and nitrate at 8°C. Similar results were obtained during the dry curing of hams at 8°C or 10°C. They concluded that nonproteolytic. nitrite.8% to 4. cooled. The number of botulinal spores was estimated to be about 0.196 Antimicrobials in Food Botulinal Challenge Tests Tests in meat and fish products. As the hams continued through the completion of curing. After curing. (1980a) studied the significance of pH on the efficacy of nitrite and sorbic acid mixtures in a chicken frank emulsion. Wagner and Busta (1983) compared the antibotulinal effect of combinations of sodium nitrite (40 and 120 µg/g). the pH of the formulation could be increased to pH 6. and potassium sorbate (0% and .44%. The nitrite (40 µg/g) also significantly increased the inhibitory effect of the sorbic acid.0 being required. The presence of sorbic acid slowed the rate of nitrite depletion.2%. The hams had a slight off-odor but no apparent putrefaction. the inhibitory effect of the nitrite was reduced. Product with low levels of toxin often appeared acceptable. and incubated at 27°C. By increasing the thermal process. competitive flora could have been destroyed. The antibotulinal efficacy of sorbic acid was pH dependent.98. (1982). (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. If a low level of nitrite (40 µg/g) was added. The pH of the fillets decreased during 27°C incubation from about pH 6. As temperature was increased to 8°C. and aw 0.2 without loss of the benefit of sorbic acid. sodium acid pyrophosphate (0% and 0. Methods and media were recommended to facilitate the recovery of both the proteolytic and the psychrotrophic nonproteolytic strains of C. 4. nitrite was more effective and an appreciable delay in toxicity was obtained with 50 and 100 µg/g of nitrite. Toxin production by a mixture of type E and nonproteolytic type B botulinal strains in liver sausage. a high level of salt (5% brine) has been required. Cuppett et al. botulinum.3%.

3 to 6. nitrite level.27 to 6. The frankfurters were made from chicken instead of beef and pork. or thermal process. although the trends in the data may not change. (1979) modified their pork slurry to a thicker consistency. sodium acid pyrophosphate was more effective than sodium hexametaphosphate or sodium tripolyphosphate. Roberts et al.27 to 6.5 to 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. One nitrite level (40 µg/g) was used throughout. 3. They found that increasing the heating from P80°C = 0. more like that of a reformed ham after heating. Roberts et al. The polyphosphates did not influence botulinal inhibition in the absence of nitrite. potassium sorbate (0. Gibson et al. and thermal process on botulinal outgrowth in pork slurries of two different pH ranges (pH 5. (1983) assessed the antibotulinal effect of three phosphates in the test system just described (Wagner and Busta. (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.2 units in the higher pH slurries.6 to 6.56. nitrate.7 and 6. 1980a). Of the polyphosphates tested. or decreasing the abuse temperature.26% potassium sorbate.4%). A separate formula was developed for slurries prepared with low-pH pork (pH 5. potassium sorbate significantly decreased toxin production. Polyphosphate increased botulinal inhibition when added to combinations of sorbic acid or potassium sorbate and a low level of nitrite (40 µg/g).7).5%. nitrite.5% salt (wt/vol water).54.26%). The pH of the pork used for the lower pH slurries ranged from 5. They found the nitrite to be less effective in slurries prepared with meat from the shoulder than meat from the leg. isoascorbate. The effect of sorbate was greater as salt levels .5%. Jarvis et al. The pH was important in all treatments tested.19. sodium acid pyrophosphate (0.03. and pH control of the emulsion was the most effective to achieve reduced nitrite levels and provide enhanced botulinal inhibition. At a given spore inoculum. The pH of the pork used for the higher pH slurries ranged from 6. Considerable variation occurred between the meat from different animals within each breed.4% sodium acid pyrophosphate caused the greatest delay in toxin production. (1981a. (1979b–e. (1982) examined the interaction of potassium sorbate in their cured pork slurry system. 1983). The increase in pH could explain the decreased inhibition in the lower pH slurries. but the reason for the increased inhibition when adding polyphosphate to the higher pH meat slurries is less evident. The combination of 40 µg/g of sodium nitrite. The addition of polyphosphate caused an increase of about 0. This increased inhibition was not observed with nitrite levels of 75 to 125 µg/g.3) and high-pH pork (pH 6. Roberts et al. except for two outliers of 5. Statistically significant reductions in toxin production occurred with increasing salt level. The pH of the four product variables was between 5.72).1 to 0.5% (wt/vol water) below the targeted values. No systematic difference in inhibition was observed with the meat from three breeds of pig.65.Nitrite 197 0.4 units in the lower pH slurries and about 0. polyphosphate. This may alter the quantitative assessment of the individual and combined effects of the additives. They used the same test system as in Sofos et al. adding isoascorbate or nitrate. although the conclusions were stated on the basis of the target values of 2. with certain phosphates being more effective than others. The levels of salt found in the slurries were often as much as 0. It is unclear whether the target or analytical values for salt were used for statistical analysis.4 and 5. There appeared to be more botulinal inhibition with the addition of sodium acid pyrophosphate.4 to 6. The combination of nitrite (40 µg/g). and 4.0.78 to 6. The test system avoided the problem of recontamination that occurs when peeling and packaging franks. 0. (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. and 0. They also found that the antibotulinal efficacy of nitrite could be increased by the addition of phosphate.65 to P80°C = 6. Nelson et al. The initial pH for the various products was 5.26%) in frankfurters.b) evaluated the interactions of salt. Tubes showing gas or other spoilage were preferentially selected for toxin assay.36 and 6.

As expected. However. Bactin was not produced when heating cysteine. and 50 kGy). the rate of nitrite depletion was influenced by storage temperature. 156. The addition of nitrate led to higher residual nitrite levels for a longer period. which otherwise prevent bacteriostatic activity. unless ferric chloride was present and the pH was 6. 100. 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. 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. Custer and Hansen (1980) reported that lactoferrin. The bacteriostatic action was reversed by the addition of cysteine. The authors suggested that Bactin formation may be responsible for the inhibition observed in Perigo-type media.5%). as pH levels decreased to below 6. After higher doses of irradiation (10. They found that the rate of depletion in their pork slurry system was influenced by many intrinsic and extrinsic factors. Pretreatment of the lactoferrin and transferrin with a protease was necessary for inhibitor formation. responded similarly. (1989) prepared ground pork slurries with 0. botulinum. 40. The amount of iron required for inhibitor formation was reportedly similar to the level found in muscle tissue. The probability of botulinal outgrowth decreased as the level of added nitrite increased above 50 µg/g. 30. The significance of microbial growth on nitrite depletion in the slurries during storage was not addressed. They suggested that the bacteriostatic effect is the result of facilitated conversion of cysteine to Bactin and a concomitant removal of thio compounds. Transferrin. an iron-binding protein in casein. They concluded that the level of residual nitrite did not influence botulinal outgrowth in their experiments. and as the storage temperature decreased. The results are intriguing and leave relatively unexplored the underlying mechanisms influencing the results. and the addition of isoascorbate. then inoculated with botulinal spores. forms an inhibitor when autoclaved in the presence of nitrite. sealed with vaspar. thioglycollate. Higher or lower levels prevented the inhibitory effect. Residual nitrite levels after irradiation were inversely related to the irradiation dose. At lower dose levels of 0 to 9 kGy. 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. and stored at 30°C for 28 days. 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. 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. and 200 µg/g of sodium nitrite. For example. 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. legislation was proposed to control the use of nitrite by monitoring the residual nitrite in cured meat products. No suggestion was offered for the increased botulinal inhibition when nitrate was added. The slurries were irradiated. there was a slight reduction of residual nitrite and the probability of botulinal outgrowth increased in only one variable (50 µg/g of sodium nitrite and the lower spore inoculum level). the addition of nitrate. In another study of Perigo factor formation. U. which resulted in rapid botulinal outgrowth. (1984) to examine the effect of various factors on the rate of nitrite depletion and botulinal outgrowth. there was a marked decrease in the residual nitrite. 20. This led Gibson et al. the . 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. and iron in culture media.5% to 3.198 Antimicrobials in Food increased (2.0. 50.

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

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

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

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. sporogenes cells and isolated proteins. and sodium nitrite on the inhibition of C. Consistent with previous reports over the past 60 years. (1987). Their results led to the conclusion that both are inactivated. They were unable to demonstrate a direct action of the compounds on the iron–sulfur proteins. (1991) and Roberts and Dainty (1991). perfringens foodborne illness involving cured meats.5%) and sodium nitrite (50. It appears that the hypothesis proposed earlier is fairly close (Tompkin et al. 1998). However. the efficacy of sodium nitroprusside was explored (Joannou et al. In a broth medium at pH 6. In the research of Duncan and Foster (1968b) such blister formation was not reported following exposure to sodium nitrite. Most cured meats have traditionally had brine levels of 3% or greater.b) examined the effect of nitrite and iron–sulfur–nitrosyl complexes on iron–sulfur clusters in C. 28°C. (1989) tested the effect of nitrite on L. and 5. Research by Payne et al. more generally. the data more strongly demonstrate that L.g.5. it is fairly certain that the mechanism of nitrite involves the inactivation of iron–sulfur proteins. the toxicity of nitrosyl compounds to bacteria. Sodium nitroprusside was found to be a very effective inhibitor of C. Additional information and views on the mechanism of nitrite are available in Roberts et al. perfringens and fecal streptococci in broth media. (1980). 120. The cell-wall surface was the primary point of attack for the nitrosyl complex associated with nitroprusside.0.0.0) found in cured meats. sporogenes.5. At pH 6. perfringens was enhanced with a decrease in pH. sodium chloride. On the basis of the research conducted by these researchers and others. The effect of nitrite on the growth of Listeria monocytogenes was examined in trypticase soy broth by Shahamat et al. In addition.5 and 6. and/or an increase in the salt concentration.. nitrite was not an effective inhibitor at pH 7. perfringens to 3% to 4% salt could explain why there have been few outbreaks in C. It can be concluded that factors (e. a decrease in temperature of incubation.0. One aspect that the more recent data suggests is that the interaction of nitric oxide with iron–sulfur complexes is irreversible. some reduction in growth was observed with the addition of 200 µg/g of sodium nitrite. The cell walls showed a 90% decrease in thiol content when compared with untreated cells. 5. monocytogenes is quite resistant to the inhibitory effect of nitrite. Buchanan et al. decreasing pH and aw) influenced the rate of death. A high salt concentration and low incubation temperatures were the more important factors influencing the inhibition of these bacteria. although the nitrite had been autoclaved with the broth. (1990a. The inhibitory effect of nitrite against C. blisters at the surface of the cells were observed when viewed under the electron microscope. botulinum was again addressed by Carpenter et al. Miscellaneous Studies in Laboratory Media Botha and Holzapfel (1987) tested the effect of nitrite on Sporolactobacillus isolates from a variety of sources.. which is more typical of cured meat and . and 200 µg/g). leading them to doubt that the inhibitory effect of nitrite is on preformed iron–sulfur proteins of the bacterial cell.5. Sodium nitrite was added to the broth before autoclaving. and 37°C) had the strongest effect on growth rate and lag-phase duration. incubation temperature. After continued research on the mechanism of compounds associated with the Perigo factor. The incubation temperature (5°C. The fecal streptococci proved to be quite resistant to nitrite.0% and 3. 19°C.. Gibson and Roberts (1986b) demonstrated the interactions of pH. monocytogenes in a broth medium at pH 7. Juntilla et al. The final pH of the products was about pH 4.202 Antimicrobials in Food The debate over whether clostridial ferredoxin or pyruvate-ferredoxin oxidoreductase is the site of nitrite inhibition for C. (1989) found the rate of death of L. comparable to the inhibitory properties of the anion of Roussin’s black salt. monocytogenes to be similar in fermented sausage manufactured with different levels of salt (3. Nitroprusside was used as a model to investigate the inhibitory mechanism of sodium nitroprusside and. At the pH values (6. such as ferredoxin and pyruvate oxidoreductase.6. The relative sensitivity of C. 1978).

monocytogenes will grow in processed meat and poultry products at refrigeration temperature.Nitrite 203 poultry products. plantarum but not with L. The depletion of nitrite was attributed to enzymatic activity (i. The lower pH meat resulted in a ham with a pH of about 6. nitrite was inhibitory. 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. Collins-Thompson and Thomson (1986) found two strains of L. buchneri (Wolf and Hammes. L. particularly homofermentative lactobacilli.5% and 4. acidophilus. can accelerate nitrite depletion. and even less by increasing the salt level (0. L. and L. lactis. CollinsThompson and Lopez (1982) and Dodds and Collins-Thompson (1984) further demonstrated that lactic acid bacteria. Subsequent tests demonstrated that a strain of L. The authors suggested that inhibitory strains produced specific antibiotic substances other than hydrogen peroxide or lactic acid. 100. the rate of nitrite depletion in cured meats can be important. The loss of residual nitrite in bologna as a result of the action of the lactic acid bacteria was estimated as 30%. vacuum packaged. demonstrate that inhibition can be dependent on the concentration of undissociated lactic acid and the degree of anaerobiosis existing at the site of growth. Under certain circumstances the level of analytically detectable residual nitrite can influence microbial inhibition. Noninhibitory strains decreased the pH of the bologna to only 5.3 within 2 weeks at 5°C. manganese). plantarum. that were resistant to the addition of 50 µg/g of sodium nitrite if grown aerobically in APT broth. the lactic acid bacteria . Fresh pork was divided into two groups: low-pH meat (5. CollinsThompson and Lopez (1982) reported the inhibition to be species dependent. Spoilage of Cured Meats Zeuthen (1980) studied the effect of meat pH on the rate of microbial growth on sliced ham. the canned hams were opened..5 to 5. nitrite may stimulate the uptake and transport of ions (e. both strains accumulated manganese in their cells. it was suggested that in trace metal metabolism. When comparing 0. Both products had a brine level of approximately 4.5 within 4 weeks. By 1988 it was established that nitrite reduction can occur in some strains of L. The meat was then used for processing as canned hams. The growth of Brochothrix thermosphacta was restricted when grown on sliced vacuum-packaged bologna with Lactobacillus brevis or L.. For example. Inhibitory species caused the pH of the bologna to decrease to 5. to a lesser extent by anaerobiosis. The hams were boned and pumped with a pickle solution containing sodium tripolyphosphate. Thus. Competitive inhibition can be a factor affecting the spoilage flora of cured meats. 50. viridescens or Leuconostoc mesenteroides. 1988).35 and a residual nitrite level of 90 µg/g.0%. After cooking and chilling.g.e.5%). viridescens. viridescens was among the most active species. The significance of these observations was discussed. The nitrite reductases generally are heme-containing enzymes. the degree of inhibition was most strongly enhanced by reducing the incubation temperature. 1985). leichmannii.7) and high-pH meat (6. and some interesting questions were posed. the rate of microbial growth was considerably slower in the sliced ham prepared from the lower pH meat. L. and 200 µg/g of sodium nitrite.0 and a residual nitrite level of 38 µg/g after processing. The higher pH meat resulted in a ham with a pH of 6. Both cultures were sensitive to the inhibitory effect of nitrite under anaerobic but not under aerobic conditions. The 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. During the 7 to 8 weeks of storage at 5°C.5). lactis was able to reduce nitrite to N2O (Dodds and CollinsThompson. plantarum. commonly involved in the spoilage of cured meats. however. however. sliced. L. and placed at 5°C. When grown anaerobically. nitrite reductase) rather than lowering the pH of the medium. The data of Grau (1980).3 to 6. 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.

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

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

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

The procedures described show promise for estimating and comparing the combined effects of multiple treatments on pathogen survival and growth.Nitrite 207 had been observed with clostridia (see Perigo Factor). As in the case of earlier research with C. 10°C) on the rate of growth of L. Australia. monocytogenes in the sliced vacuumpackaged product when stored at 4°C or 8°C.7. Sameshima et al.. (1994) inoculated a variety of vacuum-packaged cooked sliced meats with L. Low levels of salt (1% to 2%) enhanced growth. (1997) examined the effect of nitrite on three lactics in vacuum-packed cooked sausage made with and without nitrite. and the United States. F0 = 0. The model test system was then used to evaluate various combinations of heat. mesenteroides. respectively. 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. 550 µg/g). but levels above 3% were inhibitory.2 with 200 µg/g. and additives to achieve shelf stability of canned luncheon meat (Farkas and Andrássy. sausages made with nitrite and having pH 5. they found that broth adjusted to pH 5. inoculum level. viridescens. Liver paté has been implicated in outbreaks of listeriosis in the United Kingdom. Duffy et al. and 15 days for L. monocytogenes. McClure et al. as had been reported earlier for C. a combination of heat and sodium nitrite was necessary to prevent survival and growth (F0 = 0. 1997). Leuconostoc isolates seemed more sensitive to the effects of sodium nitrite and salt in comparison to the homofermentative lactobacilli. irradiation. (1995) examined the effect of sodium nitrite (0 and 200 ppm). Using C. 3%). 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. smoked flavor can be found. Farkas and Andrássy (1992a) described a test system to evaluate the combined effects of formulation. Three µM of residual undissociated nitrite doubled the time taken for a 3 log10 increase in numbers. sporogenes (9 × 105/ml) in reinforced clostridial medium. botulinum.7 with 50 µg/g). although sodium nitrite concentration was not varied. (1996) subsequently investigated the effects of salt. 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). Farber et al.8 and containing 3. In regions of Northern Europe. monocytogenes by sodium nitrite is pH dependent and related to the concentration of undissociated nitrous acid (Buchanan and Golden. Since about 1990. and sodium nitrite in laboratory medium and a predictive model for the growth of L. Buchanan et al. botulinum. salt (1%. 4. heat. 1992b). Only temperature proved to be a significant factor. An extensive series of experiments led to the conclusion that nonthermal inactivation of L. If the broth had been adjusted to pH 6. (1992). The effect of salt and nitrite on the growth of 24 lactics isolated from vacuum-packed cooked sausage was evaluated by Korkeala et al. and Enterococcus faecalis. Perhaps the high iron content of liver pâté negated the effect of sodium nitrite and sodium erythorbate in this product. they estimated the various formulations and thermal processes provided the equivalent of 4 to 7 log10 destruction of C. Fermented Meats and Dry Cured Hams Although removed from most cured meat products in the United States during the 1970s. sodium erythorbate (0.0 and a sour. some . pH. and other factors for inactivating spores and preventing the outgrowth of survivors. 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. sporogenes. temperature. and temperature of storage (4°C. Applying the procedures described by Hauschild (1982). 1995. L. These sausages are produced at temperatures below those generally used in the United States but higher than the temperatures used to manufacture traditional raw fermented dry sausage.5% sodium chloride and 50 or 200 µg/g sodium nitrite inhibited growth during 125 days storage at 37°C. monocytogenes in liver pâté. the effectiveness of sodium nitrite was significantly increased by the addition of sodium ascorbate.

complex. The basic parameters of raw fermented sausage processing in Europe are summarized from Incze (1992) in Table 6.2. Wolf et al. strongly acidic flavor. In Spain. Hungary. softer. In more modern processes. which is responsible for the reddening and other effects. Several reviews. In studies involving faster fermentation. The other group was able to reduce nitrite in the absence of hematin and yielded nitric oxide and N2O. plantarum but not P. more than 50% of the initial nitrite disappearance was attributed to conversion to .0 None Nitrate Not necessary Salty.. A few of the studies pertaining to the manufacture of fermented meats will be summarized. One group. In France. nitrate Yes Salty. 1994. Tests on 70 strains of lactic acid bacteria using cell suspensions incubated anaerobically led to two groups. The trend toward using starter cultures and slightly higher fermentation temperatures to reduce processing times is accompanied by certain negative features. published since 1987. Fernandez et al. Flores and Bermell. (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. low-temperature processing that involves long holding times and reduction of nitrate by micrococci or staphylococci to yield nitrite and other nitrogen oxides that are necessary for cure color development. The cocci also play an important role in flavor and aroma development. firm texture manufacturers in the Mediterranean countries have been shifting away from the more traditional. 1990.. was hematin dependent for nitrite reduction and yielded ammonia as the sole product. describe the procedures used and changes occurring in Europe (Incze.3°C –0. In Europe. Italy. In pursuit of more effective starter cultures for use in manufacturing dry sausage.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). 1996. Specifically. (1990) conducted further investigations on nitrite reductase activity among the lactic acid bacteria..3 0. 1997. (1994) found that sodium nitrite (80 µg/g) reduced the catalase activity of a strain of L. 1987.g. round. and the Balkan countries. thus. pentosus. Nychas and Arkoudelos. acidilactici or L. Nagy et al. Flores. these traditional raw fermented sausages have had higher pH values (e. and nonacidic. incapable of reducing nitrate or influencing flavor development. and Pediococcus pentosaceus.7% Nitrite and. loss of the traditional flavor and increased acidity has led to a decrease in consumer acceptance and declining sales. 1989. Tests by Mares et al. Lücke.208 Antimicrobials in Food TABLE 6. Scannell et al. rubbery or spreadable texture Low-Acid Sausage <12°C 14°C –16°C ~ 6.. nonacidic flavor. and 100 µg/g) in salami. 1989. Subsequent research found the lactic acid bacteria could be separated into heme-dependent and nonhemedependent catalase producers (Wolf et al. nitrate has traditionally been used for dry sausages and hams requiring long ripening times. 2001). L. 1991). In these processes micrococci convert the nitrate to nitrite. 50. Spain.8) and flavors that are more balanced. It is curious that very little information has been published during that period from researchers in North America. pentosus. and 1992. the rate of fermentation is faster and micrococci become inactive as the result of an accumulation of acid. perhaps. the rapid production of acid renders the cocci inactive and. >5. High-Acid Sausage 22°C –25°C 15°C –18°C <5. consisting of L. rich. plantarum.

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

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

allowing for short periods of elevated temperatures up to 10°C. 1999). and with refrigeration this combination is effective in inhibiting growth of C. 1997). Following a review of the literature. 2001a). shad. that combination of salt and temperature. NOCs were not detected in two studies in which nitrite had been added to smoked fish (Pelroy et al. will prevent toxin production in cold smoked fish for several weeks beyond its sensory shelf life when packaged under reduced oxygen conditions.0% salt if combined with 200 µg/g sodium nitrite (Gram. 1997). smoked fish to contain at least 3. The panel was reluctant to recommend expanded use of sodium nitrite despite evidence showing that psychrotrophic C.. although the numbers. The addition of sodium nitrite is limited to smoked salmon. It is possible.. however. particularly in combination with controlled levels of salt (Pelroy et al. In many countries there is no legal requirement for the minimum amount of salt.4°C) for at least 4 weeks. First. if necessary. Cuppett et al. a lower salt content is permitted (i. 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. 1999). Analysis demonstrated that nitrite was produced during storage. With the availability of lower cost farm-raised salmon the international market for cold smoked salmon has continued to increase. Controlling the salt in the water phase and temperature should be adequate. the salt in the water phase ranged from 2% to 4. brined shrimp). botulinum can be expected to occur in smoked fish. The panel expressed concern for the potential carcinogenic effects of nitrosamines.5% salt in the water phase). are low.. The addition of nitrite or nitrate increased the time the fish was considered acceptable by 3 or 4 weeks at 4°C. and chub. monocytogenes (Gram.. 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. a panel of experts reached the following consensus for its report to the FDA (Gram. toxin was detected in 3 to 4 weeks. Two options are available in the event commercially manufactured product is implicated in botulism.000 tons shipped internationally from 28 exporting countries to 52 importing countries (Duffes. 1982. .. Toxin production can be controlled with a combination of a moderate level of salt (3.. sablefish. 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. however. although a minimum of 3% salt in the water phase is strongly recommended (Huss et al. If packaged aerobically. 2001b). Furthermore. a minimum concentration for salt in the water phase can be established. it is highly unlikely that sodium nitrite will be adopted as an additive in smoked fish in Europe. In 1992 more than 32. In France. 1987). gravad fish. Cuppett et al.5% in the water phase) and cold storage (<4.6% (Duffes. Until there is evidence of a need. botulinum (Huss. Considering the lack of regulations and apparent variability in salt concentration in large quantities of cold smoked salmon distributed domestically and internationally. monocytogenes is common in smoked fish. sodium nitrite could be used as an additional control measure. Surveys conducted before the year 2000 indicate L. 1995). depending on the conditions of processing. lightly preserved fish products (cold smoked fish. questions still remain about “aerobic packaging” and its value for controlling botulinal outgrowth. with a production of 12. A level of 3% salt in the water phase will yield perfectly acceptable. but the panel did not consider the more recent risk assessments demonstrating the safety of dietary nitrite (see section on toxicology). This may reflect survival or contamination.380 tons of cold smoked salmon in 1997. 2001a): • • Psychrotrophic C. it would appear the risk of botulism is lower than the research has suggested. botulinum would be further inhibited. 1982. 2. but.5% salt in the water phase or 3. if present. Hyytiä et al.e. Adding potassium nitrate at 347 or 686 µg/g was equal to or more effective than sodium nitrite.. whereas with salt alone.

The growth of S. although not before some initial growth at 24°C. nitrite accelerated the death of all four strains. The authors implied that. 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. and 45 µg/g of sodium nitrate. Pelroy et al. Previous studies had found levels of 0. to produce cold smoked salmon with low levels. w/w) was much more effective and prevented growth through 50 days at 5°C when used in combination with 3% salt.7 cfu/g in positive samples of cold smoked fish from five plants. Typhimurium occurred more rapidly and to higher levels when the packages were opened. and 720 µg/g of sodium nitrate at the time of opening and inoculation. Typhimurium grew in both vacuumpackaged products when held at room temperature. nor were analytical data presented for the product tested. it uses the nitrate in the product in place of oxygen as an alternative hydrogen acceptor. Meers and Goode (1965) reported the rapid growth of S.5 to 11. Because of the relatively high tolerance of L. At the end of storage. and held at room temperature. 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. S. Typhimurium in vacuum-packaged luncheon meats.212 Antimicrobials in Food with strict adherence to good manufacturing practices. It is reasonable to assume the products were recently produced. Kittaka and Greenberg (personal communication) obtained similar results in corned beef inoculated with three different S. monocytogenes to salt and the adverse effect on flavor. Survival of the inoculum in product held at 7°C varied with the product and inoculum level. In subsequent research. 1994a). 1994. The domestic product contained 4. Chemical data were not provided for either luncheon meat. 1991.26% brine. At 5°C. Typhimurium or Salmonella anatum. because S. monocytogenes in salmon containing 3% salt in the water phase. however. Typhi has a nitrate reductase. Anatum grew in bologna but not in liver cheese. rewrapped in Saran™. After reaching 107/g. 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. Typhi in inoculated canned corned beef stored at room temperature and at 37°C. Peterson et al.. Growth occurred in both products. (1993) found the rate of growth to be similar at 5°C for salt concentrations of 3% to 5% in the water phase. coli remained flat.. S. Davidson and Webb (1973) also studied the growth of S. 15 µg/g of sodium nitrite. but a steady increase in numbers occurred at 24°C. Dehydration prevented growth if the packages were opened and left exposed at room temperature. the imported product had 4. 14 µg/g of sodium nitrite. Typhi remained after 20 months at 27°C to 38°C. viable cells remained even after 6 months of storage. Goepfert and Chung (1970) inoculated the surface of sliced bologna and liver cheese with S. Product from two sources. Peterson et al. Very erratic growth occurred on bologna at 18°C. Sodium lactate (2%. Similar results were obtained on ham. cans inoculated with C. This combination was more effective when a low inoculum level (10 cfu/g) was used as a challenge. often less than 1 cfu/g and at a low prevalence. were used for testing. Typhi strains. 1993. Viable S. The initial level of contamination is an important factor influencing growth (Guyer and Jemmi. In both products. including an isolate from the 1964 Aberdeen outbreak involving canned corned beef. one domestic and one imported. Typhimurium declined during the first 2 weeks at 5°C and then remained stable through 6 weeks. perfringens swelled within 2 days at 37°C. increasing the salt concentration above the normal range for smoked fish is an unacceptable option for control. tests were not performed to show whether the presence of nitrate influenced growth.. Mock chicken supported the best growth at both temperatures tested (24°C and 37°C). The inocula died in wieners at both 18°C and 24°C. Rørvik.13% brine. the pH of mock chicken . however. 2000). S.6% salt. Cans inoculated with E. (1994b) found the addition of sodium nitrite (190 to 200 µg/g) to be very effective in slowing the growth of L. Pelroy et al. there was a gradual decline. A lower maximum population (106 versus 108/g) and slower die-off occurred in the imported product.

Under anaerobic conditions. Enteritidis were then used to study the effect of nitrite and salt on growth in nutrient broth at 22°C. including Salmonella. It is not possible to assess the influence of nitrite from the data.2% salt and 60 µg/g of potassium nitrite and a finished brine level of about 2. the primary factor influencing growth was product pH.0. All the enteropathogens grew in one or more of the products within 24 hours at 30°C. (1979) tested the same bacteria on sliced bologna from two processors. 50. that of the chopped hams was 70% and 59%.0. perfringens did not occur under any condition. and Hafnia strains readily multiplied at all nitrite levels. The rate of growth or death was influenced by the sodium nitrite content (0.7 to 4. other than to say that fresh. 4 of 5 strains of Salmonella grew readily.0 by 160 µg/g of sodium nitrite. no data were provided for brine or nitrite levels. In the low-brine ham at 22°C. Neither of two strains of S. Fermented sausage (teewurst) was prepared with four sodium nitrite levels (0.5. perfringens. aureus.8% to 2.47 and 5. The other four species were able to grow under certain conditions. nitrite was without effect. Leistner et al. Salmonella Enteritidis was able to grow at 17°C but not at 10°C. E. The pH of the chopped ham with 70% and 59% moisture decreased to 5. It was concluded that cooked ham is a more likely source of salmonellosis if prepared with a low salt content. and 100 µg/g) and the level of inoculum (100 or 100. The primary factors influencing growth were the temperature of storage and growth of competitive flora.78. Two strains of S. but pH changes were noted. The product from one supplier remained above pH 6.58.8. At a low pH of 5. The mean pH for the ham during storage decreased to no lower than 5.7. and 130 µg/g) and two Salmonella inoculum levels (100 and 100. Both genera were prevalent in commercially produced luncheon meats and fermented sausages. Stiles et al. Although salt and nitrite values were not reported. A high-brine ham had a brine level of about 6%. growth was reduced by only about 30%. Typhimurium to utilize nitrate. given the proper circumstances. A similar response occurred with enteropathogenic E.8%.000/g). This probably reflects the quantity of fermentable carbohydrate added to the product and/or the nature of the competitive flora. viable counts of the 5 strains remained near the inoculum level. 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. 65. S. Aside from temperature of storage. respectively. cereus) and then vacuum packaged. The moisture content of the hams was 70%. Nitrite and ammonia were assimilated at comparable rates. and ammonia as the sole nitrogen source in a chemically defined minimal medium. Growth occurred in the sausage containing 0 and 65 µg/g of nitrite. coli. (1974). S. The trend in the United Kingdom toward using less salt for processing ham was studied by Akman and Park (1974). A low-brine ham was prepared with 1. (1967).0 and 5% salt. Page and Solberg (1980) found S. and 100 µg/g of sodium nitrite.000). Death occurred after 3 days in the sausage with the lower inoculum and 130 µg/g of nitrite. 75.0. The products were inoculated with five “enteropathogens” (C. No other chemical data were provided. 98. nitrite. commercially produced sliced ham and chopped ham did support the growth of the pathogens studied.6 to 6. The resistance of Salmonella to nitrite in culture media was demonstrated by Roberts and Garcia (1973). and the pH for the wieners was 4. coli. 5% salt. that of the ham was near 7. Klebsiella. pH values were followed through storage. Typhimurium. whereas the other frequently decreased to below pH 5.0. Again.Nitrite 213 and bologna was about 6. the fastest generation times occurred in . The authors did not state whether the nitrite was added before or after autoclaving the medium. nitrite values were not given. Growth of C. Stiles and Ng (1979) studied ham and chopped ham obtained fresh from two processors. At a high pH of 7. but a mixture of Enterobacter. In the high-brine ham held at 22°C. (1973) inoculated vacuum-packaged luncheon meat with a mixture of Salmonella strains and held the product at 8°C for 22 days. and B. Castellani and Niven (1955) reported S. The growth of Klebsiella and Enterobacter species on vacuum-packaged luncheon meats at 3°C was reported earlier by Hechelmann et al. Typhimurium was inhibited at pH 6. whether the nitrite was added before or after autoclaving the medium of Perigo et al.

8 and held at 24°C independent of sodium nitrite concentration. The presence of nitrite had no effect on aerobically grown cultures. Thomas et al. 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. Nitrite was only inhibitory with the highest level of nitrite and when the franks were incubated at 15°C. This research was primarily concerned with preventing the growth of Salmonella. found S. In a traditional Turkish dry sausage that is fermented and dried at temperatures .0 to 6. coli. 27°C. the inherent pH of the meat (normal pH 5. When grown anaerobically in a glucose-limited minimal medium with peptone and nitrite. Factors influencing Salmonella growth in mettwurst that was cured and stored at 20°C include the amount of nitrite and salt. (1985) identified a nitrite reductase in the cytoplasmic fraction of S. He recommended that a starter culture and/or gluconolactone be added to provide optimum safety. S. Typhimurium used nitrite as the sole nitrogen source at concentrations as high as 400 µg/g. 150. (1991). At concentrations of 500 µg/g and higher. but growth was delayed with 200 µg/g (Turanta and Ünlütürk. 1987).b) to determine the additional effect of eight different phosphates of E. the addition of sugar. Schillinger and Lücke (1989) measured Salmonella growth and/or survival in mettwurst.214 Antimicrobials in Food media containing both nitrate and ammonia. Typhimurium to be relatively resistant to sodium nitrite across the ranges of salt and pH values found in nonfermented and acidulated foods. Similar results were observed with S.8). 6.2). The franks were inoculated on the surface. except when the cultures were incubated at the lowest temperature tested (10°C). and 100 µg/g sodium nitrite. being able to use nitrite in broth culture under anaerobic conditions at 5°C.8 versus 6. 1977). 50.0. and incubated at 15°C or 27°C for 21 days. Collins-Thompson et al. When examined after 10 weeks of incubation. Hughes and McDermott (1989) used a procedure similar to that of Gibson and Roberts (1986a. Typhimurium had shorter generation times and increased cell yields compared with nitrite-free cultures. and 37°C (McClure and Roberts. a cured raw spreadable sausage. complete inhibition occurred. vacuum packaged.8. S. aureus and C. 100. The reductase required NADH and was most active at pH 8. coli. Collins-Thompson et al. and 200 µg/g) and three strains of Salmonella. 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. coli was also apparent in tests involving two-dimensional gradient plates incubated at 20°C. Isolates of Enterobacter and Serratia species were tolerant to nitrite. Page et al. Gibson and Roberts (1986a) examined the interaction of salt (1% to 10% wt/vol). None of the variables tested would likely assure the destruction of Salmonella in this product category. 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 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). and 156 µg/g) in frankfurters on a mixture of two strains of Salmonella. the effect of added phosphate was relatively minor. numbers increased during 72 hours in the presence of 0.6 to 5. Typhimurium numbers decreased in BHI broth adjusted to pH 4. 50.6. 50. Typhimurium when grown anaerobically with nitrite as the sole nitrogen source. sporogenes. Growth did not occur during fermentation or drying. 1991). coli. pH (5. At pH 5. S. using a gradient gel plate system. 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. (1984) prepared fermented sausages with sodium nitrite (0. and the presence of lactic acid-producing bacteria. The phosphates generally delayed the outgrowth of E. The presence of nitrite did not influence the survival of salmonellae. Rice and Pierson (1982) tested the effect of sodium nitrite (0. The nitrite resistance of E.2.

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

(1949) described the characteristics of Lactobacillus and Leuconostoc strains isolated from commercially cured meats with greenish discoloration. Naturally occurring nitrate in vegetables accounts for more than 85% of the dietary intake of nitrate. inoculum level. the remaining nitrite enhanced growth and aflatoxin production. 156 to 200 µg/g) initially restricted the profuse growth of mold. 3. 1957). 2. sorbate). Endogenously formed nitric oxide and oxygen radicals can play an important role in human health. 4. The name L. 1967. A system was developed to evaluate the combined effects of formulation.. 1957).g. the use of preservatives (e.5 = 12. Iwata et al..5 minutes. Niven et al.g.. Unpublished research by Shaparis and Christiansen has shown D values in minced ham to be D63 = 30. monocytogenes under certain. D68 = 5.5 to 6. . the product spoiled before aflatoxin reached detectable levels. All were salt tolerant and capable of growth at low temperature (5°C). Control of these bacteria lies in proper handling of cooked product to be reworked into succeeding lots of meat. 6. Conversion of nitrate in the saliva is the major source of nitrite in humans. The more rapidly produced products closely resemble those that have been produced in the United States over the past 30 to 40 years.5 to 15 minutes. circumstances. they are not major causes of spoilage.5 minutes. (1983). SUMMARY FOR 1990–2002 1. In addition. no aflatoxin production or mold growth occurred. 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. 1965. At 26°C and 37°C. Nitrite can contribute to the inhibition of L. viridescens was given to these bacteria (Niven and Evans. Vrchlabsky and Leistner. Increased heat resistance could be acquired by repetitive exposure to thermal processing. 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. heat. The effect of nitrite on aflatoxin production by Aspergillus parasiticus in fresh pork sausage was examined by Obioha et al. and other factors for controlling mesophilic clostridia. Gardner. The heat resistance of these bacteria has been reported (Niven et al. The role of nitrate and nitrite in the manufacture of traditional and more rapidly produced fermented meats was clarified. but not all. Green Discoloration in Cured Meats Niven et al. 1986). and D71 = 1 to 2 minutes (Tompkin. During storage at 5°C for 28 days. 1954. Evidence was presented indicating that the discoloration results from a reaction of hydrogen peroxide with the cured meat pigment. Nitrite burn is another form of green discoloration that is caused by a combination of excessive levels of nitrite and reduced pH (Deibel and Evans. The mechanism of green discoloration was further elucidated by Shank and Lundquist (1963). 5. Despite this observation. 1971). Except for a limited number of products to which nitrite and nitrate might be added. (1988). the results in pork sausage plus a test with media led to the conclusion that high levels of nitrite (e. D65. Additional information on factors affecting green discoloration by L. 1972). (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. yeasts and molds are of little or no public health importance. viridescens and other lactics has been developed by Grant and McCurdy (1986) and Grant et al..216 Antimicrobials in Food permitted. As residual nitrite decreased to less inhibitory levels. The most significant development during this period was the growing body of data that dietary nitrate and nitrite are not carcinogenic (see Toxicology).

regulations do not specify minimum nitrite levels for cured products. ASSAY METHOD Because the levels of nitrite and nitrate permitted in foods are established by regulation.177 (Code of Federal Regulations.160. The use of nitrite and nitrate in the processing of fish products is specified in 21 CFR 172. It is also common to generate a certain amount of rework during the manufacture of sausage products. 1986). the level of added nitrite is decreased. 2002a). The net effect of the U. Examples of the effect of this regulation on the maximum allowable level of nitrite in several sausage and loaf products are listed in Table 6. The status of nitrite and nitrate in cured meats and cheese in the United Kingdom was reviewed in 1978 (Ministry of Agriculture.S. The current USDA regulations controlling the use of nitrite and nitrate in meat and poultry products are specified in 9 CFR 317. 1978). and 172. The most recent summary of the regulations in different countries appears in Cassens (1990). An example of rework is the round ends of sausages that are trimmed off before slicing the product. 1978). Gerhardt. as proposed by the Expert Panel on Nitrites and Nitrosamines (Food Safety and Quality Service. 8. 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.21-23 of the federal regulations (Code of Federal Regulations. the weight of this meat is subtracted from the fresh meat portion of the formulation and the amount of nitrite added is reduced accordingly. 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. 172.3.17 and 424. 1974.S. meat and poultry regulations is that nitrite and nitrate are added on the basis of the uncured meat in the product formulation. the method of assay applicable to each country should be used. Because it has already been cured. 172. 2002b). 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.170. 1978). Braathen. Germany reduced the permitted level of nitrite in nitrite-containing curing salt by 20% (Leistner. 9. Nitrite is of relatively little value for controlling Gram-negative enteric pathogens in commercially prepared foods. A unique feature of the U. Nitrite and nitrate levels for curing meats in the United States were recommended by a USDA Expert Panel on Nitrites and Nitrosamines (Food Safety and Quality Service. This further reduces the nitrite level in the product formulation and. This material is normally used as an ingredient at a level of about 5% in the manufacture of succeeding lots of the same product. With the exception of bacon and smoked chubs. 1995). The regulations were summarized in two comprehensive surveys (Meester. and Food. As the percentage of extenders and other nonmeat ingredients is increased in the products.175. Within the United States the methods of assay appear in the Official Methods of Analysis of the Association of Official Analytical Chemists . leads to lower residual nitrite levels after processing. 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. 1979). The use of nitrate or nitrite as additives in cheese and seafood products was clarified. Other countries limit the amount of nitrite and nitrate on the basis of the total weight of the product formulation. 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. Fisheries.Nitrite 217 7.S. depending on the circumstances. 1981. U.

2002).06 mg/kg body weight. Source: Based on formulations in Pearson and Tauber (1974). with the lower doses applying to infants who are more sensitive to methemoglobinemia. as evidence of toxicity. 1995). 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.5% to 2.. estimates for the toxic dose range from 1 to 8.7 and 0 to 0. Cyanosis. and apply to all sources of intake but do not apply to infants younger than age 3 months.3 mg sodium nitrite/kg body weight (Gangolli et al. estimates of the lethal dose have ranged from 4 to 30 g (about 70 to 500 mg/kg body weight). International agreement has been reached on the specifications and analytical methods for potassium nitrate and sodium nitrite (JECFA. occurs at methemoglobin levels in excess of 10%. nonfat dry milk. For potassium nitrate. The values are expressed as nitrate and nitrite ions. 1994). 1995). whereas 30 to 60 g of sodium nitrate have been given for 2 months as an acidifying diuretic without manifestation of adverse effects. the criterion most commonly used for nitrite toxicity. TOXICOLOGY The acceptable daily intake for potassium nitrate and sodium nitrite have been reported to be 0 to 3. respectively. Two years of rat studies on nitrate showed no evidence of carcinogenicity. estimates for the lethal dose have ranged from 2 to 9 g (about 33 to 250 mg/kg body weight). 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 . respectively (JECFA. The normal range of methemoglobin levels is 0.. 1994). The no-adverseeffect dose level was estimated to be 2500 mg sodium nitrate/kg body weight/day (Gangolli et al..3 Examples of Sodium Nitrite Levels Added to Cured Meats in the United States Actual NaNO2 (µg/g) in Total Formulation if Input NaNO2 Is Reduced to 100 µg/g in the Meat 78 75 67 74 69 73 97 90 92 83 58 60 50 47 Product Bologna Bologna with NFDMa Bologna (imitation) Franks (meat)a Franks (meat)a Franks with NFDM Liver sausagea Liver sausagea Cured beef loaf Blood pudding Pickle and pimento loaf Olive loaf Family loaf Veal loaf a NaNo2 (µg/g) Added on Basis of Meat Content 156 156 156 156 156 156 156 156 156 156 156 156 78 78 Actual NaNO2 (µg/g) in Total Formulation 121 117 105 115 107 114 152 140 143 130 91 94 50 47 NFDM. 1994). Using this criterion.0%. the uppermost level being found in infants and pregnant women. and it appears that nitrate per se is not genotoxic. Data on reproductive toxicity for nitrate were limited.218 Antimicrobials in Food TABLE 6. (AOAC international. For sodium nitrite.

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

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

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

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

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

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

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

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

and food-poisoning pathogens. Growth between 0°C–30°C.. Bacillus brevis. damnosus. Obligate anaerobes. Cause spoilage of acid products. wine. Varied nisin sensitivity. Aerobe/anaerobe. mesenteroides innocua. Includes psychrotrophs. Aerobes/anaerobes. Cause blackening of canned food. Heat-resistant aerobic and facultative anaerobic spore formers. Some used for cheese production. tyrobutyricum nigrificans Enterococcus Lactobacillus faecalis. Cause spoilage of wine and beer. pasteurianum. thermophiles. Heat-resistant spore-forming thermophiles. subtilis. Can grow at low pH. casei. There have been . varians Spp. Causes food poisoning. Spore forming. M. bulgaricus. poor nutrient availability.. Gram-negative bacteria are resistant to nisin because the antimicrobial is unable to penetrate their complex cell wall to reach its site of action: the cytopl