1. Modern Food MicrobiologySixth EditionJames M. JayProfessor EmeritusWayne State UniversityDetroit, MichiganAdjunct ProfessorUniversity of Nevada Las VegasLas Vegas, NevadaAN ASPEN PUBLICATIONAspen Publishers, Inc.Gaithersburg, Maryland2000

2. The author has made every effort to ensure the accuracy of the information herein. However, appropriate informationsources should be consulted. The author, editors, and the publisher cannot be held responsible for any typographical orother errors found in this book.Library of Congress Cataloging-in-Publication DataJay, James M. (James Monroe), 1927Modern food microbiology / James M. Jay.6th ed.p. cm. (Aspen food science text series)Includes bibliographical references and index.ISBN 0-8342-1671-X1. FoodMicrobiology. I. Title. II. Series.QR115.J3 2000664'001'579dc2199-054735Copyright O 2000 by Aspen Publishers, Inc.A Wolters Kluwer Companywww.aspenpublishers.comAll rights reserved.Aspen Publishers, Inc., grants permission for photocopying for limited personal or internal use.This consent does not extend to other kinds of copying, such as copying for general distribution,for advertising or promotional purposes, for creating new collective works, or for resale.For information, address Aspen Publishers, Inc., Permissions Department,200 Orchard Ridge Drive, Suite 200, Gaithersburg, Maryland 20878.Orders: (800) 638-8437Customer Service: (800) 234-1660About Aspen Publishers For more than 40 years, Aspen has been a leading professional publisher in a variety ofdisciplines. Aspen's vast information resources are available in both print and electronic formats. We are committedto providing the highest quality information available in the most appropriate format for our customers. Visit Aspen'sInternet site for more information resources, directories, articles, and a searchable version of Aspen's full catalog,including the most recent publications: www.aspenpublishers.comAspen Publishers, Inc. The hallmark of quality in publishingMember of the worldwide Wolters Kluwer group.Editorial Services: Joan SesmaLibrary of Congress Catalog Card Number: 99-054735ISBN: 0-8342-1671-XPrinted in the United States of America2 3 4 5 3. PrefaceThe sixth edition of Modern Food Microbiol-ogy,like the previous edition, focuses on thegeneral biology of the microorganisms that arefound in foods. Thus, the contents are suitablefor its use in a second or subsequent course in amicrobiology curriculum, or as a primary foodmicrobiology course in a food science or foodtechnology curriculum. Although organic chem-istryis a desirable prerequisite, it is not neces-saryfor one to get a good grasp of the topicscovered.When used as a microbiology text, the fol-lowingsequence has been found to be suitable.A synopsis of the information in Chapter 1 willprovide students with a sense of the historicaldevelopments that have shaped this disciplineand how it continues to evolve. Memorizationof the many dates and events is not recommendedsince much of this information is presented againin the respective chapters. The material in Chap-ter2 is designed to provide a brief backgroundon microorganisms in nature with emphasis onthose that are important in foods. This materialcan be combined with the intrinsic and extrinsicparameters of growth in Chapter 3 as they existin food products and as they affect the commonfoodborne organisms. Chapters 4 to 9 deal withspecific food products and they may be coveredto the extent desired with appropriate reviews ofthe relevant topics in Chapter 3. Chapters 10 to12 cover methods for culturing and identifyingfoodborne organisms and/or their products, andthese topics may be dealt with in this sequenceor just before foodborne pathogens. The foodpreservation methods in Chapters 13 to 19 in-cludeinformation that goes beyond the usualscope of a second course. Chapters 14 and 19are new to the sixth edition. Chapter 14 consoli-datesinformation from the previous edition thatwas scattered throughout several chapters, andit contains much new information on modifiedatmosphere packaging. Chapter 19 covers highpressure and pulsed electric field processing offoods, and it contains two sections taken fromthe chapter on high temperature processing inthe previous edition.Chapters 20 and 21 deal with food sanitation,indicator organisms, and the HACCP system, andcoverage of these topics is suggested before deal-ingwith the pathogens. Chapters 22 to 31 dealwith the known (and some suspected) foodbornepathogens including their biology and methodsof control. Chapter 22 is also new to this edi-tionand it is intended to provide an overview ofthe chapters that follow. The material in this chap-terthat deals with mechanisms of pathogenesisis probably best dealt with when the specificpathogens are covered in their respective chap-ters.For most semester courses with a 3-credit lec-tureand accompanying 2 or 3 credit laboratory,only about 70% of the material in this edition is 4. likely to be covered. The remainder is meant forreference purposes. Citations for new and up-datedmaterial can be found in the Reference listsat the end of the chapters.The following individuals assisted me bycritiquing various parts or sections of the sixthedition, and I pay my special thanks to each:P. Druggan, P. Feng, R.B. Gravani, D.R. Henning,YJ. Lee, J.A. Seiter, L.A. Shelef, J.N. Sofos,A.C.L. Wong, and A.E. Yousef. Those who as-sistedme with the previous five editions are ac-knowledgedin the respective editions. 5. This page has been reformatted by Knovel to provide easier navigation. vContentsPreface .................................................................................................... xvPart I. Historical Background .............................................................. 11. History of Microorganisms in Food .......................................................... 3Historical Developments ................................................................... 4Part II. Habitats, Taxonomy, and Growth Parameters ....................... 112. Taxonomy, Role, and Significance of Microorganisms in Foods ............ 13Bacterial Taxonomy .......................................................................... 13Primary Sources of Microorganisms Found in Foods ........................ 17Synopsis of Common Foodborne Bacteria ....................................... 19Synopsis of Common Genera of Foodborne Molds .......................... 24Synopsis of Common Genera of Foodborne Yeasts ......................... 293. Intrinsic and Extrinsic Parameters of Foods That Affect MicrobialGrowth ..................................................................................................... 35Intrinsic Parameters ......................................................................... 35Extrinsic Parameters ........................................................................ 49Combined Intrinsic and Extrinsic Parameters: The HurdleConcept ...................................................................................... 53Part III. Microorganisms in Foods ...................................................... 574. Fresh Meats and Poultry ......................................................................... 59Biochemical Events That Lead to Rigor Mortis ................................. 60The Biota of Meats and Poultry ........................................................ 60Incidence/Prevalence of Microorganisms in Fresh Red Meats .......... 60Microbial Spoilage of Fresh Red Meats ............................................ 68Spoilage of Fresh Livers ................................................................... 76Incidence/Prevalence of Microorganisms in Fresh Poultry ................ 77Microbial Spoilage of Poultry ............................................................ 78Carcass Sanitizing/Washing ............................................................. 81 6. vi Contents5. Processed Meats ..................................................................................... 87Curing .............................................................................................. 87Smoking ........................................................................................... 89Sausage, Bacon, Bologna, and Related Products ............................ 89Bacon and Cured Hams ................................................................... 91Fermented Meat Products ................................................................ 936. Seafoods .................................................................................................. 101Microbiological Quality of Various Fresh and Frozen Products ......... 101Fermented Fish Products ................................................................. 104Spoilage of Fish and Shellfish .......................................................... 1057. Fermentation and Fermented Dairy Products ......................................... 113Fermentation .................................................................................... 113Dairy Products .................................................................................. 119Apparent Health Benefits of Fermented Milks ................................... 124Diseases Caused by Lactic Acid Bacteria ......................................... 1288. Fruit and Vegetable Products: Whole, Fresh-Cut, andFermented ................................................................................................ 131Fresh and Frozen Vegetables .......................................................... 131Spoilage of Fruits ............................................................................. 141Fresh-Cut Produce ........................................................................... 141Fermented Products ......................................................................... 146Miscellaneous Fermented Products .................................................. 1549. Miscellaneous Food Products ................................................................. 163Delicatessen and Related Foods ...................................................... 163Eggs ................................................................................................. 164Mayonnaise and Salad Dressing ...................................................... 167Cereals, Flour, and Dough Products ................................................. 168Bakery Products ............................................................................... 168Frozen Meat Pies ............................................................................. 168Sugar, Candies, and Spices ............................................................. 169Nutmeats .......................................................................................... 169Dehydrated Foods ............................................................................ 170Enteral Nutrient Solutions (Medical Foods) ....................................... 171Single-Cell Protein ............................................................................ 171This page has been reformatted by Knovel to provide easier navigation. 7. Contents viiPart IV. Determining Microorganisms and/or Their Products inFoods ........................................................................................ 17710. Culture, Microscopic, and Sampling Methods ........................................ 179Conventional Standard Plate Count .................................................. 179Membrane Filters ............................................................................. 182Microscope Colony Counts ............................................................... 184Agar Droplets ................................................................................... 184Dry Film and Related Methods ......................................................... 185Most Probable Numbers ................................................................... 186Dye Reduction .................................................................................. 186Roll Tubes ........................................................................................ 187Direct Microscopic Count .................................................................. 187Microbiological Examination of Surfaces .......................................... 188Metabolically Injured Organisms ....................................................... 190Viable but Nonculturable Organisms ................................................ 19411. Physical, Chemical, Molecular, and Immunological Methods ................ 201Physical Methods ............................................................................. 201Chemical Methods ............................................................................ 206Methods for Characterizing and Fingerprinting FoodborneOrganisms .................................................................................. 214Immunological Methods .................................................................... 22112. Bioassay and Related Methods .............................................................. 237Whole-Animal Assays ....................................................................... 237Animal Models Requiring Surgical Procedures ................................. 242Cell Culture Systems ........................................................................ 243Part V. Food Preservation and Some Properties ofPsychrotrophs, Thermophiles, and Radiation-ResistantBacteria ..................................................................................... 25113. Food Preservation with Chemicals .......................................................... 253Benzoic Acid and the Parabens ........................................................ 253Sorbic Acid ....................................................................................... 255The Propionates ............................................................................... 257Sulfur Dioxide and Sulfites ............................................................... 257This page has been reformatted by Knovel to provide easier navigation. 8. viii ContentsNitrites and Nitrates .......................................................................... 258NaCl and Sugars .............................................................................. 264Indirect Antimicrobials ...................................................................... 265Acetic and Lactic Acids ..................................................................... 268Antibiotics and Bacteriocins .............................................................. 268Antifungal Agents for Fruits .............................................................. 274Ethylene and Propylene Oxides ....................................................... 274Miscellaneous Chemical Preservatives ............................................. 27514. Food Preservation with Modified Atmospheres ...................................... 283Definitions ........................................................................................ 283Primary Effects of CO2 on Microorganisms ....................................... 286Food Products .................................................................................. 288The Safety of MAP Foods ................................................................. 290Spoilage of MAP and Vacuum-Packaged Meats .............................. 29315. Radiation Preservation of Foods and Nature of MicrobialRadiation Resistance ............................................................................... 301Characteristics of Radiations of Interest in Food Preservation .......... 301Principles Underlying the Destruction of Microorganisms byIrradiation ................................................................................... 303Processing of Foods for Irradiation ................................................... 305Application of Radiation .................................................................... 305Radappertization, Radicidation, and Radurization of Foods ............. 306Legal Status of Food Irradiation ........................................................ 312Effect of Irradiation on Food Quality ................................................. 313Storage Stability of Irradiated Foods ................................................. 315Nature of Radiation Resistance of Microorganisms .......................... 31516. Low-Temperature Food Preservation and Characteristics ofPsychrotrophic Microorganisms .............................................................. 323Definitions ........................................................................................ 323Temperature Growth Minima ............................................................ 324Preparation of Foods for Freezing .................................................... 324Freezing of Foods and Freezing Effects ........................................... 325Storage Stability of Frozen Foods .................................................... 327This page has been reformatted by Knovel to provide easier navigation. 9. Contents ixEffect of Freezing on Microorganisms .............................................. 327Some Characteristics of Psychrotrophs and Psychrophiles .............. 331The Effect of Low Temperatures on Microbial PhysiologicMechanisms ............................................................................... 333Nature of the Low Heat Resistance of Psychrotrophs ....................... 33617. High-Temperature Food Preservation and Characteristics ofThermophilic Microorganisms ................................................................. 341Factors Affecting Heat Resistance in Microorganisms ...................... 342Relative Heat Resistance of Microorganisms ................................... 346Thermal Destruction of Microorganisms ........................................... 348Some Characteristics of Thermophiles ............................................. 351Other Aspects of Thermophilic Microorganisms ................................ 354Canned Food Spoilage ..................................................................... 35618. Preservation of Foods by Drying ............................................................. 363Preparation and Drying of Low-Moisture Foods ................................ 363Effect of Drying on Microorganisms .................................................. 364Storage Stability of Dried Foods ....................................................... 366Intermediate-Moisture Foods ............................................................ 36719. Other Food Preservation Methods .......................................................... 375High-Pressure Processing ................................................................ 375Pulsed Electric Fields ....................................................................... 379Aseptic Packaging ............................................................................ 380Manothermosonication (Thermoultrasonication) ............................... 381Part VI. Indicators of Food Safety and Quality, Principles ofQuality Control, and Microbial Criteria ................................... 38520. Indicators of Food Microbial Quality and Safety ..................................... 387Indicators of Product Quality ............................................................ 387Indicators of Food Safety .................................................................. 388The Possible Overuse of Fecal Indicator Organisms ........................ 401Predictive Microbiology/Microbial Modeling ...................................... 40221. The HACCP System and Food Safety .................................................... 407Hazard Analysis Critical Control Point System ................................. 407Microbiological Criteria ..................................................................... 415This page has been reformatted by Knovel to provide easier navigation. 10. x ContentsPart VII. Foodborne Diseases .............................................................. 42322. Introduction to Foodborne Pathogens ..................................................... 425Introduction ...................................................................................... 425Host Invasion ................................................................................... 425Pathogenesis .................................................................................... 428Summary .......................................................................................... 43423. Staphylococcal Gastroenteritis ................................................................ 441Species of Concern in Foods ............................................................ 441Habitat and Distribution .................................................................... 443Incidence in Foods ........................................................................... 443Nutritional Requirements for Growth ................................................. 444Temperature Growth Range ............................................................. 444Effect of Salts and Other Chemicals ................................................. 444Effect of pH, Water Activity, and Other Parameters .......................... 444Staphylococcal Enterotoxins: Types and Incidence .......................... 445The Gastroenteritis Syndrome .......................................................... 453Incidence and Vehicle Foods ............................................................ 454Ecology of S. aureus Growth ............................................................ 455Prevention of Staphylococcal and Other Food-PoisoningSyndromes ................................................................................. 45524. Food Poisoning Caused by Gram-Positive SporeformingBacteria .................................................................................................... 461Clostridium perfringens Food Poisoning ........................................... 461Botulism ........................................................................................... 466Bacillus Cereus Gastroenteritis ........................................................ 47725. Foodborne Listeriosis .............................................................................. 485Taxonomy of Listeria ........................................................................ 485Growth .............................................................................................. 488Distribution ....................................................................................... 492Thermal Properties ........................................................................... 494Virulence Properties ......................................................................... 497Animal Models and Infectious Dose .................................................. 498Incidence and Nature of the Listeriosis Syndromes .......................... 500Resistance to Listeriosis ................................................................... 502This page has been reformatted by Knovel to provide easier navigation. 11. Contents xiPersistence of L. monocytogenes in Foods ...................................... 503Regulatory Status of L. monocytogenes in Foods ............................. 50426. Foodborne Gastroenteritis Caused by Salmonella and Shigella ............ 511Salmonellosis ................................................................................... 511Shigellosis ........................................................................................ 52527. Foodborne Gastroenteritis Caused by Escherichia coli .......................... 531Serological Classification .................................................................. 531The Recognized Virulence Groups ................................................... 531Prevention ........................................................................................ 543Travelers' Diarrhea ........................................................................... 54328. Foodborne Gastroenteritis Caused by Vibrio, Yersinia, andCampylobacter Species ........................................................................... 549Vibriosis (Vibrio parahaemolyticus ) .................................................. 549Other Vibrios .................................................................................... 552Yersiniosis (Yersinia enterocolitica) .................................................. 556Campylobacteriosis (Campylobacter jejuni) ...................................... 560Prevention ........................................................................................ 56329. Foodborne Animal Parasites ................................................................... 569Protozoa ........................................................................................... 569Flatworms ......................................................................................... 579Roundworms .................................................................................... 58430. Mycotoxins ............................................................................................... 595Aflatoxins .......................................................................................... 595Alternaria Toxins .............................................................................. 600Citrinin .............................................................................................. 600Ochratoxins ...................................................................................... 601Patulin .............................................................................................. 601Penicillic Acid .................................................................................... 602Sterigmatocystin ............................................................................... 602Fumonisins ....................................................................................... 602Sambutoxin ...................................................................................... 606Zearalenone ..................................................................................... 606Control of Production ........................................................................ 606This page has been reformatted by Knovel to provide easier navigation. 12. xii Contents31. Viruses and Some Other Proven and Suspected FoodborneBiohazards ............................................................................................... 611Viruses ............................................................................................. 611Bacteria and Prions .......................................................................... 616Toxigenic Phytoplanktons ................................................................. 622Appendices ............................................................................................ 629Appendix A: Relationships of Common Foodborne Genera of Gram-Negative Bacteria .................................................................................... 629Appendix B: Relationship of Common Foodborne Genera of Gram-Positive Bacteria ...................................................................................... 631Appendix C: Biofilms ...................................................................................... 633Appendix D: Grouping of the Gram-Negative Asporogenous Rods,Polar-Flagellate, Oxidase Positive, and Not Sensitive to 2.5 IUPenicillin, on the Results of Four Other Tests ......................................... 635Index ....................................................................................................... 637This page has been reformatted by Knovel to provide easier navigation. 13. PART IHistorical BackgroundThe material in this part provides a glimpseof some of the early events that ultimately led tothe recognition of the significance and role ofmicroorganisms in foods. Food microbiology asa defined subdiscipline does not have a precisebeginning. Some of the early findings and ob-servationsare noted, along with dates. The se-lectivelists of events noted for food preserva-tion,food spoilage, food poisoning, and foodlegislation are meant to be guideposts in the con-tinuingevolution and development of food mi-crobiology.An excellent and more detailed review of thehistory of food microbiology has been presentedby Hartman.Hartman, P.A. 1997. The evolution of food microbiology.In Food MicrobiologyFundamentals and Frontiers,eds. M.P Doyle, L.R. Beuchat, and TJ. Montville, 3-12.Washington, D.C.: ASM Press. 14. CHAPTER 1History of Microorganisms in FoodAlthough it is extremely difficult to pinpointthe precise beginnings of human awareness ofthe presence and role of microorganisms infoods, the available evidence indicates that thisknowledge preceded the establishment of bacte-riologyor microbiology as a science. The eraprior to the establishment of bacteriology as ascience may be designated the prescientific era.This era may be further divided into what hasbeen called the food-gathering period and thefood-producing period. The former covers thetime from human origin over 1 million years agoup to 8,000 years ago. During this period, hu-manswere presumably carnivorous, with plantfoods coming into their diet later in this period.It is also during this period that foods were firstcooked.The food-producing period dates from about8,000 to 10,000 years ago and, of course, includesthe present time. It is presumed that the prob-lemsof spoilage and food poisoning were en-counteredearly in this period. With the adventof prepared foods, the problems of disease trans-missionby foods and of faster spoilage causedby improper storage made their appearance.Spoilage of prepared foods apparently dates fromaround 6000 BC. The practice of making potterywas brought to Western Europe about 5000 BCfrom the Near East. The first boiler pots arethought to have originated in the Near East about8,000 years ago.11 The arts of cereal cookery,brewing, and food storage were either started atabout this time or stimulated by this new devel-opment.10 The first evidence of beer manufac-turehas been traced to ancient Babylonia as farback as 7000 BC.8 The Sumerians of about 3000BC are believed to have been the first great live-stockbreeders and dairymen and were amongthe first to make butter. Salted meats, fish, fat,dried skins, wheat, and barley are also known tohave been associated with this culture. Milk,butter, and cheese were used by the Egyptians asearly as 3000 BC. Between 3000 BC and 1200 BC,the Jews used salt from the Dead Sea in the pres-ervationof various foods.2 The Chinese andGreeks used salted fish in their diet, and theGreeks are credited with passing this practice onto the Romans, whose diet included pickledmeats. Mummification and preservation of foodswere related technologies that seem to have in-fluencedeach other's development. Wines areknown to have been prepared by the Assyriansby 3500 BC. Fermented sausages were preparedand consumed by the ancient Babyloniansand the people of ancient China as far back as1500 BC.8Another method of food preservation that ap-parentlyarose during this time was the use ofoils such as olive and sesame. Jensen7 has pointedout that the use of oils leads to high incidencesof staphylococcal food poisoning. The Romansexcelled in the preservation of meats other thanbeef by around 1000 BC and are known to haveused snow to pack prawns and other perishables, 15. according to Seneca. The practice of smokingmeats as a form of preservation is presumed tohave emerged sometime during this period, asdid the making of cheese and wines. It is doubt-fulwhether people at this time understood thenature of these newly found preservation tech-niques.It is also doubtful whether the role offoods in the transmission of disease or the dan-gerof eating meat from infected animals wasrecognized.Few advances were apparently made towardunderstanding the nature of food poisoning andfood spoilage between the time of the birth ofChrist and AD 1100. Ergot poisoning (caused byClaviceps purpurea, a fungus that grows on ryeand other grains) caused many deaths during theMiddle Ages. Over 40,000 deaths due to ergotpoisoning were recorded in France alone in AD943, but it was not known that the toxin of thisdisease was produced by a fungus.12 Meat butch-ersare mentioned for the first time in 1156, andby 1248 the Swiss were concerned with market-ableand nonmarketable meats. In 1276, a com-pulsoryslaughter and inspection order was is-suedfor public abattoirs in Augsburg. Althoughpeople were aware of quality attributes in meatsby the thirteenth century, it is doubtful that therewas any knowledge of the causal relationshipbetween meat quality and microorganisms.Perhaps the first person to suggest the role ofmicroorganisms in spoiling foods was A. Kircher,a monk, who as early as 1658 examined decay-ingbodies, meat, milk, and other substances andsaw what he referred to as "worms" invisible tothe naked eye. Kircher's descriptions lacked pre-cision,however, and his observations did not re-ceivewide acceptance. In 1765, L. Spallanzanishowed that beef broth that had been boiled foran hour and sealed remained sterile and did notspoil. Spallanzani performed this experiment todisprove the doctrine of the spontaneous gen-erationof life. However, he did not convince theproponents of the theory because they believedthat his treatment excluded oxygen, which theyfelt was vital to spontaneous generation. In 1837,Schwann showed that heated infusions remainedsterile in the presence of air, which he suppliedby passing it through heated coils into the infu-sion.9 Although both of these men demonstratedthe idea of the heat preservation of foods, nei-thertook advantage of his findings with respectto application. The same may be said of D. Papinand G. Leibniz, who hinted at the heat preserva-tionof foods at the turn of the eighteenth cen-tury.The event that led to the discovery of canninghad its beginnings in 1795, when the Frenchgovernment offered a prize of 12,000 francs forthe discovery of a practical method of food pres-ervation.In 1809, a Parisian confectioner,Frangois (Nicholas) Appert, succeeded in pre-servingmeats in glass bottles that had been keptin boiling water for varying periods of time. Thisdiscovery was made public in 1810, when Appertwas issued a patent for his process.6 Not being ascientist, Appert was probably unaware of thelong-range significance of his discovery or whyit worked. This, of course, was the beginning ofcanning as it is known and practiced today.5 Thisevent occurred some 50 years before L. Pasteurdemonstrated the role of microorganisms in thespoilage of French wines, a development thatgave rise to the rediscovery of bacteria. A.Leeuwenhoek in the Netherlands had examinedbacteria through a microscope and describedthem in 1683, but it is unlikely that Appert wasaware of this development, as he was not a sci-entistand Leeuwenhoek's report was not avail-ablein French.The first person to appreciate and understandthe presence and role of microorganisms in foodwas Pasteur. In 1837, he showed that the souringof milk was caused by microorganisms, and inabout 1860 he used heat for the first time to de-stroyundesirable organisms in wine and beer.This process is now known as pasteurization.HISTORICAL DEVELOPMENTSSome of the more significant dates and eventsin the history of food preservation, food spoil- 16. age, food poisoning, and food legislation arelisted below.Food Preservation1782 Canning of vinegar was introduced by aSwedish chemist.1810Preservation of food by canning waspatented by Appert in France. Peter Durand was issued a British patentto preserve food in "glass, pottery, tinor other metals or fit materials." Thepatent was later acquired by Hall,Gamble, and Donkin, possibly fromAppert.141813Donkin, Hall, and Gamble introducedthe practice of postprocessing incubationof canned foods. Use of SO2 as a meat preservative isthought to have originated around thistime.1825T. Kensett and E. Daggett were granteda U.S. patent for preserving food in tincans.1835A patent was granted to Newton in En-glandfor making condensed milk.1837Winslow was the first to can corn fromthe cob.1839Tin cans came into wide use in theUnited States.3 L.A. Fastier was given a French patentfor the use of brine bath to raise the boil-ingtemperature of water.1840 Fish and fruit were first canned.1841S. Goldner and J. Wertheimer were is-suedBritish patents for brine baths basedon Fastier's method.1842A patent was issued to H. Benjamin inEngland for freezing foods by immer-sionin an ice and salt brine.1843Sterilization by steam was first at-temptedby I. Winslow in Maine.1845 S. Elliott introduced canning to Austra-lia.1853R. Chevallier-Appert obtained a patentfor sterilization of food by autoclaving.1854 Pasteur began wine investigations. Heat-ingto remove undesirable organismswas introduced commercially in 1867-1868.1855Grim wade in England was the first toproduce powdered milk.1856 A patent for the manufacture of unsweet-enedcondensed milk was granted to GailBorden in the United States,1861 I. Solomon introduced the use of brinebaths to the United States.1865The artificial freezing offish on a com-mercialscale was begun in the UnitedStates. Eggs followed in 1889.1874The first extensive use of ice in trans-portingmeat at sea was begun. Steam pressure cookers or retorts wereintroduced.1878 The first successful cargo of frozen meatwent from Australia to England. The firstfrom New Zealand to England was sentin 1882.1880 The pasteurization of milk was begun inGermany.1882Krukowitsch was the first to note thedestructive effects of ozone on spoilagebacteria.1886A mechanical process of drying fruitsand vegetables was carried out by anAmerican, A.F. Spawn.1890The commercial pasteurization of milkwas begun in the United States. Mechanical refrigeration for fruit stor-agewas begun in Chicago.1893The Certified Milk movement was be-gunby H.L. Coit in New Jersey.1895The first bacteriological study of can-ningwas made by Russell.1907E. Metchnikoff and co-workers isolatedand named one of the yogurt bacteria,Lactobacillus bulgaricus. The role of acetic acid bacteria in ciderproduction was noted by B.TP. Barker.1908Sodium benzoate was given officialsanction by the United States as a pre-servativein certain foods. 17. 1916The quick freezing of foods wasachieved in Germany by R. Plank, E.Ehrenbaum, and K. Reuter.1917Clarence Birdseye in the United Statesbegan work on the freezing of foods forthe retail trade. Franks was issued a patent for preserv-ingfruits and vegetables under CO2.1920Bigelow and Esty published the firstsystematic study of spore heat resistanceabove 212F. The "general method" forcalculating thermal processes was pub-lishedby Bigelow, Bohart, Richardson,and Ball; the method was simplified byCO. Ball in 1923.1922 Esty and Meyer establishedz = 18F forClostridium botulinum spores in phos-phatebuffer.1928The first commercial use of controlled-atmospherestorage of apples was madein Europe (first used in New York in1940).1929A patent issued in France proposed theuse of high-energy radiation for the pro-cessingof foods. Birdseye frozen foods were placed inretail markets.1943 B.E. Proctor in the United States was thefirst to employ the use of ionizing ra-diationto preserve hamburger meat.1950The D value concept came into generaluse.1954 The antibiotic nisin was patented in En-glandfor use in certain processedcheeses to control clostridial defects,1955Sorbic acid was approved for use as afood preservative. The antibiotic chlortetracycline was ap-provedfor use in fresh poultry (oxytet-racyclinefollowed a year later). Ap-provalwas rescinded in 1966.1967The first commercial facility designedto irradiate foods was planned anddesigned in the United States. The sec-ondbecame operational in 1992 inFlorida.1988Nisin accorded GRAS (generally re-gardedas safe) status in the UnitedStates.1990Irradiation of poultry approved in theUnited States.1997The irradiation of fresh beef up to amaximum level of 4.5 kGy and frozenbeef up to 7.0 kGy was approved in theUnited States.1997Ozone was declared GRAS by the U.S.Food and Drug Administration for fooduse.Food Spoilage1659 Kircher demonstrated the occurrence ofbacteria in milk; Bondeau did the samein 1847.1680Leeuwenhoek was the first to observeyeast cells.1780 Scheele identified lactic acid as the prin-cipalacid in sour milk.1836Latour discovered the existence ofyeasts.1839Kircher examined slimy beet juice andfound organisms that formed slime whengrown in sucrose solutions.1857 Pasteur showed that the souring of milkwas caused by the growth of organismsin it.1866 L. Pasteur's Etude sur Ie Vin was pub-lished.1867Martin advanced the theory that cheeseripening was similar to alcoholic, lactic,and butyric fermentations.1873The first reported study on the micro-bialdeterioration of eggs was carried outby Gayon. Lister was first to isolate Lactococcuslactis in pure culture.1876 Tyndall observed that bacteria in decom-posingsubstances were always traceableto air, substances, or containers.1878Cienkowski reported the first micro-biologicalstudy of sugar slimes and 18. isolated Leuconostoc mesenteroidesfrom them.1887Forster was the first to demonstrate theability of pure cultures of bacteria togrow at 00C.1888Miquel was the first to study thermo-philicbacteria.1895The first records on the determinationof numbers of bacteria in milk werethose of Von Geuns in Amsterdam. S.C. Prescott and W. Underwood tracedthe spoilage of canned corn to improperheat processing for the first time.1902 The termpsychrophile was first used bySchmidt-Nielsen for microorganismsthat grow at 00C.1912The term osmophilic was coined byRichter to describe yeasts that grow wellin an environment of high osmotic pres-sure.1915Bacillus coagulans was first isolatedfrom coagulated milk by B. W. Hammer.1917Bacillus stearothermophilus was firstisolated from cream-style corn by RJ.Donk.1933Oliver and Smith in England observedspoilage by Byssochlamys fulva; firstdescribed in the United States in 1964by D. Maunder.Food Poisoning1820The German poet Justinus Kerner de-scribed"sausage poisoning" (which inall probability was botulism) and its highfatality rate.1857Milk was incriminated as a transmitterof typhoid fever by W. Taylor of Penrith,England.1870 Francesco Selmi advanced his theory ofptomaine poisoning to explain illnesscontracted by eating certain foods.1888 Gaertner first isolated Salmonella enter-itidisfrom meat that had caused 57 casesof food poisoning.1894T. Denys was the first to associatestaphylococci with food poisoning.1896Van Ermengem first discoveredClostridium botulinum.1904Type A strain of C. botulinum was iden-tifiedby G. Landman.1906 Bacillus cereus food poisoning was rec-ognized.The first case of diphylloboth-riasiswas recognized.1926 The first report of food poisoning bystreptococci was made by Linden,Turner, and Thorn.1937Type E strain of C. botulinum was iden-tifiedby L. Bier and E. Hazen.1937 Paralytic shellfish poisoning was recog-nized.1938Outbreaks of Campylobacter enteritiswere traced to milk in Illinois.1939Gastroenteritis caused by Yersiniaenterocolitica was first recognized bySchleifstein and Coleman.1945 McClung was the first to prove the etio-logicstatus of Clostridium perfringens(welchii) in food poisoning.1951 Vibrio parahaemolyticus was shown tobe an agent of food poisoning by T.Fujino of Japan.1955Similarities between cholera and Es-cherichiacoli gastroenteritis in infantswere noted by S. Thompson. Scombroid (histamine-associated) poi-soningwas recognized. The first documented case of anisakiasisoccurred in the United States.1960Type F strain of C. botulinum identifiedby Moller and Scheibel. The production of aflatoxins by As-pergillusflavus was first reported.1965Foodborne giardiasis was recognized.1969 C. perfringens enterotoxin was demon-stratedby CL. Duncan and D.H. Strong. C. botulinum type G was first isolatedin Argentina by Gimenez and Ciccarelli.1971 First U.S. foodborne outbreak of Vibriopar ahaemolyticus gastroenteritis oc-curredin Maryland. 19. First documented outbreak of E. colifoodborne gastroenteritis occurred in theUnited States.1975Salmonella enterotoxin was demon-stratedby L.R. Koupal and R.H. Deibel.1976 First U.S. foodborne outbreak of Yersiniaenterocolitica gastroenteritis occurred inNew York. Infant botulism was first recognized inCalifornia.1977The first documented outbreak ofcyclosporiasis occurred in Papua, NewGuinea; first in United States in 1990.1978 Documented foodborne outbreak of gas-troenteritiscaused by the Norwalk virusoccurred in Australia.1979Foodborne gastroenteritis caused bynon-01 Vibrio cholerae occurred in Flor-ida.Earlier outbreaks occurred in Czecho-slovakia(1965) and Australia (1973).1981 Foodborne listeriosis outbreak was rec-ognizedin the United States.1982The first outbreaks of foodborne hem-orrhagiccolitis occurred in the UnitedStates.1983Campylobacter jejuni enterotoxin wasdescribed by Ruiz-Palacios et al.1985 The irradiation of pork to 0.3 to 1.0 kGyto control Trichinella spiralis was ap-provedin the United States.1986Bovine spongiform encephalopathy(BSE) was first diagnosed in cattle inthe United Kingdom.Food Legislation1890The first national meat inspection lawwas enacted. It required the inspectionof meats for export only.REFERENCES1. Bishop, RW. 1978. Who introduced the tin can? NicolasAppert? Peter Durand? Bryan Donkin? Food Technol32(4):60-67.2. Brandly, RJ., G. Migaki, and K.E. Taylor, 1966. MeatHygiene. 3d ed., Chap. 1. Philadelphia: Lea & Febiger.1895The previous meat inspection actwas amended to strengthen its provi-sions.1906 The U.S. Federal Food and Drug Act waspassed by Congress.1910The New York City Board of Health is-suedan order requiring the pasteuriza-tionof milk.1939 The new Food, Drug, and Cosmetic Actbecame law.1954 The Miller Pesticide Chemicals Amend-mentto the Food, Drug, and CosmeticAct was passed by Congress.1957 The U.S. Compulsory Poultry and Poul-tryProducts law was enacted.1958The Food Additives Amendment to theFood Drug, and Cosmetics Act waspassed.1962The Talmadge-Aiken Act (allowing forfederal meat inspection by states) wasenacted into law.1963 The U.S. Food and Drug Administrationapproved the use of irradiation for thepreservation of bacon.1967The U.S. Wholesome Meat Act waspassed by Congress and enacted into lawon December 15.1968 The Food and Drug Administration with-drewits 1963 approval of irradiated ba-con. The Poultry Inspection Bill was signedinto law.1969The U.S. Food and Drug Administra-tionestablished an allowable level of20 ppb of aflatoxin for edible grains andnuts.1973The state of Oregon adopted microbialstandards for fresh and processed retailmeat. They were repealed in 1977.3. Cowell, N.D. 1995. Who introduced the tin can?Anew candidate. Food Technol 49(12):61-64.4. Farrer, K.T.H. 1979. Who invented the brinebath?The Isaac Solomon myth. Food Technol. 33(2):75-77. 20. 5. Goldblith, S. A. 1971. A condensed history of the sci-enceand technology of thermal processing. FoodTechnol. 25(12): 44-50.6. Goldblith, S.A., M.A. Joslyn, and J.T.R. Nickerson.Introduction to Thermal Processing of Foods, vol. 1.Westport, CT: AVI.7. Jensen, L.B. 1953. Man's Foods, chaps. 1, 4, 12.Champaign, IL: Garrard Press.8. Pederson, CS. 1971. Microbiology of Food Fermen-tations.Westport, CT: AVI.9. Schormiiller, J. 1966. Die Erhaltung der Lebensmittel.Stuttgart: Ferdinand Enke Verlag.10. Stewart, G.F., and M.A. Amerine. 1973. Introductionto Food Science and Technology, chap. 1. New York:Academic Press.11. Tanner, F. W. 1944. The Microbiology of Foods, 2d ed.Champaign, IL: Garrard Press.12. Tanner, F.W., and L.P. Tanner. 1953. Food-Borne In-fectionsand Intoxications. 2d ed. Champaign, IL:Garrard Press. 21. PART IIHabitats, Taxonomy, andGrowth ParametersMany changes in the taxonomy of foodborne or-ganismshave been made during the past decade,and they are reflected in Chapter 2 along withthe primary habitats of some organisms of con-cernin foods. The factors/parameters that affectthe growth of microorganisms are treated in Chap-ter3. See the following for more information:Deak,T., and L.R. Beuchat. 1996. Handbook of Food Spoil-ageYeasts. Boca Raton, FL: CRC Press. Detection, enu-meration,and identification of foodborne yeasts.Doyle, M.P., L.R. Beuchat, TJ. Montville, eds. 1997. FoodMicrobiologyFundamentals and Frontiers. Washing-ton,D C : ASM Press. Food spoilage as well as foodbornepathogens are covered in this 768-page work along withgeneral growth parameters.International Commission on Microbiological Specifica-tionof Foods (ICMSF). 1996. Microorganisms in Foods.5th ed. Gaithersburg, MD: Aspen Publishers, Inc. Allof the foodborne pathogens are covered in this 512-page work with details on growth parameters. Well ref-erenced. 22. CHAPTER 2Taxonomy, Role, and Significanceof Microorganisms in FoodsBecause human food sources are of plant andanimal origin, it is important to understand thebiological principles of the microbial biota as-sociatedwith plants and animals in their naturalhabitats and respective roles. Although it some-timesappears that microorganisms are trying toruin our food sources by infecting and destroy-ingplants and animals, including humans, thisis by no means their primary role in nature. Inour present view of life on this planet, the pri-maryfunction of microorganisms in nature isself-perpetuation. During this process, the het-erotrophscarry out the following general reac-tion:All organic matter(carbohydrates, proteins, lipids, etc.)iEnergy + Inorganic compounds(nitrates, sulfates, etc.)This, of course, is essentially nothing more thanthe operation of the nitrogen cycle and the cycleof other elements (Figure 2-1). The microbialspoilage of foods may be viewed simply as anattempt by the food biota to carry out what ap-pearsto be their primary role in nature. Thisshould not be taken in the teleological sense. Inspite of their simplicity when compared to higherforms, microorganisms are capable of carryingout many complex chemical reactions essentialto their perpetuation. To do this, they must ob-tainnutrients from organic matter, some of whichconstitutes our food supply.If one considers the types of microorganismsassociated with plant and animal foods in theirnatural states, one can then predict the generaltypes of microorganisms to be expected on thisparticular food product at some later stage in itshistory. Results from many laboratories show thatuntreated foods may be expected to contain vary-ingnumbers of bacteria, molds, or yeasts, andthe question often arises as to the safety of a givenfood product based on total microbial numbers.The question should be twofold: What is the to-talnumber of microorganisms present per gramor milliliter and what types of organisms are rep-resentedin this number? It is necessary to knowwhich organisms are associated with a particu-larfood in its natural state and which of the or-ganismspresent are not normal for that particu-larfood. It is, therefore, of value to know thegeneral distribution of bacteria in nature and thegeneral types of organisms normally presentunder given conditions where foods are grownand handled.BACTERIAL TAXONOMYMany changes have taken place in the classi-ficationor taxonomy of bacteria in the past de-cade.Many of the new taxa have been created asa result of the employment of molecular genetic 23. Nitrogen(Atmospheric)Nitrogen fixationAtmospheric nitrogen fixed bymany microorganisms, e.g..Rhizobium. Ctostridium, Azotobacteretc.Figure 2-1 Nitrogen cycle in nature is here depicted schematically to show the role of microorganisms. Source:From Microbiology by MJ. Pelczar and R. Reid, copyright 1965 by McGraw-Hill Book Company, used withpermission of the publisher.methods, alone or in combination with some ofthe more traditional methods: DNA homology and mol% G + C contentofDNA 23S, 16S, and 5S rRNA sequence similari-ties Oligonucleotide cataloging Numerical taxonomic analysis of totalsoluble proteins or of a battery of morpho-logicaland biochemical characteristics Cell wall analysis Serological profiles Cellular fatty acid profilesAlthough some of these have been employed formany years (e.g., cell wall analysis and serologi-calprofiles) others (e.g., ribosomal RNA [rRNA]sequence similarity) came into wide use only dur-ingthe 1980s. The methods that are the mostpowerful as bacterial taxonomic tools are out-linedand briefly discussed below.rRNA AnalysesTaxonomic information can be obtained fromRNA in the production of nucleotide catalogs andthe determination of RNA sequence similarities.DenitrificationReduction of nitrates to gaseousnitrogen by bacteria, e.g..pseudomonadsNitrate formation(Nitrification)Nitrite oxidized to nitrate bynitrobacterNitrate serves as plantfoodMany heterotropicspecies reducenitrates to ammoniavia nitritesOrganic nitrogen formation"Fixed" nitrogen utilized byplantsconverted to plant protein;plants consumed by animals.animal proteins, etc.. formedMicroorganisms utilizeammonia as nitrogensource and synthesizecellular proteinsNitrite formationAmmonia oxidized to nitrite bynitrosomonasSoil organic nitrogenExcretion products of animals,dead animals, and plant tissuedeposited in soilAmmonia formation(Ammonification)Amino acids deaminated by manymicroorganisms; ammonia one ofthe end products of this processOrganic nitrogen degradationProteins, nucleic acids, etc..attacked by a wide variety ofmicroorganisms; completebreakdown yields mixtures ofamino acids 24. First, the prokaryotic ribosome is a 70S (Sved-berg)unit, which is composed of two separatefunctional subunits: 5OS and 30S. The 50S sub-unitis composed of 23 S and 5 S RNA in addi-tionto about 34 proteins, whereas the 30S sub-unitis composed of 16S RNA plus about 21proteins.Ribosome70S/ 3OS 50S// 16S 21 34 23S + 5SProteinsThe 16S subunit is highly conserved and is con-sideredto be an excellent chronometer of bacte-riaover time.48 By use of reverse transcriptase,16S rRNA can be sequenced to produce longstretches (about 95% of the total sequence) toallow for the determination of precise phyloge-neticrelationships.26 Because of its smaller size,5 S RNA has been sequenced totally.To sequence 16S rRNA, a single-strandedDNA copy is made by use of reverse transcriptasewith the RNA as template. When the single-strandedDNA is made in the presence ofdideoxynucleotides, DNA fragments of varioussizes result that can be sequenced by the Sangermethod. From the DNA sequences, the template16S rRNA sequence can be deduced. It wasthrough studies of 16S rRNA sequences that ledWoese and his associates to propose the estab-lishmentof three kingdoms of life-forms: Eu-karyotes,Archaebacteria, and Prokaryotes. Thelast include the cyanobacteria and the eubacteria,with the bacteria of importance in foods beingeubacteria. Sequence similarities of 16S rRNAare widely employed, and some of the newfoodborne taxa were created primarily by its usealong with other information. Libraries ofeubacterial 5 S rRNA sequences also exist, butthey are fewer than for 16S.Nucleotide catalogs of 16S rRNA have beenprepared for a number of organisms, and exten-sivelibraries exist. By this method, 16S rRNA issubjected to digestion by RNAse Tl, whichcleaves the molecule at G(uanine) residues. Se-quences(-mers) of 6-20 bases are produced andseparated, and similarities SAB (Dice-type coef-ficient)between organisms can be compared. Al-thoughthe relationship between SAB and percent-agesimilarity is not good below SAB value of 0.40,the information derived is useful at the phylumlevel. The sequencing of 16S rRNA by reversetranscriptase is preferred to oligonucleotide cata-loging,as longer stretches of rRNA can be se-quenced.Analysis of DNAThe mol% G + C of bacterial DNA has beenemployed in bacterial taxonomy for several de-cades,and its use in combination with 16S and5 S rRNA sequence data makes it even moremeaningful. By 16S rRNA analysis, the gram-positiveeubacteria fall into two groups at thephylum level: one group with mol% G + C >55,and the other BHA >BHT. Conidial germination of four FusariumTable 13-3 Some GRAS Indirectly Antimicrobial Chemicals Used in FoodsCompound Primary Use Most Susceptible OrganismsButylated hydroxyanisole (BHA) Antioxidant Bacteria, some fungiButylated hydroxytoluene (BHT) Antioxidant Bacteria, viruses, fungif-Butylhydroxyquinoline (TBHQ) Antioxidant Bacteria, fungiPropyl gallate (PG) Antioxidant BacteriaNordihydroguaiaretic acid Antioxidant BacteriaEthylenediaminetetraacetic acid (EDTA) Sequestrant/stabilizer BacteriaSodium citrate Buffer/sequestrant BacteriaLaurie acid Defoaming agent Gram-positive bacteriaMonolaurin Emulsifier Gram-positive bacteria, yeastsDiacetyl Flavoring Gram-negative bacteria, fungid- and /-Carvone Flavoring Fungi, gram-positive bacteriaPhenylacetaldehyde Flavoring Fungi, gram-positive bacteriaMenthol Flavoring Bacteria, fungiVanillin, ethyl vanillin Flavoring FungiSpices/spice oils Flavoring Bacteria, fungi 268. spp. was inhibited by 200 ppm BHA or propylparaben (PP) over the pH range 4-10, but over-all,PP was more inhibitory than BHA.114Foodborne pathogens such as Bacillus cereus,Vparahaemolyticus, salmonellae, and S. aureusare effectively inhibited at concentrations 6.0 is not effec-tive.Shelf life of vacuum-packaged fish isshortened by the growth of'Photobacteriumphosphoreum16 and Shewanella putre-faciens.l In general, the gram-negative bacteria aremore sensitive to CO2 inhibition than grampositives, with pseudomonads being amongthe most sensitive and clostridia the mostresistant (Table 14-4). Upon prolonged stor-ageof meats, CO2 effects a rather dramaticshift in biota from one that is largely gramnegative in fresh products to one that islargely or exclusively gram positive. Thiscan be seen in Table 14-5 for smoked porkloins and frankfurter sausage.6 Both lag and logrithmic phases of growthare retarded. CO2 under pressure is considerably moreantimicrobial than not, and pressures of6 to 30 megapascal (mPa) can destroy bac-teriaand fungi under varying conditions (seeHigh Hydrostatic Pressure in Chapter 19).The destructive action is believed to occurwhen pressure is released suddenly.Mode of ActionAs to the mechanism of CO2 inhibition of mi-croorganisms,two explanations have been of-fered.King and Nagel60 found that CO2 blockedTable 14-4 Relative Sensitivity ofMicroorganisms to CO2 Relative to Vacuum-andModified-Atmosphere PackagingPseudomonas spp. (most sensitive)Aeromonas spp.Bacillus spp.MoldsEnterobacteriaceaeEnterococcus spp.Brochothrix spp.Lactobacillus spp.Clostridium spp. (most resistant)Source: Adapted with permission from G. Molin69, The Re-sistanceto Carbon Dioxide of Some Food Related Bacteria,European Journal of Applied Microbiology and Biotechnology,Vol. 18, pp. 214-217, 1983, Springer-Verlag New York, Inc. 288. Table 14-5 Effect of Storage on the Microbiota of Two Meats Held from 48 to 140 Days at 4Cthe metabolism of Pseudomonas aeruginosa andappeared to effect a mass action on enzymaticdecarboxylations. Sears and Eisenberg78 foundthat CO2 affected the permeability of cell mem-branes,and Enfors and Molin23 found supportfor the latter hypothesis in their studies on thegermination of Clostridium sporogenes and C.perfringens endospores. At 1 atm CO2, sporegermination of these two species was stimulated,whereas B. cereus spore germination was inhib-ited.As was shown by others, CO2 is more stimu-latoryat low pH than high. With 55 atm CO2,only 4% germination of C. sporogenes sporesoccurred, whereas with C. perfringens, 50 atmreduced termination to 4%.23 These authors sug-gestedthat CO2 inhibition was due to its accu-mulationin the membrane lipid bilayer such thatSmoked Pork Loinsincreased fluidity results. An adverse effect oncell permeability has been suggested by others.IfCO2 is dissolved in the form of carbonic acid,HCO3" would be present as a dissociation prod-uct,and thus can cause changes in cell perme-ability.17 When dissolved in water, CO2 productsare as follows:CO2 + H2O ; H2CO3^ H+ + HCO3" ; 2H+ + CO32-The antimicrobial spectrum of CO2 anddiacetyl is quite similar, and while this per sedoes not mean they possess identical modes ofaction, the striking similarities seem worthy ofnote. Diacetyl is an arginine antagonist and itsmode of action along with some other a-dicar-bonylcompounds has been discussed.54 TheLog APC/gPHDominant biota (%)O Day2.55.8Flavo (20)Arthro (20)Yeasts (20)Pseudo (11)Coryne (10)Vacuum48 Days7.65.8Lactos (52)aCO248 Days6.95.9Lactos (74)bN248 Days7.25.9Lactos (67)cFrankfurter SausageLog APC/gPHDominant biota (%)O Day1.75.9Bac (34)Coryne (34)Flavo (8)Broch (8)Vacuum98 Days9.05.4Lactos (38)CO2140 Days2.45.6Lactos (88)dN2140 Days4.85.9Lactos (88)eNote: Percentage biota represented by Weissella viridescens: a40; b72; c50; d22; e35. APC = aero-bicplate count; Flavo = Flavobacterium; Arthro = Arthrobacter; Pseudo = Pseudomonas; Coryne =Corynebacterium; Bac = Bacillus; Broch = Brochothrix.Source: Adapted from Blickstad and Molin.6 289. greater sensitivity of gram-negative bacteria toa-dicarbonyl inhibitors appears to be due to theircapacity to inactivate amino acid-binding pro-teinsof the cell's periplasm, especially the argi-nine-binding proteins. Thus, it is not inconceiv-ablethat the site of action of CO2 is the periplasm,where it interferes with the normal functioningof amino acid-binding proteins. Further discus-sionsof possible modes of action of CO2 can befound in references 18 and 21.FOOD PRODUCTSThe successful use of vacuum packaging,MAP, and CAS to extend the shelf life of a widevariety of food products is well documented, andsome of the specific antimicrobial aspects areoutlined below.Fresh and Processed MeatsAmong the first to demonstrate the effective-nessof high levels of CO2 in preserving cut-upmeats was J. Brooks in England, who in 1933studied the effect of CO2 on lean meat; E. Cal-lowin England, who studied pork and bacon;R.B. Haines, also in England, who was amongthe first to show the effect of CO2 on spoilageorganisms; and W.A. Empey in Australia, whoin 1933 applied CO2 to beef73In general, the shelf life of red meats can beextended for up to 2 months if packaged in 75%O2 + 25% CO2 and stored at -10C. The high levelof oxygen ensures that the red-meat color ismaintained. It has been shown that at least 15%CO2 is necessary to retard microbial growth onbeef steaks, and that the mixture of 15% CO2 +75% O2 + 10% N2 was more effective thanvacuum both for red-meat color and microbialquality.4 The importance of temperature of stor-ageof MAP meats was shown early by Jaye etal.,55 who found striking differences in qualitywhen ground beef was stored at 30 versus 38.They compared the use of the more gas-imper-meableSaran to the gas-permeable cellophanepacks. Earlier, Halleck et al.40 showed the dra-maticinhibitory effect of vacuum packaging andstorage at 1.1-3.3 0C. The importance of tempera-tureof storage was demonstrated in another studyusing the packaging system known as theCaptech process, which combines hygienic pro-cessing,storage at -1.50C, high CO2, low O2, andgas-impermeable packaging.39 The process wasapplied to pork loins, with the temperature ofholding for simulated retail display being raisedto 8C. Lactic acid bacteria grew withoutperceptible decrease in lag phase, and reached107/cm2 within 9 weeks. The behavior of the biotaof smoked pork loins and frankfurter sausagestored under vacuum and CO2 is presented inTable 14-5. As is typical of MAP meats, the ini-tialheterogeneous biota became homogeneousupon long-term storage under vacuum or MAPwith pH being decreased due to predominanceof lactic acid bacteria.6The relative effectiveness of MAP/vacuumpackaging of red meats can be assessed by de-terminingchanges that occur in hydration capac-ity.When fresh ground beef was stored in high-barrierbags and held at 7C for up to 13 days,the hydration capacity was essentially unchangedin comparison to the samples that were looselywrapped in foil to allow for aerobic conditions(Figure 14-1). This is reflected by extract-releasevolume (ERV) (see Chapter 4). Over the hold-ingperiod, gram-negative bacteria increased byabout 6 log10 but by only 3 log10 for the aerobi-callystored foil-wrapped and high-barrier bag-storedmeats, respectively. Similar results can beobtained by using the filter-paper press methodto measure hydration capacity.53 The increasedhydration is brought about by the preferentialgrowth of lactic acid bacteria, which depress pH.In their study of beef and pork livers and beefkidneys packaged in high-barrier bags, Hannaet al.45 found that pH decreased in each productwhen held at 2C for up to 28 days. ERV hasbeen used to assess the spoilage of vacuum-pack-agedmeats.75Overall, the storage of fresh meats undervacuum or MAP has been very successful andsafe. The latter is in large part a reflection of theexistence of lactic acid and related bacteria on 290. DAYSFigure 14-1 Lack of increase in hydration capacity of fresh ground beef stored in high-barrier bags at 7C for13 days as measured by extract-release volume (ERV). The foil-wrapped samples underwent aerobic spoilageas evidenced by increased hydration and endotoxin titers.fresh meats, and when these products are storedunder low O2 and high CO2 conditions at lowtemperatures, the normal biota prevents thegrowth of pathogens by virtue of depressed pH,competition for O2, possible production of anti-microbialsubstances, and other factors.PoultryThe effectiveness of MAP for the storage offresh poultry was demonstrated in the early1950s73 and since that time a number of studieshave been reported. Hotchkiss51 used from 60%to 80% CO2 on raw poultry in glass jars and foundan increase in shelf life to at least 35 days at2C. In another study, when high-barrier film(oxygen transmission rate [OTR] ca. 18 mL) wasused to pack cut-up or whole chicken that washeld at 5C, the chicken had lower numbers ofbacteria and kept longer than that which wasstretch-wrapped with a film that had an OTR of6,500 mL, and this is illustrated in Figure 14-2.61With poultry stored in air, the aerobic plate count(APC) of drip after 16 days at 100C was 9.40 log10,whereas in 20% CO2 the APC was 6.14 log10.92Overall, the generally higher initial pH of freshpoultry meat is primarily responsible for thisproduct's not having the MAP shelf life of prod-uctssuch as fresh beef.SeafoodsMAP/vacuum packaging has been shown toextend the shelf life of cod fillets, red snapper,rainbow trout, herrings, mackerel, sardines, cat-fish,and others. In 1933, EP. Coyne of Englandwas apparently the first person to show the pre-servativeeffects of CO2 on fish.73For fish using 80% CO2 + air, log numbersafter 14 days at 35C were approximatelyERVLOG. NOS. & TITER 291. DAYS IN STORAGE AT 5CFigure 14-2 Numbers of total aerobic mesophilicbacteria from packaged whole chickens under aero-bic(tray pack) and vacuum-pack storage. Source: Re-printedwith permission from J. Kraft et al., Micro-biologicalQuality of Vacuum Packaged Poultry withor without Chlorine Treatment, Journal of Food Sci-ence,Vol. 47, p. 381, 1982, Institute of Food Tech-nologists.6.00/cm2 compared to air controls with log num-bers>10.5 cm2. The pH of CO2-stored productsafter 14 days decreased from around 6.75 toaround 6.30, whereas controls increased toaround 7.45.74 The shelf life of rockfish andsalmon at 4.5C was extended by 20-80% CO2.8At least 1 log difference in bacterial counts overcontrols was obtained when trout and croakerwere stored in CO2 environments at 4C.42 Whenfresh shrimp or prawns were packed in ice withan atmosphere of 100% CO2, they were ediblefor up to 2 weeks, and bacterial counts after 14days were lower than air-packed controls after 7days.65 When cod fillets were stored at 2C, air-storedsamples spoiled in 6 days, with APC oflog10 7.7, whereas samples stored in 50% CO2 +50% O2 or 50% CO2 + 50% N2 or 100% CO2 didnot show bacterial spoilage until, respectively,26,34, and 34 days, with respective APCs of 7.2,6.6, and 5.5/g.88 It was suggested that the use of50% CO2 + 50% O2 is technically more feasiblethan the use of 100% CO2. Whereas the practi-calupper limit of CO2 for red meats is around20%, higher concentrations can be used with fishbecause they contain lower levels of myoglobin.The concern over the use of MAP for fisheryproducts has to do with the fact that nonproteo-lyticbotulism strains are found in waters and theycan grow at temperatures