DEVELOPMENTOFANONCUREDPORKPRODUCTWITH THEPOTENTIALFORNONREFRIGERATEDSTORAGE By LLOYDJASONWOOLSEY ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL OFTHEUNIVERSITYOFFLORIDAINPARTIALFULFILLMENT OFTHEREQUIREMENTSFORTHEDEGREEOF DOCTOROFPHILOSOPHY UNIVERSITYOFFLORIDA 1997 InlovingmemoryofDurwardW.Woolsey,Sr.,mygrandfather. ACKNOWLEDGEMENTS Glory,honorandpraisebelongtomyLordand Saviour,JesusChrist. Ihave takenrefugeinHim;Hehasneverletmebeputtoshame;HehasdeliveredmeintoHis righteousness. Hehasnothandedmeovertotheenemybuthasputmyfeetinaspacious place(Psalm31:1&8,NIV). Hehassustainedmethroughsomedifficultspiritualand emotionaltrials. Hehasusedthesetrialstodevelopperseverance,characterandhope withinme(Romans5:3&4,NIV). AppreciationisextendedtoDr.R.L.West,chairmanofthesupervisorycommittee, forthe guidance, wisdom and patiencehehas shownme during the duration ofmy program. Thanks are also extended to Dr. D.D. Johnson, Dr. F.W. Leak, Dr. S.K. Williams, Dr. J.A. LindsayandDr. S.F. O'Keefe fortheireffortsandguidanceasa supervisorycommittee. SpecialthanksareextendedtoDr.West,Dr.JohnsonandDr. Williamsforfundingthisproject. The friendships ofDebbieNeubauer, Mary BethFort and Larry Eubanks are appreciatedandhavecontributedtomyenjoymentofthisprogram. DebbieNeubauer, AnnaZometa,PamRoyerandDorisSartainareappreciatedfortheirassistanceinthe laboratory. Larry Eubanks and the Meat Laboratory crew are appreciated for their assistanceinlivestockslaughterandproductpreparation. Thefriendshipsoffellowgraduatestudents,ClaudioAlvarez,N.T.Cosby,Mike Coker,WillRhodes,JeffArseneau,MariaRivas,JoannaHarrelson,BrendaAllenand iii VictoriaD'Costahaveaddedaspecialyetunexplainabletwisttomydoctoralprogram. SpecialthanksareextendedtoMariaRivas,JoannaHarrelsonandBrendaAllenfortheir assistanceduringfootballSaturdays. Myparents,DurwardandJoAnneWoolsey,aregreatlyappreciatedforinstilling qualitiesofcommitment,persistence, honesty, integrityandservanthoodintomeasa youngman. Theirmoralandoccasionalfinancialsupportduringthisprogramhavebeen invaluabletome. Myfriends,brothersandsistersinChrist,atInterVarsityChristianFellowshipand TheFirstAssemblyofGod,Gainesville,areappreciatedfortheloveandencouragement theyhavegivenmeoverthepastseveralyears. SpecialthanksareextendedtoEric Koenig,ShadSmith,LauraFarrowandJimCapenwhooftensimplylistened. LV TABLEOFCONTENTS paLie ACKNOWLEDGEMENTS iii LISTOFTABLES ix LISTOFFIGURES xiv ABSTRACT xvi CHAPTERS 1 INTRODUCTION 1 2 LITERATUREREVIEW 7 PreservativesofInterest Nisin 7 Chemicalcharacteristics 8 Antimicrobialactivity 9 Modeofaction 14 Methodsofapplyingbacteriocinstomeat 16 Lactates 21 Chemicalcharacteristics 22 Antimicrobialactivityoflacticacid 23 Antimicrobialactivityoflactatesalts 32 Modeofaction 35 Applicationsoflacticacidon/inmeat 38 Applicationsoflactatesaltsinmeat 40 GluconicAcidandGlucono-Delta-Lactone 45 Chemicalcharacteristics 45 Antimicrobialactivity 46 Modeofaction 49 Applicationsofgluconicacidorglucono-delta-lactone inmeat 50 EthylenediaminetetraaceticAcid 51 Physicalandchemicalproperties 51 Antimicrobialactivity 52 Modeofaction 56 v page ImportantFoodbornePathogens 57 Escherichiacoli0157:H7 57 Salmonella 62 Yersiniaenterocolitica 66 Listeriamonocytogenes 71 Staphylococcusaureus 76 3 THEEFFECTSOFSODIUMLACTATE,GLUCONO-DELTA- LACTONE,DISODIUMETHYLENEDIAMINETETRAACETATE, NISAPLINANDTHEIRCOMBINATIONSONTHEGROWTHOF ESCHERICHIACOLI0157:H7 80 Introduction 80 MaterialsandMethods 82 Experimentaldesign 82 Cookedmeatmediapreparation 84 Inoculum 84 Microbialanalysis 84 Statisticalanalysis 85 ResultsandDiscussion 86 EDTAat0,0.372and0.745%(0,0.01 and0.02M) 86 EDTAat0,0.0036and0.0072% 94 Conclusions 97 4 THEEFFECTSOFSODIUMLACTATE,GLUCONO-DELTA- LACTONE,NISAPLINANDTHEIRCOMBINATIONSONTHE GROWTHOFSELECTEDFOODBORNEPATHOGENS 99 Introduction 99 MaterialsandMethods 101 Experimentaldesign 101 Cookedmeatmediapreparation 101 Inoculum 102 Microbialanalysis 102 Statisticalanalysis 103 ResultsandDiscussion 104 Additiveeffectsandinteractions 104 Gram-negativepathogens 104 Gram-positivepathogens Ill Conclusions 120 vi page 5 THEEFFECTSOFSODIUMLACTATE,GLUCONO-DELTA- LACTONEANDDISODIUMEDTAONTHEGROWTHOF ESCHERICHIACOLI0157:H7ANDSALMONELLA TYPHIMURIUMINCOOKEDMEATMEDIA 122 Introduction 122 MaterialsandMethods 124 Experimentaldesign 124 Cookedmeatmediapreparation 125 Inoculum 125 Microbialanalysis 126 pHanalysis 126 Statisticalanalysis 126 ResultsandDiscussion 127 TreatmenteffectsonpHbeforeinoculation 127 TreatmenteffectsonpHandmicrobialgrowthduring incubation 131 Potentialmodesofsynergisticinteraction 141 Conclusions 147 ComparisonofCurrentResultswithResultsObtainedinChapters3 and4 148 6 THEUTILIZATIONOFSODIUMLACTATEANDGLUCONO- DELTA-LACTONEINNONREFRIGERATEDAND REFRIGERATED,RESTRUCTUREDPORKROLLS 155 Introduction 155 MaterialsandMethods 157 Experimentaldesign 157 Porkrollprocessing 158 Cookingyields 159 Microbialanalysis 159 Chemicalanalysis 161 Sensoryanalysis 162 Statisticalanalysis 163 ResultsandDiscussion 164 Cookingyields 164 Porkrollcomposition 165 Sensoryevaluation 166 Nonrefrigeratedstorage 168 Microbialanalysis 168 Chemicalanalysis 173 vii page Refrigeratedstorage 179 Microbialanalysis 179 Chemicalanalysis 180 Conclusions 184 7 SUMMARYANDCONCLUSIONS 186 APPENDICES AMICROBIALLEVELSOFMAINEFFECTSANDTREATMENTS... 189 BSALMONELLAENRICHMENTMETHOD 195 REFERENCES 197 BIOGRAPHICALSKETCH 216 viii LISTOFTABLES Table page 2-1 ThegrowthofListeriamonocytogenesintrypticasesoybrothat variouspHvalues,totallacticacidandundissociatedlacticacid concentrations 27 2-2 InactivationofYersiniaenterocoliticaCI22-76andY7Pafter24hr ofincubationat25°Cintrypticsoybrothacidifiedwithlacticacid atvariouspHvalues,totalacidandundissociatedacid concentrations 28 2-3 ThegrowthofEscherichiacoli0157:H7ina20%slurryof ground,roastedbeefatvariouspHvalues,totallacticacidand undissociatedlacticacidconcentrations 30 2-4 USDA,FSISpathogenreductionperformancestandards 65 3-1 Probabilityvalues(P>F)fortheeffectsofglucono-delta-lactone, Nisaplin,disodiumethylenediaminetetraacetate,sodiumlactateand theircombinationsonEscherichiacoli0157:H7growthafter incubationfor1dayat37°C 87 3-2 Probabilityvalues(P>F)fortheeffectsofselectedcombinations ofglucono-delta-lactone,Nisaplin,disodiumethylenediaminetetra- acetateand sodiumlactateonEscherichiacoli0157:H7growth afterextendedincubationat37°C 92 4-1 Probabilityvalues(P>F)fortheeffectsofglucono-delta-lactone, sodiumlactate,Nisaplinandtheircombinationsonthegrowthof selectedfoodbornepathogensincookedmeatmediaafter1 dayof incubationat37°C 105 4-2 Probabilityvalues(P>F)fortheeffectsofselectedcombinations ofglucono-delta-lactone,sodiumlactateandNisaplinonthegrowth ofselectedfoodbornepathogensincookedmeatmediafor3days ofincubationat37°C 107 ix Table page 5-1 Probabilityvalues(P>F)fortheeffectsofglucono-delta-lactone, sodiumlactate,disodiumethylenediaminetetraacetateandtheir combinationsonthepHofthemediabeforeandaftercookingat day0priortoinoculationwithEscherichiacoli0157:H7and Salmonellatyphimuriumincookedmeatmedia 129 5-2 ThepHofcookedmeatmediacontainingcombinationsofglucono- delta-lactone,sodiumlactateanddisodiumethylenediamine- tetraacetatebeforeandaftersimulatedcookingto70°Cpriorto inoculationwithEscherichiacoli0157:H7(STDERR±0.011)... 130 5-3 ThepHofcookedmeatmediacontainingcombinationsofglucono- delta-lactone,sodiumlactateanddisodiumethylenediamine- tetraacetatebeforeandaftersimulatedcookingto70°Cpriorto inoculationwithSalmonellatyphimurium(STDERR±0.019).... 131 5-4 Probabilityvalues(P>F)fortheeffectsofglucono-delta-lactone, sodiumlactate,disodiumethylenediaminetetraacetateandtheir combinationsonthepHofthemediaandthegrowthofEscherichia coli0157:H7andSalmonellatyphimuriumincookedmeatmedia after1dayofincubationat37°C 132 5-5 ThepHofcookedmeatmediacontainingcombinationsofglucono- delta-lactone,sodiumlactateanddisodiumethylenediamine- tetraacetateandinoculatedwithEscherichiacoli0157:H7after1 dayofincubationat37°C(STDERR±0.006) 133 5-6 Levels(log10CFU/ml)ofEscherichiacoli0157:H7incookedmeat mediacontainingcombinationsofglucono-delta-lactone,sodium lactateanddisodiumethylenediaminetetraacetateafter1 dayof incubationat37°C(STDERR±0.11) 134 5-7 ThepHofcookedmeatmediacontainingcombinationsofglucono- delta-lactone,sodiumlactateanddisodiumethylenediamine- tetraacetateandinoculatedwithSalmonellatyphimuriumafter1day ofincubationat37°C(STDERR±0.015) 136 5-8 Levels(log,0CFU/ml)ofSalmonellatyphimuriumincookedmeat mediacontainingcombinationsofglucono-delta-lactone,sodium lactateanddisodiumethylenediaminetetraacetateafter1 dayof incubationat37°C(STDERR±0.24) 137 x