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Methods in Molecular Biology 1887

Lactic Acid Bacteria

Methods and Protocols

M ETHODSIN M OLECULAR B IOLOGY

SchoolofLifeandMedicalSciences

UniversityofHertfordshire Hatfield,Hertfordshire,AL109AB,UK

Forfurthervolumes: http://www.springer.com/series/7651

LacticAcidBacteria

MethodsandProtocols

DepartmentofFoodManagement,MiyagiUniversity,Sendai,Miyagi,Japan

MiyagiUniversity

Sendai,Miyagi,Japan

ISSN1064-3745ISSN1940-6029(electronic)

MethodsinMolecularBiology

ISBN978-1-4939-8906-5ISBN978-1-4939-8907-2(eBook) https://doi.org/10.1007/978-1-4939-8907-2

LibraryofCongressControlNumber:2018960875

© SpringerScience+BusinessMedia,LLC,partofSpringerNature2019

Thisworkissubjecttocopyright.AllrightsarereservedbythePublisher,whetherthewholeorpartofthematerialis concerned,specificallytherightsoftranslation,reprinting,reuseofillustrations,recitation,broadcasting,reproduction onmicrofilmsorinanyotherphysicalway,andtransmissionorinformationstorageandretrieval,electronicadaptation, computersoftware,orbysimilarordissimilarmethodologynowknownorhereafterdeveloped. Theuseofgeneraldescriptivenames,registerednames,trademarks,servicemarks,etc.inthispublicationdoesnotimply, evenintheabsenceofaspecificstatement,thatsuchnamesareexemptfromtherelevantprotectivelawsandregulations andthereforefreeforgeneraluse.

Thepublisher,theauthors,andtheeditorsaresafetoassumethattheadviceandinformationinthisbookarebelievedto betrueandaccurateatthedateofpublication.Neitherthepublishernortheauthorsortheeditorsgiveawarranty, expressorimplied,withrespecttothematerialcontainedhereinorforanyerrorsoromissionsthatmayhavebeenmade. Thepublisherremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations.

ThisHumanaPressimprintispublishedbytheregisteredcompanySpringerScience+BusinessMedia,LLC,partof SpringerNature.

Theregisteredcompanyaddressis:233SpringStreet,NewYork,NY10013,U.S.A.

Preface

Lacticacidbacteriahavetraditionallybeenusedinfoodproductionasimportantfermentativebacteria.Theyhavebeenusedtopreparedairyproductssuchasyogurtandcheese, alcoholicbeveragessuchaswineandwhisky,andseasoningssuchassoysauceandfish products.

In1908,Metchnikoffreportedthatlacticacidbacteriainyogurtimproveintestinal conditions.Asaresultofthatbenefit,lifespanextensioncanbeexpectedasaninfluencefrom thebacteria.ResearchintolacticacidbacteriaforprobioticuseadvancedafterMetchnikoff’s reports.Generally,hundredsofspecies,100trillioncellsofbacteria,arepresentasintestinal florainhumanintestines.Manyresearchershavereportedontheroleofbacteriaasa componentofthebacterialflorainhumanintestines.Recently,thebacteriahavebeen usedinmedicineasanintestinalmedicine.Moreover,immunobioticsusinglacticacid bacteriahavebeeninvestigatedrecentlybymanyscholars.Consequently,lacticacidbacteria areusedwidelyforhumanlife.Manyscholarshopetoknowmoreabouthowprofessional researchersevaluatethevariouslacticacidbacteriafunctions.

Thisvolumeofthe MethodsinMolecularBiology seriesprovidesacollectionofprotocols fornumerousexperimentalapproachesusedbytheauthorsforlacticacidbacteriaresearch, suchastheisolationoflacticacid,alongwithapplicationsoflacticacidforfoodproduction andhealthyfunction,in16chaptersdividedintothreeparts.Allauthorshavecontributedin theformatusedinthe MethodsinMolecularBiology series.Intheseexplanations,the Materials sectionslistallthechemicals,reagents,buffers,andothermaterialsusedforthe protocols.Furthermore,detaileddescriptionsofeveryprotocolareprovidedinthe Methods section.Theyareexpectedtoleadtothesuccessfulcompletionofeachmethod.Some emergentdifficultiesortechniquesforeachprotocolarepresentedinthe Notes section.

InPartI,weexplainbacteriametabolism,methodsofisolatinglacticacidbacteriafrom naturalsubstances,andbacteriocinasantibacterialsubstancesproducedbylacticacid bacteria.Bacteriocinisanoteworthysubstancethatisrecognizedthroughouttheworld foritsuseasanantibacterialsubstanceinfood.Therefore,methodsofselectingalacticacid bacteriastraintoproducebacteriocinandevaluationofbacteriocinproducedfromthe bacteriaaredescribed.Furthermore,lacticacidbacteriaproduceslacticacidfromsugar, butthebacteriaarealsoinastresscondition.Asaresultofthestressresponsetoacid, internalpHchangesconsiderably.Actually,internalpHisanimportantindicationforthe foodindustryandmicrobiologyusingthebacteria.Therefore,methodsofassayingpHare alsodescribedinthissection.

Secondly,wepresentanapplicationforthefoodindustryinPartII.Anauthorprovides methodsforcountingmicroorganismssuchaslacticacidbacteria,yeast,andmoldinyogurt, andmethodsforqualityanalysisortexture.Furthermore,thepolysaccharideproducedfrom lacticacidbacteriaismentionedsuccessivelyinthreechapters.Thepolysaccharidenotonly hasarelationtoyogurttexturebutalsohasaroleinimprovingimmunity.Theauthors provideevaluationmethodsandanalyticalmethodsforthepolysaccharideandtheproductionoflacticacidbacteria.Furthermore,infoodproduction,thegrowthoflacticacid bacteriaisknowntospoilthequalityoffood.Detectionofspoilageoflacticacidbacteria inbeerusingPCRmethodisdemonstratedbytheauthors.

Finally,beneficialeffectsoflacticacidbacteriaarepresentedinPartIII.Theauthors describemethodsfortheevaluationofdetoxificationbybiosorptionofheavymetalsby lacticacidbacteria,productionofimmunobioticsbythebacteria,adhesionofthebacteriato intestinalmucosa,andneutralizationoflipopolysaccharides(LPS)bythebacteria.Many lacticacidbacteriahavehealthyfunctionafteroralingestionasprebiotics.Wehopetoapply theresultstomanyresearcheffortsinthedomainsoffoodscienceandhealthscience.

Iwouldliketoacknowledgeallauthorsforkindlycontributingtheirchapters.Weare especiallygratefultotheSeriesEditorDr.JohnWalkerandtheEditorofSpringerProtocols, DavidC.Casey,fortheirassistance,andtotheinformationtechnologydepartmentfor providingtherequisiteframework,whichgreatlyenhancedthecompilationofthebook chapters.

Sendai,JapanMakotoKanauchi

Preface.. ...................................................................v Contributors

PART IIDENTIFICATIONAND METABOLISMSOF LAB

1IsolationandIdentificationofLacticAcidBacteriafrom EnvironmentalSamples ..................................................3

AkihitoEndo,YasuhiroTanizawa,andMasanoriArita

2BasicAntibacterialAssaytoScreenforBacteriocinogenicLactic AcidBacteriaandtoElementarilyCharacterizeTheirBacteriocins ............15 KensukeArakawa

3Assaying D-BodyAminoAcidsas D-AlanineandAminoAcidRacemase (AARase)ActivityUsingNADHOxidoreductionEnzymicSystem ............23 NatsukiMatsumotoandMakotoKanauchi

4IntracellularpHDeterminationfortheStudyofAcidToleranceof LacticAcidBacteria. ....................................................33 HiromuKudoandYasukoSasaki

PART IIAPPLICATIONSOF FOOD INDUSTRIESOF LABS

5YogurtProduction.. ....................................................45 SeijiNagaoka

6Purification,RheologicalCharacterization,andVisualizationof Viscous,Neutral,Hetero-exopolysaccharideProducedbyLactic AcidBacteria ...........................................................55 S.Ikeda,D.Kondoh,N.P.D.Aryantini,T.Urashima,andK.Fukuda

7StructuralAnalysisofExopolysaccharidesfromLacticAcidBacteria... ........67 GerritJ.Gerwig

8PreparationofExopolysaccharideSynthesizedbyLacticAcidBacteria. ........85 JunkoNishimura

9PCRAnalysisMethodsforDetectionandIdentification ofBeer-SpoilageLacticAcidBacteria. .....................................95 S.Asano,M.Shimokawa,andK.Suzuki

10IsolationofLacticAcidBacteriaEliminatingTrimethylamine(TMA) forApplicationtoFisheryProcessing. .....................................109 SatoshiMohriandMakotoKanauchi

11ScreeningtheLacticAcidBacteriaconvertingHydroxyFattyAcid fromUnsaturatedFattyAcid .............................................119 MakotoKanauchi

PART IIIHEALTHY FUNCTIONSOF LABS

12ScreeningandCharacterizationofImmunobioticLacticAcid BacteriawithPorcineImmunoassaySystems. ..............................131

ValeriaGarcia-Castillo,LeonardoAlbarracin,HarukiKitazawa, andJulioVillena

13BiosorptionofHeavyMetalsbyLacticAcidBacteriaforDetoxification ........145

HidekiKinoshita

14Adhesionof Lactobacillus toIntestinalMucin ..............................159

KeitaNishiyamaandTakaoMukai

15EliminatingLipopolysaccharide(LPS)UsingLacticAcidBacteria(LAB) andaFractionofitsLPS-EliminationProtein ..............................167

MakotoKanauchi,AyakaKondo,andKyokoAsami

16CloningandExpressionofLipopolysaccharideEliminationProtein(LEP) inLacticAcidBacteria ...................................................175

KyokoAsamiandMakotoKanauchi

Contributors

LEONARDO ALBARRACIN LaboratoryofImmunobiotechnology,ReferenceCentrefor Lactobacilli(CERELA-CONICET),Tucuman,Argentina;FoodandFeedImmunology Group,GraduateSchoolofAgriculturalScience,TohokuUniversity,Sendai,Japan; LaboratoryofComputingScience,FacultyofExactSciencesandTechnology,Tucuman University,Tucuman,Argentina

KENSUKE ARAKAWA GraduateSchoolofEnvironmentalandLifeScience,Okayama University,Okayama,Japan

MASANORI ARITA CenterforInformationBiology,NationalInstituteofGenetics,Mishima, ShizuokaPrefecture,Japan;RIKEN,CenterforSustainableResourceScience,Kanagawa, Japan

N.P.D.ARYANTINI DepartmentofLifeandFoodSciences,ObihiroUniversityof AgricultureandVeterinaryMedicine,Obihiro,Hokkaido,Japan

KYOKO ASAMI DepartmentofFoodManagement,MiyagiUniversity,Sendai,Miyagi,Japan

S.ASANO ResearchLaboratoriesforAlcoholBeverages,DepartmentofFermentationand MicrobiologyTechnology,AsahiBreweries,Ltd.,Ibaraki,Japan

AKIHITO ENDO DepartmentofFood,AromaandCosmeticChemistry,TokyoUniversityof Agriculture,Hokkaido,Japan

K.FUKUDA DepartmentofLifeandFoodSciences,ObihiroUniversityofAgricultureand VeterinaryMedicine,Obihiro,Hokkaido,Japan;DepartmentofAgricultureandAnimal Science,ResearchCenterforGlobalAgromedicine,Obihiro,Hokkaido,Japan

VALERIA GARCIA-CASTILLO LaboratoryofBacterialPathogenicity,FacultyofBiological Sciences,UniversityofConcepcion,Concepcion,Chile;LaboratoryofImmunobiotechnology, ReferenceCentreforLactobacilli(CERELA-CONICET),Tucuman,Argentina;Food andFeedImmunologyGroup,GraduateSchoolofAgriculturalScience,TohokuUniversity, Sendai,Japan

GERRIT J.GERWIG MicrobialPhysiology,GroningenBiomolecularSciencesand BiotechnologyInstitute(GBB),UniversityofGroningen,Groningen,TheNetherlands

S.IKEDA DepartmentofFoodScience,UniversityofWisconsin-Madison,Madison,WI,USA

MAKOTO KANAUCHI DepartmentofFoodManagement,MiyagiUniversity,Sendai,Miyagi, Japan

HIDEKI KINOSHITA LaboratoryofFoodBiochemistry,DepartmentofBioscience,Schoolof Agriculture,TokaiUniversity,Kumamoto,Japan

HARUKI KITAZAWA FoodandFeedImmunologyGroup,GraduateSchoolofAgricultural Science,TohokuUniversity,Sendai,Japan;InternationalEducationandResearchCenter forFoodandAgriculturalImmunology(CFAI),GraduateSchoolofAgriculturalScience, TohokuUniversity,Sendai,Japan

AYAKA KONDO DepartmentofFoodManagement,MiyagiUniversity,Sendai,Miyagi, Japan

D.KONDOH DivisionofBasicVeterinaryMedicine,ObihiroUniversityofAgricultureand VeterinaryMedicine,Obihiro,Hokkaido,Japan

HIROMU KUDO MeijiUniversity,Kanagawa,Japan

NATSUKI MATSUMOTO DepartmentofFoodManagement,MiyagiUniversity,Sendai, Miyagi,Japan

SATOSHI MOHRI DepartmentofFoodManagement,MiyagiUniversity,Sendai,Miyagi, Japan

TAKAO MUKAI DepartmentofAnimalScience,SchoolofVeterinaryMedicine,Kitasato University,Towada,Aomori,Japan

SEIJI NAGAOKA FermentedMilkDevelopmentDepartment,FoodDevelopmentLaboratories, R&DDivision,MeijiCo.,Ltd.,Hachiouji,Tokyo,Japan

JUNKO NISHIMURA DepartmentofLifeandEnvironmentalScience,FacultyofEngineering, HachinoheInstituteofTechnology,Hachinohe,Aomori,Japan

KEITA NISHIYAMA DepartmentofMicrobiology,SchoolofPharmacy,KitasatoUniversity, Tokyo,Japan

YASUKO SASAKI MeijiUniversity,Kanagawa,Japan

M.SHIMOKAWA ResearchLaboratoriesforAlcoholBeverages,DepartmentofFermentation andMicrobiologyTechnology,AsahiBreweries,Ltd.,Ibaraki,Japan

K.SUZUKI ResearchLaboratoriesforAlcoholBeverages,DepartmentofFermentationand MicrobiologyTechnology,AsahiBreweries,Ltd.,Ibaraki,Japan

YASUHIRO TANIZAWA CenterforInformationBiology,NationalInstituteofGenetics, Mishima,ShizuokaPrefecture,Japan

T.URASHIMA DepartmentofLifeandFoodSciences,ObihiroUniversityofAgricultureand VeterinaryMedicine,Obihiro,Hokkaido,Japan

JULIO VILLENA LaboratoryofImmunobiotechnology,ReferenceCentreforLactobacilli (CERELA-CONICET),Tucuman,Argentina;FoodandFeedImmunologyGroup, GraduateSchoolofAgriculturalScience,TohokuUniversity,Sendai,Japan

IdentificationandMetabolismsofLAB

IsolationandIdentificationofLacticAcidBacteria fromEnvironmentalSamples

AkihitoEndo,YasuhiroTanizawa,andMasanoriArita

Abstract

Isolationoflacticacidbacteria(LAB)isthefirstandcrucialsteptostudypossiblerolesofLABinthe environment,especiallyinfoodfermentation.Thisisalsoimportanttousetheorganismsforfurther application.LABarediversebacterialgroupandhavediversegrowthcharacteristics.Cultureconditionof LABisthusvaried,andselectionofasuitableculturemediumisessentialforthepurposes.Identificationis alsoanimportantstep,sincecertaindesirableandundesirablecharacteristicsaresharedwithinspecies. IdentificationwasclassicallycarriedoutbyphenotypiccharacteristicsbutisusuallyperformedbyDNA sequence-basedapproaches.16SrRNAgenesequencingisgenerallyusedforidentification,andsequencing ofhousekeepinggenesisusedwhenneeded.Inaddition,identificationbasedonwhole-genomesequence similaritiesisbecomingcommon.HerewedescribeisolationandidentificationofLABbriefly.

Keywords Lacticacidbacteria,Isolation,Culturemedium,Identification,16SrRNAgene, Housekeepinggenes,Whole-genomesequencesimilarities

1Introduction

Lacticacidbacteria(LAB)areadiversegroupofbacteriawhich phylogeneticallybelongstotheorder Lactobacillales.Thisdiverse orderincludes6families,over30generaandover300species. Thesemicroorganismsproducelactateasthemainendproducts frommetabolismofglucose,andcertainspeciesalsoproduceethanol,CO2,andacetate.Lactatemetabolismisnotcommoninthe organisms.Bifidobacteriaarenotmembersoftheorder Lactobacillales,andtheorganismsarethusnotincludedinthischapter. LABcanbeseeninwidevarietyofhabitats,includinggastrointestinaltracts,vaginaltracts,oralcavitiesofanimals,fermentedfoods, silages,plantsurfaces,andcomposts.Certainspeciesoriginated fromclinicalsamples.Becauseofthehugevarieties,cultureconditionofLABisvaried.LABrequirerichnutrientsforgrowth,e.g., carbohydrates,aminoacids,vitamins,minerals,andsometimesfatty acidsandpeptides.TheyusuallylackmostpartofTCAcycleand

MakotoKanauchi(ed.), LacticAcidBacteria:MethodsandProtocols,MethodsinMolecularBiology,vol.1887, https://doi.org/10.1007/978-1-4939-8907-2_1, © SpringerScience+BusinessMedia,LLC,partofSpringerNature2019

quinone/ubiquinonebiosynthesissystems,meaningthattheydo notconductrespiration.Oxygenthusdoesnotusuallysupport theirgrowth,andtheypreferanaerobicconditionsratherthan aerobicconditionsforgrowth.LABgenerallyinhabitinnature withothermicrobes,includingmolds,yeasts,aerobes,andanaerobes.Severalchemicals,includingantibiotics,arethususuallyused foraselectiveisolationofLABfromenvironmentalsamples.IdentificationisessentialaftertheisolationofLAB.Identificationis usuallyconductedbyphylogeneticanalysisbasedon16SrRNA genesequences,whereasspeciesincertainLABgroups,e.g., Lactobacillusplantarum group, Lactobacilluscasei group,and Enterococcusfaecium group,areknowntosharehighsequencesimilarities of16SrRNAgenewithinthegroups.Housekeepinggenesare alternativemarkersforanaccurateidentificationofsuchLAB groups.Inaddition,identificationbasedonwhole-genome sequencesimilarities,e.g.,allnucleotideidentity(ANI)andinsilico DNA-DNAhybridization(DDH),isbecomingcommoninrecent years.

2Materials

2.1CultureMedia andDilutionBuffer

MajorculturemediaforisolationofLABaredeMan,Rogosa,and Sharpe(MRS)medium, Lactobacillus selection(LBS)medium,and M17medium.Thesemediaarecommerciallyavailableinseveral producers.Moreover,acidictomato/grapemediumandfructophilicLABisolationmediumarealsousedforisolationofspecificLAB species.ThesemediausuallycontainrichnutrientsbutnotantibioticsforselectiveisolationofLAB.Supplementof10mg/Lof sodiumazideand10mg/Lofcycloheximideisusefultosuppress growthofaerobesandfungi,respectively.Calciumcarbonate (5g/L)supplementedintotheagarmediumisalsousefulfor visibledistinctionbetweenLABandothermicrobes,sinceclear zonesareformedsurroundingcoloniesbecauseoftheiracidproduction.Anaerobicculturingbyusingagas-generatingkitor anaerobicworkstationisusuallyhelpfulforisolationofLAB.

1.DeMan,Rogosa,andSharpe(MRS)medium:Weigh52gof theMRSmediumpowder(Oxoid),put1Lofwater,mix thoroughly,andweigh15gofagar(see Note1).Autoclaveat 121 Cfor15minpriortouse.Themediumisavailablein severalproducers,andrecipeforthemediumvariesbetween producers.

2. Lactobacillus selection(LBS)medium:Weigh84goftheLBS agarpowder(BecktonDickinson),put1Lofwater,andmix thoroughly(see Note2).Put1.32mLofglacialaceticacid,and heatituntilagarcompletelydissolves.Autoclaveisnotneeded forthismedium.

2.2DNAIsolation

3.M17medium:Weigh48.25goftheM17mediumpowder (BecktonDickinson),put950mLofwater,mixthoroughly, andheatittocompletelydissolvethepowder(see Note3). Autoclaveat121 Cfor15min,coolto50 C,andadd50mL ofsterile10%lactosesolution(optional).

4.Acidictomato/grapebrothmedium:Weigh10gofglucose, 5gofyeastextract,10gofpeptone,0.2gofMgSO4·7H2O, and0.05gofMnSO4·4H2O,andput750mLofwaterand 250mLofgrape/tomatojuice(see Note4).SetpHat4.8with HCl.Autoclaveat121 Cfor15minpriortouse.

5.FructophilicLAB(FLAB)isolationmedium:BasalmediausuallyusedforFLABisolationmediumareMRS,GYP,andFYP. ForpreparationofGYPandFYPmedia,weigh10gofglucose/fructose,10gofyeastextract,5gofPolypeptone,2gof sodiumacetate,0.5gofTween80,0.2gofMgSO4·7H2O, 0.01gofMnSO4·4H2O,0.01gofFeSO4·7H2O,and0.01g ofNaCl[1].SetpHat6.8withNaOH.IfMRSorGYP mediumisused,supplementationof10g/Loffructoseor pyruvateisneeded(see Note5).Autoclaveat121 Cfor 15minpriortouse.

6.Saline:Weigh8.5gofNaClandmakeupto1Lwithwater.If needed(usuallyforanaerobicbacteria),put0.5gofcysteineHCl.Autoclaveat121 Cfor15minpriortouse.

ForgeneticalidentificationofLABisolates,DNAisolationisthe firstandcrucialstep.SeveralkitsareavailableforisolationofDNA frombacterialcells.Moreover,bacterialDNAcanbeisolatedwith generaltechniquescombinedwithphysicalcelldisruptionand ethanolprecipitation.Glassbeads(approx.0.1mmindiameter) andabeatingmachineareusedforthecelldisruption.Thedetailed methodisdescribedbelow.Prepareallsolutionsusingultrapure waterorsimilargradewaterandanalyticalgradereagents.

1.Extractionbuffer:100mMTris-40mMEDTA·2Nabufferat pH9.0.Weigh12.1gofTrisand14.9gofEDTA·2Na, dissolveitwith900mLofwater,andsetpHat9.0withHCl. Makeupto1Lwithwater.Autoclaveat121 Cfor15min priortouse.Storeat4 C.

2.3MSodiumacetatesolution:Weigh246gofsodiumacetate anddissolveitwith700mLofwater.Makeupto1Lwith water.Storeat4 C.

3.TEbuffer:10mMTris-1mMEDTAbufferatpH8.0.Weigh 1.2gofTrisand0.4gofEDTA·2Na,dissolveitwith950mL ofwater,andsetpHat8.0withHCl.Makeupto1Lwith water.Autoclaveat121 Cfor15minpriortouse.Storeat 4 C.

2.3PCR, Electrophoresis, Purification, andSequencing

4.Sodiumdodecylsulfate(SDS)solution:10%(w/v)SDSsolution.Weigh10gofSDSandmakeupwith100mLwithwater. Storeatroomtemperature.

5.Benzylchloride:Storeatroomtemperature.

6.Isopropanol:Storeat4 C.

7.70%Ethanol:Mix700mLofethanoland300mLofwater. Storeat4 C.

8.Glassbeads:100mgglassbeadswith0.1mmindiameter. Weigh100mgofglassbeads(0.1mmindiameter)andsterilize at180 Cfor30min.

9.Cellcrusher:FastPrep-24(MPBiomedicals).

10.Spectrophotometer.

1.CommercialPCRkit:Storeat 20 C.Thekitusuallycontains DNApolymerase,PCRreactionbuffer,anddNTPmixture.

2.Primersusedforamplificationandsequencingof16SrRNA gene:8F(50 -AGAGTTTGATCMTGGCTCAG-30 ),15R(50 -A AGGAGGTGATCCARCCGCA-30 ),930F(50 -GCACAAGCG GTGGAGCATGTGG-30 ),520R(50 -ACCGCGGCTGCTGG C-30 ),800R(50 -CAGGACTACCAGGGTATCTAAT-30 ),and 1100R(50 -AGGGTTGCGCTCGTTG-30 ).

3.Primersusedforamplificationandsequencingofhousekeeping genes:recEXT-f(50 -GGCTATGAAACAAATTGAAAAA CAATWYGGNAARGG-30 )andrecEXT-r(50 -TGTT TAAACGGTGGAGCAACTTTRTTYTTNAC-30 )for recA gene;pheS-21-F(5’-CAYCCNGCHCGYGAYATGC-30 ), pheS-22-R(50 -CCWARVCCRAARGCAAARCC-30 ),and pheS-23-R(50 -GGRTGRACCATVCCNGCHCC-30 )for pheS gene;andrpoA-21-F(50 -ATGATYGARTTTGAAAAACC-30 ), rpoA-23-R(50 -ACHGTRTTRATDCCDGCRCG-30 ),and rpoA-22-R(50 -ACYTTVATCATNTCWGVYTC-30 )for rpoA gene.

4.TAEbuffer:Weigh4.8gofTris,1.1mLofaceticacid,and 0.074gofEDTA·2Na,anddissolveitwith900mLofwater. Makeupto1Lwithwater.

5.1%Agarosegel:Weigh1gofagaroseandput100mLofTAE buffer.Heatitwithmicrowaveuntilagarosecompletelymelts. Castagarosesolutiononagelmaker.

6.CommercialpurificationkitforPCRproducts.

7.BigDyeTerminatorCycleSequencingkit(ThermoFisherScientific):Storeat 20 C.

8.3MSodiumacetate:Prepareasdescribedabove.

9.99%Ethanol:Storeatroomtemperature.

10.70%Ethanol:Prepareasdescribedabove.Storeatroom temperature.

11.PCRmachine.

12.Electrophoresismachine.

13.DNAsequencer:AppliedBiosystemsmodel3130(Thermo FisherScientific).

3Methods

3.1IsolationofLAB

3.2DNAIsolation

IsolationofLAB,DNAextraction,andsequencing-basedidentification,including16SrRNAgene,housekeepinggenes,andwholegenomesequences,areintroducedhere.

1.Environmentalsamplesareseriallydilutedwithsalineand spreadontoappropriateagarmediumlistedinSubheading 2.

2.Themediaareincubatedat30or37 Cunderanaerobic/ aerobicconditionsfor48to72h.Incubationtemperaturesat 25and42 CareusedforisolationofmesophilicLABand thermophilicLAB,respectively.Longerincubationhour(upto 120h)isneededforisolationofspecificslowlygrowingLAB, e.g.,wineLAB O.oeni

3.Coloniesonagarmediacanbeculturedinliquidbrothwhichis thesamemediumusedforisolationbutexclusiveofagar.

1.Culturedcellsareharvested(10,000 g,5min)inplastic tubes,discardsupernatant,andsuspendinasolutioncontaining250 μLofextractionbuffer,50 μLof10%SDSsolution, and150 μLofbenzylchloride.Onehundredmilligramsof glassbeads(0.1mmindiameter)isaddedtothesuspension, andthemixtureisbeatenatmaximumspeedfor2minina beatingmachine(modelFastPrep-24,MPBiomedicals).

2.Onehundredfiftymicrolitersof3Msodiumacetatesolutionis addedtothebeatensamples,andthesamplesarecooledonice for15min.

3.Thesamplesarecentrifuged(15,000 g,10min),andthe resultantsupernatantistransferredtoanewtube.

4.Fourhundredfiftymicrolitersofisopropanolisaddedtothe supernatant.Thesamplesaremixedandcentrifuged (15,000 g,15min).

5.Supernatantisdiscardedand70%ethanolisaddedtothe samples.Thesamplesarecentrifuged(15,000 g,5min).

6.Supernatantisremovedcarefullyandthesamplesaredried. TheresultantDNAisdissolvedin50 μLofTEbuffer,and

3.3Amplification andSequencingof16S rRNAGene

concentrationandqualityofDNAaredeterminedbyspectrophotometry.DNAcanbedilutedwithTEbufferataconcentrationof10ng/μL.

1.PCRreactionmixturecomprises10pmolofeachprimer,PCR reactionbuffer,2-mMMgCl2,0.2mMeachdNTP,1.25Uof TaqDNApolymerase,and10ngoftheisolatedDNA.8Fand 15RprimersareusedforthisPCR.PCRprogramconsistedof 35cyclesof94 Cfor30s,55 Cfor30s,and72 Cfor90s withthelastextensionat72 Cfor2min.Amplificationcanbe confirmedbyagarosegel(1%)electrophoresisinTAEbuffer.

2.ThePCRproductsarepurifiedbycommercialPCRpurificationkitsaccordingtothemanufacturer’sinstruction.ThepurifiedDNAisusedforcyclesequencing.

3.Reactionmixtureforcyclesequencingcomprises1 μLofBigDyeTerminatorCycleSequencingkit,2 μLof5 sequencing buffer,1 μLofprimer(1.6pmol),5 μLofdeionizedwater,and 1 μLofthepurifiedDNA.PCRprogramconsistedof25cycles of96 Cfor10s,50 Cfor5s,and60 Cfor4min.Primers usedforamplification,i.e.,8Fand15R,canbeusedinthis step.Ifneeded,primers930F,520R,800R,and1100Rare helpfulfordeterminationofnearlywhole16SrRNAgene sequencing.

4.Theproductshavetobepurifiedforfurtheranalysis.The productsaretransferredintosteriletubes,addedwith1 μLof 3Msodiumacetateand25 μLof99%ethanol,andkeptfor 15minatroomtemperature(RT).

5.Centrifuge(15,000 g,15min,RT),discardsupernatant,and add125 μLof70%ethanol.

6.Centrifuge(15,000 g,5min,RT),discardsupernatant,and dryup.

7.SamplescanbeanalyzedinaDNAsequencer(AppliedBiosystemsmodel3130)accordingtothemanufacturer’s instructions.

8.BLASTanalysisishelpfulfordeterminationofsequencesimilaritiesbetweentheisolatesandsequencesofknownbacterial speciesdepositedindatabase.NCBIdatabase(https://blast. ncbi.nlm.nih.gov/Blast.cgi)providesthisvaluableservice. Sequencesimilaritiesover99%aregenerallyacceptablevalues forspeciesidentification[2](see Note6).

3.4Amplification

1.16SrRNAgenesequencesimilarityisnotalwayssufficientfor speciesdiscrimination.Housekeepinggenesaremorevariable andthereforehaveagreaterdegreeofresolution.Theyarethus recommendedtouseforidentificationanddescriptionofnovel species[3].Amongthehousekeepinggenes, recA, pheS,and

IsolationandIdentificationofLAB9

rpoA arewidelyusedascomplementaryphylogeneticmarkers fortaxonomyofLAB.Herethusintroducesamplificationand sequencingofthethreehousekeepinggenes(see Note7).

(a)Foramplificationof recA gene,PCRreactionmixturecomprises25pmolofeachprimer,PCRreactionbuffer,2-mM MgCl2,0.1mMeachdNTP,2UofTaqDNApolymerase, and10ngoftheisolatedDNA.PrimersrecEXT-fand recEXT-rareusedforthisPCR[4].PCRprogramconsisted ofinitialdenaturationat94 Cfor5min;35cyclesof94 C for45s,45 Cfor120s,and72 Cfor105s;andthelast extensionat72 Cfor7min.Amplificationcanbeconfirmedbyagarosegel(1%)electrophoresis.

(b)Foramplificationof pheS gene,PCRreactionmixture comprises25pmolofeachprimer,PCRreactionbuffer, 2-mMMgCl2,0.2mMeachdNTP,1.25UofTaqDNA polymerase,and10ngoftheisolatedDNA.Primers pheS-21-FandpheS-22-RareusedforthisPCR,anda primerpheS-23-RcanbereplacedwithpheS-22-Rwhen amplificationisnotseenbyusingprimerspheS-21-Fand pheS-22-R[5].PCRprogramconsistedofinitialdenaturationat95 Cfor5min;3cyclesof95 Cfor60s,46 C for135s,and72 Cfor75s;30cyclesof95 Cfor35s, 46 Cfor75s,and72 Cfor75s;andthelastextensionat 72 Cfor7min.Amplificationcanbeconfirmedby agarosegel(1%)electrophoresis.

(c)Foramplificationof rpoA gene,PCRreactionmixture comprises25pmolofeachprimer,PCRreactionbuffer, 2-mMMgCl2,0.2mMeachdNTP,1.25UofTaqDNA polymerase,and10ngoftheisolatedDNA.Primers rpoA-21-FandrpoA-23-RareusedforthisPCR,anda primerrpoA-22-RcanbereplacedwithrpoA-23-Rwhen amplificationisnotseenbyusingprimersrpoA-21-Fand rpoA-23-R[5].PCRprogramconsistedofinitialdenaturationat95 Cfor5min;3cyclesof95 Cfor60s,46 C for135s,and72 Cfor75s;30cyclesof95 Cfor35s, 46 C(or42 C)for75s,and72 Cfor75s;andthelast extensionat72 Cfor7min.Amplificationcanbeconfirmedbyagarosegel(1%)electrophoresis.

2.PurificationofthePCRproducts,cyclesequencing,purificationofcyclesequencingproducts,analysiswithaDNA sequencer,andBLASTanalysiscanbecarriedoutbythe methodasdescribedabove,exceptthatprimersforcycle sequencingarethoseusedforPCR.Thresholdforspecies identificationissomewhatunclearwhenhousekeepinggenes areusedasmarkers.Strainsclassifiedinthesamespeciesusually shareover95–97%sequencesimilaritiesbasedonthesetwo genes(see Note6).

3.5Whole-Genome SequenceSimilarities

Whole-genomerelatednessof70%experimentallydetermined usingDDHhaslongbeenagoldstandardinmicrobialspecies delineation.Recently,theuseofwhole-genome-basedmetrics suchasANIhasbeenspreadingasareplacementfortraditional DDHtechnique.ANIrepresentsmeannucleotideidentityina pair-wisesequencealignmentbetweentwogenomes.TheANI valueof95%approximatelycorrespondstotheDDHvalueof 70%andisnowbeingrecognizedasacutofflinetodemarcate species[6, 7].Inmostcases,theinterspeciesANIisbelow85%, muchlowerthanthewidelyusedthreshold,evenbetweenspecies difficulttodistinguishby16SrRNAgenesequencessuchasspecies in L.casei groupand L.plantarum group[8].Thereareseveral implementationsofANI.Amongthem,ANIbisthemoststandard method,inwhichoneofthegenomesinapair(query)iscutinto fragments,typicallyabout1000ntinlength,andtheyarealigned tothereferencegenomeusingtheBLASTNalgorithm.ANIbis calculatedasthemeanidentityofallBLASTNmatchesthatshowed morethan30%overallsequenceidentityoveranalignableregionof atleast70%oftheirlength[6].Similarly,ANImusesMUMmerfor sequencealignment[7],andmorerobustandfastermethodssuch asOrthoANIandOrthoANIuhavealsobeenproposedrecently [9].ApartfromANI,GenomeBlastDistancePhylogeny(GBDP)is anotherinsilicometricsforwhole-genomerelatedness.Itcanbe calculatedusingGenome-to-GenomeDistanceCalculator (GGDC, http://ggdc.dsmz.de),whichalsoestimatesinsilico DDHwithconfidenceintervals[10].Thespeciesthresholdofin silicoDDHis70%,sameasthewet-labDDH.

TocalculateANIorinsilicoDDH,assembleddraftorcompletegenomesequencesareused.Forcompletegenomesconsistingofmultiplerepliconsordraftgenomes,allofthesequences shouldbebundledinasinglefile(multi-FASTAformat).

Forvalididentification,comparisonagainstthegenomefroma typestrainisdesirable.Now,largeamountofgenomicdataare depositedinthepublicsequencedatabases.TheNCBIAssembly databaseisofusetoobtainreferencegenomes.Genomesfromtype strainscanbesearchedbytypingkeywordslike‘(Lactobacillus plantarum)AND“assemblyfromtypematerial”[FromType Material]’inthesearchform.GenomesequencefilesinaFASTA format(theonewithafilesuffix“genomic.fna.gz”)shouldbe downloadedfromtheNCBIFTPserverandbeunarchived.

SeveralwaystocalculateANIusingonlinetoolsareintroduced below.

3.5.1ANICalculator

ANIcalculator(http://enve-omics.ce.gatech.edu/ani/)isoperatedbyaresearchgroupthatoriginallyproposedANI[6].ANI canbecalculatedbyuploadingaquerygenomeandareference genomeinthesubmissionform.Ajobnameandane-mailaddress

3.5.2JSpeciesWS

canbespecifiedoptionally.Then,press“Submit”buttontostart calculation.Notificationwillbesentwhenthejobiscompletedifan e-mailaddressisprovided.Ofnote,ANIvaluesarelessreliable whenthenumberofalignedfragmentsisnotsufficient.Forexample,genomesfromdifferentspeciesthatsharesimilarmobileelementmightexhibithigherANIthanexpected.

JSpeciesWS(http://jspecies.ribohost.com/jspeciesws/ [11])can accommodatetwoormoregenomes(upto15).ANIcanbe calculatedinanall-against-allmannerbetweeneachpairofthe genomesorcanbecalculatedagainsteachofreferencegenomesif onegenomeisspecifiedasaquery.Thiswillbeusefulinaproposal ofnewtaxa,inwhichcomparisontoallspeciesofcloserelativesis required.Inadditiontouploadinglocalfiles,genomedatacanbe importedfromitsassociatedgenomedatabaseGenomesDB.In additiontoANIb,itcancalculateANImandcorrelationcoefficientsoftetranucleotidefrequencies(Tetra),whichisanalignmentfreemetricsforgenomerelatedness.Thestand-aloneversionJSpeciesisalsoavailablefordownloadandcanberunonalocal computer[7].

3.5.3DFAST

DDBJFastAnnotationandSubmissionTool(DFAST[8])isoriginallydevelopedbytheauthorstoassistprokaryoticgenomeannotationanddatasubmissiontoDNADataBankofJapan(DDBJ).It alsoprovidesafunctiontoassessqualityofgenomesandtaxonomic identificationusingANI(https://dfast.nig.ac.jp/analysis/assess ment/,currentlyonlyavailablefor Lactobacillus and Pediococcus). DFASTisequippedwithreferencegenomes,thusuploadingof referencegenomesisnotrequired.Allusershavetodoisupload aquerygenome.Bydefault,ANIiscalculatedagainstallofthe referencegenomes.Thetargetgenomescanbebarroweddownby specifyingorganismgroupstoreducerunningtime.Inaddition, DFASTreportscompletenessandcontaminationvaluesofthe querygenomeascalculatedusingCheckM[12].

4Notes

1.MRSmediumisdesignedforisolationofgeneralLAB[13]and isstillusedasagoldstandardforisolationofLABfromvarious environmentalsamples.Antibiotics,e.g.,sodiumazide (10mg/L)andcycloheximide(10mg/L),arehelpfulfor selectiveisolationofLAB.Supplementofcalciumcarbonate (5g/L)ishelpfultodistinguishbetweenacidproducers (mainlyLAB)andothers.

2.LBSmediumisdesignedforselectiveisolationoflactobacilli fromoralandstoolsamples[14].Themediumcontainslarge

amountsofsodiumacetate(25g/L)whichinhibitsthegrowth ofmanymicroorganisms,includingbacteriaandmolds.Acetic acidisalsoaddedtothemedium(1.32mL/L)tolowerpH, whichresultsingrowthinhibitionofnontargetmicroorganisms.Becauseofthesechemicals,autoclavingisnotneeded beforeuse.Additionoftomatojuicetothemediumishelpful toisolatespecificlactobacilli(Lactobacillusacidophilus).

3.M17mediumisusuallyusedforisolationof Lactococcuslactis and Streptococcusthermophilus fromdairyproducts,including yogurtandcheese[15].Themediumcontains1.9%(w/v) disodium-β-glycerophosphateforbufferaction,andthechemicalalsohelpsforselectiveisolationof S.thermophilus from yogurtbysuppressionof Lactobacillusdelbrueckii subsp. bulgaricus.

4.Acidictomatobrothmediumandacidicgrapemediumare usedforisolationofwineLAB,especiallyamalolacticfermenting Oenococcusoeni [16, 17].Theorganismisacidophilicand requiresaspecificgrowthfactor.Tomatoorgrapejuiceprovidesthegrowthfactor.Anaerobicculturingisessentialfor isolationof O.oeni.Antibiotics,e.g.,sodiumazide(10mg/ L)andcycloheximide(10mg/L),arehelpfulforselective isolationofLAB.

5.FLAB,including Fructobacillus spp., Lactobacilluskunkeei,and Lactobacillusapinorum,sharespecificgrowthcharacteristics: preferenceoffructosethanglucoseasagrowthsubstrateand requirementofexternalelectronacceptor(s)forglucose metabolism.Oxygen,pyruvate,andfructoseareusuallyused astheelectronacceptors.Thesemean,unliketootherLAB, aerobicculturingdramaticallyenhancesgrowthoftheseinterestingmicroorganisms.Aerobicculturingusuallyproduces maximumbiomass.SupplementofsodiumazideandcycloheximideishelpfulforselectiveisolationofFLAB.

6.Asdescribed,certainLABspeciesshareover99%sequence similaritiesbasedon16SrRNAgenesequences,andthusidentificationshouldbecarefullyconducted. L.plantarum group species,i.e., L.plantarum,L.pentosus,and L.paraplantarum, shareover99.5%sequencesimilaritiesbasedon16SrRNA genebutshare83–87%similaritiesbasedon recA gene sequences[18]. Fructobacillustropaeoli possesses99.2and 85.6%sequencesimilaritieswith Fructobacilluspseudoficulneus basedon16SrRNAgeneand recA,respectively[19].

7.Onceadraftorcompletegenomeisdetermined,nucleotide sequencesforhousekeepinggenessuchas pheS, rpoA,and recA areeasilyobtainedbyusingstandardgenomeannotationpipelineslikeDFASTandRAST[20](http://rast.nmpdr.org). However,itisnoteasytoextractafull-length16SrRNA

References

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6.GorisJ,KonstantinidisKT,KlappenbachJA, CoenyeT,VandammeP,TiedjeJM(2007) DNA-DNAhybridizationvaluesandtheirrelationshiptowhole-genomesequencesimilarities.IntJSystEvolMicrobiol57:81–91

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BasicAntibacterialAssaytoScreenforBacteriocinogenic

LacticAcidBacteriaandtoElementarilyCharacterizeTheir Bacteriocins

KensukeArakawa

Abstract

Bacteriocinsribosomallyproducedbylacticacidbacteriaareantibacterialpeptidesexpectedtobeusedasa safebiopreservativeandafermentationcontrollerinfoodindustry.Themodifiedagar-welldiffusion methodismostfrequentlyusedforantibacterialactivityassaytoscreenforpotentiallybacteriocinproducingstrainsandtoelementarilycharacterizetheirbacteriocinsandtherelatives.Here,Idescribe procedureofthemodifiedagar-welldiffusionassayinthedetails.

Keywords Lacticacidbacteria,Bacteriocin,Bacteriocinogenics,Antibacterialactivityassay,Agar-well diffusionmethod

1Introduction

Bacteriocinsareantibacterialpeptidesribosomallybiosynthesized bysomebacteriaincludinglacticacidbacteria(LAB).Uptodate, manyLABbacteriocinshavebeendiscovered,andsomeofthem havebeendeeplycharacterizedinstructure,genetics,antibacterial intensityandspectrum,modeofaction,secretionandimmunity mechanisms,andsoon.Onthebasisofsuchcharacteristics,LAB bacteriocinsarebroadlyclassifiedintotwogroups[1]:ClassI, lanthionineandtheothermodified/unusualaminoacidscontainingbacteriocins(namedaslanthibiotics),andClassII, non-lanthibioticbacteriocinsconsistingofusualproteinogenic aminoacids.ClassIIisfurtherdividedintofoursubgroups:Class IIa,pediocin-likelistericidalbacteriocins;ClassIIb,synergistically actingtwo-componentbacteriocins;ClassIIc,head-to-tailcircular bacteriocins;andClassIId,theotherClassIIbacteriocins.Most LABbacteriocinshavemuchstrongerantibacterialactivityparticularlyagainstGram-positivebacteriathananyotherLABmetabolites suchasorganicacidincludinglactateandacetate,hydrogen

MakotoKanauchi(ed.), LacticAcidBacteria:MethodsandProtocols,MethodsinMolecularBiology,vol.1887, https://doi.org/10.1007/978-1-4939-8907-2_2, © SpringerScience+BusinessMedia,LLC,partofSpringerNature2019

peroxide,acetaldehyde,diacetyl,andreuterin.Then,thebacteriocinsareheat-stableandcomparativelypHtolerantandhavenarrow-/broad-spectrumantibacterialactivityagainstfoodspoilage andpathogenicGram-positivebacteriasuchas Bacilluscereus, Listeriamonocytogenes,and Staphylococcusaureus.Inaddition,LAB bacteriocinsaregenerallyrecognizedtobesafe,becausetheirproducers,namely,LAB,arefamiliarwithhumanthroughoutlong historyandtheirpeptidebackbonesareeasytobedigestedand absorbedinthehumangastrointestinaltract.SuchcapableLAB bacteriocinsareexpectedtobeusedasasafeandeffectivebiopreservativeandafermentationcontrollerinsteadofconventional artificialadditivesinfoodindustry;andthereforemanytrialsfor foodapplicationofLABbacteriocinshavebeenperformedand reporteduntilnow.Recently,itisalsoconsideredtobethealmost truththatbacteriocinsproducedbyentericLABwouldcontribute tohumanandanimalhealthbyactingasaprobioticfactortokill pathogensandtomaintainmicrobiotabalanceinthegastrointestinaltract[2].

Ingeneralbacteriocinresearch,thefirststepisprincipallyto screenforapotentialbacteriocinproducer(abacteriocinogenicstrain)fromLABcollection.Inthescreeningwithantibacterialactivityassay,theagar-welldiffusionmethod[3]ismost frequentlyemployedaftersomemodifications.Occasionally,the crossstreakmethod[4],thespot-colonyoverlaymethod[5], spot-on-lawnmethod[6],paperdiskmethod[7],andmicroplate method[8]wouldbealsoemployedforthescreening.However, someofthesemethodscouldbeaffectedbyantibacterialactivityof organicacidsincludinglactateandacetateandbacteriophages.In themodifiedagar-welldiffusionassay,acidiceffectiseliminatedby neutralizingtestsamplessuchascell-freeculturesupernatant(CFS) beforeuse.Moreover,effectofbacteriophagesisdistinguishably reducedinthemodifiedmethod,becausehardagarusedtherefor propagationofabacteriocin-indicatormakesitdifficultforbacteriophagestodiffuseinside.Perhapsfromthesereasons,themodified agar-welldiffusionmethodismostfrequentlyusedforbacteriocin activityassay.

Afterscreeningforpotentialbacteriocinogenics,elementary characterizationofthemandtheirbacteriocins(orbacteriocinlikeinhibitorysubstances;BLIS)arenormallyperformedasthe secondstep.Forexample,relationshipbetweenbacterialgrowth ofandbacteriocin/BLISproductionfromaproducerisinvestigated,andthenheat/storagestability,pHtolerance,proteolytic susceptibility,andantibacterialspectrumofthebacteriocin/BLIS areevaluated.Alsoforthesecharacterizations,themodifiedagarwelldiffusionassayismainlyusedwithfurthermodificationsas showninlatersections.Afterthat,advancedcharacterizationis carriedoutifneeded.

AntibacterialAssayofBacteriocins17

Inthischapter,Ipresentprocedureofantibacterialactivity assayusingthemodifiedagar-welldiffusionmethod,whichisthe mostbasicandimportanttechniqueinbacteriocinresearch.

2Materials

2.1BacterialStrains

2.2Bacterial Medium

AllreagentswithoutanyindicationsarepurchasedfromNacalai Tesque(Kyoto,Japan)orFUJIFILMWakoPureChemicalCorporation(Osaka,Japan).

1. Lis.monocytogenes VTU206(see Note1):Distributedfrom DepartmentofVeterinaryPublicHealth,FacultyofAgriculture,UniversityofTokyo(Tokyo,Japan).

2. Lactobacillusdelbrueckii subsp. bulgaricus JCM1002T (see Note1):DistributedfromMicrobeDivision(JapanCollection ofMicroorganisms;JCM)inRIKENBioResourceResearch Center(RIKENBRC;Ibaraki,Japan).

3. Lactobacillussakei subsp. sakei JCM1157T (see Note1): DistributedfromJCM.

4. Lactococcuslactis subsp. lactis NBRC12007(see Note2): DistributedfromNITEBiologicalResourceCenter(NBRC) inNationalInstituteofTechnologyandEvaluation(NITE; Chiba,Japan).FormerlynamedasIFO12007andNCDO 497.

5. Lc.lactis subsp. lactis JCM7638(see Note2):Distributed fromJCM.FormerlynamedasIO-1.

6. Lc.lactis subsp. lactis NBRC100933T (see Note2): DistributedfromNBRC.

7. Lactobacillusgasseri JCM11046(see Note3):Distributed fromJCM.SameasstrainLA158.

8. Lb.gasseri JCM11657(see Note3):DistributedfromJCM. SameasstrainLA39.

9. Lb.gasseri JCM1131T (see Note3):DistributedfromJCM.

10. Leuconostocmesenteroides subsp. mesenteroides 406and213M0 (see Note4):Thesestrainswereisolatedandstockedinmy laboratory.

11. Leu.mesenteroides subsp. mesenteroides NBRC100496T (see Note4):DistributedfromNBRC.

1.DeMan,Rogosa,andSharpe(MRS)broth(see Note5):MRS brothpowder(Oxoid,Hampshire,UK)iscompletelydissolved indistilledwaterappropriately.Afterdispensing5mLofthe solutiontoeachtube,themediumissterilizedusinganautoclaveat121 Cfor15min.

2.3ReagentSolution

2.Tryptone,yeastextract,lactose,andglucose(TYLG)broth(see Note5)[9]:1.0goftryptone(Bectone,DickinsonandCompany;BD,FranklinLakes,NJ),0.5gofyeastextract(BD), 0.5goflactosemonohydrate,0.5gof D-glucose,0.01gof Lcysteinehydrochloridemonohydrate,and0.01gofTween 80arecompletelymixedanddissolvedin100mLofdistilled water,andthenthesolutionisadjustedtopH6.8–7.0with 1–6NNaOH(see Subheading 2.3, item1).Afterdispensing 5mLofthesolutiontoeachtube,themediumissterilized usinganautoclaveat121 Cfor15min.

3.GM17broth(see Note5):M17brothpowder(Oxoid)is completelydissolvedindistilledwaterappropriately.Afterdispensing4.5mLofthesolutiontoeachtube,themediumis sterilizedusinganautoclaveat121 Cfor15min.10%(w/v) lactoseand10%(w/v) D-glucosesolutionsarealsoprepared andautoclavedunderthesamecondition.250 μLofboth sugarsolutionsareasepticallyaddedtotheM17brothtubes.

4.MRSagar(see Note6):MRSagarpowder(Oxoid)ismixedin distilledwaterappropriately,andcompletelydissolvedwith boiling.Afterthat,theagarsolutionissterilizedusingan autoclaveat121 Cfor15min.Beforeuse,itiskeptat 55–60 Cinawaterbathtoavoidsolidification.

5.StandardPlateCount(SPC)agar(see Note6):SPCagar powder(NissuiPharmaceutical,Tokyo,Japan)ismixedin distilledwaterappropriatelyandcompletelydissolvedwith boiling.Afterthat,theagarsolutionissterilizedusingan autoclaveat121 Cfor15min.Beforeuse,itiskeptat 55–60 Cinawaterbathtoavoidsolidification.

1.1–6NNaOH:6NNaOHsolutionisdilutedwithdistilled waterappropriately.

2.1–6NHCl:HClreagent(12N)isdilutedwithdistilledwater appropriately.

3.Sterilephysiologicalsaline(0.85%NaClsolution):NaClis mixedanddissolvedindistilledwaterappropriately.Afterdissolving,thesolutionissterilizedusinganautoclaveat121 C for15min.

4.Enzymesolutions[9]:Catalaseandproteasesolutionsareused forenzymaticsusceptibilitytestsofabacteriocin/BLIS.Each enzymeisdissolvedinanddilutedwithdistilledwatertothe finalconcentrationat1U/mg(see Note7).

3.1Preparation ofCFSSamplesfrom LABCultures

Operationforbacterialinoculation,agarplatepreparation,well formation,sampledilution,andsampleinjectionareaseptically performedinacleanbench.

1.LABstrainsareprecultivatedtwiceinMRS(forlactobacilli) andTYLG(orGM17;forLABcocci)brothsateachoptimum temperaturesuchas25,30,or37 Cfor24h(see Note5).

2.TheLABstrainsarecultivatedunderthesameconditionsas aboveforthefollowingCFSsamplepreparation(see Note8).

3.CulturesarepH-adjustednormallyto7.0using1–6NHCl and1–6NNaOHandthencentrifugedat1600 g for20min (see Notes7, 9,and 10).

4.Aftercentrifugation,theculturesupernatantsarefiltered througha0.20 μmmembrane(syringefilter;Sartorius,Go ¨ ttingen,Germany)toprepareCFSsamples(see Notes7 and 10).

5.Ifneeded,CFSisasepticallydilutedtwofoldwithsterilephysiologicalsalineusingwellsofamicrotiterplate.

3.2Preparation ofAgarPlates Containing anIndicatorStrain andFormation ofSampleWells

1. Lis.monocytogenes VTU206, Lb.delbrueckii subsp. bulgaricus JCM1002T or Lb.sakei subsp. sakei JCM1157T usedasa indicatorinthemodifiedagar-welldiffusionassayareprecultivatedtwiceinTYLG(forVTU206)andMRS(JCM1002T andJCM1157T)brothsat30(JCM1157T)and37 C(VTU 206andJCM1002T)for24h(see Note5).

2.50 μLofculturesolutionofanindicatorstrainand20mLof SPC(forVTU206)orMRS(forJCM1002T andJCM1157T) agarsolutionataround50–55 Carepouredintoapetridish andgentlybutwellstirredbeforesolidification(see Note11).

3.Afteragarsolidification,punchwells(atmost20)inanagar plateusingametalcork-borer(6mmindiameter)whichis flame-sterilizedandcooleddownbeforeuse(see Note12).

3.3SampleInjection intoWells andIncubation oftheAgarPlate

3.4Evaluationof AntibacterialActivity

1.50 μLofCFSsamplesandthetwofolddilutionspreparedin aboveorbacteriocinsolutionareinjectedintoawell.

2.Theagarplateisincubatedat30(JCM1157T)or37 C(VTU 206andJCM1002T)for24h.

1.Afterincubation,aclearzonewithoutcellgrowthoftheindicatoraroundawellmeanspresenceofabacteriocin/BLIS. Antibacterialactivityofabacteriocin/BLISisevaluatedwith size(mmindiameter)oftheclearzone(see Notes13 and 14). Moreover,antibacterialtiterisdeterminedbyarbitraryunits (AU)whichwasdefinedasthereciprocalofthehighestdilutioninhibitingthegrowthoftheindicatorstrain(see Note15).

1. Lis.monocytogenes VTU206, Lb.delbrueckii subsp. bulgaricus JCM1002T,and Lb.sakei JCM1157T arehighlybacteriocin sensitiveandthereforeusedasanindicatorstrainontheantibacterialassay[8–10].

2. Lc.lactis subsp. lactis NBRC12007andJCM7638produce nisinsAandZ,respectively[10].Nisinisthemostfamous ClassIlanthibioticbacteriocinandisalreadypracticallyused asbiopreservativesinworldwidefoodindustry.NisinAreagent (Sigma)isalsolaunchedontothemarket.Theyareoftenused forapositivecontrolintheantibacterialassay.Ontheother hand, Lc.lactis subsp. lactis NBRC100933T,anon-bacteriocin producer,isusedasanegativecontrol.

3. Lb.gasseri JCM11046andJCM11657producegassericinsT andA,respectively[8].GassericinAisarepresentativeClassIIc circularbacteriocin.GassericinTisarepresentativeclassIIb two-componentbacteriocin.Theyareusedasapositivecontrol intheantibacterialassay,whereas Lb.gasseri JCM1131T,a non-bacteriocinproducer,isusedasanegativecontrolin thecase.

4. Leu.mesenteroides subsp. mesenteroides 406and213M0were isolatedfromMongoliantraditionalfermentedmilk,airag,in mylaboratory[9, 11].TheybothproduceanidenticalClassIIa listericidalbacteriocintomesentericinY105[12, 13]and thereforecanbeusedasapositivecontrolintheantibacterial assay.Inthecase, Leu.mesenteroides subsp. mesenteroides NBRC100496T,anon-bacteriocinproducer,shouldbeused asanegativecontrol.

5.MRSandBM17brothsaregenerallyusedtocultivateLAB rods(lactobacilli)andLABcocci,respectively.Inmylaboratory,TYLGbrothisusedtopropagateLABcocciand Lis. monocytogenes [9].

6.Inthisprocedure,twokindsofagarmediaareusedtocultivate indicatorstrainsforthemodifiedagar-welldiffusionantibacterialactivityassay.MRSagarisusedfor Lb.delbrueckii subsp. bulgaricus JCM1002T and Lb.sakei JCM1157T [8, 10].SPC agarisusedfor Lis.monocytogenes VTU206[9, 11].

7.Inthecaseofenzymaticsusceptibilitytestsofabacteriocin/ BLISusingcatalaseandseveralproteases[9],CFSsatpH2.0, 7.0,7.5,and7.8arepreparedforreactionswithpepsin (37 C),catalase(25 C),trypsin(37 C), α-chymotrypsin (25 C),andproteinaseK(37 C),respectively.Aftereach reactionfor1h,enzymesareinactivatedwithheatingat 95 Cfor10min.Then,eachenzymaticallytreatedCFSis

usedforantibacterialactivityassay.Bydecreaseofantibacterial activity,itcanbejudgedwhethertheBLISinCFSisabacteriocinwithapeptidebackboneornot.

8.Inthecaseofanalysisofrelationshipbetweenbacterialgrowth ofandbacteriocin/BLISproductionfromaproducer[9],the strainiscultivatedfor72h.Eachcultureafterincubationfor 0,2,4,8,12,18,24,48,and72hiscollectedandusedto evaluatethegrowthbymeasurementofculturepH,turbidity, andviablecellcountandtoprepareCFSforantibacterial activityassay.

9.InthecasefordeterminationactivepHrangeofabacteriocin/ BLIS[9],culturesupernatantispH-adjustedto2,3,4,5,6,7, 8,9,10,and11using1–6NHCland1–6NNaOH.AfterpH adjustment,theculturesupernatantiscentrifugedagainand followedbysterile-filtrationtoprepareCFSsamples.

10.Inthecaseofheat/storage-stabilitytestsofabacteriocin/BLIS [9],CFSsatpH4.5and7.0areprepared.TheCFSsareheated at65 Cfor15–60minor99–121 Cfor15minorstoredat 4 Cfor7daysor25 Cfor1–7days.Afterheatingand storage,allCFSsareusedassamplestomeasureantibacterial activity.

11.Inthecaseforestimationofantibacterialspectrumofabacteriocin/BLIS[9],variousspeciesbacteriasuchasotherlactic acidbacteria,food-spoilagebacteria,andfoodbornepathogens areusedastestedstrainsinsteadoftheindicatorstrains. Growthmediaandconditionsandinoculumratioaredependentoneachtestedstrain.

12.Itshouldnotbeovertime(>1h)topunchwells,because indicatorcellsgraduallygrow.Ifyoufeeldifficulttopunch wellsonlyusingasterilecork-borer,Irecommendthatresidual agargelinawellisremovedusinganeedle-liketiptweezers whichisofcourseflame-sterilizedandcooleddownbeforeuse aswellasthecork-borer.

13.Sizeofclearzonesformedarevariousamongindicatorsevenif thesamesampleisinjected.Inaddition,evenifthesame indicatorisused,thesizeisvariedamonggrowthmediaand (pre)cultivationconditionsoftheindicator.Thereforewhen youevaluateantibacterialactivityofabateriocin/BLIS,you mustuseabacterialmediumandfixedcultureconditionsforan indicator.

14.Sizeofclearzonesformeddonotcorrespondtotheantibacterialtiter,becausebacteriocins/BLISshavedifferentdiffusionabilityandpermeabilityintoagargel.Todetermine antibacterialtiter,itisnecessarytoemploythesampleCFS dilutionmethod.

Another random document with no related content on Scribd:

Fam. P.

Plethodon cinereus cinereus (Green). Sugar Grove.

Plethodon cinereus erythronotus (Green). Sugar Grove, Columbus, and Worthington. In the early part of the year this is the commonest salamander in the ravines in Franklin County. It is found generally away from water, under loose debris two or more rods from the stream.

Plethodon glutinosus (Green). Sugar Grove. This salamander is found in such localities as were mentioned for P. c. erythronotus.

Gyrinophilus porphyriticus (Green). Sugar Grove.

Spelerpes bilineatus (Green). Sugar Grove. Habits apparently aquatic.

Spelerpes longicauda (Green). Sugar Grove. This salamander is abundant in this region where it may be found in May under stones at the edge of the water together with its eggs; the eggs are attached to the under side of a hollow stone. Some individuals were found in May, 1900, away from water.

Spelerpes ruber (Daudin). Fairfield County.

Desmognathus fusca (Rafin). Sugar Grove and Perry Co. Aquatic in habits.

Fam. P.

Diemictylus viridescens miniatus (Rafin). Sugar Grove.

Fam. B.

Bufo lentiginosus (Shaw). Columbus and Knox County. This is the common toad of Central Ohio.

Bufo lentiginosus americanus LeConte. A specimen from the sand dunes of Cedar Point, Sandusky, Ohio.

Fam. H.

Acris gryllus crepitans Baird. Knox County, Central College and Columbus. The common cricket-frog of Central Ohio is this subspecies. The young resemble the species gryllus LeConte in having the under surface of the thigh reticulated and blotched.

Chorophilus triseriatus (Wied.). Sugar Grove.

Hyla versicolor LeConte. Knox County and Columbus.

Hyla pickeringii Storer. Sugar Grove.

Fam. R.

Rana virescens Kalm. Sugar Grove and Columbus.

Rana palustris LeConte. Sugar Grove.

Rana sylvatica LeConte. Knox County and Sugar Grove.

Rana clamata Daudin. Columbus.

Rana catesbiana Shaw. Columbus.

S B.—Families 8, Genera 12, Species 25.

THE PROMETHEA MOTH, CALLOSAMIA PROMETHEA.

H O.

This beautiful moth is one of the rather common species belonging to the group of silkmaking Lepidoptera. The moths appear in May or June. The female is light rusty brown and drab with a darker area across the middle of the wings, while the males are much darker, nearly black, and differ further from the females in the shape of the wings and markings as shown in the figures.

F. 1. Callosamia promethea, female. (H. O. ad. nat. 1880.)

The eggs are laid in early summer almost immediately after pairing, and hatch in course of a few days, the larvae growing through the summer. The cocoons are hung to twigs of trees by a silken cord, and quite often a leaf is utilized as the outer covering within which the elongate oval cocoon is built. In any case the cocoon bears resemblance to a withered curled leaf hanging by its petiole. In this manner cocoons hang upon the trees through the winter.

They are found most commonly on wild cherry, this being apparently the favorite food plant of the larva. They feed however on a large number of common trees and shrubs.

The figures of the moth, male and female, were drawn twenty one years ago, and having now come of age they may perhaps be trusted to make their first public appearance.

F. 2. Callosamia promethea, male. (H. O. ad. nat. 1880.)

MEETING OF THE BIOLOGICAL CLUB.

The Biological Club met in Zoological lecture room on the evening of April 1, 1901. Professor Osborn presided.

Professor Schaffner reviewed a paper entitled Zur Kenntniss der Zelltheilung bei Myriopoden, published in Archiv fur Mikroskopische Anatomie.

Dr. Morrey spoke on the subject, “Two years in Europe as a Student.” Most of the time was spent at the University of Vienna, although the University at Zurich and the Pasteur Institute at Paris were each attended for a short term.

The University of Vienna ranks among the first in the advantages offered to medical students. The hospitals of the city are noteworthy on account of the large number of cases and the great variety of diseases treated. The numerous holidays observed in Vienna seriously interrupt college work. Hardly a week passes in which there is not one or more holidays on which work is wholly suspended.

The speaker placed on the exhibition table a fine series of photographs procured during his stay abroad. These furnished a treat for those present after the regular program was completed.

J. S. H, Secretary.

NEWS AND NOTES.

The Summer Field Meeting of the Ohio State Academy of Science will be held at Wooster, Ohio, on Friday and Saturday, May 31 and June 1, 1901, under the auspices of the University of Wooster, the Ohio Experiment Station and the Wooster Field Naturalist’s Club. The plan includes Friday about the small lakes southwest of Wooster, and an evening meeting in Wooster; Saturday morning at the Experiment Station, to be followed by an excursion to North Lawrence with its mines and Fox Lake with its tamarack bog.

Prof. Charles S. Prosser in an article in the Am. Jour. of Sci. 11:191–199, 1901, discusses the names applied to the formations of the Ohio Coal measures. The following names are proposed.

Present Names.

Proposed Names.

Upper Barren Coal Measures Dunkard formation

Upper Productive Coal Measures Monongahela formation

Lower Barren Coal Measures Conemaugh formation

Lower Productive Coal Measures Allegheny formation

The Philadelphia Fleabane (Erigeron philadelphicus L.) is one of our interesting spring plants and will repay careful study. The leaves of the stem in most individuals have a decided polarity and for the most part are twisted so as to stand in a single plane. In this respect the plant is as striking as any of the so-called compass plants, although the plane in which the leaves lie may be in any direction. Another interesting adaptation is the drooping of the top of the young plant. The entire inflorescence nods at first and finally the individual heads, but one by one these assume the upright position as the flowers begin to open.

W A O.—The Ohio species of cactus, Opuntia humifusa Raf., has an interesting habit which seems to be a protective measure against cold. At the approach of Winter the flattened stems lose their upright position and press themselves closely to the surface of the ground.

The stems lose considerable of their moisture at the same time, becoming wrinkled but not at all flaccid. By the end of April they are again upright and distended.

Ohio State University

Six distinct and independent Colleges, each with a Dean and Faculty of its own.

THIRTY SEVEN DEPARTMENTS. THIRTY DISTINCT COURSES.

A, A, L, E, P, V M.

Superior facilities for education in Applied Science. Short or special courses for mature students not candidates for degrees.

One hundred and twenty one instructors. Over thirteen hundred students.

FINEST GYMNASIUM IN THE WEST.

For further information address the President,

D. W. O. T, S U, C.

Ohio Forest Trees Identified by Leaves and Fruit.

A neat pamphlet for every one who wishes to learn our native forest trees. Keys simple. Description plain. Can learn the names of the trees easily.

Price reduced from 25 cents to 10 cents.

Also, The Fourth State Catalogue of Ohio Plants.

Bound copies at cost of binding, namely 20 cents.

Gives list of scientific and common names; distribution by counties.

Teachers and others will also be interested in Prof. Kellerman’s Phyto-theca or Herbarium Portfolio, Practical Studies in Elementary Botany, Elementary Botany with Spring Flora, all published by Eldredge & Bro., Philadelphia, to whom apply.

For information or copies of Forest Trees and Catalogue or names of plant specimens of your region address

W. A. K, C, O

American Entomological Co.

1040 DE KALB AVENUE, BROOKLYN, N. Y.

Lepidoptera Price List No. 2.—Price 5 cents Refunded to Buyers I N 15th 1900.

Dealers of all kinds of ... E S

Manufacturers of the Original and Celebrated ... SCHMITT INSECT BOXES

Builders of INSECT CABINETS, ETC.

The Twentieth Century Text Books of Biology.

All by JOHN MERLE COULTER, A. M., Ph. D., Head of Dept. of Botany, University of Chicago.

They are already the preferred texts, and the reasons will be apparent on examination.

ANIMAL LIFE: A First Book of Zoology.

By DAVID S. JORDAN, M. S., M. D., Ph. D., LL. D., President of the Leland Stanford Junior University, and VERNON L. KELLOGG, M. S., Professor in Leland Stanford Junior University. 12mo. Cloth, $1.20. Now ready.

Not a book for learning the classification, anatomy, and nomenclature of animals, but to show how animals reached their present development, the effects of environment, their place in Nature, their relations to one another and to the human race. Designed for one-half year’s work in high schools. Send for sample pages.

ANIMAL FORMS: A Second Book of Zoology.

By DAVID S. JORDAN. M. S., M. D., Ph. D., LL. D., and HAROLD HEATH, Ph. D., Professor in Leland Stanford Junior University. Ready in February, 1901.

D. APPLETON AND COMPANY, Publishers, New York, Chicago, London.

R S W

In Astronomy, Dr. Simon Newcomb’s new book, published October, 1900; in Physics, the Johns Hopkins text of Professors Rowland and Ames; also in Physics for second and third year high school work, the text of Dr. Hoadley, of Swarthmore; in Physiology, the text by Drs. Macy and Norris, based on the Nervous System; also the High School Physiology indorsed by the W. C. T. U., written by Drs. Hewes, of Harvard University; in Geology, the Revised “Compend” of Dr. Le Conte, and the two standard works of Dana,—The Manual for University Work, and the New Text Book, revision and rewriting of Dr. Rice, for fourth year high school work; in Chemistry, the approved Storer and Lindsay, recommended for secondary schools by the leading colleges; in Zoology, the Laboratory Manual of

Dr. Needham, of Cornell; and the series “Scientific Memoirs” edited by Dr. Ames, of Johns Hopkins. Nine volumes ready.

The publishers cordially invite correspondence.

AMERICAN BOOK COMPANY, C

TRANSCRIBER’S NOTES

1. Silently corrected obvious typographical errors and variations in spelling.

2. Retained archaic, non-standard, and uncertain spellings as printed.

*** END OF THE PROJECT GUTENBERG EBOOK THE OHIO NATURALIST, VOL. I, NO. 7, MAY, 1901 ***

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