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NewBacillussubtilisStrainsasPromisingProbioticsFRONTIERSINIMMUNOLOGYNewBacillussubtilisStrains1CONTENTS01020304INTRODUCTIONMATERIALSANDMETHODSRESULTSANDDISCUSSIONCONCLUSIONCONTENTS01020304INTRODUCTIONMA2INTRODUCTIONINTRODUCTION3Probiotics:livemicroorganismspositiveeffectondiversefunctionsofanorganismpreventtheinvasionofvariouspathogensExperimentswithmiceprovedthatB.subtilissporeswereabletogerminate,andthecellscouldproliferateandformthesporesagaininintestinesofanimalsAutochthonouslacticacidbacteriaofthegeneraLactobacillusandBifidobacteriumareusuallyusedasprobiotics;grampositivespore-formingbacteriaofthegenusBacillusareusedasprobioticsaswell

StudieshaverevealedthatinadditiontotheresistanceoftheprobioticBacillusstrainstobileacids,theywerecapableofimmunestimulationincaseofgastrointestinaldisorders.Abilitytosynthesizeantibiotics,bacteriocins,cycliclipopeptides,andlyticenzymeswithantimicrobialactivityprovidesprobioticactivityforbacteriaofthegenusBacillus.Beingprobiotics,thestrainsofB.subtilisandB.coagulanswereshowntohaveagrowth-stimulatingandprophylacticeffectonbroilerchickens1INTRODUCTIONProbiotics:Experimentswithmi4標(biāo)題Thegoalofthepresentworkwastocharacterizethepropertiesofthesenewstrainsandtoassesstheirpotentialuseasprobiotics.01INTRODUCTION標(biāo)題01INTRODUCTION5MATERIALSANDMETHODSMATERIALSANDMETHODS6ThesubjectsofthestudywereB.subtilisstrainsGM2andGM5withhighantimicrobialactivity(Mardanovaetal.,2017).Opportunisticcoliformbacteria,SalmonellaentericaserotypeTyphimuriumATCC14028s,Klebsiellaoxytoca,andEscherichiacoli,aswellasmicromycetesFusariumavenaceum,F.oxysporum,F.redolens,andF.solani,wereusedastestcultures.

02MATERIALSANDMETHODSThesubjectsofthestudywere7Nutrientmediaandcultivationconditions.Thefollowingmediawereusedforcultivation.Thesewere:

(1)LB(Luria–Bertoni)medium(g/L):tryptone,10.0;yeastextract,5.0;NaCl,5.0;(2)LAmedium(g/L):tryptone,10.0;yeastextract,5.0;NaCl,5.0;

agar,20.0;(3)mediumno.1(g/L):maltose,20.0;peptone,10.0;CaCl2,1.0;(4)mediumno.2(g/L):soybeanmeal,30.0;NaNO3,3.0;K2HPO4,1.0;MgSO4,0.2;KCl,0.2;(5)mediumno.3(g/L):cornextract,20.0;lactose,10.0;(6)deoxycholatecitrateagarforsalmonellacultivation.Bacteriawerecultivatedinathermostatat37°CandusinganIKA?KS4000incubatorshaker(Germany)at37°Candrotationspeedof200rpm.OpticaldensityoftheculturewasmeasuredusingaBio-Radspectrophotometer(UnitedStates)atawavelengthof590nm.

02MATERIALSANDMETHODSNutrientmediaandcultivation8ThedynamicsofbacterialgrowthandsporeformationwerestudiedontheLBmediumandthemediumno.1.Bacteriawerecultivatedfor48husingtheincubatorshaker(37°C,200rpm).Bacterialculturesampleswerecollectedevery2hinordertomeasureopticaldensity(atthewavelengthof590nm)usingaspectrophotometerandtodeterminetheextracellularproteolyticactivity.ThenumbersofsporesformedwerecountedinaGoryaevchamber(OpticalMarket,Ukraine),startingafter10hofcultivationofbacteria.ThebacteriawereexaminedunderaMICROSAUSTRIAMC300microscope(Austria).

02MATERIALSANDMETHODSAbilitytogrowatvariousvaluesofambientpHwasstudiedin250-mLErlenmeyerflaskscontaining50mLoftheLBmedium(pH2–10).Bacteriawerecultivatedusingtheincubatorshaker(37°C,200rpm,aeration).Opticaldensityofthecultureswasdeterminedafter24hofgrowth.Thedynamicsofbacterialgrow9ResistanceofthesporestopHandsporecapacityforinvitrogerminationwerestudiedaccordingtothemethoddescribed(Halleretal.,2001).

Sporesofbacilliwereobtainedbyheatinga2-day-oldbacterialculturefor90minat60°Ctoeliminatethevegetativecells.Theinitialconcentrationofthesporeswas4×107CFU/mL.ThesporesuspensioninLBmediumwasincubatedatvariouspHvaluesat40°C.ThesporeswereincubatedatpH5.0for60minandconcentratedbycentrifugation(6000rpm,10min).Thesupernatantwasremoved;thesporepelletwastransferredintothesamevolumeofthefreshLBmedium(pH3.0)andincubatedfor90min.Analiquotofthesuspension(0.1mL)wastakenandheatedfor90minat60°C.AseriesofdilutionswaspreparedandplatedtoformalawnontheLAmediumforthesubsequentenumerationofCFU/mL.Theremainingsporesuspensionwasconcentratedbycentrifugation,transferredtoLBmedium(pH7.0),andincubatedfor150min.Analiquot(0.1mL)washeatedfor90minat60°CandusedtodetermineCFU/mL.Toenumeratethevegetativecellsaftersporegerminationfromtheunheatedsuspension,aseriesofdilutionswaspreparedandplatedontotheLAmedium

02MATERIALSANDMETHODSResistanceofthesporestopH10ResistanceofbacterialsporestobilewasinvestigatedinvitrousingthemethoddescribedbyCencietal.(2006).Bileofbroilerchickenswasused,whichwassterilizedbyfiltrationthroughasterileMilliporefilter(0.22μm).ThesporeswereintroducedintotheLBmediumafteradditionofbile(1and10%)andadjustingpHto7.5.Thesuspensionofsporeswasincubatedfor6hat40°CandusedtodetermineCFU/mL.

02MATERIALSANDMETHODSResistanceofbacterialspores11Proteolyticactivityinthebacterialliquidculturewasdeterminedbydigestionofazocasein(Sigma,UnitedStates)(Demidyuketal.,2006).Inhibitory

analysiswasperformedinthepresenceof1,10-phenanthroline(Serva,Germany),aspecificinhibitorofmetalloproteinases,andPMSF(Serva,Germany),aspecificinhibitorofserineproteinases.Theinhibitors(1.5mM)wereaddedtotheliquidcultureofbacilli,incubatedfor1hatroomtemperature,andresidualactivitywasdeterminedbyhydrolysisofazocasein.Theactivitywasexpressedasapercentagerelativetothecontrol(asimilaractivitywithoutinhibitors).

02MATERIALSANDMETHODSPhytate-hydrolyzingactivitywasdeterminedusingaselectivenutrientmediumforphytate-degradingbacteria,phytasescreeningmedium(PSM),accordingtoMittal’stechnique(Mittaletal.,2011).Abilitytohydrolyzesodiumphytatewasassessedbythepresenceofzonesofhydrolysisaroundbacterialcolonies.

Proteolyticactivityintheba12Determinationofvirulence,toxicity,andtoxigenicityofbacteriawascarriedoutinmiceofbothsexesoftheICR(CD-1)lineattheLaboratoryofChemicalandBiologicalResearchoftheArbuzovInstituteofOrganicandPhysicalChemistry,KazanScientificCenter,RussianAcademyofSciences.Micewerehousedunderthestandardvivariumconditions;thestandardgranulatedcombinedfeedwasused.Foreachexperimentalgroup,sixmiceofthesameageweighing16±0.5gwereselected.

02MATERIALSANDMETHODSAntagonisticactivityofB.subtilisstrainsGM2andGM5againsttestcultureswasdeterminedbythemethodsofwells,double-layeragar,andblocks(Egorov,2004).Thelevelofantagonisticactivityofbacteriaandexometaboliteswasjudgedbythediameterofzonesofinhibitionofthetestculturegrowtharoundthewellscontainingtheliquidcultureorthecoloniesofantagonisticbacteria.

Determinationofvirulence,to13RESULTSANDDISCUSSIONDynamicsofbacterialgrowth,proteinasebiosynthesis,andsporulation.ResistancetotheGITacidsandbile

AbilityofsporestogerminateatdifferentpHvaluesinvitro

Proteolyticactivityofthestrainswasdeterminedby

hydrolysisofazocaseinCapacityforphytateuse

Antagonisticactivity

RESULTSANDDISCUSSIONDynamics1403RESULTSANDDISCUSSION03RESULTSANDDISCUSSION1503課題研究過程展示03課題研究過程展示1603RESULTSANDDISCUSSION03RESULTSANDDISCUSSION1703RESULTSANDDISCUSSION03RESULTSANDDISCUSSION1803RESULTSANDDISCUSSION03RESULTSANDDISCUSSION1903RESULTSANDDISCUSSION03RESULTSANDDISCUSSION20枯草芽孢桿菌作為新的益生菌演講稿課件2103RESULTSANDDISCUSSION03RESULTSANDDISCUSSION22CONCLUSIONCONCLUSION23AntibacterialactivityofthelipopeptidefractionofB.subtilisGM2andGM5wasstudied.Thelipopeptidefractionwasisolatedfromtheliquidcultureofbacteriagrownafter72honthemediumcontainingmaltose(mediumno.1).Inhibitoryactivityoflipopeptideswasdeterminedbythediskdiffusionmethodagainstdifferentspeciesofenterobacteria,Salmonellatyphimurium,E.coli,andK.oxytoca(Table5).LipopeptidesofB.subtilisGM2andGM5showedantagonisticactivityagainstalltestculturesstudied.However,theinhibitoryeffectwashigherincaseofthestrainB.subtilisGM5,indicatingitshigherantibacterialandantifungalactivity.Thus,B.subtilisstrainsGM2andGM5isolatedfromtherhizospherearecharacterizedbyantagonisticpropertiesagainstphytopathogenicmicromycetesandpathogenicandopportunisticenterobacteria,whichmaybeduetotheeffectoflipopeptides.Thesemetabolitesofbacillicansuppresspathogenicandopportunisticmicroflora.SporesofthebacteriastudiedwereresistanttobileandawiderangeoftheambientpH.Vegetativecellsofthebacilliwereabletosynthesizeproteolyticandphytate-hydrolyzingenzymes,whichisimportantforimprovingdigestivefunctionsbyaddingthestrainstofeeds.TheresultsobtainedsuggestthatB.subtilisGM2andGM5haveprospectiveuseasprobiotics.04CONCLUSIONAntibacterialactivityofthe24感謝聆聽感謝聆聽25NewBacillussubtilisStrainsasPromisingProbioticsFRONTIERSINIMMUNOLOGYNewBacillussubtilisStrains26CONTENTS01020304INTRODUCTIONMATERIALSANDMETHODSRESULTSANDDISCUSSIONCONCLUSIONCONTENTS01020304INTRODUCTIONMA27INTRODUCTIONINTRODUCTION28Probiotics:livemicroorganismspositiveeffectondiversefunctionsofanorganismpreventtheinvasionofvariouspathogensExperimentswithmiceprovedthatB.subtilissporeswereabletogerminate,andthecellscouldproliferateandformthesporesagaininintestinesofanimalsAutochthonouslacticacidbacteriaofthegeneraLactobacillusandBifidobacteriumareusuallyusedasprobiotics;grampositivespore-formingbacteriaofthegenusBacillusareusedasprobioticsaswell

StudieshaverevealedthatinadditiontotheresistanceoftheprobioticBacillusstrainstobileacids,theywerecapableofimmunestimulationincaseofgastrointestinaldisorders.Abilitytosynthesizeantibiotics,bacteriocins,cycliclipopeptides,andlyticenzymeswithantimicrobialactivityprovidesprobioticactivityforbacteriaofthegenusBacillus.Beingprobiotics,thestrainsofB.subtilisandB.coagulanswereshowntohaveagrowth-stimulatingandprophylacticeffectonbroilerchickens1INTRODUCTIONProbiotics:Experimentswithmi29標(biāo)題Thegoalofthepresentworkwastocharacterizethepropertiesofthesenewstrainsandtoassesstheirpotentialuseasprobiotics.01INTRODUCTION標(biāo)題01INTRODUCTION30MATERIALSANDMETHODSMATERIALSANDMETHODS31ThesubjectsofthestudywereB.subtilisstrainsGM2andGM5withhighantimicrobialactivity(Mardanovaetal.,2017).Opportunisticcoliformbacteria,SalmonellaentericaserotypeTyphimuriumATCC14028s,Klebsiellaoxytoca,andEscherichiacoli,aswellasmicromycetesFusariumavenaceum,F.oxysporum,F.redolens,andF.solani,wereusedastestcultures.

02MATERIALSANDMETHODSThesubjectsofthestudywere32Nutrientmediaandcultivationconditions.Thefollowingmediawereusedforcultivation.Thesewere:

(1)LB(Luria–Bertoni)medium(g/L):tryptone,10.0;yeastextract,5.0;NaCl,5.0;(2)LAmedium(g/L):tryptone,10.0;yeastextract,5.0;NaCl,5.0;

agar,20.0;(3)mediumno.1(g/L):maltose,20.0;peptone,10.0;CaCl2,1.0;(4)mediumno.2(g/L):soybeanmeal,30.0;NaNO3,3.0;K2HPO4,1.0;MgSO4,0.2;KCl,0.2;(5)mediumno.3(g/L):cornextract,20.0;lactose,10.0;(6)deoxycholatecitrateagarforsalmonellacultivation.Bacteriawerecultivatedinathermostatat37°CandusinganIKA?KS4000incubatorshaker(Germany)at37°Candrotationspeedof200rpm.OpticaldensityoftheculturewasmeasuredusingaBio-Radspectrophotometer(UnitedStates)atawavelengthof590nm.

02MATERIALSANDMETHODSNutrientmediaandcultivation33ThedynamicsofbacterialgrowthandsporeformationwerestudiedontheLBmediumandthemediumno.1.Bacteriawerecultivatedfor48husingtheincubatorshaker(37°C,200rpm).Bacterialculturesampleswerecollectedevery2hinordertomeasureopticaldensity(atthewavelengthof590nm)usingaspectrophotometerandtodeterminetheextracellularproteolyticactivity.ThenumbersofsporesformedwerecountedinaGoryaevchamber(OpticalMarket,Ukraine),startingafter10hofcultivationofbacteria.ThebacteriawereexaminedunderaMICROSAUSTRIAMC300microscope(Austria).

02MATERIALSANDMETHODSAbilitytogrowatvariousvaluesofambientpHwasstudiedin250-mLErlenmeyerflaskscontaining50mLoftheLBmedium(pH2–10).Bacteriawerecultivatedusingtheincubatorshaker(37°C,200rpm,aeration).Opticaldensityofthecultureswasdeterminedafter24hofgrowth.Thedynamicsofbacterialgrow34ResistanceofthesporestopHandsporecapacityforinvitrogerminationwerestudiedaccordingtothemethoddescribed(Halleretal.,2001).

Sporesofbacilliwereobtainedbyheatinga2-day-oldbacterialculturefor90minat60°Ctoeliminatethevegetativecells.Theinitialconcentrationofthesporeswas4×107CFU/mL.ThesporesuspensioninLBmediumwasincubatedatvariouspHvaluesat40°C.ThesporeswereincubatedatpH5.0for60minandconcentratedbycentrifugation(6000rpm,10min).Thesupernatantwasremoved;thesporepelletwastransferredintothesamevolumeofthefreshLBmedium(pH3.0)andincubatedfor90min.Analiquotofthesuspension(0.1mL)wastakenandheatedfor90minat60°C.AseriesofdilutionswaspreparedandplatedtoformalawnontheLAmediumforthesubsequentenumerationofCFU/mL.Theremainingsporesuspensionwasconcentratedbycentrifugation,transferredtoLBmedium(pH7.0),andincubatedfor150min.Analiquot(0.1mL)washeatedfor90minat60°CandusedtodetermineCFU/mL.Toenumeratethevegetativecellsaftersporegerminationfromtheunheatedsuspension,aseriesofdilutionswaspreparedandplatedontotheLAmedium

02MATERIALSANDMETHODSResistanceofthesporestopH35ResistanceofbacterialsporestobilewasinvestigatedinvitrousingthemethoddescribedbyCencietal.(2006).Bileofbroilerchickenswasused,whichwassterilizedbyfiltrationthroughasterileMilliporefilter(0.22μm).ThesporeswereintroducedintotheLBmediumafteradditionofbile(1and10%)andadjustingpHto7.5.Thesuspensionofsporeswasincubatedfor6hat40°CandusedtodetermineCFU/mL.

02MATERIALSANDMETHODSResistanceofbacterialspores36Proteolyticactivityinthebacterialliquidculturewasdeterminedbydigestionofazocasein(Sigma,UnitedStates)(Demidyuketal.,2006).Inhibitory

analysiswasperformedinthepresenceof1,10-phenanthroline(Serva,Germany),aspecificinhibitorofmetalloproteinases,andPMSF(Serva,Germany),aspecificinhibitorofserineproteinases.Theinhibitors(1.5mM)wereaddedtotheliquidcultureofbacilli,incubatedfor1hatroomtemperature,andresidualactivitywasdeterminedbyhydrolysisofazocasein.Theactivitywasexpressedasapercentagerelativetothecontrol(asimilaractivitywithoutinhibitors).

02MATERIALSANDMETHODSPhytate-hydrolyzingactivitywasdeterminedusingaselectivenutrientmediumforphytate-degradingbacteria,phytasescreeningmedium(PSM),accordingtoMittal’stechnique(Mittaletal.,2011).Abilitytohydrolyzesodiumphytatewasassessedbythepresenceofzonesofhydrolysisaroundbacterialcolonies.

Proteolyticactivityintheba37Determinationofvirulence,toxicity,andtoxigenicityofbacteriawascarriedoutinmiceofbothsexesoftheICR(CD-1)lineattheLaboratoryofChemicalandBiologicalResearchoftheArbuzovInstituteofOrganicandPhysicalChemistry,KazanScientificCenter,RussianAcademyofSciences.Micewerehousedunderthestandardvivariumconditions;thestandardgranulatedcombinedfeedwasused.Foreachexperimentalgroup,sixmiceofthesameageweighing16±0.5gwereselected.

02MATERIALSANDMETHODSAntagonisticactivityofB.subtilisstrainsGM2andGM5againsttestcultureswasdeterminedbythemethodsofwells,double-layeragar,andblocks(Egorov,2004).Thelevelofantagonisticactivityofbacteriaandexometaboliteswasjudgedbythediameterofzonesofinhibitionofthetestculturegrowtharoundthewellscontainingtheliquidcultureorthecoloniesofantagonisticbacteria.

Determinationofvirulence,to38RESULTSANDDISCUSSIONDynamicsofbacterialgrowth,proteinasebiosynthesis,andsporulation.ResistancetotheGITacidsandbile

AbilityofsporestogerminateatdifferentpHvaluesinvitro

Proteolyticactivityofthestrainswasdeterminedby

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