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The Influence of Shock Treatment on Survival Rate of Intestinal Probiotics during Freezing

By Virginia Rose,2014-08-05 22:45
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The Influence of Shock Treatment on Survival Rate of Intestinal Probiotics during Freezingon,of,OF,Shock,Rate,The,the,THE,rate,shock

    The Influence of Shock Treatment on

    Survival Rate of Intestinal Probiotics

    during Freezing

    June2oo6JournalofNortheastAgnculturalUniversityV01.13.No.1364O

    ArticleID:10068104(2006,01003605

    eInfluenceofShockTreatmentonSurvivalRateof

    IntestinalProbioticsduringFreezing

    LIChun,ZHANGLanwei.LIULibo,FENGZhen

    (1.CollegeofFood,NortheastAgriculturalUniversity,HarbinHeilongjiang150030,PRC; 2.FoodScienceandHeredityEngineeringAcademy,HarbinInstituteofTechnology,HarbinHeilongjiang150080.PRC)

    Abstract:Probioticswhichcanprovidepotentialhealthbenefitsforconsumersandpreventdisease.

    isthemost

    importantresearchfieldforfunotionalfoodinthefurure.Itisthekeypointthathowmuchthecountofbacteriaand

    howlongitcanpreserve.Freeze

    dryingskillisoneofthebestpreservationmethods,butit'sdefectthatdamageto biologicalsystemscanbeattributedtochangesinthephysicalstateofmembranelipidsor/andchangesinthe

    structureofsensitiveproteinsandthedecreasedsurvivaIrate.Thisworkusedpretreatmentmethods-coldshockand

    hotshocktoreducethedamagetobacteriaduringfreeze

    drying.Usingunpretreatedsamplesascontro1.aspectsof

    coldshockorheatshock.TheresponseofEnterococcousfaeca/isA31wasinvestigatedduringlagphase,themiddle

ofexponentialgrowthphaseandtheterminalofexponentialgrowthphase.Theresultssugges

    tedthatwhencoldshock.

    theshockproteinconcentrationproducedbytreatedsamplewithin2hduringthemiddleofexp

    onentialgrowthphase

    washig'1erthan4,8,24h,Theconcentrationofshockproteinproducedbytreatedsampleat10

    ?washigherthan20?,

    Theconcentrationofsampleproteinatthemiddleofexponentialgrowthphasewashigherthanthatattheterminalof

    exponentialgrowthphase.Afteronemonthstorage.thesurvivalrateat10?

    wasbetterthan20?and4?

    comparatively.Thesurvivalrateat(6h)4?/24h(1agperiod)washigherthan(10h)4?

    /24h(themiddleof

    exponentialgrowthphase).andtheeffectof10?

    /8hshocktreatmentswasbestduringthemiddleofexponential growthphase,whenhotshock,Theconcentrationofheatshockproteinsproducedwasnotobvious.andsurvivaIrate

    washigherat45?.30minthanat45?.60min.

    Keywords:Enterococcousfaeca//sA31:freezedrying;shockproteins;survivalrate CLCnumber:Q939.1Documentcode:A

    Freeze——dryingisoneoftheimportantbiological

    technologies.Ithasbeenwidelyusedinfoodprocess

    ingasprobioticsstarterinrecentyearswiththerapid

    developmentoffunctionalfoods.Howeveritwilldam

    agethecellmembraneanddecreasethesurvivalrate

    ofbacteria.Itleadstothedamageoffluidmosaic

    model,membraneintegralityandthemembranekey

    enzymewhichwereresultedbyfreezedrying'.The

    freezedryingsignificancewasdiminishedwhenthe

    damageresultedinthedeclineofactivitycellandthe

    changeofbasiccharacteristicsofcel1.

    Inresponsetoasuddendecreaseintemperature, microorganismsareknowntoundergosomephysiolog- icalandbiochemicalchanges.Theseprocessesinclude thesynthesisofagroupofproteins,thecoldshockpr-

    oteins,theacquisitionofincreasedcryotoleranceto subsequentexposuretocoldtemperature.Jeroenet alfoundthattheconcentrationofcold-shockproteins producedbyStreptococcusthermophilusduringstor——

    ageat20?/4hwereapproximatelytwiceortriplicity asmuchasthoseobservedat10?,4h.andtheac

    tivityweresignificanthigherthanthelatterBroa

    dbentetalfoundthatcoldshockproteinspresented bya2hcoldshockat10oCinL.1actisssp.Lactis cellandtheconcentrationofunsaturatedfattyacids increased,lipidmehingpointfailingledtoalowerra- Receiveddate:20o5O6l0

    Foundationitem:SupportedbyNationalAculturalTranslationFund(02EFN2l230l073)

    Biography:LIChun(1977-),male,Doctor,assistantprofessor,engagedintheresearchofdair

    yprocessinganddairymicroorganism.

    Correspondenceauthor

    No.1JournalofNortheastAgiculturalUniversity?37? tioofsaturatedtounsaturatedfattyacidcontentand theincreasecryotolerance.KimetaltOlalsofoundthat samplesfromacoldshock(exposedto10?for5h)

    cuhureandnoncoldshockculture,thesurvivalrate

    oftheformerwashigher.Butthefunctionofcold

    shockproteindiminishedduringlong——termstorage.

    Prasadetalt"1reportedthatLactobacillusrham

nosll~HNO01showedsignificant(P<O.05)improve

    mentinviabilitycomparedwiththenonstressedcon——

    trolcultureafterstorageat30?inthedriedform.

    Theactivityoflactococcicouldbesignificantly(P< O.051improvedbya25minheatedshockat42?af-

    terlyophilization.Furthermore,heatedshockproteins (Hsps)inheatshockedcellscanbeendetectedafter electrophoresis,andcyclopropanefattyacidcontent 19:0werehigherthancontrolcells.Thereforethepro

    cessofheatingshockisoneofthemethodstoen——

    hancethesurvivalrateofbacteriaafterfreezedrying.

    Freezinganddryingaretwoprocessesoffreeze——

    dryingtechnology,inwhichfreezingisthemostserious damagetobacteriacel1.Thisobjectmainlydescribed howtoreducethedamagetobacteriaduringfreezeing. InthistestEnterococcousfaecalisA31wasinvestigat——

    edindifferentcoldshockorheatedshocktemperature andtime.Optimizepretreatmentconditionsbeforefree

    ze-dryingwerestudiedinordertoincreaseactivityof probioticspowderandcommercialstarter.

    1MaterialsandMethods

    1.1Bacterialstrain

    ThemicroorganismusedinthisstudywereEnte——

    rococcousfaecalisA31fromourlaboratory).Thebact- eriawastransferredtomodifiedMRSmedium(300 mg'Lcysteinesaltandl%pancreaspeptonewere added),pH7.0.EnterococcousfaecalisA31wasincu

    batedat39?.after6hincubatingtheperiodofbac- teriaattmnedlagphase,themiddleofexponential growthat10hphaseandtheterminalofexponential

growthphaseat16h.

    1.2Preparationofgelelectrophorus

    TheEnterococcousfaecalisA31was

    samplesJ

    incubatedin

    100mLmodifiedMRS.Atthelagphase,themiddle ofexponentialgrowthphase,samplesandtherestcon

    tinuetocultureuntilretainingtheterminalofexponen——

    tialgrowthphase.Sampleswerethenquicklychilledin icewaterandcellswereharvestedbycentrifugation. Thecellswerewashedby3mL20mmo1.LNa2HPO4 anddilutedto1.5mL.Thenusingultrasonictocrash thecells(15kHz,23min),thesupernatantwastrans

    ferredtomicrofugetubes,whichwerecentrifugedat l2000r?minfor15minat4?.rhesupernatant

    wascombinedwithTrisHClbufier(1:1,pH8.O),and

    thenheatedfor4minintheboilingwater,andstored at2O?untilanalysed.

    1.3Electrophoresismethodandcondition SDS——PAGEelectrophoresiswasusedtodetect thevarietyofproteins.20

    by15%seperatinggeland

    ixLofsampleweretested

    4%stackingge1.thepHof

    bufferwas8.3?O.1.thegelwasstainedwithCoomassie brilliantblue(G-250)andfadedby10%aceticacid. Iestablevoltageoftheelectrophoresiswas100-150V. Heatupthesampleintheboilingwaterbathfor4min beforetested,andthesampleswhichdonottreatedby shocktreatmentascontro1.

1.4Shocktreatmentplan

    EnterococcousfaecalisA31culturedinmodified MRSbrothwasreceivedthefollowingtreatmentsfor onebottleafter6h,(6h)4?/24h(itindicatesthe

    sampleisdepositedfor24hat4?after6h1.

    Thefollowingexpresseswerethesameasthat,it receivedthefollowingtreatmentsfor10bottlesafter 10h,(10h)45%/30min;(10h)45?/6Omin;(10h)

    10~C/2h;(10h)10%/4h;(10h)10h;(10h)10?/

    24h;(1Oh)20%/2h;(10h)20%/4h;(10h)20%/8h; (1Oh)4?/24andtakingoutthecontrolsampleafter 16h.

    1.5Detectionmethods

    Conservingtestswerecarriedouttoshockand notshocktreatmentsstoredat20?.anddetected

    theaccountoflivebacteriaatintervals,theresultsof proteinquantitativemeasurementswereobtainedby GelImagingsoftware.Detectedtheratiooftheshock proteinandtotalproteinsandanalysedthevarietyof

?

    38?JournalofNortheastAgnculturalUniversityV0l_l3 originalproteinsconcentration.

    2ResuitsandDiscussion

    BacteriawereculturedinmodifiedMRS,andit shouldensurethatthebacteriacountswere0.51.5xl09

    CFU?mLineverytreatmentmethod.thisindicatethat thedifferencesofcellcountsarenotsignificantbetween controlsamplesandtreatmentsamples,andthebacteria survivalrateislessthan.Theresultssuggestthatthe

    methodofextractingbacterialproteiniseffective. 2.1Effectofcoldshockoncryotolerance

    Fig.1showedtheproteinproducedbyEntero

    0ll223344556677

    coccolzsfaecalisA31atdifferenttreatmentconditions. Inthefigure,number2presented3proteins(23.54, 21.63,16.O1kDa),number3presentedabout7I4%, 13.52kDaproteins.furthermore,thetotalproteincon

    centrationof7piecesofstripsincreased;number4 and5presentedunsignificantchangesonproteins, number6presentedabout1.6%13.52kDaproteins. thetotalproteinconcentrationof5piecesofstripsis increasing;number8alsopresents23.54,21.63kDa proteins.

    Inaddition,number10and11alsopresented 27.06kDaproteins.andthe13.52kDaproteinalso wasseenclearly.

    889910lOlJlllO

    

    0.1denotedproteinmarkerandcontrolsample;2-11denoted10%12h,20%12h,10%/4h,20

    %14h,10%/24h,20%18h

    (6h)4%124h,(10h)4%124h,10%/8h,respectively. Fig.1Comparisonofproteinsproducedfromdifferencecoldshocktreatment

    Itcouldbeseenfromtheproteinproducedby shocktreatmentthattheshockproteinconcentration producedbytreatedsamplewithin2hduringthe middleofexponentialgrowthphasewashigherthan4, 8,24h,theshockproteinconcentrationproducedby treatedsampleat10oCwasmorethan20oC,andthe

sampleproteinconcentrationduringthemiddleofex——

    ponentialgrowthphasewasmorethanlagperiod. Theresultofconservingexperimentwasshownin Fig.2.Thesurvivalrateofmosttreatedsampleswere higherthancontrolsampleduringtheshorttimepreser- vation,thesuperorityoftheproteinproducedbyshock tobacteriapreservationwasdecreasedafteronemonth. Theeffectat10oCwasbetterthan20oCand4oCcon——

    paratively,thesurvivalrateduring(6h)4%/24h(1ag period)washigherthan(10h)4%/24h(themiddleof exponentialgrowthphase),thesurvivalrateofshock treatmentswasthehighestbytakingoutsamplesduring themiddleofexponentialgrowthphase-10oC/8h, about31.7%,itwas20%/2h,20%/8hsecondly. 2.2Effectofhotshockoncryotolerance

    Heat——shockproteinsproducedbyEnterococcusf- aeca//sA31wasnotobvious(Fig.3).Itisreported thatfactorsthataffecthotshockproteinsynthesisare mostly,suchasstrain,shocktreatmentconditions, erythromycin,sensitivetohightemperaturel9.Butit

    stillshowsthatsomeadvantagesinl0wtemperaturep

    reservationexperimentinashortperiod(Fig.4) triggedbyexposingexponentiallygrowingcellstoheat shocktreatmentwhichdealingat45oC,30mincould receivebetterresultthanat45oC,60min,survival rateattained29.1%and19.1%forcontrolsample.

    l

    

.

    懿一

    

    

    No.1JournalofNonheastAgriculturalUniversity?39? 60

    O

    Control

    lO?/8h

    20?/4h

    4~C/24h(6h1

    O

    10~C/2h

    l0~C/24h

    20oC/8h

    1530day

    10'E/4h

    20?,2h

    4~C/24h(10h1

    Fig.2SurvivalratesofEnterococcusfaecal~A31bycoldshock

    l0aabb

    a,bdenoted(10h)45T/30min,(10h)45T~/60min,respectively

    Fig.3Comparisonofproteinsproducedfrom diljrerenceheatshock

    Manyresearchershavereportedthatcold——shock

    proteinsandheat-shockproteinscanenhancecapaci——

    tytosurvivefreezingt9'.Inthisstudy,weobserved lessornokindsofshockproteinsbyEnterococcus faecalisA31aftershocktreatmentthanreportedpre-

viously.Itmaybeexplainedthatshockproteinscon

    centrationmaybeinfluencedbymanyenvironment factors,suchasshocktemperature,growingphase, compositionofmedium,exposedtoerythromycinor notandSOon,orrelatedtospeciesthatthebacteria belongedto9].Ofcourse.theseproteinsmaynotbe triggeredbyshocktreatment.

    Inanalyzingdatasofpreservationexperiment,we couldseethatheat——shockandcold——shocktreatment

    aregoodforprobioticspreservedatlowtemperature thatwasobviousinashorttime.whilewasnotobvi

    Ol5

    ControlI45?,3Omin

    30day

    ?45~C/60min

    Fig.4SurvivalratesofEnterococcusfaecal~A31 byheatshock

    OUSinalongperiod,thismechanismwasnotclear. 3Conclusions

    a.Timeandtemperatureplayakeyroleintrig

    geringbacteriatoproduceproteinswhileprolonging timeisnotinevitablygainingmorekindsofproteins andtheeffectofthetreatmentwilldecrease. b.Atdifferentgrowingphases,survivalratesof bacteriaaredifferentinpreservation. C.Inalongperiodofcoldstorage,thedifference ofsurvivalbetweentreatedsamplesandcontrolsam——

    pleswillbecutdownandprotectioneffectsofpro——

    teinswilldecline.

    d.Thereisarelationbetweenshock-proteinand

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