ENZYME IMMOBILIZATION 固定酶文档格式.docx

ENZYME IMMOBILIZATION 固定酶文档格式.docx

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Partners：

WuTing，WangTing，ZhouHan，WayneTravers，SanhanathMeadows

Date：

7th,21stOct

Abstract：

TheeffectofdifferentconditionsoffermenterslikeairflowandagitationspeedontheantimicrobialactivityofS.Warneribacteriocinswerestudiedinthisexperiment.Thebestconditionsweredeterminedbyantimicrobialactivitywhichdecidedbythezonesizeandhighestdilutiondemonstratingactivityofthebacteriocinsafterdifferentperiods.Theexperimenthasbeendonefor2weeks.10differentconditionshavebeentested.Thehighestairflow（4L/min）andagitationspeed（450RPM）weredecidedastheoptimizedoperatingcondition.

Andtheconcentrationofdissolvedoxygenandturbiditywerealsostudiedbythecurve.

Introduction/Background

Bacteriocinareantibioticpolypeptide，proteinorproteincomplexproducedbysomebacteria.In1982，Konislydefiniteditastheproteinwhichhasantimicrobialactivitiesgeneratedbytheribosomesofsomebacteria.Generally，mostofbacteriocinswillonlybetoxictothebacteriahavingclosegeneticrelationship.SourcesofbacterioncinsareverywidelikeGram-positivebacteria（G+）（E-coli）,Gram-negativebacteria（G-）,andarchaea.ThebacteriocinproducedbyLacticacidbacterialikenisinhashighgradeofsafetywhichhavebeengenerallyappliedatfood,pharmaceuticalindustrybecauseofthegreatpotentialoftheproducedbacteriocinsasfoodadditives.Inrecentyears，otherbacteriocinslikebacteriocinsproducedbystaphylococcalspecieshavebeenstudiedmoreandmore.Staphylococcusbacteriocinshavemanyadvantagessuchasdifficulttohavebacterialresistance，noresidue，non-toxicsideeffectsonbodyetc.ItismeaningfultotreattheinfectioncausedbyStaphylococus.

Differencebetweenantibioticsandbacteriocins.

Somearticlesdidn’tseparatedtheantibioticsandbacteriocins.Itwilllimitedtheappropriateapplicationofbacteriocinsinfoodindustry.Althoughtheyarebothproducedbymicroorganisms,therearemanydifferenceontheproductionmethodsandmechanismsofactionetc.1.Bacteriocinsareprimarymicrobialproductsynthesizedbyribosomeswhichbelongingtoproteinswhiletheantibioticsaresecondarymicrobialproductsynthesizedbymultienzymecomplexes.2.Inaddition,bacteriocinshaverelativelynarrowantibacterialspectrumwhichonlyaffectthebacteriahavingclosegeneticrelationshipwhiletheantibioticshavewideantibacterialspectrum.3.Antibioticsareappliedontheclinicaltherapywhilethebacteriocinsareappliedonfoodindustry.

Themechanismofactionofbacteriocins：

Bacteriocinsabsorbedtothecellsurfacenon-specificallybutthespecificbindingwithcellisdependedonthestructureofthecellwallandplasmamembrane.TheabsorptioncapacityofG-positivebacteriocinsisrelativeweak，maynotneedtocombinewithspecificreceptorandcanaffectdirectly.ComparedwithG-positivebacteriocins，G-negativebacteriocinsshouldcombinefirstlybelongingtoreceptor-mediateabsorption.G-positivebacteriocinsdestroythestructureofmembranebyformingamembranechannelontheplasmamembraneofsusceptiblecelloraffectsthestabilitywhichleadstocelldeathbytheleakageofions,aminoacids,andATP.DifferentG-positivebacteriocinsneeddifferentminimummembranepotentialtoreact.Inadditiontoformafilmchannel,someG-positivebacteriocinscanalsoinducecellautolysis.Actually,itissimilartomostofantibioticsinsomeways.

Theusageofbacteriocins

Becausebacteriocinshavemanyadvantagessuchashighefficiency,widerangeofapplication,nochangetotheflavorofproduct（goodadditives）andpathogenresistance.Sobacteriocinshavebeenappliedtofood,medicine,andfodderetc.

Infoodindustry:

Nisinhasbeenusedasimportantnaturalfoodpreservativeinworld.Itmainapplicationareasincludemilkproducts（includingfreshmilk,powderedmilk,yogurt,cheese,etc.）,cannedfood,beverage,meat,fish,andalcoholicbeveragesetc.Bacteriocinsoftencombinewithotherpreservationmethodandcangreatlyimprovetheeffectivenessofpreservative.Severalfactorsneedtobeconsideredbeforeaddingbacteriocins.Firstly,thestructureandcompositionoffoodneedtobeestimatedbecausethespecificcomponentcanaffecttheactivityofbacteriocins.Forexample,bacteriocinscanbondwithfattoaffectitsactivity.Secondly,temperature,pHandexogenousenzymes（eg.Proteases）shouldalsobeconsidered.Bacteriocinscanbebyproductsoffermentationpresentedincheese,yogurt,sausage.Itcanpreventandcontrolthecontaminationcausedbybacteria.

Inmedicine:

Duetothegrowingproblemofantibioticresistance,peopleconstantlylookingforsomethingthatcanreplacetheantibiotics.,whichbacteriocinshavebeenconsideredtohavegreatpotentials.Comparedwiththewideantibacterialspectrumofantibiotics,thebacteriocins’antibacterialspectrumisnarrow,withsomespecificity.Butitisdifficulttohavedrugresistance.Atthesametime,thespeciesofbacteriocinsaremorethanantibioticsandpeoplenormallyabletofindtherelativelybacteriocinforanykindofbacterialpathogens.Currently,thetreatmentofbacterialinfectionsusedbacteriocinsaregenerallyappliedproducingbacterialstrains.Noneofbacteriocinshasbeenusedasdrugforclinicaltreatmentdirectly,butonlyappliedinlaboratory.

Bacteriocinshavegreatpotentialincommercialproduction.Butforlarge-scaleproductionofbacteriocins,currentlytherearemanylimitations.Firstly,althoughthebacterioinshavemanyspecies,onlynisinhasbeenusedasacommodity.Secondly,thecurrentapplicationofnisinstillhasmanydifficult.1）mostofthelacticacidbacteriahasnarrowspectrum.2）Theamountofbacteriocinsarealittle.

Method

Oct7th

1.Prepared4agarplates（useTSA）

TSAquantity：

40gfor1L→2.4fgfor60ml

Put2.4gTSAand60mlintotube→4x60ml

1.Putthemintoautoclavetosterile（hightemp.andhighpressure）

2.Oncesterile，prepareGroassayplates→Add1.2mlofa50:

50mixtureoftween20waterand3mlofafreshlygrownbroth

3.Shakedthetubeuntilitisnotveryhot.

4.Pouredthecontentsintoa150mmsterilePetridish.

5.Allowedthecontentsoftheplatetosolidify.

6.Asepticallycutaseriesofwellsintheagarplateusing0.5mlvolumes.

Thegraphofinoculation.

7.PreparedasetofserialdilutionsofthesamplesusingTSB→6microtubescontaining250ulTSB/timept

8.AT13:

00,14:

00,15:

00,16:

00Repeatthefollowingsteps

preparesample→centrifugedandfilteredit.

Dilutedthesampleinto6sixtubesandmarkedthemfrom1to6

Took250ulneatintothefirsttubeandcentrifugeditfollowedbytaking250ulfromthefirsttubeintothesecondtube.Andsoon.

Usingpipettetoplace100ulofsample（neat）,nisin（aspositive）,broth（asnegative）,andaseriesofdilutionintowells.

Positivetestifthereisbacteria

Negativetestifthereiscontamination

Oct21st

TheprocedureissameastheOct7th.

Discussion

∙QUAD3：

07/10/2014pH7.0,300RPM.37℃，2L/min

PHandtemperature：

ThepHandtemperaturehadbeenmaintainallthetime.

Turbidityandoxygen：

1.Thegraphshowsthatduringthefirst1h,theturbidityclimbedslowlyfromalmost0AUto0.2Authan1h-5h.whilethepO2wasdecreasingsteadilyfrom90to80between0hand1h.→Itmaymeanthatthebacteriawasinlagphase，andoxygenwasconsumed

2.Between1hand5htheturbidityclimbedrapidlyfromalmost0.4Auto1.8Auwhendissolvedoxygendroppeddramaticallyatfirstfrom1hto2.5h.→Itmaymeansthatatearlyexponentialphase，thenumberofbacteriawasincreasingrapidlyandtheoxygenuptakerateofthebacteriawassignificanthigherthantherateoffermentersupport.

3.Between1hand5htheturbidityclimbedrapidlyfromalmost0.4Auto1.8Au，whendissolvedoxygenreachedequilibriumfrom2.5hto5handhadremainedstableatalowlevel.→Itmeansthatwhenthebacteriawasduringtheexponentialphase（1h-5h），thenumberofbacteriawasincreasingrapidlyandbacteriagrowthrequiredalotofdissolvedoxygen.

Inthefermentationprocess,whenthedissolvedoxygenintheculturemedium（CL）washigherthanthecriticaloxygenneededbycellgrowth,metabolicactivitiesandrespirationofcell,therateofgrowthwasn’taffectedandbacteriagrowthrequiresalotofdissolvedoxygen.

4.After5h，turbiditygrowslowlyandsteadilyfrom1.8Auto2.2Auwhenthedissolvedoxygentrenddidn’tchange.→Itmaymeanthatbacteriawasduringthedecelerationandstationaryphase，alotofthedissolvedoxygenwasrequiredbybacteriagrowth.

∙QUAD2：

21/10/2014pH7.0,300RPM,37℃，4L/min

PHandtemperature:

Turbidityandoxygen:

1.Thegraphshowsthatbetween0hand3h，theturbidityslightlyroseandthepO2slightlydecreased.→Itmaymeanthatthebacteriawasduringthelagphaseanditneededoxygen.

2.Between3hto12h,theturbiditygrewrapidlyfromalmost0AUto2.6AUandthepO2increasedrapidlyduring3h-4h,thenthecurvebecamesteady.→Itmaymeanthatthebacteriawasinexponentialphaseandneededalotofoxygenatfirstbutafter4h,thegrowthtrendwassteadyandpO2didn’taffecttherateanymore.

3.Between12hand20h，theturbiditywerebothsteadyandthepO2wasfluctuatedslightly.→Itmeansthatthebacteriawasindecelerationandstationaryphase.

4.Between20hand24h，theturbiditydroppedalittlewhenthepO2didn’tchange.→Itmayrepresentthatthebacteriawasinthedeathordecline