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外文文献

MediumoptimizationforglutathioneproductionbySaccharomycescerevisiae

Chi-HsienLiu,

Chin-FaHwang,

Chii-CherngLiao

FoodIndustryResearchandDevelopmentInstitute.P.O.Box246,Hsinchu,Taiwan,ROC

Received28April1997.Revised25March1998.Accepted28March1998.Availableonline19August1999.

http:

//dx.doi.org/10.1016/S0032-9592（98）00055-7,HowtoCiteorLinkUsingDOI

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Abstract

Glucose,peptoneandmagnesiumsulphatewerefoundtobesuitablecomponentsforthecellgrowthandglutathione（GSH）productionintheyeaststrain.SaccharomycescerevisiaeCCRC21727.TheBox–Behnkendesignandresponsesurfacemethodologywereemployedtoderiveastatisticalmodeltoinvestigatetheeffectsofglucose,peptoneandmagnesiumsulphateconcentrationsonGSHproduction.Neuralnetworkswerecomparedwithasecond-order-polynomialmodelinpredictingtheeffectsofcomponentconcentrationsontheproductionofGSHanddrycellweight（DCW）.NeuralnetworkmodelscanpredictcellgrowthandGSHproductionmorepreciselythansecond-order-response-surfacemodels.

Keywords

mediumoptimization;

glutathione;

neuralnetworkprediction;

response-surfacemodel

1.Introduction

Glutathione（GSH）isatripeptideconsistingofl-glutamate,l-cysteineandglycine.In1921,HopkinsisolatedGSHfromyeasts,animalliversandmuscle[1].GSHwidelyaffectsoxidationandreductioninvivo.Itisusedasamedicineforliverandasascavengeroftoxigen[2].HighlevelsofGSHcontentexistinsomestrainsofyeastswhicharethemicroorganismscommonlyusedforcommercialGSHproduction[3] and [4].

Neuralnetworks（NNs）,computermodelsystems,matchthefunctionalityofthebraininafundamentalmanner.NNshaverecentlybeenusedinmanyareasoffermentationsuchasbrewing[5],[6] and [7]andprocessstatevariableestimate[8],[9] and [10].Kennedyetal.comparedtheuseofNNstofactorialdesignastoolsformediumdesignandreportedthatNNscouldhetrainedonlessdataandpredictedtheexperimentswithaccuracyformediumdesign[11].

Responsesurfacemethodology（RSM）,anexperimentalstrategyforseekingtheoptimumconditionsforamultivariablesystem,isamuchmoreefficienttoolforoptimizationthantheone-factor-at-a-timeapproach[12].RSMhasbeensuccessfullyappliedforoptimizingmediagradientsinshake-flasksystems[13],[14],[15] and [16].Thesteepestascentmethod（SAM）wasusedtoevaluatetheinfluenceofcomponentconcentrationsontheproductviatheregressionmodel[12],[13] and [17].Inthisstudy,aGSH-producingstrainofSaccharomycescerevisiae,wasused.TheauthorsadoptedBox–Behnkendesign[12]andRSMtodeterminetheoptimalmediumforGSHproductionandcellgrowth.Neural-networkmodelswerecomparedwithsecond-order-response-surfacemodelstopredictGSHproductionanddrycellweight（DCW）alongthesteepestascentpaths.

2.Materialsandmethods

2.1.Organismandcultivationconditions

SaccharomycescerevisiaeCCRC21727（ATCC7754）fromtheCultureCollectionandResearchCenterofFoodIndustryResearchandDevelopmentInstituteinTaiwanwasmaintaincdonYMAgar（DifcoLaboratories,MI,USA）andgrowninYMBroth（DifcoLaboratories）at30°Cand150rpmfor18hastheinoculum.Forallexperiments,a4%inoculumwasusedina50mlitreHinton-flaskcontaining10mlitreculturemediumandincubatedat30°Cand150rpmfor24h.Eachtrialwasconductedintriplicate.

2.2.Experimentaldesignandstatisticalanalysis

Variouscarbon,nitrogenandsaltsourcesweretestedtodeterminethebestcomponentsforGSHproductionandcellgrowth.Themostimportantcomponentswerethenchosenforoptimizationofconcentrations.Thethree-variableBox–Behnkendesignwith13combinationsandthreereplicationsofthecentrepointwasused[18].Eachtrialwasconductedintriplicate.TheaveragedataofGSHproductionandDCWwereanalysedbySASsoftware[19]toconstructthesecond-ordermodelsandusedtotrainNNssoftware[20].AsetofexperimentsdesignedbySAMwasusedtoevaluatetheaccuracyofthesecond-ordermodelsandthetrainedNNs.

TheartificialNNhasbeeninspiredbybiologicalmodelsandthebuildingblocksofartificialNNareprocessingelements（PEs）.PEsarecombinedintolayers,theinputisreceivedandweightedaccordingtoweightingfactorsandtheresultingquantitiesaresummed.PEsthenexecuteatransferfunctionontheweightedsum,andpassthevalueontothenextPE.ThetransferfunctionmapsthepossiblyinfinitesummationofPEinputtoapredefinedrange,theoutput.ThelearningortrainingphaseofaNNrequirespairedinput-outputdata.Theinputisfedintothenetwork,transferredthroughthenetworklayers.Ultimately,thenetworkcalculatesapredictedoutput.Thispredictedoutputissubsequentlycomparedwiththeactualoutput,andtheconnectionweightsbetweenthePEsaremodifiedtominimizethedeviationbetweenthepredictedoutputandtheactualone.Thelearningrulespecifieshowconnectionweightsarechangedduringthelearningprocess.Theprocedurecontinuesuntiladefinedaccuracyhasbeenreached.Thisistheconceptofback-propagation[24].

Inthisstudy,twoback-propagationNNsconsisting,offourlayers,oneinputlayer,twohiddenlayersandoneoutputlayer,wereused.ThenumbersofPEsinthesetwohiddenlayersweresixandthree,respectively.Thehyperbolictangentfunctionanddeltarulewerechosenforthetransferfunctionandlearningrule.

2.3.Assays

Afterfermentationthebrothwasboiledfor5min,ice-cooledandthencentrifugedat12 000rpmfor3min,theGSHcontentinthesupernatantwasdeterminedaccordingtothepublishedmethods[21] and [22]bymeasuringtheabsorbanceofreactionsolutionsat412nmusingaspectrophotometer（BeckmanInc.,DU7400Spectrophotometer,CA,USA）.CellgrowthwasdeterminedbymeasuringtheDCW.DCWwasdeterminedafteryeastcellswerecentrifugedat12 000rpmfor3minandwashedtwicewithdeionizedwaterbeforedryingat70°Cfor24h.

3.Resultsanddiscussion

TheeffectsofvariouscarbonsourcesonGSHproductionandcellgrowthinS.cerevisiaecultureareillustratedinTable1.GlucosewasfoundtobethebestcarbonsourceforGSHproductionwhilefructoseandsucrosewerethetwomostsuitablecarbonsourcesforgrowthofS.cerevisiae.Shimizuandcoworkersreportedthatwhenglucoseratherthanethanolwasusedassolecarbonsource,bothspecificgrowthrateandGSHspecificproductionratewerehigher[3].CellgrowthwassimilarbutGSHproductionwas92%higherwhenglucosewasthecarbonsourceascomparedtoethanol.Withglycerolascarbonsource,theratioofGSHtoDCWwasthehighestamongallthecarbonsourcestestedbutGSHandDCWproductionswas56·9and25·8%ofthosewhenglucosewasusedascarbonsource.Whenlactoseormannitolwasused,GSHandDCWproductionwaspoor.Glucosewasusedasthecarbonsourceforfurtherexperiments.

Table1.EffectsofvariouscarbonsourcesonrelativeGSHproductionandrelativeDCWinS.cerevisiaeculturea

Carbonsource

RelativeGSHproduction（%）

RelativeDCW（%）

RatioofGSHtoDCW（%）

Glucose

100·0

100·0

0·48

Lactose

22·8

21·6

0·51

Fructose

77·1

117·3

0·32

Sucrose

87·9

115·3

0·37

Glycerol

56·9

25·8

1·06

Mannitol

24·0

28·9

0·40

Ethanol

51·7

100·0

0·25

a

Themediumcontained1%yeastextract,andthecarbonsourceswereaddedataconcentrationof1%（w/v）.GSHproductionandDCWwhichequateto100%are28·26mglitre−1and5·88glitre−1,respectively.

Full-sizetable

TheeffectsofnitrogensourcesonGSHproductionandcellgrowthareshowninTable2.ThehighestGSHcontentperunitofDCWwasobtainedwhentryptonewasusedasnitrogensource,althoughtheamountsofGSHandDCWwere61·4and57·6%ofthosewhenpeptonewasused.PeptoneandyeastextractwerethetwobestnitrogensourcesforGSHproductionbyS.cerevisiae.Therewasatwo-foldincreaseingrowthwhenyeastextractwasusedinsteadofpeptoneasnitrogensource,resultinginlowerGSHcontentperunitofDCW.Peptonewasthuschosenasthenitrogensourceforfurtherexperiments.

Table2.EffectsofvariousnitrogensourcesonrelativeGSHproductionandrelativeDCWinS.cerevisiaeculturea

Nitrogensource

RelativeGSHproduction（%）

RelativeDCW（%）

RatioGSHtoDCW（%）

Peptone

100·0

100·0

0·97

Tryptone

61·4

57·6

1·04

Meatextract

80·7

112·8

0·70

Yeastextract

100·0

202·8

0·48

Maltextract

10·7

42·8

0·24

a

Themediumcontained1%glucose,andthenitrogensourceswereaddedataconcentrationof1%（w/v）.GSHproductionandDCWwhichequateto100%are28·26mglitre−1and2·90glitre−1,respectively.

Full-sizetable

MetalionssuchasK+,Mg+2andCa+2directlyinfluencefermentativemetabolisminyeasts[25]and（NH4）2SO4andNH4ClstimulateethanolproductionbyS.cerevisiae[27].Khanetal.reportedthat（NH4）2SO4wasasuitablenitrogensourcefortheproductionofyeastbiomass[26].TheeffectsofvarioussaltsourcesonGSHproductionandcellgrowthareshowninTable3.AllsaltsexceptMgSO4showedinhibitiononbothGSHproductionandcellgrowth.TherewasnopreviousreportontheeffectofMgSO4onGSHproductioninyeast.Millsetal.reportedthatmagnesiumdeficientdietinhibitsbiosynthesisofbloodGSHinrat[23].AsMgSO4couldstimulateGSHproductionandcellgrowth,itwaschosenforfurtherstudy.

Table3.EffectsofvarioussaltsonrelativeGSHproductionandrelativeDCWinS.cerevisiaeculturea

Saltsource

RelativeGSHproduction（%）

RelativeDCW（%）

RatioofGSHtoDCW（%）

Controlb

100·0

100·0

0·97

CaCl2

39·0

87·2

0·44

KH2PO4

46·2

75·2

0·60

（NH4）2SO4

43·7

73·4

0·58

NH4CL

41·4

43·1

0·94