第二章-培养基及其制备-发酵工艺课件

Chapter2PreparationofCultureMedium

Chapter2Unit1IndustrialFermentationMedium

Thebasicingredientsofmediaincludecarbonsource、nitrogensource、inorganicsalt、growthfactoranddistilledwateretc.Unit1IndustrialFermentatioMediumDevelopmentTomaintaineconomiccompetitiveness,low-costcrudematerialsarefrequentlyusedLevelsofmineralsandgrowthfactorsmaybecriticalMediumDevelopmentTomaintainKindsofCultureMedia

ThesourceofmediaconstituentComplexmediaSyntheticordefinedmediaSemi-definedmediumPhysicsstatesLiquidmediumSolidmediumSemi-solidmediumKindsofCultureMediaThesouForuseBasicmediumEnrichedmediumDifferentialmediumSelectivemediumManufactureintentionSeedmediumFermentmediumForuseManufactureintentionNutrientsourcesforindustrialfermentationCarbon&energysource+nitrogensource+O2+otherrequirements→Biomass+Product+byproducts+CO2+H2O+heatNutrientsourcesforindustriaNutrientRawmaterialCarbonmolasses,starchNitrogencornsteepliquor,soybeanmeal,pureammoniaorammoniumsalts,urea,nitratesalts,phosphatesaltsVitaminsandgrowthfactorsbiotin,yeastextract,beefextract,cornsteepliquor,wheatgermmealFermentationmediaNutrientRawmaterialCarbonmola1.Carbonsources

(1)Starch(corn,wheat,potato)WidelyusedinfermentationindustryStarch

dextringlucoseAdvantages:cheaperthanglucose1.Carbonsources(2)MolassesByproductofcaneorbeetsugarproductionAdarkviscoussyrupcontaining50%-75%fermentablesugars(mainlysucrose)with2%nitrogen,vitaminsandmineralsCheaper(2)Molasses第二章-培养基及其制备-发酵工艺课件2.Nitrogensource

(1)Inorganicnitrogensource

Ammonia,ammonium

andnitrateMicrobescanutilizeitfaster.Afteritisutilized,thepHofmediumwillbechanged.(NH4)2SO4

2NH3+H2SO4NaNO3+4H2

NH3+2H2O+NaOH2.Nitrogensource(2)OrganicnitrogensourcesUreaYeastextractPeptonesCornsteepliquorSoybeancakepowderPeanutpowderBranhydrolysisliquidcornsteepliquorpowder(2)Organicnitrogensourcesco3.TraceelementsFe,Mn,B,Zn,Cl,Mo,Cu4.Growthfactors

Smallamountoforganiccompoundsnecessaryformicrobialgrowth.e.g.aminoacids,vitamins,biotinSources:normallyorganicnitrogensource

e.g.cornsteepliquor3.Traceelements

CornsteepLiquor,CSLCornsteepliquoristhewaterextractby-productresultingfrom

thesteeping

ofcornduringthecommercialproductionofcorn

starchandothercornproducts.CornsteepLiquor,CSLCornstDifferentmediumfordifferentfoodproductsChinesedistilledspirit：solidmedium

Fruitspirit：fruitjuiceorfruitsauce

Beer：wort

Alcohol

:starchmash

Aminoacid:

starchmash

Citricacid：starchmash

Lacticacid：starchmashDifferentmediumfordifferentUnit2PreparationofStarchSugar

Starchisalargemoleculethatismadeofbranchingchainsofglucosemolecules.Itcanbebrokendownintoglucosebyhydrolysis.Unit2PreparationofStarcStarchfoundingrains,potatoes,beans,peas,cassavaTypically20-30%amylose,remainderamylopectinStarchfoundingrains,potatoCharacteristicsofstarchInsolubleinhotwaterBluecomplexcompoundbyreactionwithiodine.Insolublein30%ofethanolsolutionorabove.Hydrolyzedbyacidandenzyme,theultimateproductisglucose.CharacteristicsofstarchstarchgranulestarchgranuleIV.A.ii.-20Starch:AmyloseAmyloseisalinearglucanwith[1,4]glycosidiclinkagesIV.A.ii.-20Starch:AmyloseAmyIV.A.ii.-21Starch:AmylopectinAmylopectinisabranchedglucanwith[1,4]and[1,6]glycosidiclinkagesIV.A.ii.-21Starch:AmylopectiStarch:Amylopectinlessextended(morecompact)thanamyloselargerMW[1,6]linkages

[1,6]branchpointsresultintree-likestructureStarch:Amylopectin[1,6]linWhyshouldstarchbehydrolyzedtoglucose?Mostmicrobescouldnotutilizestarchdirectly.2.Toobtainsugarfromthe

starchofmanydifferentplants,ratherthanjustsugarbeetsorsugarcane.WhyshouldstarchbehydrolyzeConversionofstarchtofermentablesugars

1.Acidhydrolysismethod

StarchslurryisacidifiedtoapHvalueandthenheatedinaconverterunderpressure.(C6H10O5)n→(C6H10O5)x→

C12H22O11→C6H12O6

amylum→dextrin→oligosaccharides→glucoseConversionofstarchtofermenDuetoheatandacid,thereare…Glucosecombinationreaction

and

decompositionreactionDuetoheatandacid,therear

starchglucose

oligosaccharidecolourcontent(difficulttoutilize)(difficulttodecolor)

Totallostofglucose7％<1％DecompositionreactionacidCombinationreaction

Howtoavoidcombinationanddecompositionreactionsoccur?ConcentrationofstarchConcentrationofhydrolysisacid

ReactiontemperaturenottoohighHowtoavoidcombinationanAdvantageSimpleTimesavingDisadvantageMoreby-product,DE

valueonly90％Requiringfinestarch

NeedcorrosionresistantmaterialsNeedmoreenergyforheatingAdvantageExtentofhydrolysismeasuredusingdextroseequivalents(DE)value

reducingsugarsDE

value＝

100％

totalsolids

ExtentofhydrolysismeasuredThehighertheDE,themoresugarsandlessdextrinsarepresent.TheDEvalueofstarchiszeroandthatofdextroseis100.ThehighertheDE,themoresuABC

DEtimeABCDEtimeStarchandhydrochloricacidremoveafterminutescooladdsodiumbicarbonatetestwithBenedict’stestsamplewithiodineStarchandhydrochloricacidre2.Enzymehydrolysismethod

Whichenzymeisusedtobreakdownstarchintosugarsyrup?AmylaseGlucoamylaseAlsocalled:doubleenzymehydrolysis

2.Enzymehydrolysismethodamylaseattacks[1,4]bondstoproducevariousoligosaccharidesEndoenzymespullulanaseattacks[1,6]bondstoproducevariousoligosaccharides(amylosefragments)LowerMW!amylaseEndoenzymespullulanas

-amylaseattacks[1,4]bondsatnon-reducingend

toproducemaltoseExoenzymes(non-reducingend)glucoamylaseattacks[1,4]bondsatnon-reducingendtoproduceglucoseSweet!HighMW!-amylaseExoenzymes(non-reducEnzymeSourceAction

α-amylaseBacillussubtilis

Onlyα-1,4-oligosaccharidelinksarecleavedtogivedextrinswithmaltoseandupto50%(w/w)glucose

β-AmylaseMaltedbarleyOnlyα-1,4-linksarecleaved,fromnon-reducingends,togivelimitdextrinsandmaltoseGlucoamylaseAspergillusniger

α-1,4andα-1,6-linksarecleaved,fromthenon-reducingendstogiveglucosePullulanaseB.acidopullulyticus

Onlyα-1,6-linksarecleavedtogivestraight-chainmaltodextrinsEnzymesinprocessingstarchEnzymeSourceAction

α-amylasTherearetwobasicstepsinenzymaticstarchconversion:liquefactionandsaccharificationTherearetwobasicstepsineCornStarchSlurryProductionofGlucoseSyrupsfromStarchLiquefactionThermostablea-amylaseGelatinization(105°C,5min)Dextrinization(95°C,2h)LiquefiedStarchDE10-15SaccharificationGlucoamylase(60°C,pH4.0-4.5,24-72h)GlucoseSyrupsDE95-96CornStarchSlurryProduction

1)LiquefactionHighertemperaturehydrolysisofstarchinvolveastarchgelatinizationprocess,dissolutionofthenanogram-sizedstarchgranulestoformaviscoussuspension.

1)LiquefactionDuringliquefaction,long-chainedstarchmoleculesarepartialhydrolysisintosmallerbranchedandlinearchainsofglucoseunits(dextrins),withconcomitantlossinviscosity.Enzyme:α-amylase

End-point:DEvaule2030(why?)iodide→brownDuringliquefaction,long-Whyshouldbetwosteps,liquefactionandsaccharification?LowDEvalueSubstancesizeWhyshouldbetwosteps,lique2)Saccharification

Involvingtheproductionofglucoseandalittlemaltosebyfurtherhydrolysis.Enzyme:glucoamylaseEnd-point:DEisthehighest2)Saccharification第二章-培养基及其制备-发酵工艺课件AdvantageHigherqualitySaveenergyProtectenvironmentDisadvantageComplexoperationTimeconsumingAdvantageHowtomakehigh-fructosecornsyrup?Need3stepsand3enzymesalfa-amylase->shorteningthestarch(Liquefaction)glucoamylase->producesglucose(saccharification)glucoseisomerase->converttofructose(isomerization)

Howtomakehigh-fructosecorn

3.

Acid-enzymecombinationmethod(Two-stagehydrolysis)

Slurryisonlypartiallyconvertedbyacidandthentreatedwithanappropriateenzymeorenzymesuntiltheconversioniscomplete.

3.Acid-enzymecombinationme1)Acid-enzymehydrolysisAcid---liquefaction

Glucoamylase-----saccharificationSuitablefor:starchgranuleishard1)Acid-enzymehydrolysis2)Enzyme-acidhydrolysisα-Amylase(thermostable)---liquefaction

Acid-----saccharificationSuitablefor:starchgranulesizeisirregular2)Enzyme-acidhydrolysisProductionofGlucosefromStarchItemsacidacid/enzymeenzymeDE

value919598Color100.30.2ProcessingconditionHTPHTPNTPEnergyconsuminghighhighlowBy-productmoremoderatelittleCycletimeshortmoderatelongProductionofGlucosefromStaReviewQuestions1.Whichkindofcarbonsourceandnitrogensourceusuallyusedinindustry?Citetheadvantagesanddisadvantagesofthedifferentchoices.2.Whatisthedefinitionofgrowthfactoranditsmainsource?3.Whatisthesignificanceofgettingsugarbyhydrolyzingstarch?4.Comparethethreemethodstoproduceglucosefromstarchandthedifferencebetween

combinationreactionanddecompositionreaction,liquefactionandsaccharification.ReviewQuestions1.WhichkindoInductionRepression1.Itturnstheoperonon.1.Itturnstheoperonoff.2.Itstartstranscriptionandtranslation.2.Itstopstranscriptionandtranslation.3.Itiscausedbyanewsubstance,whichneedsenzymestogetused.3.Itiscausedbyanexcessofexistingmetabolite.4.Itoperatesinacatabolicpathway.4.Itoperatesinananabolicpathway.5.Repressorispreventedbytheinducerfromjoiningtheoperatorgene.5.Repressorisenabledbyaco-repressortojointheoperatorgene.InductionRepression1.ItturnsFeedbackrepressionFeedbackinhibitionRegulationlevelgenetic:RNAtranscriptioncellular:activityofenzymeComplexformedendproduct+repressorendproduct+enzymeEffectoperatoronDNAtemplateoccupiedbythecomplexreducedenzymeactivityConsequenceblockedtranscriptionTherespectivereactionisinhibited.FeedbackrepressionFeedbackin

Chapter2PreparationofCultureMedium

Chapter2Unit1IndustrialFermentationMedium

Thebasicingredientsofmediaincludecarbonsource、nitrogensource、inorganicsalt、growthfactoranddistilledwateretc.Unit1IndustrialFermentatioMediumDevelopmentTomaintaineconomiccompetitiveness,low-costcrudematerialsarefrequentlyusedLevelsofmineralsandgrowthfactorsmaybecriticalMediumDevelopmentTomaintainKindsofCultureMedia

ThesourceofmediaconstituentComplexmediaSyntheticordefinedmediaSemi-definedmediumPhysicsstatesLiquidmediumSolidmediumSemi-solidmediumKindsofCultureMediaThesouForuseBasicmediumEnrichedmediumDifferentialmediumSelectivemediumManufactureintentionSeedmediumFermentmediumForuseManufactureintentionNutrientsourcesforindustrialfermentationCarbon&energysource+nitrogensource+O2+otherrequirements→Biomass+Product+byproducts+CO2+H2O+heatNutrientsourcesforindustriaNutrientRawmaterialCarbonmolasses,starchNitrogencornsteepliquor,soybeanmeal,pureammoniaorammoniumsalts,urea,nitratesalts,phosphatesaltsVitaminsandgrowthfactorsbiotin,yeastextract,beefextract,cornsteepliquor,wheatgermmealFermentationmediaNutrientRawmaterialCarbonmola1.Carbonsources

(1)Starch(corn,wheat,potato)WidelyusedinfermentationindustryStarch

dextringlucoseAdvantages:cheaperthanglucose1.Carbonsources(2)MolassesByproductofcaneorbeetsugarproductionAdarkviscoussyrupcontaining50%-75%fermentablesugars(mainlysucrose)with2%nitrogen,vitaminsandmineralsCheaper(2)Molasses第二章-培养基及其制备-发酵工艺课件2.Nitrogensource

(1)Inorganicnitrogensource

Ammonia,ammonium

andnitrateMicrobescanutilizeitfaster.Afteritisutilized,thepHofmediumwillbechanged.(NH4)2SO4

2NH3+H2SO4NaNO3+4H2

NH3+2H2O+NaOH2.Nitrogensource(2)OrganicnitrogensourcesUreaYeastextractPeptonesCornsteepliquorSoybeancakepowderPeanutpowderBranhydrolysisliquidcornsteepliquorpowder(2)Organicnitrogensourcesco3.TraceelementsFe,Mn,B,Zn,Cl,Mo,Cu4.Growthfactors

Smallamountoforganiccompoundsnecessaryformicrobialgrowth.e.g.aminoacids,vitamins,biotinSources:normallyorganicnitrogensource

e.g.cornsteepliquor3.Traceelements

CornsteepLiquor,CSLCornsteepliquoristhewaterextractby-productresultingfrom

thesteeping

ofcornduringthecommercialproductionofcorn

starchandothercornproducts.CornsteepLiquor,CSLCornstDifferentmediumfordifferentfoodproductsChinesedistilledspirit：solidmedium

Fruitspirit：fruitjuiceorfruitsauce

Beer：wort

Alcohol

:starchmash

Aminoacid:

starchmash

Citricacid：starchmash

Lacticacid：starchmashDifferentmediumfordifferentUnit2PreparationofStarchSugar

Starchisalargemoleculethatismadeofbranchingchainsofglucosemolecules.Itcanbebrokendownintoglucosebyhydrolysis.Unit2PreparationofStarcStarchfoundingrains,potatoes,beans,peas,cassavaTypically20-30%amylose,remainderamylopectinStarchfoundingrains,potatoCharacteristicsofstarchInsolubleinhotwaterBluecomplexcompoundbyreactionwithiodine.Insolublein30%ofethanolsolutionorabove.Hydrolyzedbyacidandenzyme,theultimateproductisglucose.CharacteristicsofstarchstarchgranulestarchgranuleIV.A.ii.-72Starch:AmyloseAmyloseisalinearglucanwith[1,4]glycosidiclinkagesIV.A.ii.-20Starch:AmyloseAmyIV.A.ii.-73Starch:AmylopectinAmylopectinisabranchedglucanwith[1,4]and[1,6]glycosidiclinkagesIV.A.ii.-21Starch:AmylopectiStarch:Amylopectinlessextended(morecompact)thanamyloselargerMW[1,6]linkages

[1,6]branchpointsresultintree-likestructureStarch:Amylopectin[1,6]linWhyshouldstarchbehydrolyzedtoglucose?Mostmicrobescouldnotutilizestarchdirectly.2.Toobtainsugarfromthe

starchofmanydifferentplants,ratherthanjustsugarbeetsorsugarcane.WhyshouldstarchbehydrolyzeConversionofstarchtofermentablesugars

1.Acidhydrolysismethod

StarchslurryisacidifiedtoapHvalueandthenheatedinaconverterunderpressure.(C6H10O5)n→(C6H10O5)x→

C12H22O11→C6H12O6

amylum→dextrin→oligosaccharides→glucoseConversionofstarchtofermenDuetoheatandacid,thereare…Glucosecombinationreaction

and

decompositionreactionDuetoheatandacid,therear

starchglucose

oligosaccharidecolourcontent(difficulttoutilize)(difficulttodecolor)

Totallostofglucose7％<1％DecompositionreactionacidCombinationreaction

Howtoavoidcombinationanddecompositionreactionsoccur?ConcentrationofstarchConcentrationofhydrolysisacid

ReactiontemperaturenottoohighHowtoavoidcombinationanAdvantageSimpleTimesavingDisadvantageMoreby-product,DE

valueonly90％Requiringfinestarch

NeedcorrosionresistantmaterialsNeedmoreenergyforheatingAdvantageExtentofhydrolysismeasuredusingdextroseequivalents(DE)value

reducingsugarsDE

value＝

100％

totalsolids

ExtentofhydrolysismeasuredThehighertheDE,themoresugarsandlessdextrinsarepresent.TheDEvalueofstarchiszeroandthatofdextroseis100.ThehighertheDE,themoresuABC

DEtimeABCDEtimeStarchandhydrochloricacidremoveafterminutescooladdsodiumbicarbonatetestwithBenedict’stestsamplewithiodineStarchandhydrochloricacidre2.Enzymehydrolysismethod

Whichenzymeisusedtobreakdownstarchintosugarsyrup?AmylaseGlucoamylaseAlsocalled:doubleenzymehydrolysis

2.Enzymehydrolysismethodamylaseattacks[1,4]bondstoproducevariousoligosaccharidesEndoenzymespullulanaseattacks[1,6]bondstoproducevariousoligosaccharides(amylosefragments)LowerMW!amylaseEndoenzymespullulanas

-amylaseattacks[1,4]bondsatnon-reducingend

toproducemaltoseExoenzymes(non-reducingend)glucoamylaseattacks[1,4]bondsatnon-reducingendtoproduceglucoseSweet!HighMW!-amylaseExoenzymes(non-reducEnzymeSourceAction

α-amylaseBacillussubtilis

Onlyα-1,4-oligosaccharidelinksarecleavedtogivedextrinswithmaltoseandupto50%(w/w)glucose

β-AmylaseMaltedbarleyOnlyα-1,4-linksarecleaved,fromnon-reducingends,togivelimitdextrinsandmaltoseGlucoamylaseAspergillusniger

α-1,4andα-1,6-linksarecleaved,fromthenon-reducingendstogiveglucosePullulanaseB.acidopullulyticus

Onlyα-1,6-linksarecleavedtogivestraight-chainmaltodextrinsEnzymesinprocessingstarchEnzymeSourceAction

α-amylasTherearetwobasicstepsinenzymaticstarchconversion:liquefactionandsaccharificationTherearetwobasicstepsineCornStarchSlurryProductionofGlucoseSyrupsfromStarchLiquefactionThermostablea-amylaseGelatinization(105°C,5min)Dextrinization(95°C,2h)LiquefiedStarchDE10-15SaccharificationGlucoamylase(60°C,pH4.0-4.5,24-72h)GlucoseSyrupsDE95-96CornStarchSlurryProduction

1)LiquefactionHighertemperaturehydrolysisofstarchinvolveastarchgelatinizationprocess,dissolutionofthenanogram-sizedstarchgranulestoformaviscoussuspension.

1)LiquefactionDuringliquefaction,long-chainedstarchmoleculesarepartialhydrolysisintosmallerbranchedandlinearchainsofglucoseunits(dextrins),withconcomitantlossinviscosity.Enzyme:α-amylase

End-point:DEvaule2030(why?)iodide→brownDuringliquefaction,long-Whyshouldbetwosteps,liquefactionandsaccharification?LowDEvalueSubstancesizeWhyshouldbetwosteps,lique2)Saccharification

Involvingtheproductionofglucoseandalittlemaltosebyfurtherhydrolysis.Enzyme:glucoamylaseEnd-point:DEisthehighest2)Saccharification第二章-培养基及其制备-发酵工艺课件AdvantageHigherqualitySaveenergyProtectenvironmentDisadvantageComplexoperationTimeconsumingAdvantageHowtomakehigh-fructosecornsyrup?Need3stepsand3enzymesalfa-amylase->shorteningthestarch(Liquefaction)glucoamylase->producesglucose(saccharification)glucoseisomerase->converttofructose(isomerization)

Howtomake