PreparationofCultureMedium

Unit1IndustrialFermentationMedium

Thebasicingredientsofmediaincludecarbonsource、nitrogensource、inorganicsalt、growthfactoranddistilledwateretc.MediumDevelopmentTomaintaineconomiccompetitiveness,low-costcrudematerialsarefrequentlyusedLevelsofmineralsandgrowthfactorsmaybecriticalKindsofCultureMedia

ThesourceofmediaconstituentComplexmediaSyntheticordefinedmediaSemi-definedmediumPhysicsstatesLiquidmediumSolidmediumSemi-solidmediumForuseBasicmediumEnrichedmediumDifferentialmediumSelectivemediumManufactureintentionSeedmediumFermentmediumNutrientsourcesforindustrialfermentationCarbon&energysource+nitrogensource+O2+otherrequirements→Biomass+Product+byproducts+CO2+H2O+heatNutrientRawmaterialCarbonmolasses,starchNitrogencornsteepliquor,soybeanmeal,pureammoniaorammoniumsalts,urea,nitratesalts,phosphatesaltsVitaminsandgrowthfactorsbiotin,yeastextract,beefextract,cornsteepliquor,wheatgermmealFermentationmedia1.Carbonsources

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

dextrin

glucoseAdvantages:cheaperthanglucose(2)MolassesByproductofcaneorbeetsugarproductionAdarkviscoussyrupcontaining50%-75%fermentablesugars(mainlysucrose)with2%nitrogen,vitaminsandmineralsCheaper2.Nitrogensource

(1)Inorganicnitrogensource

Ammonia,ammonium

andnitrateMicrobescanutilizeitfaster.Afteritisutilized,thepHofmediumwillbechanged.(NH4)2SO4

2NH3+H2SO4NaNO3+4H2

NH3+2H2O+NaOH(2)OrganicnitrogensourcesUreaYeastextractPeptonesCornsteepliquorSoybeancakepowderPeanutpowderBranhydrolysisliquidcornsteepliquorpowder3.TraceelementsFe,Mn,B,Zn,Cl,Mo,Cu4.Growthfactors

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

e.g.cornsteepliquor

CornsteepLiquor,CSLCornsteepliquoristhewaterextractby-productresultingfrom

thesteeping

ofcornduringthecommercialproductionofcorn

starchandothercornproducts.DifferentmediumfordifferentfoodproductsChinesedistilledspirit：solidmedium

Fruitspirit：fruitjuiceorfruitsauce

Beer：wort

Alcohol

:starchmash

Aminoacid:

starchmash

Citricacid：starchmash

Lacticacid：starchmashUnit2PreparationofStarchSugar

Starchisalargemoleculethatismadeofbranchingchainsofglucosemolecules.Itcanbebrokendownintoglucosebyhydrolysis.Starchfoundingrains,potatoes,beans,peas,cassavaTypically20-30%amylose,remainderamylopectinCharacteristicsofstarchInsolubleinhotwaterBluecomplexcompoundbyreactionwithiodine.Insolublein30%ofethanolsolutionorabove.Hydrolyzedbyacidandenzyme,theultimateproductisglucose.starchgranuleIV.A.ii.-20Starch:AmyloseAmyloseisalinearglucanwith

[1,4]glycosidiclinkagesIV.A.ii.-21Starch:AmylopectinAmylopectinisabranchedglucanwith

[1,4]and

[1,6]glycosidiclinkagesStarch:Amylopectinlessextended(morecompact)thanamyloselargerMW

[1,6]linkages

[1,6]branchpointsresultintree-likestructureWhyshouldstarchbehydrolyzedtoglucose?Mostmicrobescouldnotutilizestarchdirectly.2.Toobtainsugarfromthe

starchofmanydifferentplants,ratherthanjustsugarbeetsorsugarcane.Conversionofstarchtofermentablesugars

1.Acidhydrolysismethod

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

C12H22O11→C6H12O6

amylum→dextrin→oligosaccharides→glucoseDuetoheatandacid,thereare…Glucosecombinationreaction

and

decompositionreaction

starchglucose

oligosaccharidecolourcontent(difficulttoutilize)(difficulttodecolor)

Totallostofglucose7％<1％DecompositionreactionacidCombinationreactionHowtoavoidcombinationanddecompositionreactionsoccur?ConcentrationofstarchConcentrationofhydrolysisacid

ReactiontemperaturenottoohighAdvantageSimpleTimesavingDisadvantageMoreby-product,DE

valueonly90％Requiringfinestarch

NeedcorrosionresistantmaterialsNeedmoreenergyforheatingExtentofhydrolysismeasuredusingdextroseequivalents(DE)value

reducingsugarsDE

value＝

100％

totalsolids

ThehighertheDE,themoresugarsandlessdextrinsarepresent.TheDEvalueofstarchiszeroandthatofdextroseis100.ABC

DEtimeStarchandhydrochloricacidremoveafterminutescooladdsodiumbicarbonatetestwithBenedict’stestsamplewithiodine2.Enzymehydrolysismethod

Whichenzymeisusedtobreakdownstarchintosugarsyrup?AmylaseGlucoamylaseAlsocalled:doubleenzymehydrolysis

amylaseattacks[1,4]bondstoproducevariousoligosaccharidesEndoenzymespullulanaseattacks[1,6]bondstoproducevariousoligosaccharides(amylosefragments)LowerMW!

-amylaseattacks

[1,4]bondsatnon-reducingend

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

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

1)LiquefactionHighertemperaturehydrolysisofstarchinvolveastarchgelatinizationprocess,dissolutionofthenanogram-sizedstarchgranulestoformaviscoussuspension.

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

End-point:DEvaule20

30(why?)iodide→brownWhyshouldbetwosteps,liquefactionandsaccharification?LowDEvalueSubstancesize2)Saccharification

Involvingtheproductionofglucoseandalittlemaltosebyfurtherhydrolysis.Enzyme:glucoamylaseEnd-point:DEisthehighestAdvantageHigherqualitySaveenergyProtectenvironmentDisadvantageComplexoperationTimeconsumingHowtomakehigh-fructosecornsyrup?Need3stepsand3enzymesalfa-amylase->shorteningthestarch(Liquefaction)glucoamylase->producesglucose(saccharification)glucoseisomerase->converttofructose(isomerization)

3.

Acid-enzymecombinationmethod(Two-stagehydrolysis)

Slurryisonlypartiallyconvertedbyacidandthentreatedwithanappropriateenzymeorenzymesuntiltheconversioniscomplete.

1)Acid-enzymehydrolysisAcid---liquefaction

Glucoamylase-----saccharificationSuitablefor:starchgranuleishard2)Enzyme-acidhydrolysisα-Amylase(thermostable)---liquefaction

Acid-----saccharificationSuitablefor:starchgranulesizeisirregularProductionofGlucosefromStarchItemsacidacid/enzymeenzymeDE

value919598Color100.30.2ProcessingconditionHTPHTPNTPEnergyconsuminghighhighlowBy-productmoremoderatelittleCycletimeshortmoderatelongReviewQuestions1.Whichkindofcarbonsourceandnitrogensourceusuallyusedinindustry?Citetheadvantagesanddisadvantagesofthedifferentchoices.2.Whatisthedefinitionofgrowthfactoranditsmainsource?3.Whatisthesignificanceofgettingsugarbyhydrolyzingstarch?4.Comparethethreemethodsto