4微生物培養(yǎng)基Culturalmedium

4.微生物培育基CulturalmediaAculturalmediumisthesubstanceinwhichaspecificorganismlivesandgrows.Itmustcontaintheessentialnutrientsneededforthemicrobetogrow.Agoodmediumisveryimportanttothesuccessofanindustrialfermentation.Themediumsuppliesnutrientsforgrowth,energy,buildingofcellsubstance,andbiosynthesisoffermentationproducts.4.1培育基的類型及功能

Classificationsofmediaandtheirfunctions按純度分類：--合成培育基chemicallydefinedmedia:compoundidentityandconcentrationofallcomponentsareknown.Syntheticmedia--天然培育基chemicallyundefinedmedia:ingredientsincludeoneormorechemicallycomplexsubstancessuchasproteinhydrolysatesandextracts.CrudemediaThechoiceofdefinedorundefinedmediumisdependentuponitsapplication.---Chemicallydefinedmediaareusefulinbiochemicalormetabolicstudiesoforganisms.--Generallaboratorygrowthmediaandindustrialmediaareoftenchemicallycomplex.Suchas花生餅、蛋白胨等。普通不需外加微量元素(traceelements)、維生素等。

按形狀分類：-Solidmedia：suitabletocultureandpreservespeciesandspores;cultivatingfungi.-Liquidmedia:80%-90%water,industrialfermentationmedia.-Semi-solidmedia:liquidmedium+agar主要用于鑒定細菌、察看細菌運動特征等。按用途分類孢子培育基、種子培育基和發(fā)酵培育基1.孢子培育基sporemedia要求：使菌體迅速生長，產生較多的優(yōu)質孢子，不易引起菌種發(fā)生變異(variation)。根本配制要求：⑴notrichinnutrition,especiallyorganicnitrogensoure.⑵optimalmineralsaltconcentration⑶optimalpHandhumidity消費上常用孢子培育基：麩皮、大米、小米培育基；由葡萄糖、蛋白胨、牛肉膏和食鹽等配制的瓊脂斜面培育基。大米和小米常用作霉菌孢子培育基，因它們含氮少、疏松、外表積大，是較好的孢子培育基。水分控制在21％－25％。種子培育基inoculummedia:種子培育基是供孢子發(fā)芽、生長和大量繁衍菌絲體，并使菌絲體長得粗壯，成為活力強的‘種子’。Inoculummediaareusuallylessnutritiousthanareproductionmedia,andtheyusuallycontainaconsiderablylowerlevelofthemainnutritivecarbonsource.普通種子培育基常包括有機氮源和無機氮源最后一級的種子培育基的成分最好接近發(fā)酵培育基。發(fā)酵培育基productionmedia發(fā)酵培育基是供菌體生長、繁衍和合成產物之用。要求：既要使種子接種后能迅速生長，到達一定的菌絲濃度，又要使長好的菌體迅速合成所需產物。配制要求：其組成除有菌體生長所必需的元素和化合物外，還需有合成產物所需的特定元素、前體和促進劑等。Inadditiontothecarbonandnitrogencompounds,amediumcontainsinorganicsalts,vitamins,growthfactors,precursorsoffermentationproducts.Microbialgrowthunderindustrialfermentationconditionsusuallyutilizesa“l(fā)uxurymetabolism〞oftheorganisms.Thus,goodyieldsoffermentationproductsoccuronlyifrelativelylargeamountsofcarbon,andpossiblynitrogen,arechanneledthroughthemetabolicpathwaysofthemicroorganism.4.2發(fā)酵培育基的成分及來源

CompositionofmediaandtheirsourcesTheparticularcompositionofafermentationmediumcanbesimpletocomplexdependingontheparticularmicroorganismanditsfermentation.Autotrophic(自養(yǎng)的)microorganismsrequireonlythesimplestofinor-ganicmedia.Heterotrophic(異養(yǎng)的)microorganismslacktheabilitytosynthesizemanyoftheirsustenanceandgrowthrequirements.4.2.1碳源carbonsourcesCarbonsourceisoneofmaincompositionofmedia.Thefunctionsare:providingcarbonsandenergy;thecomponentsforsynthesisofproducts.Thesecanincludesimplesugars,complexcarbohydrates,alcohols,aminoandotherorganicacids,andshort-chainlipids.常用的碳源有糖類、油脂、有機酸和低碳醇。蛋白質、氨基酸也可用作碳源。4.2.1.1糖類sugarsSugarsusedmainly:glucose,molasses(糖蜜),dextrin(糊精).Glucose:mostmicrobescanutilizeit.速效碳源、highconcentrationspeedrespirationupDOnoenoughintermediates(中間產物〕accumulationinhibitinggrowthmolasses(糖蜜)Beet(甜菜)andcanemolassesareby-productsofthesugarindustry.containing50%-75%fermentablesugars,mainlysucrose.Alsocontainingnitrogenouscompounds,vitaminsandminerals.CheaperStarchanddextrin(糊精)Starch:----widelyusedinfermentationindustry---starchhydrolyzeusingmicrobialamylasedextringlucose-Mostcommonusedstarches:corn,wheat,potatostarches.Alsousingcornflour.--advantages:緩效碳源、cheaperthanglucose油和脂肪oilsandfatsOilandfatsarealsousedascarbonsourcesbecausesomemicrobescontainlipase(脂肪酶)Fatglycerol(甘油)andfattyacids

oxidizeCO2+water,releasingmuchenergyProvidingenoughO2isimportant.常用的油：豆油、菜子油、豬油、魚油、棉子油等。有機酸organicacidsSomemicrobescanuseorganicacidsascarbonsources,suchaslacticacid,citricacid,aceticacid.pHofculturalmediumwillincrease.CH3COONa+O22CO2+H2O+NaOH烴和醇類hydrocarbonandethanol石油及裂解產物如正烷烴也可用作carbonsources.自然界中能同化乙醇的微生物和能同化糖質的微生物一樣普遍。4.2.2氮源nitrogensources氮源主要用于構成細胞物質〔氨基酸、蛋白質、核酸等〕和含氮代謝物。有機氮源organicnitrogensources常用的:花生餅、棉子餅、玉米漿、玉米蛋白粉、蛋白胨、酵母粉、魚粉、蠶蛹粉、尿素、廢菌絲體和酒糟等。organicnitrogensourcesmicrobialproteinaseaminoacidsmetabolizedbymicrobes.organicnitrogensourcescontainrichproteins,peptides,aminoacids,vitaminsandgrowthfactors.在含有機碳源的培育基中，微生物常表現出生長旺盛、菌絲或細胞濃度增長迅速的特點。有些微生物對氨基酸有特殊的需求。如纈氨酸可提高紅霉素的發(fā)酵單位。玉米漿CornsteepLiquorCornsteepliquoristhewaterextractby-productresultingfromthesteeping(浸泡)ofcornduringthecommercialproductionofcornstarchandothercornproducts.Ofthe50%solidsofcornsteepliquor,nearlyhalfislacticacid.Therestincludesaminoacids,glucoseandotherreducingsugars,salts,vitamins,andprecursors(前體)suchasthoseforthepenicillinmolecule.Thehighlacticacidcontentofcornsteepliquorresultsfromthegrowthoflacticacidbacteriaduringitsmanufacture.Thisvariationincomposition,attimes,canleadtopoorreproducibilityofanindustrialfermentation.尿素ureaUreaisalsoacommonlyusednitrogensource.Itsfeature:---singlecomponent,nonutritionalfeatureofcomplexnitrogensources;---cheaper,oftenusedinproductionofpenicillinandglutamicacid.yeastextractpeptones有機氮源除了作為菌體生長繁衍的營養(yǎng)外，有的還是產物的前體。纈氨酸、半胱氨酸和-是合成青霉素和頭孢菌素的主要前體；甘氨酸可作為L-絲氨酸的前體等。無機氮源inorganicnitrogensource常用的：氨水(ammonia)、銨鹽(ammonium)和硝酸鹽(nitrate)Microbescanutilizeitfaster.Afteritisutilized,thepHofmediumwillbechanged.(NH4)2SO42NH3+H2SO4NaNO3+4H2NH3+2H2O+NaOH生理酸性物質：NH4酸性物，硫酸銨生理堿性物質：NH4堿性物，硝酸銨正確運用生理酸堿性物質Exceptusingasanitrogensource,ammoniaisoftenusedtoadjustpHofmedia.Stirringisnecessary.4.2.3無機鹽及微量元素

mineralsaltsandtraceelementsMineralsaltsandtraceelementsareneededwhenmicrobialcellsgrowandreproduce.Mostcommonmineralsaltsandtraceelements:磷(phosphorus)、鎂(magnesium)、硫(sulphur)、鉀(potassium)、鈉(sodium)、鐵(iron)、氯(chlorine)、錳(manganese)、鋅(zinc)、鈷(cobalt)、鈣等作為微生物細胞生理活性物質組成或生理活性作用的調理物。Whentheirconcentrationislow,theyshowastimulationfunctionforcellgrowandproductsynthesis.Whentheirconcentrationishigh,theywillinhibitthecellgrowth.Theiroptimalconcentrationsdependonthespeciesandstrains.無機鹽成分普通所用的濃度范圍成分濃度g/L成分濃度g/LKH2PO4MgSO4·7H2OKClCaCO3FeSO4·4H2O1.0~4.00.25~3.00.5~12.05~170.01~0.1ZnSO4·8H2OMnSO4·H2OCuSO4·5H2ONa2MoO4·2H2O0.1

~1.00.01~0.10.003~0.010.01~0.1Theseelementsareaddedintoculturalmediaintheformoftheirsalts.Exceptsyntheticmedia,cobalt鈷,copper,iron,manganese錳,zinc,molybdenum鉬arenotaddedintocrudemedia.Phosphoruselementisoneofnucleicacidsandproteinscomposition,onecomponentofATP,energytransfer.Phosphoruscanstimulatemicrobialgrowthandmetabolism.Butitinhibitscellgrowthwhenitsconcentrationishigh.許多次級代謝過程對磷酸鹽濃度的接受限制比生長繁衍過程低，故必需嚴厲控制。Magnesiumdoesnotinvolvethecellinstitution.ButMgistheactivatorformanyimportantenzymes.如己糖磷酸化酶、檸檬酸脫氫酶等鎂離子可提高抗生素產生菌的抗生素耐受才干，如鏈霉素、卡那霉素等產生菌。常以硫酸鎂的方式參與培育基。硫存在于細胞的蛋白質中，是含硫氨基酸的組分和某些輔酶的活性基，如輔酶A(coenzymeA),谷胱甘肽等。硫是某些產物如青霉素、頭孢菌素等分子的組成部分，在培育基中參與Na2SO4等含硫化合物作硫源。鐵(iron)是細胞色素、細胞色素氧化酶和過氧化氫酶的成分，因此鐵是菌體有氧氧化必不可少的元素。Iniron-madefermentor,generallyironionconcentrationcanbe30μg/ml.Soironcontainingcompoundsarenotnecessaryforindustrialmedia.Chlorinedoesnothavenutritionalfunctionsformostmicroorganisms,butitisnecessaryforcellgrowthofhalophile.一些產含氯代謝物如金霉素和灰黃霉素等的發(fā)酵中，除天然含有外，通常還需參與0.1%氯化鉀。在啤酒消費中，20－60mg/ml的氯對酶和酵母有一定的促進作用。Sodium,potassium,calciumarenotcomponentsofcellconstitution,theyarestillnecessarycompositionforculturalmedia.鈉、鉀離子與維持細胞的浸透壓有關。鉀離子是許多酶的激活劑，能促進糖代謝。鈣是某些酶(如蛋白酶)的激活劑，還參與細胞膜通透性的調理。培育基中鈣鹽過多時，會構成磷酸鈣沉淀。。鋅、鎂、鈷、錳等是某些酶的輔基或激活劑。鎂離子還可提高抗生素消費菌對本人所產生抗生素的耐性；鈷既是一些酶的激活劑，又是VB12的組成元素，發(fā)酵中參與一定量的鈷鹽，能使VB12的產量提高數倍；錳對于羧化作用是必需的，糖代謝中許多酶的活性都與錳有關。4.2.4WaterMaincompositionofmediaFunctions:---joindirectlysomemetabolisms---solvents---providingaphysiologicalenvironmentsforcellgrowthandsynthesisofproducts.Effectofwaterqualityonfermentation4.2.5生長因子、前體、產物促進劑(1)生長因子growthfactorsDefinition:smallamountoforganiccompoundsnecessaryformicrobialgrowth.Features:nosynthesisbyitself.Examples：aminoacids,purines,vitaminsSources:normallyorganicnitrogensource.Cornsteepliquor.(2)前體precursorsDefinition:substancesaddedpriortoorsimultaneouslywiththefermentation,whichareincorporatedwithoutanymajorchangeintothemoleculeofthefermentationproduct,andwhichgenerallyservetoincreasetheyieldorimprovethequalityoftheproduct.前體是指某些化合物參與到發(fā)酵培育基中，能直接被微生物在生物合成過程中結合到產物分子中去，而其本身的構造并沒有多大變化，但是產物的產量卻因參與前體而有較大的提高。Examples：苯乙酸青霉素G苯氧乙酸青霉素V發(fā)酵過程中所用的一些前體物質產品前體產品前體青霉素G青霉素V金霉素灰黃霉素紅霉素苯乙酸苯氧乙酸氯化物氯化物正丙醇核黃素類胡蘿卜素L-異亮氨酸L-色氨酸L-絲氨酸丙酸鹽β-紫羅酮

-氨基丁酸鄰氨基苯甲酸甘氨酸(3)產物促進劑產物促進劑productstimulatorDefinition:substancesareneithernutrientsnorprecursors,andincreaseproductyieldafteraddingthem.Examples:Tween(0.1%)amylase(淀粉酶),cellulase(纖維素酶)大豆酒精提取物cellulaseMechanisms:notveryclearPossiblereasons:---enzymeinducers誘導劑---improvementofcellpermeability浸透性---protectiononenzymeactivity4.3培育基的設計及優(yōu)化

mediadesignanditsoptimization

ImportanceofmediadesignCurrentsituationsofmediadesign:manyfactorsempirical閱歷+scientific4.3.1培育基成分選擇的原那么(1)菌體的同化才干：largemolecules+enzymessmallmoleculesSelectionofcarbonsources:

glucose,starches不同糖化工藝所得糖液質量的比較項目酸法酸酶法雙酶法葡萄糖值(DE值)葡萄糖含量％灰分％色度羥甲基糠氨％葡萄糖收率91861.610.00.3095930.40.30.008較酸法高5％98970.10.20.003較酸法高10％Selectionofnitrogensources:---Ifthemicrobeproducesproteolyticenzymes,avarietyofcrudenitrogensourcessuchassoybeanmealcanbeused.--Ifthemicrobelackofproteases,theorganicnitrogensourceshouldbehydrolyzed.豆餅粉＋water+hydrochloricacid鹽酸,pH1.0100°Caminoacids.(2)代謝物的阻遏和誘導---葡萄糖效應---combinationoforganicandinorganicnitrogen/carbonsources---enzymeagentproduction:Formanyinducedenzymes,starchanddextrinarecommoncarbonsource.

碳源對生長和產酶的影響碳源地衣芽孢桿菌細胞量g/l

淀粉酶活/(U/ml)glucose