发酵工程--抗生素发酵生产技术概述(ppt 34页).ppt

1,韩北忠中国农业大学食品学院HanBei-zhong,Prof.PhDCollegeofFoodScience&NutritionalEngineeringChinaAgriculturalUniversity,抗生素发酵生产技术AntibioticsFermentationProduction,2,概述生产前期研究生产菌改良生产工艺过程发酵条件的控制提取和精制,IntroductionResearchinLabStrainimprovementProductionprocessingFermentationcontrolIsolation&purification,抗生素发酵生产技术AntibioticsFermentationProduction,3,抗生素是生物在其生产活动过程中所产生，并能在低微浓度下有选择性地抑制或杀灭其他微生物或肿瘤细胞的有机物。,AntibioticsAnyorganicsubstancesproducedbyvariousorganismsthathavethepowerofarrestingthegrowthofothermicroorganismsorofdestroyingthem.,1.概述Introduction,4,历史1929年，英国弗莱明发现青霉素二战期间，大规模发酵生产世界上最早用于临床的抗菌素,HistoryPenicillinwasdiscoveredbyFlemingin1929.WorldWarII,fermentedinlarge-scaleThefirstmajorantibiotictobecommercialized,1.概述Introduction,5,根据抗生素的作用机制分类(ModesofActionofAntibiotics)抑制细胞壁合成(CellWallSynthesis)：青霉素抑制细胞膜功能(CellMembraneDamage)：多烯类抗生素抑制蛋白质合成(ProteinSynthesis)：四环素抑制核酸合成(NucleicAcidSynthesis)：丝裂霉素,分类Classification,1.概述Introduction,6,复制,翻译,转录,7,根据抗生素的化学结构分类-内酰胺类(-lactam)：青霉素类(penicillins)头孢菌素类(cephalosporins)氨基糖苷类(aminoglycosides)：链霉素、庆大霉素大环内酯类(macrolides)：红霉素、麦迪加霉素四环类(tetracyclines)：四环素、土霉素多肽类(polymyxin)：多粘菌素,分类Classification,1.概述Introduction,8,naturalpenicillinR=,CH2-CO-,9,-内酰胺环,10,两性霉素,Antibioticsaresocomplextheycanonlybesynthesizedinalivingsystem,11,产生菌的筛选抗菌性试验提取、精制、鉴定毒性试验药理和临床试验,ScreeningofastrainAntimicrobialtrialIsolation,purificationandidentificationToxicologicaltrialMedicinal&clinicaltrial,2生产前期研究ResearchinLab,12,Agardiffusionassay,agarplate琼脂培养基,13,分析仪器Analyticalinstruments,现代分析仪器：核磁共振毛细管电泳气相色谱高效液相色谱质谱,Modernanalyticalinstruments:Nuclearmagneticresonance(NMR)Capillaryelectrophoresis(CE)Gaschromatography(GC)Highperformanceliquidchromatography(HPLC)Massspectroscopy(MS)AutomationSensitivityAccuracy自动灵敏准确,14,人工诱变法：紫外线、X-线、r-线照射亚硝基胍、亚硝酸、秋水仙素、氮芥等诱变剂处理,ConventionalstrainselectionPhysicalmutageneg.UltravioletRadiationChemicalmutageneg.Nitrosoguanidine(NTG)Improvedstraincanbeselectedempirically,3.抗生素生产菌改良StrainImprovement,15,基因工程GeneticEngineering,3.抗生素生产菌改良StrainImprovement,RecombinantDNATechnology（DNA重组技术）,克隆,16,StrainImprovementScale-up,Strain出发菌株,Agarplate平板技术,PrimaryshakeflaskEvaluation摇瓶培养,Secondaryshakeflaskevaluationdifferentmedia二级摇瓶培养各种培养基,LabfermentorevaluationProductionCondition小型发酵罐模拟生产条件,Mastercellbank,Workingcellbank,InoculumDevelopment菌种扩大培养,PilotplantEvaluation中试生产,ProductionEvaluation生产,6000,300,30,3,1,Possiblerecycle,17,18,Scale-up（比拟放大）,models,ELGaddenJr,模拟,19,摇瓶发酵Shake-flaskfermentation,10-50mlin125-500mlflaskBatchonlyLimitedcontrol:temperatureSlowmetabolizingcarbohydrateAmbientpressureBuffersneededtocontrolpHIn-processsamplingdifficultVolumedecreasebyevaporationSolidgrowthonsidewallsAntifoamnotneededAgitationlimited:shakerspeedNocontrolofdissolvedoxygen,装液量：10%(250ml)间歇培养温控受限制代谢碳水化合物慢常压缓冲液控制pH在线取样难容量减少瓶壁上结垢无需消泡搅拌受限制无法控制溶解氧,20,搅拌罐发酵Stirredtankfermentation,10-100,000升容量间歇和补料培养连续控制：pH，温度需要铵盐或氨气2大气压无需缓冲液在线取样易补料,容量增加生长均匀一致需消泡多种搅拌控制溶解氧:通气,加压,10-100,000litervesselsBatchandfeedpossibleContinuouscontrol:pH,temperatureAmmoniumsaltsorammonianeededPressureattwoatmospherespossibleNobuffersnecessaryIn-processsamplingeasyVolumeincreasebysugarfeedVeryuniformgrowthAntifoamoftenrequiredWidevarietyofimpellersandbafflesDissolvedoxygencontrolledbyaeration,pressure,21,摇瓶发酵Shake-flask,装液量：10%(250ml)间歇培养温控受限制代谢碳水化合物慢常压缓冲液控制pH在线取样难容量减少瓶壁上结垢无需消泡搅拌受限制无法控制溶解氧,10-100,000升容量间歇和补料培养连续控制：pH，温度需要铵盐或氨气2大气压无需缓冲液在线取样易补料,容量增加生长均匀一致需消泡多种搅拌控制溶解氧:通气,加压,搅拌罐发酵Stirredtank,22,菌种种子制备发酵发酵液预处理提取及精制成品包装,StrainPreparationofseedFermentationTreatmentofbrothIsolation&purificationPackageofproduct,4.抗生素生产工艺过程AntibioticsProductionProcessing,23,Aschemeofthematerialflowinanantibioticproduction,24,Aschemeofthematerialflowinanantibioticproduction,连消,发酵罐,缓冲罐,种子罐,25,发酵生产原料MaterialsforFermentation,Carbonsource:Beet&canemolassesGlucose,lactose,starchEthanol,methanolGlycerolMaltosesyrup,cornsyrupCottonseedoil,peanutoil,soyoilLardoilMethyloleateTallow,碳源:甜菜和甘蔗糖蜜葡萄糖，乳糖，淀粉乙醇，甲醇甘油麦芽糖浆，玉米糖浆绵籽油，花生油，豆油猪油油酸甲酯油脂,26,氮源:绵籽粉玉米粉和玉米浸出液大豆粉和大豆面花生粉菜籽粉牛血，酪蛋白水解液乳清鱼粉酵母酵母膏肉粉和骨粉,Nitrogensource:CottonseedmealCornmeal&steepliquorSoybeanmeal&flourPeanutmealRapeseedmealBeefblood,CaseinhydrolysateWheyFishmealWholeyeastYeastextractMeat&bonemeal,发酵生产原料MaterialsforFermentation,27,培养基MediaforCellGrowth&Finalproduction,ComponentsRange%组成Glucose/sucrose3.0-5.0葡萄糖/蔗糖Starch淀粉3.0-5.0Cornsteepliquor3.0-5.0Calciumcarbonate0.5-1.0碳酸钙Phosphate磷酸盐0.1-0.5Ammoniumsulphate0.1-1.0硫酸胺Urea尿素0.1-0.5Oil油0.1-0.5,Fermentation发酵培养基,ComponentsRange%组成Glucose0.5-1.0Starch0.5-5.0Cornsteepliquor5.0-8.0玉米浸提液Soyflour1.0-5.0Phosphate0.1-1.0Ammoniumsulphate0.5-1.0andfedperiodicallyOil1.0andfedcontinuouslyCornsyrupfedcontinuously玉米糖浆连续加料,Tankseed种子培养基,28,StirredtankFermentation搅拌发酵,29,5.发酵控制FermentationControl,SampleAnalysispHDOSugarAmmoniaPhosphateSulphateProductsPrecursorsContamination,Pressureprobe,Levelprobe,pHprobe,Tbe,DOprobe,Antifoam,Acid/Base,Cooling,Air/agitation,Sugar/Oilfeed,30,Scale,WeightSignal,PumpSpeed,Amplifier,Controller,SupervisoryComputer,FlowRateControl流量控制,31,发酵控制Fermentationcontrol,32,菌种(strain)：产黄青霉菌(Penicilliumchrysogenum)孢子培养(sporecultivation)：25C,6-7d种子培养(seedcultivation)：25C，2-3d发酵培养(fermentation)：22-26C,6-7d