10抗生素生产工艺(4)

AntibioticsFermentationProduction抗生素发酵生产技术1.概述2.生产前期研究3.生产菌改良4.生产工艺过程5.发酵条件的控制6.提取和精制1.Introduction2.ResearchinLab3.Strainimprovement4.Productionprocessing5.Fermentationcontrol6.Isolation&purification抗生素发酵生产技术AntibioticsFermentationProduction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-scaleThefirstmajorantibiotictobecommercialized1.概述Introduction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Classification1.概述Introduction复制翻译转录根据抗生素的化学结构分类β-内酰胺类(β-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Classification1.概述IntroductionHNONSCH3CH3HCOO-H-lactamringβ-内酰胺环RnaturalpenicillinR=CH2-CO-β-内酰胺环两性霉素Antibioticsaresocomplextheycanonlybesynthesizedinalivingsystem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&clinicaltrial2．生产前期研究ResearchinLabAgardiffusionassayagarplate琼脂培养基lawnoftestbacteria测试菌苔filterpaperssoakedwithtestcompounds含药物滤纸zonesofinhibition(nogrowth)抑菌圈分析仪器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)Improvedstraincanbeselectedempirically3.抗生素生产菌改良StrainImprovement基因工程GeneticEngineering3.抗生素生产菌改良StrainImprovementRecombinantDNATechnology（DNA重组技术）克隆StrainImprovementScale-upStrain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MastercellbankWorkingcellbankInoculumDevelopment菌种扩大培养PilotplantEvaluation中试生产ProductionEvaluation生产60003003031PossiblerecycleScale-up（比拟放大）modelsELGaddenJr10dm3GoodproductGoodproductivity200m3•Sameproductivity?•Limitations?模拟摇瓶发酵Shake-flaskfermentation•10-50mlin125-500mlflask•Batchonly•Limitedcontrol:temperature•Slowmetabolizingcarbohydrate•Ambientpressure•BuffersneededtocontrolpH•In-processsamplingdifficult•Volumedecreasebyevaporation•Solidgrowthonsidewalls•Antifoamnotneeded•Agitationlimited:shakerspeed•Nocontrolofdissolvedoxygen•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•10-100,000litervessels•Batchandfeedpossible•Continuouscontrol:pH,temperature•Ammoniumsaltsorammonianeeded•Pressureattwoatmospherespossible•Nobuffersnecessary•In-processsamplingeasy•Volumeincreasebysugarfeed•Veryuniformgrowth•Antifoamoftenrequired•Widevarietyofimpellersandbaffles•Dissolvedoxygencontrolledbyaeration,pressure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菌种→种子制备→发酵→发酵液预处理→提取及精制→成品包装Strain→Preparationofseed→Fermentation→Treatmentofbroth→Isolation&purification→Packageofproduct4.抗生素生产工艺过程AntibioticsProductionProcessingAschemeofthematerialflowinanantibioticproductionAschemeofthematerialflowinanantibioticproduction连消发酵罐缓冲罐种子罐发酵生产原料MaterialsforFermentationCarbonsource:•Beet&canemolasses•Glucose,lactose,starch•Ethanol,methanol•Glycerol•Maltosesyrup,cornsyrup•Cottonseedoil,peanutoil,soyoil•Lardoil•Methyloleate•Tallow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:•Cottonseedmeal•Cornmeal&steepliquor•Soybeanmeal&flour•Peanutmeal•Rapeseedmeal•Beefblood,Caseinhydrolysate•Whey•Fishmeal•Wholeyeast•Yeastextract•Meat&bonemeal发酵生产原料MaterialsforFermentation培养基MediaforCellGrowth&FinalproductionComponentsRange%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0.1-0.5Fermentation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StirredtankFermentation搅拌发酵tpogoCgFogiColollkACVorolsClcVtoloC5.发酵控制FermentationControlSampleAnalysis•pH•DO•Sugar•Ammonia•Phosphate•Sulphate•Products•Precursors•ContaminationPressureprobeLevelprobepHprobeTemp.probeDOprobeAntifoamAcid/BaseCoolingAir/agitationSugar/OilfeedScaleWeightSignalPumpSpe