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1、1,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,IntroductionResearch in LabStrain improvementProduction processingFermentation controlIsolation&purification,抗 生 素 发 酵 生 产 技 术Antibiotics Fermentation Production,2,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。,Antibiotics Any organic substances produced by va
2、rious organisms that have the power of arresting the growth of other microorganisms or of destroying them.,1.概述 Introduction,3,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,History Penicillin was discovered by Fleming in 1929.World War II,fermented in large-scale The first major antibiotic to be comme
3、rcialized,1.概述 Introduction,4,根据抗生素的作用机制分类(Modes of Action of Antibiotics)抑制细胞壁合成(Cell Wall Synthesis)：青霉素抑制细胞膜功能(Cell Membrane Damage)：多烯类抗生素抑制蛋白质合成(Protein Synthesis)：四环素抑制核酸合成(Nucleic Acid Synthesis)：丝裂霉素,分类 Classification,1.概述 Introduction,5,复制,翻译,转录,6,根据抗生素的化学结构分类-内酰胺类(-lactam)：青霉素类(penicillins
4、)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,7,natural penicillin R=,CH2-CO-,8,-内酰胺 环,9,两性霉素,Antibiotics are so complex they can only be synthesized in a living system,10,产生菌的筛选抗菌性
5、试验提取、精制、鉴定毒性试验药理和临床试验,Screening of a strainAntimicrobial trialIsolation,purification and identificationToxicological trial Medicinal&clinical trial,2生产前期研究 Research in Lab,11,Agar diffusion assay,agar plate琼脂培养基,12,分 析 仪 器Analytical instruments,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,Modern analytical instrume
6、nts:Nuclear magnetic resonance(NMR)Capillary electrophoresis(CE)Gas chromatography(GC)High performance liquid chromatography(HPLC)Mass spectroscopy(MS)AutomationSensitivityAccuracy自动灵敏准确,13,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,Conventional strain selectionPhysical mutagen eg.Ultraviolet Radiati
7、onChemical mutagen eg.Nitrosoguanidine(NTG)Improved strain can be selected empirically,3.抗生素生产菌改良 Strain Improvement,14,基因工程Genetic Engineering,3.抗生素生产菌改良 Strain Improvement,Recombinant DNA Technology（DNA重组技术）,克隆,15,Strain Improvement Scale-up,Strain出发菌株,Agar plate平板技术,Primaryshake flaskEvaluation摇瓶
8、培养,Secondary shake flask evaluationdifferent media二级摇瓶培养各种培养基,Lab fermentor evaluationProductionCondition小型发酵罐模拟生产条件,Mastercell bank,Workingcell bank,InoculumDevelopment菌种扩大培养,Pilot plantEvaluation中试生产,ProductionEvaluation生产,6000,300,30,3,1,Possiblerecycle,16,17,Scale-up（比拟放大）,models,EL Gadden Jr,模
9、拟,18,摇瓶发酵 Shake-flask fermentation,10-50 ml in 125-500 ml flaskBatch onlyLimited control:temperatureSlow metabolizing carbohydrateAmbient pressureBuffers needed to control pHIn-process sampling difficultVolume decrease by evaporationSolid growth on side wallsAntifoam not neededAgitation limited:shak
10、er speedNo control of dissolved oxygen,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,19,搅 拌 罐 发 酵 Stirred tank fermentation,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,10-100,000 liter vesselsBatch and feed possibleContinuous control
11、:pH,temperatureAmmonium salts or ammonia neededPressure at two atmospheres possibleNo buffers necessary In-process sampling easyVolume increase by sugar feedVery uniform growthAntifoam often requiredWide variety of impellers and bafflesDissolved oxygen controlled by aeration,pressure,20,摇 瓶 发 酵 Shak
12、e-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 Stirred tank,21,菌种 种子制备 发酵 发酵液预处理 提取及精制 成品包装,Strain Preparation of seed Fermentation Treatment of broth Isolation&purificatio
13、n Package of product,4.抗生素生产工艺过程Antibiotics Production Processing,22,A scheme of the material flow in an antibiotic production,23,A scheme of the material flow in an antibiotic production,连消,发酵罐,缓冲罐,种子罐,24,发酵生产原料 Materials for Fermentation,Carbon source:Beet&cane molassesGlucose,lactose,starchEthano
14、l,methanol GlycerolMaltose syrup,corn syrupCottonseed oil,peanut oil,soy oilLard oil Methyl oleate 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,25,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,Nitrogen source:Cotton seed meal Corn meal&steep liquorSoybean meal&flour Peanut meal
15、 Rapeseed mealBeef blood,Casein hydrolysate Whey Fish mealWhole yeastYeast extractMeat&bone meal,发酵生产原料 Materials for Fermentation,26,培 养 基Media for Cell Growth&Final production,Components Range%组 成Glucose/sucrose 3.0-5.0 葡萄糖/蔗糖Starch 淀粉 3.0-5.0Corn steep liquor 3.0-5.0Calcium carbonate 0.5-1.0 碳酸钙P
16、hosphate 磷酸盐 0.1-0.5Ammonium sulphate 0.1-1.0 硫酸胺Urea 尿素 0.1-0.5Oil 油 0.1-0.5,Fermentation 发酵培养基,Components Range%组 成Glucose 0.5-1.0Starch 0.5-5.0Corn steep liquor 5.0-8.0 玉米浸提液Soy flour 1.0-5.0Phosphate 0.1-1.0Ammonium sulphate 0.5-1.0 and fed periodicallyOil 1.0 and fed continuouslyCorn syrup fed
17、continuously 玉米糖浆 连续加料,Tank seed 种子培养基,27,Stirred tank Fermentation 搅拌发酵,28,5.发 酵 控 制Fermentation Control,Sample AnalysispHDOSugarAmmoniaPhosphateSulphateProductsPrecursorsContamination,Pressure probe,Level probe,pH probe,Temp.probe,DO probe,Antifoam,Acid/Base,Cooling,Air/agitation,Sugar/Oilfeed,29,
18、Scale,Weight Signal,Pump Speed,Amplifier,Controller,SupervisoryComputer,Flow Rate Control 流量控制,30,发 酵 控 制 Fermentation control,31,菌种(strain)：产黄青霉菌(Penicillium chrysogenum)孢子培养(spore cultivation)：25 C,6-7 d种子培养(seed cultivation)：25 C，2-3 d 发酵培养(fermentation)：22-26 C,6-7 d加糖控制(sugar feeding)：残糖降至0.6,青 霉 素 发 酵(1)Penicillin Fermentation,32,补氮(nitrogen feeding)：氨控制在 0.01-0.05前体(precursor)：残余苯乙酰胺 0.05-0.08温度控制(temperature)：前期 25-26 C 后期 23 C通气与搅拌(aeration&stir)：1:0.8 VVM泡沫(foam)：天然油脂,如豆油,青 霉 素 发 酵(2)Penicillin Fermentation,33,Penicillin Fermentation Profile 变化曲线,
