09第九章 发酵工程各论3--抗生素发酵工艺学

1、韩 北 忠 Han Bei-zhong, Prof. PhDCollege of Food Science & Nutritional EngineeringChina Agricultural University抗 生 素 发 酵 生 产 技 术Antibiotics Fermentation Production1概述生产前期研究生产菌改进生产工艺过程发酵条件的控制提取和精制IntroductionResearch in LabStrain improvementProduction processingFermentation controlIsolation & purificati

2、on 抗 生 素 发 酵 生 产 技 术Antibiotics Fermentation Production2抗生素是生物在其生产活动过程中所产生，并能在卑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 various organisms that have the power of arresting the growth of other microorganisms or of destroying them.1. 概述 Introduction3历史1929年，英国弗莱明发现青霉

3、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 commercialized1. 概述 Introduction4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

4、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 Classification1. 概述 Introduction5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) 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 (polymyxi

5、n) ： 多粘菌素 分类 Classification1. 概述Introduction7HNONSCH3CH3HCOO-H-lactam ring -内酰胺 环Rnatural 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 system10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Screening of a strainAntimicrobial trialIsolation, purification and ide

6、ntificationToxicological trial Medicinal & clinical trial 2生产前期研究 Research in Lab11 Agar diffusion assayagar plate琼脂培养基lawn of test bacteria测 试 菌 苔filter papers soakedwith test compounds含 药 物 滤 纸zones of inhibition(no growth)抑菌圈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 analyt

7、ical instruments: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. Ult

8、raviolet RadiationChemical mutagen eg. Nitrosoguanidine (NTG)Improved strain can be selected empirically 3. 抗生素生产菌改进 Strain Improvement14基因工程Genetic Engineering 3. 抗生素生产菌改进 Strain ImprovementRecombinant DNA Technology （DNA重组技术）克隆15Strain Improvement Scale-upStrain出发菌株Agar plate平板技术Primaryshake flask

9、Evaluation摇瓶培养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 bankWorkingcell bankInoculumDevelopment菌种扩大培养Pilot plantEvaluation中试生产ProductionEvaluation生产60003003031Possiblerecycle1617Scale-up（比较放大）models EL Gadden Jr 200 m3S

10、ame productivity ?Limitations ?模 拟18摇瓶发酵 Shake-flask fermentation10-50 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 n

11、ot neededAgitation limited: shaker 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 fermentation10-100,000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 fee

12、d possibleContinuous control: 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 aer

13、ation, pressure20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 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 b

14、roth Isolation & purification Package of product4. 抗生素生产工艺过程Antibiotics Production Processing22A scheme of the material flow in an antibiotic production 23A 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 FermentationCarbon source:Beet & cane molassesGlucose

15、, lactose, starchEthanol, 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 liquorSoyb

16、ean meal & flour Peanut meal Rapeseed mealBeef blood, Casein hydrolysate Whey Fish mealWhole yeastYeast extractMeat & bone meal 发酵生产原料 Materials for Fermentation26 培 养 基Media for Cell Growth & Final productionComponents Range % 组 成Glucose/ sucrose 3.0-5.0 葡萄糖/ 蔗糖Starch 淀粉 3.0-5.0Corn steep liquor 3.

17、0-5.0Calcium carbonate 0.5-1.0 碳酸钙Phosphate 磷酸盐 0.1-0.5Ammonium sulphate 0.1-1.0 硫酸胺Urea 尿素 0.1-0.5Oil 油 0.1-0.5Fermentation 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

18、 and fed continuouslyCorn syrup fed continuously 玉米糖浆 连续加料 Tank seed 种子培养基27Stirred tank Fermentation 搅拌发酵285. 发 酵 控 制Fermentation Control Sample AnalysispHDOSugarAmmoniaPhosphateSulphateProductsPrecursorsContaminationPressure probeLevel probepH probeTemp. probeDO probeAntifoamAcid/BaseCoolingAir/agitationSugar/Oilfeed29ScaleWeight SignalPump SpeedAmplifierControllerSupervisoryComputer Flow Rate Control 流量控制30发 酵 控 制 Fermentation control31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