抗生素发酵生产工艺学.ppt

1、1,抗 生 素 发 酵 生 产 技 术 Antibiotics Fermentation Production,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,Introduction Research in Lab Strain improvement Production processing Fermentation control Isolation & purification,抗 生 素 发 酵 生 产 技 术 Antibiotics Fermentation Production,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

2、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 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,History Penicillin was discovered by Fleming in 1929. Wor

3、ld War II, fermented in large-scale The first major antibiotic to be commercialized,1. 概述 Introduction,5,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,

4、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,natural penicilli

5、n R,CH2-CO,9,内酰胺 环,10,两性霉素,Antibiotics are so complex they can only be synthesized in a living system,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,Screening of a strain Antimicrobial trial Isolation, purification and identification Toxicological trial Medicinal & clinical trial,2生产前期研究 Research in Lab,12,A

6、gar diffusion assay,agar plate 琼脂培养基,13,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 instruments: Nuclear magnetic resonance (NMR) Capillary electrophoresis(CE) Gas chromatography (GC) High performance liquid chromatography (HPLC) Mass spectroscopy (MS) Automati

7、onSensitivityAccuracy 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,Conventional strain selection Physical mutagen eg. Ultraviolet Radiation Chemical mutagen eg. Nitrosoguanidine (NTG) Improved strain can be selected empirically,3. 抗生素生产菌改良 Strain Improvement,15,基因工程 Genetic Engineering,3. 抗

8、生素生产菌改良 Strain Improvement,Recombinant DNA Technology （DNA重组技术,克隆,16,Strain Improvement Scale-up,Strain 出发菌株,Agar plate 平板技术,Primary shake flask Evaluation 摇瓶培养,Secondary shake flask evaluation different media 二级摇瓶培养 各种培养基,Lab fermentor evaluation Production Condition 小型发酵罐 模拟生产条件,Master cell bank,W

9、orking cell bank,Inoculum Development 菌种扩大培养,Pilot plant Evaluation 中试生产,Production Evaluation 生产,6000,300,30,3,1,Possible recycle,17,18,Scale-up（比拟放大,models,EL Gadden Jr,模 拟,19,摇瓶发酵 Shake-flask fermentation,10-50 ml in 125-500 ml flask Batch only Limited control: temperature Slow metabolizing carbo

10、hydrate Ambient pressure Buffers needed to control pH In-process sampling difficult Volume decrease by evaporation Solid growth on side walls Antifoam not needed Agitation limited: shaker speed No 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

11、无法控制溶解氧,20,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 vessels Batch and feed possible Continuous control: pH, temperature Ammonium salts or ammonia needed Pressure at two atmospheres possible No

12、buffers necessary In-process sampling easy Volume increase by sugar feed Very uniform growth Antifoam often required Wide variety of impellers and baffles Dissolved oxygen controlled by aeration, 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

13、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,22,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 & purification Package of product,4. 抗生素生产工艺过程 Antibiotics Production

14、Processing,23,A scheme of the material flow in an antibiotic production,24,A scheme of the material flow in an antibiotic production,连消,发酵罐,缓冲罐,种子罐,25,发酵生产原料 Materials for Fermentation,Carbon source: Beet & cane molasses Glucose, lactose, starch Ethanol, methanol Glycerol Maltose syrup, corn syrup C

15、ottonseed oil, peanut oil, soy oil Lard 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,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,Nitrogen source: Cotton seed meal Corn meal & steep liquor Soybean meal & flour Peanut meal Rapeseed meal

16、Beef blood, Casein hydrolysate Whey Fish meal Whole yeast Yeast extract Meat & bone meal,发酵生产原料 Materials for Fermentation,27,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 3.0-5.0 Corn steep liquor 3.0-5.0 Calcium carbonate 0.5-1.0 碳酸

17、钙 Phosphate 磷酸盐 0.1-0.5 Ammonium sulphate 0.1-1.0 硫酸胺 Urea 尿素 0.1-0.5 Oil 油 0.1-0.5,Fermentation 发酵培养基,Components Range % 组 成 Glucose 0.5-1.0 Starch 0.5-5.0 Corn steep liquor 5.0-8.0 玉米浸提液 Soy flour 1.0-5.0 Phosphate 0.1-1.0 Ammonium sulphate 0.5-1.0 and fed periodically Oil 1.0 and fed continuously

18、 Corn syrup fed continuously 玉米糖浆 连续加料,Tank seed 种子培养基,28,Stirred tank Fermentation 搅拌发酵,29,5. 发 酵 控 制Fermentation Control,Sample Analysis pH DO Sugar Ammonia Phosphate Sulphate Products Precursors Contamination,Pressure probe,Level probe,pH probe,Temp. probe,DO probe,Antifoam,Acid/Base,Cooling,Air/agitation,Sugar/Oil f