09第九章 發(fā)酵工程各論3--抗生素發(fā)酵工藝學(xué)

1、韓 北 忠 Han Bei-zhong, Prof. PhDCollege of Food Science & Nutritional EngineeringChina Agricultural University抗 生 素 發(fā) 酵 生 產(chǎn) 技 術(shù)Antibiotics Fermentation Production1概述生產(chǎn)前期研究生產(chǎn)菌改進(jìn)生產(chǎn)工藝過程發(fā)酵條件的控制提取和精制IntroductionResearch in LabStrain improvementProduction processingFermentation controlIsolation & purificati

2、on 抗 生 素 發(fā) 酵 生 產(chǎn) 技 術(shù)Antibiotics Fermentation Production2抗生素是生物在其生產(chǎn)活動(dòng)過程中所產(chǎn)生，并能在卑微濃度下有選擇性地抑制或殺滅其他微生物或腫瘤細(xì)胞的有機(jī)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歷史1929年，英國(guó)弗萊明發(fā)現(xiàn)青霉

3、素二戰(zhàn)期間，大規(guī)模發(fā)酵生產(chǎn)世界上最早用于臨床的抗菌素 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根據(jù)抗生素的作用機(jī)制分類 (Modes of Action of Antibiotics) 抑制細(xì)胞壁合成 (Cell Wall Synthesis) ： 青霉素抑制細(xì)胞膜功能 (Cell Membrane Damage) ： 多

4、烯類抗生素抑制蛋白質(zhì)合成 (Protein Synthesis) ： 四環(huán)素抑制核酸合成 (Nucleic Acid Synthesis) ： 絲裂霉素 分類 Classification1. 概述 Introduction5復(fù)制翻譯轉(zhuǎn)錄6根據(jù)抗生素的化學(xué)結(jié)構(gòu)分類-內(nèi)酰胺類 (-lactam)： 青霉素類 (penicillins) 頭孢菌素類 (cephalosporins)氨基糖苷類 (aminoglycosides) ： 鏈霉素、慶大霉素大環(huán)內(nèi)酯類 (macrolides) ： 紅霉素、麥迪加霉素四 環(huán) 類 (tetracyclines) ： 四環(huán)素、土霉素多 肽 類 (polymyxi

5、n) ： 多粘菌素 分類 Classification1. 概述Introduction7HNONSCH3CH3HCOO-H-lactam ring -內(nèi)酰胺 環(huán)Rnatural penicillin R =CH2-CO-8-內(nèi)酰胺 環(huán)9兩性霉素Antibiotics are so complex they can only be synthesized in a living system10產(chǎn)生菌的篩選抗菌性試驗(yàn)提取、精制、鑒定毒性試驗(yàn)藥理和臨床試驗(yàn)Screening of a strainAntimicrobial trialIsolation, purification and ide

6、ntificationToxicological trial Medicinal & clinical trial 2生產(chǎn)前期研究 Research in Lab11 Agar diffusion assayagar plate瓊脂培養(yǎng)基lawn of test bacteria測(cè) 試 菌 苔filter papers soakedwith test compounds含 藥 物 濾 紙zones of inhibition(no growth)抑菌圈12分 析 儀 器Analytical instruments現(xiàn)代分析儀器：核磁共振毛細(xì)管電泳氣相色譜高效液相色譜質(zhì)譜Modern analyt

7、ical instruments:Nuclear magnetic resonance (NMR)Capillary electrophoresis(CE)Gas chromatography (GC)High performance liquid chromatography (HPLC)Mass spectroscopy (MS) AutomationSensitivityAccuracy自動(dòng)靈敏準(zhǔn)確13人工誘變法：紫外線、X-線、r-線照射亞硝基胍、亞硝酸、秋水仙素、氮芥等誘變劑處理Conventional strain selectionPhysical mutagen eg. Ult

8、raviolet RadiationChemical mutagen eg. Nitrosoguanidine (NTG)Improved strain can be selected empirically 3. 抗生素生產(chǎn)菌改進(jìn) Strain Improvement14基因工程Genetic Engineering 3. 抗生素生產(chǎn)菌改進(jìn) Strain ImprovementRecombinant DNA Technology （DNA重組技術(shù)）克隆15Strain Improvement Scale-upStrain出發(fā)菌株Agar plate平板技術(shù)Primaryshake flask

9、Evaluation搖瓶培養(yǎng)Secondary shake flask evaluationdifferent media二級(jí)搖瓶培養(yǎng)各種培養(yǎng)基Lab fermentor evaluationProductionCondition小型發(fā)酵罐模擬生產(chǎn)條件Mastercell bankWorkingcell bankInoculumDevelopment菌種擴(kuò)大培養(yǎng)Pilot plantEvaluation中試生產(chǎn)ProductionEvaluation生產(chǎn)60003003031Possiblerecycle1617Scale-up（比較放大）models EL Gadden Jr 200 m3S

10、ame productivity ?Limitations ?模 擬18搖瓶發(fā)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裝液量：10% (250ml)間歇培養(yǎng)溫控受限制代謝碳水化合物慢常壓緩沖液控制pH在線取樣難容量減少瓶壁上結(jié)垢無需消泡攪拌受限制無法控制溶解氧19攪 拌 罐 發(fā) 酵 Stirred tank fermentation10-100,000升容量間歇和補(bǔ)料培養(yǎng)連續(xù)控制：pH，溫度需要銨鹽或氨氣2 大氣壓無需緩沖液在線取樣易補(bǔ)料,容量增加 生長(zhǎng)均勻一致需消泡多種攪拌 控制溶解氧:通氣,加壓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搖 瓶 發(fā) 酵 Shake-flask 裝液量：10% (250ml)間歇培養(yǎng)溫控受限制代謝碳水化合物慢常壓緩沖液控制pH在線取樣難容量減少瓶壁上結(jié)垢無需消泡攪拌受限制無法控制溶解氧10-100,000升容量間歇和補(bǔ)料培養(yǎng)連續(xù)控制：pH，溫度需要銨鹽或氨氣2 大氣壓無需緩沖液在線取樣易補(bǔ)料,容量增加 生長(zhǎng)均勻一致需消泡多種攪拌 控制溶解氧:通氣,加壓攪 拌 罐 發(fā) 酵 Stirred tank 21菌種 種子制備 發(fā)酵 發(fā)酵液預(yù)處理 提取及精制 成品包裝Strain Preparation of seed Fermentation Treatment of b

14、roth Isolation & purification Package of product4. 抗生素生產(chǎn)工藝過程Antibiotics Production Processing22A scheme of the material flow in an antibiotic production 23A scheme of the material flow in an antibiotic production 連消發(fā)酵罐緩沖罐種子罐24發(fā)酵生產(chǎn)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，甲醇甘油麥芽糖漿，玉米糖漿綿籽油，花生油，豆油豬油油酸甲酯油脂25氮源:綿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，酪蛋白水解液乳清魚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 發(fā)酵生產(chǎn)原料 Materials for Fermentation26 培 養(yǎng) 基Media for Cell Growth & Final productionComponents Range % 組 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 碳酸鈣Phosphate 磷酸鹽 0.1-0.5Ammonium sulphate 0.1-1.0 硫酸胺Urea 尿素 0.1-0.5Oil 油 0.1-0.5Fermentation 發(fā)酵培養(yǎng)基 Components Range % 組 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 玉米糖漿 連續(xù)加料 Tank seed 種子培養(yǎng)基27Stirred tank Fermentation 攪拌發(fā)酵285. 發(fā) 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發(fā) 酵 控 制 Fermentation control31菌種 (strain)：產(chǎn)黃青霉菌 (Penicillium chrysogenum)孢子培養(yǎng) (spore cultivation)：25 C, 6-7 d種子培養(yǎng) (seed cultivation)： 25 C， 2-3 d 發(fā)酵培養(yǎng) (fermentation)： 22-26 C, 6-7 d加糖控制