發(fā)酵工程抗生素發(fā)酵生產(chǎn)技術(shù)概述

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1、Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,*,*,韓 北 忠,,中國農(nóng)業(yè)大學(xué) 食品學(xué)院,Han Bei-zhong,,,Prof.,PhD,College of Food Science & Nutritional Engineering,China Agricultural University,抗 生 素 發(fā) 酵 生 產(chǎn) 技 術(shù),Antibiotics Fermentation Produc

2、tion,1,概述,生產(chǎn)前期研究,生產(chǎn)菌改良,生產(chǎn)工藝過程,發(fā)酵條件的控制,提取和精制,Introduction,Research in Lab,Strain improvement,Production processing,Fermentation control,Isolation & purification,抗 生 素 發(fā) 酵 生 產(chǎn) 技 術(shù),Antibiotics Fermentation Production,2,抗生素,是生物在其生產(chǎn)活動過程中所產(chǎn)生，并能在低微濃度下有選擇性地,抑制或殺滅,其他微生物或腫瘤細胞的,有機物,。,,Antibiotics,,Any,organic

3、substances,produced by various organisms that have the power of,arresting,the,,growth of,other microorganisms,or of,destroying,them.,1.,概　述,Introduction,3,歷史,1929,年，英國弗萊明發(fā)現(xiàn)青霉素,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,,

4、The first major antibiotic to be commercialized,,1.,概　述,Introduction,4,根據(jù)抗生素的作用機制分類,(Modes of Action of Antibiotics),,抑制細胞壁合成,(Cell Wall Synthesis),,： 青霉素,抑制細胞膜功能,(Cell Membrane Damage),,： 多烯類抗生素,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,,

5、,分　類,Classification,1.,概　述,Introduction,5,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,β-,內(nèi)酰胺類,(β-lactam),： 青霉素類,(p,enicillins),,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,(t,etracyclines),： 四環(huán)素、土霉素,多 肽 類,(polymyxin),：

6、多粘菌素,,,分　類,Classification,1.,概　述,Introduction,7,,H,N,O,N,S,CH,3,CH,3,H,COO-,H,?,-,lactam ring,β-,內(nèi)酰胺 環(huán),R,natural penicillin,R =,CH,2,-CO-,8,β-,內(nèi)酰胺 環(huán),9,兩性霉素,Antibiotics are,,so complex,they can only be synthesized,,in a living system,,10,產(chǎn)生菌,的,的篩選,抗菌性,試,試驗,提取、,精,精制、,鑒,鑒定,毒性試,驗,驗,藥理和,臨,臨床試,驗,驗,Scree

7、ningofastrain,Antimicrobial trial,Isolation, purificationandidentification,Toxicological trial,Medicinal&clinicaltrial,2,．,生產(chǎn)前,期,期研究,ResearchinLab,11,Agar diffusionassay,agar plate,瓊脂培,養(yǎng),養(yǎng)基,lawn of test bacteria,測 試 菌 苔,filter papers soaked,with test compounds,含 藥 物 濾 紙,,zones of inhibition,(,no gro

8、wth),,抑菌圈,,12,分 析,儀,儀,器,器,Analytical instruments,現(xiàn)代分,析,析儀器,：,：,核磁共,振,振,毛細管,電,電泳,氣相色,譜,譜,高效液,相,相色譜,質(zhì)譜,Modernanalyticalinstruments:,Nuclear magnetic resonance(NMR),Capillaryelectrophoresis(CE),Gaschromatography(GC),High performance liquidchromatography(HPLC),Mass spectroscopy(MS),,AutomationSensitivit

9、yAccuracy,自　動,靈,靈　敏,準,準　確,13,人工誘,變,變法：,紫外線、,X-,線、,r-,線照射,亞硝基,胍,胍、亞硝,酸,酸、秋,水,水仙素,、,、氮芥,等,等誘變,劑,劑處理,Conventionalstrain selection,Physicalmutagen,eg.UltravioletRadiation,Chemicalmutagen,eg.Nitrosoguanidine(NTG),Improvedstraincan be selected empirically,3.,抗生素,生產(chǎn)菌,改,改良,StrainImprovement,14,基因工,程,程,Genet

10、ic Engineering,,,3.,抗生素,生產(chǎn)菌,改,改良,StrainImprovement,RecombinantDNATechnology,（,DNA,重組技,術(shù),術(shù),）,克隆,15,StrainImprovementScale-up,Strain,出發(fā)菌,株,株,Agar plate,平板技,術(shù),術(shù),Primary,shakeflask,Evaluation,搖瓶培,養(yǎng),養(yǎng),Secondary,shakeflask,evaluation,differentmedia,二級搖,瓶,瓶培養(yǎng),各種培,養(yǎng),養(yǎng)基,,Labfermentor,evaluation,Production,Co

11、ndition,小型發(fā),酵,酵罐,模擬生,產(chǎn),產(chǎn)條件,Master,cell bank,Working,cell bank,Inoculum,Development,菌種擴,大,大培養(yǎng),Pilotplant,Evaluation,中試生,產(chǎn),產(chǎn),Production,Evaluation,生產(chǎn),6000,300,30,3,1,Possible,recycle,16,17,Scale-up,（,比擬放,大,大）,models,EL Gadden Jr,<,10,dm,3,Good product,Good productivity,> 200 m,3,Same productivity ?,Li

12、mitations ?,模,擬,擬,18,搖　瓶,發(fā),發(fā),酵,酵,Shake-flaskfermentation,10-50mlin125-500 ml flask,Batchonly,Limited control: temperature,Slow metabolizingcarbohydrate,Ambient pressure,Buffers neededtocontrol pH,In-process sampling difficult,Volumedecreasebyevaporation,Solidgrowthonsidewalls,Antifoamnotneeded,Agita

13、tionlimited:shaker speed,Nocontrolofdissolvedoxygen,裝,液,液,量,量,：,：,10%(~250ml),間,歇,歇,培,培,養(yǎng),養(yǎng),溫,控,控,受,受,限,限,制,制,代,謝,謝,碳,碳,水,水,化,化,合,合,物,物,慢,慢,常,壓,壓,緩,沖,沖,液,液,控,控,制,制,pH,在,線,線,取,取,樣,樣,難,難,容,量,量,減,減,少,少,瓶,壁,壁,上,上,結(jié),結(jié),垢,垢,無,需,需,消,消,泡,泡,攪,拌,拌,受,受,限,限,制,制,無,法,法,控,控,制,制,溶,溶,解,解,氧,氧,19,攪,拌,拌,罐,罐,發(fā),發(fā),酵,酵,Stir

14、redtankfermentation,10-100,000,升,容,容,量,量,間,歇,歇,和,和,補,補,料,料,培,培,養(yǎng),養(yǎng),連,續(xù),續(xù),控,控,制,制,：,：,pH,，,溫,度,度,需,要,要,銨,銨,鹽,鹽,或,或,氨,氨,氣,氣,2,大,氣,氣,壓,壓,無,需,需,緩,沖,沖,液,液,在,線,線,取,取,樣,樣,易,易,補,料,料,,,容,量,量,增,增,加,加,生,長,長,均,均,勻,勻,一,一,致,致,需,消,消,泡,泡,多,種,種,攪,攪,拌,拌,控,制,制,溶,溶,解,解,氧,氧,:,通,氣,氣,,,加,壓,壓,,10-100,000litervessels,Batcha

15、ndfeedpossible,Continuouscontrol:pH,temperature,Ammoniumsaltsorammonianeeded,Pressureattwoatmospherespossible,Nobuffersnecessary,In-processsamplingeasy,Volumeincreasebysugarfeed,Veryuniformgrowth,Antifoamoftenrequired,Widevarietyofimpellersandbaffles,Dissolvedoxygencontrolledby,aeration,pressure,20,

16、搖,瓶,瓶,發(fā),發(fā),酵,酵,Shake-flask,裝,液,液,量,量,：,：,10%(~250ml),間,歇,歇,培,培,養(yǎng),養(yǎng),溫,控,控,受,受,限,限,制,制,代,謝,謝,碳,碳,水,水,化,化,合,合,物,物,慢,慢,常,壓,壓,緩,沖,沖,液,液,控,控,制,制,pH,在,線,線,取,取,樣,樣,難,難,容,量,量,減,減,少,少,瓶,壁,壁,上,上,結(jié),結(jié),垢,垢,無,需,需,消,消,泡,泡,攪,拌,拌,受,受,限,限,制,制,無,法,法,控,控,制,制,溶,溶,解,解,氧,氧,10-100,000,升,容,容,量,量,間,歇,歇,和,和,補,補,料,料,培,培,養(yǎng),養(yǎng),連,續(xù),

17、續(xù),控,控,制,制,：,：,pH,，,溫,度,度,需,要,要,銨,銨,鹽,鹽,或,或,氨,氨,氣,氣,2,大,氣,氣,壓,壓,無,需,需,緩,沖,沖,液,液,在,線,線,取,取,樣,樣,易,易,補,料,料,,,容,量,量,增,增,加,加,生,長,長,均,均,勻,勻,一,一,致,致,需,消,消,泡,泡,多,種,種,攪,攪,拌,拌,控,制,制,溶,溶,解,解,氧,氧,:,通,氣,氣,,,加,壓,壓,攪,拌,拌,罐,罐,發(fā),發(fā),酵,酵,Stirredtank,21,菌,種,種,→,種,子,子,制,制,備,備,→,發(fā),酵,酵,→,發(fā),酵,酵,液,液,預(yù),預(yù),處,處,理,理,→,提,取,取,及,及,精,

18、精,制,制,→,成,品,品,包,包,裝,裝,Strain,→,Preparationofseed,→,Fermentation,→,Treatmentofbroth,→,Isolation&purification,→,Packageofproduct,4.,抗,生,生,素,素,生,生,產(chǎn),產(chǎn),工,工,藝,藝,過,過,程,程,AntibioticsProductionProcessing,22,A schemeofthematerialflow inanantibiotic production,23,A schemeofthematerialflow inanantibiotic produ

19、ction,連消,發(fā)酵罐,緩沖罐,種子罐,24,發(fā)酵生產(chǎn),原,原料,Materials for Fermentation,Carbon source:,Beet& canemolasses,Glucose,lactose, starch,Ethanol,methanol,Glycerol,Maltosesyrup, cornsyrup,,Cottonseed oil,peanut oil,soyoil,Lardoil,Methyl oleate,Tallow,碳源,:,甜菜和甘,蔗,蔗糖蜜,葡萄糖，,乳,乳糖，淀,粉,粉,乙醇，甲,醇,醇,甘油,麥芽糖漿,，,，玉米糖,漿,漿,,綿,籽,油，花

20、生,油,油，豆油,豬油,油酸甲酯,油脂,25,氮源,:,綿,籽,粉,玉米粉和,玉,玉米浸出,液,液,大豆粉和,大,大豆面,花生粉,菜籽粉,,牛血，酪,蛋,蛋白水解,液,液,乳清,魚粉,酵母,酵母膏,肉粉和骨,粉,粉,Nitrogensource:,Cottonseedmeal,Cornmeal&steepliquor,Soybeanmeal&flour,Peanutmeal,Rapeseedmeal,,Beefblood,Caseinhydrolysate,Whey,Fishmeal,Wholeyeast,Yeastextract,Meat&bonemeal,發(fā),酵,酵,生,生,產(chǎn),產(chǎn),原,原

21、,料,料,MaterialsforFermentation,26,培,養(yǎng),養(yǎng),基,基,MediaforCellGrowth&Finalproduction,ComponentsRange%,組,成,成,Glucose/sucrose3.0-5.0,葡,萄,萄,糖,糖,/,蔗,糖,糖,Starch,淀,粉,粉,3.0-5.0,Cornsteepliquor3.0-5.0,Calciumcarbonate0.5-1.0,碳,酸,酸,鈣,鈣,Phosphate,磷,酸,酸,鹽,鹽,0.1-0.5,Ammoniumsulphate0.1-1.0,硫,酸,酸,胺,胺,Urea,尿,素,素,0.1-0.5

22、,Oil,油,0.1-0.5,,Fermentation,發(fā),酵,酵,培,培,養(yǎng),養(yǎng),基,基,ComponentsRange%,組,成,成,Glucose0.5-1.0,Starch0.5-5.0,Cornsteepliquor5.0-8.0,玉,米,米,浸,浸,提,提,液,液,Soyflour1.0-5.0,Phosphate0.1-1.0,Ammoniumsulphate0.5-1.0,andfedperiodically,Oil1.0,andfedcontinuously,Cornsyrupfedcontinuously,玉,米,米,糖,糖,漿,漿,連,連,續(xù),續(xù),加,加,料,料,,Ta

23、nkseed,種,子,子,培,培,養(yǎng),養(yǎng),基,基,27,StirredtankFermentation,攪,拌,拌,發(fā),發(fā),酵,酵,28,5.,發(fā),酵,酵,控,控,制,制,FermentationControl,SampleAnalysis,pH,DO,Sugar,Ammonia,Phosphate,Sulphate,Products,Precursors,Contamination,,Pressureprobe,Levelprobe,pHprobe,Temp.probe,DOprobe,Antifoam,Acid/Base,Cooling,Air/agitation,Sugar/Oil,fe

24、ed,29,,Scale,WeightSignal,PumpSpeed,Amplifier,Controller,Supervisory,Computer,FlowRateControl,流,量,量,控,控,制,制,30,發(fā),酵,酵,控,控,制,制,Fermentationcontrol,31,菌,種,種,(strain),：,產(chǎn),產(chǎn),黃,黃,青,青,霉,霉,菌,菌,(,Penicilliumchrysogenum,),孢,子,子,培,培,養(yǎng),養(yǎng),(sporecultivation),：,25,oC,,6-7d,種,子,子,培,培,養(yǎng),養(yǎng),(seedcultivation),：,25,oC,，

25、,2-3d,發(fā),酵,酵,培,培,養(yǎng),養(yǎng),(fermentation),：,22-26,oC,,6-7d,加,糖,糖,控,控,制,制,(sugarfeeding),：,殘,殘,糖,糖,降,降,至,至,0.6,％,,青,霉,霉,素,素,發(fā),發(fā),酵,酵,(1),PenicillinFermentation,32,補,氮,氮,(nitrogenfeeding),：,氨,氨,控,控,制,制,在,在,0.01-0.05,％,前,體,體,(precursor),：,殘,殘,余,余,苯,苯,乙,乙,酰,酰,胺,胺,0.05-0.08,％,溫,度,度,控,控,制,制,(temperature),：,前,前,期,

26、期,25-26,oC,后,期,期,23,oC,通,氣,氣,與,與,攪,攪,拌,拌,(aeration&stir),：,1:0.8VVM,泡,沫,沫,(foam),：,天,天,然,然,油,油,脂,脂,,,如,豆,豆,油,油,,青,霉,霉,素,素,發(fā),發(fā),酵,酵,(2),PenicillinFermentation,33,PenicillinFermentationProfile,變,化,化,曲,曲,線,線,34,9,、,靜,靜,夜,夜,四,四,無,無,鄰,鄰,，,，,荒,荒,居,居,舊,舊,業(yè),業(yè),貧,貧,。,。,。,。12,月,月-2212,月,月-22,Thursday,December29

27、,2022,10,、,雨,雨,中,中,黃,黃,葉,葉,樹,樹,，,，,燈,燈,下,下,白,白,頭,頭,人,人,。,。,。,。04:48:2004:48:2004:48,12/29/20224:48:20AM,11,、以,我,我獨,沈,沈久,，,，愧,君,君相,見,見頻,。,。。12,月,月-2204:48:2004:48,Dec-2229-Dec-22,12,、故,人,人江,海,海別,，,，幾,度,度隔,山,山川,。,。。04:48:2004:48:2004:48,Thursday,December29,2022,13,、乍見翻,疑,疑夢，相,悲,悲各問年,。,。。12月-2212月-2204

28、:48:2004:48:20,December29,2022,14,、他鄉(xiāng)生,白,白發(fā)，舊,國,國見青山,。,。。29 十,二,二月 20224:48:20,上,上午04:48:2012月-22,15,、比不,了,了得就,不,不比，,得,得不到,的,的就不,要,要。。。十二月224:48 上,午,午12月-2204:48,December29,2022,16,、行動,出,出成果,，,，工作,出,出財富,。,。。2022/12/29 4:48:2004:48:20,29December2022,17,、做,前,前，,能,能夠,環(huán),環(huán)視,四,四周,；,；做,時,時，,你,你只,能,能或,者,者最,

29、好,好沿,著,著以,腳,腳為,起,起點,的,的射,線,線向,前,前。,。,。4:48:20,上,上午4:48,上,上,午,午04:48:2012,月,月-22,9,、沒有,失,失敗，,只,只有暫,時,時停止,成,成功！,。,。12月-2212月-22,Thursday,December29, 2022,10,、很多事,情,情努力了,未,未必有結(jié),果,果，但是,不,不努力卻,什,什么改變,也,也沒有。,。,。04:48:2004:48:2004:48,12/29/2022 4:48:20 AM,11,、成,功,功就,是,是日,復(fù),復(fù)一,日,日那,一,一點,點,點小,小,小努,力,力的,積,積累,

30、。,。。12,月,月-2204:48:2004:48,Dec-2229-Dec-22,12,、,世,世,間,間,成,成,事,事,，,，,不,不,求,求,其,其,絕,絕,對,對,圓,圓,滿,滿,，,，,留,留,一,一,份,份,不,不,足,足,，,，,可,可,得,得,無,無,限,限,完,完,美,美,。,。,。,。04:48:2004:48:2004:48,Thursday,December29,2022,13,、不,知,知香,積,積寺,，,，數(shù),里,里入,云,云峰,。,。。12,月,月-2212,月,月-2204:48:2004:48:20,December29,2022,14,、意志堅,強,強的

31、人能,把,把世界放,在,在手中像,泥,泥塊一樣,任,任意揉捏,。,。29 十,二,二月 20224:48:20,上,上午04:48:2012月-22,15,、楚塞,三,三湘接,，,，荊門,九,九派通,。,。。。十二月224:48 上,午,午12月-2204:48,December29,2022,16,、少年十,五,五二十時,，,，步行奪,得,得胡馬騎,。,。。2022/12/29 4:48:2004:48:20,29 December2022,17,、空山新,雨,雨后，天,氣,氣晚來秋,。,。。4:48:20,上,上午4:48,上,上午04:48:2012月-22,9,、,楊,楊,柳,柳,散,

32、散,和,和,風(fēng),風(fēng),，,，,青,青,山,山,澹,澹,吾,吾,慮,慮,。,。,。,。12,月,月-2212,月,月-22,Thursday,December29,2022,10,、閱讀,一,一切好,書,書如同,和,和過去,最,最杰出,的,的人談,話,話。04:48:2004:48:2004:48,12/29/2022 4:48:20AM,11,、越,是,是沒,有,有本,領(lǐng),領(lǐng)的,就,就越,加,加自,命,命不,凡,凡。12,月,月-2204:48:2004:48,Dec-2229-Dec-22,12,、越是無,能,能的人，,越,越喜歡挑,剔,剔別人的,錯,錯兒。04:48:2004:48:2004

33、:48,Thursday, December 29, 2022,13,、知人者,智,智，自知,者,者明。勝,人,人者有力,，,，自勝者,強,強。12月-2212月-2204:48:2004:48:20,December29,2022,14,、意,志,志堅,強,強的,人,人能,把,把世,界,界放,在,在手,中,中像,泥,泥塊,一,一樣,任,任意,揉,揉捏,。,。29,十,十,二,二月20224:48:20,上,上午04:48:2012,月,月-22,15,、,最,最,具,具,挑,挑,戰(zhàn),戰(zhàn),性,性,的,的,挑,挑,戰(zhàn),戰(zhàn),莫,莫,過,過,于,于,提,提,升,升,自,自,我,我,。,。,。,。十,

34、二,二,月,月224:48,上,上,午,午12,月,月-2204:48,December29,2022,16,、,業(yè),業(yè),余,余,生,生,活,活,要,要,有,有,意,意,義,義,，,，,不,不,要,要,越,越,軌,軌,。,。2022/12/294:48:2004:48:20,29December2022,17,、一個,人,人即使,已,已登上,頂,頂峰，,也,也仍要,自,自強不,息,息。4:48:20 上,午,午4:48 上,午,午04:48:2012月-22,,MOMODA POWERPOINT,Lorem ipsum dolor sit amet, consectetur adipiscing elit. Fusce id urna blandit, eleifend nulla ac, fringilla purus. Nulla iaculis tempor felis ut cursus.,感謝您,的,的下載,觀,觀看,專家告,訴,訴,