纳他霉素发酵工艺的研究
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摘要
本论文首先确定了纳他霉素的检测方法—HPLC法；然后对纳他霉素产生菌发酵性能做了初步研究。对其发酵工艺进行了优化，并根据其合成机制，对其代谢调控进行了初步研究。最后在5L自动罐上研究了纳他霉素的间歇补料分批发酵。
确定了HPLC法检测发酵液中纳他霉素的各项参数：在Phenomenex prodigy 5μ ODS3 100A(250mm×4.60mm i.d.，5μm)色谱柱上，以甲醇-水-磷酸(体积比85:15:0.15)为流动相，流速为1mL/min，紫外检测波长为303nm。
在研究菌株发酵性能的基础上，优化了发酵培养基配方：葡萄糖36g/L，大豆蛋白胨20g/L，酵母粉4.5g/L，pH7.9。
根据纳他霉素的合成途径，研究了前体物及代谢调节物对发酵的影响。结果表明，亚铁氰化钾和生物素都对纳他霉素的合成无促进作用；在初始培养基中加入0.5％的磷酸镁并在快速生长末期加入0.1％的丙酸钠能提高纳他霉素的产量，使之达到2.79g/L。
以模糊理论为基础，以生长生产耦合度定量表征了菌株生长和生产的同步程度。发现在发酵过程中生长和生产不同步，耦合度仅为4.09％。
在5L机械搅拌发酵罐中进行了控制pH间歇流加培养实验。确定的工艺参数为：pH控制为45h前保持6.5，45h后保持5.5；从第45h开始流加50％葡萄糖，以发酵液中葡萄糖水平为控制指标，维持葡萄糖浓度20g/L；培养时间为93h。最终纳他霉素产量达到2.97g/L。
The determination methods of natamycin-HPLC was studied. Based on the study of strain's fermentation ability, the medium was optimized and the metabolic control of No5 was studied. Finally, fed-batch fermentation was studied to improve the yield of natamycin in 5L auto-fermentor.
The parameters of HPLC were determined. The operating conditions were Phenomenex prodigy 5 ?ODS3 100A(250mm?.60mm i.d., 5 ?m), at room temperature, methanol-water-phosphoric acid (85:15:0.15, V/V) as mobile phase with a flow rate of l.0mL/min and UV detection at 303nm.
Based on the study of its fermentation ability, the optimum medium for natamycin production was demonstrated (g/L): glucose 36, soybean peptone 20, yeast extract powder 4.5 and pH 7.9.
Based on natamycin's synthesis way, the influence of precursor and regulator on natamycin production was explored. The result showed: K4Fe(CN)6-3H2O and biotin have no positive effect on natamycin production. Adding 0.5% magnesium phosphate at the beginning and 0.1% sodium propionate before the end of logarithmic growth phase can improve natamycin production of flask to 2.739g/L.
Based on the fuzzy theory, ration of coupling growth and expression was introduced to show the extent of biomass growth and natamycin production in fermentation. It was shown that the growth and production were not synchronous in the original process and the ration of coupling was only 4.09%.
The fed-batch fermentation with pH control was determined based on the studies of batch fermentation result in flask. pH was controlled at 6.5 before 45h and 5.5 after that. 50% glucose began to be added by pulsed feeding at 45h, and the glucose concentration was maintained about 20g/L during fermentation process. The fermentation time was 93h. The yield of natamycin under these conditions was 2. 97g/L
确定了HPLC法检测发酵液中纳他霉素的各项参数：在Phenomenex prodigy 5μ ODS3 100A(250mm×4.60mm i.d.，5μm)色谱柱上，以甲醇-水-磷酸(体积比85:15:0.15)为流动相，流速为1mL/min，紫外检测波长为303nm。
在研究菌株发酵性能的基础上，优化了发酵培养基配方：葡萄糖36g/L，大豆蛋白胨20g/L，酵母粉4.5g/L，pH7.9。
根据纳他霉素的合成途径，研究了前体物及代谢调节物对发酵的影响。结果表明，亚铁氰化钾和生物素都对纳他霉素的合成无促进作用；在初始培养基中加入0.5％的磷酸镁并在快速生长末期加入0.1％的丙酸钠能提高纳他霉素的产量，使之达到2.79g/L。
以模糊理论为基础，以生长生产耦合度定量表征了菌株生长和生产的同步程度。发现在发酵过程中生长和生产不同步，耦合度仅为4.09％。
在5L机械搅拌发酵罐中进行了控制pH间歇流加培养实验。确定的工艺参数为：pH控制为45h前保持6.5，45h后保持5.5；从第45h开始流加50％葡萄糖，以发酵液中葡萄糖水平为控制指标，维持葡萄糖浓度20g/L；培养时间为93h。最终纳他霉素产量达到2.97g/L。
The determination methods of natamycin-HPLC was studied. Based on the study of strain's fermentation ability, the medium was optimized and the metabolic control of No5 was studied. Finally, fed-batch fermentation was studied to improve the yield of natamycin in 5L auto-fermentor.
The parameters of HPLC were determined. The operating conditions were Phenomenex prodigy 5 ?ODS3 100A(250mm?.60mm i.d., 5 ?m), at room temperature, methanol-water-phosphoric acid (85:15:0.15, V/V) as mobile phase with a flow rate of l.0mL/min and UV detection at 303nm.
Based on the study of its fermentation ability, the optimum medium for natamycin production was demonstrated (g/L): glucose 36, soybean peptone 20, yeast extract powder 4.5 and pH 7.9.
Based on natamycin's synthesis way, the influence of precursor and regulator on natamycin production was explored. The result showed: K4Fe(CN)6-3H2O and biotin have no positive effect on natamycin production. Adding 0.5% magnesium phosphate at the beginning and 0.1% sodium propionate before the end of logarithmic growth phase can improve natamycin production of flask to 2.739g/L.
Based on the fuzzy theory, ration of coupling growth and expression was introduced to show the extent of biomass growth and natamycin production in fermentation. It was shown that the growth and production were not synchronous in the original process and the ration of coupling was only 4.09%.
The fed-batch fermentation with pH control was determined based on the studies of batch fermentation result in flask. pH was controlled at 6.5 before 45h and 5.5 after that. 50% glucose began to be added by pulsed feeding at 45h, and the glucose concentration was maintained about 20g/L during fermentation process. The fermentation time was 93h. The yield of natamycin under these conditions was 2. 97g/L
引文
[1] Harry Brik. Natamycin [J]. Analytical Profiles of Drug Substances.1994, 10: 514-557.
[2] P.Michael Davidson, Craig H. Doan. Natamycin [J]. University of Idaho.1993, 7:395-407.
[3] J.G.Ostendorp. Natamycin [J]. Antonie van Leeuwenhoek. 1981, 47:170-171.
[4] 方金瑞主编．抗生素[M]．科学社会出版社．1988，45-53，93．
[5] 尤新主编．功能性发酵制品[M]．中国轻工业出版社．2000，350-361．
[6] Lueck-E, Jager-M. Antimicrobial food additives: characteristics, uses, effects.214-218.
[7] Cirigliano-MC, McKenna-RT, Rothenberg-PJ. Method of preserving tea containing beverages[P]. US Patent 5 895 681, 1999.10.
[8] 袁亦丞．纳他霉素[J]．中国食品用化学品．1997，1(3)：37-41．
[9] 陈森，郑鹏然．霉克对沙拉酱抑制霉菌效果观察[J]．中国食品用化学品．1997，1(6)：17-18．
[10] 凌关庭，许胜明．纳他霉素对广式月饼防霉效果的试验[J]．食品工业．1998，1(3)：20-22．
[11] Kaliuzhnaia LD, Murzina EA. An evaluation of the efficacy of Pimaricin in candidiasis of skin and mucous membranes[J]. Lik. Sprava. 1997,10(5): 158-160.
[12] Jani BK, Rinaldi MG, Reinhart WJ. An unusual case of fungal keratitis: Metarrhizium anisopliae [J]. Cornea. 2001, 20(7):765-768.
[13] O'Day DM, Head WS. Advances in the management of keratomycosis and acanthamoeba keratitis [J]. Cornea. 2000, 19(5):681-687.
[14] Domniz Y et al.Successful treatment of paecilomyces lilacinus endophthalmitis after foreign body trauma to the cornea [J]. Cornea. 2001,20(1):109-111.
[15] 熊宗贵．发酵工艺原理[M]．中国医药科技出版社．1995．
[16] 储矩．特殊前体在抗生素生物合成中的作用[J]．国外医药抗生素分册．1999，20(5)：202-212．
[17] 倪孟祥等．柱晶白霉素发酵工艺的代谢调控研究[J]．中国药科大学学报．2000，31(6)：466-469．
[18] 刘刚等．磷酸镁对螺旋霉素发酵的影响[J]．中国抗生素杂志．1997，22(3)：231-233．
[19] 陈长华等．前体对螺旋霉素发酵的影响[J]．中国抗生素杂志．1995，20(5)：392-393．
[20] 张蔚文，焦瑞身．丙酸盐对利福霉素生物合成的调控[J]．中国抗生素杂志．1995，20(4)：268-272．
[21] M．A．艾森申克等．纳他霉素的种菌制备和繁殖方法[P]．CN 1071460A，1993．4．
[22] M．A．艾森申克等．通过控制发酵培养基的pH值以提高纳他霉素的生产速率[P]．CN 1072959A，1993．6．
[23] P．T．奥尔森．连续发酵法生产纳他霉素[P]．CN 1070688A，1993．4．
[24] Eisenschink, Michael Allen. Fermentation process for producing natamycin with additional carbon and nitrogen [P]. P. 262804, 1994.6.
[25] Enshasy HA, Farid MA. Influence of inoculum type and cultivation conditions on natamycin production by Streptomyces natalensis[J]. J. Basic Microbial. 2000, 40(5-6):333-342.
[26] Enshasy HA, Farid MA. Optization of the cultivation medium for natamycin production by Streptomyces natalensis[J].J.Basic Microbial.2000,40(3):157-166.
[27] Aparicio JF, Colina AJ et al. The biosynthetic gene cluster for the 26-membered ring plyene macrolide pimaricin[J]. J Biol Chem.1999,274(15): 10133-10139.
[28] Aparicio JF, Fouces R et al. A complex multienzyme system encoded by five polyketide synthase genes is involved in the biosythesis of the 26-membered plyene macrolide pimaricin in Streptomyces natalensis[J]. Chem. Boil. 2000,7(11):895-905.
[29] Marta V Mendes, Jesus F Aparicio. Complete Nucleotide Sequence and Characterization of pSNA1 from Pimaricin-Producing Streptomyces natalensis That Replicates by a Rolling Circle Mechanism[J]. Plasmid. 2000,43:159-165.
[30] Marta V Mendes, Eliseo Recio. Engineered biosynthesis of novel polyenes:a pimaricin derivative produced by targeted gene disruption in Streptomyces natalensis[J]. Chemistry & Biology. 2001, 8:635-644.
[31] Bordan et al. Process for natamycin recovery[P]. US Patent 5942611, 1999.8.
[32] 杜连祥等编著．工业微生物学实验技术[M]．科学技术出版社．1992．
[33] 邬行彦，雄宗贵，胡章助主编．抗生素生产工艺学[M]．化学工业出版社．1987．
[34] 王克利等．HPLC法测定沙拉酱及干酪中纳他霉素含量方法的研究[J]．中
国卫生检验杂志．1998，8(6)：380-381．
[35]Fletouris-DJ, Botsoglou-NA, Mantis-AJ.Rapid spectrophotometric method for analyzing natamycin in cheese and cheese rind[J]. Journal of AOAC International. 1995, 78(4):1024-1029.
[36]Hutchinson C R, et al. J. Med Chem. 1989, 32(5):929-937.
[37]马逢时，何良材等．应用概率统计．高等教育出版社，1989
[38]董大钧．SAS统计分析．辽宁科学出版社．1996
[39]Gee-Dong Lee, Joong-Ho Kwon. The use of response surface methodology to optimize the Maillar reaction to produce melanoidins with high antioxidative and antimutagenic activities.J Food Sci. Tech. 1998, 33: 375-383
[40]Gawande N. Patkar Y. Biotechnology Bioengineering.1999, 20(2): 58-61
[41]胡运权，张宗浩．试验设计方法．哈尔滨工业大学出版社．1997．
[42]S.J. Kalil, F. Maugeri, et al. Process Biochemistry, 2000, 35: 539-550.
[43]A Konya,A Jekkel,J Suto.J.of Industrial Microbiology Biotechnology,1998, 20: 150-152.
[44]方柏山等．生物工程学报，2000，6(5)：648-650．
[45]Capitan Vallvey, et al. Rapid ultraviolet spectrophotometric and liquid chromatographic methods for the determination of Natamycin in lactoserum matrix[J]. Journal of AOAC International. 2000, 83(4): 802-806.
[46]惠大丰等．统计分析系统SAS软件实用教程[M]．北京航空航天大学出版社．1996．
[47]李颖辉．生物防腐剂纳他霉素发酵生产工艺的研究．天津轻工业学院硕士学位论文，2002．
[48]朱明军．红发夫酵母Phaffia rhodozyma培养生产虾青素的研究．华南理工大学工学博士学位论文，2001．
[49]何艳玲．生物防腐剂纳他霉素发酵工艺的研究．天津轻工业学院硕士学位论文，2002．
[50]李志勇，郭祀远，李琳．藻类对微量元素的生物富集及其应用[J]．微生物学通报．1997，3：12-14．
[51]苗志奇，未作君，元英进．水杨酸在紫杉醇生物合成中诱导作用的研究[J]．生物工程学报．2000，16(4)：509-513．
[52]张磊．基因工程α-2b干扰素新型发酵技术及结构修饰与新制剂研究．天津大学博士学位论文，2003．
[2] P.Michael Davidson, Craig H. Doan. Natamycin [J]. University of Idaho.1993, 7:395-407.
[3] J.G.Ostendorp. Natamycin [J]. Antonie van Leeuwenhoek. 1981, 47:170-171.
[4] 方金瑞主编．抗生素[M]．科学社会出版社．1988，45-53，93．
[5] 尤新主编．功能性发酵制品[M]．中国轻工业出版社．2000，350-361．
[6] Lueck-E, Jager-M. Antimicrobial food additives: characteristics, uses, effects.214-218.
[7] Cirigliano-MC, McKenna-RT, Rothenberg-PJ. Method of preserving tea containing beverages[P]. US Patent 5 895 681, 1999.10.
[8] 袁亦丞．纳他霉素[J]．中国食品用化学品．1997，1(3)：37-41．
[9] 陈森，郑鹏然．霉克对沙拉酱抑制霉菌效果观察[J]．中国食品用化学品．1997，1(6)：17-18．
[10] 凌关庭，许胜明．纳他霉素对广式月饼防霉效果的试验[J]．食品工业．1998，1(3)：20-22．
[11] Kaliuzhnaia LD, Murzina EA. An evaluation of the efficacy of Pimaricin in candidiasis of skin and mucous membranes[J]. Lik. Sprava. 1997,10(5): 158-160.
[12] Jani BK, Rinaldi MG, Reinhart WJ. An unusual case of fungal keratitis: Metarrhizium anisopliae [J]. Cornea. 2001, 20(7):765-768.
[13] O'Day DM, Head WS. Advances in the management of keratomycosis and acanthamoeba keratitis [J]. Cornea. 2000, 19(5):681-687.
[14] Domniz Y et al.Successful treatment of paecilomyces lilacinus endophthalmitis after foreign body trauma to the cornea [J]. Cornea. 2001,20(1):109-111.
[15] 熊宗贵．发酵工艺原理[M]．中国医药科技出版社．1995．
[16] 储矩．特殊前体在抗生素生物合成中的作用[J]．国外医药抗生素分册．1999，20(5)：202-212．
[17] 倪孟祥等．柱晶白霉素发酵工艺的代谢调控研究[J]．中国药科大学学报．2000，31(6)：466-469．
[18] 刘刚等．磷酸镁对螺旋霉素发酵的影响[J]．中国抗生素杂志．1997，22(3)：231-233．
[19] 陈长华等．前体对螺旋霉素发酵的影响[J]．中国抗生素杂志．1995，20(5)：392-393．
[20] 张蔚文，焦瑞身．丙酸盐对利福霉素生物合成的调控[J]．中国抗生素杂志．1995，20(4)：268-272．
[21] M．A．艾森申克等．纳他霉素的种菌制备和繁殖方法[P]．CN 1071460A，1993．4．
[22] M．A．艾森申克等．通过控制发酵培养基的pH值以提高纳他霉素的生产速率[P]．CN 1072959A，1993．6．
[23] P．T．奥尔森．连续发酵法生产纳他霉素[P]．CN 1070688A，1993．4．
[24] Eisenschink, Michael Allen. Fermentation process for producing natamycin with additional carbon and nitrogen [P]. P. 262804, 1994.6.
[25] Enshasy HA, Farid MA. Influence of inoculum type and cultivation conditions on natamycin production by Streptomyces natalensis[J]. J. Basic Microbial. 2000, 40(5-6):333-342.
[26] Enshasy HA, Farid MA. Optization of the cultivation medium for natamycin production by Streptomyces natalensis[J].J.Basic Microbial.2000,40(3):157-166.
[27] Aparicio JF, Colina AJ et al. The biosynthetic gene cluster for the 26-membered ring plyene macrolide pimaricin[J]. J Biol Chem.1999,274(15): 10133-10139.
[28] Aparicio JF, Fouces R et al. A complex multienzyme system encoded by five polyketide synthase genes is involved in the biosythesis of the 26-membered plyene macrolide pimaricin in Streptomyces natalensis[J]. Chem. Boil. 2000,7(11):895-905.
[29] Marta V Mendes, Jesus F Aparicio. Complete Nucleotide Sequence and Characterization of pSNA1 from Pimaricin-Producing Streptomyces natalensis That Replicates by a Rolling Circle Mechanism[J]. Plasmid. 2000,43:159-165.
[30] Marta V Mendes, Eliseo Recio. Engineered biosynthesis of novel polyenes:a pimaricin derivative produced by targeted gene disruption in Streptomyces natalensis[J]. Chemistry & Biology. 2001, 8:635-644.
[31] Bordan et al. Process for natamycin recovery[P]. US Patent 5942611, 1999.8.
[32] 杜连祥等编著．工业微生物学实验技术[M]．科学技术出版社．1992．
[33] 邬行彦，雄宗贵，胡章助主编．抗生素生产工艺学[M]．化学工业出版社．1987．
[34] 王克利等．HPLC法测定沙拉酱及干酪中纳他霉素含量方法的研究[J]．中
国卫生检验杂志．1998，8(6)：380-381．
[35]Fletouris-DJ, Botsoglou-NA, Mantis-AJ.Rapid spectrophotometric method for analyzing natamycin in cheese and cheese rind[J]. Journal of AOAC International. 1995, 78(4):1024-1029.
[36]Hutchinson C R, et al. J. Med Chem. 1989, 32(5):929-937.
[37]马逢时，何良材等．应用概率统计．高等教育出版社，1989
[38]董大钧．SAS统计分析．辽宁科学出版社．1996
[39]Gee-Dong Lee, Joong-Ho Kwon. The use of response surface methodology to optimize the Maillar reaction to produce melanoidins with high antioxidative and antimutagenic activities.J Food Sci. Tech. 1998, 33: 375-383
[40]Gawande N. Patkar Y. Biotechnology Bioengineering.1999, 20(2): 58-61
[41]胡运权，张宗浩．试验设计方法．哈尔滨工业大学出版社．1997．
[42]S.J. Kalil, F. Maugeri, et al. Process Biochemistry, 2000, 35: 539-550.
[43]A Konya,A Jekkel,J Suto.J.of Industrial Microbiology Biotechnology,1998, 20: 150-152.
[44]方柏山等．生物工程学报，2000，6(5)：648-650．
[45]Capitan Vallvey, et al. Rapid ultraviolet spectrophotometric and liquid chromatographic methods for the determination of Natamycin in lactoserum matrix[J]. Journal of AOAC International. 2000, 83(4): 802-806.
[46]惠大丰等．统计分析系统SAS软件实用教程[M]．北京航空航天大学出版社．1996．
[47]李颖辉．生物防腐剂纳他霉素发酵生产工艺的研究．天津轻工业学院硕士学位论文，2002．
[48]朱明军．红发夫酵母Phaffia rhodozyma培养生产虾青素的研究．华南理工大学工学博士学位论文，2001．
[49]何艳玲．生物防腐剂纳他霉素发酵工艺的研究．天津轻工业学院硕士学位论文，2002．
[50]李志勇，郭祀远，李琳．藻类对微量元素的生物富集及其应用[J]．微生物学通报．1997，3：12-14．
[51]苗志奇，未作君，元英进．水杨酸在紫杉醇生物合成中诱导作用的研究[J]．生物工程学报．2000，16(4)：509-513．
[52]张磊．基因工程α-2b干扰素新型发酵技术及结构修饰与新制剂研究．天津大学博士学位论文，2003．