枯草芽胞杆菌T-500产脂肽类抗生素的摇瓶发酵工艺优化
|
摘要
[目的]枯草芽胞杆菌(Bacillus subtilis)T-500是1株对水稻纹枯病和稻瘟病均有良好防治效果的生防菌,通过优化其摇瓶发酵工艺,从而提高发酵液中脂肽类抗生素的含量,为T-500菌株生防制剂的开发提供技术支撑。[方法]以水稻纹枯病菌(Rhizoctonia solani)为指示菌,利用酸沉淀法提取T-500菌株发酵液中的脂肽类抗生素,并进行脂肽抗生素粗提液的抑菌效果分析,筛选影响抑菌效果的发酵培养基主成分;随后通过Plackett-Burman试验设计、中心组合试验设计和响应曲面法,优化T-500菌株高产脂肽类抗生素的发酵培养基成分和发酵条件。[结果]T-500菌株高产脂肽类抗生素的最佳培养基为:黄豆饼粉7.00 g·L~(-1),蛋白胨4.92 g·L~(-1),酵母粉1.90 g·L~(-1),小麦粉5.00 g·L~(-1),玉米糊5.00 g·L~(-1),NaCl 1.00 g·L~(-1),MgSO_40.20 g·L~(-1),MnSO_45.0 mg·L~(-1),FeSO_40.5 mg·L~(-1)。最佳发酵培养条件为:装液量500 mL三角瓶装105 mL,接种量0.87%,发酵时间41.35 h,温度28℃,转速180 r·min~(-1)。利用最佳摇瓶发酵工艺,T-500菌株所产生的脂肽类抗生素对纹枯病菌的抑菌带最宽,达(11.23±0.15)mm,菌含量达(7.41±1.18)×109CFU·mL~(-1)。经摇瓶发酵试验和抑菌活性验证,理论预测值与实际值无显著差异。质谱和色谱检测表明:优化发酵工艺后产生的Surfactin含量较基础培养基提高了48.2%,Iturin含量较基础培养基提高了180.9%;优化发酵工艺后检测到了Fengycin的产生,但优化前未发现Fengycin的产生。[结论]利用响应曲面法成功优化了枯草芽胞杆菌T-500产脂肽类抗生素的摇瓶发酵工艺;优化后,T-500产脂肽类抗生素产量增加,抑菌活性增强。
[Objectives]Bacillus subtilis T-500 showed strong biocontrol activities to rice sheath blight and rice blast bacteria. Here,the fermentation process was optimized to improve the yield of lipopeptide antibiotics,which will provide technical support for the development of biocontrol agent containing T-500. [Methods]According to the inhibition zoom of lipopeptide extraction against Rhizoctonia solani,the main nutritional components that are suitable for the production of lipopetides in Bacillus subtilis T-500 were screened by comparing the effects of 10 media that are commonly used for cycle lipopetide antibiotics production by Bacillus. After that,the Plackett-Burman method,the central composite design and response surface methodology were used to obtain optional medium and fermentation condition. [Results]The optimum medium of T-500 was soybean powder 7.00 g·L~(-1),peptone 4.92 g·L~(-1),yeast extract 1.90 g·L~(-1),wheat powder 5.00 g·L~(-1),corn powder 5.00 g·L~(-1),NaCl 1.00 g·L~(-1),MgSO_40. 20 g·L~(-1),MnSO_45. 0 mg·L~(-1),FeSO_40.5 mg·L~(-1). The optimum culture condition was 28 ℃,inoculation volume 0. 87% and the filling volume 105 mL medium in 500 mL flask for 41.35 h at 180 r·min~(-1). Under the optimum fermentation medium and culture conditions,the width of inhibition zoom was about(11.23±0.15) mm,and cell amount was up to(7. 41 ± 1. 18) × 109 CFU·mL~(-1). Fermentation experiments with shake flasks verified that there was no statistical difference between real and forecasted yields. The results of mass spectrometry and chromatography showed that Surfactin content increased by 48.2% and the content of Iturin increased by 180.9% compared with the basal medium after optimization of the fermentation process. Fengycin was detected after optimization of the fermentation process,but not in the basal medium. [Conclusions]Response surface methodology was used in the optimization of the fermentation of T-500 for production of lipopeptide. After optimization,the yield and the antifungal activity of lipopeptide was significantly enhanced respectively.
[Objectives]Bacillus subtilis T-500 showed strong biocontrol activities to rice sheath blight and rice blast bacteria. Here,the fermentation process was optimized to improve the yield of lipopeptide antibiotics,which will provide technical support for the development of biocontrol agent containing T-500. [Methods]According to the inhibition zoom of lipopeptide extraction against Rhizoctonia solani,the main nutritional components that are suitable for the production of lipopetides in Bacillus subtilis T-500 were screened by comparing the effects of 10 media that are commonly used for cycle lipopetide antibiotics production by Bacillus. After that,the Plackett-Burman method,the central composite design and response surface methodology were used to obtain optional medium and fermentation condition. [Results]The optimum medium of T-500 was soybean powder 7.00 g·L~(-1),peptone 4.92 g·L~(-1),yeast extract 1.90 g·L~(-1),wheat powder 5.00 g·L~(-1),corn powder 5.00 g·L~(-1),NaCl 1.00 g·L~(-1),MgSO_40. 20 g·L~(-1),MnSO_45. 0 mg·L~(-1),FeSO_40.5 mg·L~(-1). The optimum culture condition was 28 ℃,inoculation volume 0. 87% and the filling volume 105 mL medium in 500 mL flask for 41.35 h at 180 r·min~(-1). Under the optimum fermentation medium and culture conditions,the width of inhibition zoom was about(11.23±0.15) mm,and cell amount was up to(7. 41 ± 1. 18) × 109 CFU·mL~(-1). Fermentation experiments with shake flasks verified that there was no statistical difference between real and forecasted yields. The results of mass spectrometry and chromatography showed that Surfactin content increased by 48.2% and the content of Iturin increased by 180.9% compared with the basal medium after optimization of the fermentation process. Fengycin was detected after optimization of the fermentation process,but not in the basal medium. [Conclusions]Response surface methodology was used in the optimization of the fermentation of T-500 for production of lipopeptide. After optimization,the yield and the antifungal activity of lipopeptide was significantly enhanced respectively.
引文
[1]Beneduzi A,Ambrosini A,Passaglia L M.Plant growth-promoting rhizobacteria(PGPR):their potential as antagonists and biocontrol agents[J].Genetics and Molecular Biology,2012,35(4):1044-1051.
[2]Rainer B.Use of Plant-Associated Bacillus Strains as Biofertilizers and Biocontrol Agents in Agriculture[M].Heidelberg:Springer,2011.
[3]陈志谊,许志刚,高泰东,等.水稻纹枯病拮抗细菌的评价及利用[J].中国水稻科学,2000,14(2):98-102.Chen Z Y,Xu Z G,Gao T D,et al.Evaluation and utilization of antagonistic bacteria against rice sheath blight[J].Chinese Journal of Rice Science,2000,14(2):98-102(in Chinese with English abstract).
[4]陈志谊,刘荣,刘永锋.水稻纹枯病拮抗细菌B-916的选育[J].中国生物防治,2003,19(1):15-18.Chen Z Y,Liu R,Liu Y F.The screening of antagonistic bacteria strain B-916 against rice sheath blight[J].Chinese Journal of Biological Control,2003,19(1):15-18(in Chinese with English abstract).
[5]Luo C,Liu X,Zhou H,et al.Nonribosomal peptide synthase gene clusters for lipopeptide biosynthesis in Bacillus subtilis 916 and their phenotypic functions[J].Applied and Environmental Microbiology,2015,81(1):422-431.
[6]Chen X H,Koumoutsi A,Scholz R,et al.Comparative analysis of the complete genome sequence of the plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42[J].Nature Biotechnology,2007,25(9):1007-1014.
[7]Luo C,Zhou H,Zou J,et al.Bacillomycin L and surfactin contribute synergistically to the phenotypic features of Bacillus subtilis 916 and thebiocontrol of rice sheath blight induced by Rhizoctonia solani[J].Applied Microbiology and Biotechnology,2015,99:1897-1910.
[8]张荣胜,梁雪杰,刘永锋,等.解淀粉芽胞杆菌Lx-11生物发酵工艺优化[J].中国生物防治学报,2013,29(2):254-262.Zhang R S,Liang X J,Liu Y F,et al.Optimization of fermentation process of Bacillus amyloliquefaciens Lx-11[J].Chinese Journal of Biological Control,2013,29(2):254-262(in Chinese with English abstract).
[9]王美英,王芳,韩金志,等.Paenibacillus polymyxa JSa-9发酵培养基优化及其在黄瓜枯萎病中的应用研究[J].南京农业大学学报,2016,39(4):673-680.DOI:10.7685/jnau.201512010.Wang M Y,Wang F,Han J Z,et al.Optimization fermentation medium and application in cucumber fusarium wilt of Paenibacillus polymyxa JSa-9[J].Journal of Nanjing Agricultural University,2016,39(4):673-680(in Chinese with English abstract).
[10]刘永锋,陈志谊,于俊杰,等.一株枯草芽胞杆菌及其应用:CN201210328716.5[P].2013-11-06.Liu Y F,Chen Z Y,Yu J J,et al.The application of a Bacillus subtilis strain:CN201210328716.5[P].2013-11-06(in Chinese).
[11]刘永锋,孟祥坤,尹小乐,等.一种枯草芽胞杆菌的干悬浮剂及其制备方法:CN201310301606.4[P].2014-12-24.Liu Y F,Meng X K,Yin X L,et al.The preparation method of a Bacillus subtilis dry suspension concentration:CN201310301606.4[P].2014-12-24(in Chinese).
[12]向亚萍,周华飞,刘永锋,等.解淀粉芽胞杆菌B1619脂肽类抗生素的分离鉴定及其对番茄枯萎病菌的抑制作用[J].中国农业科学,2016,49(15):2935-2944.Xiang Y P,Zhou H F,Liu Y F,et al.Isolation and identification of lipopeptide antibiotics produced by Bacillus amyloliquefaciens B1619 and the inhibition of the lipopeptide antibiotics to Fusarium oxysporum f.sp.lycopersici[J].Scientia Agricultura Sinica,2016,49(15):2935-2944(in Chinese with English abstract).
[13]Ongena M,Jacques P.Bacillus lipopeptides:versatile weapons for plant disease biocontrol[J].Trends in Microbiology,2008,16(3):115-125.
[14]Ongena M,Jourdan E,Adam A,et al.Surfactin and fengycin lipopeptides of Bacillus subtilis as elicitors of induced systemic resistance in plants[J].Environmental Microbiology,2007,9(4):1084-1090.
[15]代志凯,张翠,阮征.试验设计和优化及其在发酵培养基优化中的应用[J].微生物学通报,2010,37(6):894-903.Dai Z K,Zhang C,Ruan Z.The application of experimental design and optimization techniques in optimization of microbial medium[J].Microbiology China,2010,37(6):894-903(in Chinese with English abstract).
[16]郝学财,余晓斌,刘志钰,等.响应面方法在优化微生物培养基中的应用[J].食品研究与开发,2006,27(1):38-41.Hao X C,Yu X B,Liu Z Y,et al.The application of response surface methodology in optimization of microbial media[J].Food Research and Development,2006,27(1):38-41(in Chinese with English abstract).
[17]刘京兰,蔡勋超,薛雅蓉,等.生防解淀粉芽胞杆菌CC09菌株合成Iturin A条件的响应面优化[J].中国生物防治学报,2016,32(2):235-243.Liu J L,Cai X C,Xue Y R,et al.Optimization of Iturin A synthesis by Bacillus amyloliquefaciens CC09 using response surface technique[J].Chinese Journal of Biological Control,2016,32(2):235-243(in Chinese with English abstract).
[18]Sen R,Swaminathan T.Response surface modeling and optimization to elucidate and analyze the effects of inoculum age and size on surfactin production[J].Biochemical Engineering Journal,2004,21:141-148.
[19]方传记,陆兆新,孙力军,等.淀粉液化芽胞杆菌抗菌脂肽发酵培养基及发酵条件优化[J].中国农业科学,2008,41(2):533-539.Fang Z J,Lu Z X,Sun L J,et al.Optimization of fermentation technology for lipopeptides producing bacteria Bacillus amyloliquefaciens ES-2-4[J].Scientia Agricultura Sinica,2008,41(2):533-539(in Chinese with English abstract).
[20]权春善,王军华,徐洪涛,等.一株抗真菌解淀粉芽胞杆菌的分离鉴定及其发酵条件的初步研究[J].微生物学报,2006,46(1):7-12.Quan C S,Wang J H,Xu H T,et al.Identification and characterization of a Bacillus amyloliquefaciens with high antifungal activity[J].Acta Microbiologica Sinica,2006,46(1):7-12(in Chinese with English abstract).
[21]信珊珊,祁高富,朱发银,等.一株解淀粉芽胞杆菌发酵条件的优化及其对油茶炭疽病的防效[J].华中农业大学学报,2011,30(4):411-415.Xin S S,Qi G F,Zhu F Y,et al.Optimization of fermentation condition for Bacillus amyloliquefaciens WH1 and its biological control effect on Colletotrichum gloeosporioides[J].Journal of Huazhong Agricultural University,2011,30(4):411-415(in Chinese with English abstract).
[2]Rainer B.Use of Plant-Associated Bacillus Strains as Biofertilizers and Biocontrol Agents in Agriculture[M].Heidelberg:Springer,2011.
[3]陈志谊,许志刚,高泰东,等.水稻纹枯病拮抗细菌的评价及利用[J].中国水稻科学,2000,14(2):98-102.Chen Z Y,Xu Z G,Gao T D,et al.Evaluation and utilization of antagonistic bacteria against rice sheath blight[J].Chinese Journal of Rice Science,2000,14(2):98-102(in Chinese with English abstract).
[4]陈志谊,刘荣,刘永锋.水稻纹枯病拮抗细菌B-916的选育[J].中国生物防治,2003,19(1):15-18.Chen Z Y,Liu R,Liu Y F.The screening of antagonistic bacteria strain B-916 against rice sheath blight[J].Chinese Journal of Biological Control,2003,19(1):15-18(in Chinese with English abstract).
[5]Luo C,Liu X,Zhou H,et al.Nonribosomal peptide synthase gene clusters for lipopeptide biosynthesis in Bacillus subtilis 916 and their phenotypic functions[J].Applied and Environmental Microbiology,2015,81(1):422-431.
[6]Chen X H,Koumoutsi A,Scholz R,et al.Comparative analysis of the complete genome sequence of the plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42[J].Nature Biotechnology,2007,25(9):1007-1014.
[7]Luo C,Zhou H,Zou J,et al.Bacillomycin L and surfactin contribute synergistically to the phenotypic features of Bacillus subtilis 916 and thebiocontrol of rice sheath blight induced by Rhizoctonia solani[J].Applied Microbiology and Biotechnology,2015,99:1897-1910.
[8]张荣胜,梁雪杰,刘永锋,等.解淀粉芽胞杆菌Lx-11生物发酵工艺优化[J].中国生物防治学报,2013,29(2):254-262.Zhang R S,Liang X J,Liu Y F,et al.Optimization of fermentation process of Bacillus amyloliquefaciens Lx-11[J].Chinese Journal of Biological Control,2013,29(2):254-262(in Chinese with English abstract).
[9]王美英,王芳,韩金志,等.Paenibacillus polymyxa JSa-9发酵培养基优化及其在黄瓜枯萎病中的应用研究[J].南京农业大学学报,2016,39(4):673-680.DOI:10.7685/jnau.201512010.Wang M Y,Wang F,Han J Z,et al.Optimization fermentation medium and application in cucumber fusarium wilt of Paenibacillus polymyxa JSa-9[J].Journal of Nanjing Agricultural University,2016,39(4):673-680(in Chinese with English abstract).
[10]刘永锋,陈志谊,于俊杰,等.一株枯草芽胞杆菌及其应用:CN201210328716.5[P].2013-11-06.Liu Y F,Chen Z Y,Yu J J,et al.The application of a Bacillus subtilis strain:CN201210328716.5[P].2013-11-06(in Chinese).
[11]刘永锋,孟祥坤,尹小乐,等.一种枯草芽胞杆菌的干悬浮剂及其制备方法:CN201310301606.4[P].2014-12-24.Liu Y F,Meng X K,Yin X L,et al.The preparation method of a Bacillus subtilis dry suspension concentration:CN201310301606.4[P].2014-12-24(in Chinese).
[12]向亚萍,周华飞,刘永锋,等.解淀粉芽胞杆菌B1619脂肽类抗生素的分离鉴定及其对番茄枯萎病菌的抑制作用[J].中国农业科学,2016,49(15):2935-2944.Xiang Y P,Zhou H F,Liu Y F,et al.Isolation and identification of lipopeptide antibiotics produced by Bacillus amyloliquefaciens B1619 and the inhibition of the lipopeptide antibiotics to Fusarium oxysporum f.sp.lycopersici[J].Scientia Agricultura Sinica,2016,49(15):2935-2944(in Chinese with English abstract).
[13]Ongena M,Jacques P.Bacillus lipopeptides:versatile weapons for plant disease biocontrol[J].Trends in Microbiology,2008,16(3):115-125.
[14]Ongena M,Jourdan E,Adam A,et al.Surfactin and fengycin lipopeptides of Bacillus subtilis as elicitors of induced systemic resistance in plants[J].Environmental Microbiology,2007,9(4):1084-1090.
[15]代志凯,张翠,阮征.试验设计和优化及其在发酵培养基优化中的应用[J].微生物学通报,2010,37(6):894-903.Dai Z K,Zhang C,Ruan Z.The application of experimental design and optimization techniques in optimization of microbial medium[J].Microbiology China,2010,37(6):894-903(in Chinese with English abstract).
[16]郝学财,余晓斌,刘志钰,等.响应面方法在优化微生物培养基中的应用[J].食品研究与开发,2006,27(1):38-41.Hao X C,Yu X B,Liu Z Y,et al.The application of response surface methodology in optimization of microbial media[J].Food Research and Development,2006,27(1):38-41(in Chinese with English abstract).
[17]刘京兰,蔡勋超,薛雅蓉,等.生防解淀粉芽胞杆菌CC09菌株合成Iturin A条件的响应面优化[J].中国生物防治学报,2016,32(2):235-243.Liu J L,Cai X C,Xue Y R,et al.Optimization of Iturin A synthesis by Bacillus amyloliquefaciens CC09 using response surface technique[J].Chinese Journal of Biological Control,2016,32(2):235-243(in Chinese with English abstract).
[18]Sen R,Swaminathan T.Response surface modeling and optimization to elucidate and analyze the effects of inoculum age and size on surfactin production[J].Biochemical Engineering Journal,2004,21:141-148.
[19]方传记,陆兆新,孙力军,等.淀粉液化芽胞杆菌抗菌脂肽发酵培养基及发酵条件优化[J].中国农业科学,2008,41(2):533-539.Fang Z J,Lu Z X,Sun L J,et al.Optimization of fermentation technology for lipopeptides producing bacteria Bacillus amyloliquefaciens ES-2-4[J].Scientia Agricultura Sinica,2008,41(2):533-539(in Chinese with English abstract).
[20]权春善,王军华,徐洪涛,等.一株抗真菌解淀粉芽胞杆菌的分离鉴定及其发酵条件的初步研究[J].微生物学报,2006,46(1):7-12.Quan C S,Wang J H,Xu H T,et al.Identification and characterization of a Bacillus amyloliquefaciens with high antifungal activity[J].Acta Microbiologica Sinica,2006,46(1):7-12(in Chinese with English abstract).
[21]信珊珊,祁高富,朱发银,等.一株解淀粉芽胞杆菌发酵条件的优化及其对油茶炭疽病的防效[J].华中农业大学学报,2011,30(4):411-415.Xin S S,Qi G F,Zhu F Y,et al.Optimization of fermentation condition for Bacillus amyloliquefaciens WH1 and its biological control effect on Colletotrichum gloeosporioides[J].Journal of Huazhong Agricultural University,2011,30(4):411-415(in Chinese with English abstract).