一株产γ-PGA的芽孢杆菌的分离鉴定及发酵条件的优化
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摘要
目的:筛选一株高产γ-聚谷氨酸(γ-PGA)的菌株,并优化其发酵条件。方法:从豆腐作坊周边土地取样,采用平板稀释涂布法,筛选高产γ-PGA的菌株,通过菌落形态、分子生物学方法对其进行鉴定;以γ-PGA产量为响应值,在单因素实验的基础上,以温度、pH、转速、底物浓度为实验因素,采用Box-Behnken法设计四因素三水平试验进行响应面优化,确定其产γ-PGA最佳发酵条件。结果:筛选获得一株高产γ-PGA的芽孢杆菌B-6578,鉴定为暹罗芽孢杆菌(Bacillus siamensis);通过单因素和响应面最终获得该芽孢杆菌发酵产γ-PGA的最佳条件为:温度37.5℃,pH7.48,转速240 r/min,底物浓度52.70 g/L,摇瓶发酵36 h,γ-PGA的产量达到24.82 g/L,γ-PGA转化率为47.10%,比优化前提高了25.19%。结论:采用响应面法优化得到的发酵条件方便可行,利于γ-PGA的进一步开发利用。
Objective: A strain with high-yield of the γ-PGA( γ-polyglutamic acid) was isolated and purified,and the fermentation conditions for the γ-PGA production were optimized. Methods: A strain of high-yield of γ-PGA production was isolated and identified from the soil around the tofu workshop,the strain was isolated and purified by plate dilution method,and then the strain was identified by the morphological and its 16 S rDNA sequence.On the basis of single factor experiment,with temperature,pH,rotating speed and substrate concentration as experimental factors,the Box-Behnken method was adopted to optimize the fermentation conditions.Results: The strain was named as Bacillus siamensis.By the experiments of single factor and Box-Behnken design,the optimal culture conditions were as follows: Temperature 37.5 ℃,p H7.48,speed 240 r/min,substrate concentration 52.70 g/L,in the condition of shaking flask fermentation 36 h,γ-PGA yield was of 24.82 g/L,the substrate utilization of monosodium glutamate was 47.10%,increased by 25.19% than before optimization.Conclusion: It was convenient and feasible to optimize the fermentation conditions by the response surface method,which is beneficial to the further development and utilization of the γ-PGA.
Objective: A strain with high-yield of the γ-PGA( γ-polyglutamic acid) was isolated and purified,and the fermentation conditions for the γ-PGA production were optimized. Methods: A strain of high-yield of γ-PGA production was isolated and identified from the soil around the tofu workshop,the strain was isolated and purified by plate dilution method,and then the strain was identified by the morphological and its 16 S rDNA sequence.On the basis of single factor experiment,with temperature,pH,rotating speed and substrate concentration as experimental factors,the Box-Behnken method was adopted to optimize the fermentation conditions.Results: The strain was named as Bacillus siamensis.By the experiments of single factor and Box-Behnken design,the optimal culture conditions were as follows: Temperature 37.5 ℃,p H7.48,speed 240 r/min,substrate concentration 52.70 g/L,in the condition of shaking flask fermentation 36 h,γ-PGA yield was of 24.82 g/L,the substrate utilization of monosodium glutamate was 47.10%,increased by 25.19% than before optimization.Conclusion: It was convenient and feasible to optimize the fermentation conditions by the response surface method,which is beneficial to the further development and utilization of the γ-PGA.
引文
[1]阮文辉,杨家志,姚俊,等.γ-聚谷氨酸合成菌株的筛选与优化培养[J].中国酿造,2011(5):66-69.
[2]Kunioka M. Biosynthesis and chemical reactions of poly(amino acids)s from microorganisms[J]. Applied Microbiology and Biotechnology,1997,47(5):469-475.
[3]Shih I L,Van Y T.The production of poly-(γ-glutamic acid)from microorganism and its various applications[J]. Bioresource Technology,2001,79(3):207-225.
[4]Ashiuchi M,Misono H.Biopolymers[M].Weinheim:WileyVCH,2002,7:123-126.
[5]Shih I L,Wu P J,Shieh C J.Microbial production of a poly(γ-glutamic acid)derivative by Bacillus subtilis[J]. Process Biochemistry,2005,40(8):2827-2832.
[6]Pereira A E S,Sandoval-Herrera I E,Zavala-Betancourt S A.γ-Polyglutamic acid/chitosan nanoparticles for the plant growth regulator gibberellic acid:Characterization and evaluation of biological activity[J]. Carbohydrate Polymers,2017,157(10):1862-1873.
[7]Pillarisetti S,Maya S,Sathianarayanan S. Tunable p H and redox-responsive drug release from curcumin conjugatedγ-polyglutamic acid nanoparticles in cancer microenvironment[J]. Colloids and Surfaces B Biointerfaces,2017,159(1):809-819.
[8]徐虹,欧阳平凯,李霜,等.利用枯草芽孢杆菌NX-2制备γ-聚谷氨酸及其盐和谷胱甘肽及其前体:中国,1425762[P].2003-06-25.
[9]胡荣章,叶海峰,金丽,等.γ-聚谷氨酸高产菌株筛选及发酵条件优化[J].中国生物工程杂志,2005,25(12):62-65.
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[11]何剑,雍晓宇,周俊,等.一株γ-多聚谷氨酸生产菌的分离筛选与鉴定[J].生物加工过程,2014,12(4):87-93.
[12]贺杨扬,曾伟,王青龙,等.响应面法优化枯草芽孢杆菌产γ-聚谷氨酸发酵工艺[J].食品科学,2014,35(9):147-151.
[13]孙梦桐,马欢欢,吕欣然,等.响应面法优化复合乳酸菌抑制荧光假单胞菌作用[J].食品工业科技,2017,38(17):119-124.
[14]赵月菊,刘阳,邢福国,等.一株暹罗芽孢杆菌及其在防治禾谷镰刀菌方面的应用:中国,104342394[P]. 2015-02-11.
[15]陈倩倩,刘波,王阶平,等.芽孢杆菌FJAT-28592抗真菌脂肽的研究[J].农业生物技术学报,2016,24(2):261-269.
[16]王瑶,赵月菊,邢福国,等.禾谷镰刀菌拮抗菌株的筛选及鉴定[J].核农学报,2017,31(6):1128-1136.
[17]黄福祥,马建彬,朱西儒,等.地衣芽孢杆菌菌株鉴定和生化特征的研究[C]//全国生物农药研讨会,2004:111-117.
[18]徐艳萍,王树英,李华钟,等.聚γ-谷氨酸高产突变株的选育及摇瓶发酵条件[J].无锡轻工大学学报,2004,23(5):6-10.
[19]冯志彬,程仕伟,缪静,等.γ-聚谷氨酸生产菌的选育及培养条件研究[J].生物加工过程,2010,8(1):40-44.
[20]万红兵,田洪涛,马乐辉,等.保加利亚乳杆菌番茄复合汁增菌培养基的优选研究[J].中国乳品工业,2006,34(8):14-17.
[21]卢彦梅.γ-氨基丁酸产生菌的选育及发酵工艺研究[D].无锡:江南大学,2008.
[22]梁金钟,王风.微生物发酵法合成高分子聚合物γ-PGA的研究[J].北京工商大学学报,2011,29(1):24-29.
[23]Feng S,Zhinan X,Peilin C.Efficient production of poly-γ-glutamic acid by Bacillus subtilis ZJU-7[J].Applied Biochemistry and Biotechnology,2006,133(3):271-281.
[24]潘龙,靳魁奇,刘宇鹏.静息细胞转化赤藓糖醇生产L-赤藓酮糖条件优化[J].食品与发酵工业,2014,40(12):72-76.
[25]刘晓鸥,乔长晟,李睿颖,等.枯草芽孢杆菌TKPG011聚谷氨酸发酵培养基的优化[J].现代食品科技,2010,26(3):253-255.
[2]Kunioka M. Biosynthesis and chemical reactions of poly(amino acids)s from microorganisms[J]. Applied Microbiology and Biotechnology,1997,47(5):469-475.
[3]Shih I L,Van Y T.The production of poly-(γ-glutamic acid)from microorganism and its various applications[J]. Bioresource Technology,2001,79(3):207-225.
[4]Ashiuchi M,Misono H.Biopolymers[M].Weinheim:WileyVCH,2002,7:123-126.
[5]Shih I L,Wu P J,Shieh C J.Microbial production of a poly(γ-glutamic acid)derivative by Bacillus subtilis[J]. Process Biochemistry,2005,40(8):2827-2832.
[6]Pereira A E S,Sandoval-Herrera I E,Zavala-Betancourt S A.γ-Polyglutamic acid/chitosan nanoparticles for the plant growth regulator gibberellic acid:Characterization and evaluation of biological activity[J]. Carbohydrate Polymers,2017,157(10):1862-1873.
[7]Pillarisetti S,Maya S,Sathianarayanan S. Tunable p H and redox-responsive drug release from curcumin conjugatedγ-polyglutamic acid nanoparticles in cancer microenvironment[J]. Colloids and Surfaces B Biointerfaces,2017,159(1):809-819.
[8]徐虹,欧阳平凯,李霜,等.利用枯草芽孢杆菌NX-2制备γ-聚谷氨酸及其盐和谷胱甘肽及其前体:中国,1425762[P].2003-06-25.
[9]胡荣章,叶海峰,金丽,等.γ-聚谷氨酸高产菌株筛选及发酵条件优化[J].中国生物工程杂志,2005,25(12):62-65.
[10]任尚美,马霞,王海波,等.γ-聚谷氨酸的发酵条件优化及其初步表征分析[J].中国酿造,2008(19):44-48.
[11]何剑,雍晓宇,周俊,等.一株γ-多聚谷氨酸生产菌的分离筛选与鉴定[J].生物加工过程,2014,12(4):87-93.
[12]贺杨扬,曾伟,王青龙,等.响应面法优化枯草芽孢杆菌产γ-聚谷氨酸发酵工艺[J].食品科学,2014,35(9):147-151.
[13]孙梦桐,马欢欢,吕欣然,等.响应面法优化复合乳酸菌抑制荧光假单胞菌作用[J].食品工业科技,2017,38(17):119-124.
[14]赵月菊,刘阳,邢福国,等.一株暹罗芽孢杆菌及其在防治禾谷镰刀菌方面的应用:中国,104342394[P]. 2015-02-11.
[15]陈倩倩,刘波,王阶平,等.芽孢杆菌FJAT-28592抗真菌脂肽的研究[J].农业生物技术学报,2016,24(2):261-269.
[16]王瑶,赵月菊,邢福国,等.禾谷镰刀菌拮抗菌株的筛选及鉴定[J].核农学报,2017,31(6):1128-1136.
[17]黄福祥,马建彬,朱西儒,等.地衣芽孢杆菌菌株鉴定和生化特征的研究[C]//全国生物农药研讨会,2004:111-117.
[18]徐艳萍,王树英,李华钟,等.聚γ-谷氨酸高产突变株的选育及摇瓶发酵条件[J].无锡轻工大学学报,2004,23(5):6-10.
[19]冯志彬,程仕伟,缪静,等.γ-聚谷氨酸生产菌的选育及培养条件研究[J].生物加工过程,2010,8(1):40-44.
[20]万红兵,田洪涛,马乐辉,等.保加利亚乳杆菌番茄复合汁增菌培养基的优选研究[J].中国乳品工业,2006,34(8):14-17.
[21]卢彦梅.γ-氨基丁酸产生菌的选育及发酵工艺研究[D].无锡:江南大学,2008.
[22]梁金钟,王风.微生物发酵法合成高分子聚合物γ-PGA的研究[J].北京工商大学学报,2011,29(1):24-29.
[23]Feng S,Zhinan X,Peilin C.Efficient production of poly-γ-glutamic acid by Bacillus subtilis ZJU-7[J].Applied Biochemistry and Biotechnology,2006,133(3):271-281.
[24]潘龙,靳魁奇,刘宇鹏.静息细胞转化赤藓糖醇生产L-赤藓酮糖条件优化[J].食品与发酵工业,2014,40(12):72-76.
[25]刘晓鸥,乔长晟,李睿颖,等.枯草芽孢杆菌TKPG011聚谷氨酸发酵培养基的优化[J].现代食品科技,2010,26(3):253-255.