解淀粉芽孢杆菌高密度发酵条件的优化
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摘要
该文探讨解淀粉芽孢杆菌(B.amyloliquefaciens)1841的高密度工业发酵工艺优化,为解淀粉芽孢杆菌的工业化生产提供依据。以平板菌落直径、及培养后的活菌浓度为指标,对种子的保存方法、活化方式和培养基、发酵培养基中的酵母粉和大豆蛋白粉用量以及发酵的后期调控进行筛选和优化。筛选出最佳的工业菌种保存条件为30%甘油为保护剂,-64℃保存;最佳活化方法为采用PDA培养基活化,并经过一次48 h活化和24 h二次活化;最佳的转菌时间为在种子培养后17~18 h。最佳的酵母粉和大豆蛋白粉浓度分别为20 g/L和40 g/L,发酵的后期调控需要进行补料和流加氨水将p H控制在7.0±0.2,发酵时间延长至72 h。通过对发酵的基本条件进行筛选和优化,得到可在100 L发酵罐上进行高密度发酵的体系,发酵浓度可达常规发酵的100倍(>1×1014CFU/m L)。
This study explored the high-density fermentation conditions of Bacillus amyloliquefaciens1841 by using colony diameter and culturing concentration as index. The optimal strain persevation conditions were using 30% glycerol as protective agent stored at-64 ℃. The optimal strain activation was on the PDA medium with the first activation for 48 hours followed by the second for24 hours. By taking into accounting of fermentation cost and the product concentration, the culture after 17-18 hours of seed fermentation was transferred to the fermentation tank of the optimal industrial fermentation medium, in which 20 g/L yeast and 40 g/L soy protein powder were added into the basic medium. By feeding ammonia water to maintain p H at 7.0 ±0.2 in the late fermentation stage and fermentation time extended to 72 hours, high-density fermentation could be achieved in the 100 L fermentation tank with cells >1 ×1014CFU/m L, more than 100 times of the conventional fermentation.
This study explored the high-density fermentation conditions of Bacillus amyloliquefaciens1841 by using colony diameter and culturing concentration as index. The optimal strain persevation conditions were using 30% glycerol as protective agent stored at-64 ℃. The optimal strain activation was on the PDA medium with the first activation for 48 hours followed by the second for24 hours. By taking into accounting of fermentation cost and the product concentration, the culture after 17-18 hours of seed fermentation was transferred to the fermentation tank of the optimal industrial fermentation medium, in which 20 g/L yeast and 40 g/L soy protein powder were added into the basic medium. By feeding ammonia water to maintain p H at 7.0 ±0.2 in the late fermentation stage and fermentation time extended to 72 hours, high-density fermentation could be achieved in the 100 L fermentation tank with cells >1 ×1014CFU/m L, more than 100 times of the conventional fermentation.
引文
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[9]王美玲,吴骏晨,朱赟程.解淀粉芽孢杆菌在动物生产中应用的研究进展[J].2017,53(2):19-23.
[10]车晓曦,李社增,李校堃,等.1株解淀粉芽孢杆菌发酵培养基的设计及发酵条件的优化[J].安徽农业科学,2010,38(18):9402-9405,9437.
[11]王卉,游成真,秦宇轩,等.解淀粉芽孢杆菌L-S60生物学特性及其固态发酵工艺研究[J].2016,21(9):133-142.
[12]LIU X Y,REN B,GAO H.Optimization for the production of surfactin with a new Syner gistic antifungal activity[J].PLo S One,2012,7(5):1-9.
[13]王帅,高圣风,高学文,等.枯草芽孢杆菌脂肽类抗生素发酵和提取条件[J].2007,23(4):342-347.
[14]金敏,王忠彦.产胶原酶地衣芽孢杆菌菌种的分离,筛选及发酵条件研究[J].四川大学学报(自然科学版),2000,37(5):764-767.
[15]舒丹,李宏,严建华,等.高温芽孢杆菌碱性蛋白酶发酵条件及酶性质研究[J].四川大学学报(自然科学版),2004,41(4):856-860.
[2]冯金龙,杨成德,陈秀蓉,等.解淀粉芽孢杆菌S27D的鉴定、生物学功能测定及其对马铃薯病原真菌的拮抗作用研究[J].甘肃农业大学学报,2013,48(4):56-61.
[3]CHEN X,ZHANG Y,FU X,et al.Isolation and chara cterization of Bacillus amyloliquefaciens PG12 for the biological control of apple ring rot[J].Postharvest Biology and Technology,2016(115):113-121.
[4]陈哲,黄静,赵佳,等.解淀粉芽孢杆菌抑菌机制的研究进展[J].生物技术通报,2015,31(6):37-41.
[5]JASIM B,BENNY R,SABU R,et al.Metabolite and mechanistic basis of antifungal property exhibited by endophytic Bacillus amyloliquefaciens[J].Applied biochemistry and biotechnology,2016,179(5):1-16.
[6]NAM H,OH B J,KIM Y C.Biological control potential of Bacillus amyloliquefaciens KB3 isolated from the feces of allomyrinadichotoma larvae[J].The Plant Pathology Journal,2016,32(3):273-280.
[7]WANG N,LIU M,GUO L,et al.A novel protein elicitor(Pe BA1)from Bacillus amylolique faciens NC6Induces systemic resistance in tobacco[J].International Journal of Biological Sciences,2016,12(6):757-767.
[8]SRIVASTAVA S,BIST V,SRIVASTAVA S,et al.Unraveling aspects of Bacillus amyloliquefaciens mediated enhanced production of rice under biotic stress of Rhizoctonia solani[J].Frontiers in Plant Science,2016(7):587.
[9]王美玲,吴骏晨,朱赟程.解淀粉芽孢杆菌在动物生产中应用的研究进展[J].2017,53(2):19-23.
[10]车晓曦,李社增,李校堃,等.1株解淀粉芽孢杆菌发酵培养基的设计及发酵条件的优化[J].安徽农业科学,2010,38(18):9402-9405,9437.
[11]王卉,游成真,秦宇轩,等.解淀粉芽孢杆菌L-S60生物学特性及其固态发酵工艺研究[J].2016,21(9):133-142.
[12]LIU X Y,REN B,GAO H.Optimization for the production of surfactin with a new Syner gistic antifungal activity[J].PLo S One,2012,7(5):1-9.
[13]王帅,高圣风,高学文,等.枯草芽孢杆菌脂肽类抗生素发酵和提取条件[J].2007,23(4):342-347.
[14]金敏,王忠彦.产胶原酶地衣芽孢杆菌菌种的分离,筛选及发酵条件研究[J].四川大学学报(自然科学版),2000,37(5):764-767.
[15]舒丹,李宏,严建华,等.高温芽孢杆菌碱性蛋白酶发酵条件及酶性质研究[J].四川大学学报(自然科学版),2004,41(4):856-860.