布拉酵母高密度发酵培养基及发酵工艺优化
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摘要
为实现布拉酵母高密度培养,对其高密度发酵培养基和发酵工艺进行优化。采用Plackett-Burman试验筛选培养基中的显著因素,并进行中心组合设计。通过人工神经网络(artificial neural network,ANN)和响应面试验建立菌体布拉酵母产量与培养基之间的关系模型,利用遗传算法(genetic algorithm,GA)进行全局寻优。结果表明,ANN模型有较好的数据拟合能力和预测能力,更适合处理复杂的非线性问题。GA优化获得最佳培养基组合:葡萄糖40.52 g/L、蛋白胨36.8 g/L、玉米浆17.32 g/L、硝酸钾14 g/L、酵母营养盐1.5 g/L、磷酸二氢钾0.6 g/L、硫酸镁0.8 g/L。利用该培养基进行摇瓶培养,菌体布拉酵母产量可达到8.21 g/L,比优化前提高1.39倍。在此基础上利用1 L发酵罐培养确定最佳发酵工艺:温度30℃、接种量10%、pH 5.0、溶氧40%。利用50 L发酵罐进行扩大培养,流加葡萄糖和蛋白胨控制发酵液中葡萄糖3 g/L、氨氮0.06 g/L,菌体布拉酵母产量达到51.21 g/L。
For high cell density cultivation of Saccharomyces boulardii, we attempted to optimize medium composition and culture conditions. Plackett-Burman design was used to recognize the signi?cant medium components. Subsequently,response surface methodology and arti?cial neural network(ANN) based on central composite design(CCD) were applied to model the relationship between dry biomass production and medium composition. The optimization of medium composition was carried out using genetic algorithm(GA). The results showed that the ANN model, more suitable for complex and nonlinear modeling, had better goodness of ?t and prediction performance. The optimal medium composition was obtained as follows(g/L): glucose 40.52, peptone 36.8, corn steep liquor 17.32, KNO_3 14, yeast nutrients 1.5, KH_2PO_4 0.6 and MgSO4 0.8 shake ?ask cultivation using the optimized medium gave a dry biomass yield of 8.21 g/L, which was 2.39 folds higher than that obtained from the original medium. Based on these results, we determined the optimal high cell density culture conditions for S. boulardii cultivated in a 1-L fermentor as follows: temperature 30 ℃, 10% inoculum, pH 5.0 and40% dissolved oxygen. Furthermore, we scaled up the culture process in a 50-L fermentor with the addition of glucose and peptone to maintain the glucose concentration at 3 g/L and the ammonia nitrogen concentration at 0.06 g/L. The dry biomass yield of S. boulardii reached 51.21 g/L in the large scale experiment.
For high cell density cultivation of Saccharomyces boulardii, we attempted to optimize medium composition and culture conditions. Plackett-Burman design was used to recognize the signi?cant medium components. Subsequently,response surface methodology and arti?cial neural network(ANN) based on central composite design(CCD) were applied to model the relationship between dry biomass production and medium composition. The optimization of medium composition was carried out using genetic algorithm(GA). The results showed that the ANN model, more suitable for complex and nonlinear modeling, had better goodness of ?t and prediction performance. The optimal medium composition was obtained as follows(g/L): glucose 40.52, peptone 36.8, corn steep liquor 17.32, KNO_3 14, yeast nutrients 1.5, KH_2PO_4 0.6 and MgSO4 0.8 shake ?ask cultivation using the optimized medium gave a dry biomass yield of 8.21 g/L, which was 2.39 folds higher than that obtained from the original medium. Based on these results, we determined the optimal high cell density culture conditions for S. boulardii cultivated in a 1-L fermentor as follows: temperature 30 ℃, 10% inoculum, pH 5.0 and40% dissolved oxygen. Furthermore, we scaled up the culture process in a 50-L fermentor with the addition of glucose and peptone to maintain the glucose concentration at 3 g/L and the ammonia nitrogen concentration at 0.06 g/L. The dry biomass yield of S. boulardii reached 51.21 g/L in the large scale experiment.
引文
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[9]雷张藤.保拉迪酵母菌粉的制备及稳定性研究[D].西安:陕西科技大学,2017:39-42.
[10]杜晓蒙,陶如玉,圣志存,等.培养基组成对布拉氏酵母生长的影响及其优化[J].食品研究与开发,2014,35(5):105-108.DOI:10.3969/j.issn.1005-6521.2014.05.030.
[11]王学锋,苑伟,刘延琳.培养基的主要成分对优选酿酒酵母生物量的影响[J].中国酿造,2010(8):18-21.DOI:10.3969/j.issn.0254-5071.2010.08.006.
[12]王子辉.布拉迪酵母菌高生物量及高产海藻糖菌株的筛选[D].济南:山东轻工业学院,2011:44-46.
[13]陆震鸣,何喆,许泓瑜,等.基于人工神经网络-遗传算法的樟芝发酵培养基优化[J].生物工程学报,2011,25(12):1773-1779.DOI:10.13345/j.cjb.2011.12.007.
[14]王莹,栾天齐,朴美子.基于神经网络和遗传算法的醋酸发酵培养基优化[J].中国食品学报,2012,12(5):88-94.DOI:10.3969/j.issn.1009-7848.2012.05.015.
[15]TIAN H,LIU C,GAO X D.et al.Optimization of auto-induction medium for G-CSF production by Escherichia coli using artificial neural networks coupled with genetic algorithm[J].World Journal of Microbiology&Biotechnology,2013,29(3):505-513.DOI:10.1007/s11274-012-1204-1.
[16]SINGH V,KHAN M,KHAN S,et al.Optimization of actinomycin Vproduction by Streptomyces triostinicus using artificial neural network and genetic algorithm[J].Applied Microbiology Biotechnology,2009,82(2):379-385.DOI:10.1007/s00253-008-1828-0.
[17]LAN Z,ZHAO C,GUO W,et al.Optimization of culture medium for maximal production of spinosad using an artificial neural networkgenetic algorithm modeling[J].Journal of Molecular Microbiology and Biotechnology,2015,25(4):253-261.DOI:10.1159/000381312.
[18]王军峰,杜吉泉,储炬,等.应用神经网络和遗传算法优化利福霉素B发酵培养基[J].中国抗生素杂志,2006,31(5):278-280.DOI:10.3969/j.issn.1001-8689.2006.05.006.
[19]彭锋,李安明.BP神经网络和遗传算法在红发夫酵母培养基优化中的应用[J].环境应用与微生物学报,2008,14(6):834-837.DOI:10.3724/SP.J.1145.2008.00834.
[20]姚博,贠建民,艾对元,等.基于径向基神经网络和遗传算法的聚谷氨酸发酵培养基优化[J].食品科学,2018,39(6):107-115.DOI:10.3321/j.issn:1002-6630.2006.10.069.
[21]李妍,赵文静,高鹏飞,等.优化益生菌Lactobacillus casei Zhang高密度培养条件[J].微生物学通报,2009,36(2):181-186.DOI:10.13344/j.microbiol.china.2009.02.001.
[22]洪厚胜,窦冰然,郭会明.基于面包酵母中海藻糖提取工艺优化及菌株筛选的高密度培养工艺[J].食品科学,2018,39(6):122-129.DOI:10.7506/spkx1002-6630-201806020.
[23]周旻.夫西地酸发酵工艺的初步研究[D].石家庄:河北科技大学,2012:16-17.
[24]刘开放,王敏,郑君,等.骆驼凝乳酶工程菌发酵培养基的优化[J].现代食品科技,2007,32(13):170-176.DOI:10.13982/j.mfst.1673-9078.2017.12.026.
[25]FRODA A,FITZGERALD G F.Biotechnological approaches to the understanding and improvement of mature cheese flavor[J].Current Opinion in Biotechnology,2000,11(5):484-489.DOI:10.1016/S0958-1669(00)00130-0.
[26]冯培勇,赵彦宏,张丽.响应面法优化黑曲霉产纤维素酶发酵条件[J].食品科学,2009,30(23):335-339.DOI:10.3321/j.issn:1002-6630.2009.23.076.
[27]ADHIKARI K,HEYMANN H,HUFF H E.Textural characteristics of lowfat,fullfat and smoked cheeses:sensory and instrumental approaches[J].Food Quality and Preference,2003,14(3):211-218.DOI:10.1016/s0950-3293(02)00067-8.
[28]周景文,徐建,陈守文,等.基于径向基神经网络和遗传算法的聚谷氨酸发酵培养基优化[J].食品科学,2006,27(10):288-292.DOI:10.3321/j.issn:1002-6630.2006.10.069.
[29]ZHAO Z Y,MA S S,LI A,et al.Effects of trophic modes,carbon sources,and salinity on the cell growth and lipid accumulation of tropic ocean oilgae strain Desmodesmus sp.WC08[J].Applied Biochemistry and Biotechnology,2016,180(3):452-463.DOI:10.1007/s12010-016-2109-5.
[30]BI F,MENT D,LURIA N,et al.Mutation of AREA affects growth,sporulation,nitrogen regulation,and pathogenicity in Colletotrichum gloeosporioides[J].Fungal Genetics and Biology,2017,99:29.DOI:10.1016/j.fgb.2016.12.006.
[31]王丽君,梁万秋,李华乔,等.发酵过程玉米浆质量研究[J].科技创新导报,2011(6):6.DOI:10.3969/j.issn.1674-098X.2011.06.005.
[2]BERNI C R,CUCCHIARA S,CUOMO R,et al.Saccharomyces boulardii:a summary of the evidence for gastroenterology clinical practice in adults and children[J].European Review for Medical and Pharmacological Sciences,2011,15(7):809-822.DOI:10.1016/j.euroneuro.2010.11.003.
[3]MOMBELLI B,GISMONDO M R.The use of probiotics in medical practice[J].International Journal of Antimicrobial Agents,2000,16(4):531-536.DOI:10.1016/S0924-8579(00)00322-8.
[4]BUTS J P.Twenty-five years of research on Saccharomyces boulardii trophic effects:updates and perspectives[J].Digestive Diseases and Sciences,2009,54(1):15-18.DOI:10.1007/s10620-008-0322-y.
[5]DOROTA C,PATRICK R.Experimental effects of Saccharomyces boulardii on diarrheal pathogens[J].Microbes and Infection,2002,4(7):733-739.DOI:10.1016/s1286-4579(02)01592-7.
[6]GIANG H H,VIET T Q,OGLE B,et al.Growth performance,digestibility,gut environment and health status in weaned piglets fed a diet supplemented with a complex of lactic acid bacteria alone or in combination with Bacillus subtilis and Saccharomyces boulardii[J].Livestock Science,2012,143(2):132-141.DOI:10.1016/j.livsci.2010.01.010.
[7]OEZTUERK H,SCHROEDER B,BEYERBACH M,et al.Influence of living and autoclaved yeasts of Saccharomyces boulardii on in vitro rminal microbial metabolism[J].Journal of Dairy Science,2005,88(7):2594-2600.DOI:10.3168/jds.S0022-0302(05)72935-0.
[8]楚杰,张大伟,郝永任,等.布拉氏酵母菌对肠道菌群紊乱的调整作用研究[J].中国畜牧兽医,2008,35(7):87-89.
[9]雷张藤.保拉迪酵母菌粉的制备及稳定性研究[D].西安:陕西科技大学,2017:39-42.
[10]杜晓蒙,陶如玉,圣志存,等.培养基组成对布拉氏酵母生长的影响及其优化[J].食品研究与开发,2014,35(5):105-108.DOI:10.3969/j.issn.1005-6521.2014.05.030.
[11]王学锋,苑伟,刘延琳.培养基的主要成分对优选酿酒酵母生物量的影响[J].中国酿造,2010(8):18-21.DOI:10.3969/j.issn.0254-5071.2010.08.006.
[12]王子辉.布拉迪酵母菌高生物量及高产海藻糖菌株的筛选[D].济南:山东轻工业学院,2011:44-46.
[13]陆震鸣,何喆,许泓瑜,等.基于人工神经网络-遗传算法的樟芝发酵培养基优化[J].生物工程学报,2011,25(12):1773-1779.DOI:10.13345/j.cjb.2011.12.007.
[14]王莹,栾天齐,朴美子.基于神经网络和遗传算法的醋酸发酵培养基优化[J].中国食品学报,2012,12(5):88-94.DOI:10.3969/j.issn.1009-7848.2012.05.015.
[15]TIAN H,LIU C,GAO X D.et al.Optimization of auto-induction medium for G-CSF production by Escherichia coli using artificial neural networks coupled with genetic algorithm[J].World Journal of Microbiology&Biotechnology,2013,29(3):505-513.DOI:10.1007/s11274-012-1204-1.
[16]SINGH V,KHAN M,KHAN S,et al.Optimization of actinomycin Vproduction by Streptomyces triostinicus using artificial neural network and genetic algorithm[J].Applied Microbiology Biotechnology,2009,82(2):379-385.DOI:10.1007/s00253-008-1828-0.
[17]LAN Z,ZHAO C,GUO W,et al.Optimization of culture medium for maximal production of spinosad using an artificial neural networkgenetic algorithm modeling[J].Journal of Molecular Microbiology and Biotechnology,2015,25(4):253-261.DOI:10.1159/000381312.
[18]王军峰,杜吉泉,储炬,等.应用神经网络和遗传算法优化利福霉素B发酵培养基[J].中国抗生素杂志,2006,31(5):278-280.DOI:10.3969/j.issn.1001-8689.2006.05.006.
[19]彭锋,李安明.BP神经网络和遗传算法在红发夫酵母培养基优化中的应用[J].环境应用与微生物学报,2008,14(6):834-837.DOI:10.3724/SP.J.1145.2008.00834.
[20]姚博,贠建民,艾对元,等.基于径向基神经网络和遗传算法的聚谷氨酸发酵培养基优化[J].食品科学,2018,39(6):107-115.DOI:10.3321/j.issn:1002-6630.2006.10.069.
[21]李妍,赵文静,高鹏飞,等.优化益生菌Lactobacillus casei Zhang高密度培养条件[J].微生物学通报,2009,36(2):181-186.DOI:10.13344/j.microbiol.china.2009.02.001.
[22]洪厚胜,窦冰然,郭会明.基于面包酵母中海藻糖提取工艺优化及菌株筛选的高密度培养工艺[J].食品科学,2018,39(6):122-129.DOI:10.7506/spkx1002-6630-201806020.
[23]周旻.夫西地酸发酵工艺的初步研究[D].石家庄:河北科技大学,2012:16-17.
[24]刘开放,王敏,郑君,等.骆驼凝乳酶工程菌发酵培养基的优化[J].现代食品科技,2007,32(13):170-176.DOI:10.13982/j.mfst.1673-9078.2017.12.026.
[25]FRODA A,FITZGERALD G F.Biotechnological approaches to the understanding and improvement of mature cheese flavor[J].Current Opinion in Biotechnology,2000,11(5):484-489.DOI:10.1016/S0958-1669(00)00130-0.
[26]冯培勇,赵彦宏,张丽.响应面法优化黑曲霉产纤维素酶发酵条件[J].食品科学,2009,30(23):335-339.DOI:10.3321/j.issn:1002-6630.2009.23.076.
[27]ADHIKARI K,HEYMANN H,HUFF H E.Textural characteristics of lowfat,fullfat and smoked cheeses:sensory and instrumental approaches[J].Food Quality and Preference,2003,14(3):211-218.DOI:10.1016/s0950-3293(02)00067-8.
[28]周景文,徐建,陈守文,等.基于径向基神经网络和遗传算法的聚谷氨酸发酵培养基优化[J].食品科学,2006,27(10):288-292.DOI:10.3321/j.issn:1002-6630.2006.10.069.
[29]ZHAO Z Y,MA S S,LI A,et al.Effects of trophic modes,carbon sources,and salinity on the cell growth and lipid accumulation of tropic ocean oilgae strain Desmodesmus sp.WC08[J].Applied Biochemistry and Biotechnology,2016,180(3):452-463.DOI:10.1007/s12010-016-2109-5.
[30]BI F,MENT D,LURIA N,et al.Mutation of AREA affects growth,sporulation,nitrogen regulation,and pathogenicity in Colletotrichum gloeosporioides[J].Fungal Genetics and Biology,2017,99:29.DOI:10.1016/j.fgb.2016.12.006.
[31]王丽君,梁万秋,李华乔,等.发酵过程玉米浆质量研究[J].科技创新导报,2011(6):6.DOI:10.3969/j.issn.1674-098X.2011.06.005.
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