Optimization of Conditions for γ-Aminobutyric Acid Yield by Co-fermentation of Enterococcus faecium with Saccharomyces cerevisiae and Mechanism Research
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摘要: 为提高γ-氨基丁酸(γ-aminobutyric acid,GABA)产量,以产GABA屎肠球菌(Enterococcus faecium)AB157与酿酒酵母(Saccharomyces cerevisiae)SC-125为研究对象,通过单因素实验和响应面法优化共发酵条件;同时分析最优条件下共发酵和单菌发酵体系谷氨酸脱羧酶(glutamate decarboxylase,GAD)的酶活力及通过添加无细胞上清液(cell-free supernatant,CFS)探究高产GABA机制。结果表明:当总接种量2%(V/V),发酵温度为35 ℃、屎肠球菌AB157和酿酒酵母SC-125的接种比例为5:1(V/V)、L-谷氨酸钠浓度为12 g/L、发酵96 h时,共发酵体系GABA产量最高,达6.55 g/L,较单菌发酵体系产量提高到1.78倍;GAD酶活力分析表明,共发酵可显著提高GAD酶活;添加屎肠球菌AB157或酿酒酵母SC-125的CFS可显著提升GABA产量。本研究为屎肠球菌和酿酒酵母共发酵提高GABA产量及高产GABA机制的探讨提供了一定的理论参考。Abstract: In order to improve the yield of γ-aminobutyric acid (GABA), the co-fermentation conditions of Enterococcus faecium AB157 and Saccharomyces cerevisiae SC-125 were optimized by one-factor-at-a-time method and response surface methodology (RSM). Simultaneously, the enzyme activity of glutamate decarboxylase (GAD) was analyzed under optimal conditions in co-fermentation and single strain fermentation systems, and the mechanism of high GABA yield was explored by adding cell-free supernatant (CFS). The optimization results showed that when the overall quantity of inoculum was 2% (V/V), the optimal co-fermentation conditions were as follows: The fermentation temperature was 35 ℃, the inoculum proportions of E. faecium AB157 and S. cerevisiae SC-125 was 5:1 (V/V), and the L-monosodium glutamate concentration was 12 g/L with shaking fermentation for 96 h. In addition, the yield of 6.55 g/L GABA was 1.78 times higher than in single strain fermentation systems. The GAD enzyme activity analysis showed that co-fermentation could significantly improve GAD enzyme activity. Meanwhile, GABA yield could be significantly increased by adding CFS of E. faecium AB157 or S. cerevisiae SC-125. This study served as a theoretical foundation for the discussion of the co-fermentation of E. faecium and S. cerevisiae to increase GABA yield and the mechanism of high GABA yield.
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表 1 因素水平表
Table 1. The factors and levels
水平 因素 A 发酵温度(℃) B 接种比例(V/V) C L-谷氨酸钠(g/L) −1 33 3:1 10 0 35 4:1 12 1 37 5:1 14 表 2 响应面试验设计与结果
Table 2. Response surface experimental design and results
A 发酵温度(℃) B 接种比例(V/V) C L-谷氨酸钠(g/L) GABA产量(g/L) 1 33 3:1 12 5.55 2 35 5:1 14 6.52 3 35 4:1 12 6.55 4 33 5:1 12 5.98 5 35 4:1 12 6.55 6 37 4:1 10 5.33 7 35 4:1 12 6.54 8 35 4:1 12 6.51 9 37 4:1 14 6.21 10 37 3:1 12 5.86 11 37 5:1 12 6.26 12 35 5:1 10 5.85 13 33 4:1 10 5.26 14 33 4:1 14 5.69 15 35 3:1 10 5.45 16 35 4:1 12 6.50 17 35 3:1 14 6.1 表 3 回归模型方差分析
Table 3. Analysis of variance of regression model
方差来源 平方和 自由度 均方 F值 P值 显著性 模型 3.50 9 0.39 178.69 < 0.0001 ** A 0.17 1 0.17 77.56 < 0.0001 ** B 0.34 1 0.34 157.86 < 0.0001 ** C 0.87 1 0.87 398.46 < 0.0001 ** AB 2.403E-004 1 2.403E-004 0.11 0.7495 AC 0.051 1 0.051 23.19 0.0019 ** BC 6.006E-005 1 6.006E-005 0.028 0.8728 A2 1.04 1 1.04 476.00 < 0.0001 ** B2 0.082 1 0.082 37.74 0.0005 ** C2 0.78 1 0.78 358.01 < 0.0001 ** 残差 0.015 7 2.178E-003 失拟项 0.012 3 3.864E-003 4.23 0.0987 净误差 3.655E-003 4 9.137E-004 总离差 3.52 16 决定系数 R2=0.9957 R2Adj=0.9901 注:**表示差异极显著P<0.01。 -
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