来源于益生菌FGM的aga2基因在黄芪发酵过程中的表达

Analysis of aga2 Gene Expression Derived from Probiotics FGM in Astragalus membranaceus Fermentation

通过对益生菌FGM所产α-半乳糖苷酶aga2基因在黄芪发酵不同阶段的表达水平分析,为α-半乳糖苷酶在黄芪发酵过程中的作用及其益生机制探究提供依据。绘制益生菌FGM在黄芪发酵过程中的生长及发酵液pH变化曲线,同源序列法克隆aga2基因,运用SYBR Green I实时荧光定量PCR技术检测aga2基因在黄芪发酵不同阶段表达水平。结果表明,在发酵6h内FGM菌株处于对数生长期,大量产酸使pH由初始7.2迅速下降到5.3;12h后细菌进入稳定期,pH下降到4.5;48h至发酵结束pH稳定在4.0左右。成功克隆到一段648bp的aga2基因片段(GenBank登录号KC202825),序列相似性最高达98%。发酵24h内aga2基因表达逐渐上调并达到最高;36h表达水平下降明显,仅是初始发酵水平的1.28倍,之后至60h又呈上升趋势。上述结果提示,益生菌FGM在黄芪发酵中可产生α-半乳糖苷酶以分解黄芪粉中的抗营养因子α-半乳糖苷类,其分解产物半乳糖通过Leloir途径中间产物UDP-半乳糖可能间接参与胞外多糖合成,为黄芪发酵产物中多糖得率增加起了一定作用。

The present study was conducted to investigate the possible functions of ^-galactosidase derived from Streptococcus alactolyticus strain FGM in Astragalus membranaceus fermentation. The a-galactosidase aga2 gene was cloned with homology-based cloning and its expression during A. membranaceus fermentation was estimated using the determined ideal reference gene ldh through real-time reverse transcription quantitative PCR （RT-qPCR）. The results showed that the strain FGM was in exponential growth phase within 6 h and there was a rapid decline of pH value from initial 7.2 to 5.3. The bacteria stable growth phase was post 12 h, pH value was 4.5 and pH value changes were not observed from 48 h to the end of fermentation （72 h）. A nucleo- tide segment of 648 bp was successfully obtained （GenBank No. KC202825） and its highest se- quence identity was 98 %. Aga2 gene expression was significantly up-regulated （P〈0.05） within 24 h and reached a maximum at 24 h （6.04-fold）. The aga2 gene expression was only 1.3-fold higher at 36 h. However, it showed a gradual decrease from 36 h to 60 h and was down-regulated at 72 h when it reached a minimum （0.68-fold）. The results suggested that a-galactosidase pro- duced by S. alactolyticus strain FGM not only showed some ability to hydrolyze some anti-nutri- tional factors a-galactosides in A. membranaceus, but also might be responsible for exopolysaccha- rides biosynthesis indirectly by the Leloir pathway of galactose metabolism.