高产谷胱甘肽酵母菌株筛选及其发酵条件优化

Screening of Glutathione High Yielding Yeast Strains and Optimization of Fermentation Conditions

研究发现并分离获得高产谷胱甘肽(GSH)的微生物资源。项目通过筛选培养、平板涂布划线分离获得两株高产GSH野生酵母菌株,通过28S rDNA序列分析两菌株分别为毕赤酵母(Pichia occidentalis,P.occ)和库兹曼氏菌(Kurtzmaniellaquercitrusa,K.que)。项目通过发酵条件优化,结合内源性GSH的乙醇提取法和ALLOXAN试剂衍生测定法最终确定了上述两个菌株的最佳发酵条件。研究结果表明,K.que菌株最优发酵培养基组分为:每100 mL中含有蔗糖1 g、(NH_(4))_(2)SO_(4)1 g、胰蛋白胨1 g、KH_(2)PO_(4)0.6 g、MgSO_(4)0.3 g、FeSO_(4)和ZnSO_(4)各0.001 g、pH 5.0;装液量40 mL/250 mL、种龄16 h、接种量10%、发酵温度28℃、摇床转速240 r/min、发酵时间72 h。P.occ菌株最优发酵培养基组分为:每100 mL中含有葡萄糖3 g、(NH_(4))_(2)SO_(4)1 g、牛肉粉1.5 g、KH 2 PO_(4)0.6 g、MgSO_(4)0.3 g、FeSO_(4)和ZnSO_(4)各0.001 g、pH 6.0;装液量30 mL/250 mL、种龄16 h、接种量15%、发酵温度28℃、摇床转速240 r/min、发酵时间48 h。酵母内源性GSH提取条件为:乙醇提取浓度60%、提取时间40 min、物料比6∶1(K.que)与4∶1(P.occ)。通过发酵条件优化后,K.que菌株内源性GSH产量由81.5 mg/L提升至144.48 mg/L,提高了1.77倍;P.occ菌株内源性GSH产量由108.2 mg/L提升至193.92 mg/L,提高了1.79倍。本研究为高产GSH酵母菌株的诱变育种奠定前期研究基础,也为后续开发富化内源性GSH产品提供备选菌株。

This study is to identify and isolate microbial strains with the capacity to produce glutathione(GSH)with high quantities.Two high-yield GSH wild yeast strains were isolated successfully through a comprehensive screening process involving culture,plate coating and stripe isolation techniques.The alignment analysis of the 28S rDNA sequences revealed that the two strains were identified as Pichia occidentalis(P.occ)and Kurtzmaniellaquercitrusa(K.que)yeasts,respectively.The optimal fermentation conditions of the two strains were determined through the optimization of fermentation parameters,incorporating the ethanol extraction technique for endogenous GSH and the ALLOXAN reagent derivation measurement method.The results demonstrated that the optimal fermentation medium for K.que strain was as follows:each 100 mL solution included 1 g of sucrose,1 g of(NH_(4))_(2)SO_(4),1 g of tryptone,0.6 g of KH_(2)PO_(4),0.3 g of MgSO_(4),and trace amounts(0.001 g)of FeSO_(4) and ZnSO_(4),with a pH level of 5.0.The fermentation was conducted for 72 hours at a temperature of 28℃,shaker rotary speed of 240 r/min,and liquid volume ratio of 40 mL/250 mL.The seed age was set to 16 hours with an inoculum concentration of 10%.The optimal fermentation medium for P.occ strain was as follows:each 100 mL solution included 3 g of glucose,1 g of(NH_(4))_(2)SO_(4),1.5 g of beef powder,0.6 g of KH_(2)PO_(4),0.3 g of MgSO_(4),and trace amounts(0.001 g)of FeSO_(4) and ZnSO_(4) and with a pH level of 6.0.The fermentation was conducted for 48 hours at a temperature 28℃,shaker rotary speed of 240 r/min,and liquid volume ratio of 30 mL/250 mL.The seed age was set to 16 hours with an inoculum concentration of 15%.The extraction conditions for endogenous GSH in yeast were as follows:ethanol concentration of 60%,extraction time of 40 minutes,material ratio of 6:1(K.que)and 4:1(P.occ).After the optimization of the fermentation conditions,there was a significant enhancement in endogenous glutathione production for both K.que and P.occ strains with levels increasing from initial concentrations of 81.5 mg/L and 108.2 mg/L to final concentrations of 144.48 mg/L and 193.92mg/L respectively,resulting in respective fold increases of approximately 1.77 and l.79.This study laid a foundation for preliminary research on the mutation breeding of high-GSH producing yeast strains,and provided alternative strains for the subsequent development of endogenous GSH enriched products.