将唾液乳杆菌在有氧条件下进行高密度发酵,检测其活菌数,并以活菌数作为指标,研究唾液乳杆菌有氧呼吸的条件,提高活菌数和存活时间,使其更好的适应大规模工业发酵。从实验室保藏菌株中选择乳杆菌GDW1994,并进行16S rDNA测序鉴定,确定为唾液乳杆菌(Lactobacillus salivarius)。通过对菌株GDW1994进行酸和H2O2的耐性检测并研究其有氧呼吸发现其呼吸链的激活需要同时外加血红素(heme)和四烯甲萘醌(VK2)。优化后的培养基配方为蛋白胨10 g/L、酵母膏2.5 g/L、葡萄糖20 g/L、牛肉膏15 g/L、无水乙酸钠2 g/L、MgSO4 0.6 g/L、MnSO4·H2O 0.2 g/L。培养温度37 ℃、接种量4%、pH值6.4、转速为160 r/min、过氧化氢酶质量浓度为30 g/L。随着血红素与四烯甲萘醌的获得及O2的参与,菌株GDW1994在4 ℃保存可长达30 d,活菌数为108 CFU/mL,存活率是没有加血红素与四烯甲萘醌和O2参与的100倍,是外源过氧化氢酶的20倍。
High-density fermentation of Lactobacillus salivarius was conducted under aerobic conditions and the number of viable bacteria was measured.Using total viable count as the indicator to study the optimal conditions for aerobic respiration of L.salivarius,the number of viable bacteria and the survival time were improved for large-scale industrial fermentation.A Lactobacillusstrain named GDW1994 was screened and identified as L.salivarius by 16S rDNA sequencing.The effect of acid and hydrogen peroxide on the growth of strain L.salivarius GDW1994 revealed that the activation of its respiratory chain required the simultaneous addition of heme and tetraene naphthalene (VK2).The optimized medium was peptone 10 g/L,yeast extract 2.5 g/L,glucose 20 g/L,beef paste 15 g/L,anhydrous sodium 2 g/L,MgSO4 0.6 g/L,MnSO4·H2O 0.2 g/L.The optimal conditions were 37℃,inoculation volume 4%,pH 6.4,160 r/min and catalase 30 g/L.With the acquisition of heme and tetraene methanone,and the participation of oxygen,the GDW 1994 strain could be preserved for up to 30 d at 4 ℃.The total viable count reached 108 CFU/mL and survival rate was 100 times higher than that of the medium without heme,tetraene naphthalene and oxygen.It was 20 times higher than that of the culture medium added with exogenous catalase.
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