枸杞果汁发酵过程复合乳酸菌的实时定量检测

Real-time quantitative detection of multiple lactic acid bacteria during wolfberry juice fermentation

利用植物乳植杆菌(Lactiplantibacillus plantarum)、发酵粘液乳杆菌(Limosilactobacillus fermentum)和瑞士乳杆菌(Lactobacillus helveticus)混合菌种进行枸杞果汁发酵。该研究对乳酸菌亚致死修复液的组成和修复温度进行优化,分别针对3株乳酸菌设计特异性引物,利用叠氮溴化丙锭(PMA)-荧光定量聚合酶链式反应(fq PCR)的方法,实时荧光定量检测枸杞果汁发酵过程中乳酸菌活菌数。结果表明,优化修复液的组成为蛋白胨1 g/L、牛肉浸出粉0.3 g/L、氯化钠0.5 g/L、吐温80 0.10 g/L、丙酮酸钠0.09 g/L、过氧化氢酶0.04 g/L、Mg Cl_(2)3 mmol/L、Na_(2)HPO_(4)1 mmol/L、MnCl_(2)2 mmol/L和Fe Cl_(2)2 mmol/L。在27℃条件下培养15 min,乳酸菌亚致死细胞修复率达到97%。利用该方法实现了枸杞果汁发酵过程不同乳酸菌的实时定量检测,为今后枸杞果汁发酵生产过程中的微生物动态监测提供了方法。

Wolfberry(Lycium barbarum)juice was fermented using multiple lactic acid bacteria of Lactiplantibacillus plantarum,Limosilactobacillus fermentum and Lactobacillus helveticus.In this study,the composition of sublethal repair solution and repair temperature of lactic acid bacteria were optimized,the specific primers were designed for 3 strains of lactic acid bacteria,and the counts of viable lactic acid bacteria during wolfberry juice fermentation was detected with real time fluorescence quantification by propidium monoazide(PMA)-fluorescence quantitative polymerase chain reaction(fqPCR)method.The results showed that the optimal composition of the repair solution was as follows:peptone 1 g/L,beef extract powder 0.3 g/L,sodium chloride 0.5 g/L,Tween 800.10 g/L,sodium pyruvate 0.09 g/L,catalase 0.04 g/L,MgCl_(2)3 mmol/L,Na_(2)HPO41.00 mmol/L,MnCl_(2)2 mmol/L and FeCl_(2)2 mmol/L.When cultured at 27℃for 15 min,the repair rate of sublethal cells of lactic acid bacteria reached 97%.The real-time quantitative detection of different lactic acid bacteria during wolfberry juice fermentation was realized,which provided a method for the dynamic monitoring of microorganism during wolfberry juice fermentation production in the future.