罗伊氏乳杆菌原生质体的制备与再生条件的研究  

Studies on Protoplasts Preparation and Regeneration Condition of Lactobacillus Reuteri

作　　者：韩璞[1] 田洪涛[1] 苑社强[1] 郭红敏[1] 郭兴华[2] 罗云波[3] 

机构地区：[1]河北农业大学食品科技学院,河北保定071001 [2]中国科学院微生物研究所,北京100080 [3]中国农业大学食品科技与营养工程学院,北京100083

出　　处：《中国食品学报》2010年第1期10-18,共9页Journal of Chinese Institute Of Food Science and Technology

基　　金：国家高技术研究发展计划(863计划)项目(No.2006AA10Z317)

摘　　要：研究细胞培养和溶菌酶酶解条件对罗伊氏乳杆菌原生质体形成率的影响以及罗伊氏乳杆菌原生质体在多种再生培养基中的再生条件,结果表明罗伊氏乳杆菌05-1和05-2原生质体形成的最适条件:在MRS培养基中接种量5%(两菌株活菌数分别为4.96×108 cfu/mL和8.04×107 cfu/mL),37℃静置培养至OD600值1.0(两菌株活菌数分别为2.36×1012 cfu/mL和6.56×109 cfu/mL)左右,酶解pH8.0、酶质量浓度20 mg/mL、酶解时间45min。在此最适条件下,两菌株原生质体形成率分别为90.2%和92.8%;在最适再生培养基RM1中,罗伊氏乳杆菌05-1和05-2原生质体的再生率分别为30.6%和23.2%;RM1中,胎牛血清(BSA)是罗伊氏乳杆菌原生质体再生不可获缺的物质。本研究为构建益生乳杆菌食品级基因克隆和表达系统,实现原生质体的转化及细胞融合育种与基因组洗牌分子育种,提供理论依据。The effect of protoplasts formation ratio in LactobaciUus reuteri have been researched with cell culture and lysozyme conditions in this paper.Regeneration of protoplasts in Lactobacillus reuteri have been explored on multimedia. The results showed that Lactobacillus reuteri 05-1 and 05-2 protoplast formation of the optimum conditions were as follows：5% of incubation in MRS media （The viable cells counts of two strains were respectively 4.96×10^8 cfu/mL and 8.04×10^7 cfu/mL）, strains were cultured at 37 ℃ with an optical density about 1.0 at 600 nm （The viable cells counts of two strains were respectively 2.36×10^12 cfu/mL and 6.56×10^9 cfu/mL）,range of pH 8.0, enzyme concentration of 20 mg/mL, treating time of 45min, in the optimum conditions, the two strains protoplast formation rates were reached to 90.2% and 92.8% respectively. The ratio of regeneration of Lactobacillus reuteri 05-1 and 05-2 were 30.6% and 23.2% in the optimum renewable medium RM1. In the renewable medium RM1, fetal calf serum （BSA） was the indispensable material to the regeneration in Lactobacillus reuteri. This research provide practical basis, in order to construct of the food-grade gene cloning and expression system for Lactobacillus, realize the protoplasts transformation, and screen good stains by fusion of Lactobacillus and genome shuffling.

关 键 词：罗伊氏乳杆菌 原生质体 制备 再生 

分 类 号：TS201.3[轻工技术与工程—食品科学]