乳酸菌原生质体制备与再生研究

Study on Protoplast Formation and Regeneration in Lactobacillus

从市售酸乳中分离嗜热链球菌和保加利亚乳杆菌,并对其进行原生质体制备和再生。采用溶菌酶对嗜热链球菌和保加利亚乳杆菌进行处理,脱去细胞壁以探讨原生质体形成和恢复与菌龄、酶浓度、酶解时间及酶解温度之间的关系,利用四因素三水平的正交试验选出原生质体形成和恢复较适宜的条件。试验结果显示嗜热链球菌原生质体制备和恢复的较优条件为,菌龄16h,酶浓度1mg/ml,酶解时间40min,酶解温度42℃,此时原生质体形成率为98.75%,再生率为23.8%;而保加利亚乳杆菌原生质体制备和恢复的较优条件为,菌龄16h,酶浓度10mg/ml,酶解时间30min,酶解温度36℃,此时原生质体形成率为82.05%,再生率为27.1%。本文研究为这两种乳酸菌原生质体的制备、再生条件及其基因操作和相关研究提供技术思路和实验条件。

Object： To make protoplast and regenerate from Streptococcus thermophillus and Lactobacillus bulgaricus. The factors are consided that the protoplast are formed with the fungus age, the enzyme density, between the enzymolysis time and the enzymolysis temperature. To select the better conditions of the protoplast production with four factors three levels of the orthogonal test method. Results： The optimum conditions of the Streptococcus thermophilus are as follows： 16 hours of incubation times, concentration of lysozyme lmg/ml, the hydrolyzing temprature 42℃, treating time 40 minutes. Under the optimum conditions, the ratio of formation protoplast are 98.75%, the ratio of regeneration are 23.8%; and the Lactobacillus bulgaricus are as follows： 16 hours, 10mg/ml, 36℃, 30min, 82.05%, 27.1%.Conclusion： The technique of this article could provide the effective data to genetic engineer and its protoplast fusion of the two bacteria.