考察了长双歧杆菌WBL001菌体生长状态、酶解浓度、时长、温度以及不同渗透压稳定剂等因素,获得长双歧杆菌WBL001原生质体制备及再生的优化条件:长双歧杆菌以3%接种量活化至MRS培养基,培养至对数中期(OD600∶1),5μg/mL变溶菌素,37℃孵育30 min,以原生质体稳定液SMM为反应缓冲液,原生质体生成率达到81%,其再生率达到48%;在此基础上,通过聚乙二醇PEG融合,将穿梭表达载体pBAX转入双歧杆菌原生质体,成功建立双歧杆菌转化体系。

Six factors involved in the preparation and regeneration of Bifidobacteria longum protoplast were investigated,including state of growing cells,Mutanolysin concentration,enzymolysis time,hydrolysis temperature,and osmotic stabilizers.The optimum conditions were as follows: a log phase(19 h) culture of the recipient strain was grown in MRS medium,Mutanolysin concentration was 5 μg/mL,enzymolysis time was 30 min,SMM was used as reaction buffer.Using these optimum conditions,the protoplast formation rate reached 81%,and the ratio of regeneration was 48% in the optimum regeneration medium.On this basis,a Bifidobacteria transformation system of pBAX with PEG was established.