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.