Protoplast preparation and regeneration of the dominant aerobic bacteria isolated from feces of animals

In this study,bacterium suitable for protoplast preparation and regeneration was selected from dominant aerobic intestinal bacteria isolated from different animals.The optimal conditions for protoplast preparation and regeneration of the selected predominant aerobic intestinal bacterium were determined,the study of which will provide research basis for the usage of the protoplast of the selected bacterium as one parent in protoplast fusion with that of an anaerobic bacterium.The result showed that only 5 of the 20 predominant aerobic intestinal bacteria were relatively sensitive to lysozyme,which included bacterial strains M3,M4,M5 and M6 isolated from intestinal tract of ICR mouse,and Z5 isolated from pig.Further studies indicated that among all of the 5 lysozyme sensitive bacteria,strain M6(i.e.Escherichia fergusonii M6) had the highest protoplast preparation rate and protoplast regeneration rate.Therefore,bacterium Escherichia fergusonii M6 was chosen for further study.Different factors,including bacterial age,enzyme concentration,enzymolysis time,osmotic pressure stabilizer and addition of different chemical compounds were studied.The optimal conditions for the protoplast preparation and regeneration of strain M6were as follows : bacterial age 5h,lysozyme concentration 20mg/mL,enzymolysis time 45min;and in the regeneration medium,osmotic pressure stabilizer 0.7mol/L D-sorbitol,0.8%bovine serum albumin and 0.1mol/L N-acetylglucosamine were added.The average protoplast preparation and regeneration rate was 87.80% and 85.42% under the optimal conditions.