Protoplasting, regeneration and fusion of lactobacilli

Cells of two Lactobacillus strains were protoplasted by treatment with mutanolysin or combinations of mutanolysin and lysozyme for various incubation times. Almost all protoplastization treatments tested were suitable for reducing the number of osmotically stable cells two log cycles. Protoplasts were successfully regenerated on a complex medium containing McCl₂, CaCl₂, gelatin, raffinose and bovine serum albumin (BSA). Maximal regeneration frequencies ranged from 6 to 10 % for L. casei subsp. rhamnosus and L. lactis, respectively. The use of agar overlays did not affect the regeneration ability of either strain. Treatment of protoplasts of both strains with 40 % polyethylene glycol (PEG) resulted in a regeneration frequency decrease of several hundredfold. Using the different resistance levels of both strains to the antibiotics kanamycin and penicillin as selected genetic markers, attempts to fuse protoplasts of these lactobacilli in the presence of 40 % PEG were not successful.