Abstract:

Primary factors affecting protoplast formation and regeneration of Lactobacillus bulgaricus and Streptococcus
thermophilus were investigated using orthogonal array design to obtain fusants with bacteriophage resistance and superior
fermentation performance. The results showed that the optimal preparation conditions for L. bulgaricus protoplasts
were achieved by treatment with 1.0 mg/mL lysozyme for 30 min at 36 ℃ in the presence of hypertonic solution
with phosphate buffer and mannitol. Under these conditions, the protoplast formation and regeneration rates reached
(89.02 ± 2.31)% and (4.62 ± 0.22)%, respectively. The protoplast formation and regeneration rates of bacteriophage-resistant
S. thermophilus were up to (99.15 ± 0.23)% and (5.79 ± 0.17)% after treatment with 0.1 mg/mL lysozyme for 30 min in
the presence of hypertonic solution with Tris-HCl buffer and sucrose. The fusion rate of protoplasts between L. bulgaricus
and S. thermophilus reached (1.85 ± 0.12) × 10-6 after infusion for 2 min at 40 ℃ in the presence of 400 g/L PEG6000,
0.01 mol/L CaCl2 and 0.02 mol/L MgCl2. The performance of the fusant is stable and it is applicable to yoghurt production.

Key words: Lactobacillus bulgaricus, bacteriophage-resistant mutant of Streptococcus thermophilus, protoplast formation, protoplast fusion