保加利亚乳杆菌与嗜热链球菌抗噬菌体菌株的原生质体制备及融合

Protoplast Formation and Fusion between Lactobacillus bulgaricus and Bacteriophage-Resistant Mutant of Streptococcus thermophilus

为获得具有抗噬菌体功能且发酵性能优良的乳酸菌融合子,采用单亲灭活及正交分析方法,研究了保加利亚乳杆菌与嗜热链球菌抗噬菌体菌株的原生质体制备及融合条件。结果表明:保加利亚乳杆菌原生质体制备的最适条件是以磷酸盐缓冲液和甘露醇制作的高渗溶液为原生质体稳定剂,1.0 mg/m L的溶菌酶36℃处理30 min,原生质体的形成率为(89.02±2.31)%,再生率为(4.62±0.22)%。嗜热链球菌抗噬菌体菌株原生质体制备的最适条件是以Tris-HCl和蔗糖制作的高渗溶液为原生质体稳定剂,0.1 mg/m L的溶菌酶42℃处理30 min,原生质体的形成率为(99.15±0.23)%,再生率为(5.79±0.17)%。单亲灭活保加利亚乳杆菌与嗜热链球菌抗噬菌体菌株原生质体融合的最适条件为聚乙二醇6000(质量浓度为400 g/L,添加0.01 mol/L Ca Cl2、0.02 mol/L Mg Cl2)40℃促融2 min,融合率可达(1.85±0.12)×10-6。所得融合子各项性能优良,适合于酸奶生产。

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/m L 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-resistantS. thermophilus were up to（99.15 ± 0.23）% and（5.79 ± 0.17）% after treatment with 0.1 mg/m L lysozyme for 30 min in the presence of hypertonic solution with Tris-HCl buffer and sucrose. The fusion rate of protoplasts between L. bulgaricusand 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 Ca Cl2 and 0.02 mol/L Mg Cl2. The performance of the fusant is stable and it is applicable to yoghurt production.