凝结芽孢杆菌原生质体的制备、再生及灭活

Protoplasts preparation,regeneration and inactivation of Bacillus coagulans

凝结芽孢杆菌是乳酸工业化生产的主要发酵菌株之一,但在发酵结束时,发酵液中往往会残留(6~8)g/L的残糖。为了降低发酵液中的残糖含量,可以通过灭活原生质体融合技术引入外源可降解残糖的酶基因来改善凝结芽孢杆菌降解残糖的能力。以凝结芽孢杆菌(Bacillus coagulans)C菌株为供试菌株,考察了酶浓度、酶解温度和酶解时间对凝结芽孢杆菌原生质体制备与再生的影响。结果表明,在菌体OD_(620)为10时,最适的酶解条件为:溶菌酶浓度0.14 mg/mL,酶解温度37℃,酶解时间10 min。在此条件下,原生质体的制备率为95.98%~97.64%,再生率达到13.49%~15.62%。在此基础上,分别考察了紫外致死和高温致死对凝结芽孢杆菌原生质体再生的影响。当紫外照射360 s和70℃高温处理25 min时,原生质体的致死率都能达到100%,这为后续的原生质体融合奠定了基础。

Bacillus coagulans is one of the main industrial strains for lactic acid production, although there is normally（6-8）g/L total sugar remaining in the broth along with the end of lactic acid fermentation. In order to reduce residual sugar content, it is feasible to improve the residual sugar degradation ability of B. coagulans by inactivated protoplast fusion with introducing the enzyme gene of degrading residual sugar. In this study, the effects of the concentration, temperature and action time of lysozyme on protoplast preparation and regeneration by B. coagulans C were investigated. The results showed that the optimal conditions for preparation and regeneration of protoplasts were treating cells with 0.14 mg/mL lysozyme for 10 min at 37 ℃ when the cell biomass was adjusted to OD620 of 10. Under these conditions, the protoplast formation and regeneration rates were 95.98%-97.64% and 13.49%-15.62%, respectively. On this basis, the effects of heating treatment and ultraviolet（UV） irradiation treatment on the inactivated protoplast rate of B. coagulans C were studied. As a result, the inactivation conditions for B. coagulans C protoplast were heating treatment at 70 ℃ for 25 min and UV irradiation for 360 s, respectively, which could reach to 100% inactivation rate of protoplast. This study played a fundamental role in subsequent protoplast fusion.