保加利亚乳杆菌H+-ATPase缺陷型菌株的筛选

【目的】从传统乳制品中筛选具有新霉素抗性的H+-ATPase缺陷的德氏乳杆菌保加利亚亚种自发突变株，为最终开发弱后酸化的酸奶发酵剂奠定基础。【方法】利用API 50 CH细菌鉴定系统和16s rRNA基因序列分析对菌株进行鉴定。新霉素作为筛选压力，筛选具有新霉素抗性自发突变菌株，比较亲本和突变菌株的H+-ATPase活力及其代谢情况。【结果】从内蒙古地区的传统发酵酸奶中分离鉴定出一株德氏乳杆菌保加利亚亚种（Lactobacillus delbrueckii subsp. bulgaricus），并命名为KLDS 1.9201。以此为出发菌株，筛选出两株H+-ATPase缺陷的自发突变株，分别命名为KLDS 1.9201-1、KLDS 1.9201-4，它们的H+-ATPase活力分别比亲本KLDS 1.9201降低了46%和60%。在MRS培养基中生长24 h后，KLDS 1.9201、KLDS 1.9201-1和KLDS 1.9201-4对初始葡萄糖的代谢率分别为65%、41%和31%，终产物中乳酸的浓度分别为26g/L、18g/L和15g/L，突变菌株的生物量均低于亲本。【结论】H+-ATPase活力降低的德氏乳杆菌保加利亚亚种的自发突变株具有较低的生长速率和弱产酸能力，它们可被用于制作弱后酸化的酸奶发酵剂。

Screening of H+-ATPase deficient mutant of Lactobacillus delbrueckii subsp. Bulgaricus

Objective] Wes identified a neomycin-resistant mutant with reduced membrane-bound H+-ATPase activity from L. delbrueckii subsp. bulgaricus originated from the traditional dairy product to develop a yoghurt starter culture with low post-acidification capacity. Methods] API 50 CH identification system and 16s rDNA sequence analysis were applied to identify the strain isolated from the indigenous yoghurt. Neomycin was used to screen spontaneous neomycin-resistant mutants. H+-ATPase activity and metabolic dynamics were evaluated between the parent strain and mutants. Results] One strain was identified as L. delbrueckii subsp. bulgaricus by API 50 CH identification system and 16s rDNA sequence analysis, coded as KLDS 1.9201. Two mutant strains were mutated from KLDS 1.9201 and coded as KLDS 1.9201-1, KLDS 1.9201-4. Compared with the parent strain, the H+-ATPase activity of the mutants KLDS 1.9201-1 and KLDS 1.9201-4 decreased by 46% and 60% respectively. After cultured in MRS broth for 24 h, metabolic efficiency of the initial glucose for the parent strain was 65%, the mutants were 41% and 31%, the lactic acid concentration in the culture for the parent strain was 26g/L, the mutants were 18g/L and 15g/L respectively. The cell density of the mutants was lower than that of the parent strain. Conclusion] The mutants of L. delbrueckii subsp. bulgaricus with reduced membrane-bound H+-ATPase activity had low growth and acid production, which could be applied to develop yoghurt starter culture with lower post-acidification.