利用pGhost系统筛选抗后酸化保加利亚乳杆菌

Construction of Anti-postacidification Mutants of Lactobacillus delbrueckii subsp. bulgaricus Using pGhost System

目的:利用p Ghost系统构建抗后酸化保加利亚乳杆菌。方法:利用载体p Ghost9:ISS1经复制型转座构建突变体,之后在p H 4.3条件下,突变细胞用含终质量浓度2 000μg/m L青霉素处理5 h。将处理后的菌液涂布在含终质量浓度2μg/m L红霉素的MRS平板上,长出的菌落分别点种p H 4.3和p H 6.5的MRS平板进行筛选,在低p H值条件下生长不良或不生长的菌株即目的菌株。经进一步检验,筛选发酵性能与出发菌株相当,而贮藏期后酸化能力大为减弱的菌株。将筛选出的抗后酸化菌株通过低温刺激,使转座后的载体重组,载体部分从染色体上切除,并经高温培养后丢失。剔除载体后的突变型菌落是食品级突变体,为红霉素敏感型。结果:获得3株抗后酸化保加利亚乳杆菌菌株,其生物学性状稳定,回复突变率低。

The aim of this study was to obtain anti-postacidification mutants of Lactobacillus delbrueckii subsp. bulgaricus using pGhost system. Methods： After the construction of the mutants gained by replication translocation using plasmid pGhost9：ISS1, these strains were treated for 5 h on MRS agar plates containing 2 000 μg·mL-1penicillin and pH4.3. Then the samples were diluted and plated on MRS agar plates containing 2 μg·mL-1Em at 42 ℃ for 48-72 h. Subsequently the colonies were transferred with toothpicks to low pH 4.3 and high pH 6.5 MRS agar plates and the strains which were poor growth or no growth under the low pH were purpose strains. After further tests, the strains were similar to the starting strain in fermentation performance but had a significant reduction in postacidification ability. At last all the wanted strains were restructured with the stimulation of temperature shift and the efficient excision of the replicon, which gave rise to stable food-grade mutant strains sensitive to Em. Results： Through this study we screened out 3 anti-postacidification mutants with stable biological characters and low recovery mutation rate.