枯草芽胞杆菌信号分子的分离纯化

为获得较高纯度的B.subtilis分泌的信号分子,并研究其对L.paracasei HD1.7产细菌素的作用,本研究采用中空纤维系统与离子交换层析对信号分子进行分离纯化。利用SDS-PAGE检测信号分子的纯度及表观分子量,并采用抑菌试验分析信号分子对L.paracasei HD1.7产细菌素的作用。结果表明以pH4.0的Na Ac+Na Cl溶液作为洗脱液时出现3个洗脱峰,抑菌试验表明添加洗脱峰P3的L.paracasei HD1.7抑菌能力较原始菌株提高了11.31%,而添加洗脱峰P1和P2的L.paracasei HD1.7抑菌能力较原始菌株没有提高。因此,P3为含有信号分子的目的洗脱峰,SDS-PAGE电泳验证洗脱峰P3为单个条带。最佳层析条件:离子交换层析洗脱液(B液)为pH 4.0的Na Ac+Na Cl溶液,平衡液(A液)为pH 4.0的Na Ac溶液,洗脱流速为2 m L/min。SDS-PAGE验证表明在5 k Da左右出现单个条带,抑菌试验结果表明层析液中的B.subtilis信号分子能促进L.paracasei HD1.7产细菌素。

Separation and purification of the signal molecules of Bacillus subtilis

To obtain high- purity signal molecules secreted by B. subtilis and to explore the effect on the bacteriocin produced by L. paracasei HD1.7, the signal molecules were separated and purified by hollow fiber system and ion exchange chromatography in this study. SDS-PAGE was used to detect the purity and apparent molecular weight of signal molecules. The effect of signal molecules on L. paracasei HD1.7 bacteriocin was analyzed by antibacterial test. The results showed that three elution peaks occurred when pH 4.0 NaAc + NaCl solution was used as eluent, and the antimicrobial activity of L. paracasei HD1.7 added with elution peak P3 was higher than that of the original strain by 11.31%. However, the addition of elution peak P1 and P2 did not increase the antibacterial activity of L. paracasei HD1.7 compared with the original strain. Thus, P3 was the target elution peak containing the signal molecule, and the elution peak P3 was a single band detected by SDSPAGE. The optimal chromatographic condition was: ion exchange chromatography eluent (B eluent) was NaAc+ NaCl solution with pH 4.0, equilibrium eluent (A eluent) was NaAc solution with pH 4.0, the flow rate was 2 mL/min. SDS- PAGE analysis showed that a band appeared with molecule mass about 5 kDa. Subsequent antibacterial tests indicated that B. subtilis signal molecular in the chromatography solution could promote L. paracasei HD1.7 producing barteriocins.