基于气相色谱-飞行时间质谱联用技术分析枯草芽孢杆菌对变异链球菌的抑制作用

Measurement of inhibitory effect of Bacillus subtilis on Streptococcus mutans by gas-chromatography-time of flight-mass spectrometry

摘要:

【背景】龋病危害着人类健康，变异链球菌(Streptococcus mutans)是公认的主要致龋菌。我们前期的研究从口腔中分离出一株枯草芽孢杆菌(Bacillus subtilis)，其能抑制S. mutans的生长，但尚未明确其抑菌作用的组分。【目的】利用非靶向代谢组学技术检测分析B. subtilis zh78抑制S. mutans生长可能的小分子代谢物，以评估其在龋病防治研究及应用中的前景。【方法】取B. subtilis zh78经生长0 h、7 h、12 h、5 d时的菌液，利用牛津杯法将冷甲醇萃取后的代谢物对S. mutans进行抑菌实验；采用气相色谱-飞行时间质谱联用技术(GC-TOF-MS)对代谢物进行检测；利用主成分分析(Principal component analysis，PCA)、正交偏最小二乘判别分析(Orthogonal partial least squares discriminant analysis，OPLS-DA)和皮尔森相关分析(Pearson correlation analysis)等方法进行数据分析。【结果】B. subtilis zh78生长5 d时的代谢产物对S. mutans具有明显抑菌作用，其代谢物中木糖醇、氨基酸类及有机酸类等36种物质与其对S. mutans的抑制作用显著相关。【结论】B. subtilis zh78可能产生木糖醇、氨基酸类及有机酸类等可能促进B. subtilis zh78抑制S. mutans生长的小分子代谢产物，具有一定的防龋应用研究前景。

Abstract:

[Background] Dental caries is harmful to human health. Streptococcus mutans is recognized as the main cariogenic bacteria. In our previous studies, we found the Bacillus subtilis (Bacillus subtilis zh78) isolated from oral cavity could inhibit the growth of S. mutans. However, its bacteriostatic components were still ambiguous. [Objective] This study aimed to (1): determine the possible small molecular metabolite of B. subtilis zh78 that may inhibit the growth of S. mutans by untargeted metabolomics approach; (2) evaluate the prospect of B. subtilis zh78 in caries prevention and treatment. [Methods] Firstly, we used the cold methanol to extract the metabolites in the bacteria solutions from the B. subtilis zh78 growth at 0 hour, 7 hours, 12 hours and 5 days respectively. Oxford cup method was used to test the antibacterial activity of the extracted metabolites against S. mutans. Then, gas-chromatography-time of flight-mass spectrometry (GC-TOF-MS) was utilized to metabonomics detection. Lastly, we used principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA) and Pearson correlation analysis to process the data. [Results] The metabolites at 5 days of B. subtilis zh78 showed the most obvious inhibitory effect on S. mutans. 36 kinds of substances, such as xylitol, amino acids and organic acids produced by B. subtilis zh78 were significantly related to its inhibitory effect on S. mutans. [Conclusion] The findings suggested that the B. subtilis zh78 could produce certain kinds of metabolites (e.g. xylitol, amino acids and organic acids) which might exert the antibacterial effects on S. mutans. The B. subtilis zh78 displayed its potential in oral probiotic application and the related studies.