枯草芽孢杆菌斯氏亚种胞外产物的抑菌活性及其特性分析

Antibacterial activity and characterization of extracellular products of Bacillus subtilis subsp. spizizenii

摘要:

【目的】 研究枯草芽孢杆菌斯氏亚种(Bacillus subtilis subsp. spizizenii) Bspi2104胞外抑菌物质的理化特性及抗菌活性组分。【方法】 选取具有广谱抗菌活性的枯草芽孢杆菌斯氏亚种Bspi2104菌株和无抗菌作用的枯草芽孢杆菌斯氏亚种BspiL6菌株，结合牛津杯法和代谢组学检测技术对菌株胞外产物的抑菌活性和理化特性及其抗菌活性组分进行研究。【结果】 胞外产物理化稳定性试验表明，胰蛋白酶、木瓜蛋白酶、蛋白酶K、胃蛋白酶和脂肪酶处理对胞外产物的抑菌活性无显著影响；胞外产物经80 ℃和100 ℃处理后丧失抑菌活性；pH 9.0和pH 11.0条件下胞外产物的抑菌活性下降，pH值越大抑菌活性越弱；经过紫外处理的胞外产物与未经紫外处理的胞外产物抑菌活性无明显差异(P>0.05)。60%、70%和80%饱和度下的硫酸铵沉淀产物具有抑菌活性，硫酸铵饱和度为70%时抑菌活性最强；菌株胞外产物进行盐酸沉淀后，分别通过甲醇抽提提取、乙酸乙酯萃取提取和氯仿萃取提取后的产物均有抑菌活性，其中菌株Bspi2104的乙酸乙酯萃取提取产物抑菌效果最好。通过LC-MS/MS检测技术对菌株Bspi2104和BspiL6不同提取方式下枯草芽孢杆菌胞外产物组分进行分析，差异代谢物筛选表明，两株菌的不同提取方式产物中共有的差异代谢物有35种；差异代谢产物主要分属于羧酸及其衍生物、脂肪酸类、有机含氧化合物、有机含氮化合物、甾体及其衍生物、孕烯醇酮脂类、酚类、生物碱及其衍生物、甘油磷脂类、异黄酮类、苯及其取代衍生物等37类物质，包括苦参素、表面活性素B等多种抑菌物质。【结论】 枯草芽孢杆菌斯氏亚种Bspi2104胞外产物的理化稳定性较好，在多种蛋白酶和脂肪酶、pH 1.0−11.0、−20−60 ℃和紫外照射3 h处理下仍然具备较高的抑菌活性。枯草芽孢杆菌斯氏亚种Bspi2104胞外产物的硫酸铵沉淀、盐酸沉淀甲醇抽提、乙酸乙酯萃取和氯仿萃取提取物均有抑菌活性，其中70%硫酸铵沉淀提取物和乙酸乙酯萃取提取物抑菌效果最好，菌株具有多种类型的抑菌活性物质。相关研究结果为芽孢杆菌抑菌活性组分的发现和筛选提供了理论参考，相关代谢产物具有一定的研究前景。

Abstract:

[Objective] To study the physicochemical properties and active components of extracellular antibacterial substances from Bacillus subtilis subsp. spizizenii Bspi2104. [Methods] The Oxford cup method was employed to determine the antibacterial activities and physicochemical properties of B. subtilis subsp. spizizenii Bspi2104 with broad-spectrum antibacterial activity and B. subtilis subsp. spizizenii BspiL6 without antibacterial activity. Metabolomics was employed to detect the antibacterial components of the extracellular products of the strain Bspi2104. [Results] The treatment with trypsin, papain, protease K, pepsin, and lipase had no significant effect on the antibacterial activity of extracellular products. Extracellular products lost the antibacterial activity after treatment at 80 ℃ and 100 ℃ and presented decreased antibacterial activity at pH 9.0 and pH 11.0, especially at higher pH values. There was no significant difference in the antibacterial activity between the extracellular products treated with and without UV (P>0.05). The ammonium sulfate precipitates at 60%, 70%, and 80% saturation exerted antibacterial activities, which was the strongest at the saturation of 70%. The extracellular products of the two strains were extracted by hydrochloric acid precipitation, combined with methanol extraction, ethyl acetate extraction, and chloroform extraction, and all the extracts showed antibacterial activities. The ethyl acetate extract had the strongest antibacterial activity. LC-MS/MS was employed to analyze the composition of extracellular products of Bspi2104 and BspiL6 extracted with different methods. There were 35 common differential metabolites in the extracellular products of the two strains extracted with different methods. The differential metabolites belonged to 37 categories of compounds, including carboxylic acids and derivatives, fatty acids, organic oxygen-containing compounds, organic nitrogen-containing compounds, steroids and derivatives, pregnenolone lipids, phenols, alkaloids and derivatives, glycerol phosphates, isoflavonoids, and benzene and substituted derivatives. Some of these compounds, such as kurarinone, and surfactin B, had antibacterial activities. [Conclusion] The extracellular products of B. subtilis subsp. spizizenii Bspi2104 had good physicochemical stability and maintained high antibacterial activity after treatment with various proteases and lipases, and at −20 ℃ to 60 ℃, pH 1.0–11.0, and UV irradiation for 3 h. The ammonium sulfate precipitates and the extracts from hydrochloric acid precipitation combined with methanol extraction, ethyl acetate extraction, and chloroform extraction of the extracellular products of B. subtilis subsp. spizizenii Bspi2104 had antibacterial activities. Among them, the 70% ammonium sulfate precipitate and ethyl acetate extract had the best antibacterial effects. The extracellular products of the strain contained diverse categories of antibacterial compounds. The findings provide theoretical reference for the discovery and screening of antibacterial components of Bacillus, and the related metabolites have research prospects.