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

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.

Key words:Bacillus subtilis;extracellular products;physical and chemical stability;antibacterial components