一株蜂粮源拮抗细菌的分离鉴定及其抑菌物质特性

Isolation, identification of an antagonistic bacteria from bee bread and characteristics of antimicrobial substances

【目的】为发掘和利用蜂粮中拮抗菌资源,对分离获得的拮抗细菌菌株PC2进行分类鉴定,并测定其发酵液抑菌物质基本特性。【方法】采用改良牛津杯双层平板法测定菌株发酵液抑菌谱及温度、p H、紫外线和蛋白酶对其抑菌活性稳定性的影响,菌株鉴定结合形态学、生理生化特征和16S r RNA基因序列分析,硫酸铵沉淀法和盐酸沉淀有机溶剂提取法进行抑菌活性物质的初步分离。【结果】从3种蜂粮中分离筛选得到17株拮抗菌株,其中1株细菌PC2以马铃薯葡萄糖液体培养基发酵制备的无菌发酵液对7种供试菌株具有较强抑制作用,经形态、生理生化特征及16S r RNA基因序列分析,将其初步鉴定为解淀粉芽胞杆菌(Bacillus amyloliquefaciens)。菌株发酵液抑菌活性对温度、酸和紫外线具有较强的稳定性,对蛋白酶K、胃蛋白酶、碱性蛋白酶处理敏感。菌株发酵液存在抑菌蛋白和脂肽类物质。【结论】菌株PC2在食品保鲜和农业生防中具有潜在的开发应用价值。

[Objective] To explore potent antagonistic microorganisms in bee bread, an antagonistic strain PC2 isolated from bee bread was identified, and its antimicrobial characteristics were evaluated. [Methods] Antagonistic spectrum and effect of thermal, p H, UV irradiation and protease on fermentation broth inhibitory activity were assessed by Oxford cup plate assay; strain identification was based on morphology, physiological and biochemical characteristics and 16 S r RNA gene sequence analysis. The antimicrobial protein and lipopeptide compounds were separated by ammonium sulfate precipitation and acid-precipitate organic solvent extraction method, respectively. [Results] A total of 17 antagonistic microorganisms were isolated from 3 different bee bread samples. Strain PC2 fermentation broth cultured in Potato Dextrose Broth showed excellent antimicrobial activity against 7 tested microorganisms. Strain PC2 was preliminary identified as Bacillus amyloliquefaciens by morphological, physiological and biochemical characteristics combined with 16 S r RNA gene sequence analysis. The antimicrobial activity of fermentation broth was not sensitive to temperature, acidity and UV irradiation. However, fermentation broth treated with pepsin, alkaline proteinase and protease K lost antimicrobial activity. Proteins and lipopeptides were the main antimicrobial compounds. [Conclusion] Strain PC2 isolated from bee bread has potent applications in food preservation and agricultural biological control.