甲基营养型芽孢杆菌Z21发酵条件优化及抑菌活性分析

Fermentation Optimization and Antifungal Activity of Bacillus methylotrophicus Z21

【目的】芽孢杆菌Z21发酵液对黑曲霉、康氏木霉等真菌有拮抗性,探究Z21菌抑菌活性物质稳定性及抑菌活性分析,可为开发天然防腐剂提供参考。【方法】通过牛津杯法,以康氏木霉为指示菌,优化Z21菌产抑菌活性物质的发酵条件,探讨不同温度、紫外线、pH、作用时间、酶和金属离子对Z21菌抑菌活性物质稳定性的影响;测定硫酸铵沉淀法提取的粗提物MIC及对康氏木霉孢子萌发和菌丝生长的影响。【结果】当发酵条件为500 mL锥形瓶装液体培养基150 mL、接种量3%、发酵时间60 h、发酵温度30℃、转速140 r·min^(-1)时,150μL无菌发酵液对康氏木霉菌丝生长的抑制率为72%左右,粗提物的MIC为25 mg·mL^(-1)。在紫外光波长为260 nm和280 nm时,经25 mg·mL^(-1)的粗取物处理后的康氏木霉菌液紫外吸光值升高,说明菌丝的核酸和蛋白质有泄露。Z21菌发酵液耐热,耐酸、碱,对紫外线、许多酶和金属离子不敏感。扫描电镜观察显示:Z21菌处理后的康氏木霉菌丝比较纤细,不分化孢子梗。【结论】Z21菌发酵液对康氏木霉的生长和繁殖均受到抑制,抑菌活性物质稳定性良好。

【Objective】Fermentation process of Bacillus methylotrophicus Z21,which exhibited antifungal activities on Aspergillus niger and Trichoderma koningii among others in a preliminary study,was optimized,and the stability and antibacterial properties of the broth as a nature preservative examined.【Method】The fermentation conditions,including temperature,UV,pH,time,enzymes,and metal ions,were optimized based on the antifungal activity of the broth against T.koningii determined by the Oxford cup method.Effects of the ammonium sulfate-precipitated crude extract on the MIC,inhibition rate,spore germination,and hypha growth of T.koningii were also examined.【Result】The optimized process was conducted in a 500 mL flask to hold 150 mL liquid medium with 3%inoculum for the 60 h fermentation at 30℃under constant 140 r·min^(-1) stirring.The inhibition rate of the 150μL sterile fermentation broth on the hypha growth was 72%.The increased UV absorbances of the T.koningii culture liquid at OD260 and OD280 by the treatment of the extract at MIC of 25 mg·mL^(-1) indicated a leakage of nucleic acids and proteins into the liquid from the mycelia.The Z21 fermentation broth was resistant to heat,acid,and alkali but insensitive to UV,enzymes,and metal ions.The scanning electron microscopic images showed that the hyphae of T.koningii treated by the broth became slender without spore stems.【Conclusion】The growth and reproduction of T.koningii were significantly inhibited by the presence of B.methylotrophicus Z21 fermentation broth.The antifungal activity of the broth remained stable under elevated temperature and/or acidic or alkali conditions.