摘要
【背景】近年来,从植物内生菌的发酵产物中挖掘抑菌活性成分已成为寻找新型生物活性成分的重要途径。【目的】寻找新的具有高效抗菌活性的植物内生菌,并探究其产生抗菌活性物质的最优发酵条件和抑菌机制。【方法】利用组织分离法和划线法从柠檬皮和籽核中分离内生菌株,按照滤纸片和牛津杯法筛选对病原菌具有较强抑制作用的菌株,通过形态观察和16S rRNA基因序列分析确定活性菌株的分类地位,基于微孔板法检测活性菌株发酵液提取液(B02c)的抑菌效果,以单因素试验和响应面试验设计对发酵条件进行优化,并通过碘化丙啶/SYTO-9探针标记结合流式细胞术检测B02c对检定菌细胞膜的影响。【结果】从柠檬籽核中筛选出一株对金黄色葡萄球菌(Staphylococcus aureus)具有显著抑制作用的菌株Z10,鉴定为芽孢杆菌(Bacillus sp.);菌株Z10的最佳发酵条件为柑橘皮渣添加量1.6 g/100 mL、pH 6.8、接种量10.0%、转速172 r/min、培养时间36 h,优化后B02c的抑菌率达到84.36%,较优化前提升16.36%;B02c对金黄色葡萄球菌的最低抑制浓度(minimum inhibitory concentration,MIC)为3.125 mg/mL,其能够破坏金黄色葡萄球菌细胞膜的完整性。【结论】柠檬内生菌Z10在优化发酵条件后获得的B02c对金黄色葡萄球菌具有良好的抑菌效果,在新型、高效的天然抑菌活性物质筛选及其工业化生产中具有一定应用前景。
[Background]In recent years,mining antimicrobial components from the fermentation products of endophytes has become an important way to discover new bioactive components.[Objective]To find new endophytes with high antimicrobial activity,explore the optimum fermentation conditions for the production of bioactive components,and decipher their antimicrobial mechanisms.[Methods]The endophytes were isolated from lemon peels and seeds by tissue culture and streaking method.The strains with strong inhibitory effects on pathogenic microorganisms were screened by filter paper and Oxford cup methods and then identified by morphological observation and 16S rRNA gene sequencing.The antimicrobial effect of the fermentation liquid extract(B02c)of the active strain was examined by the microplate method.Single factor tests and response surface experiment were carried out to optimize the fermentation conditions.Propidium iodide/SYTO-9 probe marking combined with flow cytometry were employed to measure the effect of B02c on the cell membrane of the tested strains.[Results]A strain Z10 with significant inhibitory effect on Staphylococcus aureus was isolated from lemon seeds and identified as Bacillus sp.The optimal fermentation conditions of strain Z10 were as follows:addition of orange peel residue at 1.6 g/100 mL,pH 6.8,inoculum amount of 10.0%,rotation speed of 172 r/min,and culture for 36 h.After optimization,the obtained B02c showed the inhibition rate reaching 84.36%,increased by 16.36%compared with that of the fermentation liquid obtained at the original conditions.B02c showed the MIC of 3.125 mg/mL against S.aureus and destroyed the cell membrane integrity of S.aureus at this dose.[Conclusion]B02c of the lemon endophyte Z10 under optimized fermentation conditions demonstrates a strong inhibitory effect on S.aureus,serving the screening and industrial production of new and efficient natural bioactive substances.
作者
钱程
叶景
Khadija EI Galai
张琴
高梦蝶
杨新宇
喻艳玲
唐松
QIAN Cheng;YE Jing;Khadija EI Galai;ZHANG Qin;GAO Mengdie;YANG Xinyu;YU Yanling;TANG Song(College of Biological and Food Engineering,Anhui Polytechnic University,Wuhu 241000,Anhui,China)
出处
《微生物学通报》
CAS
CSCD
北大核心
2024年第5期1641-1658,共18页
Microbiology China
基金
安徽省重点研究与开发项目(201904a07020003)
安徽省自然科学基金(2108085MB34)
安徽省工业微生物分子育种工程实验室开放基金(ELMB-02)。
关键词
分离鉴定
芽孢杆菌
内生菌
抑菌机制
发酵优化
isolation and identification
Bacillus sp.
endophyte
antimicrobial mechanism
fermentation condition optimization
