以夏黑葡萄为研究对象,利用平板划线法分离纯化培养得到附生细菌,通过提取基因组DNA、PCR扩增16S rDNA、基因序列测序,构建系统发育树,进行聚类分析,结合形态学观察,确定菌种的种属地位。在此基础上,以抑菌活性为指标,筛选具有抑菌活性的附生细菌。将筛选到的目标株菌发酵后,采用溶剂萃取得到发酵粗提物,经过电喷雾(electrospray ionization mass spectrometry,ESI-MS)对发酵产物进行分析,初步确定发酵产物的结构。共从夏黑葡萄中分离鉴定了9株附生细菌,其中BDG-5和BDD-2具有显著的抗菌活性,分别为副假单胞菌(Pseudomonas paralactis)和贝莱斯芽孢杆菌(Bacillus velezensis),ESI-MS分析表明,P.paralactis发酵产物中含有环脂肽surfactin和iturin C,B.velezensis可产生脂肽surfactin、iturin A和bacillomycin D,它们是抑菌活性的主要物质。
In this study, plated streaks were used to separate and purify cultures to obtain epiphytic bacteria, genomic DNA was extracted, 16S rDNA was amplified by PCR, the gene was sequenced, phylogenetic tree was constructed for cluster analysis, and morphological observation was performed to determine the species of strains. The epiphyte bacteria with antibacterial activity were screened. After fermenting the selected target strains, crude extracts were obtained by solvent extraction, and the fermentation products were analyzed by electrospray ionization mass spectrometry (ESI-MS) to determine their structures. The results showed that nine strains of epiphyte were isolated and identified from the Xiahei grapes. Among them, BDG-5 and BDD-2 had significant antibacterial activities. They were Pseudomonas paralactis and Bacillus velezensis, respectively. ESI-MS showed that P.paralactis fermentation products contained cyclolipopeptides surfactin and iturin C. B.velezensis produced lipopeptides surfactin, iturin A, and bacillinomycin D. These cyclolipopeptides were the main substances possessed antibacterial activity.
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