培养时间对蛹拟青霉(Paecilomyces militaris)菌丝体中代谢产物的影响

Influence of incubation time on metabolites in mycelia of Paecilomyces militaris

摘要:

摘要:【目的】通过测定不同培养时间蛹拟青霉(Paecilomyces militaris)菌丝体中次级代谢产物的变化，分析蛹拟青霉次级代谢产物与培养时间之间的关系。【方法】液体种子接入SDAY固体培养基;培养温度为25℃，培养周期为9 d，从第2 天开始每天取样;用甲醇与乙酸乙酯分别提取菌丝体中次级代谢产物，离心、过滤后合并提取液，用液质联用仪进行分析，用MetaboAnalyst software软件进行数据采集分析。【结果】主成分分析结果表明供试菌株在不同培养时间内其菌丝中次级代谢产物差异显著。聚类分析结果显示，供试拟青霉对生物碱、肽类和核苷等易形成阳离子类物质的代谢物可分为前中后三个阶段。供试拟青霉对糖类和有机酸等易形成阴离子类物质的代谢主要分为前后两个阶段。差异代谢物及热图分析结果表明，在培养的第2和第3天含量显著增加的代谢产物种类较多，主要有酯类及其水解产物、细胞破坏素B和拟青霉素，以及多种尚未鉴定的含氮化合物等;在培养第4和第5天含量显著增加的差异代谢物质种类较少，主要有细胞破坏素A和团囊虫草素等肽类抗菌杀虫物质;在培养第6天至第9天含量显著增加的代谢物种类较多，除多种白僵菌交酯和细胞破坏素等肽类抗生素外，显著增加的还有多种脂肪酸、氨基酸、鼠李糖、海藻糖、脑苷脂类化合物和核黄素等物质。【结论】培养时间对蛹拟青霉菌丝中次级代谢产物的产生有显著影响。在培养初期，菌体中酯类及细胞破坏素B和拟青霉素等含氮化合物的合成旺盛。在培养中期，次生代谢物明显减少，但细胞破坏素A2和团囊虫草素等肽类抗生素的合成却仍显著增多。在培养后期，供试蛹拟青霉除代谢出多种白僵菌交酯等肽类抗生素外，还大量产生有机酸、氨基酸和海藻糖等物质。虽然在整个培养过程中都有肽类抗生素产生，但这些抗生素并不相同，同时后一阶段新的抗生素的产生常常伴随着前一阶段的抗生素减少，因此前期的抗生素可能是后期产生的抗生素的前体。

Abstract:

Abstract:［Objective］ To determine the secondary metabolites production in mycelia of Paecilomyces militaris．［Methods］Mycelia were cultured in plates with sabouraud dextrose agar yeast medium at 25℃ for 9 days． Sampling was done every day from the second to the ninth day． The secondary metabolites in the mycelia of Paecilomyces militaris were extracted with either methanol or ethyl acetate．The extracts were blended and analyzed by liquid chromatography-mass spectrometry (LC-MS)．LC-MS data were collected and analyzed by MetaboAnalyst software．［Result］ Principal component analysis indicates different secondary metabolites accumulation with incubation times． Hierarchical clustering analysis shows that the metabolic process of cationic compounds such as alkaloids，peptides and nucleosides can be divided into three stages，and that the metabolic process of anionic compounds such as organic acids and saccharides can be divided into two stages． Metabolites difference and heat map analysis show that:(1) The number of metabolites with significant increased contents was raised significantly in mycelia of Paecilomyces militaris on the second and third incubation days．The main species with increased contents were esters and their hydrolized products，destruxin B，variotin and some unidentified nitrogin contained compounds．(2) The number of metabolites with significant raised contents was decreased significantly on the fourth and fifth incubation days．The main species with increased contents were ophiocordin and destruxin A．(3) Apart from peptide antibiotics such as several beauverolides，the content increased metabolites included also several organic acids，amino acids，rhamnose，trehalose，cerebroside and riboflavine during the sixth to ninth incubation days．［Conclusion］ The secondary metabolites in mycelia of Paecilomyces militaris were related significantly to the incubation time.