南极大磷虾肠道曲霉菌Aspergillus sydowii NJF7的分离与代谢产物的研究

Isolation of Aspergillus sydowii NJF7 from Euphausia superba intestines and study on its metabolites

通过对2017年第34次“雪龙号”南极考察采集的南极大磷虾(Euphausia superba)共复生微生物进行分离,并对有价值菌株的次级代谢产物进行了研究。采用在3种选择性培养基中添加不同抗生素的稀释平板涂布法,从南极大磷虾中分离出一株有价值的真菌进行扩大培养,运用TLC、柱色谱(正相硅胶、C18硅胶、MCI、Sephadex LH-20凝胶)和半制备型HPLC等色谱方法对其代谢产物进行高效快速地分离纯化,结合现代波谱学手段(ESI HR MS、1H-NMR),鉴定次级代谢产物结构。首先经过形态学、18S rDNA-ITS序列分析,鉴定分离到一株真菌为曲霉Aspergillus sp.NJF7;从曲霉NJF7中分离到13个代谢产物,分别鉴定为环(脯氨酸-酪氨酸)(1)、环(色氨酸-酪氨酸)(2)、环(脯氨酸-苯丙氨酸)(3)、环(缬氨酸-脯氨酸)(4)、环(缬氨酸-色氨酸)(5)、环(异亮氨酸-色氨酸)(6)、环(谷氨酰胺-色氨酸)(7)、对羟基肉桂酸甲酯(8)、尿嘧啶(9)、对羟基苯乙胺(10)、苯乙胺(11)、N-甲基酪胺(12)、大麦芽碱(13),其中化合物7为新的天然产物。

Antarctic krill(Euphausia superba)are rich in DHA/EPA,phospholipids,and a variety of active psychrophilic enzymes,etc.Intestinal tract of E.superba is an important organ for digestion and absorption.There are numerous intestinal microbiotas with complex structure,which are relative to nutrition metabolism,genetics,development and evolution,immune defense and other important life processes of the host.Some scholars found that there were a large number of new species microorganisms in the intestinal tract of E.superba.Then,the secondary metabolites and biological activities of these strains were studied.48 compounds were identified and found to have various biological activities,such as anti-marine virus,anti-marine pathogen and anti-fouling.Therefore,it is necessary to study the general biology of Euphausia superba in order to extremely expand comprehensive utilization of this resource in China.In this study,the secondary metabolites of a valuable fungus strain obtained firstly from E.superba were studied.First the fungus strain was isolated by serial dilutions and plate coating method in which different antibiotics were added to Rose Bengal Agar,PDA Broth and YPD Broth selective media.Then the fungi were subjected to batch culture.The metabolites were separated and purified efficiently and rapidly by TLC,column chromatography(normal phase silica gel,C18 silica gel,MCI,Sephadex LH-20 gel)and semi-preparative HPLC,and with combined modern spectroscopic methods(ESI HR MS,1H-NMR),their structures were identified.The strain was identified as Aspergillus sp.NJF7 by the homologies and genetic distances for the ITS region and 18S rDNA-ITS sequence analysis.Thirteen compounds were isolated from Aspergillus sp.NJF7,which were elucidated as cyclo(Pro-Tyr)(1),cyclo(try-tyr)(2),cyclo(Pro-Phe)(3),cyclo(Val-Prol)(4),cyclo(Val-Trp)(5),cyclo(Ile-Trp)(6),cyclo(Gln-Trp)(7),methyl p-hydroxycinnamate(8),uracil(9),p-hydroxy-phenylethylamine(10),phenylethylamine(11),N-methyltyramine(12),hordenine(13).Among them,compound 7 was isolated as a new natural product for the first time.The bioactivity of cyclopeptides including antitumor,antibacterial and antifungal bioactivities will be further studied.The secondary metabolites of Aspergillus sp.NJF7 were mainly aromatic acids,alkaloids,and diketoperazines,and their structures were relatively simple.There were many factors contributing to this result.The fermentation medium used in this experiment was one of the elements.The second was the culture condition.Therefore,in the future,the medium types and culture conditions will be optimized in order to obtain novel compounds with better biological activities and even natural products with prospects for new drug development.