伊犁野生郁金香内生烟曲霉的分离鉴定及活性

Isolation, identification, and activity of endophytic Aspergillus fumigatus from wild tulip in Ili

摘要:

【背景】对郁金香的研究主要集中在种质资源、引种栽培、扩繁育种及化学成分分析方面，而关于伊犁野生郁金香内生菌的研究尚未见报道。【目的】从伊犁野生郁金香中筛选出内生真菌并对其进行抑菌及抗氧化活性研究。【方法】采用组织块培养法和平板划线法对伊犁野生郁金香内生菌进行分离纯化；用斜面低温保存法对内生菌进行保存；以形态学方法和分子生物学方法对分离出的内生真菌进行鉴定；通过液体发酵得到次级代谢产物，对乙酸乙酯萃取发酵产物进行滤纸片抑菌分析。使用Fe³⁺总还原能力法、2-2'二苯基-1-三硝基苯肼(2,2'-diphenyl-1-picrylhydrazyl，DPPH)自由基法、2'-联氨-双-3-乙基苯并噻唑啉-6-磺酸[2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonicacid,ABTS]自由基法及羟基自由基法比较菌株乙酸乙酯层和水层的抗氧化活性。【结果】从伊犁野生郁金香中分离获得一株内生真菌，经鉴定为曲霉属(Aspergillus)烟曲霉(Aspergillus fumigatus)，简称为YGL-1。YGL-1对金黄色葡萄球菌(Staphylococcus aureus)、大肠埃希氏菌(Escherichia coli)、枯草芽孢杆菌(Bacillus subtilis)、白色念珠菌(Candida albicans)均具有显著的抑菌活性。经抗氧化活性实验发现有机溶剂层和水层都具有还原Fe³⁺和清除DPPH自由基、ABTS自由基、羟基自由基的能力，尤其是清除DPPH自由基和ABTS自由基，抗氧化活性的能力均随着浓度的增高而增强，属于剂量依赖型。当两相溶剂萃取粗提物浓度为2.0mg/mL时，有机溶剂层清除DPPH自由基效率为91.45%，清除ABTS自由基效率为99.84%。YGL-1的发酵产物具有较强的抗氧化活性，主要分布在有机溶剂层，其次分布于水层。【结论】从伊犁野生郁金香中分离纯化出一株内生真菌，为曲霉属(Aspergillus)烟曲霉(Aspergillus fumigatus)。研究结果预示郁金香内生烟曲霉是产生抑菌、抗氧化活性物质的重要资源，YGL-1菌株可作为进一步的实验研究对象。

Abstract:

[Background] There has been an explosion of research on the germplasm resources, introduction, expansion and breeding, and chemical components of tulip, but no report on the endophytes of wild tulip in Ili is available. [Objective] To screen endophytic fungi from wild tulip in Ili and study their antibacterial and antioxidant activities. [Methods] The endophytic fungi of Ili wild tulip were isolated and purified by tissue culture and streak plate method. The endophytes were preserved with the slant cryopreservation method. The isolated endophytic fungi were identified based on morphological observation and molecular biology methods. The secondary metabolites were obtained by liquid fermentation, and the antibacterial activity of the fermentation product extracted with ethyl acetate was analyzed with the filter paper method. The total Fe³⁺-reducing power, 2,2'-diphenyl-1-picrylhydrazyl (DPPH)-scavenging ability, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonicacid) (ABTS)-scavenging ability, and hydroxyl radical-scavenging ability of the aqueous fraction and ethyl acetate fraction were determined to compare the antioxidant activity. [Results] An endophytic strain was isolated and identified as Aspergillus fumigatus YGL-1. YGL-1 showed significant inhibitory activity against Staphylococcus aureus, Escherichia coli, Bacillus subtilis, and Candida albicans. Both the aqueous fraction and the ethyl acetate fraction can reduce Fe³⁺, and scavenge DPPH, ABTS, and hydroxyl radicals, especially DPPH and ABTS. The antioxidant activity was enhanced with increase in concentration. When the concentration of the crude extract was 2.0 mg/mL, the ethyl acetate fraction can scavenge 91.45% of DPPH and 99.84% of ABTS. In summary, the fermentation products of YGL-1 had strong antioxidant activity, particularly the organic solvent fraction, followed by the aqueous fraction. [Conclusion] A. fumigatus was isolated from wild tulip in Ili. This study is expected to serve as a reference for the comprehensive utilization of endophytic fungi in plant resources and the development of antibacterial and antioxidant drugs.