瓦布贝母内生真菌Fusarium redolens 6WBY3多糖的理化性质及抗氧化活性

Physicochemical property and antioxidant activity of exopolysaccharide produced by endophytic fungal Fusarium redolens 6WBY3 isolated from Fritillaria unibracteata var. wabuensis

摘要:

[目的]对瓦布贝母内生真菌芳香镰孢菌6WBY3胞外多糖及其抗氧化活性进行研究。[方法]利用液体发酵，有机溶剂脱脂，乙醇沉淀、大孔吸附树脂除色素，酶法和sevag法结合除蛋白、透析除盐等小分子物质、纤维素DE-52阴离子交换柱对胞外多糖进行分离；通过高效凝胶色谱-蒸发光检测器（HPGPC-ELSD）、紫外光谱（UV）、扫描电镜（SEM）、PMP柱前衍生高效液相色谱（PMP-HPLC）、傅立叶变换红外光谱（FT-IR）和氢核磁共振谱（¹H NMR）等方法对胞外多糖的理化特征进行研究；利用DPPH和ABTS自由基清除实验，铁离子螯合实验对其抗氧化活性进行研究。[结果]从菌株6WBY3发酵液中分离出2个均一的胞外多糖6WBY3EPS-3和6WBY3EPS-4，分子量分别约为17.41×10⁶ Da和8.84×10⁵ Da，主要单糖组分及摩尔比分别为甘露糖：葡萄糖：半乳糖=8.16：4.96：10.00，甘露糖：鼠李糖：葡萄糖：半乳糖=8.08：1.71：6.32：10.00；SEM特征前者呈现不规则多边体结构，后者呈现规则四边体结构；FT-IR和¹H NMR结果表明6WBY3EPS-3含有丰富的半乳呋喃糖和甘露呋喃糖及少量的α-D-吡喃葡萄糖的酸性多糖，而6WBY3EPS-4同样主要为吡喃环糖，并且二者均含有α-和β-糖苷构型异头氢，均以α-构型为主。抗氧化研究结果表明多糖6WBY3EPS-3和6WBY3EPS-4有较弱的DPPH自由基清除能力，中等的ABTS自由基清除能力和中等的铁离子螯合能力。[结论]首次对瓦布贝母内生真菌6WBY3的胞外多糖进行了研究，该研究表明内生真菌6WBY3的胞外多糖具有良好的抗氧化活性，具有开发成抗氧化剂应用于医药食品等工业的潜力。同时本研究也可为其他内生真菌胞外多糖的研究提供理论参考。

Abstract:

[Objective] To study the exopolysaccharide (EPS) of endophytic fungal Fusarium redolens 6WBY3 isolated from Fritillaria unibracteata var. wabuensis (FUW) including its antioxidant activities.[Methods] We isolated the EPS from the culture medium of strain 6WBY3 using the methods of degreasing with organic solvent, precipitation with ethanol, decoloration with macroporous resins, deproteinization with protease in combination with sevag reagent, desalination with dialysis and separation with DEAE-cellulose ion exchange chromatography. Then, we analyzed the EPS fractions using high-performance gel-permeation chromatography with evaporative light scattering detector (HPGPC-ELSD), ultra violet (UV) spectra, scanning electron microscope (SEM), PMP precolumn derivatization with high performance liquid chromatography (PMP-HPLC), fourier transform infrared (FT-IR) spectroscopy and ¹H-nuclear magnetic resonance (¹H NMR) methods. We evaluated the antioxidant activity of the EPS using DPPH and ABTS radical scavenging activities and iron ion chelating ability methods.[Results] We obtained two homogeneous EPSs, namely 6WBY3EPS-3 and 6WBY3EPS-4, with the molecular weight (Mw) of 17.41×10⁶ and 8.84×10⁵ Da, respectively. 6WBY3EPS-3 was composed of mannose, glucose and galactose in a molar ratio of 8.16:4.96:10.00, while 6WBY3EPS-4 was composed of mannose, rhamnose, glucose and galactose in a molar ratio of 8.08:1.71:6.32:10.00. The results of SEM showed that 6WBY3EPS-3 was the irregular multilateral body, and 6WBY3EPS-4 was rules of quadrilateral body. The results of FT-IR and ¹H NMR analysis exhibited that 6WBY3EPS-3 was acidic polysaccharose with abundant galactofuranose, mannofuranose and a few α-D-glucopyranose, while 6WBY3EPS-4 possessed pyranose ring mainly. Those two EPSs had anomeric hydrogen with α-(main) and β-glycosidic configuration. Furthermore, the results of antioxidant activity suggested that both 6WBY3EPS-3 and 6WBY3EPS-4 had a weak DPPH radical scavenging ability, a moderate ABTS radical scavenging activity and a moderate iron ion chelating effect.[Conclusion] The two EPSs from the endophytic fungus 6WBY3 were firstly obtained and investigated. Our investigation indicated that the EPS from 6WBY3 had the application potential as an antioxidant in medicine and food industries. In addition, this work can also provide theory reference for improving development of EPS from other endophytic fungi.