金福菇多糖TLH-G的结构鉴定及其抗氧化活性研究

发布时间：2018-09-17 15:24

【摘要】：金福菇(Tricholoma lobayense),是一种味道鲜美的食用菌。本文的目的是筛选高抗氧化活性的金福菇多糖,进而从不同种金福菇子实体中筛选到一种新型的高抗氧化活性金福菇多糖TLH-G,并且对其物理化学性质和结构特征进行了深入研究,结合实验室前期对金福菇多糖的构效关系的研究,进一步分析了导致金福菇多糖高抗氧化活性的因素,结果如下:1.不同品种金福菇菌种的筛选在宏观上采用菌种平板培养法分析菌种之间存在明显的拮抗作用,采用ITS序列分析技术分析了三种金福菇菌种之间的亲缘关系,发现它们与Tricholoma giganteum(金福菇,登录号:KF360838.1,KF360837.1,AF042591.1)相似度高达99%以上,通过对进化树的构建,确定它们为不同种的金福菇。经多糖提取及HPLC分析,三种不同金福菇子实体多糖组分分布不一,具有品种多样性,这与金福菇自身的遗传因素密切相关,此外组分分布与实验室前期研究的金福菇多糖组分分布一致。通过前期大量的活性比较研究发现,高邮金福菇子实体小分子量的多糖组分具有较高的抗氧化活性。结果表明高邮金福菇子实体多糖具有优越的抗氧化能力。所以选择高邮金福菇子实体多糖进行下一步实验。2.金福菇多糖TLH-G的理化性质及抗氧化活性研究(1)采用闪式提取金福菇粗多糖,然后Savage除蛋白及超滤法获得多糖TLH-G。经HPLC-ELSD检测证明TLH-G为纯品,分子量分别为4.1×103 Da,TLH-G的提取率为2.8%。其总糖含量可以高达99%,糖醛酸含量为13.85%,从而导致好的水溶性,紫外光谱分析TLH-G中没有蛋白和核酸的吸收峰,红外检测结果表明TLH-G具有多糖的典型特征吸收峰,并且只有吡喃环的存在。(2)体外抗氧化结果表明:在清除DPPH及总抗氧化能力的两个体系中,金福菇多糖TLH-G均表现出较高的抗氧化能力。关于DPPH自由基的清除能力,TLH-G的表现出的IC50值为430 μg/mL;总抗氧化能力测定中TLH-G的IC50值大于500μg/mL。以DPPH为考核指标,研究比较9种常见真菌多糖的抗氧化活性,TLH-G具有最小的IC50值,结果说明其是一种天然高效的抗氧化剂。3.对分离纯化得到的金福菇多糖TLH-G进行物理化学性质及结构鉴定,结合TLH-3的抗氧化活性与结构的关系,进一步深入剖析金福菇多糖的构效关系。(1)经HPLC测定金福菇多糖TLH-G的分子量为4.1×103Da,再次证明了在金福菇多糖中,分子量越小,抗氧化能力越强。同时,金福菇多糖TLH-G与LTH-3均具有较强的水溶性,也进一步说明金福菇多糖抗氧化活性与其水溶性密切相关。(2)单糖组成结果表明:TLH-G中共7种单糖,其摩尔比为甘露糖:鼠李糖:阿拉伯糖:葡萄糖醛酸:半乳糖醛酸:葡萄糖:半乳糖=1.00:0.56:0.14:0.33:0.10:14.13:2.92。与TLH-3的单糖组成相比,分析发现葡萄糖和半乳糖的绝对含量以及一定量的鼠李糖是金福菇多糖TLH-3和TLH-G的高抗氧化活性的重要因素。更重要的是,半乳糖和半乳糖醛酸含量直接关系到TLH-3和TLH-G活性的强弱,是金福菇多糖抗氧化活性的重要影响因素。(3)甲基化GC-MS图谱结果表明:TLH-G中主要是由→1)-G1cp-(6→构成,另外→1)-Ga1p-6→有占有一定比例,TLH-G的分支度分别为0.21,属于小分支的多糖,而TLH-3分支度为0.74,是一种高度分支的多糖。故分支度是影响金福菇多糖TLH-G和TLH-3抗氧化活性的又一因素,分支度越高的金福菇多糖,抗氧化活性越强。(4)经NMR检测预测出TLH-G的结构,主链主要是(1→6)-β-D-G1cp和(1→6)-β-D-Ga1p,分支点有一个:(1→3,6)-α-D-Manp。该结果与甲基化GC-MS结果基本相吻合。结果与TLH-3相比较,多元化的糖苷键类型是金福菇多糖高抗氧化活性的重要原因;这两种多糖活性的差异进一步解释了 TLH-3中→3,6)-α-D-G1cp-(1→,→4)-α-D-G1cp-(1→和→3)-β-D-Ga1p-(1→糖苷键对于维持多糖的高抗氧化活性具有重要作用;另外TLH-G中的(1→6)-β-D-G1c和特殊的结构特征使得TLH-G的具有很强抗氧化活性。综上所述,从高邮菌种栽培所获得的子实体中筛选分离到一种新型多糖TLH-G,研究发现具有非常好的抗氧化作用,并结合TLH-3的构效关系研究,进一步解析了分子量,水溶性等物理化学性质以及单糖组成,分支度,糖苷键类型等对于金福菇多糖抗氧化活性有着重要影响。本课题为金福菇多糖构效关系的构建提供理论支撑,对于开发高活性金福菇多糖具有重要意义。
[Abstract]:Tricholoma Lobayense is a kind of edible mushroom with delicious taste.The purpose of this paper is to screen polysaccharides with high antioxidant activity,and then to screen a new type of polysaccharide TLH-G with high antioxidant activity from different fruiting bodies of Tricholoma lobayense. The results are as follows: 1. The screening of different strains of Jinfu mushroom has obvious antagonistic effect on the analysis of strains by plate culture method, and ITS sequence analysis technique is used to analyze the antioxidant activity of Jinfu mushroom polysaccharides. The phylogenetic relationship among the three strains of Jinfu mushroom was studied. The similarity between them and Tricholoma giganteum (KF360838.1, KF360837.1, AF042591.1) was more than 99%. The results showed that the low molecular weight polysaccharides from the fruiting body of Jinfu mushroom from Gaoyou had higher antioxidant activity. The polysaccharides from the fruiting body of Jinfu mushroom of Minggaoyou have superior antioxidant capacity. Therefore, the polysaccharides from the fruiting body of Jinfu mushroom of Gaoyou were selected for the next experiment. 2. The physicochemical properties and antioxidant activity of the polysaccharides TLH-G from Jinfu mushroom were studied. (1) The crude polysaccharides from Jinfu mushroom were extracted by flash extraction, and then the polysaccharides TLH-G was TLH-G was proved to be pure with a molecular weight of 4.1 *103 Da and a TLH-G extraction rate of 2.8%. The total sugar content of TLH-G could reach 99% and the content of glucuronic acid was 13.85%. As a result, there was no absorption peak of protein and nucleic acid in TLH-G by ultraviolet spectroscopy. Infrared detection results showed that TLH-G had typical characteristic absorption peak of polysaccharide, and only one was found. (2) The results of antioxidant activity in vitro showed that the polysaccharide TLH-G showed high antioxidant activity in both DPPH scavenging and total antioxidant capacity systems. The antioxidant activity of nine common fungal polysaccharides was compared with that of TLH-G. TLH-G had the lowest IC50 value. The results showed that TLH-G was a natural and efficient antioxidant. The structure-activity relationship of the polysaccharide was analyzed. (1) The molecular weight of the polysaccharide TLH-G was 4.1 6550 (2) The results of monosaccharide composition showed that there were seven monosaccharides in TLH-G. The molar ratio was mannose: rhamnose: arabinose: glucuronic acid: galacturonic acid: glucose: galactose = 1.00:0.56:0.14:0.33:0.10:14.13:2.92. Compared with the monosaccharide composition of TLH-3, the absolute content of glucose and galactose and a certain amount of them were found. The content of galactose and galacturonic acid is directly related to the activity of TLH-3 and TLH-G, and is an important factor affecting the antioxidant activity of the polysaccharides. (3) The results of GC-MS analysis showed that the content of TLH-G is mainly from 1) - G1cp - (6). The branching degree of TLH-G is 0.21, which belongs to small branching polysaccharides, while the branching degree of TLH-3 is 0.74, which is a highly branching polysaccharide. Therefore, branching degree is another factor affecting the antioxidant activity of Jinfu mushroom polysaccharides TLH-G and TLH-3. The higher the branching degree, the higher the antioxidant activity of Jinfu mushroom polysaccharides. Strong. (4) The structure of TLH-G was predicted by NMR. The main chain of TLH-G was (1_6) - beta-D-G1cp and (1_6) - beta-D-Ga1p. There was one branching point: (1_3,6) - a-D-Manp. The results were basically consistent with the results of methylated GC-MS. Compared with TLH-3, the polyglycoside bond type was the important reason for the high antioxidant activity of Jinfu mushroom polysaccharide. The difference in sugar activity further explains that TLH-3_3,6) - alpha-D-G1cp - (1_4) - alpha-D-G1cp - (1_and 3) - beta-D-Ga1p - (1 glycoside bond) plays an important role in maintaining the high antioxidant activity of polysaccharides; in addition, the (1_6) - beta-D-G1c and special structural characteristics of TLH-G make TLH-G have strong antioxidant activity. A new polysaccharide TLH-G was isolated from the fruiting bodies obtained from the cultivation of postmortem fungi. It was found that the polysaccharide TLH-G had very good antioxidant activity. Combined with the structure-activity relationship of TLH-3, the antioxidant activity of the polysaccharide was further analyzed by analyzing the physical and chemical properties such as molecular weight, water-solubility, monosaccharide composition, branching degree, glycoside bond type and so on. This study provides theoretical support for the construction of the structure-activity relationship of the polysaccharides and is of great significance for the development of high-activity polysaccharides.
【学位授予单位】：安徽大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q936

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