發(fā)布時(shí)間：2018-09-17 15:24

【摘要】：金福菇(Tricholoma lobayense),是一種味道鮮美的食用菌。本文的目的是篩選高抗氧化活性的金福菇多糖,進(jìn)而從不同種金福菇子實(shí)體中篩選到一種新型的高抗氧化活性金福菇多糖TLH-G,并且對(duì)其物理化學(xué)性質(zhì)和結(jié)構(gòu)特征進(jìn)行了深入研究,結(jié)合實(shí)驗(yàn)室前期對(duì)金福菇多糖的構(gòu)效關(guān)系的研究,進(jìn)一步分析了導(dǎo)致金福菇多糖高抗氧化活性的因素,結(jié)果如下:1.不同品種金福菇菌種的篩選在宏觀上采用菌種平板培養(yǎng)法分析菌種之間存在明顯的拮抗作用,采用ITS序列分析技術(shù)分析了三種金福菇菌種之間的親緣關(guān)系,發(fā)現(xiàn)它們與Tricholoma giganteum(金福菇,登錄號(hào):KF360838.1,KF360837.1,AF042591.1)相似度高達(dá)99%以上,通過對(duì)進(jìn)化樹的構(gòu)建,確定它們?yōu)椴煌N的金福菇。經(jīng)多糖提取及HPLC分析,三種不同金福菇子實(shí)體多糖組分分布不一,具有品種多樣性,這與金福菇自身的遺傳因素密切相關(guān),此外組分分布與實(shí)驗(yàn)室前期研究的金福菇多糖組分分布一致。通過前期大量的活性比較研究發(fā)現(xiàn),高郵金福菇子實(shí)體小分子量的多糖組分具有較高的抗氧化活性。結(jié)果表明高郵金福菇子實(shí)體多糖具有優(yōu)越的抗氧化能力。所以選擇高郵金福菇子實(shí)體多糖進(jìn)行下一步實(shí)驗(yàn)。2.金福菇多糖TLH-G的理化性質(zhì)及抗氧化活性研究(1)采用閃式提取金福菇粗多糖,然后Savage除蛋白及超濾法獲得多糖TLH-G。經(jīng)HPLC-ELSD檢測(cè)證明TLH-G為純品,分子量分別為4.1×103 Da,TLH-G的提取率為2.8%。其總糖含量可以高達(dá)99%,糖醛酸含量為13.85%,從而導(dǎo)致好的水溶性,紫外光譜分析TLH-G中沒有蛋白和核酸的吸收峰,紅外檢測(cè)結(jié)果表明TLH-G具有多糖的典型特征吸收峰,并且只有吡喃環(huán)的存在。(2)體外抗氧化結(jié)果表明:在清除DPPH及總抗氧化能力的兩個(gè)體系中,金福菇多糖TLH-G均表現(xiàn)出較高的抗氧化能力。關(guān)于DPPH自由基的清除能力,TLH-G的表現(xiàn)出的IC50值為430 μg/mL;總抗氧化能力測(cè)定中TLH-G的IC50值大于500μg/mL。以DPPH為考核指標(biāo),研究比較9種常見真菌多糖的抗氧化活性,TLH-G具有最小的IC50值,結(jié)果說明其是一種天然高效的抗氧化劑。3.對(duì)分離純化得到的金福菇多糖TLH-G進(jìn)行物理化學(xué)性質(zhì)及結(jié)構(gòu)鑒定,結(jié)合TLH-3的抗氧化活性與結(jié)構(gòu)的關(guān)系,進(jìn)一步深入剖析金福菇多糖的構(gòu)效關(guān)系。(1)經(jīng)HPLC測(cè)定金福菇多糖TLH-G的分子量為4.1×103Da,再次證明了在金福菇多糖中,分子量越小,抗氧化能力越強(qiáng)。同時(shí),金福菇多糖TLH-G與LTH-3均具有較強(qiáng)的水溶性,也進(jìn)一步說明金福菇多糖抗氧化活性與其水溶性密切相關(guān)。(2)單糖組成結(jié)果表明:TLH-G中共7種單糖,其摩爾比為甘露糖: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。與TLH-3的單糖組成相比,分析發(fā)現(xiàn)葡萄糖和半乳糖的絕對(duì)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關(guān)系到TLH-3和TLH-G活性的強(qiáng)弱,是金福菇多糖抗氧化活性的重要影響因素。(3)甲基化GC-MS圖譜結(jié)果表明:TLH-G中主要是由→1)-G1cp-(6→構(gòu)成,另外→1)-Ga1p-6→有占有一定比例,TLH-G的分支度分別為0.21,屬于小分支的多糖,而TLH-3分支度為0.74,是一種高度分支的多糖。故分支度是影響金福菇多糖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,抗氧化活性越強(qiáng)。(4)經(jīng)NMR檢測(cè)預(yù)測(cè)出TLH-G的結(jié)構(gòu),主鏈主要是(1→6)-β-D-G1cp和(1→6)-β-D-Ga1p,分支點(diǎn)有一個(gè):(1→3,6)-α-D-Manp。該結(jié)果與甲基化GC-MS結(jié)果基本相吻合。結(jié)果與TLH-3相比較,多元化的糖苷鍵類型是金福菇多糖高抗氧化活性的重要原因;這兩種多糖活性的差異進(jìn)一步解釋了 TLH-3中→3,6)-α-D-G1cp-(1→,→4)-α-D-G1cp-(1→和→3)-β-D-Ga1p-(1→糖苷鍵對(duì)于維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和特殊的結(jié)構(gòu)特征使得TLH-G的具有很強(qiáng)抗氧化活性。綜上所述,從高郵菌種栽培所獲得的子實(shí)體中篩選分離到一種新型多糖TLH-G,研究發(fā)現(xiàn)具有非常好的抗氧化作用,并結(jié)合TLH-3的構(gòu)效關(guān)系研究,進(jìn)一步解析了分子量,水溶性等物理化學(xué)性質(zhì)以及單糖組成,分支度,糖苷鍵類型等對(duì)于金福菇多糖抗氧化活性有著重要影響。本課題為金福菇多糖構(gòu)效關(guān)系的構(gòu)建提供理論支撐,對(duì)于開發(fā)高活性金福菇多糖具有重要意義。
[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.
【學(xué)位授予單位】：安徽大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q936

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