【作者】 王涛；

【导师】 梅余霞； 梁运祥； 陈振民；

【作者基本信息】 华中农业大学 ， 工程硕士（专业学位）， 2018， 硕士

【摘要】 香菇,是一种重要的食药用真菌,由于其味道鲜美、营养丰富,在东方国家广泛种植,在中国和日本分别叫做“Xianggu”和“Shiitake”。香菇中最重要的活性物质是香菇多糖,具有免疫调节、抗肿瘤、抗氧化等生物学活性。目前,对子实体多糖的研究一直是人们关注的重点,并且主要侧重于抗肿瘤活性的研究,而关于液态发酵菌丝体多糖的研究较少。虽然香菇子实体已实现人工大面积培养,但是存在着培养周期长、占地面积大等弊端,液态发酵具有生长周期短、占地面积小、生长速度快等优点,短期内可获得大量的菌丝体。因此,本研究采用液态发酵法生产香菇菌丝体,分离纯化菌丝体多糖,并对其结构特征和免疫调节活性进行研究,以期为菌丝体多糖的开发利用提供相关的理论依据,促进香菇资源的工业化加工与应用。主要研究结果如下:1、利用实验室保藏的香菇菌株,通过单因素实验对香菇液态发酵培养基组成及培养条件进行优化,得到优化后的液态发酵培养基配方为:葡萄糖20 g/L,M 60 g/L,酵母提取物5 g/L。调节初始pH值7.0,在装液量100/250 m L、接种量5%、摇床转速160 r/min的条件下,采用生长10 d的种子液接种,发酵12 d,香菇菌丝生物量可达34.27±0.06 g/L,与优化前相比,提高了202.47%。2、采用热水浸提法提取香菇菌丝体多糖,经除淀粉、除蛋白、醇沉、冷冻干燥等步骤得到初步纯化的粗香菇多糖(Crude Lentinus edodes polysaccharides,cLEP),随后用DEAE-52纤维素柱和Sephadex G-200凝胶柱进一步纯化,得到香菇多糖组分1(L.edodes polysaccharide fraction-1,LEP1)和香菇多糖组分2(L.edodes polysaccharide fraction-2,LEP2)。经检测,LEP1组分的多糖含量、蛋白质含量、糖醛酸含量分别为85.84%、0.11%和2.47%,LEP2三种组分的含量分别为85.15%、3.44%和4.51%。利用FTIR、GC分析对LEP1和LEP2进行结构解析,结果表明LEP1主要由甘露糖(27.88%)、葡萄糖(30.17%)、半乳糖(31.46%)组成,还含有少量的鼠李糖、阿拉伯糖、岩藻糖;LEP2主要由甘露糖(31.78%)、葡萄糖(35.05%)、岩藻糖(17.58%)组成,还含有少量的鼠李糖和半乳糖。3、通过体外实验研究cLEP、LEP1和LEP2对DPPH自由基、羟基自由基的清除能力以及铁离子还原能力,以此来评价香菇多糖的抗氧化效果。结果表明,在一定浓度范围内,三种多糖组分均表现出一定的DPPH自由基、羟基自由基清除能力以及铁离子还原能力。DPPH自由基清除能力表现为LEP2强于cLEP和LEP1;羟基自由基清除能力表现为cLEP>LEP2>LEP1;与阳性对照Vc相比,三种多糖还原力表现整体较弱,表现为cLEP>LEP2>LEP1。4、以小鼠单核巨噬细胞RAW264.7为模型,通过体外实验研究了香菇多糖对巨噬细胞增殖、吞噬中性红、分泌NO以及细胞因子TNF-α、IL-6的影响。结果显示,巨噬细胞在香菇多糖的刺激下,吞噬中性红、分泌NO以及细胞因子TNF-α、IL-6的能力显著增强,并且还可以增强iNOS、TNF-α和IL-6基因在mRNA水平的表达,这种促进作用是通过MAPKs信号途径实现的。更多还原

【Abstract】 Lentinus edodes is an important edible and medicinal fungus,Because of its delicious taste and rich nutrition,L.edodes is widely grown in eastern countries and called“Xianggu”and“Shiitake”in China and Japan respectively.The most important active components of L.edodes is lentinan,which has many biological activities,such as immunomodulatory,anti-tumor,and anti-oxidant.At present,L.edodes polysaccharides from fruiting bodies have been focused on,especially on the anti-tumor activity.There are few studies on liquid fermentation mycelium polysaccharides.Although the fruiting body of L.edodes has been artificially cultivated in large areas,there are disadvantages such as long culture period and large area.Liquid fermentation has the advantages of short growth cycle,small area,and high growth rate.In the short term,a large number of mycelia can be obtained.In this study,polysaccharides of L.edodes mycelia obtained by liquid fermentation were isolated and purified,and its structural characteristics and immunomodulatory activities were studied subsequently,The main results are as follows:This study was aimed to improve mycelial biomass of L.edodes preserved in our laboratory by liquid fermentation,Through the single factor experiment,the medium and conditions of the liquid fermentation of L.edodes were optimized.The optimized liquid fermentation medium was composed of glucose(20 g/L),M(60 g/L),yeast extract(5 g/L).And the fermentation conditions were determined as follows:initial pH 7.0,medium volume 100/250 m L,inoculum volume 5%,rotation speed 160 r/min.The maximal mycelial yield was 34.27±0.06 g/L after 12 d fermentation,which is 202.47%higher than the previous result.Extraction of polysaccharides from L.edodes mycelia was performed by hot water extraction.The crude polysaccharide of L.edodes(cLEP)were purified by removing starch and protein,alcohol precipitation and freeze-drying.Further purification was conducted using DEAE-52 column and Sephadex G-200 gel column to obtain L.edodes polysaccharide fraction-1(LEP1)and L.edodes polysaccharide fraction-2(LEP2).The contents of carbohydrate,protein and uronic acid in LEP1 were 85.84%,0.11%and 2.47%,respectively,and the contents in LEP2 were 85.15%,3.44%and 4.51%,respectively.Structural analysis of LEP1 and LEP2 by infrared spectroscopy and gas chromatography showed that LEP1 is mainly composed of mannose(27.88%),glucose(30.17%)and galactose(31.46%),and also contains a small amount of rhamnose,arabinose,fucose;LEP2 mainly consists of mannose(31.78%),glucose(35.05%),fucose(17.58%),and trace of rhamnose and galactose.In this study,the antioxidant activity,including DPPH free radical,hydroxyl radical scavenging ability and iron ion reduction ability were valuated.The results showed that in a certain concentration range,the three polysaccharide components showed certain DPPH radicals,hydroxyl radical scavenging ability and iron ion reduction ability.LEP2 exhibited the highest scavenging effect on DPPH radicals among all fractions,followed by cLEP and LEP1;cLEP exhibited the highest scavenging effect on hydroxyl radicals among all fractions,followed by LEP2,while LEP1 had the weakest scavenging ability of hydroxyl radical.Compared with the positive control Vc,the reduction performance of the three polysaccharides was weak overall,showing that cLEP was better than LEP2,and LEP1 had the weakest effect.The in vitro immunomodulatory assays demonstrated that LEP1 and LEP2 could evidently promote the phagocytosis and stimulate the production of nitric oxide(NO),tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)in RAW264.7 cells,and promote the mRNA expression levels of inducible nitric oxide synthase(iNOS),TNF-αand IL-6.Furthermore,western blotting analysis demonstrated that the phosphorylated extracellular signal-regulated kinase(ERK),phosphorylated c-Jun N-terminal kinase(JNK)were all remarkably increased in LEP1 and LEP2-treated RAW264.7 cells.更多还原