光帽鳞伞菌丝体锌多糖的制备及其抗衰老、降血脂、保护肝脏功效的研究

发布时间：2019-07-07 19:39

【摘要】：锌是人体内执行生理功能最多的必须微量元素,与300多种酶的活性密切相关。机体锌缺乏fB会导致机体出现长发育迟缓、免疫功能紊乱、抗氧化防御功能紊乱、学习记忆能力下降和皮肤不完全角化等一系列临床症状。据报道,世界上50%的人口面临锌摄入量不足的风险。食药用真菌多糖是从食药用真菌子实体、菌丝体或发酵液中提取的大分子活性物质,具有提高免疫力、抗氧化、降血脂、降血糖、保护肝脏、抗肿瘤等多种生物学功效。本研究以我国广泛栽培的丝膜菌科(Cortinariaceae)食用真菌—光帽鳞伞(Pholiota nameko)SW-03为富锌载体,通过液体深层发酵的途径生产光帽鳞伞菌丝体锌多糖(Mycelia zinc polysaccharide,MZPS),以期开发出兼具多糖和有机锌两者生物活性的新型补锌产品及功能食品。(1)运用原生质体诱变育种技术筛选光帽鳞伞菌丝体多糖(Mycelia polysaccharide,MPS)高产菌株。实验结果表明,用含2%蜗牛酶和2%纤维素酶的复合酶,对培养7d的菌丝进行酶解,酶解温度为30℃,酶解pH值为6.5,酶解时间为3h,稳渗剂为0.6mol/L的硫酸镁,在此条件下所得原生质体的产量最高,为3.92×106个/mL。筛选出的16号诱变菌株(记为SW-03),MPS产量较出发菌株提高了33.43%,并具有遗传稳定性。(2)研究了光帽鳞伞液体发酵过程中对锌元素的富集能力和耐受能力。实验结果表明,培养基中最优锌添加量为80mg/L,上罐发酵168h后,富锌菌丝体生物量为2.28g/L,MZPS产量为41.31mg/L。并且,制备的MZPS中锌元素含量为16.39±0.72mg/g,较MPS中的10.76±0.41mg/g有了显著提高(P0.1)。(3)运用Plackett-Burman实验设计和响应面实验设计对MZPS的提取工艺进行优化。实验结果表明,加水倍数为25.00,pH值为8.00,醇沉时间为22.00h是提取MZPS的最优工艺条件。用优化后的工艺条件提取光帽鳞伞MZPS,MZPS的提取率为2.98%。(4)采用DEAE-52纤维素阴离子交换柱层析和Sephadex G-100葡聚糖凝胶柱层析对MZPS进行分离纯化,得到三个多糖组分:MZPS-1a、MZPS-2a和MZPS-3a,其中MZPS-2a为MZPS的主要成分。高效凝胶渗透色谱法(High-performance gel permeation chromatography,HPGPC)的实验结果表明,MZPS-2a为均一多糖,重均分子量(Weight-average molecularweight,Mw)为1.36×104Da。傅立叶红外光谱(Fourier transform infrared spectroscopy,FT-IR)的分析结果表明MZPS-2a具有多糖的特征吸收峰。柱前衍生高效液相色谱(High performance liquid chromatography,HPLC)法的实验结果表明MZPS-2a含有葡萄糖、甘露糖、葡萄糖醛酸、半乳糖、半乳糖醛酸和阿拉伯糖,且摩尔比为172.59:5.29:4.61:4.20:1.01:1.00。(5)通过体外抗氧化实验和体内抗氧化实验分析MZPS的抗衰老能力。体外抗氧化实验表明,MZPS及其组分具备还原力和清除超氧阴离子自由基、羟基自由基和DPPH自由基的能力。并且,在MZPS的组分中,MZPS-2a的抗氧化能力最强,这可能与MZPS具备较低的分子量,较高的糖醛酸含量以及葡萄糖在单糖组成中所占比例较低的结构特征相关。体内抗氧化实验证明MZPS可以显著提高衰老小鼠体内超氧化物歧化酶(Superoxide dismutase,SOD)活性和总抗氧化能力(Total antioxidant capability,T-AOC),降低脂质过氧化物(Lipid peroxide,LPO)和丙二醛(Malondialdehyde,MDA)含量。特别是MZPS较MPS表现出了更强的降低LPO和MDA含量的能力,这可能与锌的抗氧化功能有关。实验结果说明,MZPS是具有延缓衰老功效的天然抗氧化剂。(6)通过建立高血脂症小鼠模型,分析MZPS降血脂、保护肝脏的能力。实验结果表明,MZPS可以显著降低高脂血症小鼠血清中的总胆固醇(Total cholesterol,TC)、甘油三酯(Triacylglycerols,TG)、低密度脂蛋白胆固醇(Low-density lipoprotein cholesterol,LDL-C)和极低密度脂蛋白胆固醇(Very low-density lipoprotein cholesterol,VLDL-C)水平,升高高密度脂蛋白胆固醇(High-density lipoprotein cholesterol,HDL-C)水平,并且降低高脂血症小鼠肝脏中的TC和TG水平。同时,小鼠血清和肝脏中的SOD活性和T-AOC水平显著上升,LPO和MDA含量显著下降。另外,形态学观察表明MZPS可以减轻高脂血症小鼠的肝脏损伤。实验结果说明,MZPS具有降血脂,预防高脂饮食引起的非酒精性脂肪肝的功效。
[Abstract]:Zinc is the most important trace element in human body, which is closely related to the activity of more than 300 enzymes. The lack of fB in the body of the body can lead to a series of clinical symptoms such as long development delay, immune function disorder, anti-oxidation defense function disorder, reduced learning and memory ability, and incomplete skin. It is reported that 50 per cent of the world's population is at risk of insufficient zinc intake. The edible fungus polysaccharide is a macromolecular active substance extracted from a medicinal fungus fruiting body, a mycelium or a fermentation liquid, and has various biological effects of improving immunity, resisting oxidation, reducing blood fat, reducing blood sugar, protecting liver, and resisting tumor. In this study, a zinc-rich carrier, Mycelia zincascharide (MZPS), was produced by submerged fermentation of the Corinariaceae (Corinariaceae) edible fungus, which is a zinc-rich carrier. In order to develop a novel zinc-supplementing product with both polysaccharide and organic zinc, and a functional food. (1) The high-yield strains of Mycelia polyacide (MPS) were screened by protoplast-induced mutation breeding. The experimental results showed that the mycelium of 7 days of culture was subjected to enzymolysis with 2% snail enzyme and 2% cellulase, the temperature of the enzyme was 30 鈩，

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