【摘要】：鋅是人體內(nèi)執(zhí)行生理功能最多的必須微量元素,與300多種酶的活性密切相關(guān)。機體鋅缺乏fB會導(dǎo)致機體出現(xiàn)長發(fā)育遲緩、免疫功能紊亂、抗氧化防御功能紊亂、學(xué)習(xí)記憶能力下降和皮膚不完全角化等一系列臨床癥狀。據(jù)報道,世界上50%的人口面臨鋅攝入量不足的風(fēng)險。食藥用真菌多糖是從食藥用真菌子實體、菌絲體或發(fā)酵液中提取的大分子活性物質(zhì),具有提高免疫力、抗氧化、降血脂、降血糖、保護肝臟、抗腫瘤等多種生物學(xué)功效。本研究以我國廣泛栽培的絲膜菌科(Cortinariaceae)食用真菌—光帽鱗傘(Pholiota nameko)SW-03為富鋅載體,通過液體深層發(fā)酵的途徑生產(chǎn)光帽鱗傘菌絲體鋅多糖(Mycelia zinc polysaccharide,MZPS),以期開發(fā)出兼具多糖和有機鋅兩者生物活性的新型補鋅產(chǎn)品及功能食品。(1)運用原生質(zhì)體誘變育種技術(shù)篩選光帽鱗傘菌絲體多糖(Mycelia polysaccharide,MPS)高產(chǎn)菌株。實驗結(jié)果表明,用含2%蝸牛酶和2%纖維素酶的復(fù)合酶,對培養(yǎng)7d的菌絲進行酶解,酶解溫度為30℃,酶解pH值為6.5,酶解時間為3h,穩(wěn)滲劑為0.6mol/L的硫酸鎂,在此條件下所得原生質(zhì)體的產(chǎn)量最高,為3.92×106個/mL。篩選出的16號誘變菌株(記為SW-03),MPS產(chǎn)量較出發(fā)菌株提高了33.43%,并具有遺傳穩(wěn)定性。(2)研究了光帽鱗傘液體發(fā)酵過程中對鋅元素的富集能力和耐受能力。實驗結(jié)果表明,培養(yǎng)基中最優(yōu)鋅添加量為80mg/L,上罐發(fā)酵168h后,富鋅菌絲體生物量為2.28g/L,MZPS產(chǎn)量為41.31mg/L。并且,制備的MZPS中鋅元素含量為16.39±0.72mg/g,較MPS中的10.76±0.41mg/g有了顯著提高(P0.1)。(3)運用Plackett-Burman實驗設(shè)計和響應(yīng)面實驗設(shè)計對MZPS的提取工藝進行優(yōu)化。實驗結(jié)果表明,加水倍數(shù)為25.00,pH值為8.00,醇沉?xí)r間為22.00h是提取MZPS的最優(yōu)工藝條件。用優(yōu)化后的工藝條件提取光帽鱗傘MZPS,MZPS的提取率為2.98%。(4)采用DEAE-52纖維素陰離子交換柱層析和Sephadex G-100葡聚糖凝膠柱層析對MZPS進行分離純化,得到三個多糖組分:MZPS-1a、MZPS-2a和MZPS-3a,其中MZPS-2a為MZPS的主要成分。高效凝膠滲透色譜法(High-performance gel permeation chromatography,HPGPC)的實驗結(jié)果表明,MZPS-2a為均一多糖,重均分子量(Weight-average molecularweight,Mw)為1.36×104Da。傅立葉紅外光譜(Fourier transform infrared spectroscopy,FT-IR)的分析結(jié)果表明MZPS-2a具有多糖的特征吸收峰。柱前衍生高效液相色譜(High performance liquid chromatography,HPLC)法的實驗結(jié)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,且摩爾比為172.59:5.29:4.61:4.20:1.01:1.00。(5)通過體外抗氧化實驗和體內(nèi)抗氧化實驗分析MZPS的抗衰老能力。體外抗氧化實驗表明,MZPS及其組分具備還原力和清除超氧陰離子自由基、羥基自由基和DPPH自由基的能力。并且,在MZPS的組分中,MZPS-2a的抗氧化能力最強,這可能與MZPS具備較低的分子量,較高的糖醛酸含量以及葡萄糖在單糖組成中所占比例較低的結(jié)構(gòu)特征相關(guān)。體內(nèi)抗氧化實驗證明MZPS可以顯著提高衰老小鼠體內(nèi)超氧化物歧化酶(Superoxide dismutase,SOD)活性和總抗氧化能力(Total antioxidant capability,T-AOC),降低脂質(zhì)過氧化物(Lipid peroxide,LPO)和丙二醛(Malondialdehyde,MDA)含量。特別是MZPS較MPS表現(xiàn)出了更強的降低LPO和MDA含量的能力,這可能與鋅的抗氧化功能有關(guān)。實驗結(jié)果說明,MZPS是具有延緩衰老功效的天然抗氧化劑。(6)通過建立高血脂癥小鼠模型,分析MZPS降血脂、保護肝臟的能力。實驗結(jié)果表明,MZPS可以顯著降低高脂血癥小鼠血清中的總膽固醇(Total cholesterol,TC)、甘油三酯(Triacylglycerols,TG)、低密度脂蛋白膽固醇(Low-density lipoprotein cholesterol,LDL-C)和極低密度脂蛋白膽固醇(Very low-density lipoprotein cholesterol,VLDL-C)水平,升高高密度脂蛋白膽固醇(High-density lipoprotein cholesterol,HDL-C)水平,并且降低高脂血癥小鼠肝臟中的TC和TG水平。同時,小鼠血清和肝臟中的SOD活性和T-AOC水平顯著上升,LPO和MDA含量顯著下降。另外,形態(tài)學(xué)觀察表明MZPS可以減輕高脂血癥小鼠的肝臟損傷。實驗結(jié)果說明,MZPS具有降血脂,預(yù)防高脂飲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，

本文編號：2511394