双酶协同加压热水法提取与纯化香菇多糖

摘要

Lentinan which has the biological activity of enhancing immune system,anti-cancer,antihypertensive and hypolipidemic activities,was widely used in medicine and food industry.The enzyme-assisted (papain and cellulose) pressured hot water extraction was applied to extract lentinan from fresh Lentinus edodes in the present study.The fresh Lentinus edodes was firstly prehydrolyzed with papain and cellulase,and followed by pressured hot water extraction.Afterwards,gel chromatography with Superose 6 PG was applied for the purification of lentinan.The optimal conditions of enzymatic prehydrolysis were as follows:cellulase of 1.00 g/g (calculated by dry mass of Lentinus edodes),papain of 0.024 g/g,temperature of 45 ℃,pH of 4.5 and enzymatic prehydrolysis time of 10 h.After that,the pressured hot water extraction was used to extract lentinan for 80 min at 115 ℃ with a solid/liquid ratio of 1∶50 (W/V).Under the optimal conditions,the extraction yield of lentinan was up to 23.6％.Then,purification of crude lentinan was carried out on Superose 6 PG with degassed purified water as eluent,and the flow rate and column temperature were 0.6 mL/min and 48 ℃,respectively.A homogeneous polysaccharide fraction was obtained by the two-step gel chromatography.The purified lentinan was identified by anthrone-H2SO4 method,periodate oxidation method and infrared spectroscopy.The weight-average molecular weight of the purified lentinan was 3.75 × 104,which was measured by gel permeation chromatography.A red shift phenomenon was observed when the Congo red reaction was used to analyse the purified lentian,which suggested the helix conformation was existed in the purified lentinan.The results of present research will supply a new approach for extraction and purification of lentian from fresh Lentinus edodes.%香菇多糖具有增强免疫、抗肿瘤、降血压、降血脂等功效,广泛应用于食品与医药领域.以新鲜香菇为原料,采用木瓜蛋白酶和纤维素酶双酶协同加压热水法提取香菇多糖,优化双酶预水解的工艺条件.在此基础上,以凝胶层析法纯化香菇多糖,并鉴定纯化的香菇多糖.研究结果表明:纤维素酶加入量为1.00 g/g(以1 g绝干香菇质量计)和木瓜蛋白酶加入量为0.024 g/g(以1 g绝干香菇质量计),在pH 4.5和45℃的条件下,双酶协同预水解香菇原料10 h,接着采用加压热水法提取香菇多糖,在固液比1∶50 (g/mL)和提取温度115℃条件下,提取80 min,香菇多糖得率可达23.6％.采用凝胶层析法纯化香菇多糖,以Superose 6 PG为分离介质,流动相为脱气纯水,在流速0.6 mL/min和柱温48℃的条件下,分离纯化香菇多糖粗品,经过两步分离纯化后,获得均一的多糖样品.以蒽酮-硫酸法、高碘酸钠法和红外光谱法鉴别纯化组分为香菇多糖;以凝胶渗透色谱法测定香菇多糖的重均分子量为3.75×104;以刚果红法分析香菇多糖,香菇多糖与刚果红形成络合物,最大吸收波长移向长波长,发生红移现象,表明纯化的香菇多糖中存在螺旋构象.研究结果为香菇多糖的高效提取与分离纯化提供了新的路线.