摘要
本文以香菇(Lentinus edodes)为原料,系统研究了香菇多糖(lentinan)的提取、分离、纯化、纯度鉴定、理化性质、化学结构,并对不同分子量香菇多糖的体外抗乙型肝炎病毒活性进行了测定。现有制备高纯香菇多糖的技术比较复杂,产品价格非常高,针对香菇多糖的应用前景和开发现状,本论文将开发从香菇中分离纯化高纯香菇多糖的工业技术作为研究重点,旨在得到易于工业化的流程短、工艺简单、经济、高回收率和高纯香菇多糖的工艺路线。主要研究内容与结论如下:
1.对香菇多糖的提取方法进行了系统的研究,分别考察了热水浸提、超声波辅助热水浸提、微波提取、酶法辅助热水浸提四种提取方法,优化了提取工艺。研究证明:热水浸提得到的香菇多糖提取得率为4.53%,产品纯度23.2%;超声波辅助热水浸提方法多糖提取得率为4.48%,产品纯度21.5%;微波提取多糖提取得率4.44%,多糖纯度24.2%;酶法辅助提取多糖提取得率为6.63%,多糖纯度32.1%。和热水浸提法相比,超声波辅助热水浸提法提取温度低,提取时间短,微波提取能大大缩短提取时间,酶法辅助热水提取能显著提高多糖提取率,所得粗产品纯度高。酶法提取是一种较理想的多糖提取方法。
2.采用壳聚糖对多糖提取液进行沉降,除去溶液中悬浮颗粒、胶状物质,部分色素和蛋白质杂质,提高溶液的澄清度,降低溶液的粘度。实验优化了壳聚糖沉降提取液的工艺条件。多糖溶液中色素降低26%,蛋白质浓度降低35%,溶液粘度降低15%。
3.采用超滤方法对香菇多糖提取液进行分离与分级,选用两种不同截留分子量的陶瓷膜(50kDa,300kDa)超滤分离,得到三种不同分子量的香菇多糖Le1、Le2和Le3,Le1、Le2和Le3的纯度分别为70.5%,52.3%,26.3%。Le1为基本不含蛋白质的低分子量多糖,Le2为含少量蛋白质的中等分子量多糖,Le3为含大量蛋白质的大分子量多糖。超滤分离最佳操作压力为0.15MPa,温度35℃,pH7.0。对膜污染机理和膜清洗方法进行了探讨,通过凝胶极化模型对超滤分离香菇多糖的膜阻力进行分析,阻力模型很好的拟合凝胶极化模型。
4.首次采用八种离子交换树脂和大孔吸附树脂对香菇多糖溶液脱色,发现大孔吸附树脂DA201-C具有最好的脱色效果,同时对蛋白质也有一定的分离效果,对DA201-C大孔树脂分离香菇多糖中的色素工艺进行了优化,多糖溶液的脱色率为87.7%,蛋白质的去除率为42.0%,多糖的损失率为15.0%。经树脂脱色后,三种多糖Le1、Le2和Le3的纯度分别为85.3%、72.9%、49.6%。
5.首次采用阴离子交换树脂分离香菇多糖中的蛋白质,优化了717型阴离子交换树脂除蛋白的工艺条件,酸度是影响蛋白质吸附效果的最关键因素,最佳吸附酸度为8.8-9.2。树脂可用0.5mol/L的HCl溶液再生,经过再生后,树脂的吸附率可恢复90%。分离蛋白质杂质之前,多糖Le1、Le2和Le3的蛋白质含量分别为:5.4%,15.3%和38.7%,分离蛋白后,蛋白质含量分别降为0.5%,1.6%和6.4%,多糖纯度分别为95.1%、89.5%、86.4%。
6.对蛋白质吸附动力学研究表明,717型阴离子交换树脂分离多糖提取液中的蛋白质的吸附过程是单分子层的离子交换过程,是放热的吸附过程,低温有利于吸附。膜扩散速率是离子交换树脂吸附过程的控速步骤,蛋白质的吸附等温线可用Langmuir方程来模拟。
7.研究了Le1、Le2和Le3的理化性质。经Sephadex G-200柱层析、琼脂糖凝胶电泳分析表明:Le1、Le2和Le3均为单一多糖,三种多糖纯度分别为99.2%,97.8%和96.5%。单糖组成分析表明:Le1、Le2和Le3均由阿拉伯糖、木糖、甘露糖、葡萄糖和半乳糖五种单糖组成,其中葡萄糖为主要组分,Le1、Le2和Le3中阿拉伯糖,木糖,甘露糖,半乳糖,葡萄糖的摩尔比分别为0.15:0.52:1.00:1.20:7.20;0.21:0.68:1.00:1.02:11.56;0.29:0.42:1.00:0.85:16.20。采用凝胶柱层析测定多糖分子量表明,Le1、Le2和Le3的平均分子量分别为4.02×10~4、2.16×10~5、8.93×10~5。红外光谱和核磁共振光谱表明:Le1主要是含a糖甙键的葡聚糖,Le3主要是含β糖甙键的葡聚糖,而Le2是既含有a糖甙键,又含β糖甙键的葡聚糖。
8.测定了三种香菇多糖Le1、Le2和Le3的体外抗乙型肝炎病毒活性,结果表明三种香菇多糖都有一定的抗病毒活性,其中Le3活性最强,Le2活性次之。表明香菇多糖的生物活性与多糖的分子量大小有一定关系。
In this dissertation, research of extraction, isolation, purification, physicochemical properties and structure of lentinan were made, a comparison of in vivo anti-HBV activity was also carried between three lentinan with different molecular weight. The purification methods of lentinan are complex at present, and the product is expensive. Aimed at the application prospect and development situation, in this thesis, the studied emphasis was to develop the technology of extraction and purification of high purity lentinan, in order to obtain the short process, simplicity and economic technology of high purification and recovery yield based on industrialization. The research expressed that the design of process route was reasonable, the method was proper and the purity and recovery yield of product reached higher lever. The main contents and conclusions are listed as follows:
1. The extraction methods of lentinan were systematacially studied, the method of leaching with hot water, ultrasonic-assisted extraction, microwave extraction and enzymatic extraction were investigated respectively, and these processes were optimized. The results showed that: the extraction yield of lentinan extracted by hot water was 4.53%, the lentinan purity was 23.2%; the extraction yield by ultrasonic-assisted extraction was 4.48%, the lentinan purity was 21.5%,; the extraction yield by microwave extraction was 4.44%, the lentinan purity was 24.2%; the extraction yield by enzymatic extraction was 6.63%, the lentinan purity was 32.1%. Compared with the method of extracting by hot water, ultrasonic-assisted extraction could reduce the extraction temperature and time, microwave extraction could reduce the extraction time very much, enzymatic extraction could improve the extraction yield and purity of the product obviously. So enzymatic extraction mothod is the best extraction mothod.
2. The lentinan extraction solution was settled with chitosan for the first time, the suspended particulate, gelatinoid, partial pigment and protein could be eliminated effectively, at the same time the solution clarification degree could be improved and the solution viscosity could be decreased. The process was optimized. The pigment in solution decreased about 26%, protein concentration decreased 35% and the solution viscosity decreased 15% about also.
3. The ultrafiltration technology was used to isolate and classify the leached solution of lentinan for the first time. Two kinds of ceramic membrane of molecular weight cut-off 50kDa and 300kDa were selected, the lentinan was separated to three parts with different molecular weight: Le1, Le2, Le3, the purity of them were 70.5%, 52.3%, 26.3%. Le1 was low molecular weight lentinan with low content protein, Le2 was middle molecular weight lentinan including little protein, Le3 was macromolecular lentinan with high content protein. The optimized separation conditions of ultrafiltration were as follows: temperature 35℃, pH value 7.0, 0.15MPa pressure. The mechanism of membrane fouling and method of membrane cleaning were investigated. The membrance resistance of ultrafiltration of lentinan solution was analyzed with gel polarization model, the membrance resistance model could fit the gel polarization model well.
4. The application of ion exchange resin and macroporous adsorption resin to purify lentinan was studied for the first time, it was found that macroporous adsorption resin DA201-C had the best decolorizing effect of lentinan, meanwhile, it could also remove protein at some degree. The technology process of lentinan isolation by DA201-C was optimized. The decolorizing ratio of lentinan was 87.7%, the removal ratio of protein in lentinan was 42%, and lentinan loss ratio was 15%. The purity of Le1, Le2 and Le3 after decolorizing by macroporous adsorption resin DA201-C were 85.3%, 72.9%, 49.6% separately.
5. The application of ion exchange resin to removal protein was studied for the first time. The technology processing of reproteinization from lentinan by 717 anion- exchange resin was optimized, the acidity was the major factor influencing the adsorption effect, the best pH of lentinan solution was 8.8～9.2. The content of protein in Le1, Le2 and Le3 were 5.4%, 15.3% and 38.7% before removing protein by 717 anion-exchange resin, after removing protein, the content of protein in Le1, Le2 and Le3 were declined to 0.5%, 1.6% and 6.4%, and the content of lentinan were 95.1%, 89.5%, 86.4% separatery. The resin could regenerate by 0.5mol/L HCl solution, and the adsorption efficiency could return to 90% above.
6. Adsorption dynamics research of protein indicated that adsorption rate of protein was governed by membrane diffusion rate, the adsorption isotherm of protein could be described with the equation of Langmuir, adsorption reaction was exothermic reaction at the same time.
7. The physicochemical properties of three kinds of lentinan Le1, Le2 and Le3 were studied, the molecular weight of Le1, Le2 and Le3 was proved to be homogeneous by gel filtration chromatography of Sephadex G-200 and agargel electrophoresis. The polysaccharides component were determined by gas chromatography, three lentinans were composed of glucose (Glu), arabinose (Ara), xylose (Xyl), mannose (Man) and galactose (Gla). The neutral saccharide molar ratios Ara : Xyl : Man : Gal : Glu are as follows: Le1, 0.15 : 0.52 : 1.00 : 1.20 : 7.20; Le2, 0.21 : 0.68 : 1.00 : 1.02 : 11.56; Le3, 0.29 : 0.42 : 1.00 : 0.85 : 16.20. The average molecular weight (M_w) of the three polysaccharides Le1, Le2 and Le3 were determined to be 4.02×10~4, 2.16×10~5, 8.93×10~5, respectively. The ~1HNMR, C~(13)NMR and IR spectra confirmed that the sugar residue of Le1 wasα-glycosidically linked, the sugar residue of Le3 wasβ-glycosidically linked, Le2 containedα-glycosidically linked andβ-glycosidically linked component at the same time.
8. The anti-HBV activity of Le1, Le2 and Le3 was determined in vivo. The results proved all these lentinan had anti-HBV activity, Le3 showed highest anti-HBV activity, and Le1 showed lowest anti-HBV activity, it demonstrated the bio-activity of lentinan had some connection with its molecular weight.
引文
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