黑木耳多糖的提取及其工艺研究

编号：四川农业大学本科毕业论文（2013届）题目：黑木耳多糖的提取工艺研究学院：风景园林学院专业：生物技术教育学生姓名：柯鳗峻学号：20099065指导教师：罗傲雪职称：副教授完成日期：2013年5月4日四川农业大学教务处制目录1前言······································································41.1黑木耳的资源分布·························································41.2黑木耳的生长习性·························································41.3黑木耳中多糖的活性·······················································52研究前景···································································62.1材料与试剂·······························································62.2仪器·····································································63实验方法···································································63.1标准曲线的制备···························································63.1.1对照品溶液的制备·······················································63.1.2标准曲线的制备·························································63.2黑木耳多糖（APS）的提取工艺流程···········································73.3APS的提取率的计算························································73.4APS的成分分析····························································74结果与分析··································································74.1黑木耳多糖提取工艺条件的研究·············································74.1.1标准曲线的结果及绘制···················································74.1.2葡萄糖标准品测定的结果················································74.1.3葡萄糖标准品测定结果曲线的绘制·········································84.1.4单因素实验结果························································8料液比对多糖得率的影响·····················································8提取温度对多糖得率的影响···················································9提取时间对多糖得率的影响···················································94.1.5正交试验设计及结果·····················································104.2对APS最佳工艺条件的确定················································114.3黑木耳多糖的成分分析·····················································115结论······································································12参考文献····································································12致谢········································································13黑木耳多糖的提取及其工艺研究姓名：柯鳗峻（四川农业大学风景园林学院，生物技术专业，611830）导师：罗傲雪（四川农业大学风景园林学院，园林植物系，611830）摘要：目的：研究提取多糖的最佳条件。方法：本文通过酶法来提取黑木耳多糖，从而来确定最佳提取条件。结果：利用中性蛋白酶来提取多糖，其最佳提取条件为：料水比1:40,浸提温度60℃，浸提时间2.5h，多糖的得率为4.96%。关键词：黑木耳多糖提取正交设计StudyonextractionofpolysaccharidefromauriculariaAuricularanditsprocessName:KeManjun(CollegeofSichuanagriculturalUniversitylandscape,biologicaltechnology,611,830)Instructor:LuoAoxue(CollegeofSichuanagriculturalUniversitylandscape,landscapeplants,611,830)Abstract:Objective:AuriculariaAuricula,andtodeterminethebestextractioncondition.Theresult:theutoresearchtheoptimalconditionsofextractingpolysaccharides.Method:thisseofneutralproteaseextractedpolysaccharidesanditsoptimalextractionCondition:water1:40,extractiontemperature60℃,theextractiontime2.5h..Keywords:proteaseofAuricularia；Auricula；polysaccharide；extract.1前言1.1黑木耳资源的分布黑木耳多糖是黑木耳子实体中的重要活性物质，除具有一般的生化性质外，还具有调节免疫功能、抗血栓、抑菌、降血糖、抗溃疡、抗肝炎和抗突变、降血脂和促进血清蛋白生物合成等多种功效[1]木耳生长于栎、杨、榕、槐等120多种阔叶树的腐木上，单生或群生。真菌学分类属担子菌纲，木耳目，木耳科。国内有9个种，黑龙江拥有现有的全部8个品种，云南现有7个种、河南卢氏县有一种。野生黑木耳主要分布在大小兴安岭林区、秦巴山脉、伏牛山脉等。湖北房县、随州、四川青川、云南文山、红河、保山、德宏、丽江、大理、西双版纳、曲靖等地州市和河南省卢氏县是中国木耳的生产区。我国是世界上主要的木耳生产国，年产量占世界总产量的90%以上[2].黑木耳是著名的山珍，可食、可药、可补，中国老百姓餐桌上久食不厌，有“素中之荤”之美誉，世界上被称之为“中餐中的黑色瑰宝”。而黑木耳培植方法，在世界农艺、园艺、菌艺史上，都堪称一绝。1.2黑木耳的生长习性黑木耳属于腐生性中温型真菌。菌丝在6～36℃之间均可生长，但以22～32℃最适宜；15～27℃都可分化出于实体，但以20～24℃最适宜，河南省卢氏县就是因为良好的天然环境造就了卢氏黑木耳为其地理保护产品。菌丝在含水量60%～70%的栽培料及段木中均可生长，子实体形成时要求耳木含水量达70%以上，空气相对湿度90%～95%[3]。暗中能正常生长，子实体生长期需250～1000lx的光照强度。为好气性真菌，pH5～5.6最适宜。黑木耳栽培方法有段木栽培与塑料袋代料栽培等多种。黑木耳，其性味甘平，是我国传统保健佳品，也是世界公认的保健品。它是我国珍贵的药用和食用胶质真菌，属于木耳系真菌类胆子菌纲。黑木耳营养丰富，食味鲜美，含义人体不可缺少的蛋白质、脂肪、碳水化合物，钙、铁、胡萝卜素、维生素B1、B2等。其具有益气强身、补气血、润肺、止血、止痛、通便等功效。可防止动脉硬化、高血压和眼底出血，增强抵抗力，延年益寿[4-5]。黑木耳多糖有抗凝血、抗血栓、增强机体免疫功能及降血脂等多种功效[6]。黑木耳多糖为黑木耳的重要活性成分，近年来，随着人们营养保健意识的增强，对黑木耳的研究也越来越深入，结果表明，黑木耳的重要生理功能都是与其多糖组分密切相关的［7］。1.3黑木耳中多糖的活性多糖是由多个相同或不相同的单糖基以糖苷键相连而成的聚合度超过10的高聚物。其基本结构是一种或多种单糖，由一个糖的还原性端基与另一个糖的C2、C3、C4或C6的-OH彼此脱水缩合而成的大分子化合物，结构复杂，不具有原来单糖的性质。多糖还原性小，有旋光性，分子量一般较大，不溶于有机溶剂[8],量通常在数万至数百万之间，多糖纯品实质上是一定分子量范围的均一组分。迄今为止，人类对核酸和蛋白质的研究取得了重大进展，推动了生命科学及相关科学领域的发展，多糖将成为最后一类有待攻破的生物活性大分子。多糖的来源大致分为植物来源多糖、动物来源多糖和微生物来源多糖，即细菌多糖和真菌多糖[9]多糖是一种具有合成高分子所没有的特殊生物活性的物质，因其化学结构中的糖基数量相对单糖和双糖为多而得名。在自然界中多糖的种类繁多，它们在动物、植物、微生物中均有存在且履行一系列不同的功能。微生物产生的多糖可以分为胞内多糖、细胞壁多糖和胞外多糖三类。根据多糖化学结构中糖基的种类，又可将其分为同聚多糖和杂聚多糖，前者为相同单糖所组成，如阿拉伯胶、淀粉等，后者为一种以上单糖所组成，如半纤维素，粘多糖[10],用真菌多糖是微生物多糖的重要组成部分。食药用真菌多糖在国际上称为“生物反应调节剂”（Biologicalresponsemodifier,BRN），它已成为目前医药保健品开发的热点现代研究表明，黑木耳多糖是一种较全面的保健食品和保健药品，黑木耳多糖、酸性杂多糖组分β—D—葡聚糖组分都有可能分别作为药物的有效成分而单独使用或作为辅助药物使用。例如：目前临床常用的抗溃疡药物不良反应突出，长期服用难以忍受，黑木耳多糖对溃疡病有效，且未见不良反应。它很可能是一种新的低毒抗溃疡药物。另外，其β—D—葡聚糖组分抗肿瘤活性很突出，可与放疗或抗肿瘤药物合用，对肿瘤的治疗有协同作用，并可降低副作用。酸性杂多糖的升白细胞活性很显著而且毒性低，有望将其开发为一种低毒、高效的升白细胞药物。而黑木耳细胞壁关键组分几丁质和β-葡聚糖，质地坚韧不易被人体消化，所以黑木耳细胞壁内所含有的其他多种有效成分也很难透过细胞壁被人体所吸收[11]。1.4研究前景近年来，随着糖科学和糖技术的发展，黑木耳多糖已日益被人们所重视，它将会为人类的健康和安全提供更为有利的帮助，黑木耳多糖将具有更广阔的发展前景。多糖常用的提取方法有：热水浸提法、酸浸提法、碱浸提法、酶法等[12-15].传统黑木耳多糖提取采用水体系，需要多次浸提，操作时间长，收率低。酸浸提法和碱浸提法容易使部分多糖发生水解，破坏多糖的活性结构、减少多糖得率。本文采用酶法提取黑木耳多糖，确