樟树籽蛋白质的提取与分析

武夷学院2008级生物技术应用专业毕业论文1武夷学院毕业设计(论文)樟树籽蛋白质的提取与分析院系：武夷学院专业（班级）：生物技术及应用姓名：林开鹏学号:20082501015指导教师：李卫林职称：讲师完成日期：2011年6月3日武夷学院教务处制樟树籽蛋白质的提取和分析樟树籽蛋白质的提取与分析摘要：植物蛋白的提取方法有等电点法、离子交换法及超滤法、乙醇沉淀蛋白法等本实验采用了乙醇沉淀蛋白,其优点是避免蛋白质变性,尽可能减少杂质。本实验采用磷酸盐缓冲液溶解蛋白质再用乙醇为脱水剂破坏蛋白质胶体质点的水化层，使其沉淀析出进而对樟树籽蛋白进行提取,并用紫外光吸收法测定蛋白质的浓度,SDS-聚丙烯酰胺凝胶电泳法测定蛋白质的相对分子质量，对樟树籽蛋白进行分析关键词：樟树籽，蛋白质，提取，分析武夷学院2008级生物技术应用专业毕业论文3目录1前言·············································································11.1樟树籽简介·································································································11.2油类作物蛋白提取方法···················································································11.2.1水相萃取法···························································································21.2.2有机相萃取法························································································21.2.3水相酶解法···························································································21.3蛋白质分析检测方法·····················································································31.3.1Folin-酚试剂法(Lowry法)·······································································31.3.2紫外吸收法···························································································32材料和方法····································································32.1材料··········································································································32.1.1实验材料······························································································32.1.2试剂····································································································32.2器材··········································································································42.3实验方法····································································································42.3.1提取方法······························································································42.3.2蛋白质分离方法·····················································································52.3.3蛋白质的定性························································································52.3.4蛋白质的等电点·····················································································62.3.5紫外光吸收法测定蛋白质浓度····································································62.3.6SDS聚丙烯酰胺凝胶电泳法—蛋白质的分子量测定·········································73结果与讨论····································································93.1前处理·······································································································93.2蛋白质的提取······························································································93.3SDS聚丙烯酰胺凝胶电泳法············································································104结论·············································································11参考文献·········································································11致谢························································错误!未定义书签。武夷学院2008级生物技术应用专业毕业论文11前言1.1樟树籽简介樟树属樟科(Lauraceae)植物,是一种生长在亚热带地区四季常青的树种,在我国长江以南地区及台湾省有大量种植。樟树籽,又名樟犁、香樟子、大木姜子、樟子等，其氨基酸含量丰富,品质优良,但是、目前,樟树籽除极少量被采摘入药外,大部分均自生自灭,没有被很好地利用.由于食物资源供需矛盾的日益突出,开发利用新的蛋白质资源已成当务之急,尤其是植物性蛋白质源的开发利用已成为世界各国解决食物资源匮乏的重要突破口.大豆蛋白、花生蛋白的应用已日趋普遍,但对樟树籽蛋白的利用还没有引起足够的重视，樟树(CinnamomumCamphora)是樟科属的常绿乔木植物,高20～30m。主要生长在热带和亚热带地区,全世界有45个属约2500余种,在我国有约20个属近430种,其中我国特有的有355余种,樟树是我国特产珍贵木材和经济林树种,被誉为江南宝树。樟树3～4月开花,花小呈绿色或淡黄色,长大约2mm,花被6裂,椭圆形。花期能散发出淡淡的清香,这段时期大约一周至半月左右。每年10～12月樟树籽基本成熟,但这时的樟树籽很少自行脱落,其果皮依然是与叶面相同的绿色。再往后果皮的颜色逐渐加深,到第二年1～2月樟树籽果皮已由绿色逐渐变成棕黑色或黑色。樟树是樟科属的常绿乔木植物中经济价值最大的树种之一,是天然樟脑、芳香油、油脂、优质木材、医药等类的重要资源,其木材、根、叶、果实皆含精油,以往主要是对根、茎、叶进行利用与加工,近年来对樟树籽的利用研究增多,发现其核仁含脂肪油,含油率高达40%以上。油脂中脂肪酸组成以C10、C12脂肪酸为主,占90%左右。樟树籽,又名樟梨、香樟大木姜子、樟子等。成熟的樟树籽呈扁球形,直径大约5～9mm,每千粒重大约是200g,果皮肉质薄,能散发出芬芳的清香。我国很早就将樟树籽作为药物用于治病,并认为樟树籽有驱风散寒、行气止痛之功效。癸酸(C10)已应用于医药行业合成鱼腥草素等消炎药,主要含有癸酸(C10)的樟树籽脂肪油已被试制成碳酸甘油脂,用于治疗脂肪代谢紊乱病症,并能降血脂及胆固醇。1.2油类作物蛋白提取方法油类作物蛋白营养价值和功能特性已被广泛接受，但国内外仍没有工业化产品。由于油类中抗营养因子的存在，过去很长一段时间内，油脂工业只注重油类作物榨油，忽略了油类作物蛋白的利用，造成植物蛋白资源的浪费。当今世界蛋樟树籽蛋白质的提取和分析2白资源供求日趋紧张，所以，各国学者们开始转向油类作物蛋白的研究。目前，从油类作物中提取蛋白质的方法主要有以下几种。1.2.1水相萃取法水相萃取法是采用不同水相将菜籽蛋白提取，再分离(常用等电点法)，得到菜籽分离蛋白或者是通过水相将菜籽饼粕中硫苷、植酸、可溶性糖等非蛋白组分溶出，获得菜籽浓缩蛋白的方法。常用的水相有：水、稀酸、稀碱、NaCl溶液、六偏磷酸钠溶液等。刘大川等人(2005年)以脱皮脱脂双低菜籽粕为原料，在pH值11，同液比1：12，浸提温度50℃下，用NaOH溶液浸提4次，每次35min，得到产品的蛋白含量86．12％，蛋白得率24．35％。金晶等人(2009年)选用双低菜籽粕为原料，采用超声微波辅助水相法，在超声频率40kHz，功率50w，微波功率73w，料液比1：28，时间8min下，提取3次，获得蛋白含量为83．65％的产品，蛋白得率38．76％。水相萃取法工艺简单、成本低，但蛋白得率不高，易污染环境。蛋白得率偏低的主要原冈是菜籽蛋白组成复杂，相对分子量差异大，使得菜籽蛋白溶解曲线不能形成“U”型，而出现2个或多个等电点区域。1.2.2有机相萃取法采用醇、酮等有机溶剂提取菜籽饼粕中植酸、多酚、残油、色素等组分，制取菜籽浓缩蛋白的方法。曾晓波等人(2001年)以双低脱壳菜籽饼粕为原料，采用丙酮浸提法，在丙酮体积分数为78％。85％，NaCI质量分数为7％，pH值为4.0下提取，所得浓缩蛋白的蛋白质质量分数为70％，产品色浅味淡，功能性质较好，可用作食品添加剂。金青哲等人(2006年)…I以双低菜籽为材料，采用超声波辅助乙醇浸提法，在超声频率20kHz，功率100kW，乙醇体积分数为70％，液同比3．5：1的条件下，提取3次，每次20min，所得浓缩蛋白蛋白质量分数大于60％，氨基酸平衡，硫苷、植酸、单宁等抗营养因子含量低。有机相萃取法具有产品色泽好，脱毒率高等优点，但工艺复杂，成本高，易引起蛋白变性，影响蛋白营养效价。1.2.3水相酶解法水相酶解法是在水相萃取法基础上，增加了酶水解过程，以提高菜籽蛋白收率和品质的方法。常用的酶有：碱性蛋白酶、中性蛋白酶、酸性蛋白酶和木瓜蛋白酶、风味蛋白酶、果胶酶、纤维素酶和半纤维素酶等。刘志强等人(2004年)旧以双