【作者】 姜薇；

【导师】 殷涌光；

【作者基本信息】 吉林大学 ， 农业机械化工程， 2014， 博士

【摘要】 目前，我国是世界上的鹿产业大国，而吉林省的特色产业之一就是养鹿业。鹿角冒(Antler base)是梅花鹿或马鹿鹿茸根部骨质部分，鹿茸被锯掉后，于第二年的春天新鹿茸长出后脱落。它长得像一个圆盘，故命名为鹿角盘或鹿角冒。鹿角冒在2000年前就被记载在中国药典《神农本草经》上，认为其具有强筋健体、补肾、行血消肿和滋阴的功效。在中国，鹿角冒被广泛地用于中药中，用于治疗一系列的疾病，如乳腺炎、乳腺增生、恶性溃疡、儿童流行性腮腺炎及子宫肿瘤等。多年以来，很多研究人员主要集中在鹿茸的药理价值上。相对于鹿角冒蛋白的研究较少，范围较窄，严重阻碍了鹿角冒产业的发展。因此，本文以鹿角冒为原料，更广泛、更深入的研究鹿角冒蛋白，以促进鹿角冒产业的开发，带来更高的经济和药用价值。本文通过三条途径分别考察了鹿角冒中肽聚糖识别蛋白的提取、分离及性能检测；抗氧化活性多肽的制备及精制；胶原蛋白的提取及性能检测等。主要研究内容分为以下五个部分：(1)鹿角冒识别蛋白1(cnPGRP1)是从梅花鹿鹿角冒中提取分离出的一种新的抗微生物蛋白，它在切掉鹿茸的创面上高度表达。文中采用预冷的5mM的乙酸钠缓冲溶液进行匀浆提取鹿角冒可溶性蛋白，可溶性蛋白经两倍体积的乙醇沉淀除杂，40℃旋转蒸发除乙醇后，冻干得鹿角冒粗多肽。并采用凝胶葡聚糖G-25、CM阳离子吸附树脂及高效液相色谱柱对粗多肽进行分离纯化。纯化后的多肽经SDS-PAGE电泳分析，其分子量为17.2kDa。鹿角冒多肽经MALDI-TOF-MS鉴定为肽聚糖识别蛋白1，匹配序列为RLYEIIQKWPHYRA。我们定义该纯化蛋白为cnPGRP1。在本文的体外抑菌试验中，我们提取分离纯化的cnPGRP1，在浓度为50-250μg/mL，作用4h时，均能在不同程度上杀死革兰氏阳性菌和革兰氏阴性菌。通过微生物绑定试验表明，鹿角冒肽聚糖识别蛋白能够绑定到革兰氏阳性菌(金黄色葡萄球菌和枯草杆菌)、革兰氏阴性菌(大肠杆菌和志贺氏菌)以及真菌(酵母菌)。cnPGRP1是直接从组织中提取、分离纯化得到的单体，与通过克隆、分离纯化的小鼠及人类的PGRP1不同，其杀菌活性不依赖于Zn2+等阳离子，类似于牛科的PGRP1。(2)采用碱性蛋白酶、中性蛋白酶、复合蛋白酶、菠萝蛋白酶和胰蛋白酶五种酶酶解鹿角冒蛋白，制备抗氧化活性多肽。文中探讨了各种酶的水解能力，及产物的抗氧化活性，其中碱性蛋白酶的水解能力和酶解后产物的抗氧化活性均较高，最终选择碱性蛋白酶作为最佳用酶。根据单因素试验结果，对影响鹿角冒蛋白水解工艺的酶浓度(X1)、温度(X2)和pH值(X3)三个因素进行优化，并以酶解液对DPPH自由基的清除率为响应值(Y)，进行三因素五水平的响应面分析，得出显著性水平α﹤0.05下的数学模型：Y=73.45+3.49X1-1.18X2-1.21X1X2-2.47X1X3-3.89X21-4.03X22-2.57X23。在模型的基础上，计算得出最佳酶解工艺参数：底物浓度15%、加酶量6%、温度53℃、pH8.5的参数条件下酶解4h，在此条件下，酶解液清除DPPH·自由基的能力为74%(5mg/ml)。(3)抗氧化活性多肽的精制主要体现在脱色和脱盐两个方面。通过研究各脱色因素(多肽的pH、脱色温度、脱色时间、活性炭用量)对活性碳脱色效果的影响，确定了活性碳的脱色最佳条件为活性碳用量2%，pH5，脱色温度50℃时，脱色3h，在此最优条件下，活性炭对鹿角冒多肽的脱色率达到79.86±0.26%。通过研究各吸附因素(pH、温度、浓度、料液比)对DA201-C大孔树脂吸附能力的影响，确定了树脂的静态吸附的最优条件：取pH4.0，温度25℃，多肽浓度10mg/mL，料液比1:5，振荡吸附24h，在此条件下，树脂对鹿角冒多肽的吸附率达到78.52±1.05%。根据DA201-C树脂的动态吸附与解析试验对多肽进行脱盐处理，多肽的脱盐率达到98.64±2.45%，多肽的回收率为86.31±0.97%。采用抗氧化活性试验检测脱盐后多肽的抗氧化活性，得出多肽经DA201-C树脂除盐后，鹿角冒多肽仍具有较高的抗氧化活性。所以，采用DA201-C树脂对鹿角冒多肽的除盐和初步的粗分具有良好的工业前景。(4)通过研究加酶量、电场强度及脉冲数三个因素对提取鹿角冒中胶原蛋白的影响，并以提取胶原蛋白的浓度为试验指标，并联合提取液的电泳条带，确定了胶原蛋白的最佳提取条件为电场强度20kV/cm，脉冲数为8，胃蛋白酶的浓度2%，在此最佳条件下胶原蛋白的提取率为73.41±1.07%，电泳条带较纯，没有小分子多肽条带。对提取的胶原蛋白进行体外成纤维及变性温度检测，得出所提取的胶原蛋白具有很好的体外成纤维能力，其变性温度为36℃。红外检测表明，所提取蛋白具有胶原蛋白的特征。更多还原

【Abstract】 Antler base is the ossified and rudimental antler on the pedicle of male sika deer(Cervus nippon) after sawing off the velvet antler, which then falls off by itself everyspring when the new velvet antler begins to germinate. It looks like a plate, so it istherefore named ‘antler base’. Antler base has high medicinal value according to theShen Nong Ben Cao Jing and is believed to tonify the liver and kidneys, andinvigorate the circulation of blood and detumescence. Over years, many researchershave focused on the antler base pharmacological value, which less than the researchof antler base protein. The range of research was narrow, that seriously hindered thedevelopment of antler base at industry. In this paper, antler base as the object of study,a broader and more in-depth study of antler base protein were carried out, to promotethe development of industry, and bring more economic and medicinal value.Through three channels to research the antler base protein: extraction, isolationand characteristic of antler base PRGPs; preparation and purification of antler baseantioxidant peptide; extraction and characteristic of antler base collagen. The researchcontents are as follows:(1) CnPGRP1is a novel antimicrobial protein from antler base of the sika deerCervus nipp, expressed at high levels to protect the wound of velvet antler frombacterial infections after removal. The samples were ground with10volumes ofprecooled solvent A (5mM sodium acetate, pH4.5) by using a colloidal mill. Theimpurity was removed from peptide by ethanol precipitation. The supernatant wasthen removed ethanol using a rotary evaporator at40℃, and lyophilized. The antlerbase antimicrobial proteins (AAP) were subjected to consecutive chromatographicmethods connected to Sephadex G-25gel filtration column,(CM) anion exchangecolumn, and RP-HPLC. The molecular weight of cnPGRP1was17.2kDa underSDS-PAGE, and peptide mass fingerprint analysis by MALDI-TOF-MS aspeptidoglycan recognition protein1matched to dasypus novemcinctus. The matchedamino acids sequences were RLYEIIQKWPHYRA.In a standard liquid assay in vitro, both Gram-positive bacteria andGram-negative bacteria are killed by cnPGRP-S, at a dose of50-250μg/mL withrange at4hours of incubation. Our results indicate that cnPGRP binds toGram-positive bacteria (S. aureus and B. subtilis), Gram-negative bacteria (E. coli andS. dysenteriae), and fungus (S. cerevisia). The requirement for Zn2+or Ca2+for microbicidal activity may explain why previously murine and human recombinantPGRP-S was only bacteriostatic, and not microbicidal. Our isolated cnPGRP from theantler base of the sika deer, a native protein, was the same as bovine PRGP-S, and theprobable retained Zn2+that was likely bound to them in vivo.(2) The protein of antler base was enzymatic hydrolyzed by alkaline protease,neutral protease, compound protease, pineapple protease and trypsin, and topreparated antioxidant peptides. The enzyme hydrolysis ability and antioxidantactivity of peptide were discussed, the hydrolysis active of alkaline protease and theantioxidant active of the peptide which were hydrolyzed by alkaline protease werehigher than other proteases, so the alkaline protease was chosen to hydrolyze antlerbase.According to the results of single factor experiment, enzyme concentration (X1),reaction temperature (X2) and pH (X3) were chosen to optimize the conditions ofenzymatic hydrolysis of antler base protein by three factors five levels of responsesurface analysis, and the clearance ratio of DPPH free radical by peptides as theresponse value (Y). The mathematical model was established based on the test result,as follow: Y=73.45+3.49X1-1.18X2-1.21X1X2-2.47X1X3-3.89X21-4.03X22-2.57X23, forthe significant level α<0.05. The optimal conditions of enzymatic hydrolysis as follow:the substrate concentration15%, enzyme concentration6%, temperature53℃, pH8.5,the clearance ratio of DPPH free radical by peptides was74%.(3) The dispose of antioxidant peptides mainly reflected in two aspects ofdecoloring and desalination. The peptide pH, reaction temperature, reaction time andactive carbon concentration were studied about the factors of decolorizing of effectson the decoloring of active carbon. The best conditions of active carbon decoloringwere determined as follow: the peptide pH5, reaction temperature50℃, reaction time3h and active carbon concentration4%, the decoloring rate of antler base peptides byactive carbon was79.86±0.26%.The pH, reaction temperature, peptide concentration and the ratio of peptide tomaterial were studied about the factors of adsorption of effects on the adsorptioncapacity of macroporous resin DA201-C. The best conditions of static adsorption ofmacroporous resin DA201-C were determined as follow: the peptide pH4, reactiontemperature25℃, peptides concentration10mg/mL the ratio of peptide to material5:1and adsorption time24h. Under this optimal condition, the adsorption rate ofantler base peptides by macroporous resin DA201-C was78.52±1.05%.According to the dynamic adsorption and elution experiment of resin DA201-Cof desalination processing of antler base peptides, the desalination rate of peptideswas98.64±2.45%, and the recovery of peptides was86.31±0.97%. The antioxidant activity of peptides which were desalted by resin DA201-C, still had high antioxidantactivity. So the resin DA201-C was suitable for desalting and purification of antlerbase peptides, and has a good industrial prospect.(4) The effects of enzyme concentration, electric field intensity and pulse numberon extraction of antler base collagen were studied, with the collagen concentration astest indexes, and jointed the collagen banding in the SDS-PAGE. The optimalconditions of extraction of collagen were confirmed as follow: electric filed intensity20kV/cm, pulse number8and pepsin concentration2%, under this optimal conditionof extraction, the extraction rate of antler base collagen was73.41±1.07%, and thebandings of collagen were more pure, there was not peptide banding under thecollagen bandings.The denaturation temperature and in vitro fibrillogenesis test of antler base collagen werestudied, the test showed that the denaturation temperature of collagen was36℃, the in vitrofibrillogenesis was good. The infrared spectrum showed that the extraction wascollagen.更多还原