【作者】 曲敏；

【导师】 冯叙桥；

【作者基本信息】 沈阳农业大学 ， 食品科学， 2012， 博士

【摘要】 大鲵（Andrias davidianus）属两栖纲、有尾目、隐鳃鲵科，是我国特有的珍稀特产，属国家二类保护动物,主要分布于长江中上游、珠江中上游及汉水上游的溪流中。上世纪90年代以来，湖南、湖北、陕西及广西都建立了大量的大鲵驯养繁殖企业。仅湖南省就有大鲵驯养繁殖企业35家左右，年繁殖能力已达4-5万尾。随着养殖规模的扩大，大鲵资源的精深加工利用成为一个关系大鲵产业持续发展的紧迫课题。至今还没有一种经济效益显著的大鲵精深加工利用技术，这严重制约了大鲵养殖业的发展。大鲵体表无鳞，多皮肤腺，在生长过程中不断分泌黏液。大鲵作为有3亿5千万年的物种，除了具有寿命长、能自我修复的特点，还具有包括体表黏液在内的独特的非特异性免疫系统。大鲵在长期进化过程中体表黏液具备了抵御环境变化的能力和丰富的生物学活性。因此，大鲵黏液具有更大的利用价值和利用的可能性。如果能够对大鲵体表黏液进行开发利用，可以起到既利用大鲵资源，又不危害大鲵生长繁殖的效果，是实现大鲵资源可持续利用的最理想状态。本文利用海洋曲霉菌酸性蛋白酶（Aspergillus sp. acid protease）酶解大鲵体表黏液获得了大鲵低聚糖肽，研究了大鲵低聚糖肽的制备工艺、物理化学特性、生物学活性，并研究了它在果汁饮料与化妆品中的应用。研究结果对大鲵体表黏液的加工利用具有理论和实际意义,主要研究结果如下：1.通过正交试验，得到海洋曲霉菌酸性蛋白酶酶解大鲵体表黏液的最优酶解条件。各因素对酶解大鲵黏液影响的大小顺序为：酶解时间>E/S>温度>pH。海洋酸性蛋白酶酶解大鲵粘液最优酶解条件为：55oC，pH：2.0， E/S：0.03%,，酶解时间：3h。通过对大鲵酶解物溶液共晶点、冻融过程以及冷冻浓缩过程的研究，得到了一些可以提高冷冻干燥大鲵酶解物溶液效率的措施。即冷冻干燥操作前，大鲵酶解物溶液在-8.6～-13.6℃进行预冻结，来提高大鲵酶解物溶液的浓度。2.大鲵黏液酶解物经飞行质谱检测后被确认为分子量低于3.5kDa的大鲵低聚糖肽。大鲵低聚糖肽总蛋白含量较高,为80.01%，总糖含量为15.65%。总糖中盐酸氨基葡萄糖最高，半乳糖醛酸其次，之后依次为葡萄糖醛酸和唾液酸，分别占大鲵低聚糖肽3.39%、2.45%、0.65%、0.60%，占总糖含量的22.38%、16.17%、4.29%和3.69%。经测定大鲵低聚糖肽中不含硫酸糖。大鲵低聚糖肽中所含有18种氨基酸，除色氨酸未测出外，人体其它7种必需氨基酸的含量较高,其中苏氨酸（Thr）含量最为丰富，高达13.1%,其次是天冬氨酸/酰胺（Asx）和谷氨氨酸/酰胺（Glx）,分别为11.2%和12.5%，而丝氨酸（Ser）含量最低,仅为0.2%。用β-消旋反应与血细胞凝集法测定糖肽键连接方式表明，大鲵低聚糖肽中存在O-连接糖肽键。对大鲵低聚糖肽进行nano-ESI-MS/MS分析，串联质谱图经Micromass专用软件处理后，用Spectrum List通过Mascot查询NCBI、SWISSPROT等数据库，经MasSeq软件分析，得到3个序列为：KAPILSDSSCKSC、KLQGTVSWGSGCQAKNC和VVHSLVQVTANKVMVRM。3.大鲵低聚糖肽具有清除羟基自由基、DPPH自由基和超氧阴离子自由基的作用。随着大鲵低聚糖肽浓度的升高，其清除自由基的能力逐渐增强。当浓度达到1mg/mL时，羟基自由基清除率达到54.69%，DPPH自由基清除率高达92.25%，超氧阴离子自由基清除率达到52%。大鲵低聚糖肽具有较好的ACE抑制活性。HPLC测试表明，大鲵低聚糖肽在40mg/mL时对ACE抑制活性最高，达90.82%。4.研究不同剂量的大鲵低聚糖肽对小鼠免疫功能影响的结果表明，按体质量将昆明种小鼠分成对照组（0g/kg·bw）和低（0.01g/kg·bw）、中（0.05g/kg·bw）、高（0.1g/kg·bw）3个剂量组，大鲵低聚糖肽饲喂小鼠8d后，各剂量组小鼠血清中IgG含量、吞噬鸡红细胞的巨噬细胞数以及T淋巴细胞活性与对照组相比均显著提高。将大鲵低聚糖肽按低浓度（100mg/kg体重）和高浓度（150mg/kg体重）两个浓度剂量组连续饲喂小鼠，并每天按剂量0.1mL/16g体重灌胃（对照组灌服相同体积生理盐水），15d后进行小鼠负重游泳试验。结果显示大鲵低聚糖肽高浓度组小鼠的游泳竭力增加到78.5min（P<0.01）。服用高浓度大鲵低聚糖肽的小鼠比空白组小鼠的肝糖原浓度增加了100mg/mL，增加率达到30.3%,肌乳酸浓度减少了43.2mg/mL，减少率为39.2%；尿素减少了26mg/mL，减少率高达68.4%。选昆明种雄性小鼠，随机分成空白对照组，模型对照组和联苯双酯阳性对照组[200mg/（kg.d）]，大鲵低聚糖肽低浓度[200mg/（kg.d）]、中浓度[400mg/（kg.d）]、高浓度[800mg/（kg.d）]剂量组。各给药组每天给药灌胃1次，连续给药7d后，测定大鲵低聚糖肽对小鼠CCl₄肝损伤的保护作用。结果表明中、高大鲵低聚糖肽剂量组可显著抑制小鼠肝脏组织中由CCl₄造成的血清谷草转氨酶（AST）、谷丙转氨酶（ALT）活性升高，明显降低丙二醛（MDA）含量，并且提高超氧化物岐化酶（SOD）活性。光镜检查结果表明，CCl₄模型组肝细胞明显肿胀变形，肝细胞索紊乱，伴有炎症细胞侵润。大鲵低聚糖肽各剂量组的肝细胞索排列整齐、肝细胞排列规则，较模型组肝组织损伤明显减轻。5.大鲵低聚糖肽对280-315nm的紫外线有一定吸收。小鼠紫外损伤试验表明涂抹大鲵低聚糖肽的小鼠耳指数（耳片质量/小鼠质量）与未进行紫外线损伤照射的涂抹食用植物油的小鼠耳指数相当，说明小鼠耳部皮肤涂抹大鲵低聚糖肽可以避免紫外照射损伤。6. Caco-2细胞吸收试验表明，大鲵低聚糖肽可以被Caco-2细胞吸收。大鲵低聚糖肽可以被HaCaT细胞吸收，同时大鲵低聚糖肽具有促进HaCaT细胞生长的作用。MTT法表明，HaCaT细胞在大鲵低聚糖肽浓度0.098mg/mL中培养72h的存活率,是对照组的145%。7.以pH值、沉淀率、感官评分和固形物含量为指标，通过单因素和正交实验确定大鲵低聚糖肽果汁保健饮料的最佳制备工艺为：黑加仑果汁和蓝莓果汁的比例为1:3（15mL:45mL）、大鲵低聚糖肽和牡蛎多糖的比例1:3（1.5mL:4.5mL），柠檬汁为10mL（总体积200mL）和果胶含量为0.1%，杀菌温度95℃，杀菌时间10min为最佳，产品的色泽、风味和稳定效果较好。在低湿度条件（RH=43%）下，大鲵低聚糖肽的吸湿率达3.8%；在高湿度条件（RH=81%）下，大鲵低聚糖肽吸湿率达15.6%。以大鲵低聚糖肽制备的润肤霜稳定性符合中华人民共和国行业标准QB/T1684-93的要求。按照2007年卫生部制定的化妆品卫生规范进行皮肤变态反应试验，涂抹大鲵低聚糖肽化妆品的豚鼠皮肤没有发生任何的变态反应，30例人体受试者没有发生任何不良反应。更多还原

【Abstract】 Giant salamander （Andrias davidianus） belongs to the class of Amphibia, order ofCaudata, family of Cryptobranchidae. It is a rare precious animal protected as Chinesenational second grade animal, mainly distributed in the upstream of Yangtze River,Zhujiang River, and Hanshui River. A large number of the domestication and breedingenterprises have been set up since1990s in Hunan, Hubei, Shanxi and Guangxi provinces.There are about35taming and breeding enterprises in Hunan province and about40,000-50,000giant salamanders are cultured every year. Deep processing and utilization ofgiant salamander need to be developed immediately. Therefore, fast creation of thetechnology for deep processing and utilization of giant salamander is an urgent task.Giant salamander is that there are many skin glands, instead of scales, on the bodysurface. These skin glands secrete a lot of mucus during the process of growth. If the mucuscan be used, sustainable utilization of giant salamander resources can be realized. Use ofmucus secreted by the skin glands of giant salamanders will exert no injuries to their growthand reproduction.As a species existed for350million years, Giant salamander has remarkablecharacteristics of longevity and self-repairs with a unique non-specific immune systempresented as in their mucus. Materials with biological activities for environmental changesare, after long-term evolution, contained in the mucus of giant salamander, which render themucus potential utilization in food industry.In this study, giant salamander glycopeptides （GSGPs） were obtained by using theAspergillus sp. acid protease from the marine organism to hydrolyze the mucus.. Thepreparation process, physical and chemical properties, biological activities of GSGPs andtheir application in juice and cosmetics are studied. The results are of theoretical andpractical significance for the processing and utilization of this mucus for the benefits ofhuman beings. Results are summerized as follows:1. The optimum conditions of marine-derived Aspergillus acid protease forhydrolyzing giant salamander skin mucus are obtained by the orthogonal test. The effectorder of hydrolysis is: hydrolysis time> E/S> temperature>pH. The optimum conditionsare:55C，pH:2.0，E/S:0.03%,，hydrolysis time:3h. Eutectic point, frozen-thaw processand freeze concentration process of the giant salamander hydrolysates are studied, and some of measures are obtained to improve the efficiency of freeze-drying of giant salamanderhydrolysates. Before freeze-drying operation, the giant salamander hydrolysates ispre-freezed in temperature range-8.6to-13.6C, meanwhile the solution concentration isimproved by the approaches of frozen-thaw and freeze concentration.2. The hydrolysates of giant salamander mucus （GSGPs） detected byMALDI-TOF-MS. Total protein and carbohydrate contents of GSGPs were estimated to be80.01%and15.15%, respectively. GSGPs contain3.39%glucosamine,0.65%glucuronicacid,2.45%galacturonic acid and0.60%sialic acid. While the sugar contents are22.38%、16.17%、4.29%and3.69%in total sugar respectively. Sulfuric acid sugar has not beendetected in GSGPs.18amino acids have been determined in GSGPs. In addition totryptophan not be detected, the other8essential amino acids （EAA） for human being arehigher. Amino acid of GSGPs was rich in Thr （13.1%）, Pro, Ala, Leu, Arg and Phe.The O-glycosidic linkage of GSGPs is demonstrated by β-elimination reaction andblood cell agglutination test.GSGPs are performed peptide sequencing by nano-electrospray ionization massspectrometry/mass spectrometry（nano-ESI-MS/MS）．The database search is finished withthe Mascot search engine （http://www．matrixscience．co．uk） using the data processedthrough MasSeq． Amino acid sequences of3peptides are obtained as follows:KAPILSDSSCKSC, KLQGTVSWGSGCQAKNC and VVHSLVQVTANKVMVRM,respectively.3. The hydroxyl radical, DPPH free radical and superoxide anion radicals scavengingactivities are detected in GSGPs. The free radical scavenging capacity is graduallyenhanced with GSGPs concentration increasing. GSGPs at the concentration of1.0mg/mlexhibited54.69%scavenging activity against hydroxyl radical,92.25%against DPPHradical and52%against superoxide radical.GSGPs have showed an ACE inhibitory activity by HPLC. An ACE inhibitory activityratio attains90.82%at GSGPs concentration of40mg/mL.4. The impacts of different doses of GSGPs on immune functions of mice indicate that:the mice were divided into three groups （0.01g/kg bw,0.05g/kg bw,0.1g/kg bw）compared with control group （0g/kg bw） according to the body weight, and fed withGSGPs at three levels for8d, respectively. GSGPs at three levels obviously improved theamounts of antibody, enhanced the phagocytic capacity of macrophages and increased the amounts of positive ANAE cell compared with the control. Therefore, the immunologicalfunction of mice is improved by GSGPs.GSGPs were injected into the stomach of the mice once a day for15consecutive days.The mice are divided into2groups according to different GSGPs concentrations （lowconcentration of100mg/mL, high concentration of150mg/mL）. Each group daily givendose is0.1mL/16g bw compared with control group with the same volume of saline bygavage. After intragastric administration for15d，the mice were forced swimming to deathwith5%body weight loaded. The results show that compared with control group，theswimming time prolonged significantly, up to103min （P <0.01）; the concentration ofhepatic glycogen increased100mg/mL, by30.3%up to; the level of lactic acid in musclereduced43.2mg/mL, by39.2%; the level of urea reduced26mg/mL, by68.4%.Kunming male mice are divided into6groups: blank control group, model controlgroup, DDB control group [200mg/（kg d）], low GSGPs concentration [200mg/（kg d）],middle GSGPs concentration [400mg/（kg d）] and high GSGPs concentration [800mg/（kg d）]. GSGPs are given to mice once a day for7consecutive days by gavage. Theprotection of mice liver damaged by CCl₄has been detected. The results indicate that theactivity increases of AST and ALT caused by CCl₄are significantly inhibited by middleGSGPs concentration and high GSGPs concentration. In mice liver, the MDA content ofhigh GSGPs concentration has significantly been lowered, as well as SOD activityenhanced.Microscopic observations show that: in the CCl₄model group, liver cells are markedlyswelled and deformed, liver cell cords inordinate and accompanied by the inflammatory cellinfiltration. At low, middle and high GSGPs concentration，liver cell cords are arranged inorder, liver cells are regular arranged, and the damage is significantly reduced comparedwith the model group.5. The function of the glycopeptides in resisting the ultraviolet （UV） rays was strongerunder the wavelength from280to315nm. UV damaged test in mice demonstrates that themouse ear index of smearing GSGPs group is similar with the index of smearing ediblevegetable oil group without UV irradiation reveals smearing GSGPs on the skin of mouseear may avoid UV injury.6. Caco-2cell absorption test reveals that GSGPs can be absorbed by Caco-2cell.GSGPs can be absorbed by HaCaT cell; meanwhile GSGPs have the ability of thegrowth-promoting for HaCaT cell. MTT colorimetric assay indicates that compared with control group, the livability ratio of HaCaT cell cultured for72h in the GSGPs of0.098mg/mL is145%.7. As indicators of pH, precipitation rate, sensory score, and solid content, throughsingle factor and orthogonal experiments the optimum production technology conditions ofhealth beverage containing GSGPs are: the ratio of blackcurrant juice and blueberry juice1:3（15mL:45mL）, the ratio of GSGPs and oyster polysaccharide1:3（1.5mL:4.5mL），lemonjuice10mL, pectin0.1%, sterilization temperature95C，sterilization time10min. Theproduct manufactured by the optimum technology has better color, flavor and stability.The cream with GSGPs has capability of hygroscopicity under the low humiditycondition（RH=43%）, which is3.8%; under the high humidity condition（RH=81%） thehygroscopicity rate is15.6%. The stability of the cream made of GSGPs meets therequirements of Chinese Industry Standard QB/T1684-93. There is no allergic reactionappeared on the skin of guinea pigs smeared by GSGPs cream, similar with the skin ofhuman subjects.The work is supported by National Natural Science Foundation Project （31071612）、Hunan Provincial Science and Technology Plan Project （2011FJ4098） and Science andTechnology Plan Project of Zhangjiajie （2011ZD16）.更多还原