【作者】 许金光；

【导师】 刘长江；

【作者基本信息】 沈阳农业大学 ， 食品科学， 2012， 博士

【摘要】 东北野生软枣猕猴桃是一种珍稀的野果资源，软枣猕猴桃（Actinidia arguta Sieb.etZucc）为猕猴桃科猕猴桃属，又名软枣子、藤枣、藤瓜和猕猴梨。分布在我国的华北、东北、西北及长江流域，其中东北南部长白山脉一带山区较多。软枣猕猴桃的根、茎、叶及果实均可以入药。根味淡、微涩、有健胃、清热、利湿等功能,茎可以做手杖及烟袋杆,叶中含有大量叶绿素、胡萝卜素、钾、钠等物质，其果实含有SOD、氨基酸、糖类等功能活性成分，具有生津润肺、滋补强身等作用，对高血脂、高血压、心绞痛等有一定的疗效。多肽是目前研究的一个热点，目前，国内外对软枣猕猴桃果实的系统研究尚处于起步阶段，见诸报道的仅有果实中的SOD、氨基酸、酶的研究，尚无对其中的蛋白及多肽进行系统研究。本研究为东北野生软枣猕猴桃的综合开发利用奠定理论基础，具有重要的科研价值。本文对软枣猕猴桃多肽的制备、纯化及生物活性进行了系统研究，以提取率为考察指标，以碱法为提取方法，通过响应面分析确定了总蛋白的最佳提取条件；以蛋白质水解度为指标，以响应面法为手段，确定了酶法制备软枣猕猴桃多肽的最佳条件。接着，采用了超滤、Sephadex G-25凝胶色谱、DEAE-32离子交换层析及RP-HPLC方法对软枣猕猴桃多肽进行了分离纯化，通过体内、体外试验综合评价软枣猕猴桃多肽的抗氧化能力；并用醋酸纤维素薄膜电泳鉴定了纯度，对其稳定性进行了研究。在试验中，通过抑菌谱的测定，评价了软枣猕猴桃多肽的抑菌能力；通过淋巴细胞转化试验、半数溶血值（HC50）的测定、吞噬指数、免疫器官指数和小鼠足趾肿胀度的测定研究软枣猕猴桃多肽的免疫增强功能。以多孔淀粉为芯材，分别采用明胶和β-环状糊精对软枣猕猴桃多肽进行包埋，研究了超声时间、超声温度、超声功率对包埋效果的影响，并模拟了微胶囊在体内环境下的释放。在对软枣猕猴桃多肽的安全性评价上，采用了急性毒理试验、Ames试验、小鼠骨髓嗜多染红细胞微核试验、小鼠睾丸精母细胞染色体畸变试验、彗星试验等方法，明确了软枣猕猴桃多肽的安全性。本文主要研究结论如下：1.软枣猕猴桃中鲜果中粗蛋白含为1.25%（鲜果）,干果的含量为7.5%，碱法提取软枣猕猴桃总蛋白最佳条件为：料液比为1：19，提取液pH为7.9，温度50℃，提取时间为2.3h，蛋白质的提取率可达53.23%。2、以胰蛋白酶、木瓜蛋白酶、中性蛋白酶为筛选范围，以水解度为考察指标，对软枣猕猴桃蛋白进行酶解，结果表明胰蛋白酶的水解效果优于其他两种酶，通过响应面法进行优化，最佳酶解工艺理论条件为：pH7.14，酶活添加量为7500u/g料，温度56.43℃，底物浓度1.86%，在此条件下对软枣猕猴桃蛋白进行水解，水解度为23.32%，多肽含量为60.36，经氨基酸自动分析仪分析，酶解物中含有17种氨基酸，其中含有7种人体必需氨基酸。3.对软枣猕猴桃多肽进行超滤，研究了较优的条件为：操作压力0.06Mpa,温度30℃，流速2.25ml/s,浓缩倍数2倍，操作时间4min。以10K Da为界点，将软枣猕猴桃多肽分成两个组分，10KDa以下的为组分Ⅰ，10KDa以上的为组分Ⅱ，结果表明两个组分均具有抗氧化性，且抗氧化性由于Vc,但组分Ⅰ的抗氧化性较好。采用Sephadex G-25对组分Ⅰ进行层析，获得3个分离峰，其中第3个峰抗氧化活性最强；采用DEAE-32纤维素对第三个峰分离，获得4个峰，其中第4个峰的抗氧化活性最强；采用RT-HPLC对第4个峰进行分离，分理出两个主峰，其中第1个峰具有较高的抗氧化活性，对超氧阴离子和自由基的清除能力分别达76.1%和85.9%，对其进一步进行醋酸纤维薄膜试验，测得纯化后的多肽含量可达98.36%。4、抑菌试验表明，软枣猕猴桃多肽对细菌具有广谱抑菌效果，对酵母与霉菌未见明显抑制作用。5、免疫试验表明，软枣猕猴桃多肽能够延长小鼠负重游泳时间、增加小鼠肝糖原储备量、增强动物运动耐力、缓解动物体力疲劳的作用。6、稳定性试验表明，软枣猕猴桃多肽在pH6-8范围内较稳定，温度越高，稳定性越差。丙三醇、蔗糖、乳糖、甘露醇对软枣猕猴桃多肽的热变性有明显的保护作用，其中8%的丙三醇保护效果最好。在人工模拟胃液消化试验中，胃蛋白酶和胰蛋白酶均能降低软枣猕猴桃多肽的抗氧化活性，其中胃蛋白酶的作用更为明显。7、对软枣猕猴桃多肽进行微胶囊处理，结果表明，以多孔淀粉为芯材，β-环状糊精为壁材效果较好，其抗氧化性保持较好，且不易吸潮。8、安全性试验表明：软枣猕猴桃多肽小鼠急性毒性试验，LD50>20g/kg，属无毒级。骨髓细胞微核试验、Ames试验、睾丸精母细胞染色体畸变试验、彗星试验结果均为阴性，表明软枣猕猴桃多肽是一种无毒物质，初步证明软枣猕猴桃多肽作为保健食品的食用安全性。更多还原

【Abstract】 The northeast wild Actinidia arguta is an important wild fruit resource. Actinidia argutaSieb.et Zucc belongs to Actinidiaceae of actinidia. Actinidia arguta is named that cane jujube、cane malon and actinidia pear. This wild plant distributes in the North China, Northeast China,Northeast China and Yangtze river, and chiefly concentrated in the Changbai mountains in theNortheast China. Its root, stem, leaves and fruit are all useful for us in their medical value andhealthy function. The root has the effect on the Promotingdigestion，clear heat and diuresis,the leaves contains lots of chlorophyll, carotene, potassium, sodium and etc. the fruitscontains lots of SOD, amino acid, sugars and etc. meanwhile, the fruits has the effect on thehyperlipidemia, hypertension, and stenocardia.Polypeptides is the study of a hot spot, at present, The researchers have no furtherresearch and study in the actinidia arguta. We can only search the reports of fruit SOD,Amino acids, and Enzymes. This article offer the important value on the total exploitation ofactinidia arguta.In this paper, preparation, purification and biological activity of actinidia argutapolypeptide was systematic studied. The extraction yield index as index, the actinidia argutaprotein extraction was used by alkali method, the optimum extraction conditions weredetermined through the response surface analysis method. The degree of hydrolysis as index,the actinidia arguta polypeptides preparation process was used by enzyme method, theoptimum conditions were determined through the response surface analysis method.Then, separate and purify the polypeptides of actinidia arguta by using the ultrafiltration,Gel chromatography of Sephadex G-25, ion-exchange column chromatography of DEAE-32and RP-HPLC. Identify the purity by using the cellulose acetate membrance electrophoresis.The antioxidant capacity of actinidia arguta polypeptide was comprehensive evaluated by invivo and vitro test.in the next step, and then have researched the stability of actinidia arguta.The Restrain bacterial capacity of actinidia arguta was comprehensive evaluated bydetermine the Antibacterial spectrum.The function of immune enhancement of actinidiaarguta polypeptide were evaluated by lymphocyte transformation test, determination of halfhemolysis value, phagocytic index, immune organ index and toes degree of mice.Use the multihole starch as the core materials, we encase the polypeptides of actinidiaarguta individual by gelatin and β-cyclodextrine. In the Safety evaluation on actinidia arguta.we use the Acute toxicological method, Ames method, PCE method, comets method and soon.ensuring the safety of the polypeptides of actinidia arguta.This paper makes a study of the conclusions are as follows:1.The protein content is1.25%in the fresh fruit of actinidia arguta.in the dried fruits,thecontent is7.5%.The optimum extraction conditions of alkali method are as follows: solid-liquid ratio of1:19, pH7.9, temperature50℃, time2.3h, protein extraction rate is53.23%.2.Use the degree of hydrolysis as index, we enzymolysis the protein of actinidia argutaby trypsin，papain and Neutral protease, the result showed that trypsin is better than the other.the optimum enzymolysis conditions were determined through the response surface analysismethod, the results as follows: pH7.14, enzyme activity7500u/g material, temperature56.43℃, Substrate concentration1.86%, under these conditions for hydrolysis of actinidia argutaprotein, degree of hydrolysis is23.32%, peptide yield is60.36%. there is17kinds of aminoacid by the automatic analyzer analysis, Contains7kinds of human body essential aminoacids.3．Ultrafiltration were carried out to separate actinidia arguta protein hydrolysate,thebetter conditions are:pressure0.06Mpa, temperature30℃, rate of velocity2.25ml/s.Theboth compontes of polypeptides has function on antioxidant.But the less than10KDamolecular weight is stronger SephadexG-25column chromatography was used to separatelow molecular weight of actinidia arguta protein hydrolysate, three separate peaks were got,of which the third peak with the strongest antioxidant activity. DEAE-32cellulose columnwas used to separate the active component purified by the SephadexG-25, four separate peakswere got, of which the fourth peak with the strongest antioxidant activity. RT-HPLC was usedto separate the active component purified by the DEAE-32cellulose column, two peaks wereisolated, of which the first has higher antioxidant activity. After the series of purification, thecontent of peptide is98.36%.4. Bacteriostatic experiment shows that the polypeptides of actinidia arguta has thebroadly function on restrain the activity of bacteria,but except the yeast and mold.5. The immune experiment shows that polypeptides of actinidia arguta can prolong theswimming time of weighted mice, increase the content of glycogen, enhance the enduranceof mice and easing the fatigue of mice.6. The Stability experiments showed that the polypeptides of actinida arguta keep thestability at pH6-8, The higher of the temperature,the more stability. The glycerin, sucrose,lactose, mannitol all can enhance the activity of stability.of which the glycerin is better. In theexperiments of Simulation gastric juice to digest, both the pepsin and trypsin can reduce theactivity of antioxidant.7. The Microcapsule experiments shows: the better process is Use the multihole starch asthe core materials and use the β-cyclodextrine as the material of wall.The capsule maintainedthe activity of antioxidant and keep dried.8. Toxicity test show that actinidia arguta peptide in mice acute toxicity test, LD50>20g/kg, is non-toxic class. Results of mouse bone marrow micronucleus test, Ames test, thechromosome aberration test of mouse primary spermatocyte and comet assay were negative. Actinidia arguta peptide is a non-toxic material. The present studies thus provide preliminaryproof of actinidia arguta peptide for the application of health food safety.更多还原