【作者】 胡筱波；

【导师】 吴谋成；

【作者基本信息】 华中农业大学 ， 农产品加工及贮藏工程， 2007， 博士

【摘要】 油菜是我国的主要油料作物，因而油菜蜂花粉是高产价廉的蜂花粉。油菜蜂花粉中蛋白质含量高达20％-25％，足以与大豆、豌豆相媲美，其氨基酸组成比例合适，必需氨基酸含量较高。利用酶法水解蛋白质制备油菜花粉肽，目前还未见报道。我们以油菜花粉为原料，首次系统研究了油菜花粉蛋白各组分，对谷蛋白进行酶解，得到油菜花粉谷蛋白酶解肽（PRPG），并采用现代分子生物学技术研究了PRPG的抗肿瘤作用及其作用机制。主要结果如下：1油菜花粉蛋白组分的研究新油菜花粉与陈油菜花粉的各营养素含量相差不大，而且陈油菜花粉更有利于破壁；破壁脱脂后的油菜花粉蛋白质含量为22.42％，破壁脱脂后的油菜花粉中谷蛋白和清蛋白为油菜蜂花粉中主要的蛋白组成，分别占总蛋白的55.7％和39.0％，其它依次为球蛋白（3.2％）和醇溶蛋白（2.1％）。2油菜花粉蛋白的营养价值评价油菜花粉蛋白的理化性质研究表明，清蛋白具有较好的吸水性、吸油性以及良好的乳化性和乳化稳定性等，可广泛应用于食品、饮料中。在清除·OH试验中，清蛋白对羟基自由基的抑制率为54.35％，而碱溶谷蛋白只有25.08％。氨基酸检测表明花粉蛋白氨基酸组成独特，平衡良好，必需氨基酸含量高。由氨基酸比值系数法（SRC）分析结果表明，四种蛋白中碱溶谷蛋白的SRC最高，故其营养价值最好，但由于它在人体胃酸性环境下生物利用率很低，所以我们以谷蛋白为后续实验材料，经酶法改性后，不仅生物利用率提高，且能使它的生物活性提高。通过单因素试验和正交试验分析确定了油菜花粉谷蛋白的最佳提取条件为：料液比为1∶20、温度70℃、pH11、浸提时间75分钟、氢氧化钠浓度0.3％。3油菜花粉谷蛋白酶解肽的制备以水解度为指标，选择碱性蛋白酶（alcalase）来水解制备花粉谷蛋白酶解物。通过对水解条件的系统研究发现，碱性蛋白酶（alcalase）能够有效地水解花粉谷蛋白，其最佳酶解条件确定为底物浓度6％，pH值9.0，水解温度50℃，酶底比1460U／g蛋白。在此条件下水解2h，酶解液水解度可达24.5％左右。以盐水作为洗脱液，用葡聚糖凝胶Sephadex G-25层析分离PRPG，获得三个级分（P1、P2和P3）。经过层析分级后，总回收率为60.21％，三个级分在总量中所占比例依次为15.7％、70.2％、14.1％。酶解肽粗品及经分离后得到的各组分在2mg／mL时对羟基自由基均有较好的清除作用，但只有含量最低的P3对羟基的清除能力高于同等浓度下的酶解肽粗品，P1和P2都没有酶解肽粗品的抗氧化能力强，P1甚至低于相同浓度时的清蛋白和球蛋白的活性。4油菜花粉谷蛋白酶解肽的抗氧化活性经体内体外试验，从不同层次水平系统研究了PRPG的抗氧化活性，对PRPG的抗氧化作用与机理进行了研究。测定了油菜花粉酶解物PRPG在化学模拟体系、小鼠肝线粒体、小鼠红细胞以及小鼠肝匀浆中的抗氧化作用。结果显示：在化学反应体系中，PRPG具有较强的还原能力以及清除·OH羟基自由基的能力。在体外实验中，PRPG能抑制小鼠肝线粒体膜的肿胀，抑制小鼠红细胞自氧化溶血，抑制小鼠红细胞、肝组织匀浆中MDA的生成。在体内实验中，灌胃PRPG组小鼠（150mg／kg·bw·d）与对照组相比，肝匀浆和红细胞中MDA的降低显著。以上实验表明，PRPG在体内外均具有明显的抗氧化作用，高的还原能力是PRPG抗氧化的机制。5油菜蜂花粉谷蛋白酶解肽的抗肿瘤活性研究通过体外抑瘤试验表明，不同浓度的油菜花粉蛋白的酶解产物对S180以及Hela肿瘤细胞都有一定的抑制作用。其中，终浓度为1mg／mL的抑瘤效果最明显，对S180以及Hela肿瘤细胞的抑瘤率分别为35.72％和31.70％。体内抑瘤实验中，PRPG对S₁₈₀肿瘤细胞有较强的抑制作用，当灌胃剂量为100mg／Kg·d时，抑瘤效果较好，抑瘤率达45.44％。PRPG对荷瘤小鼠有较好的免疫增强作用，它可以提高荷瘤小鼠免疫器官的重量，增强荷瘤鼠巨噬细胞吞噬能力、脾淋巴细胞转化能力、NK细胞杀伤力、DNFB诱导的迟发性超敏反应，还可显著增强荷瘤鼠脾细胞抗体生成能力，表明PRPG对荷瘤鼠的特异性和非特异性免疫功能均有明显增强作用。PRPG能显著提高荷瘤鼠体内抗氧化酶SOD、GSH-Px的活性，降低荷瘤小鼠血清LDH活性，减少脂质过氧化产物MDA的含量，揭示PRPG可提高机体抗氧化能力而发挥抗肿瘤作用。小鼠接种S180细胞后对其外周血象有明显影响，白细胞明显增多，红细胞、血红蛋白数降低。经灌服PRPG后可使外周血象基本恢复至正常状况。PRPG与环磷酰胺合用有一定协同作用，PRPG（50mg／kg·d）可使环磷酰胺的抑瘤率提高至55.06％，且对环磷酰胺的毒副作用有一定的拮抗作用，可在一定程度上缓解环磷酰胺对荷瘤鼠机体免疫器官的伤害。采用动物抑制性肿瘤法，以环磷酰胺为对照，观察PRPG对荷瘤鼠肿瘤、肝、肾、脾组织形态的影响，结果表明PRPG有较好的抗肿瘤效果，对荷瘤鼠肝、肾等组织无明显毒副作用，且能缓解环磷酰胺对机体组织器官的伤害。6 PRPG对荷瘤鼠脾细胞因子分泌及其mRNA表达的影响采用双抗夹心ELISA法检测结果表明PRPG可明显促进荷瘤鼠脾细胞TNF-a、IL-2和IL-6的分泌，逆转录-聚合酶链反应检测结果表明PRPG可显著提高TNF-a和IL-6 mRNA的表达，且在一定浓度范围内存在较好的浓度依赖关系，说明PRPG可通过提高荷瘤鼠细胞因子mRMA表达来达到抗肿瘤的目的。更多还原

【Abstract】 Rapeseed is one of the most important oilseed crops cultivated in the world, thus rape pollen is the cheap high-yielding pollen. However, because the rape pollen has a special unpleasant flavor that can not be accepted by young people, it is not popular to consumers directly as a commodity for sale, leading to large bulks of storage. Protein content is as high as 20%-25% in Rape pollen, which is comparable to soybeans and peas, suitable proportion of its amino acid composition, with higher contents of essential amino acids, much peptides of higher quality physiological activity were expected to be obtained after the enzymatic modification. But no disquisition on preparation of rape pollen peptides with the proteinase has been reported as yet. Cell-fragmentated and defatted rape pollen was used as material in this study. A research on preparing rape pollen protein fractionation and part of their functional properties were performed for the first time. Based on this, rape pollen gluntelins was hydrolyzed with alcalase and the peptides of rape pollen gluntelins（PRPG） were obtained. The antitumor activity and mechanisms of PRPG were also studied here. The main results were shown as follows:1 Classification of rape pollen protein isolateAll the nutrient contents of fresh rape pollen is not much different from the stale one, and the stale rape pollen is much more conducive to breaking cell. The protein contents in rape pollen account for 22.4% after being defatted and cell-fragmentated. The results showed that glutelins and albumins were found to be the predominant proteins in rape pollen, comprising 55.7% and 39.0% of total proteins, While globulins and prolamins were 3.2% and 2.1% respectively.2 Nutritive value evaluation of pollen protein isolatesThe physico-chemical properties of pollen protein were investigated, including water absorption, oil absorption, emulsifying activity and emulsion stability. Albumins have better function, including water absorption, oil absorption, emulsifying activity and emulsion stability. So albumins can be widely applied in the food and beverage industry. The scavenging hydroxyl radical capability of pollen protein was studied, the inhibition rate .OH of glutelins was and of albumins was 54.35%. Amino acid profile of the isolates indicated that the total essential amino acid of each protein fraction was high. Amino acid ratio coefficient （SRC） is to evaluate the protein quality by dispersion that all essential amino acids diverge from the amino acid model, SRC is highly correlated with the biological value and also is closer to biological value in number. It also showed that the SRC of glutelins was the highest, therefore its nutritional value is the best. Glutelins in human gastric acid environment was of very low bioavailability, therefore, Glutelins was used as the experimental material for the follow-up experiments, and then enzymatic modification not only could increase its bioavailability, but also increase its biological activities.Through orthogonal test, the optimum conditions of extraction of glutelins were obtained on the basis of single factor test, as follow: ratio of solid-liquid 1:20, NaOH concentration 0.30%, pH value 11, time 75min, temperature 70℃.3 Preparation of peptides of rape pollen glutelinsDegree （DH） were used as response values in analysis of response surface regression （RSREG）, the optimum conditions of alcalase enzymatic hydrolysis have been determined by mono-factor analysis and response surface methodology as follows: pH=9, hydrolyzing temperature 50℃, enzyme concentration 1460U per gram of substrate, concentration of substrate 6%, hydrolyzing time 2h. And now DH could reach 24.5%.PRPG was graded by Sephadex G-25 column with salt solution as eluant and fractions were pooled into three major groups（P1, P2 and P3）. The overall recovery rate was 60.21% and the proportion of P1、P2、P3 were 15.7%、70.2%、14.1%. The antioxidant capability of the groups was different. Crude enzymolysis peptides（2mg/mL） and its three separate components （2mg/mL） all could scavenge hydroxyl free radicals effectively, and P3 which was 14.1% can scavenge hydroxyl free radicals more effectively than at the same concentration of the crude peptides. P1 even have lower antioxidant activity than the same concentration of albumins and globulins.4. Antioxidant activities of PRPGThe antioxidant effects of PRPG were studied in some modified chemical systems, with mice liver mitochondria and mice red blood cell （RBC）, mice serum and liver homogenate in vitro and mice in vivo. The deoxidation was measured by K₃[Fe（CN）₆]system and the content of malondialdehyde（MDA） was analysed by the reagent kits. The results showed that PRPG had higher reductive activity and could scavenge hydroxyl radicals in some modified chemical systems. In vitro, PRPG could inhibit the swelling of mice liver mitochondria and the hemolysis of mice RBC induced by H₂O₂, also, could heighten the antioxidant capability of mice serum, and inhibit the formation of malondialdehyde（MDA） in mice liver mitochondria and mice liver homogenate. In vivo, there was a significant difference in MDA value of mice liver homogenate between the control and the groups treated with PRPG（150mg/kg·bw·d）. All the above indicated that PRPG had evident antioxidant function.5. Study on the antitumor activity of PRPGThe results showed PRPG of different concentration could inhibit the growth of S180 and Hela cell in vitro, and the significant result appears from the eventually concentration of 1mg/mL, of which the inhibition rate on S180 and Hela is 35.72% and 34.70%.The results in vivo showed that PRPG could significantly inhibit the growth of S180 cell, the inhibition rate of 100 （mg/kg.d） dosage group was respectively 45.44%. PRPG could significantly increase the weight of immune organ, phagocytic function of monocytes, T lymphocyte transformation test, and NK cell activity, restore DTH response and antibody product, which showed that PRPG could increase both specific and non-specific immune function in tumor-bearing mice.The antioxidant capacity of mice greatly decline after they were inoculated with S180 cell. PRPG could remarkably increased SOD and GSH-Px activity and MDA content in serum, which indicated that PRPG could enhance its antioxidant capacity in tumor-bearing mice.Peripheral blood of tumor-bearing mice were greatly abnormal. white blood greatly increased, and red blood cell and hemoglobin famously deceased. PRPG could importantly make them regained.Cy had notable damage on immune organ. PRPG used in combination with Cy could display synergism. It could make antitumor activity of Cy raised to 55.06%%, and reduce toxicity of Cy on immune organ.The effect of PRPG on histomorphology of tumor, liver, kidney and spleen were tested through transplantable animal tumor, compared with Cy. The results of histomorphology showed the PRPG had no obvious toxicity on liver and kidney and spleen in tumor-bearing mice, and could protect them. The result also expressed that PRPG used in combination with Cy could display synergism and reduced Cy toxicity on immune organ. 6 Effect of PRPG on production and mRNA expression of cytokines in spleen cells of tumor-bearing miceThe results of two-antibody-sandwich ELISA indicated that PRPG could remarkably improve the production of TNF-a, IL-2 and IL-6 in spleen cells of tumor-bearing mice, and the results of reverse transcription-polymerase chain reaction showed that PRPG could great raised mRNA expression of TNF-a and IL-6, and induced dose-dependence, which announced that the function of anti-tumor of PRPG was due to enhance cytokine mMRA expression.更多还原