【作者】 李鸿梅；

【导师】 张学忠；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 分别以碳酸氢钠脱脂后的玉米蛋白粉和乙醇浸提的玉米醇溶蛋白为原料,以一种工业用碱性蛋白酶为催化剂,制备了主要分子量为400-600道尔顿、富含谷氨酸、丙氨酸和亮氨酸的CPS和ZPS两种玉米肽产品。利用Sephedax G-15、G-10葡聚糖凝胶层析法和HPLC法对CPS和ZPS进行了分离纯化,得到PCPS、PZPS、HPCPS和HPZPS四种主要组分。检测了其中抗氧化能力最高的活性组分—PCPS和PZPS的分子量分布情况和氨基酸组成情况。以α-生育酚为阳性对照,研究了CPS、ZPS、PCPS、PZPS、HPCPS和HPZPS对DPPH自由基、超氧阴离子自由基、羟自由基的清除作用及对亚油酸氧化的抑制作用,证明玉米肽具有抗氧化性。通过对以上各组分还原力的测定进一步证明了玉米肽的还原剂性质。采用Vallee&Hoch法,通过离体实验和动物实验,研究了玉米肽对乙醇代谢的两种酶—AND和ALDH的影响。通过建立酒精氧化性肝损伤模型,证明了玉米肽对氧化性肝损伤的辅助保护作用,初步探讨了玉米肽迅速调节乙醇代谢的机理为:提高了AND和ALDH的活力及结合了乙醇代谢的中间产物—乙醛。同时研究了玉米肽抗疲劳功效和对乳腺癌细胞增殖的影响。在以上实验结果的基础上,对玉米功能肽的制备方法进行了放大实验研究,采用微滤和超滤技术对玉米肽粗制品进行了精制处理,利用正交设计方法研制了玉米肽饮料配方。通过功能性检验、卫生学检验、毒理学实验和理化性质分析,证明了玉米肽产品的功效性、功效成分的稳定性和食用安全性,为其规模化生产提供了理论依据。更多还原

【Abstract】 Free radicals are the dissociative molecules, atoms or ions with unpaired electron,having an active chemical property. They always attact on protein and lipid, subsequently form new other free radicals and lipid peroxide, which damage the organism. Under the normal physiological condition, free radicals are constantly produced and constantly scavenged by the antioxidants in the organism, occupying the state of dynamic equilibrium. Once the equilibrium is broken by the augment of oxygen free radicals, or the descent of antioxidant level and activity, the organism is then injuried. Therefore, it is necessary to supply the exogenous oxygen free radical scaverger to keep the equilibrium. Recently, more and more researchers pay close attention to natural exogenous antioxidants.In the present study, CPS (corn peptides) and ZPS (zein peptides) were prepared from defatted corn gluten meal and zein respectively by proteolysis with an alkaline protease. The activest members of CPS and ZPS, PCPS and PZPS were separated by Sephadex G15, G10 and HPLC column method. Their antioxidant abilities were confirmed by their scavenging effects of the 2,2-diphenyl-1- picrylhydrazyl (DPPH) radical, superoxide anion radical, hydroxyl radical and inhibition of linoleic acid autooxidation. Their reducing powers were also analyzed. The EC50 of PCPS and PZPS of scavenging DPPH were 1.05mg/ml and 1.04mg/ml. The EC50 of scavenging superoxide anion radical of them were 10.94mg/ml and 10.85mg/ml. The EC50 of scavenging hydroxyl radical of them were 0.73mg/ml and 0.73mg/ml. The results indicated that PCPS and PZPS possessed the similar antioxidant abilities.The molecular-mass distributions of CPS, ZPS, PCPS and PZPS were determined and compared by MS. The main molecular-mass distributions of them were from 400 to 600 Da. Their amino acid compositions were also analyzed by HPLC. Glutamic acid, leucine and alanine were rich in CPS, ZPS, PCPS and PZPS.8.38% fat in the corn gluten meal delayed the process of super-filtration and increased the cost during preparation of CPS. Sodium carbonate and sodium bicarbonate were chosen as the defatting reagent to avoid the high cost and hazard of organic solvent and supercritical CO2 extraction technology. The best defatting technology of sodium bicarbonate by orthogonal test was determined: 1g sifted(250μm)corn gluten meal powder was mixed with 12ml 80g/l sodium bicarbonate for 20min at 40℃, the defatting rate was 81.73%.At present, alcohol abuse and alcoholism are serious health and socioeconomic problems throughout the world. Drunkenness is a symptom that transfer function of central nerve system is broken induced by the increase of the concentration of ethanol in the plasma. Therefore, study on sobering medicament is very important.It is well known that the principal organ responsible for the elimination of ingested alcohol is the liver. Alcohol is metabolized by alcohol dehydrogenase (ADH) enzyme system-catalyzed oxidative processes. ADH, as the first enzyme in alcohol metabolism process, its levels and activities has very important effect on the metabolism of alcohol in the organism. Therefore, the accelerating effects of CPS, ZPS, PCPS and PZPS to ADH activities in vitro were determined in the present study when using glutathione (GSH) as the positive control by Vallee & Hoch. The results indicated that the accelerating effect of PCPS to ADH activities was super to GSH. The results also indicated that PCPS possessed good ability of increasing ADH activities in the presence of pyrazole, the inhibitor of ADH. The results of in vivo experiments also indicated that CPS possessed good ability of increasing ADH activitiesThe results of influence of CPS to aldehyde dehydrogenase (ALDH) activities in vitro did not proved CPS accelerating ALDH activities since PCPS reacted to aldehyde. But the results of in vivo experiments indicated that CPS possessed good ability of increasing ALDH activities. The data showed that PCPS accelerated activities of ALDH.Using Haiwangjinzun, a sobering medicament, as positive control, the protective effect of CPS against acute hepatic injuries induced by alcohol was verified in NH mice that were fed with different dosage of CPS for 30 days and given an acute dose of alcohol orally subsequently.As a result, CPS reduced both hepatic malondialdehyde and triacylglycerol levels along with enhanced hepatic GSH level relative to the control. Hepatic histological changes were also observed. The result indicates that CPS is capable of attenuating ethanol-induced hepatic injury. The possible cause was that CPS enhanced ADH and ALDH activities and consequently accelerared alcohol metabolism, subsequently protected liver.Many researchers reported anti-fatigue of CPS. therefore, Using ginsenoside as positive control, effect of alleviate fatigue of CPS was also determined in the present study. The data showed that CPS extended swimming time, increased level of liver glycogen, reduced levels of plasma urea and plasma lactates of NH mice significantly.Yamaguch et al reported that CPS can inhibit process of breast carcinoma, the influence of CPS to proliferation of breast human carcinoma cell line MCF-7 was also analyzed in the present study. The result indicated that CPS did not inhibit proliferation of breast human carcinoma cell line MCF-7. It was possible that CPS scavenged the free radicals, which participate in the course of cancerous treatment. Consequently, process of breast carcinoma was inhibited.Finally, the above preparation of active CPS was scaled up. The physical and chemical indicators of corn peptides were determined. The stability of functional component, peptide, was also proved, including antioxidant properties. The histological observation on toxicity of CPS indicated that CPS did not do harm to heart, liver, spleen, lung, kidney, stomach, duodenum, testicle and ovary. The formula of corn peptides beverage determined by orthogonal test was: 20g/l corn peptides,80g/l xylitol and 2g/l malic acid.All in all, the results obtained in the present study can lay academic and practical foundation for its application to pharmaceutical and food systems as a functional additive in the future.更多还原