【作者】 汪敬科；

【导师】 赵新淮；

【作者基本信息】 东北农业大学 ， 食品科学， 2011， 硕士

【摘要】 高血压是引发心血管疾病如心力衰竭、中风、冠心病和心肌梗塞的重要危险因素。高血压在发病前是没有任何征兆的,所以它十分危险。据统计,全世界每年因高血压引发的心脑血管疾病而死亡的人数超过1200万,高血压已经成为人类健康的第一杀手,成为全球性的重大公共卫生问题。近年来的研究结果表明,酪蛋白除具有一定的营养功能之外还具有重要的生理功能,是生物活性肽的重要来源。其中食物蛋白源ACE抑制肽安全性高、副作用小、易吸收,已经成为活性肽领域研究热点,这些ACE抑制肽在开发具有降血压功能的食品中具有广阔的应用前景。本研究在添加外源性氨基酸的情况下对酪蛋白水解物进行类蛋白反应修饰,以期得到高活性的肽。利用Alcalase 2.4L FG蛋白酶水解酪蛋白,制备生物活性肽。酪蛋白水解物的ACE抑制活性最高值出现的水解时间为6h,其相应的水解度为12.4%、IC₅₀值为42.2μg/mL、DPPH自由基清除率为41.8%。酪蛋白水解物的ACE抑制活性和水解度之间有一定的关系:当酪蛋白水解物的水解度较低时,酪蛋白中具有ACE抑制活性的片段未被充分释放出来,导致酪蛋白水解物ACE抑制活性低;当酪蛋白水解物的水解度过高时,酪蛋白ACE抑制肽进一步酶解,从而失去它们的ACE抑制性。利用中心组合设计和响应面分析法,优化类蛋白反应条件。固定酪蛋白水解物的浓度为35%（m/m）,反应时间为6 h,以反应体系中游离氨基减少量为响应值,考察氨基酸添加量、酶添加量、反应温度及3种外源性氨基酸对类蛋白反应程度的影响。研究发现:（1）氨基酸添加量、反应温度、氨基酸种类对类蛋白反应的影响显著,而酶添加量对类蛋白反应的影响不大。（2）在分别添加亮氨酸、苯丙氨酸、缬氨酸后,同样研究反应条件对酪蛋白水解物进行类蛋白反应的影响,发现所得到研究结果是相似的。得到在3种外源性氨基酸添加下酪蛋白水解物类蛋白反应的最适反应条件为:氨基酸添加量0.6 mol/mol、酶添加量3 kU/g蛋白、反应温度40℃;（3）对类蛋白反应影响的最主要因素为氨基酸添加量、反应温度。分别添加苯丙氨酸、亮氨酸、缬氨酸于酪蛋白水解物进行类蛋白反应,制备3个酪蛋白水解物的修饰产物,并测定它们的ACE抑制活性、IC₅₀值和抗氧化活性。结果表明,酪蛋白水解物的ACE抑制活性随修饰程度的增加而提高,并且都高于未经修饰的酪蛋白水解物,其IC₅₀降低至21.0²5.1μg/mL,表明添加外源氨基酸可提高修饰产物的ACE抑制活性,但添加不同种氨基酸的影响不显著。类蛋白反应修饰后,产物的抗氧化活性明显提高,且远高于酪蛋白水解物。添加不同种外源性氨基酸对抗氧化活性的影响不同:添加苯丙氨酸的修饰产物的DPPH和还原能力最高,添加亮氨酸的修饰产物羟自由基清除活性最高。更多还原

【Abstract】 Hypertension is a serious risk factor for cardiovascular disease such as heart failure, stroke, coronary heart disease and myocardial infarction. It is especially dangerous because it often has no warning signs or symptoms. It was reported that more than 12000 thousands of peoples died from the cardio- and cerebro-vascular diseases resulted from hypertension every year in the world. Hypertension has now become the first killer to people’s health and is the great public problem. Recently, many researches demonstrated that casein is not only sources of nutrients, but may also be ones of biologieally active peptides, among which ACE-inhibitory peptides have received the most attention for its safe and no side effects nature in the therapy of hypertension. These pepetides show great promise in the development of a novel physiologically functional food for preventing hypertension as well as for therapeutic purposes. In order to prepare bioactivie peptides with high activity from milk proteins, casein hydrolysates were modified by plastein reaction with Alcalase in the presence of extrinsic amino acids in the present work.The bioactive peptides were prepared by hydrolyzing casein with Alcalase, an alkaline protease from Alcalase 2.4L FG. Casein hydrolysates were prepared by incubating casein with Alcalase for 6 h, and exhibited the highest ACE-inhibitory activity (IC₅₀=42.2μg/mL) with a DH 12.4% and a DPPH radical scavenging activity of 41.8%. The ACE-inhibitory activity of the casein hydrolysates prepapred changed as the DH of casein hydrolysates increased during hydorlysis. When the DH of casein hydrolysates was too low, the hydrolysates only had little ACE-inhibitory activity because the ACE inhibitory peptides were not fully released from the casein. When the DH of casein hydrolysates was too high, the ACE-inhibitory peptides generated were destroyed by protease, which made them lost their ability to inhibit the ACE.Four reaction conditions including addition level of amino acids, enzyme addition, reaction temperature and the types of amino acids for the Plastein reaction of the casein hydrolysates were optimized by a central composite design and response surface methodology analysis with the decrease of free amino groups in the reaction mixture as the response. When the concentration of casein hydrolysates and reaction time were fixed at 35% （w/w） and 6 h, the practical results indicated that the addition level of amino acids, reaction temperature and the types of amino acid added had significant impacts on the plastein reaction of the casein hydrolysates, while the addition level of Alcalase only had little influence. The effects of these reaction condictions on the Plastein reaction of the casein hydrolysates behaved similar profiles when added amino acid was phenylalanine, leucine or valine, respectively. The optimal conditions were thus selected to be an addition level of amino acid of 0.6 mol/mol free amino groups （hydrolysates）, enzyme addition of 3 kU/g proteins and reaction temperature of 40℃. The factors impacted Plastein reaction of the casein hydrolysates significantly was in the order of addition level of amino acids and reaction temperature.Three modified casein hydrolysates were prepared with the selected suitable conditions in the presence of phenylalanine, leucine and valine, respectively. The analysis results indicated that the ACE-inhibitory or antioxidant activities of the modified casein hydrolysates increased as the extent of modification increased. The IC₅₀ values of the modified hydrolysates were in range of 21.0 to 25.1μg/mL, which indicates that the ACE-inhibitory activities of the modified hydrolysates were improved by the Plastein reaction and addition of amino acids. The types of amino acids were not important to the activity of the modified hydrolysates. Antioxidant activity of the modified casein hydrolysates was also significantly improved. The antioxidant activity of the modified casein hydrolysates was different when different extrinsic amino acids were added to the casein hydrolysates. DPPH radical scavenging and reducing activity of the modified casein hydrolysates had the highest value when phenylalanine was added to the casein hydrolysates, while hydroxyl radical scavenging of the modified casein hydrolysates had the highest value when leucine was added to the casein hydrolysates.更多还原