【作者】 张伟；

【导师】 孙智达； 徐志宏；

【作者基本信息】 华中农业大学 ， 食品科学， 2007， 硕士

【摘要】 本文以花生粕为原料，运用二次回归正交旋转组合设计对花生蛋白的提取工艺进行了优化；以血管紧张素转化酶(Angiotensin converting enzyme，ACE)抑制率为指标，从六种商业蛋白酶中筛选出碱性蛋白酶进行酶解，制备高活性的ACE抑制肽；选用凝胶层析法进行纯化分离；用凝胶电泳研究了分子量的分布；使用高效液相和质谱联用对产物结构进行了初步分析；选用自发性高血压大鼠(Spontaneously hypertensive rat，SHR)进行了降血压的体内动物实验。研究结果如下：1．二次浸提对蛋白的提取率有显著的影响。采用碱提酸沉淀法，结合DPS软件分析，研究了温度、pH和料液比对蛋白质提取率的影响。结果表明：花生蛋白质提取条件较优的范围为：温度为52-55℃，pH为9.3-9.6，料液比为1：9.7-1：10.3，在相同的条件下，进行二次浸提，每次浸提2h。2．采用响应面法筛选出碱性蛋白酶(Alcalase)酶解花生蛋白制备ACE抑制肽，以ACE抑制率为响应值，研究了温度、底物浓度和酶与底物浓度比对ACE活性的影响。结果表明：生产ACE抑制肽的最优组合为温度53.7℃，底物浓度7.72％，酶与底物浓度比4.18％，pH8.0，水解时间为120min。3．经凝胶层析的方法对ACE抑制肽进行了分离和纯化，通过Tricine-SDS-PAGE电泳法分析了各组分的分子量的分布，结果表明：分子量小于1500D的小肽具有较高的ACE抑制率。4．对于高活性的ACE抑制肽通过高效液相和质谱分析表明：经凝胶柱分离的单一组分仍然是由分子量不同的许多肽组成的混合物，其分子量主要分布在300-700D。5．通过自发性高血压大鼠的急性降压实验表明：在给药后120min血压开始明显下降，在给药后120min、150min、180min、210min时间段的血压与给药前比较均有明显的降压效果，平均动脉血压分别下降了9.9％、12.2％、14.0％和13.1％；慢性降压实验表明：连续灌服ACE抑制肽后，高血压大鼠血压呈现下降趋势，其中给药后2h、第2d、5d、7d血压明显低于模型组。更多还原

【Abstract】 Extraction process of peanut protein from peanut dregs was optimized by means oforthogonally rotational combination design in this paper. Monitored by Angiotensin convertingenzyme (ACE) inhibitory rate, the alkali proteinase was selected from six commercialproteinases and used for enzymolysis to produce highly active ACE inhibitory peptides;the gel chromatography method was used for the separation and purification, the gelelectrophoresis was used to investigate the distribution of molecular weights, HPLC-MSwas used to preliminarily analyze the product structure, and SHR was selected to testhypotensive activity in animal in vivo. The experimental results were as follows:1. Twice digestion and extraction had a significant effect on protein extraction rate.The alkali treatment and acid precipitation method was adopted and the effects of itstemperature, pH and liquid and material ratio on the extraction rate of protein wereinvestigated in combination with the DPS software analysis. The results indicated that theoptimized range of peanut protein extraction conditions were temperature 32-35℃, pH9.3-9.6, material and liquid ratio 1: 9.7-1: 10.3, twice digestion was performed under thesame conditions with extraction time of 2h each time.2. Alcalase was selected for enzymolysis of peanut protein by the method of responsesurface to produce ACE inhibitory peptides. The effects of temperature, substrateconcentration and the ratio of enzyme and substrate concentration on ACE activity wereinvestigated by using the ACE inhibitory rates as the response value. The results indicatedthat the proper conditions were temperature 53.7℃, substrate concentration 7.72%, theratio of enzyme and substrate concentration 4.18%, pH8.0, and hydrolysis time 120min.3. ACE inhibitory peptides were separated and purified by the method of gelchromatography, and the distributions of molecular weights of different components wereanalyzed by Tricine-SDS-PAGE electrophoresis. The results indicated that the short chainpeptides with the molecular weights of less than 1500D had higher inhibitory rate onACE.4. The highly active ACE inhibitory peptides were analyzed by HPLC-MS and theresults indicated that the single component separated from gel column was the mixture ofpeptides of different molecular weights, and its molecular weights were mainlydistributed in the range of 300-700D.5. The result of acute antihypertensive experiments with spontaneously hypertensiverats (SHR) indicated that blood pressures began to decrease significantly 120min afteroral administration. Compared with the blood pressure before oral administration, theblood pressures 120min, 150min, 180min, and 210min after oral administration were decreased significantly, with the mean arterial pressure (MAP) reduction of 9.9%, 12.2%,14.0% and 13.1%, respectively; and chronic antihypertensive experiments with SHRindicated that the blood pressure of SHR tended to decrease after consecutive oraladministrations, and the blood pressures 2h after oral administration, on day 2, day 5, andday 7 were significantly lower than that of the model group.更多还原