【作者】 陈亚房；

【导师】 石波；

【作者基本信息】 中国农业科学院 ， 农产品加工及贮藏工程， 2009， 硕士

【摘要】 大豆分离蛋白(SPI)作为一种植物源蛋白质,具有很高的营养价值,通过蛋白酶酶解SPI后得到的大豆低聚肽作为一种去抗营养因子、并具有一定生理活性的功能性食品基料,具有广泛的应用空间。利用低聚肽的特殊配体特征,开发新的微量元素添加剂,是增加大豆低聚肽应用范围的有益探索。本文在考虑大豆低聚肽利用方式的前提下,对利用木瓜蛋白酶和菠萝蛋白酶酶解大豆分离蛋白制备的大豆低聚肽的成分、酶解工艺优化条件、酶解产物体外ACE(血管紧张素转化酶)抑制活性组分筛选及其微量元素螯合物的制备工艺进行了研究,得到的具体结论如下:酶解产物SDS-PAGE、HPLC和质谱分析结果表明两种酶的酶解产物分子量为1000Da左右,多数为低聚肽。酶解反应初始阶段酶浓度、底物浓度、酶解反应温度对SPI水解率影响的研究表明,在反应初始阶段存在着产物和底物对反应速率的抑制现象,此外反应温度对反应初始阶段速率有一定的提高效果。木瓜蛋白酶酶解SPI的Km值为0.1404g/L,Vmax为7.51 g·L-1·min-1,菠萝蛋白酶酶解SPI的Km值为0.1173g/L,Vmax为4.95 g·L-1·min-1。通过对影响SPI酶解产物DH的主要因素:酶解时间、酶解温度、加酶量、pH、底物浓度的单因素和双因素实验,得出木瓜蛋白酶酶解SPI的最佳工艺为:酶解时间3h、酶解温度60℃、加酶量0.35g、pH7.5、底物浓度5g/100mL;菠萝蛋白酶酶解SPI的最佳工艺为:酶解时间10h、酶解温度40℃、加酶量0.35g、pH7.5、底物浓度5g/100mL。对影响大豆低聚肽ACE活性相关因素的研究,通过研究得出木瓜蛋白酶酶解SPI的时间基本不影响低聚肽ACE抑制活性,但酶解72h以后的产物抑制活性最强;菠萝蛋白酶酶解SPI制备的大豆低聚肽抑制ACE活性随时间增加而减小。而底物中的小分子糖类会降低ACE抑制活性,此外通过研究得出木瓜蛋白酶和菠萝蛋白酶酶解制备的大豆低聚肽的IC50约在70μg/mL左右。通过HPLC和SphadexG-25对大豆低聚肽的分离和抑制活性的研究,得到高ACE抑制活性抑制组分,并对其进行了初步的分离纯化,得到出峰时间为29.527min的高活性组分。通过研究影响大豆低聚肽金属离子螯合物的制备因素,得出大豆低聚肽铜螯合物的最佳工艺条件为:铜离子浓度0.67.0×10-4mol/L、时间10min、反应温度50℃、pH4.0、加入沉淀剂比例为1:2,最终螯合物收率为76.06%。大豆低聚肽锌螯合物的最佳工艺条件为:锌离子浓度0.67×10-4mo/Ll、时间10min、反应温度50℃、pH6.0、加入沉淀剂比例为1:1.75,最终收率为74.83%。铜低聚肽螯合物具有一定酸稳定性,当pH为3~4时,能够较好的以螯合物的形式存在。更多还原

【Abstract】 The soy protein isolates (SPI) have high nutritional values. Soy oligo-peptides, which are the products of SPI hydrolysis by enzyme, are kinds of functional food-bases. The metal-chelate complex of soy oligo-peptides can increase the soy oligo-peptide application scopes on food and feed. In this paper, the main study is to found a method to preparie soy oligo-peptide from SPI with papain and bromelain.The enzyme hydrolysis process conditions, including the in vitro ACE (Angiotensin-converting enzyme) inhibitory activity of hydrolysate are the research contents . Seperation of the high activie components and preparation of soy oligo-peptides’metal-chelate complex are included in this paper. The results of the experiments were as follows:a)Through the preliminary enzyme hydrolysis experiments, the enzyme hydrolysis products of SPI were analyzed by SDS-PAGE, HPLC and mass spectrometry, the results showed that the majority molecular weight of oligo-peptides of the two enzymes hydrolysis are about 1000Da. Among the relationship of the enzyme concentration, the substrate concentration, the reaction temperature and the hydrolysis degree of SPI,the hydrolysis products and the reaction substrates could inhibit the reaction rates during the initial reaction times. However the reaction temperature could increase the reaction rate at the same time. The Km of hydrolyzing SPI by Papain is 0.1404g/L and the Vmax is 7.51 g·L-1·min-1.The Km of hydrolyzing SPI by Bromelain is 0.1173g/L and the Vmax is 4.95 g·L-1·min-1.b) The impact of the main factors on the enzyme reaction of SPI: enzymolysis time, hydrolysis temperature, enzyme concentration, pH, substrate concentration was studied according to the single-factor and two-factor experiment. The best process for papain were that recation time was 3h, hydrolysis temperature was 60℃, the amount of enzyme was 0.35g, pH was 7.5, substrate concentration was 5g/100mL. The best enzyme technology for bromelain were that enzymolysis time was 10h, digestion temperature was 40℃, the amount of enzyme was 0.35g, pH was 7.5, substrate concentration was 5g/100mL.c)The study on related factors of impacting ACE inhibitory activity had shown that the proteinase hydrolysis time did not affect the ACE inhibitory activity of soy oligo-peptides deeply by papain and reached the peak at 72 hours, but the soy oligo-peptides by bromelain reducing the ACE inhibitory activity with increasing time. the small molecule carbohydrates would decreased ACE inhibitory activity. In addition, through the study of soy oligo-peptides prapared by papain and bromelain, the IC50 of hydrolysate were around 70μg/mL. Through separated soy oligo-peptide by HPLC and SphadexG-25 and study of inhibitory activity, A kind of high ACE inhibitory activity components was separated from the soy oligo-peptides by HPLC and SphadexG-25. The high active component was at 29.527min of HPLC. d)The preparation of copper chelate and zinc chelate of soy oligo-peptides were studied. For copper chelate of soy oligo-peptides, the optimum reaction conditions were that the copper ion concentration was 0.67×10-4mol/L, the reactin time was 10min, the reaction temperature was 50℃, the pH was 4.0, the precipitation agent (ethanol) ratio was 1:2. The recovery ratio is 76.06%. For chelate zinc of soy oligo-peptides, the optimum reaction conditions were that the zinc ion concentration was 0.67×10-4mol/L, the reaction time was 10min, the reaction temperature was 50℃, the pH was 6.0, the precipitation agent (ethanol) ratio was 1:1.75, the recovery ratio was 74.83%.The copper chelate of Oligo-peptides shew that it had a certain degree of resistance to acid. The compound could exist in the form of chelate when the solvent pH was between 3 ~ 4.更多还原