【作者】 徐微；

【导师】 赵新淮；

【作者基本信息】 东北农业大学 ， 食品科学， 2010， 硕士

【摘要】 酪蛋白不仅营养价值丰富,而且具有良好的功能性质,如溶解性、保水性、吸油性、乳化性、凝胶性等功能特性,还是生物活性肽(如:抗菌活性、抗氧化活性、降血压活性等)的重要来源,使酪蛋白广泛应用于食品及其它行业。利用酪蛋白为原材料,改善酪蛋白相关产品的功能特性与生物活性的研究,已经成为食品科学中的热点问题之一。本研究采用三种蛋白酶对酪蛋白进行水解,制备酪蛋白水解产物,并分别对三种酪蛋白水解产物进行Fenton反应修饰和类蛋白反应修饰,测定其抗氧化活性和血管紧张素转化酶抑制活性的变化。主要研究结果如下:(1)利用Alcalase 2.4L FG蛋白酶水解酪蛋白,制备酪蛋白水解产物。选取底物浓度5%,pH8.0,酶添加量0.3AU/g蛋白质,50℃水浴中水解2 h得到水解度为10.0%的水解产物,其清除DPPH自由基的IC50值为2.802±0.072 mg/mL。对水解产物进行Fenton反应修饰,结果表明Fenton试剂添加量对水解产物羟基化程度影响显著,而反应时间对其影响不大。修饰产物的抗氧化活性提高,其清除DPPH自由基的IC50值降至0.891±0.017 mg/mL。(2)采用Neutrase 0.8L蛋白酶水解酪蛋白,制备水解度为13.0%的酪蛋白水解产物,其对ACE的体外抑制活性IC50为40.35μg/mL。利用Neutrase 0.8L蛋白酶对水解物进行类蛋白反应修饰,并研究酶添加量、底物浓度、反应温度和时间对修饰反应的影响。结果表明,修饰反应体系中合成反应占优势,表现为游离氨基含量减少;酶添加量、底物浓度、反应时间对修饰反应的影响显著,而反应时间影响不大。利用中心组合设计和响应面分析法,优化类蛋白反应条件,以其游离氨基含量变化为响应值。软件分析得到游离氨基含量变化与酶添加量、底物浓度和反应时间的二次回归方程,最适反应条件为:反应温度为20℃,酶添加量3 kU/g蛋白质、底物浓度为62%(m/m)、反应时间6.3 h,此条件下反应体系中游离氨基含量变化达到210.01μmol/g蛋白质。对酪蛋白水解物进行不同程度的类蛋白反应修饰,得到5个不同修饰程度的修饰产物,它们的ACE抑制活性测定结果表明,修饰产物的ACE抑制活性随修饰程度的增加而提高,当反应体系中游离氨基减少量为210.01μmol/g蛋白质时,修饰产物的IC50值降低至14.72±0.22μg/mL。(3)采用碱性蛋白酶水解酪蛋白,制备水解度为13.5%的酪蛋白水解物,其对ACE的体外抑制活性IC50为45.23μg/mL。利用中性蛋白酶对水解物进行类蛋白反应修饰,并研究酶添加量、底物浓度、反应温度和时间对修饰反应的影响。结果表明:修饰反应体系中水解反应为优势反应,表现为游离氨基含量增加;酶添加量、底物浓度、反应时间对修饰反应的影响显著,而反应温度的影响不大。在低酶添加量、高底物浓度和短反应时间下,修饰反应体系的游离氨基的增加幅度减少,水解反应相对降低。制备6个不同反应程度的修饰产物,ACE抑制活性分析结果显示,修饰产物的IC50降至15.56~19.98μg/mL,表明中性蛋白酶催化的类蛋白反应修饰,可以提高碱性蛋白酶制备的酪蛋白水解物的ACE抑制活性。更多还原

【Abstract】 Casein is not only rich in nutrition, but also has good functional properties such as water-holding capacity, oil absorption, emulsification, gelation and the important source of bioactive peptides including antibiosis peptide, antioxidant peptide, angiotensin-I-converting enzyme inhibitory peptide etc. , so that it is highly and widespread applied in food and other fields. At present, the researches of the improvement in functional properties and biological properties of casein hydrolysates are popular in food science.In this study, casein hydrolysates were prepared by hydrolysis of casein with three kinds of protease and modified by Fenton reaction and plastein reaction, then, the antioxidant activity and angiotensin-converting enzyme inhibitory activity changes were measured. The results obtained in this study are as follow.(1) Casein was hydrolyzed with Alcalase 2.4L FG protease to prepared Casein hydrolysates. Casein hydrolysates that had the degree of hydrolysis of 10% and the values of IC50 about 2.802±0.072 mg / mL on DPPH radical were hydrolyzed with the substrate concentration 5%(w/v),the addition level of neutral proteinase 11.0 kU/g proteins, the temperature 50℃and the time 4 hours then modified with Fenton reaction. The results showed that, the addition level of Fenton reagent had a significantly influence on the degree of hydroxylation, while little influence on reacetion time. After modification by the Fenton reaction, the values of IC50 about antioxidation activity on DPPH radical had reduced to 0.891±0.017 mg/mL.(2) Casein hydrolysates that had the degree of hydrolysis of 13.0 % and value of IC50 on angiotensin-converting enzyme (ACE) inhibitory activity about 40.35μg/mL were prepared from casein with a protease Neutrase 0.8 L, and then modified by plastein reaction with the same protease. The effects of the addition level of Neutrase, the concentration of casein hydrolysates, temperature and time on the plastein reaction of casein hydrolysates were studied. The results indicated that synthesis reaction was the dominant reaction in the Plastein reaction system as the content of free amino groups in reaction system decreased in all conditions. The addition level of Neutrase, the concentration of casein hydrolysates and reaction time all had significant impact on the plastein reaction of casein hydrolysates, while reaction temperature gave some influence. Three reaction conditions, enzyme addition, concentration of casein hydrolysates and reaction temperature, for Plastein reaction were optimized by a central composite design and response surface methodology analysis with the changes of free amino groups in the reaction mixture as response. quadratic regression equation was obtained therefore, which could reflect the relationship of the changes of free amino groups and enzyme addition, concentration of casein hydrolysates and reaction time. The optimal reaction conditions were that enzyme addition level was 3 KU/g casein hydrolysates, concentration of hydrolysates was 62%(m/m) and reaction time was 6.3 h when the reaction temperature was at 20℃.At this condition the modified casein hydrolysates had a decrease of free amino groups of 210.01μmol/g proteins.Five modified casein hydrolysates with different modification extents were prepared and their ACE inhibitory activity and IC50 values were determined. The analysis results indicated that the ACE inhibitory activity of the modified casein hydrolysates increased as the extent of modification increased. ACE inhibitory activities of the modified casein hydrolysates were all higher than that of the origin casein hydrolysates. When the modified casein hydrolysates had a decrease of free amino groups of 210.01μmol/g proteins, whose IC50 value would decrease to 14.72±0.22μg/mL.(3) Casein hydrolysates that had the degree of hydrolysis of 13.5% and value of IC50 angiotensin-converting enzyme (ACE) inhibitory activity about 45.23μg/mL were prepared from casein with a protease Alcalase, and then modified by plastein reaction with another protease Neutrase. The effects of the addition level of Neutrase, the concentration of casein hydrolysates, temperature and time on the plastein reaction of casein hydrolysates were studied. The results indicated that hydrolysis reaction was the dominant reaction in the Plastein reaction system as the content of free amino groups in reaction system increased in all conditions. The addition level of Neutrase, the concentration of casein hydrolysates and reaction time all had significant impact on the plastein reaction of casein hydrolysates, while reaction temperature gave some influence. Under lower addition level of Neutrase, higher concentration of casein hydrolysates and shorter reaction time, the increase extent of free amino groups in Plastein reaction system showed a trend to decrease which implied that hydrolysis reaction were weakened relatively. Six modified casein hydrolysates with different reaction extents were prepared and their ACE inhibitory activities were determined. The results showed that the values of IC50 of the modified products were in range of 15.56 to 19.98μg/mL, indicating that the ACE inhibitory activity of casein hydrolysates could be improved significantly by plastein reaction catalyzed by Neutrase.更多还原