苯丙氨酸添加下酪蛋白类蛋白反应修饰物的抗氧化性质

采用木瓜蛋白酶水解酪蛋白制备水解度为9．6％、DPPH·清除率为38．7％的水解物,添加苯丙氨酸于水解物并利用响应面法优化木瓜蛋白酶催化的类蛋白反应条件。在反应时间固定为5h下得到适宜的条件为：温度30℃、底物浓度50％（w／v）、酶添加量3kU／g蛋白质、氨基酸添加量0．74mol／mol水解物游离氨基。此条件下分别制备出5个修饰产物．以不添加苯丙氨酸的修饰产物或水解物和苯丙氨酸混合物为对照,评价它们的抗氧化活性。结果表明．与酪蛋白水解物或混合物或不添加苯丙氨酸的修饰产物相比,添加苯丙氨酸的修饰产物的DPPH·清除能力、还原能力和．OH清除能力显著提高;但是抗氧化性质与所采用的氨基酸添加量、反应时间之间的关系不显著。     

高活性酪蛋白抗氧化肽的制备方法研究

采用木瓜蛋白酶对酪蛋白进行水解,得到抗氧化活性较好的酪蛋白水解物,并且水解物在木瓜蛋白酶作用下进行类蛋白反应制备出高活性酪蛋白抗氧化肽。第一步制备酪蛋白水解时酶添加量为500 U/g酪蛋白、温度45℃、底物浓度5%、反应时间2 h。第二步类蛋白反应的最优条件为：酶添加量为500 U/g水解物、温度30℃,底物浓度50%、作用时间5.5 h。毛细管电泳结果确认,类蛋白反应修饰后抗氧化肽的组成情况发生变化。抗氧化活性分析结果表明,类蛋白反应修饰后的酪蛋白抗氧化肽对两种自由基的清除能力显著提高。     

鹰嘴豆蛋白水解物类蛋白反应修饰与抗氧化活性研究

以碱性蛋白酶水解鹰嘴豆蛋白,制备水解度为20.03%的蛋白水解物,以该蛋白水解物为底物进行类蛋白反应修饰,制备抗氧化活性肽。以游离氨基减少量为试验指标,在分析反应温度、加酶量、底物质量分数对类蛋白反应影响的基础上,采用中心组合设计建立酶添加量、底物质量分数和p H值与游离氨基减少量的回归模型,优化类蛋白反应条件。试验结果表明,所建回归模型具有统计学意义,可以预测类蛋白反应规律。p H值对游离氨基减少量影响最大,其次是加酶量,底物质量分数影响较小。类蛋白反应的优化条件是:酶添加量417.4 U/g,底物质量分数49.8%和p H 3.48。抗氧化活性分析表明,类蛋白反应修饰产物的还原力和·OH清除率显著提高。     

银鲳酶解物抗氧化活性研究

选用胃蛋白酶、胰蛋白酶、碱性蛋白酶和中性蛋白酶对银鲳蛋白进行酶解以制备蛋白酶解物,以羟基自由基清除活性为指标确定银鲳最佳水解酶。结果显示,碱性蛋白酶的水解物抗氧化活性最强。实验对碱性蛋白酶水解银鲳的酶解条件(时间、温度、pH、酶添加量和固液比)进行正交实验设计,并对最佳水解条件下所获得的酶解物进行抗氧化活性测试。结果表明,银鲳蛋白碱性蛋白酶水解物对DPPH自由基和羟基自由基具有清除作用,其自由基清除效果呈现剂量依赖性,而且银鲳蛋白水解物还具有明显还原能力。所有这些体外抗氧化数据说明,银鲳蛋白水解物有明显的抗氧化效力。     

酶解制备苦荞蛋白抗氧化肽及其分离纯化研究

以苦荞麦粉为原料,提取苦荞蛋白,分别采用碱性蛋白酶、胃蛋白酶、胰蛋白酶对蛋白进行酶解,采用DPPH法比较不同酶解产物的抗氧化活性,从而筛选水解制备苦荞蛋白抗氧化肽的最适酶。以水解度为指标,利用单因素试验和响应面法优化酶解工艺条件。结果表明,不同蛋白酶酶解产物的抗氧化活性大小为:胃蛋白酶胰蛋白酶碱性蛋白酶,其中胃蛋白酶酶解产物的DPPH自由基清除率最高,为68.47%。胃蛋白酶最佳水解工艺条件为:时间2.5 h、温度38℃、pH 2.0,在此条件下苦荞蛋白水解度为32.68%。采用超滤对苦荞蛋白水解物进行分离纯化,结果表明,分子量3 kDa的水解物具有显著的抗氧化活性;经凝胶过滤色谱进一步分离得到3个峰,小分子量峰组分显示出最强的抗氧化活性。     

酪蛋白类蛋白物的溶剂分级和酶水解对ACE抑制活性的影响

利用响应面法优化类蛋白反应条件修饰酪蛋白水解物制备酪蛋白类蛋白物.酪蛋白类蛋白物的ACE抑制活性高于酪蛋白水解物,IC50值从52.6 mg/L降低到14.9 mg/L.利用乙醇-水混合溶剂对酪蛋白水解物和类蛋白物进行分级,结果表明,极性最低的溶剂得到的上清液部分活性较高,而沉淀部分活性较低.4种蛋白酶水解酪蛋白类蛋白物的分级产物,导致活性下降,除碱性蛋白酶外,木瓜蛋白酶、胃蛋白酶和胰蛋白酶的水解产物ACE抑制活性为31.5％～46.8％,但是仍然高于酪蛋白水解物的ACE抑制活性(27.8％),表明类蛋白反应提高了酪蛋白水解物对一些蛋白酶的体外抵抗能力.     

三种氨基酸添加下酶法修饰酪蛋白水解物的ACE抑制活性

采用碱性蛋白酶水解酪蛋白,制备水解度为12.4%、IC50为42.19μg/mL的酪蛋白水解物。在添加外源氨基酸的情况下对水解物进行类蛋白反应修饰,并响应面法研究氨基酸添加量、酶添加量、反应温度及3种氨基酸的影响。结果表明:氨基酸添加量、反应温度、氨基酸种类对修饰反应影响显著,而酶添加量的影响不大;分别添加苯丙氨酸、亮氨酸、缬氨酸制备3个酪蛋白水解物修饰产物,其IC50降低至21.03～25.13μg/mL,表明添加外源氨基酸可提高修饰产物的体外ACE抑制活性,但添加不同氨基酸的影响不显著。     

大豆蛋白的酶水解及类蛋白反应对其抗氧化活性的影响

采用Alcalase 2.4L FG 蛋白酶水解大豆蛋白,筛选并制备出ABTS+·清除率最高的水解物,其水解度为14.0%,对ABTS+·清除率为43.6%。以此水解物为底物,以修饰产物的游离氨基减少量为指标,应用响应面分析得到类蛋白反应的优化条件为：酶添加量1037U/g pro、底物质量浓度30g/100mL、温度20℃。在此条件下反应6h,水解物的修饰反应程度和抗氧化活性均为最大。制备反应程度不等的3 个修饰产物,进一步抗氧化活性分析表明：大豆蛋白水解物及其修饰产物的抗氧化活性好于大豆蛋白;修饰产物与水解物的DPPH 自由基清除能力、还原力、超氧阴离子自由基(O2 － ·)清除率差别不显著,但是对羟自由基(·OH)清除率差别显著。     

美拉德反应对醋蛋多肽抗氧化活性的影响

采用醋蛋多肽与葡萄糖发生美拉德反应来改善醋蛋多肽的抗氧化活性。结果表明:优化的美拉德反应条件为醋蛋多肽与葡萄糖质量比1:2、反应初始pH10、反应温度100℃、反应时间120min。在此反应条件下,反应产物的抗氧化活性与未反应的醋蛋多肽相比,DPPH自由基清除率从12.7%增加到64.8%,Fe3+还原能力(A700nm)从0.107增加到0.718。在此反应过程中,反应产物的抗氧化活性随着反应体系pH值降低、中间产物(A294nm)和褐变程度(A420nm)以及接枝度的增加而不断增强。     

不同处理条件对苜蓿叶蛋白水解物抗氧化活性的影响

以苜蓿叶蛋白为原料,通过酶解制备苜蓿叶蛋白水解物。以DPPH自由基和还原力为评价指标,采用不同的处理条件对苜蓿叶蛋白水解物的抗氧化活性进行研究。结果表明:加热处理和添加大多数食品添加剂有利于提高水解物的抗氧化活性,而添加柠檬酸和酒石酸能降低其抗氧化活性;碱性p H条件不利于提高DPPH自由基清除率,而酸性p H条件有利于提高DPPH自由基清除率;添加金属离子对抗氧化活性影响较大;巴氏灭菌、煮沸灭菌和高压蒸汽灭菌条件均有利于清除DPPH自由基,但会在一定程度上降低还原力。     

Modification of Soybean Protein Hydrolysates by Alcalase-Catalyzed Plastein Reaction and the ACE-Inhibitory Activity of the Modified Product In Vitro

Soybean protein hydrolysates were prepared by hydrolyzing soybean protein isolates with a protease alcalase to a degree of hydrolysis of 16.6%, and then modified by alcalase-catalyzed plastein reaction to reveal the impact of plastein reaction on the ACE-inhibitory activity of the modified product in vitro. The suitable conditions of plastein reaction of soybean protein hydrolysates were selected based on the results of response surface methodology with the decreased amount of the free amino groups of the modified product as response. When reaction temperature was fixed at 30°C, the selected conditions were as follows: concentration of soybean protein hydrolysates of 45% (w/w), addition level of alcalase of 275 U/g peptides, and reaction time of 3 to 4 h. Soybean protein hydrolysates and eight modified products were evaluated for their ACE-inhibitory activities in vitro. The assay results highlighted that plastein reaction improved the ACE-inhibitory activity of the modified product. The IC₅₀ of the modified products ranged from 0.64 to 1.11 mg/mL, while that of soybean protein hydrolysates was 1.45 mg/mL. The decreased amount of the free amino groups of the modified product showed influence on the ACE-inhibitory activity in vitro. Analysis results from size exclusion chromatography confirmed that some plasteins with higher molecular weights were formed in the modified product. Our results showed that alcalase-catalyzed plastein reaction could be applied as a potential approach to enhance the ACE-inhibitory activity of soybean protein hydrolysates in vitro.     

Coupled Neutrase–Catalyzed Plastein Reaction Mediated the ACE-Inhibitory Activity In Vitro of Casein Hydrolysates Prepared by Alcalase

Casein hydrolysates with a degree of hydrolysis of 13.5% were prepared by hydrolyzing casein with an alkaline protease Alcalase, and showed ACE-inhibition in vitro with an IC₅₀ value of 45.2 μg/mL. The hydrolysates were modified by plastein reaction catalyzed by a neutral protease Neutrase to reveal the impact of the coupled Neutrase-catalyzed plastein reaction on the ACE-inhibition of the casein hydrolysates. The effects of addition level of Neutrase, substrate concentration, reaction temperature, and time on the plastein reaction of the casein hydrolysates were studied with the varying amount of free amino groups of the modified hydrolysates as index. The results illustrated that the amount of free amino groups of the modified hydrolysates increased in all occasions, and the addition level of Neutrase, substrate concentration, and reaction time had a clear impact on the plastein reaction. Six modified hydrolysates were prepared at a substrate concentration of 40% (by weight), Neutrase addition level of 3 kU/g peptides, reaction temperature of 35°C, and different reaction time. The assay results highlighted that the coupled Neutrase-catalyzed plastein reaction improved the ACE-inhibition of six modified hydrolysates with IC₅₀ values ranging from 15.6 to 20.0 μg/mL. Size exclusion chromatography analysis showed that some plasteins with a molecular weight of about 68 kDa existed in the modified hydrolysates. The results also demonstrated that it was the coupled Neutrase-catalyzed plastein reaction but not further hydrolysis of casein hydrolysates that enhanced the ACE-inhibition of the modified casein hydrolysates.     

酪蛋白水解物的酶法修饰与ACE抑制活性变化

利用枯草杆菌碱性蛋白酶水解酪蛋白制备酪蛋白水解物,其水解度为11.2%,IC50为47.1μg/mL。再应用相同的酶对酪蛋白水解物进行类蛋白反应修饰,考察底物浓度、温度和酶添加量对类蛋白反应的影响,并制备5个不同的修饰产物测定其ACE抑制活性和IC50值。结果表明,修饰产物的ACE抑制活性随修饰程度(游离氨基减少量)的增加而提高,并且都高于未经修饰的酪蛋白水解物。当游离氨基减少量为154.65μmol/g(蛋白)时,修饰产物的IC50值可降至0.6μg/mL。毛细管电泳分析结果显示类蛋白修饰后水解物的多肽组成情况发生明显变化。研究结果证明酪蛋白水解物的ACE抑制活性可以通过类蛋白反应的修饰作用而提高。     

An approach to improve ACE-inhibitory activity of casein hydrolysates with plastein reaction catalyzed by Alcalase

The preparation method of casein hydrolysates with high ACE-inhibitory activity was studied by Alcalase-catalyzed hydrolysis coupled with plastein reaction. Casein hydrolysates with an IC₅₀ value of about 47 μg mL⁻¹ were first prepared by hydrolysis of casein with Alcalase and then modified with plastein reaction catalyzed by the same enzyme. The impacts of four reaction conditions on plastein reaction of casein hydrolysates were studied, and then optimal conditions were determined using response surface methodology with the decrease of free amino groups in the reaction mixture as response. When the concentration of casein hydrolysates was fixed at 35% by weight, the maximum decrease of free amino groups in the reaction mixture of 181.8 μmol g⁻¹ proteins was obtained. The optimum conditions for the above decrease were found to be an E/S ratio of 7.7 kU g⁻¹ proteins, reaction temperature of 42.7 °C and reaction time of 6 h. Analysis results showed that ACE-inhibitory activity of casein hydrolysates prepared could be improved significantly by plastein reaction. When casein hydrolysates were modified by plastein reaction, with a decrease of free amino groups in the mixture of about 154.7 μmol g⁻¹ proteins and 181.8 μmol g⁻¹ proteins, their IC₅₀ values could be decreased to 0.6 and 0.5 μg mL⁻¹.     

耦合Plastein反应的大豆蛋白降压肽酶法制备技术

利用碱性蛋白酶酶解大豆分离蛋白,制备出水解度为16.6% 的大豆蛋白水解物,随后对水解物进行Plastein反应修饰。利用响应面分析优化修饰反应条件,得到适宜参数：底物质量分数45%、酶添加量275U/g 蛋白质、反应时间3～4h、温度30℃。制备修饰反应程度不同的9 种修饰产物并评价其体外ACE 抑制活性,发现修饰产物的IC50 值为0.64～1.30mg/mL,均小于大豆蛋白水解物IC50 值(1.45mg/mL)。排阻色谱分析结果确认,修饰产物中有更多的高分子质量肽段存在。结果显示,大豆蛋白的酶解以及耦合Plastein 反应修饰,是一种制备高ACE抑制活性大豆蛋白降压肽的新技术。     

Evaluation Of Antioxidant Properties In Vitro of Plastein-Reaction-Stressed Soybean Protein Hydrolysate

Alcalase was used in the present study to carry out an enzymatic hydrolysis of soybean protein isolate and a plastein reaction of the prepared hydrolysate in vitro, aiming to investigate the influence of the plastein reaction on the antioxidant properties of the modified hydrolysate. Soybean protein hydrolysate was prepared in a degree of hydrolysis of 14.0%, exhibited a scavenging activity of 43.6% on ABTS radical in vitro, and thus was used as the substrate of the plastein reaction to prepare the plastein-reaction-stressed hydrolysate. Response surface methodology was applied to select suitable reaction conditions as follows: enzyme addition level 1037 U/g peptides, substrate concentration 29.7% (w/v), reaction temperature 20.3°C. The stressed hydrolysate showed the highest scavenging activity on ABTS radical (about 47.9%) or maximal reaction extent when reaction time was 6 h. Three stressed hydrolysates with different reaction extents were prepared and evaluated for other antioxidant activities. Compared to the original hydrolysate, the stressed hydrolysate with lower reaction extent exhibited a similar (P > 0.05) scavenging activity on DPPH (or superoxide) radical and reducing power, but a significant higher activity (P < 0.05) on hydroxyl radical. The stressed hydrolysate with the highest reaction extent behaved as these investigated antioxidant properties were significantly higher (P < 0.05) than the original hydrolysate except for scavenging activity on DPPH radical. The results of the present study highlight that the alcalase-catalyzed plastein reaction appears to be capable of improving antioxidant properties of soybean protein hydrolysate.     

Antioxidative peptides derived from milk proteins

Antioxidants may function by preventing the formation of radicals or by scavenging radicals or hydrogen peroxide and other peroxides. Milk contains several antioxidant factors, like vitamins and enzymes. Possible antioxidant activity of milk proteins and hydrolysates has also been shown. Peptides generated from the digestion of milk proteins are reported to have antioxidative activities. Milk-derived antioxidative peptides are composed of 5–11 amino acids including hydrophobic amino acids, proline, histidine, tyrosine or tryptophan in the sequence. The structure–activity relationship or the antioxidant mechanism of peptides is not fully understood. Antioxidant activity of the hydrolysates seems to be inherent to the characteristic amino acid sequences of peptides derived, depending on the protease specificity. The results suggest that the hydrolysates from milk proteins could be used as natural antioxidants in enhancing antioxidant properties of functional foods and in preventing oxidation reaction in food processing. Further studies are needed to elucidate the role of antioxidative peptides in the protective function in humans.