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。     

乙醇-水体系中酪蛋白水解物的类蛋白反应及ACE抑制活性的变化

采用Neutrase 0.8L蛋白酶水解酪蛋白,制备水解度为13.6%的酪蛋白水解产物,测得其对血管紧张素转化酶(ACE)的体外抑制活性IC50为(46.92±0.27)mg/L。在乙醇溶剂中,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、底物质量分数、反应温度、反应时间和乙醇浓度对修饰反应的影响。在优化条件下的类蛋白反应体系中,游离氨基浓度减少,说明合成反应占优势;酶添加量、底物质量分数、乙醇质量分数对修饰反应的影响显著,而反应时间和温度影响不大。通过单因素实验确定类蛋白反应的最适反应条件为:44%乙醇水溶液、反应温度为40℃,酶添加量为3 kU/g蛋白质、底物质量分数40%、反应时间6.0 h。此条件下,反应体系中游离氨基浓度变化达到202.19μmol/g蛋白质,修饰产物的IC50值降低至(25.96±0.29)mg/L,降低44.7%。     

酪蛋白水解物的Plastein反应修饰及ACE抑制活性变化

采用中性蛋白酶水解酪蛋白,一定条件下制备水解度为13.0%、IC50质量浓度为40.4 mg/L的酪蛋白水解物。利用中性蛋白酶对所制备出的水解物进行plastein反应修饰,以反应体系的游离氨基量的变化为评价指标,通过单一因素试验研究酶添加量、底物质量分数、反应时间和反应温度对修饰反应的影响。结果表明,适宜的反应条件为中性蛋白酶添加量3 kU/g蛋白质、底物质量分数60%、反应时间6 h、反应温度20℃。制备5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14.7~31.1 mg/L,表明中性蛋白酶催化的plastein反应修饰提高酪蛋白水解物的ACE抑制活性,且ACE抑制活性的提高程度与plastein反应程度有关。     

酪蛋白水解物的类蛋白反应修饰及其产物ACE抑制活性特征

采用碱性蛋白酶水解酪蛋白,制备水解度为10.9%、IC50值为52.6μg/mL的酪蛋白水解物,并利用响应面法优化碱性蛋白酶催化的类蛋白反应修饰条件。修饰反应时间固定为6h时,适宜的条件为酶添加量3.1kU/g pro、底物质量浓度50g/100mL、反应温度25℃。制备9个修饰程度不同的修饰产物,结果显示：修饰产物ACE抑制活性均提高,并且活性最高的修饰产物的IC50降低至14.9μg/mL。该修饰产物离心分级后,上清液部分和沉淀部分的ACE抑制活性分别低于和高于修饰产物,表明沉淀部分是提高ACE抑制活性的主要原因;Tricine-SDS-PAGE电泳分析表明,修饰产物及沉淀部分有较大分子质量的肽分子生成;该修饰产物和上清液部分、沉淀部分的进一步酶水解处理则显示,酶水解会导致它们的ACE抑制活性降低,但是仍然高于最初的酪蛋白水解物。     

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。     

酪蛋白水锯物的酶法修饰与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154.65 μmol/g(蛋白)时,修饰产物的IC50值可降至0.6 μg/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.     

乙醇-水相中酪蛋白水解物修饰及对2个性质的影响

利用Alcalase水解酪蛋白,制备水解度为11.6％、IC5值为42.8 mg/L的酪蛋白水解物.在乙醇-水体系中采用Alcalase催化类蛋白反应修饰酪蛋白水解物,固定反应时间6h,优化得到酶添加量、乙醇体积分数、底物质量浓度、反应温度分别为:8.36 kU/g,56.8％,568 g/L,37.5℃.制备不同反应程度的修饰产物,评估其ACE抑制活性及Zn2+螯合能力变化,发现修饰产物的ACE抑制活性得到改善,抑制最高达到62.5％(IC50达到27.7 mg/L),但是与反应程度有关;Zn2+螯合能力则由4.22 mg/g降低至1.97～3.86 mg/g.修饰产物的Zn2+螯合能力与类蛋白反应程度无关,与ACE抑制活性也不存在相关性.     

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.     

酪蛋白双酶水解物ACE抑制肽的分离纯化

采用胃蛋白酶和胰蛋白酶依次对酪蛋白进行双酶水解,制备ACE抑制肽。水解物经截留分子质量6ku的超滤膜初步分离,再通过Sephadex G-15进一步纯化,体外测定各分离产物ACE活性的半数抑制质量浓度(IC50值)。纯化得到的各组分经毛细管电泳分析肽谱、Q-TOF LC/MS检测分子质量范围。结果显示：双酶水解产物IC50值为560μg/mL,超滤流出物IC50值为250μg/mL;Sephadex G-15分离得到3个组分,组分Ⅰ的IC50值为123.41μg/mL,含有19个肽段;组分Ⅱ的IC50值为66.67μg/mL,含有14个肽段;组分Ⅲ的IC50值为64.29μg/mL,含有5个肽段。Q-TOF LC/MS测得纯化组分的分子质量范围为400～800u。     

脯氨酸存在下酪蛋白ACE抑制肽的Plastein反应修饰

利用枯草杆菌碱性蛋白酶水解酪蛋白制备ACE 抑制肽,其IC50 为47.1μg/mL;采用相同酶催化ACE 抑制肽和脯氨酸进行Plastein 反应,对ACE 抑制肽进一步修饰,并用响应面分析法优化反应条件。在ACE 抑制肽质量分数为35%,反应时间为6h,以反应体系游离氨基减少量为指标,得到适宜的反应条件为：温度为47.8℃、脯氨酸比例为0.54、酶添加量为9.5kU/g pro,此条件下体系游离氨基减少量约195.7μmol/g pro。在适宜条件下改变反应时间对酪蛋白ACE 抑制肽进行同程度的Plastein 反应修饰,制备出6 个修饰程度不同的多肽混合物并测定它们的ACE 抑制活性、计算其IC50 值。结果表明：修饰产物的ACE 抑制活性随修饰程度的增加不规则变化,当反应体系的游离氨基减少量为195.7μmol/g pro 时,修饰产物的IC50 降低至0.2μg/mL。     

ACE-inhibitory and antihypertensive properties of a bovine casein hydrolysate

The aim of this study was to investigate the potential angiotensin converting enzyme (ACE)-inhibitory activity and the antihypertensive effect, after a single oral administration, of a pepsin hydrolysed bovine casein (HBC) and a fraction with molecular mass lower than 3000 Da (HBC < 3000). ACE-inhibitory activity was measured by spectrophotometric assay. These products were orally administered by gastric intubation. The systolic (SBP) and the diastolic blood pressure (DBP) were measured in spontaneously hypertensive rats by the tail cuff method before administration and also 2, 4, 6, 8, and 24 h post-administration. HBC showed a potent ACE-inhibitory activity. This activity was 10 times higher in HBC < 3000. HBC and HBC < 3000 decreased the arterial blood pressure of the rats. The decrease in the SBP observed for HBC (400 mg/kg) or HBC < 3000 (200 mg/kg) was less pronounced than that caused by 50 mg/kg of captopril (antihypertensive positive control). However, the maximal decreases in DBP caused by HBC or HBC < 3000 were as high as the maximum decrease observed for captopril. The antihypertensive effect of these products was transient and reverted 24 h after the administration. HBC and HBC < 3000 exert antihypertensive effect caused by small peptides with ACE-inhibitory activity.     

酪蛋白非磷肽的理化性质及ACE抑制活性

对酪蛋白非磷肽的溶解性、相对黏度、相对分子质量分布和氨基酸组成进行分析,并验证其具有较强的ACE抑制活性.经体外消化实验发现,CNPPs在消化道酶作用后,ACE抑制活性从52.05%提高到57.49%;肽谱分析显示出CNPPs中肽的种类和组成非常复杂.     

Production of whey protein isolate hydrolysate fractions with enriched ACE-inhibitory activity

A study on the enrichment of angiotensin-converting enzyme (ACE) inhibitory activity in whey protein isolate (WPI) hydrolysate fractions is presented. A previously identified low molecular mass fraction (1 kDa permeate) of an enzymatically hydrolysed heat-treated WPI with elevated ACE-inhibition (IC₅₀ = 0.23 g L⁻¹) was subjected to cascade membrane ultrafiltration (UF) and diafiltration steps at lab-scale. Assaying for ACE-inhibition revealed that the 1 kDa retentate demonstrated the highest ACE-inhibitory activity (IC₅₀ = 0.17 g L⁻¹). Isoelectric focussing (IEF) of the hydrolysate fraction further increased ACE-inhibition in fractions collected within the pH range 6.1–6.6. Overall, both UF and IEF enriched the ACE inhibitory activity in the original fraction by ∼52%, demonstrating the potential for enrichment of bio-functional activities in enzymatic hydrolysates of whey proteins.     

酪蛋白的胰蛋白酶水解物分离及其抗氧化性研究

采用胰蛋白酶水解酪蛋白,使用D201阴离子交换树脂对酪蛋白酶解液进行分离,并收集5个多肽片段（P1、P2、P3、P4、P5）,评价酪蛋白酶解液及分离所获得的酪蛋白多肽的抗氧化性。结果表明,酪蛋白多肽的还原力和DPPH自由基清除力均显著高于酪蛋白酶解液（P＜0.05）;多肽P2的还原力最高,为0.234,然后是多肽P3,为0.057;多肽P4的DPPH自由基清除活力最高,达到（23.8±0.18）%,然后是多肽P3,为（4.5±0.16）%;多肽P5的Fe2+螯合率最高,为（99.80±0.12）%,多肽P3的Fe2+螯合率最低,为（2.50±0.14）%,而酶解液的Fe2+螯合率为（31.27±5.80）%;多肽P2和P3不具备脂质过氧化抑制力。多肽P1、P4、P5具有较好的抗氧化性。     

Preparation of Alcalase-catalyzed casein plasteins in the presence of proline addition and the ACE-inhibitory activity of the plasteins in vitro

Casein hydrolysates were prepared by hydrolysis of casein with alkaline protease Alcalase for 6 h and showed the highest ACE-inhibitory activity in vitro with an IC₅₀ value of 47.1 μg mL⁻¹. Casein hydrolysates prepared were subjected to Alcalase-catalyzed plastein reaction in the presence or absence of proline addition to prepare casein plasteins. Some optimal reaction conditions of plastein reaction in the presence of proline addition were studied using response surface methodology with the decrease in free amino groups in the casein plasteins as response. When the concentration of casein hydrolysates was fixed at 35% (w w⁻¹) and reaction time at 6 h, the optimal conditions were reaction temperature 48 °C, addition level of proline 0.54 mol/mol free amino groups of casein hydrolysates and addition level of Alcalase 9.5 kU g⁻¹ proteins. With these conditions, the maximal decrease in free amino groups in casein plasteins was 195.7 μmol g⁻¹ proteins. The ACE-inhibitory activities of twelve casein plasteins in vitro, prepared in the presence or absence of proline addition with different reaction extents, were evaluated and compared. The results showed that the ACE-inhibitory activity of the casein plasteins prepared in the presence of proline addition changed irregularly, different to that of the casein plasteins prepared in the absence of proline addition, and might relate to the different linking of proline to the peptides in casein hydrolysates during plastein reaction. When the casein plasteins prepared in the presence of proline addition had a decrease in free amino groups 195.7 μmol g⁻¹ proteins, the IC₅₀ value of the casein plasteins was lowered to 0.2 μg mL⁻¹.     

酪蛋白酶解特性的研究

用胰蛋白酶酶解酪蛋白,用正交实验得出的最佳酶解条件进行酶解。取不同酶解时间的酶解物,分析酶解物的水解度、氨基氮含量、可溶性氮含量、肽氮含量及pH值随时间的变化趋势,水解度和氨基氮含量在12h内出现显著性增长,可溶性氮含量和肽氮含量在1h内出现及其显著的增加,pH值在10h内表现出明显的下降趋势。从不同角度分析和推测酪蛋白酶解过程。     

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.     

Purification and identification of an ACE-inhibitory peptide from walnut protein hydrolysate

Angiotensin I-converting enzyme (ACE) is a dipeptidyl carboxypeptidase. It plays an important physiological role in regulating blood pressure in human bodies. ACE-inhibitory peptides inhibit the activity of ACE, thereby decreasing the tension of blood vessels and the blood volume, thus lowering blood pressure. ACE-inhibitory peptides derived from food proteins due to their safety properties and beneficial effects on human health have attracted more and more attentions on their ACE-inhibitory activity. In the present study, a novel ACE-inhibitory peptide, P-1a1, was homogeneously purified from walnut protein hydrolysate by ultrafiltration, consecutive column chromatography and high performance liquid chromatography. The purified peptide was characterized by Edman degradation, matrix-assisted laser desorption ionization time-of-flight mass spectrophotometer and a liquid-phase peptide sequencer. The amino acid sequence of P-1a1 was determined to be LPGRPPIKPWPL. The potent ACE-inhibitory peptide showed a high ACE-inhibitory activity with the IC₅₀ value of 128.98 μg/mL (95.2 μmol/L). The purified peptide could be used in functional food products as a bioactive component with good ACE-inhibitory activity.     

糖基化改性对酪蛋白酶解产物抗氧化活性的影响

为了提高酪蛋白酶解产物的抗氧化活性,将酶解产物分别与葡萄糖、木糖、果糖进行糖基化改性。在酶解产物与糖质量比为12的条件下,研究改性条件对产物抗氧化活性的影响。结果表明,酶解产物与木糖、果糖改性后的产物,在pH 7.0时羟基自由基清除率达到最大,分别为38.63%,32.82%（均高于对照）;pH 7.0时,葡萄糖、木糖糖基化产物的还原能力最强。随着反应温度升高,酶解产物与3种糖的糖基化产物的抗氧化活性不断增强,在120,130 ℃时,3种糖的糖基化产物的羟基自由基清除率和还原力均高于对照。在反应4 h时,酶解产物与木糖、果糖及葡萄糖糖基化后的羟基自由基清除率都达到最大（分别为66.57%,83.33%,42.00%,均高于对照）,还原力最大分别为1.12,1.14,1.05（均高于对照）。糖基化改性可以提高酪蛋白酶解产物的抗氧化活性,其中酶解产物与木糖的糖基化产物的抗氧化活性最高,其最佳糖基化反应条件为pH 7.5、温度130 ℃、时间4 h。