花生分离蛋白水解物中血管紧张素转化酶抑制肽的分离纯化与结构鉴定

利用碱性蛋白酶Alcalase对花生分离蛋白进行水解,制备花生分离蛋白水解物,并测定不同水解时间所得产物对血管紧张素转化酶(ACE)的抑制作用。未水解的花生分离蛋白没有ACE抑制活性,而利用碱性蛋白酶Alcalase水解所得的水解物具有很强的ACE抑制活性,水解30 min时水解物活性最高,其半抑制浓度为(IC50)0.56 mg/mL。通过超滤、Sephadex G-15凝胶过滤层析、反相高效液相色谱(RP-HPLC)、基质辅助激光解吸电离飞行时间串联质谱(MALDI-TOF-MS/MS)和氨基酸组成分析等分析手段从水解30 min所得的水解物中分离鉴定出两种新的ACE抑制肽,氨基酸序列为Gln-Gly-Gly-Ser-Gly-Met-Thr-Leu-Ala-Phe-Pro-Leu-Pro-Lys和Lys-Ile-Phe-Leu-Arg-Leu-Ser,其IC50值分别为10.6μmol/L和36.6μmol/L。     

基于生物信息学,QSAR及分子对接的菜籽ACE抑制肽筛选

本研究的目的是筛选菜籽蛋白来源的高活性ACE抑制肽。研究中先是利用生物信息学工具对菜籽蛋白进行计算机辅助酶解,建立样品肽集,再运行QSAR模型预测IC50值进行初筛,而后结合NANO-Q-TOF质谱技术寻找出6条目标多肽,并将目标多肽与ACE的Zn2+活性中心进行水合分子对接,结果表明多肽GRD具有高活性,其预测IC50为7.54μmol/L,是一条未报道的新ACE抑制肽,对ACE的Zn2+活性中心形成竞争性抑制。序列比对结果显示,GRD的来源蛋白是一种Napin蛋白(oleosin-like protein,NCBI中ID:ABD14345),其胃蛋白酶Pepsin酶切位点为ABD14345/50。     

油菜蜂花粉ACE抑制活性酶解物的制备及分离

采用4 种蛋白酶水解油菜蜂花粉蛋白制备ACE 抑制活性物质,高效液相色谱法测定油菜蜂花粉蛋白水解物对ACE 的抑制率。结果表明：油菜蜂花粉蛋白酶水解物具有ACE 抑制活性,水解物对ACE 的抑制活性差异显著(P ＜ 0.05),其中碱性蛋白酶＞中性蛋白酶＞木瓜蛋白酶＞酸性蛋白酶,碱性蛋白酶水解物的IC50 为0.35mg/mL。4 种蛋白酶水解物经Bio P-2 凝胶分离后,ACE 抑制活性较强的组分主要集中在保留时间70～120min,在此区间碱性蛋白酶水解物分离组分对ACE 的抑制率达到90% 以上,分子质量在376.4～1355D 之间。     

棉籽蛋白ACE抑制肽的酶法制备及其体外稳定性研究

食源性血管紧张素转化酶(angiotensin-I-converting enzyme, ACE)抑制肽因安全、无副作用、易吸收等优点,对防治高血压、提高居民健康水平具有重要作用。利用碱性蛋白酶、复合蛋白酶、风味蛋白酶、木瓜蛋白酶和菠萝蛋白酶水解棉籽蛋白制备ACE抑制肽,通过单因素实验优化水解条件,分离纯化水解物并鉴定其活性肽段组成,评价水解物的体外消化吸收稳定性。结果表明,棉籽蛋白经复合蛋白酶(1500U/g)在pH值为8.0和55℃下水解5h,蛋白回收率为39.8%,ACE抑制率可达93.7%。棉籽蛋白复合蛋白酶水解物经分离得到5个组分,其中组分4(F4)的ACE抑制活性最高(IC₅₀=220.1μg/mL)。从该组分中发现3条新型ACE抑制肽:VFNNNPQE、LLSQTPRY和VFPGCPET,其中LLSQTPRY的活性最高(IC₅₀=105.2μmol/L)。经人工胃肠液消化和Caco-2细胞单层膜吸收后,棉籽蛋白复合蛋白酶水解物仍具有一定ACE抑制活性,分别为53.8%和20.5%。研究结果表明,棉籽蛋白的复合蛋白酶水解物有望作为一种功能性食品配料,用于开发降压食品。     

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。     

两种来源于牛皮胶原蛋白的新型ACE抑制肽

从牛皮胶原蛋白中鉴定分离出抑制血管紧张素Ⅰ转换酶(ACE)的多肽片段。首先优化牛皮胶原蛋白水解方法,应用胃蛋白酶和碱性蛋白酶获得活性最高的水解产物(抑制ACE的IC50为0.168 mg/m L)。经MW 5000超滤分离后,得到水解液中活性最高的部分(IC50为0.079 mg/m L)。对分离后低于MW 5000的部分经RP-HPLC进一步分离,发现含有大量ACE抑制肽,并应用RP-HPLC-MS/MS鉴定出两种新型ACE抑制肽,氨基酸序列分别为ISVPGPM和LGPVGNPGPA,IC50值分别为0.017 mg/m L(24.3μmol/L)和0.077 mg/m L(87.8μmol/L)。最后,本文模拟了两种抑制肽在体内生理环境下的消化,发现均可被部分降解,且消化产物具有与原始多肽相近的ACE抑制活性。     

HPLC-ESI-MS对珠蛋白酶解液中的血管紧张素Ⅰ转换酶抑制肽的分离鉴定

本实验利用ESI-MS/MS对反相高效液相色谱分离的具有血管紧张素Ⅰ转换酶(ACE)抑制活性的珠蛋白小肽的结构进行鉴定.结果表明此肽的序列为Val-Val-Tyr-Pro-Trp-Thr(VVYPWT),位于猪的血红蛋白β链的34-39氨基酸序列片断,它对ACE有很好的抑制活性,其IC50为6.02μmol/L.     

酶解法制备绵羊乳酪蛋白ACE抑制肽的工艺优化及其抑制机制

为优化酶解法制备绵羊乳酪蛋白ACE抑制肽的工艺条件以及筛选和鉴定一种新的ACE抑制肽,选用5种蛋白酶水解酪蛋白,以水解度、分子质量分布和ACE抑制率为指标筛选最适蛋白酶,采用单因素和响应面试验优化工艺,采用三合一质谱仪(Orbitrap Fusion Lumos Tribrid MS)方法鉴定分子质量小于3 ku组分的氨基酸序列,筛选潜在ACE抑制肽,进行人工合成,测定其IC50值。采用Linewaver-Burk作图确定酶抑制动力学,结合分子对接解析肽段的抑制机制。结果表明:碱性蛋白酶水解酪蛋白的最佳条件为pH 6、底物含量8%、酶添加量4%、温度55 ℃、水解时间90 min,此时酪蛋白水解液ACE抑制率为99.1%。验证具有ACE抑制活性的肽段10条,筛选出一条新颖的降血压肽——LFRQFY(源自αs1-酪蛋白),其ACE抑制活性的IC50为(7.9±1.7)μmol/L,酶抑制动力学为混合抑制模式。分子对接结果表明:LFRQFY能与ACE的氨基酸残基Ala354(活性口袋S1)、His353(活性口袋S2)形成氢键,具有显著的体外降血压活性。     

从小麦胚芽蛋白中分离和鉴定血管紧张素转化酶抑制肽的研究

用碱性蛋白酶水解小麦胚芽蛋白得到的水解物对血管紧张素转化酶有强的抑制活性(IC50=0.014mg/ml),小麦胚芽蛋白水解物经SephadexG-15纯化、制备RP-HPLC分离,得一对ACE有强烈抑制作用的组分X(IC50=5.46μmol/L),X氨基酸分析、电喷雾质谱确定了X的序列为Ala-Met-Tyr。     

蚕蛹蛋白源血管紧张素转换酶抑制肽抑制机理研究

以纯化得到的两条具有ACE抑制活性的肽(P1和P2)为基础,通过基质辅助激光解吸电离飞行时间质谱(MALDI TOF MS)确定P1、P2的氨基酸序列,并采用固相合成法合成,随后对它们的抑制动力学进行研究,最后用Sybyl软件分别将其与ACE进行分子对接。结果表明:P1的氨基酸序列为Gly-Asn-Pro-Trp-Met(IC50值为12.61μg/m L),为非竞争性抑制肽,抑制常数Ki,1=0.037 mmol/L;P2的氨基酸序列为Asn-Arg-Tyr-Leu-Arg(IC50值为14.68μg/m L),为竞争性抑制肽,其抑制常数Ki,2=0.020 mmol/L;P1、P2都能与ACE活性位点氨基酸残基形成氢键。P1、P2可以作为预防高血压的功能性食品,为蚕蛹蛋白的进一步开发利用提供理论基础。     

Primary and secondary structure of novel ACE-inhibitory peptides from egg white protein

The primary structure of novel angiotensin converting enzyme (ACE) inhibitory peptide from egg white protein was investigated, and secondary structure of the peptide was explored for the first time. The potential effects of bioactive peptides were submitted to bioactivity screening with ACE inhibitory activity, antioxidant property, and anticoagulation activity. Bioactive peptides from egg white protein were characterized by LC tandem mass spectrometric, and secondary structures of those peptides were investigated by FT-IR. Our results showed that total 11 bioactive peptides with three new and eight known structures were identified with LC/MS/MS, which then were synthesized by Fmoc solid phase method. Peptide Thr-Asn-Gly-Ile-Ile-Arg (TNGIIR) exhibited higher activity against ACE to other two new peptides. The concentration of the peptide TNGIIR, necessary to inhibit 50% the activity of ACE was 70 μM. Results also suggested that the secondary structural differences between peptides could also influence the ACE inhibition capacity. Thus, it appears that primary and secondary structure of peptide plays the potential role inhibiting the ACE activity.     

麦胚蛋白的双酶分步水解及其产物的抗氧化活性研究

前期实验确定中性蛋白酶为水解麦胚蛋白制备抗氧化肽的最适单酶,在此基础上进行了双酶组合水解麦胚蛋白的酶解工艺及酶解产物的抗氧化活性的探讨性研究。结果表明,双酶最佳组合为中性蛋白酶和碱性蛋白酶。经中性蛋白酶酶解270min的酶解液加入碱性蛋白酶后,水解至300min和420min时酶解产物还原能力和肽含量有不同程度增加,但DPPH·清除率始终是呈下降趋势。因此双酶水解比单酶水解麦胚蛋白没有明显优势。     

Indigenous marine diatoms as novel sources of bioactive peptides with antihypertensive and antioxidant properties

Several bioactive compounds from microalgae have demonstrated diverse biological activities with positive effects on human health. However, the potential of bioactive peptides as functional foods is still undervalued. Therefore, the exploration of microalgae strains as sources of bioactive peptides could reveal strong and unique bioactivities, especially when these marine sources have never been explored before. For this aim, protein extracts from six indigenous marine diatoms were subjected to enzymatic hydrolysis using four proteases (flavourzyme, pepsin, papain and trypsin). The hydrolysates were then tested for angiotensin converting enzyme (ACE)-inhibitory, antioxidant and antihypertensive properties. Results showed that papain hydrolysates from all microalgae strains exhibited strong ACE-inhibitory activities and antioxidant properties. In particular, protein hydrolysates from Bellerochea malleus were found to reduce blood pressure properties of 17 mmHg after 5 days of oral administration to SHR animals. These results revealed the potential of bioactive peptides from indigenous marine diatoms for use as functional foods or nutraceuticals.     

胰蛋白酶制备鹰嘴豆抗氧化肽的条件优化

以碱溶酸沉法制备的鹰嘴豆分离蛋白为酶解底物,以超氧阴离子自由基清除率为响应值,采用响应面法优化胰蛋白酶制备鹰嘴豆抗氧化活性肽的条件。结果表明：在整个酶解过程中,所制备的鹰嘴豆抗氧化肽具有显著的抗氧化活性,胰蛋白酶制备鹰嘴豆抗氧化肽的最佳酶解条件为：底物质量分数2%、加酶量（[E]/[S]）1 800 U/g、温度32 ℃、pH 7.0、时间35 min。此条件下所制备的鹰嘴豆抗氧化肽的超氧阴离子自由基清除率为67.59%。鹰嘴豆分离蛋白水解液经超滤膜系统纯化获得了截留相对分子质量1 kD以下的水解液,并通过液相色谱-质谱联用分析,发现了一种相对分子质量为668.40、序列为RQLPR的亲水性五肽。     

大米蛋白的木瓜酶酶解及其水解物的抗氧化活性

以大米蛋白为原料,研究其酶解工艺及其水解物的抗氧化活性.选取底物浓度、加酶量、酶解pH、酶解温度为考察因素,进行了酶解工艺的单因素及正交试验.试验结果表明,底物浓度([S])10%,加酶量([E] /[S])5%,酶解pH 6.0,酶解温度60℃,酶解时间90 min为最佳酶解参数,在此条件下大米蛋白水解物的固形物含量为25.6mg/mL,对DPPH自由基清除率为54.5%.抗氧化试验显示,大米蛋白水解物具有一定的清除DPPH自由基和羟自由基能力,其IC50分别为1.738和0.238mg/mL.大米蛋白水解物同样也具有较强的还原能力.由此得出,大米蛋白水解物是一种天然的抗氧化肽.     

乳清蛋白源生物活性肽段序列及其功能

乳清蛋白经酶解产生的肽类除了具有极高的营养价值之外．而且还具有一定的潜在生物活性。目前发现的乳清蛋白源生物活性肽主要有阿片肽、抗高血压肽、抗茵肽和免疫调节肽。综述了上述4种肽的氨基酸序列及其主要功能；对酶解过程中应注意的因素，如酶的类型、水解度、预处理方式、酶的灭活方式、底物纯度和水解过程等理化参数进行了归纳总结。     

Angiotensin-I converting enzyme inhibitory and antioxidant activities of egg protein hydrolysates produced with gastrointestinal and nongastrointestinal enzymes

Egg is a well-known rich source of bioactive peptides. In this study, egg protein (egg white and egg yolk proteins) hydrolysates were produced with gastrointestinal enzymes (pepsin and pancreatin) or nongastrointestinal enzymes (thermolysin and alcalase), and fractionated by ultrafiltration and cation exchange chromatography. Angiotensin-I converting enzyme (ACE) inhibitory and antioxidant activities, amino acid composition and molecular weight distribution were studied, and the physicochemical properties were related with the bioactivities. Our results showed that egg protein hydrolysates produced with non-GI enzymes (thermolysin and alcalase) showed significantly higher ACE inhibitory activity, whereas similar or even lower antioxidative activities, than those of hydrolysates produced with GI enzymes. ACE-inhibitory activity significantly correlated with the amino acid composition, especially the proportion of positively charged amino acid, whereas antioxidant activities correlated with the proportion of low molecular weight peptides under 500 Da. Understanding the relationship between the bioactivities and physicochemical properties of the hydrolysates/fractions is important to facilitate the development technologies for preparing fractions with improved bioactivities.     

酶法水解麦胚蛋白及其产物的抗氧化活性研究

选择碱性蛋白酶、中性蛋白酶、胰酶、木瓜蛋白酶和风味蛋白酶5种蛋白酶对麦胚蛋白进行水解,并考察其水解产物的抗氧化活性。结果表明,中性蛋白酶为制备麦胚抗氧化肽的最适蛋白酶,其最佳水解条件为:底物质量分数4%,酶添加量6 000U/g,酶解温度50℃,pH值7.5,水解至270min时抗氧化活性最大。     

鲤鱼蛋白控制酶解及其酶解产物抗氧化研究

以鲤鱼为原料,利用枯草杆菌蛋白酶对鲤鱼蛋白进行控制酶解,制取富含多肽的酶解液,并对其抗氧化性进行研究。以水解度、多肽含量、氨基酸含量、抗氧化性为指标,对自由基清除率评价其抗氧化性。通过单因素试验,研究酶解温度、pH值、酶用量、酶解时间、料液比等因素对酶解过程的影响,并进行三元二次回归设计,对最佳的酶解工艺条件进行优化。结果表明：以枯草杆菌蛋白酶对鲤鱼蛋白进行酶解,最佳工艺参数为酶解温度62℃、酶用量100U/g、料液比1:2(g/mL)、pH7.0、酶解4h。此条件下,酶解液多肽含量为0.704mg/mL,对羟自由基、超氧阴离子自由基的清除率分别为73.41%和59.78%,酶解液有很好的抗氧化性。     

A new approach for identification of novel antihypertensive peptides from egg proteins by QSAR and bioinformatics

Many protein-derived bioactive peptides were identified after extensive activity-guided purification, which is labor-intensive and costly. Furthermore, the rationale behind the selection of a substrate protein and a protease over others has not been justified in literature. The purpose of the study was to explore the rationale behind the selection of conditions for the production of potent angiotensin I converting enzyme (ACE) inhibitory peptides from egg proteins. Based on in silico digestion and quantitative structure and activity relationship (QSAR) model prediction, thermolysin-pepsin digestion of ovotransferrin was chosen as the best condition due to the presence of three potent peptides, Ile-Arg-Try, Leu-Lys-Pro and Ile-Gln-Try. To our surprise, sequences of Ile-Arg-Try-Cys-Thr, Leu-Lys-Pro-Ile and Ile-Gln-Try-Cys-Ala, but not Ile-Arg-Try, Leu-Lys-Pro and Ile-Gln-Try, were present in the hydrolysate. Further study showed that sonication or reducing agent pre-treatments could improve the activity of hydrolysates over 20 times and the predicted peptides were successfully released from sonication-treated ovotransferrin hydrolysate.