大豆生理活性肽的研究(Ⅱ)--抗氧化性和ACE抑制活性的初步研究

采用AS1.398和Alcalase两种蛋白酶,制备了水解度为10%～24%的大豆多肽,对其抗氧化性、ACE抑制活性和相对分子质量的分布进行了研究,结果表明采用AS1.398酶水解的DH为12%的产品抗氧化活性最高,添加量为6 mg/mL时使亚油酸的氧化诱导期延长2.92倍,其相对分子质量分布在1 000以上的组分较多;采用Alcalase酶水解的DH为14%的大豆多肽产品,ACE抑制活性最高,IC50为0.144 mg/mL,其相对分子质量分布大多在200～600之间.     

Biological activity of camel milk casein following enzymatic digestion

The aim of this study was to investigate the effects of enzymatic hydrolysis with digestive enzymes of camel whole casein and beta-casein (β-CN) on their antioxidant and Angiotensin Converting Enzyme (ACE)-inhibitory properties. Peptides in each hydrolysate were fractionated with ultra-filtration membranes. The antioxidant activity was determined using a Trolox equivalent antioxidant capacity (TEAC) scale. After enzymatic hydrolysis, both antioxidant and ACE-inhibitory activities of camel whole casein and camel β-CN were enhanced. Camel whole casein and β-CN showed significant ACE-inhibitory activities after hydrolysis with pepsin alone and after pepsinolysis followed by trypsinolysis and chymotrypsinolysis. Camel β-CN showed high antioxidant activity after hydrolysis with chymotrypsin. The results of this study suggest that when camel milk is consumed and digested, the produced peptides start to act as natural antioxidants and ACE-inhibitors.     

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.     

Proteolysis and antioxidant activity of peptic,tryptic and chymotryptic hydrolysates of cow,buffalo, goat and camel caseins

Caseins of cow, buffalo, goat and camel milks were hydrolysed using pepsin, trypsin and chymotrypsin. The rate and degree of casein hydrolysis and the antioxidant activity (AA) of the casein hydrolysates (CH) were followed. Camel casein showed the highest rate and degree of hydrolysis with pepsin and trypsin, while cow casein was more rapidly hydrolysed with chymotrypsin than other caseins studied. The AA of all CH increased to a maximum after 24 h of hydrolysis. The AA of tryptic hydrolysates was higher (P < 0.002) than that of peptic hydrolysates. Camel CH exhibited higher AA than hydrolysates of other caseins.     

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。     

Antidiabetic,angiotensin-converting enzyme inhibitory and anti-inflammatory activities of fermented camel milk and characterisation of novel bioactive peptides from lactic-fermented camel milk with molecular interaction study

This study was to separate and purify antidiabetic and angiotensin-converting enzyme (ACE) inhibitory peptides from Lactiplantibacillus plantarum KGL3A fermented camel milk. After 48 h of fermentation at 37°C, ɑ-amylase inhibition, ɑ-glucosidase inhibition, lipase inhibition and ACE inhibitory activities were 80.94%, 64.45%, 63.93%, and 77.53%, respectively in fermented camel milk. Optimisation of growth condition for the evaluation of maximum peptide production was evaluated by measuring proteolytic activity (O-phthalaldehyde, OPA method) with different inoculation rates and incubation times and highest proteolytic activity (9.21 mg/mL) was observed after 48 h of fermentation at 2.5% rate of inoculation. The antidiabetic and ACE inhibitory activity of 3 kDa permeate fraction were higher as compared with other fractions. Purification of antidiabetic and ACE inhibitory peptides from fermented camel milks was performed through sodium dodecyl-sulphate polyacrylamide gel electrophoresis, and 2-dimensional polyacrylamide gel electrophoresis and maximum number of protein bands were present in between 25 and 10 kDa. The generated peptide sequences were matched with antihypertensive peptide database (AHTPDB) and BIOPEP databases for confirming the antidiabetic and ACE inhibitory activity. Peptides, that is. TDVMPQWW and MMSLVSLLLVGILFPTIQAK were having highest peptide ranker score among the all sequences. Furthermore, anti-inflammatory activity (TNF-α, IL-6 and IL-1β) of fermented camel milk was evaluated in macrophage cell line RAW 264.7 (Ralph and William's cell line). Furthermore, peptides were predicted to have improved binding affinity against Human Angiotensin converting enzyme (hACE) through molecular docking.     

酶解与体外模拟胃肠消化对脱脂羊奶粉ACE抑制活性的影响

本研究旨在探究商业蛋白酶酶解与体外模拟胃肠消化对高消化率的脱脂羊奶粉释放ACE抑制肽的影响,以评价脱脂羊奶粉酶解必要性及所需酶解程度。采用中性蛋白酶、碱性蛋白酶、复合蛋白酶和风味蛋白酶对脱脂后的圭山羊奶粉进行酶解反应,并对脱脂羊奶粉及其酶解物进行体外模拟胃肠消化,测定酶解液及模拟胃肠消化液的多肽含量、ACE抑制活性以及分子量分布。研究发现,羊奶粉经过体外模拟胃肠消化后,消化液的多肽含量与ACE抑制率分别达到20.81 mg/mL和62.55%;而经过碱性蛋白酶和风味蛋白酶的适度酶解,其模拟胃肠消化液多肽含量与ACE抑制活性反而下降;经过复合蛋白酶的适度酶解,其模拟胃肠消化液的多肽含量与ACE抑制活性小幅度提高,分别达到22.67 mg/mL和69.29%;经过中性蛋白酶的适度酶解,其模拟胃肠消化液多肽含量与ACE抑制活性均显著提高,分别达到23.76 mg/mL和81.10%。分子量分布结果显示,经过体外模拟胃肠消化后,<1000 Da的小分子肽由酶解液中的70.77%提高到90%。综上,圭山羊奶粉经过体内胃肠消化就可以释放出较多的ACE抑制肽,而经过中性蛋白酶酶解至水解度8%后...     

菜籽水解蛋白不同组分的体外功能性质研究

初步分析了水酶法工艺得到的菜籽水解蛋白的组成、体外抗氧化活性和抑制ACE活性,并用凝胶过滤色谱法分离水解蛋白得到不同组分,探讨了不同组分的体外抗氧化活性和抑制ACE活性与组分氨基酸组成、分子量分布的关系。结果表明,水解蛋白具有良好的体外功能性质,经Sephadex G-25分离后的9个峰中,峰2、峰4和峰5具有较好的体外功能性质,它们的分子量分布中90%以上低于1000,且含有较高量的疏水性氨基酸。     

Evaluation of angiotensin I-converting enzyme (ACE) inhibitory activities of hydrolysates generated from byproducts of freshwater clam

Solid wastes of freshwater clam in food processing, including mainly mantle, were used as a raw material for the recovery of bioactive peptides related to angiotensin I-converting enzyme (ACE) inhibitor. Among the primary hydrolysates of dried mantle (DM), the peptides corresponding to hydrolysates using 2 crude peptidases exhibited a strong ACE inhibitory activity (IC₅₀, 0.23 mg/mL), and recovery efficiency of soluble materials and their protein content were considerably large with 42.65% and 468.6 mg/g, respectively. The ACE inhibitory activity of all secondary hydrolysates digested by pepsin and trypsin was significantly increased as compared to primary hydrolysates. Furthermore, the peptic secondary hydrolysates were fractionated by gel filtration and reverse phase-HPLC (RP-HPLC) and characterized by matrix-assisted laser desorption-ionization time of flight/mass spectrometry (MALDI-TOF/MS). These peptides with molecular weight of less than 1 kDa possessed the stronger ACE inhibitory effect, and their inhibitory pattern was found to be competitive. The results showed that the DM hydoplysates might be utilized as a rich source of bioactive peptide.     

Camel milk protein hydrolysates with improved technofunctional properties and enhanced antioxidant potential in in vitro and in food model systems

Camel milk protein hydrolysates (CMPH) were generated using proteolytic enzymes, such as alcalase, bromelain, and papain, to explore the effect on the technofunctional properties and antioxidant potential under in vitro and in real food model systems. Characterization of the CMPH via degree of hydrolysis, sodium dodecyl sulfate-PAGE, and HPLC revealed that different proteins in camel milk underwent degradation at different degrees after enzymatic hydrolysis using 3 different enzymes for 2, 4, and 6 h, with papain displaying the highest degradation. Technofunctional properties, such as emulsifying activity index, surface hydrophobicity, and protein solubility, were higher in CMPH than unhydrolyzed camel milk proteins. However, the water and fat absorption capacity were lower in CMPH compared with unhydrolyzed camel milk proteins. Antioxidant properties as assessed by 2,2-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) and 2,2-diphenyl-1-picrylhydrazyl radical scavenging activities and metal-chelating activity were enhanced after hydrolysis, in contrast to ferric-reducing antioxidant power which showed a decrease after hydrolysis. The CMPH were also tested in real food model systems for their potential to inhibit lipid peroxidation in fish mince and grape seed oil-in-water emulsion, and we found that papain-produced hydrolysate displayed higher inhibition than alcalase- and bromelain-produced hydrolysates. Therefore, the CMPH demonstrated effective antioxidant potential in vitro as well as in real food systems and showed enhanced functional properties, which guarantees their potential applications in functional foods. The present study is one of few reports available on CMPH being explored in vitro as well as in real food model systems.     

In vitro antidiabetic and antihypercholesterolemic activities of camel milk protein hydrolysates derived upon simulated gastrointestinal digestion of milk from different camel breeds

Milk protein hydrolysates derived from 4 camel breeds (Pakistani, Saheli, Hozami, and Omani) were evaluated for in vitro inhibition of antidiabetic enzymatic markers (dipeptidyl peptidase IV and α-amylase) and antihypercholesterolemic enzymatic markers (pancreatic lipase and cholesterol esterase). Milk samples were subjected to in vitro simulated gastric (SGD) and gastrointestinal digestion (SGID) conditions. In comparison with intact milk proteins, the SGD-derived milk protein hydrolysates showed enhanced inhibition of α-amylase, dipeptidyl peptidase IV, pancreatic lipase, and cholesterol esterase as reflected by lower half-maximal inhibitory concentration values. Overall, milk protein hydrolysates derived from the milk of Hozami and Omani camel breeds displayed higher inhibition of different enzymatic markers compared with milk protein hydrolysates from Pakistani and Saheli breeds. In vitro SGD and SGID processes significantly increased the bioactive properties of milk from all camel breeds. Milk protein hydrolysates from different camel breeds showed significant variations for inhibition of antidiabetic and antihypercholesterolemic enzymatic markers, suggesting the importance of breed selection for production of bioactive peptides. However, further studies on identifying the peptides generated upon SGD and SGID of milk from different camel breeds are needed.     

Bioactivity of hydrolysates obtained from bovine casein using artichoke (Cynara scolymus L.) proteases

The objective of this work was to obtain casein hydrolysates with aspartic proteinases present in extracts from the artichoke flower (Cynara scolymus L.) and evaluate their antioxidant, antimicrobial, and angiotensin-I converting enzyme (ACE) inhibitory activity in vitro. The casein hydrolysates produced by the action of C. scolymus had elevated antihypertensive and antioxidant activity due to their high hydrophobic peptide content (93.84, 96.58, and 90.54% at 2, 4, and 16 h of hydrolysis, respectively). Hydrolysis time and molecular weight (<3 kDa) had a significant influence on the hypertensive and antioxidant activity of the hydrolysates, which were greater at hydrolysis times of 4 and 16 h and corresponding to the <3 kDa fractions. The <3 kDa fraction of the 16 h hydrolysate had an ACE inhibitory activity with a half-maximal inhibitory concentration (IC₅₀) of 71.77 µg peptides per mL; DPPH and ABTS^•+ radical scavenging activities of 6.27 µM and 6.21 mM Trolox equivalents per mg of peptides, respectively; and iron (II) chelation activity with an IC₅₀ of 221.49 µg of peptides per mL. Antimicrobial activity against Enterococcus faecalis was also observed in the hydrolysates. From the peptide sequences identified in the hydrolysates, we detected 22 peptides (from the BIOPEP database) that were already in their bioactive form (AMKPWIQPK, AMKPWIQPKTKVIPYVRYL, ARHPHPHLSFM, DAQSAPLRVY, FFVAPFPEVFGK, GPVRGPFPII, KVLPVPQK, LLYQEPVLGPVRGPFPIIV, MAIPPKKNQDK, NLHLPLPLL, PAAVRSPAQILQ, RELEELNVPGEIVESLSSSEESITR, RPKHPIKHQ, RPKHPIKHQGLPQEVLNENLLRF, SDIPNPIGSENSEK, TPVVVPPFLQP, VENLHLPLPLL, VKEAMAPK, VLNENLLR, VYPFPGPIH, VYQHQKAMKPWIQPKTKVIPYVRY, VYQHQKAMKPWIQPKTKVIPYVRYL) and are reported to display antioxidant, antimicrobial, and ACE inhibitory activity. We also identified 12,116, 14,513, and 25,169 peptide sequences in the hydrolysates at 2, 4, and 16 h, respectively, that were contained in the primary sequence, and these are reported to display ACE inhibitory, antioxidant, dipeptidyl peptidase IV inhibition, antithrombotic, opioid, immunomodulation, antiamnesic, anticancer, chelating, and hemolytic bioactivity.     

谷朊粉酶解物的制备及其ACE抑制肽的分离鉴定

以谷朊粉为原料,以血管紧张素转换酶（ACE）抑制率为指标,比较4种蛋白酶的酶解效果。采用超滤、葡聚糖凝胶色谱、反相高效液相色谱（RP-HPLC）的方法分离纯化ACE抑制肽并用电喷雾飞行时间质谱联用(ESI-TOF-MS)鉴定其结构。结果表明：碱性蛋白酶酶解3 h的谷朊粉酶解物具有较高的ACE抑制活性;超滤后,分子质量Mr<1 ku的组分具有较高的ACE抑制率;分离纯化后得到小肽的ACE抑制率高达（81.03±1.20）%;由ESI-TOF-MS质谱图得出该小肽的m/z为630.89,氨基酸序列为Trp-Phe-Gln-Pro（WFQP）。     

Influence of ultrasonic pretreatment on structural properties and biological activities of lupin protein hydrolysate

This study examined how application of ultrasound pretreatment (UP) (400 W, 20 kHz for 5, 10 min) influenced the structural characteristics, molecular weight distribution, amino acid composition and biological activity of hydrolysate prepared from lupin protein using protamex enzyme. Circular dichroism (CD) and Fourier transform infrared (FTIR) techniques revealed changes in secondary structure after UP, while changes in molecular weight pattern were revealed by SDS-PAGE and MALDI-TOF analyses. Also, the crystallinity and surface hydrophobicity of the hydrolysates were affected by UP. The amino acid compositions of the hydrolysate varied, especially glutamic acid, arginine and aspartic acid. In vitro biological assays showed that antioxidant, α-glucosidase, α-amylase and ACE inhibitory activities significantly improved in sonicated hydrolysates compared with non-sonicated samples, and this may be linked to the structural modifications caused by UP. Hence, the application of UP to lupin protein could en hance the biological activity of its hydrolysate.     

燕麦麸蛋白ACE抑制肽的制备及性质研究

以燕麦麸为原料制备了燕麦麸蛋白,并利用蛋白酶对其酶解。测定了不同蛋白酶的水解产物的ACE抑制活性,以Alcalase和Trypsin酶解物活性最强。在此基础上进一步考察了Alcalase和Trypsin酶解物的DH对ACE抑制活性的影响,结果表明DH11.0%的Alcalase酶解物和DH12.2%的Trypsin酶解物有最大ACE抑制活性,分别达92.31%和86.36%。两种酶解物的肽组分相对分子质量均较低,大部分都低于1 000u。氨基酸分析表明ACE抑制肽中富含Glu、Leu、Pro和Phe。为了达到活性组分富集的目的,超滤技术是一种切实可行的方案。     

鲣鱼蛋白肽谱效关系研究

采用鲣鱼为原料,以蛋白质回收率为指标,酶解制备不同工艺条件的样品。通过测定其黄嘌呤氧化酶(XOD)抑制活性、ORAC值、分子质量分布图谱和总氨基酸含量,构建鲣鱼蛋白肽体外化学活性与其分子质量分布图谱、总氨基酸含量图谱之间的谱效关系。结合超高效液相-质谱联用(LC-MS/MS)方法研究其降尿酸肽和抗氧化肽的分布。研究表明,鲣鱼蛋白肽的XOD抑制活性与其ORAC值具有显著的相关性。由多肽分子质量<1ku所占含量的分布区段和脂肪族氨基酸的含量可初步预测鲣鱼蛋白肽的化学活性。质谱分析表明,纯化组分的质谱基峰图在不同时间段的信号强度与鲣鱼蛋白肽的降尿酸活性和抗氧化活性具有明显的相关性。     

体外模拟消化对大米谷蛋白结构及水解产物生物活性的影响

通过体外模拟消化,研究不同消化时间下谷蛋白结构与抗氧化和血管紧张素转换酶(angiotensin-converting enzyme,ACE)抑制活性的关系.利用内源荧光光谱和傅里叶变换红光谱测定谷蛋白水解过程结构变化.通过测定水解产物抗氧化能力和ACE抑制率表征酶解产物的活性,利用四极杆串联飞行时间质谱鉴定具有抗氧化...     

条斑紫菜蛋白酶解物降血压活性

研究了以条斑紫菜为原料,采用木瓜蛋白酶制备紫菜蛋白活性寡肽,以ACE抑制率为评价指标,研究不同水解条件下酶解液的降血压活性,优化酶解工艺条件并测定酶解物的分子量。优化后的木瓜蛋白酶酶解工艺条件为pH7.5,温度50℃,底物浓度30 mg/mL,酶添加量600 U/g,在此条件下,水解4 h的酶解产物抑制活性达30%以上,ACE抑制肽的半抑制浓度IC50值为4.48 mg/mL。将制备的酶解液进行层析分析,测得具有降血压活性的紫菜蛋白酶解产物是相对分子质量小于2 000的活性肽。     

酶解鲢肉制取ACE抑制肽工艺条件的优化

采用复合风味酶水解鲢(Hypophthalmichthys molitrix)肉制取血管紧张素转化酶(ACE)抑制肽,通过正交试验对水解工艺进行优化,并用SephadexG-15凝胶柱对酶解产物进行分离。结果表明:复合风味酶在温度50℃、pH8.0、料液比1:6、加酶量5000U/g.pr、水解12h的条件下,酶解产物的水解程度及对ACE的抑制率最高,其水解度为34.40%,氮利用率为92.01%,抑制率为66.52%;该酶解产物中相对分子质量较大和较小部分的ACE抑制活性偏低,只有相对分子量在一定范围内的短肽才对ACE具有较好的抑制作用,其中在第3洗脱峰处得到的ACE抑制肽活性最高,其ACE抑制率为63.04%。     

酪朊酸钠制备ACE抑制肽的研究

采用商业化的胰蛋白酶对底物浓度为10％的酪朊酸钠进行可控酶解，将酶解液的上清液经截留分子量为10ku的超滤膜分离后利川紫外分光光度法测定各组分ACE体外抑制活性，结果表明酪朊酸钠的胰蛋白酶酶解液具有较强的ACE抑制活性，超滤分离可提高产品的ACE体外抑制活性，为开发新一代降血压保健食品提供广阔的前景。