Fractionation of angiotensin I converting enzyme inhibitory activity from pea and whey protein in vitro gastrointestinal digests

In vitro gastrointestinal digestion of pea and whey protein produced high angiotensin I converting enzyme (ACE) inhibitory activity with IC₅₀ values of 0.070 and 0.041 mg protein ml^?1 respectively. Ultrafiltration/centrifugation using a membrane with a molecular weight cut‐off of 3000 Da decreased the IC₅₀ value to 0.055 mg protein ml^?1 for pea permeate and 0.014 mg protein ml^?1 for whey permeate. Further fractionation by reverse phase HPLC gave IC₅₀ values as low as 0.016 mg protein ml^?1 for pea and 0.003 mg protein ml^?1 for whey. Consequently, these purification steps enriched the ACE inhibitory activity of the pea digest more than four times and that of the whey digest more than 13 times. HPLC profiles after digestion and ultrafiltration indicate that high ACE inhibitory activity is due to short and more hydrophobic peptides. The results also suggest that potent ACE inhibitory peptides were present alongside low active peptides in whey hydrolysate, while all peptides had more or less the same ACE inhibitory activity in pea hydrolysate. In addition, the hydrolysates and enriched fractions will resist in vivo gastrointestinal digestion after oral administration. Hence these ACE inhibitory peptides, as part of functional foods, can play significant roles in the prevention and treatment of hypertension. Copyright © 2004 Society of Chemical Industry     

食源蛋白制备血管紧张素抑制肽的研究进展

血管紧张素转化酶(angiotensin I converting enzyme,简称ACE)对于人体血压的调节有着十分重要的作用。血管紧张素转化酶抑制肽(ACEIP)是从食源性蛋白质中提取的功能性肽,有着抑制ACE的作用,从而降低人体血压,达到治疗高血压的目的。本文主要从ACE抑制肽的降压原理、来源、结构、分类及作用特点和前景展望这几方面对其进行总结阐述。     

海洋蛋白源ACE抑制肽研究进展

血管紧张素转化酶(ACE)抑制肽在血压调节中起着重要作用,食源性ACE抑制肽具有较强降血压效果,是调节血压的潜在活性成分。海洋蛋白源ACE抑制肽是食源性活性肽的重要组成部分,是未来研究的热点。本文综述了海洋蛋白源ACE抑制肽的制备、纯化、结构鉴定、体外和体内活性研究及构效关系等研究进展,同时对海洋蛋白源ACE抑制肽的研究和应用前景进行展望。      

Angiotensin I‐converting enzyme inhibitory peptides FQPSF and LKYPI identified in Bacillus subtilis A26 hydrolysate of thornback ray muscle

Angiotensin I‐converting enzyme (ACE) inhibitory peptides have been searched in thornback ray (Raja clavata) muscle hydrolysed with Bacillus subtilis A26 proteases until a hydrolysis degree of 18.35%. The hydrolysate showed an IC₅₀ of 0.83 mg mL^?1. To identify peptides responsible for this activity, the extract was eluted through size‐exclusion chromatography and fractions collected. The highest ACE inhibitory activity was found for fractions F2 and F3 which had IC₅₀ of 0.42 and 0.51 mg mL^?1, respectively. These fractions were analysed by nano‐liquid chromatography coupled to tandem mass spectrometry (nLC‐MS/MS). A total of 131 and 108 peptide sequences mainly derived from actin, myosin heavy chain and procollagen alpha 1 chain proteins were identified in fractions F2 and F3, respectively. FQPSF and LKYPI showed the best results with an IC₅₀ of 12.56 and 27.07 μM, respectively. These results prove the potential of thornback ray muscle hydrolysate as a source of ACE inhibitory peptides.     

醋蛋多肽血管紧张素转化酶抑制活性的稳定性研究

研究了温度、pH、金属离子、食盐及食品中常见糖类和防腐剂对醋蛋多肽血管紧张素转化酶(ACE)抑制活性的影响。结果表明:醋蛋多肽的ACE抑制活性对热不稳定,对pH稳定;当金属离子浓度达到5mmol/L时,醋蛋多肽的ACE抑制活性有较明显的下降,其影响大小顺序为K+>Zn2+>Cu2+>Ca2+>Mg2+;食盐能降低醋蛋多肽的ACE抑制活性;在实验浓度范围内,葡萄糖、蔗糖、乳糖、苯甲酸和山梨酸对醋蛋多肽ACE抑制活性影响不大。     

Identification of angiotensin converting enzyme inhibitory and antioxidant peptides in a whey protein concentrate hydrolysate produced at semi‐pilot scale

Antioxidant and angiotensin converting enzyme (ACE) inhibitory peptides were identified in a 5 kDa ultrafiltration permeate of a whey protein hydrolysate generated at semi‐pilot scale. Further laboratory scale ultrafiltration of this 5 kDa permeate resulted in a 0.65 kDa permeate with antioxidant, (1.11 ± 0.074 μmol TE per mg dry weight, oxygen radical absorbance capacity, ORAC) and ACE inhibitory (ACE IC₅₀ 0.215 ± 0.043 mg mL^?1) activities. Semi‐preparative (SP) reverse phase high‐performance liquid chromatography (RP‐HPLC) of the 0.65 kDa permeate resulted in a fraction (SP_F3) with a 4.4‐fold increase in ORAC activity (4.83 ± 0.45 μmol TE mg dry weight) and a 1.3‐fold increase in ACE inhibitory activity (84.35 ± 1.36% inhibition when assayed at 0.28 mg mL^?1). Peptides within SP_F3 were identified using UPLC‐ESI‐MS/MS. Met‐Pro‐Ile had the highest ORAC activity (205.75 ± 12.08 μmol TE per mmol peptide) while Met‐Ala‐Ala and Val‐Ala‐Gly‐Thr had the highest ACE inhibitory activities (IC₅₀:515.50 ± 1.11 and 610.30 ± 2.41 μm , respectively).     

Angiotensin I‐converting enzyme inhibitory activity of protein hydrolysates prepared from three freshwater carps (Catla catla,Labeo rohita and Cirrhinus mrigala) using Flavorzyme

Fish protein hydrolysates from three freshwater carps, Catla catla, Labeo rohita and Cirrhinus mrigala with different degree of hydrolysis (DH) (5%, 10%, 15% and 20%), were prepared using Flavorzyme enzyme and designated as HCF, HRF and HMF, respectively. The angiotensin I‐converting enzyme (ACE) inhibitory activity of hydrolysates was found to vary from 43 ± 2% to 71 ± 3%. Based on ACE inhibitory activity, HRF with DH‐15% was taken up for further study. The mode of ACE activity inhibition by HRF‐DH 15% was mixed type as revealed by Lineweaver–Burk plot. Sequential digestion of HRF‐DH 15% using pepsin and pancreatin decreased the ACE inhibitory activity from 76% to 63%. Partial purification of HRF‐DH 15% by size exclusion chromatography gave three different fractions designated as F‐1, F‐2 and F‐3 with the molecular mass in the range of 6456–407 Da. Fraction 2 had significantly higher ACE inhibitory activity than the other fractions.     

Isolation,purification and characterisation of angiotensin I‐converting enzyme–inhibitory peptides derived from catfish (Clarias batrachus) muscle protein thermolysin hydrolysates

The angiotensin I‐converting enzyme (ACE)‐inhibitory activities of catfish (Clarias batrachus) muscle protein hydrolysates were investigated. Thermolytic digests of C. batrachus sarcoplasmic and myofibrillar proteins exhibited inhibitory activity towards ACE and were purified with the aim of ultrafiltration, gel filtration and reversed‐phase high‐performance liquid chromatography (RP‐HPLC). The amino acid sequences of hydrolysates with the highest ACE‐inhibitory activities were determined using electrospray quadrupole time‐of‐flight tandem mass spectrometry (ESI‐TOFQ MS/MS). The sequences of GPPP (IC₅₀ = 0.86 μm ) and IEKPP (IC₅₀ = 1.2 μm ) corresponding to the fragments 986–989 and 441–445 of myosin‐I heavy chain were identified for the sarcoplasmic and myofibrillar protein hydrolysates, respectively. Peptide GPPP exhibited a mixed‐type inhibition whereas peptide IEKPP could only bind to the active sites of ACE. The results demonstrate that hydrolysates of C. batrachus muscle proteins obtained by thermolysin may contain bioactive peptides.     

Angiotensin I‐converting enzyme inhibitory and Ca‐binding activities of peptides prepared from tuna cooking juice and spleen proteases

Tuna cooking juice is a by‐product from the tuna canning industry. In this study, tuna cooking juice was hydrolysed by proteases extracted from the spleen. Tuna cooking juice showed the highest ACE inhibitory and Ca‐binding activities after hydrolysis for 270 and 180 min, respectively. The hydrolysate was further fractionated by ultrafiltration. The permeate exhibited highest ACE inhibitory and Ca‐binding activities when passed through 1 and 5 kDa cut‐off membranes, respectively. Gel filtration chromatography was used to determine the MW of bioactive peptides that exhibited highest ACE inhibitory and Ca‐binding activities. Those peptides that exhibited highest ACE inhibitory and Ca‐binding activities were the MW range of 238–829 Da and 1355–1880 Da, respectively. These results suggest that the tuna cooking juice and the spleen protease extract are a potential source of bioactive peptides that can be utilised as bioactive ingredients in functional food and nutraceuticals.     

酪蛋白源ACE抑制肽干燥工艺的探讨

喷雾干燥技术和冷冻干燥技术对酪蛋白源ACE抑制肽的抑制活性影响差异不显著(P>0.05),通过比较不同的喷雾干燥工艺条件,确定其进口温度范围为140～160℃,出口温度范围为70～90℃。1L小试和10L小试放大试验结果证实,肽得率稳定,为29%左右,半抑制质量浓度(IC50)为0.53g/L。     

复合酶解乳清蛋白制备降血压肽的研究

采用碱性蛋白酶、胰蛋白酶水解乳清蛋白制备ACE抑制肽,通过体外检测法测定其ACE抑制率。通过正交试验,得出最优水解条件,即碱性蛋白酶和胰蛋白酶[E1/E2]之比为5:4,温度为45℃,pH值为8.0,时间为150min,水解度11.92%的条件下,乳清蛋白肽对ACE的抑制能力最强,达到60.19%。     

Proteolytic and angiotensin‐converting enzyme‐inhibitory activities of selected probiotic bacteria

This study was carried out to examine the proteolytic and angiotensin‐converting enzyme (ACE‐I) activities of probiotic lactic acid bacteria (LAB) as influenced by the type of media, fermentation time, strain type and media supplementation with a proteolytic enzyme (Flavourzyme^®). Lactobacillus casei (Lc210), Bifidobacterium animalis ssp12 (Bb12), Lactobacillus delbrueckii subsp. bulgaricus (Lb11842) and Lactobacillus acidophilus (La2410) were grown in 12% of reconstituted skim milk (RSM) or 4% of whey protein concentrates (WPC‐35) with or without combination (0.14%) of Flavourzyme^® for 12 h at 37 °C. All the strains were able to grow in both media depending on type of strains used and fermentation time. All the strains showed higher proteolytic activity and produced more antihypersensitive peptides when grown in RSM medium at 12 h, when compared to WPC. Combination with Flavourzyme^® also increased LAB growth and proteolytic and ACE‐I activities. Of the four strains used, Bb12 and La2410 outperformed Lc210 and Lb11842. The highest ACE‐I activity and proteolytic activity were found in B. animalis ssp12 combined with Flavourzyme^®. Flavourzyme^® led to an increase in the production of bioactive peptides with ACE‐I activity during 12 h at 37 °C.     

Influence of degree of hydrolysis on functional properties and angiotensin I‐converting enzyme‐inhibitory activity of protein hydrolysates from cuttlefish (Sepia officinalis) by‐products

BACKGROUND: In Tunisia the cuttlefish‐processing industry generates large amounts of solid wastes. These wastes, which may represent 35% of the original material and constitute an important source of proteins, are discarded without any attempt at recovery. This paper describes some functional properties and the angiotensin I‐converting enzyme (ACE)‐inhibitory activity of protein hydrolysates prepared by hydrolysis of cuttlefish (Sepia officinalis) by‐products with crude enzyme extract from Bacillus licheniformis NH1. RESULTS: Cuttlefish by‐product protein hydrolysates (CPHs) with different degrees of hydrolysis (DH 5, 10 and 13.5%) were prepared. All CPHs contained 750–790 g kg^?1 proteins. Solubility, emulsifying capacity and water‐holding capacity increased while fat absorption and foaming capacity decreased with increasing DH. All hydrolysates showed greater fat absorption than the water‐soluble fraction from undigested cuttlefish by‐product proteins and casein. CPHs were also analysed for their ACE‐inhibitory activity. CPH3 (DH 13.5%) displayed the highest ACE inhibition (79%), with an IC₅₀ value of 1 mg mL^?1. CONCLUSION: Hydrolysis of cuttlefish by‐product proteins with alkaline proteases from B. licheniformis resulted in a product with excellent solubility over a wide pH range and high ACE‐inhibitory activity. This study suggests that CPHs could be utilised to develop functional foods for prevention of hypertension. Copyright © 2010 Society of Chemical Industry     

Purification and characterisation of angiotensin I converting enzyme inhibitory peptides from lysozyme hydrolysates

Hen egg white lysozyme (HEWL) was hydrolysed with trypsin, papain and a combination of the two. The prepared hydrolysates exhibited ACE inhibitory activity. The hydrolysates were fractionated using ultrafiltration and reverse phase-high performance liquid chromatography (RP-HPLC). Three fractions, which showed the highest ACE inhibitory activities, were purified by RP-HPLC. They were the F₇ (from papain-trypsin hydrolysate), F₈ (from papain hydrolysate) and F₃ (from trypsin hydrolysate) fractions. The IC₅₀ values were 0.03, 0.155 and 0.23 mg/ml for F₇, F₈ and F₃, respectively. The F₇ fraction was the most potent ACE inhibitor peptide, and was composed of 12 amino acids, Phe-Glu-Ser-Asn-Phe-Asn-Thr-Gln-Ala-Thr-Asn-Arg (MW: 1428.6 Da). Lineweaver-Burk plots suggest that the F₇ peptide acts as an uncompetitive inhibitor against ACE. The kinetic parameters (K_m, V_max, and K_i) for the F₇ peptide were measured and compared to the control.     

Grass carp peptides hydrolysed by the combination of Alcalase and Neutrase: Angiotensin‐I converting enzyme (ACE) inhibitory activity,antioxidant activities and physicochemical profiles

In this study, grass carp peptides were prepared by enzymatic hydrolysis of grass carp protein using the combination of Alcalase and Neutrase, and angiotensin‐I converting enzyme (ACE) inhibitory activity in vitro, antihypertensive activity in vivo, antioxidant activities, and physicochemical properties of peptides achieved from grass carp protein were characterised after ultrafiltration and desalted processes using mixed ion exchange resins. The purified peptides exhibited strong ACE inhibitory activity (IC₅₀ = 105 μg mL^?1), antihypertensive activity with the maximal drop for systolic blood pressure (SBP) of 43 mmHg at a dosage of 100 mg per kg body weight in spontaneously hypertensive rat (SHR), and antioxidant activities indicated by thiobarbituric acid‐reactive substance values in a liposome‐oxidising system, radical‐scavenging activity and chelation of metal ions (Fe²⁺). The molecular weight of peptides was <1000 Da. Compared to grass carp protein, the peptides separated from enzymatic hydrolysates possessed similar amino acid compositions, but contained higher concentrations of essential amino acids. Moreover, the peptides exhibited excellent solubility at a wide range of pH values from 2 to 10, and lower apparent viscosity than the protein. The peptides separated from enzymatic hydrolysates might be used as a promising ingredient in antihypertensive functional foods and nutraceuticals.     

Effect of angiotensin I converting enzyme inhibitory peptide purified from skate skin hydrolysate

Our objective was to evaluate the angiotensin I converting enzyme (ACE) inhibitory activity of skate skin protein hydrolysates and its corresponding fractions. The skate skin hydrolysates were obtained by enzymatic hydrolysis using alcalase, α-chymotrypsin, neutrase, pepsin, papain, and trypsin. Amongst the six hydrolysates, the α-chymotrypsin hydrolysate had the highest ACE inhibitory activity compared to other hydrolysates. The amino acid sequences of the purified peptides were identified to be Pro–Gly–Pro–Leu–Gly–Leu–Thr–Gly–Pro (975.38 Da), and Gln–Leu–Gly–Phe–Leu–Gly–Pro–Arg (874.45 Da). The purified peptides from skate skin had an IC₅₀ value of 95 μM and 148 μM, respectively, and the Lineweaver–Burk plots suggest that they act as a non-competitive inhibitor against ACE. Our study suggested that novel ACE inhibitory peptides derived from skate skin protein may be beneficial as anti-hypertension compounds in functional foods.     

Effect of seaweed flakes addition on the development of bioactivities in functional Camembert‐type cheese

The effect of adding Palmaria palmata or Saccharina longicruris to Camembert‐type cheese on both the antioxidant capacity (ORAC) and Angiotensin I‐converting enzyme (ACE)‐inhibitory activity has been studied with the aim of developing a functional food. The nutritional composition showed that P. palmata had the highest total protein and carbohydrate contents while S. longicruris demonstrated the highest total fibre and minerals contents. The bioactivities determined in the S. longicruris soluble extract were the highest. Three cheese model curds were studied: cheese control (CC), 2% of P. palmata (C2PP) and 2% of S. longicruris (C2SL). During ripening (20 days), the curd pH of all treatments was significantly similar, as their ORAC values, starting at 0 and reaching 41.28 mmol TE g^?1. The ACE‐inhibitory activity of the three treatments was significantly similar, at day 0 (13.20%) and day 20 (58.27%). The feasibility of including these two seaweeds into Camembert‐type cheeses was validated through the adequate development of bioactivities during ripening supporting promising food applications.     

Isolation and characterization of angiotensin I‐converting enzyme inhibitory peptides from wheat gliadin hydrolysate

Angiotensin I‐converting enzyme (ACE) inhibitory peptide was isolated from wheat gliadin hydrolysate prepared with acid protease. Consecutive purification methods were used for peptide isolation including ion‐exchange chromatography, size‐exclusion chromatography, and reverse‐phase high‐performance liquid chromatography. The amino acid sequence of this peptide was identified as Ile‐Ala‐Pro, and the ACE inhibitory activity (IC₅₀ value) was 2.7 μM . The hypotensive activity of Ile‐Ala‐Pro on spontaneously hypertensive rats was investigated. This peptide inhibited the hypertensive activity of angiotensin I with intravenous injection, and decreased the blood pressure significantly with intraperitoneal administration.     

乳清蛋白酶解ACE抑制肽分离纯化技术的研究

采用6000 u的超滤膜对乳清蛋白水解物中的ACE抑制肽进行了初步分离,确定了超滤的条件,并测定超滤前后水解物的ACE抑制率,结果表明通过超滤可以富积乳清蛋白水解物中的ACE抑制肽.采用阳离子交换树脂对水解物进行脱盐,通过测定水解物的氮回收率、脱盐率以及ACE抑制率,表明采用阳离子交换树脂脱盐率可以达到80%以上,氮的回收率达到90%以上,而水解物经过阳离子交换树脂基本上对其ACE抑制率没有影响.     

Fermentation with Bacillus spp. as a bioprocess to enhance anthocyanin content,the angiotensin converting enzyme inhibitory effect,and the reducing activity of black soybeans

Food possessing anthocyanins, angiotensin converting enzyme (ACE) inhibitory activity or reducing activity show beneficial effect on human health. To develop healthy food, black soybeans were fermented with either Bacillus subtilis BCRC 14715 or Bacillus sp. CN11, or a mixture of both Bacillus spp. in the present study. The anthocyanin content, the ACE inhibitory activity and the reducing power of the fermented black soybean were then examined. It was found that the ACE inhibitory activity of the extracts of bean and viscous material from the fermented black soybeans varied with extraction solvents and starter organism, yet increased as the fermentation period was extended, regardless of starter organism. After 18 h of fermentation, the water extract of bean showed less ACE inhibitory activity than did the respective 80% ethanol extract. While the water extract of viscous material showed a higher ACE inhibitory activity than the respective ethanol extract. With respect to extraction yield, it was found that the ACE inhibitor in the fermented black soybean could be extracted more efficiently with water than 80% ethanol. Fermentation with B. subtilis BCRC 14715 was also found to increase the anthocyanin content of black soybean and the reducing activity of the extracts. Finally, the 80% ethanol extract showed a higher reducing activity than the water extract.