Identification of antioxidant and ACE‐inhibitory peptides in fermented milk

The angiotensin‐converting enzyme (ACE)‐inhibitory activity and antioxidant properties of a commercial fermented milk from Europe were evaluated. This dairy product showed moderate ACE‐inhibitory activity and ABTS^?+ radical‐scavenging capacity. The peptides from most active fractions collected by reverse phase high‐performance liquid chromatography (RP‐HPLC) were sequenced by RP‐HPLC–tandem mass spectrometry. This technique allowed rapid identification of peptides included in the most active fractions, and various potentially active peptides were recognised according to previous studies of structure–activity relationship. Three of the identified sequences had previously been described as potent ACE inhibitors. The structure of some sequences substantiated the presence of peptides with ACE‐inhibitory, antioxidant and immunomodulatory activities. Copyright © 2005 Society of Chemical Industry     

Improvement of the ACE‐inhibitory and DPPH radical scavenging activities of soya protein hydrolysates through pepsin pretreatment

This study aimed to investigate the effect of pepsin pretreatment on the ACE‐inhibitory and DPPH radical scavenging activities of soya protein hydrolysates prepared with alcalase and protamex. The protein recovery, TCA‐soluble peptide content, surface hydrophobicity, particle size and zeta potential were evaluated. Results showed that the hydrolysates exhibited varying ACE‐inhibitory (i.e. the highest value 72.6% by alcalase and 84.3% by protamex) and DPPH radical scavenging activities (i.e. the highest value 51.9% by alcalase and 51.7% by protamex). Pepsin pretreatment could make soya proteins more susceptible for hydrolysis, and the results showed that the ACE‐inhibitory and DPPH radical scavenging activities of the resultant hydrolysates were improved. A highly significant positive correlation between ACE‐inhibitory and DPPH radical scavenging activities was observed in the alcalase hydrolysates, while no significant correlation among other treatments. The physical properties of hydrolysates, that is surface hydrophobicity, particle size and zeta potential, could influence their ACE‐inhibitory and DPPH radical scavenging activities.     

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。      

Glutaminase‐induced deamidation and hydrolysis of casein and metal‐chelating or ACE‐inhibitory activity of the hydrolysates in vitro

Glutaminase (EC 3.5.1.2) was applied in this work to induce deamidation and hydrolysis of casein. Some reaction conditions based on casein deamidation were studied. Three casein hydrolysates with degree of deamidation of 2.8%, 5.8% and 8.5%, or degree of hydrolysis of 2.5%, 3.4% and 4.9%, respectively, were prepared at casein concentration 5% (w/v), glutaminase addition level 400 U kg^?1 casein, reaction temperature 37 °C and reaction times 6, 12 and 24 h, respectively. Evaluation results showed that when iron (II) was added at 60 μm , iron (II)‐chelating powers of three hydrolysates were 41.1, 45.4 and 55.3%, while that of original casein and EDTA were 36.1 and 13.6%. Calcium (II)‐chelating power of three hydrolysates was 1.23, 1.41 and 1.49 mmol g^?1 casein, whereas that of original casein was 1.05 mmol g^?1 casein. Three hydrolysates also had ACE‐inhibitory activity in vitro, with IC₅₀ values from 0.75 to 2.34 mg mL^?1.     

Preparation,characterisation and structure of rabbit (Hyla rabbit) skin gelatine

Rabbit skin was used as a raw material to prepare gelatine. The yield of extracted rabbit skin gelatine was 8.49 ± 0.91% based on a wet weight basis. The bloom strength of 6.67% rabbit gelatine was 457.57 ± 3.302 g, and its gelling and melting temperatures were 27.8 and 34.4 °C, respectively. Sodium dodecyl sulphate‐polyacrylamide gel electrophoresis analysis demonstrated that the content of the subunit components (α chain, β and γ components) of the rabbit skin gelatine achieved 97.915%. Amino acid composition analysis indicated that the total amino acid contents (proline plus hydroxyproline) in the rabbit gelatine were 222 residues per 1000 residues. The high content of the subunit components and amino acids could contribute to the gelatine's suitable gel properties. Rabbit gelatine had nearly all of the characteristic absorption peaks of collagen from rabbit skin, and the loss of the triple helical structure and damage to part of the hydrogen bonds occurred during the conversion of collagen to gelatine.     

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.     

Purification and identification of angiotensin converting enzyme inhibitory peptides from beef hydrolysates

Sarcoplasmic protein extracts from beef rump (biceps femoris) were hydrolyzed (for 0, 4, 8, 12, and 24 h) with three enzymes or their paired combinations. Ultrafiltration, gel-filtration, and RP-HPLC were used to separate angiotensin converting enzyme (ACE) inhibitory peptides from the hydrolysates. The highest ACE inhibitory activity of enzyme hydrolysates resulted from 4 h incubation with enzymes or their paired combinations. The activities of gel filtrated fractions from these hydrolysates were assayed in vitro, demonstrating that the 3rd peak of enzyme thermolysin + proteinase A hydrolysate had the highest ACE inhibition activity (52.8%). The 3rd peak of this hydrolysate was separated by RP-HPLC into five peaks, of which peak 3 showed 30.1% ACE inhibition activity. Its peptide sequence was determined to be Val-Leu-Ala-Gln-Tyr-Lys. The results suggested that this peptide may be a potent ACE inhibitor which might perhaps be used to develop beef with a bioactive peptide to lower blood pressure.     

Characterisation of microemulsion nanofilms based on Tilapia fish skin gelatine and ZnO nanoparticles incorporated with ginger essential oil: meat packaging application

Microemulsion nanofilms based on fish skin gelatine (FSG) and 3% (w/w, gelatine) ZnO nanoparticles (ZnONP) incorporated with ginger essential oil (GEO) at different concentration (10%, 20%, 40%, 80%, w/w, gelatine) were developed and characterised. Film thickness and elongation increased, while film tensile strength and gel strength decreased as GEO level increased (P < 0.05). The water vapour permeability was reduced (P < 0.05) by GEO. Microstructural study revealed a dispersion system with GEO lipid droplet embedded into FSG matrix and some aggregation. The developed films showed strong antibacterial activity against food spoilage bacterial of psychrotrophs, mesophiles and lactobacillus spp. (LAB) in situ, and food pathogenic bacteria of Escherichia coli and Listeria monocytogenes in vitro. Together with the antioxidant activity towards lipid oxidation, the release of total volatile bases nitrogen (TVB‐N) was subsequently reduced during fresh meat storage. In conclusion, the incorporation of GEO provided the potential to make FSG‐ZnONP films more available for meat storage.     

Angiotensin I-converting enzyme inhibitory activity of low-molecular-weight peptides from Atlantic salmon (Salmo salar L.) skin

Collagen extracted from Atlantic salmon (Salmo salar L.) skin (which is normally discarded in the process of manufacture) was hydrolyzed with Alcalase and papain, and treated by multistage separation. The salmon skin collagen peptides (SSCP) obtained had high protein content (91.20 ± 1.03%) and low molecular weights, 90.79% of which were less than 1000 Da. SSCP was then separated by reversed-phase high performance liquid chromatography. Eleven major fractions were collected and their angiotensin I-converting enzyme (ACE) inhibitory activity was assayed. Fractions 5 and 7 displaying higher ACE inhibitory activity were subjected to mass spectrometer to identify the ACE inhibitory peptides. A total of eleven peptide sequences were identified, and two dipeptides, Ala-Pro and Val-Arg, were selected for further ACE inhibitory activity analysis. The ACE inhibitory activities of Ala-Pro (IC₅₀ = 0.060 ± 0.001 mg/ml) and Val-Arg (IC₅₀ = 0.332 ± 0.005 mg/ml) were found to be approximately 20- and 4-fold higher than that of SSCP (1.165 ± 0.087 mg/ml), respectively.     

海蜇生殖腺酶解肽的抗氧化活性和ACE抑制活性研究

为实现海蜇加工副产物生殖腺的高值化利用,以海蜇性腺脱脂粉为原料,选用中性蛋白酶、木瓜蛋白酶、胰蛋白酶和碱性蛋白酶分别对海蜇生殖腺进行水解,以酶解产物（JGHP）的水解度、DPPH清除活性和ACE抑制活性为指标,选择制备海蜇生殖腺活性肽的工具酶。经超滤膜将酶解产物分成JGPH-P1（大于3000 u）、P2（1000³000 u）、P3（小于1000 u）三个部分,分别测定三部分的DPPH清除活性和ACE抑制活性。结果表明,中性蛋白酶水解所得产物的水解度、DPPH清除率和ACE抑制活性均优于其他三种蛋白酶;JGHP超滤后,其中组分JGHP-P3的活性较高。利用电子自旋共振（ESR）技术检测JGHP-P3对DPPH·、·OH、O-2·的清除活性。其ACE抑制活性IC50为1.06 mg/m L,DPPH·、·OH、O-2·的清除活性IC50分别为0.38、0.63、0.59 mg/m L。由此可见,经中性蛋白酶水解得到的JGPH中小于1000 u的组分具有较高的抗氧化和ACE抑制活性。      

Extraction and characterisation of pepsin‐solubilised collagens from the skin of bigeye snapper (Priacanthus tayenus and Priacanthus macracanthus)

BACKGROUND: Fish collagen has been paid increasing attention as an alternative to the mammalian counterpart owing to the abundance of fish skin as a processing by‐product. Generally, the low yield of collagen extracted using the typical acid solubilisation process has led to the use of mammalian pepsin as an aid for increasing the yield. Alternatively, fish pepsin, especially from tuna stomach, can be used for the extraction of pepsin‐solubilised collagen (PSC). Therefore the objective of this study was to extract and characterise PSC from the skin of bigeye snapper, a fish widely used for surimi production in Thailand. RESULTS: PSCs from the skin of two species of bigeye snapper, Priacanthus tayenus and Priacanthus macracanthus, were extracted with the aid of tongol tuna (Thunnus tonggol) pepsin and porcine pepsin. PSCs from the skin of both species extracted using porcine pepsin had a higher content of β‐chain but a lower content of α‐chains compared with those extracted using tuna pepsin. All PSCs contained glycine as the major amino acid and had an imino acid (proline and hydroxyproline) content of 189–193 residues per 1000 residues. Transition temperatures of PSCs were in the range 30.6–31.3 °C. Fourier transform infrared spectra revealed some differences in molecular order between PSCs extracted using porcine pepsin and tuna pepsin. Nevertheless, the triple‐helical structure of PSCs was not affected by pepsin digestion. Zeta potential analysis indicated that PSCs from P. tayens and P. macracanthus possessed zero net charge at pH 7.15–7.46 and 5.97–6.44 respectively. CONCLUSION: Tongol tuna pepsin could be used as a replacement for mammalian pepsin in PSC extraction. However, a slight difference in PSC properties was found. Copyright © 2009 Society of Chemical Industry     

Umami and umami-enhancing peptides from myofibrillar protein hydrolysates in low-sodium dry-cured Spanish mackerel (Scomberomorus niphonius) under the action of Lactobacillus plantarum

Lactobacillus plantarum was involved in producing low-salt (2%, w/w) dry-cured Spanish mackerel, and its effect on the degradation of myofibrillar protein and production of umami peptides was investigated. Results showed that the dominant microbiome of the sample was Lactobacillus plantarum, accounting for 95.7% of the microbial community. The umami peptides in this dry-cured mackerel were extracted and separated by ultra-filtration and gel filtration chromatography and further identified by Nano-LC–MS/MS. Five umami-enhancing peptides, including TFYNELRV (1040 Da), TFYNELR (941 Da), TLFQPSF (838 Da), LYANNVL (805 Da) and FAGDDAPRAVFPS (1348 Da), were identified to be derived from myofibrillar protein, and the percentage of their hydrophobic amino acid were 42.85%, 33.33%, 57.14%, 57.14% and 53.84%, respectively. The synthetic peptides of these five umami peptides showed an umami-enhancing threshold value ranging from 0.34 to 0.45 mg mL⁻¹ through sensory evaluation. The SDS-PAGE analysis exhibited that actin band intensity was decreased during the processing of dry-cured mackerel. Therefore, Lactobacillus plantarum has the potential to improve the hydrolysis of myofibrillar protein to produce umami peptides in dry-cured mackerel.     

超声波辅助酶解对玉米醇溶蛋白酶解产物抗氧化及ACE抑制活性的影响

作为玉米加工的副产物,玉米醇溶蛋白可以在酶的作用下形成具备生物活性的多肽物质,应用于药物的生产加工过程.而常规的酶解工艺存在酶的利用率低下、产物转化率低、耗费时间长等缺陷,本文研究了超声波辅助酶解对玉米醇溶蛋白酶解产物抗氧化及ACE抑制活性的影响.研究结果表明,超声功率为200W,超声时间为40min,料液比为1 ∶1...     

Angiotensin‐converting enzyme‐inhibitory activity in protein hydrolysates from normal and anthracnose disease‐damaged Phaseolus vulgaris seeds

BACKGROUND: Bean seeds are an inexpensive source of protein. Anthracnose disease caused by the fungus Colletotrichum lindemuthianum results in serious losses in common bean (Phaseolus vulgaris L.) crops worldwide, affecting any above‐ground plant part, and protein dysfunction, inducing the synthesis of proteins that allow plants to improve their stress tolerance. The aim of this study was to evaluate the use of beans damaged by anthracnose disease as a source of peptides with angiotensin‐converting enzyme (ACE‐I)‐inhibitory activity. RESULTS: Protein concentrates from beans spoiled by anthracnose disease and from regular beans as controls were prepared by alkaline extraction and precipitation at isolelectric pH and hydrolysed using Alcalase 2.4 L. The hydrolysates from spoiled beans had ACE‐I‐inhibitory activity (IC₅₀ 0.0191 mg protein mL^?1) and were very similar to those from control beans in terms of ACE‐I inhibition, peptide electrophoretic profile and kinetics of hydrolysis. Thus preparation of hydrolysates using beans affected by anthracnose disease would allow for revalorisation of this otherwise wasted product. CONCLUSION: The present results suggest the use of spoiled bean seeds, e.g. anthracnose‐damaged beans, as an alternative for the isolation of ACE‐I‐inhibitory peptides to be further introduced as active ingredients in functional foods. © 2012 Society of Chemical Industry     

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.