Antioxidant activity of yoghurt peptides: Part 2 – Characterisation of peptide fractions

The aim of the present study was to elucidate previous findings showing that peptide fractions isolated from yoghurt had antioxidant effects. Therefore, peptides and free amino acids released during fermentation of milk were characterised. Yoghurt samples were stripped from sugars and lactic acid and subsequently fractionated by ultra filtration using membranes with cut off sizes of 30, 10 and 3 kDa. The peptides in these fractions were identified by LC–MS/MS. The identified peptides comprised a few N-terminal fragments of αs₁-, αs₂-, and κ-casein, and several fragments from β-casein. Almost all the peptides identified contained at least one proline residue. Some of the identified peptides included the hydrophobic amino acid residues Val or Leu at the N-terminus and Pro, His or Tyr in the amino acid sequence, which is characteristic of antioxidant peptides. In addition, the yoghurt contained a considerable amount of free amino acids such as His, Tyr, Thr and Lys, which have been reported to have antioxidant properties. Thus, our findings confirm that the antioxidant effects of the peptide fractions from yoghurt are due to the presence of certain peptides and free amino acids with recognised antioxidant activity in these fractions.     

Bioactive peptides in ovine and caprine cheeselike systems prepared with proteases from Cynara cardunculus

The potential angiotensin-converting enzyme (ACE)-inhibitory and antioxidant activities of peptides in water-soluble extracts, obtained from raw and sterilized ovine and caprine cheeselike systems coagulated with enzymes from the plant Cynara cardunculus, were assessed. Prior to the assay, the 3,000-Da permeate from 45-d-old cheeselike systems was fractionated by tandem chromatographic techniques. Several peaks were obtained in each chromatogram, but only some were associated with ACE-inhibitory or antioxidant activity or both. Peptides Tyr-Gln-Glu-Pro, Val-Pro-Lys-Val-Lys, and Tyr-Gln-Glu-Pro-Val-Leu-Gly-Pro-* from β-casein, as well as Arg-Pro-Lys and Arg-Pro-Lys-His-Pro-Ile-Lys-His-* from α_s1-casein exhibited ACE-inhibitory activity. Peptides released upon cleavage of the peptide bond Leu190-Tyr191 (either in ovine or caprine β-casein), and corresponding to the β-casein sequence Tyr-Gln-Glu-Pro-*, possessed antioxidant activity.     

Purification and identification of ACE inhibitory peptides from Haruan (Channa striatus) myofibrillar protein hydrolysate using HPLC–ESI-TOF MS/MS

Haruan myofibrillar protein was hydrolysed with proteinase K and thermolysin to isolate Angiotensin converting enzyme (ACE) inhibitory peptides. The thermolysin hydrolysate of myofibrillar protein with the highest ACE inhibition activity (IC₅₀ = 0.033 mg/ml) was fractionated by ultrafiltration and size exclusion chromatography to three fractions. Fraction F2 with higher ACE inhibitory activity was separated into five fractions (A–E) using reversed-phased high performance liquid chromatography (RP-HPLC). Fraction C showed 81% inhibition activity and was subjected to HPLC coupled to electrospray ionisation-time-of-flight mass spectrometry (ESI-TOF MS/MS). Two peptide sequences for the most abundant fragments were identified as VPAAPPK (IC₅₀ = 0.45 μM) at 791.155 m/z and NGTWFEPP (IC₅₀ = 0.63 μM) at 1085.841 m/z. The presence of two proline residues at the C-terminal sequence is responsible for the high ACE inhibitory activity of these peptides. The results suggest that Haruan meat protein hydrolysate is a potent ACE inhibitor and may be used to decrease blood pressure.     

Isolation and identification of angiotensin-converting enzyme inhibitory peptides from egg white protein hydrolysates

The aim of this study was to identify potential angiotensin I-converting enzyme (ACE) inhibitory peptide from egg white protein. The protein was hydrolysed by Alcalase and the hydrolysates were isolated with Gel filtration to get the high activity fraction. The fraction was identified by LC tandem mass spectrometric 4000 Q Trap MS. In the current work, 19 peptides were discovered in the fractions, five of which sourced from ovotransferrin and were synthesised by Fmoc solid phase method. ACE-inhibitory activity was measured by HPLC assay. The 50% inhibitory concentrations of Arg–Val–Pro–Ser–Leu (RVPSL) was 20 μM. Based on this remarkable ACE-inhibitory activity, it is suggested that RVPSL may have potential applications as a functional food, which could be used as nutraceutical compounds.     

Isolation of antioxidative and ACE inhibitory peptides from protein hydrolysate of skipjack (Katsuwana pelamis) roe

Bioactive peptides from protein hydrolysate of defatted skipjack (Katsuwonus pelamis) roe with 5% degree of hydrolysis (DH) prepared by Alcalase digestion were isolated and characterised. Two active fractions with ABTS radical scavenging activity (973.01–1497.53 μmol TE/mg sample) and chelating activity (0.05–0.07 μmol EE/mg sample) from consecutive purification steps including ultrafiltration, cation exchange column chromatography and reverse phase high performance liquid chromatography (RP-HPLC), were subjected to analysis of amino acid sequence by LC–MS/MS. Seven dominant peptides with 6–11 amino acid residues were identified as DWMKGQ, MLVFAV, MCYPAST, FVSACSVAG, LADGVAAPA, YVNDAATLLPR and DLDLRKDLYAN. These peptides were synthesised and analysed for ACE-inhibitory activity and antioxidative activities. MLVFAV exhibited the highest ACE inhibitory activity (IC₅₀ = 3.07 μM) (p < 0.05) with no antioxidative property, whilst DLDLRKDLYAN showed the highest metal chelating activity, ABTS radical and singlet oxygen scavenging activities. Therefore, peptides prepared from skipjack roe could be further employed as a functional food ingredient.     

Fractionation and identification of ACE-inhibitory peptides from α-lactalbumin and β-casein produced by thermolysin-catalysed hydrolysis

The thermolysin catalysed hydrolysates of α-lactalbumin and β-casein were fractionated by size-exclusion chromatography (SEC) and reversed-phase high performance liquid chromatography (RP-HPLC) in order to identify the peptides responsible for the high ACE-inhibitory activity of these hydrolysates. The SEC fractionation separated many co-eluting peptides into different fractions allowing individual peptides to be isolated in one or two subsequent semi-preparative RP-HPLC fractionation steps. Five potent ACE-inhibitory peptides from α-lactalbumin were isolated. They all contained the C-terminal sequence -PEW, corresponding to amino acid residues 24–26 in α-lactalbumin, and had IC₅₀ values of 1–5 μm. From one SEC fraction of the β-casein hydrolysate two potent ACE-inhibitory peptides were isolated and identified as f58-76 and f59-76 of β-casein A2. They both contained IPP as the C-terminal sequence and had IC₅₀ values of 4 and 5 μm. From another SEC fraction a new but less ACE-inhibitory peptide from β-casein was identified (f192–196; LYQQP).     

Two Novel Bioactive Peptides from Antarctic Krill with Dual Angiotensin Converting Enzyme and Dipeptidyl Peptidase IV Inhibitory Activities

Inhibition of dipeptidyl peptidase IV (DPP‐IV) and angiotensin converting enzyme (ACE) are considered useful in managing 2 often associated conditions: diabetes and hypertension. In this study, corolase PP was used to hydrolyze Antarctic krill protein. The hydrolysate (AKH) was isolated by ultrafiltration and purified by size‐exclusion chromatography, ion exchange chromatography and reversed‐phase high‐performance liquid chromatography (RP‐HPLC) sequentially. The in vitro inhibitory activities of all AKHs and several fractions obtained against ACE and DPP‐IV were assessed. Two peptides, purified with dual‐strength inhibitory activity against ACE and DPP‐IV, were identified by TOF‐MS/MS. Results indicated that not all fractions exhibited dual inhibitory activities of ACE and DPP‐IV. The purified peptide Lys‐Val‐Glu‐Pro‐Leu‐Pro had half‐maximal inhibitory concentrations (IC₅₀) of 0.93±0.05 and 0.73±0.04 mg/mL against ACE and DPP‐IV, respectively. The other peptide Pro‐Ala‐Leu had IC₅₀ values of 0.64±0.05 and 0.88±0.03 mg/mL against ACE and DPP‐IV, respectively. This study firstly reported the sequences of dual bioactive peptides from Antarctic krill proteins, further provided new insights into the bioactive peptides responsible for the ACE and DPP‐IV inhibitory activities from the Antarctic krill protein hydrolysate to manage hypertension and diabetes.     

Isolation and identification of antioxidative peptides from hydrolysate of threadfin bream surimi processing byproduct

The objective of this study was to determine the antioxidant activity and possible mode of action of partially purified peptides derived from threadfin bream surimi byproduct. The frame, bone and skin (FBS) byproduct, which was obtained from a deboning process, was hydrolyzed by Virgibacillus sp. SK33 proteinase and fractionated using anion exchange and size exclusion chromatography. Three fractions, i.e., B1, B2 and B3, were obtained, and the amino acid sequences of the peptides in all 3 fractions were determined using LC-MS/MS. Fractions B2 and B3 contained higher amounts of Trp, Met, Cys and Tyr residues than fraction B1. Fraction B3 exhibited high ABTS radical scavenging activity and ferric reducing antioxidant power (FRAP), while metal chelation and hydroxyl radical scavenging ability were principle modes of action of peptides in fraction B2 and B3. In addition, fraction B1 and a synthetic peptide selected from the pooled de novo peptides of fraction B3, FLGSFLYEYSR, had a cellular radical scavenging effect when HepG2 cells were treated with H₂O₂.     

Optimization of sour milk fermentation for the production of ACE-inhibitory peptides and purification of a novel peptide from whey protein hydrolysate

The effect of fermentation conditions on the production of angiotensin-I converting enzyme (ACE) inhibitory peptide in sour milk fermented by Lactobacillus helveticus LB10 was investigated using response-surface methodology. Optimal conditions to produce the maximum production of ACE-inhibitory peptides were found to be 4% (v/w) inoculum, 7.5 initial pH of medium and 39.0 °C. The fermented milk resulted in 75.46% inhibition in ACE activity. The cell-envelope proteinase, assisted by X-prolyldipeptidyl aminopeptidase of Lb. helveticus LB10 produced the ACE-inhibitory peptides. A novel ACE-inhibitory peptide from whey protein hydrolysate produced by crude proteinases of Lb. helveticus LB10 was purified. The separations were performed with Sephadex^® G-75 and Sephadex G-15 gel filtration chromatography and reversed-phase, high-performance liquid chromatography. The peptide with the RLSFNP sequence was isolated from β-lactoglobulin hydrolysate and its IC₅₀ while inhibiting ACE activity was 177.39 μm.     

Antioxidant activity of ovine casein hydrolysates: identification of active peptides by HPLC–MS/MS

This paper shows the potential role of different ovine casein fractions and their hydrolysates to exert antioxidant activity. ABTS^·+ decolorization assay was used to evaluate the antioxidant activity of the casein fractions (β-, κ- and α_s-caseins) before and after their hydrolysis by pepsin, trypsin and chymotrypsin. Although the antioxidant activity increased in all the fractions after hydrolysis, the effect was particularly remarkable in the κ-casein fraction, which increased its antioxidant activity almost threefold. Further assays in a linoleic acid oxidation system showed that κ-casein hydrolysate inhibited lipid peroxidation. Analysis of the ovine κ-casein hydrolysate by RP-HPLC–MS/MS allowed the identification of 12 peptide sequences with potential antioxidant properties. One of the most abundant peptides, the fragment HPHPHLSF [f(98–105)] was chemically synthesized. Results showed that this κ-casein-derived peptide was a potent inhibitor of linoleic acid oxidation with an activity similar to that obtained with the synthetic antioxidant BHT. Although other peptides might also contribute, HPHPHLSF was the one most likely to be responsible for the activity found in the κ-casein hydrolysate.     

Novel probiotic-fermented milk with angiotensin I-converting enzyme inhibitory peptides produced by Bifidobacterium bifidum MF 20/5

In previous research, we have demonstrated that Bifidobacterium bifidum MF 20/5 fermented milk possessed stronger angiotensin converting enzyme (ACE) inhibitory activity than other lactic acid bacteria, including Lactobacillus helveticus DSM 13137, which produces the hypotensive casokinins Ile-Pro-Pro (IPP) and Val-Pro-Pro (VPP). The aim of this study is to investigate the ACE-inhibitory peptides released in B. bifidum MF 20/5 fermented milk. The novel ACE-inhibitory peptide LVYPFP (IC₅₀ = 132 μM) is reported here for the first time. Additionally, other bioactive peptides such as the ACE-inhibitor LPLP (IC₅₀ = 703 μM), and the antioxidant VLPVPQK were identified. Moreover, the peptide and amino acid profiles, the ACE-inhibitory activity (ACEi), pH, and degree of hydrolysis of the fermented milk were determined and compared with those obtained in milk fermented by L. helveticus DSM 13137. The sequences of the major bioactive peptides present in fermented milk of B. bifidum and L. helveticus were identified and quantified. B. bifidum released a larger amount of peptides than L. helveticus but no IPP or VPP were detected in B. bifidum fermented milk. Also the lactotripeptide concentrations and ACEi were higher in L. helveticus fermented milk when the pH was maintained at 4.6. This may represent a technical advantage for B. bifidum that reduces the pH at a slow enough rate to facilitate the peptide generation without the need for pH control. Thus these findings show the potential for the use of this probiotic strain to produce fermented milk with a wider range of health benefits including reduction of blood pressure.     

Angiotensin I-converting enzyme-inhibitory peptide fractions from albumin 1 and globulin as obtained of amaranth grain

A number of biopeptides promoting health benefits have been isolated from food-protein hydrolysates and can be released during enzymatic digestion. Antihypertensive peptides can be part of protein fractions from amaranth grain. The objective of this work was to obtain ACE-inhibitory peptide fractions from albumin 1 and the globulin of amaranth (Amaranthus hypochondriacus) grain. Albumin 1 and globulin were hydrolysed with alcalase; hydrolysis was monitored by proteolytic degradation and by ACE-inhibitory activity. The highest ACE-inhibitory activity was 40% and 35% as obtained after 18 and 15 h hydrolysis for albumin 1 and globulin, respectively. Further separation and purification of the ACE-inhibitory peptide fractions were carried out by gel filtration and C₁₈ RP–HPLC. The IC₅₀ was 0.35 ± 0.02 mg/ml for albumin 1 peptide fraction and 0.15 ± 0.03 mg/ml for globulin peptide fraction. Albumin 1 peptide fraction showed an competitive mode of ACE inhibition, whereas the globulin peptide fraction was competitive. The globulin peptide fraction may have one of the most active naturally-occurring ACE-inhibitory peptides.     

Potential ACE-inhibitory activity and nanoLC-MS/MS sequencing of peptides derived from aflatoxin contaminated peanut meal

Our lab has developed a process for sequestering aflatoxin from contaminated peanut meal (PM) using commercial bentonite clays while protein is simultaneously extracted and hydrolyzed by a commercial protease. The objectives of this study were to sequence generated peptides and evaluate their potential ACE-inhibitory properties. Aflatoxin in the unprocessed PM was 610 μg kg⁻¹ compared to 9.7 μg kg⁻¹ on a dry weight basis in the 120 min hydrolysate. This hydrolysate displayed significant ACE-inhibitory activity with an IC₅₀ of 295.1 μg mL⁻¹. Ultrafiltration and size exclusion chromatography (SEC) improved the ACE-inhibitory properties, with the SEC fraction containing the smallest peptides having an IC₅₀ = 44.4 μg mL⁻¹. Additionally, 271 unique peptides were identified by nanoLC-MS/MS, of which 147 belonged to major seed storage proteins. This advanced characterization data will ultimately allow for more efficient production of hydrolysates with ACE-inhibitory activity or other bioactivities of interest from PM.     

Anti-oxidative capacity of enzymatically released peptides from soybean protein isolate

Shelf-life of dietary products and the need for health supporting functional ingredients often require components with high anti-oxidative properties. The study aimed at the characterisation of peptides with antioxidant properties from enzymatically hydrolysed soybean protein isolate. Soybean protein isolate was hydrolysed with a food grade pancreatic trypsin/chymotrypsin for industrial applications, ultrafiltered and the permeate was fractionated by size exclusion chromatography. Anti-oxidative properties of these fractions were tested in a Trolox^®-equivalent-anti-oxidative capacity assay, which is based on the absorbance suppression of 2,2′-azinobis(3-ethyl benzothiazoline-6-sulfonate) radical cations by anti-oxidative substances at λ 414 nm. The amino acid compositions and sequences of the peptide fractions were determined by liquid chromatography and mass spectrometry analysis. Compared to the intact soybean protein isolate a peptide fraction with an anti-oxidative capacity of 113 mg TEAC/g was obtained. This fraction was shown to be enriched in peptides smaller than 1 kDa with prevailing aromatic amino acid residues. Nine peptides derived from glycinin and β-conglycinin were sequenced by tandem mass spectrometry. The particular sequences were synthesized and assayed. The synthetic peptide analogue TTYY (β-conglycinin fragment 291–294) revealed significant radical scavenging properties of 13.6 up to 59.6% with 0.18–18 mM in a concentration dependent manner. Peptides from soybean protein isolate were shown to possess anti-oxidative capacities which might be linked to the presence of carboxy terminal tyrosine. These findings offer new functional application possibilities in nutrition, cosmetics and pharmaceuticals.     

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     

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.     

Activities and sequences of the angiotensin I‐converting enzyme (ACE) inhibitory peptides obtained from the digested lentil (Lens culinaris) globulins

The aim of this study was the identification of potentially bioaccessible ACE‐inhibitory peptides obtained by in vitro gastrointestinal digestion of lentil globulins. ACE‐inhibitory peptides were purified by ion exchange chromatography and gel filtration. After the first step of purification, three peptide fractions with potential antihypertensive properties were obtained and the highest inhibitory activity was determined for the fraction 5 (IC₅₀ = 0.02 mg mL^?1). This fraction was separated on Sephadex G10, and six peptide fractions were obtained. The peptides of fraction (5‐F) with the highest potential antihypertensive activity (IC₅₀ = 0.13 mg mL^?1) were identified using ESI‐MS/MS. The sequences of peptides were KLRT, TLHGMV and VNRLM. Based on Lineweaver–Burk plots for the fraction 5‐F, the kinetic parameters as K_m (1.24 mm ), V_max (0.012 U min^?1), K_i (0.12 mg mL^?1) and mode of inhibition were determined.     

Angiotensin-converting enzyme inhibitory activity of peptides derived from caprine kefir

In this study, a potent angiotensin-converting enzyme (ACE)-inhibitory activity was found in a commercial kefir made from caprine milk. The low molecular mass peptides released from caseins during fermentation were mainly responsible for this activity. Sixteen peptides were identified by HPLC-tandem mass spectrometry. Two of these peptides, with sequences PYVRYL and LVYPFTGPIPN, showed potent ACE-inhibitory properties. The impact of gastrointestinal digestion on ACE-inhibitory activity of kefir peptides was also evaluated. Some of these peptides were resistant to the incubation with pepsin followed by hydrolysis with Corolase PP. The ACE-inhibitory activity after simulated digestion was similar to or slightly lower than unhydrolyzed peptides, except for peptide β-casein f(47-52) (DKIHPF), which exhibited an activity 8 times greater after hydrolysis.     

Purification and identification of novel antioxidant peptides from enzymatic hydrolysates of sardinelle (Sardinellaaurita) by-products proteins

In order to utilise sardinelle (Sardinellaaurita) protein by-products, which is normally discarded as industrial waste in the process of fish manufacturing, heads and viscera proteins were hydrolysed by different proteases to obtain antioxidative peptides. All hydrolysates showed different degrees of hydrolysis and varying degrees of antioxidant activities. Hydrolysate generated with crude enzyme extract from sardine (Sardinapilchardus) displayed high antioxidant activity, and the higher DPPH radical-scavenging activity (87 ± 2.1% at 2 mg/ml) was obtained with a degree of hydrolysis of 6%. This hydrolysate was fractionated by size exclusion chromatography on a Sephadex G-25 into eight major fractions (P₁–P₈). Fraction P₄, which exhibited the highest DPPH scavenging activity, was then fractionated by reversed-phase high performance liquid chromatography (RP-HPLC). Seven antioxidant peptides were isolated. The molecular masses and amino acids sequences of the purified peptides were determined using ESI-MS and ESI-MS/MS, respectively. Their structures were identified as Leu-His-Tyr, Leu-Ala-Arg-Leu, Gly-Gly-Glu, Gly-Ala-His, Gly-Ala-Trp-Ala, Pro-His-Tyr-Leu and Gly-Ala-Leu-Ala-Ala-His. The first peptide displayed the highest DPPH radical-scavenging activity (63 ± 1.57%; at 150 μg/ml) among these peptides.     

榛子粕抗氧化肽的分离纯化及序列分析

为研究榛子粕抗氧化肽的抗氧化性和氨基酸组成的关系,本实验通过中性蛋白酶水解榛子粕蛋白得到榛子粕抗氧化肽,以DPPH自由基清除率为指标,采用超滤分离和葡聚糖凝胶纯化,获得DPPH自由基清除率高的榛子粕抗氧化肽P2,采用超高压液相色谱串联四级杆质谱(LC-MS/MS)测定榛子粕抗氧化肽的氨基酸序列。P2中7个短肽的氨基酸序列分别为His-ILe/Leu-Pro(362 Da)、ILe/Leu-Val-Asp-Glu(475 Da)、Thr-Pro-Pro-His-Lys(588 Da)、His-ILe/Leu-His-Ser-Ala-Thr(662 Da)、Cys-His-Ser-ILe/Leu-Met-ILe/Leu(701 Da)、Thr-His-Ala(327 Da)、Phe-ILe/Leu(279 Da)。由此得出结论,疏水性氨基酸和芳香性氨基酸可以提高榛子粕抗氧化肽的抗氧化性。