Evaluation of Antioxidant Activities of Zein Protein Fractions

Zein protein was extracted from the by‐product corn gluten meal. The obtained zein protein was 1st hydrolyzed by 4 different proteases. The antioxidant activities of the hydrolysates or peptides were evaluated by free radical scavenging activity, metal ion chelating activity, and lipid peroxidation inhibitory capacity. Among hydrolysates produced, alkaline protease hydrolysates exhibited the highest antioxidant activity. A regression model was established by uniform design to optimize the alkaline protease hydrolysis conditions. The hydrolysates with molecular weight < 3 kDa obtained from ultrafiltration showed the highest antioxidant activities in all relevant assays. The hydrolysates with molecular weight <3 kDa were subsequently purified by gel filtration chromatography, and fraction F3 exhibited the highest antioxidant activities. Two peptides were identified from fraction F3 using LC‐ESI‐Q‐TOF MS/MS as Pro‐Phe (263.13 Da) and Leu‐Pro‐Phe (375.46 Da). These peptides exhibited good free radical scavenging activity and lipid peroxidation inhibitory effect. The results clearly indicated that zein protein fractions are good sources for the development of natural antioxidants for the food industry.     

A novel antioxidant and antimicrobial peptide from hen egg white lysozyme hydrolysates

Hen egg white lysozyme (HEWL) was hydrolyzed with papain, trypsin and a combination of the two to isolate antioxidant peptides. The prepared hydrolysates were evaluated for antioxidant activity using DPPH and ABTS radical scavenging, metal ion chelation and lipid peroxidation inhibition. The obtained hydrolysate by a combination of the two enzymes exhibited the highest antioxidant activity compared to other hydrolysates and elected for isolation of antioxidant peptides by reverse-phase high-performance liquid chromatography (RP-HPLC). A most potent fraction namely F2 fraction, identified to be NTDGSTDYGILQINSR (MW: 1753.98 ± 0.5 Da) using tandem mass spectrometry. The antimicrobial activity of the F2 peptide was tested using radial diffusion assay (RDA). Our results showed that this peptide has inhibitory effects on both Gram-negative and Gram-positive bacteria. Minimum inhibition concentration (MIC) values of the F2 peptide against Escherichia coli and Leuconostoc mesenteroides bacteria were 355.64 (±2.2) and 442.25 (±2.8) μg/ml, respectively.     

Isolation and characterization of three antioxidant pentapeptides from protein hydrolysate of monkfish (Lophius litulon) muscle

In the current study, an efficient method had been developed to acquire the antioxidant hydrolysate of monkfish muscle protein (MPH) using trypsin by an orthogonal (L₉(3)⁴) test. Under the optimum conditions of enzymolysis time 4 h, enzyme-to-substrate ratio (E/S) 2%, enzymolysis temperature 40 °C and pH 8.0, the DH (Degree of hydrolysis) and hydroxyl radical scavenging activity of MPH reached 19.83 ± 0.82% and 58.05 ± 3.01%, respectively. By using ultrafiltration, gel filtration chromatography and reversed phase high performance liquid chromatography (RP-HPLC), three antioxidant pentapeptides were isolated from MPH, and their amino acid sequences were identified as Glu-Trp-Pro-Ala-Gln (MPH-P1), Phe-Leu-His-Arg-Pro (MPH-P2), and Leu-Met-Gly-Gln-Trp (MPH-P3) with molecular weights of 629.68 Da, 668.80 Da, and 633.77 Da, respectively. MPH-P1, MPH-P2, and MPH-P3 exhibited good scavenging activities on hydroxyl radical (EC₅₀ 0.269, 0.114 and 0.040 mg/ml), DPPH radical (EC₅₀ 2.408, 3.751, and 1.399 mg/ml), and superoxide anion radical (EC₅₀ 0.624, 0.101, and 0.042 mg/ml) in a dose-dependent manner. MPH-P3 was also effective against lipid peroxidation in the model system. The antioxidant activities of MPH-P1, MPH-P2, and MPH-P3 were due to their small sizes and the presence of antioxidant and hydrophobic amino acid residues within their sequences. The results of this study suggested that the protein hydrolysate and/or its isolated peptides might be effectively used as food additives for retarding lipid peroxidation occurring in foodstuffs.     

In vitro antioxidant activity of protein hydrolysates prepared from corn gluten meal

BACKGROUND: Corn gluten meal (CGM), a major by‐product of corn wet milling, is mainly used as forage in China. Because of its particular amino acid composition, in which there are large amounts of hydrophobic amino acids such as leucine, alanine and phenylalanine, CGM protein was thought to be a good resource to obtain antioxidant peptides. CGM protein was hydrolysed with a biochemical grade alcalase and the derived hydrolysates were assessed for their antioxidant properties in different in vitro assay systems, including inhibiting activity on lipid peroxidation, by reducing power, hydroxyl radical scavenging activity, and DPPH radical scavenging activity. The effects of concentration and molecular weight (MW) of hydrolysates on antioxidant activity were investigated. RESULTS: The results showed that CGM hydrolysates were effective antioxidants, and there was a dose‐dependent relationship between hydrolysate concentration and antioxidant activity; the highest antioxidant activity was found in peptides 500–1500 Da, and the antioxidant activity of peptides below 500 Da or peptides above 1500 Da were all lower than that of total hydrolysates. CONCLUSIONS: The finding showed that the antioxidant activity of CGM hydrolysates was related to molecular weight and hydrolysate concentration, and the active antioxidant fraction should be in the peptides fraction of 500–1500 Da. CGM protein hydrolysates can be a source of natural antioxidant and used as a food additive. Copyright © 2008 Society of Chemical Industry     

Release of multifunctional peptides by gastrointestinal digestion of sea cucumber (Isostichopus badionotus)

Sea cucumber is a benthic marine organism distributed worldwide and used as food in several Asian countries. The species Isostichopus badionotus is captured intensively off the Yucatan Peninsula, Mexico. Boiled I. badionotus was subjected to in vitro simulated gastrointestinal digestion using pepsin and a pepsin–Corolase PP^® mixture. ACE-inhibitory and radical scavenging activities, iron reducing capacity and cytotoxic effects against colorectal cancer cells were evaluated in the hydrolysates and their ultrafiltered fractions. ACE-inhibitory activity was potent in fractions containing peptides <3000 Da, an effect augmented with combined action of gastric (pepsin) and intestinal (Corolase PP^®) enzymes (IC₅₀ = 0.038 ± 0.004 mg/mL). Antioxidant activity was exerted by peptides with low and high molecular weights, depending on hydrolysis method. This is the first report of cytotoxic capacity against colorectal HT-29 cells in peptides from sea cucumber. Sea cucumber hydrolysates and ultrafiltered fractions are potential ingredients for development of functional foods.     

In vitro antioxidant study of vegetable oils containing conjugated linolenic acid isomers

The study aimed to evaluate the antioxidant activity (in vitro) of vegetable oils containing conjugated linolenic acid (CLnA) isomers such as α-eleostearic and punicic acid and also to evaluate the comparative effectiveness of these vegetable oils due to presence of cis–trans isomers in variable amount. Different in vitro methods were used to evaluate the free radical scavenging activity, hydroxyl radical scavenging activity, metal chelating activity and reducing activity of oils at different concentrations of CLnA isomers such as 125, 250 and 375 μg/mL. Inhibition on lipid peroxidation was evaluated by measuring thiobarbituric acid responsive substance (TBARS) and conjugated diene formation at 125 and 250 μg/mL concentrations of CLnA. Both the oils showed potent free radical scavenging activity at 375 μg/mL concentration. In contrary, these oils showed higher hydroxyl radical scavenging, metal chelation and reducing activity at lower concentration i.e. 125 μg/mL. TBARS formation and conjugated diene formation was lower i.e. inhibition of lipid peroxidation was maximum at 125 μg/mL of both CLnA isomers. Overall, both the oils showed better antioxidant activity at lower concentration due to better oxidative stability and bitter gourd oil showed more prominent antioxidant activity than snake gourd oil due to presence of higher trans content.     

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.     

Antioxidant activities of enzymatic rapeseed protein hydrolysates and the membrane ultrafiltration fractions

In this study, rapeseed protein isolate was hydrolyzed with various proteases to obtain hydrolysates that were separated by membrane ultrafiltration into four molecular size fractions (<1, 1–3, 3–5, and 5–10 kDa). Alcalase hydrolysis significantly (p < 0.05) produced the highest yield of protein hydrolysate while Flavourzyme produced the least. The <1 kDa fraction was the most abundant after the membrane ultrafiltration of the protein hydrolysates, which indicates that the proteases were efficient at reducing the native rapeseed proteins into low molecular weight peptides. Antioxidant properties of the resulting hydrolysates and membrane fractions were characterized and results showed the Pepsin + Pancreatin (P + P) protein hydrolysate had significantly highest (p < 0.05) scavenging activity against DPPH radical among the unfractionated enzymatic hydrolysates. But the P + P hydrolysate was not as effective as other hydrolysates during long-term inhibition of linoleic acid oxidation. For most of the samples, fractionation into the <1 kDa peptides significantly (p < 0.05) improved DPPH and superoxide scavenging properties when compared to the unfractionated protein hydrolysates. Only the <1 kDa fraction showed ferric reducing antioxidant power and the effect was dose-dependent. Overall, Alcalase and Proteinase K seem to be more efficient proteases to release antioxidant peptides from rapeseed proteins when compared to P + P, Flavourzyme and Thermolysin.     

Antioxidant activity of protein hydrolysates and purified peptides from Zizyphus jujuba fruits

In recent years, there has been a considerable interest in bioactive peptides derived from food proteins which might have beneficial effects on human health. Zizyphus jujuba is a medicinal plant with well-demonstrated biological functions for which various bioactive compounds except antioxidant peptides have been reported. Therefore, the aim of this study was to determine the antioxidant activity of Z. jujuba-derived protein hydrolysates and the purified peptides. Based upon this study, it was revealed that the hydrolysates prepared from Z. jujuba fruit possessed antioxidant effects. Among the prepared hydrolysates, trypsin hydrolysate with the highest antioxidant activity was fractionated using reverse-phase high-performance liquid chromatography (RP-HPLC). The most potent antioxidant peptides, named fractions F3 and F6, identified as VGQHTR (MW: 678.36 ± 0.3 Da) and GWLK (MW: 482.27 ± 0.3 Da), respectively, using tandem mass spectrometry. This study demonstrated that the derived hydrolysates and the purified peptides from Z. jujuba proteins can prevent oxidative reactions and might be underutilised for food preservation and medicinal purposes. However, more detailed studies are required to explore their antioxidant abilities in vivo.     

Antioxidant activity of peanut seed testa and its antioxidative component,ethyl protocatechuate

The antioxidant activity of ethanolic extracts of peanut seed testa (EEPST) and its antioxidative component, ethyl protocatechuate (EP), was examined. It was found that EEPST and EP showed a dose-dependent activity on the inhibition of liposome peroxidation. EEPST and EP in the range of 50–500 mg/l were effective in protecting protein against oxidative damage. EEPST and EP at 100 mg/l showed 92.6% and 84.6% scavenging effect, respectively, on α,α-diphenyl-β-picrylhydrazyl radical, indicating that they act as a primary antioxidant. In addition, EEPST and EP, at a dose of 200 mg/l, showed 70.6% and 67.7% scavenging effect, respectively, on the hydroxyl radical. EEPST also exhibited a metal-binding ability, while EP did not. The inhibitory effect of EEPST on linoleic peroxidation correlated with their polyphenolic content. These results suggest that the antioxidant mechanism, for both EEPST and EP, could possibly be due to their scavenging effect on free radical and hydroxyl radical. In addition, its metal binding ability may contribute to antioxidant activity of EEPST.     

Purification and identification of antioxidative peptides from loach (Misgurnus anguillicaudatus) protein hydrolysate by consecutive chromatography and electrospray ionization-mass spectrometry

Loach protein was hydrolyzed by papain to obtain antioxidative peptides. The results showed that the loach protein hydrolysate (LPH) could scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) (IC₅₀ = 17.0 ± 0.54 mg/mL) and hydroxyl radicals (IC₅₀ = 2.64 ± 0.29 mg/mL). It could chelate cupric ion and inhibit the lipid peroxidation in a linoleic acid emulsion system. The hydrolysate was isolated and purified by ultrafiltration and consecutive chromatographic methods including ion-exchange chromatography, gel filtration chromatography and a two-step reverse high-performance liquid chromatography (RP-HPLC). The purified antioxidant peptide was identified as Pro-Ser-Tyr-Val (464.2 Da) using RP-HPLC connected on-line to an electrospray ionization (ESI) mass spectrometer. The purified peptide showed a 9.14-fold higher scavenging activity for hydroxyl radical compared with the crude LPH. Therefore, it is possible to produce natural antioxidative peptides from loach protein by enzymatic hydrolysis and purification.     

Effect of degree of hydrolysis on the antioxidant activity of loach (Misgurnus anguillicaudatus) protein hydrolysates

Loach (Misgurnus anguillicaudatus) proteins were hydrolysed by papain and Protamex, the antioxidant activity of loach protein hydrolysates (LPH) was investigated. The results demonstrated that extensive hydrolysis by papain and Protamex led to the browning of the hydrolysates. When the degree of hydrolysis (DH) was 23%, hydrolysates prepared by papain (HA) exhibited the strongest antioxidant activity. The maximum values of the hydroxyl, 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical scavenging activities and the reducing power were 56.1%, 95.5%, 2.80 mM and 1.46, respectively. The hydrolysates prepared by Protamex (HB) showed the strongest hydroxyl radical scavenging activity (55.0%) at DH 28%, DPPH radical scavenging activity (92.2%) and ABTS radical scavenging activity (2.81 mM) at DH 23%, and the reducing power (1.17) at DH 33%. At the same DH value, there were significant (p < 0.05) differences between HA and HB. Several antioxidant amino acid residues, especially Trp and His, contributed significantly to the antioxidant activity of the hydrolysates. An increase of peptides with molecular weight below 500 Da was observed as the DH increased for all LPH. The above results indicated that DH and protease greatly influenced the molecular weight and amino acid residue composition of LPH, and further influenced the antioxidant activity.Industrial relevanceLoach has long been employed as a traditional Chinese medicine for the treatment of many kinds of diseases. From our previous work, loach was determined to be a good source of protein (accounts for approximately 17% (w/w) of the body weight). In this work, loach proteins were hydrolyzed by papain and Protamex to specific extent. The effect of DH on the antioxidant activities of hydrolysates was investigated. The results indicated that loach protein hydrolysates were potent antioxidants which were significantly affected by DH. This research is helpful for extensive development of loach product.     

Antioxidative properties of peptides prepared from tuna cooking juice hydrolysates with orientase (Bacillus subtilis)

The antioxidative activity of isolated peptides from of tuna cooking juice hydrolysates by orientase was determined. The results showed that, among the hydrolysates obtained under different hydrolysis time, the highest antioxidative activity and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging capacity were presented at 60 min. Then, the protein hydrolysate was subjected to a Sephadex G-25 gel filtration chromatography, and five major fractions were obtained. Peptide fractions ranging from 400 to 1500 Da showed the highest antioxidative activity among all fractions. The peptide fractions were further isolated using the two-step high performance liquid chromatography (HPLC-1 and HPLC-2), and six and three fractions were obtained, respectively. The final three antioxidative peptides comprised 4–10 amino acids sequences were observed, and the structures of the peptides were Pro-Val-Ser-His-Asp-His-Ala-Pro-Glu-Tyr (1305 Da), Pro-Ser-Asp-His-Asp-His-Glu (938 Da), and Val-His-Asp-Tyr (584 Da), respectively. We thus conclude that antioxidative hydrolysates from tuna cooking juice may be useful ingredients in food and nutraceutical applications.     

Effects of combined high-pressure homogenization and enzymatic treatment on extraction yield,hydrolysis and function properties of peanut proteins

Peanut protein isolate (PPI) was extracted by high-pressure homogenization (HPH) under 0.1 MPa (atmospheric pressure) and 40 or 80 MPa (high pressure). Effects of Alcalase (a proteolytic enzyme) on the enzymatic hydrolysis of PPI and the antioxidant activity of the PPI hydrolysates were investigated. The molecular weight distributions of the PPI hydrolysates were analyzed using Sephadex G-25 gel filtration chromatography while the antioxidant activities, including reducing power, 1,1-dipheny-2-picrylhydrazyl (DPPH) radical-scavenging activity and hydroxyl free radical-scavenging activity of the PPI hydrolysates were evaluated. The extraction yields of PPI by HPH under 0.1, 40 and 80 MPa were 16.84, 30.65 and 39.86%, respectively, which showed that HPH treatment improved the PPI extraction. The HPH treatment increased the degree of hydrolysis of PPI and significantly increased the reducing power and hydroxyl radical­scavenging activity. Furthermore, the molecular weight distributions of the PPI hydrolysates appeared principally over the range of 1000–5000 Da, while the HPH treatment enhanced the production of small peptides, which was in agreement with the high PPI hydrolysis degree. These results suggest that HPH treatment in combination with enzymatic hydrolysis could modify PPI properties and increase the antioxidant activities of the PPI hydrolysates.Industrial relevanceThis study was focused to evaluate the effects of high-pressure homogenization (HPH) in combination with enzymatic hydrolysis on extraction yield and enzymatic hydrolysis of PPI and antioxidant activity of the PPI hydrolysates. This study indicated the possibility of improving the availability of PPI by HPH treatment via increasing extraction yield and enzymatic hydrolysis of the PPI, which can provide a better utilization of the peanut by-product.     

Antiphotoaging effect and purification of an antioxidant peptide from tilapia (Oreochromis niloticus) gelatin peptides

In this study, the effect of tilapia gelatin peptides (TGP) on UV-induced damages to mice skin was evaluated. The antioxidant indicators, lipid, collagen and glycosaminoglycan of mice skin were determined and histological changes of the collagen were depicted. The results showed TGP could alleviate the UV-induced abnormal changes of antioxidant indicators, and the protective effect was in dose-dependent manners. TGP could protect skin lipid and collagen from the UV radiation damages, and the change of glycosaminoglycan was also recovered significantly. The action mechanisms of TGP mainly involved the antioxidant properties and the repairing to endogenous collagen synthesis. Therefore, the key antioxidant peptide was further purified from TGP. Finally, one antioxidant peptide was identified and the amino acid sequence was Leu-Ser-Gly-Tyr-Gly-Pro (592.26 Da). The IC₅₀ value of this peptide on hydroxyl radical scavenging activity was 22.47 μg/mL. TGP could be a novel antiphotoaging agent from natural resources.     

The effect of time and origin of harvest on the in vitro biological activity of Palmaria palmata protein hydrolysates

The effect of starting protein composition, and the origin and time of harvesting on the in vitro tyrosinase, dipeptidyl peptidase (DPP) IV and angiotensin converting enzyme (ACE) inhibitory and antioxidant activity of Palmaria palmata protein hydrolysates were investigated. Electrophoretic profiles showed significant differences in aqueous protein extracts obtained from the red macroalga Palmaria palmata harvested at different times of the year. No significant difference was observed in aqueous protein profiles extracted from wild and aquacultured samples of Palmaria palmata. Protein extracts from Palmaria palmata samples harvested from wild plants in April, July and October, and samples cultivated on longlines and harvested in April were hydrolysed with Alcalase 2.4 L and Corolase PP. The hydrolysates, when tested at 10 mg/ml, were shown to inhibit tyrosinase by 37–56%. The oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power (FRAP) values of the hydrolysates ranged from 323–494 and 8.9–19.9 μmol trolox equivalents per gram, respectively. The Palmaria palmata hydrolysates inhibited DPP-IV (IC₅₀: 1.60–4.24 mg/mL) and ACE (IC₅₀: 0.14–0.35 mg/mL) activities. The starting protein composition had a significant effect on the tyrosinase inhibitory activity, while protein composition and hydrolytic enzyme preparation had a significant effect on DPP-IV inhibitory and antioxidant activities. In general, the origin of the samples, wild or cultivated, had no effect on the in vitro biological activity of the protein hydrolysates. The results show that Palmaria palmata hydrolysates may have potential applications as health enhancing ingredients and as food preservatives due to their antioxidant and tyrosinase inhibitory effects.     

ABTS+ scavenging potency of selected flavonols from Hypericum perforatum L. by HPLC-ESI/MS QQQ: Reaction observation,adduct characterization and scavenging activity determination

Hypericum perforatum L. (HPL) has been used as a beneficial herb on menopause related syndromes for many years due to its antioxidant activity by inhibiting lipid oxidation. The major bioactive gradients of HPL are flavonoids. To date, extensive researches have been focused on its antioxidant effects, however, their exact antioxidant properties remain unclear. For this purpose, this study monitored the antioxidant ability of four common and representative HPL flavonols (rutin, hyperoside, quercitrin and quercetin) to interfere with the reaction of 2, 2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) free radical (ABTS⁺) based on the kinetic spectrophotometric free radical processing measurement method. In addition, to characterize the antioxidant reaction paths, the above-mentioned products were identified by high performance liquid chromatography–electrospray ionization/mass spectrometer triple quad (HPLC–ESI/MS QQQ) analysis, which was superior to gas chromatography–mass spectrometer (GC–MS) in that no derivation resulted. After the adduct products were identified in the MS2 scan mode, a multiple reaction monitoring (MRM) based LC–MS method for the ABTS⁺ scavenging determination was developed and validated. The results of free radical scavenging kinetic evaluation showed that the ABTS⁺ scavenging process of the four flavonols was relatively slow and steady. The degradation products of reactions between flavonols and ABTS⁺ were mainly a combination of flavonols with the ABTS molecule by reducing 3-ethyl-6-benzothiazolinone imine. Flavonols substituted by glycosides tended to follow a RDA cleavage fragmentation path. With a good linearity regression coefficient (r² = 0.995) and a good accuracy of 96.5–106.2%, the free radical scavenging ability of rutin, hyperoside, quercitrin and quercetin was 19.0%, 36.1%, 40.2% and 39.5%, respectively. The kinetic scavenging features and specific fragment features deduced here of the four flavonols in vitro could shed light on HPL's antioxidant properties.     

Purification and identification of three novel antioxidant peptides from protein hydrolysate of bluefin leatherjacket (Navodon septentrionalis) skin

Skin protein from a bluefin leatherjacket (Navodon septentrionalis) processing by-product was hydrolyzed by trypsin, flavourzyme, neutrase, papain, alcalase, and pepsin, and protein hydrolysate (BSH) prepared using alcalase showed the highest DPPH, HO, and O₂⁻ · scavenging activities among all hydrolysates. Using ultrafiltration and consecutive chromatography, three novel peptides with strong antioxidant properties were purified from BSH, and their sequences were determined as Gly-Ser-Gly-Gly-Leu (GSGGL, BSP-A), Gly-Pro-Gly-Gly-Phe-Ile (GPGGFI, BSP-B), and Phe-Ile-Gly-Pro (FIGP, BSP-C) with molecular weights of 389.41, 546.63, and 432.52 Da, respectively. BSP-C exhibited the highest scavenging activities on DPPH (EC₅₀ 0.118 mg/ml), HO (EC₅₀ 0.073 mg/ml), and O₂⁻ · (EC₅₀ 0.311 mg/ml) among the three peptides. In addition, BSP-C could effectively inhibit autooxidation in a linoleic acid model system. The antioxidant activities of BSP-A, BSP-B, and BSP-C might be due to the small molecular sizes and the hydrophobic and/or aromatic amino acid residues in their amino acid sequences. The present results suggested that peptides purified from the skin protein hydrolysate of bluefin leatherjacket were excellent antioxidants and could be effectively used as food ingredients and additives, and pharmaceuticals.     

Identification and characterization of papain-generated antioxidant peptides from palm kernel cake proteins

Novel peptides with antioxidant activity were isolated and identified from papain generated palm kernel cake (PKC) proteolysate. The proteolysate was fractionated into individual peptides based on hydrophobicity and isoelectric point using reversed-phase high-performance liquid chromatography and isoelectric focusing techniques. The individual peptides were identified by tandem mass spectrometry and their respective antioxidant activities were evaluated using 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity and metal chelating activity assays. Peptide sequences, AWFS, WAF, and LPWRPATNVF showed the highest radical scavenging activities of 71%, 56%, and 50%, respectively, while peptide sequences GGIF, YGIKVGYAIP and YLLLK showed the highest metal chelating activities of 56%, 53%, and 50%, respectively. However, the best IC₅₀ values of peptides measured by DPPH assay were displayed by GIFE, GVQEGAGHYALL and GGIF at 0.02 μM, 0.09 μM and 0.35 μM, respectively, while the best half maximal inhibitory concentration values measured using metal chelating activity were shown by LPWRPATNVF, AWFS and YGIKVGYAIP at 0.001 μM, 0.002 μM and 0.087 μM, respectively. It can be concluded that the peptides derived from PKC proteolysate were more potent and distinctive compared to those previously reported from other plant protein sources.     

Intracellular ROS scavenging and antioxidant enzyme regulating capacities of corn gluten meal-derived antioxidant peptides in HepG2 cells

The objective of this study was to investigate the intracellular reactive oxygen species (ROS) scavenging activities and antioxidant enzyme regulating capacities of corn gluten peptide fractions (CPFs) in HepG2 cells. A cellular antioxidant activity (CAA) assay was used to assess their antioxidant activities and revealed that both CPF1 (molecular weight < 1 kDa) and CPF2 (molecular weight between 1 and 3 kDa) exhibited high cellular antioxidant activities with EC₅₀ values of 2.85 ± 0.19 mg/mL and 5.05 ± 0.32 mg/mL, respectively. Both CPFs also exhibited cytoprotective effects and intracellular ROS scavenging activities in HepG2 cells subjected to oxidative stress by oxidation with H₂O₂. In addition, at concentrations of 2.50 mg/mL, the CPFs increased the activity levels of superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GR), as well as the total glutathione (GSH) levels in oxidized HepG2 cells (from 86.54% to 114.14% (CPF1) or 109.72% (CPF2) for SOD activity; from 71.91% to 107.64% (CPF1) or 106.50% (CPF2) for CAT activity; from 70.52% to 103.01% (CPF1) or 104.10% (CPF2) for GR activity; and from 81.39% to 114.00% (CPF1) or 108.82% (CPF2) for total GSH levels). These results suggested that both CPF1 and CPF2 exhibited positive effects on the activities of the intracellular antioxidant enzymes SOD, CAT and GR, as well as on the total GSH levels in HepG2 cells under conditions of oxidative stress. Furthermore, size exclusion gel chromatography and MALDI-TOF/TOF mass spectrometry revealed that the molecular weights of the antioxidant peptides in CPF1 were between 500 Da to 900 Da, and a novel antioxidant peptide consisting of GLLLPH (Gly-Leu-Leu-Leu-Pro-His) was identified in CPF1.