Purification,characterization, and in vitro digestion of novel antioxidant peptides from chicken blood hemoglobin

The study aimed to purify and characterize antioxidant peptides from chicken blood hemoglobin hydrolysate. The fraction M₂ (< 3 KDa) with the strongest antioxidant activity was isolated by ultrafiltration, and its DPPH (1,1-diphenyl-2-picryl-hydrazyl radical) free radical scavenging rate, ABTS [2,2′-Azinobis-(3-ethylbenzthiazoline-6-sulphonate)] free radical scavenging rate, and iron ion chelation activity were 82.91%, 77.49%, and 80.99%, respectively. After in vitro digestion, the antioxidant capacity of chicken blood hydrolysate was significantly higher than that before digestion (p < 0.05). M₂ exhibited the strongest antioxidant activity after stomach digestion, with a DPPH radical scavenging rate and iron ion chelating power of 82.91% and 79.61%, respectively. Component A was purified from M₂ by Sephadex G-25 gel chromatography. The peptide sequences were identified by LC-MS/MS from fraction A, and four peptides, AEDKKLIQ (944.54 Da), APAPAAK (625.36 Da), LSDLHAHKL (1033.57 Da), and LSNLHAYNL (1044.54 Da) were synthesized using the solid-phase peptide method, among which APAPAAK was a novel antioxidant peptide. Molecular docking was used to simulate the binding of these four peptides to the key active site of Keap1 via hydrogen bonding. This study suggests that chicken blood may provide a new natural source of antioxidant peptides.     

Antioxidant activities of enzymatic‐hydrolysed proteins of dromedary (Camelus dromedarius) colostrum

This work investigated the antioxidant activities of dromedary colostrum proteins before and after hydrolysis by pepsin, trypsin, α‐chymotrypsin, pancreatin and papain. The enzymatic hydrolysis affected the degrees of hydrolysis, electrophoretic profiles, molecular weight distribution and hydrophobic/hydrophilic properties of the generated peptides. The antioxidant activities were evaluated using four antioxidant assays, including 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) and 2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS) radical‐scavenging activities, ferric reducing power and ferrous ion chelating activity. Interestingly, the antioxidant activities of dromedary colostrum proteins were enhanced after enzymatic hydrolysis. The highest antioxidant potential was obtained by pancreatic hydrolysates (P ≤ 0.05). These results suggest that dromedary colostrum protein hydrolysates are an important source of natural antioxidant peptides.     

Identification of radical scavenging peptides (<3 kDa) from Burgos-type cheese

The aim of the present study was to identify low molecular weight peptides with radical scavenging activity from cheese made using different types of rennet: batch 1, animal rennet (95% chymosin and 5% bovine pepsin), batch 2, rennet of plant origin (Cynara cardunculus) and batch 3, microbial rennet (Mucor miehei). After preparation of the peptide extracts (<3 kDa), antioxidant activity was assayed by their DPPH radical scavenging and metal chelating a6ctivity. All of the batches showed antioxidant activity, which could be dependent on the peptides which are present in extracts: Batch 2 and 3 showed the highest values for DPPH inhibition and chelating effect. Fourteen fractions out of the total peptide fractions collected after RP-HPLC analysis showed radical scavenging activity using the DPPH inhibition method. Free amino acids and peptides were identified from these fractions. One of the peptides, derived from α_s1-casein, was a potential new antioxidant peptide. These antioxidant peptides were present in a lower content in extracts obtained from animal rennet cheese in comparison with the other extracts.     

Production and characterisation of duck albumen hydrolysate using enzymatic process

Influences of different ultrasound treatments combined with heat pretreatment on enzymatic hydrolysis, emulsifying properties and antioxidant activities of hydrolysates from duck egg albumen were studied. Heat pretreatment at 95 °C for 30 min inhibited both serine and cysteine protease inhibitors effectively. Ultrasonication of heated duck albumen at 60% amplitude for 10 min yielded the highest surface hydrophobicity. Coincidentally, aforementioned pretreatment rendered the hydrolysate with highest degree of hydrolysis (DH) than other pretreatments when Alcalase was used. The resulting hydrolysate showed the highest antioxidant activities including DPPH radical and ABTS radical cation scavenging activities and ferric reducing antioxidant power as well as emulsifying properties when hydrolysis time of 90 min was used. The hydrolysate possessed the peptides with molecular weight of 219–255 Da with the highest ABTS radical scavenging activity. Thus, heat pretreatment, followed by ultrasonication of duck albumen under appropriate condition could increase DH, antioxidant activities and emulsifying properties of duck albumen hydrolysate.     

Evaluation of antioxidant activities and total phenolic contents of typical malting barley varieties

Fourteen typical malting barley varieties from China were evaluated for their DPPH radical, ABTS radical cation and superoxide anion radical scavenging activities, reducing power, metal chelating activities, and total phenolic contents (TPC). All barley samples exhibited significant antioxidant activities determined by different assays, and contained significant levels of phenolic compounds. Gan4 and Wupi1 barley exhibited the highest DPPH radical scavenging activity, ABTS radical cation scavenging activity and reducing power. Gan4 and Humai16 barley showed the highest TPC, whereas the highest superoxide anion radical scavenging activity and metal chelating activity were found in Huaimai19 and Ken3 barley, respectively. The Pearson correlation analysis revealed that the TPC showed strong correlations with DPPH radical scavenging activity, ABTS radical cation scavenging activity, and reducing power (P < 0.01), whereas its correlations with superoxide anion radical scavenging activity and metal chelating activity were poor (P > 0.05). Moreover, DPPH radical scavenging activity, ABTS radical cation scavenging activity and reducing power were well positively correlated with each other (P < 0.01). Principal component analysis (PCA) was applied to understand the interrelationships among the measured antioxidant activity evaluation indices, and to gain an overview of the similarities and differences among the 14 barley varieties.     

Biochemical properties and antioxidant activity of myofibrillar protein hydrolysates obtained from patin (Pangasius sutchi)

Antioxidant activities of myofibrillar protein hydrolysates (MPH) prepared from patin (Pangasius sutchi) using papain and Alcalase^® 2.4 L with different degrees of hydrolysis (DH) were investigated. With a DH of 65.83%, the hydrolysate prepared with papain exhibited the maximum of 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) radical‐scavenging activity (71.14%) with a reducing power of 0.310. At a concentration of 1 mg mL^?1, the papain‐MPH exhibited a Trolox equivalent antioxidant capacity (TEAC) of 70.50 ± 1.22 μmol g^?1 protein. With a DH of 83.6%, the Alcalase‐MPH had the highest metal‐chelating activity. Low molecular weight peptides showed higher antioxidant activities than high molecular weight peptides. Both papain‐MPH and Alcalase‐MPH contained high amounts of the essential amino acids (48.71% and 48.10%, respectively) with glutamic acid, aspartic acid and lysine as the dominant amino acids. These results suggest that the protein hydrolysates derived from patin may be used as an antioxidative ingredient in both functional food and nutraceutical applications.     

Purification and identification of antioxidant peptides from corn gluten meal

Corn gluten meal was hydrolyzed by alkaline protease and Flavourzyme to obtain the antioxidant peptides. The antioxidant activities of the hydrolysates or peptides were evaluated by free radical scavenging capacity (1,1-diphenyl-2-picrylhydrazyl/2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt/hydroxyl radical/superoxide radical anion), metal ion (Fe²⁺/Cu²⁺) chelating activity and lipid peroxidation inhibitory capacity. The hydrolysates were separated by ultrafiltration, and those with molecular weight <10 kDa exhibited highest antioxidant activity in all relevant assays. The hydrolysates were subsequently purified by gel filtration chromatography, and fraction F3 showed the highest antioxidant activity. Three peptides were identified from fraction F3 using LC–ESI–Q–TOF MS/MS as Leu-Pro-Phe (375.46 Da), Leu-Leu-Pro-Phe (488.64 Da) and Phe-Leu-Pro-Phe (522.64 Da). These peptides exhibited good free radical scavenging activity and lipid peroxidation inhibitory effect. Thus, corn gluten meal may be used as a potential source of antioxidant peptides for food and nutraceutical applications.     

Antioxidant activities of red pepper (Capsicum annuum) pericarp and seed extracts

In this study, we examined the antioxidant activities of red pepper (Capsicum annuum, L.) pericarp and red pepper seed extracts. The extracts were evaluated by various antioxidant assays, including 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) radical scavenging, superoxide anion radical scavenging, hydroxyl radical scavenging, [2,2′‐azino‐bis(3‐ethylbenzthiazoline‐6‐sulphonic acid)] (ABTS) radical scavenging, ferrous chelating activity, superoxide dismutase (SOD) activity and reducing power, along with the determination of total phenolic and flavonoid contents. All the extracts showed strong antioxidant activity by the testing methods. The red pepper pericarp extract exhibited strong ferrous chelating activity and high scavenging activity against free radicals, including both the hydroxyl and DPPH radicals, but it exhibited weaker scavenging activity for the superoxide anion radical and for SOD. In contrast, the red pepper seed extract exhibited strong SOD activity and high scavenging activity against the superoxide anion radical, but showed weaker ferrous chelating activity, hydroxyl radical scavenging, and DPPH radical scavenging. We observed that the reducing power level and ABTS radical scavenging activity of the red pepper seed were higher than those of the red pepper pericarp at the highest tested concentration. Most of the test results for the red pepper seed and red pepper pericarp extracts increased markedly with increasing concentration; however, the metal chelating, SOD and ABTS radical scavenging activities did not increase with the concentration. Highest total phenolic and flavonoid contents were obtained from the red pepper pericarp extracts. Overall, the red pepper seed and red pepper pericarp extracts were highly effective for the antioxidant properties assayed, with the exceptions of ferrous chelating activity, hydroxyl radical scavenging and SOD activity.     

Antioxidant function of tea dregs protein hydrolysates in liposome–meat system and its possible action mechanism

Tea dregs possess abundant proteins, and the objective of this study was to investigate the antioxidant activity of tea dregs protein hydrolysate with limited hydrolysis by protamex and its possible action mechanism. Tea dregs protein was hydrolysed by alcalase, protamex or neutrase. The hydrolysis condition was optimised, and the hydrolysate was characterised for 1,1‐diphenyl‐2‐picryl hydrazyl (DPPH) radical‐scavenging activity, hydroxyl radical‐scavenging activity and antioxidant activity in linoleic acid (LA) system and in chicken products. Tea dregs protein hydrolysate (TDPH) was formulated (0.1%, 0.5%, 1.0%, w/w) into chicken products to determine in situ antioxidant efficacy. Thiobarbituric acid‐reactive substances (TBARS) and peroxide value (POV) formed in chicken products during storage (4 °C, 0–7 days) were analysed. Results showed that the optimum hydrolysis condition was at 50 °C, pH 7.0 for 20 min, and the concentration of tea dregs protein was 1.5%; ratio of protamex to substrate was 6000 U g^?1. The radical‐scavenging ratio of TDPH to 1,1‐diphenyl‐2‐picryl hydrazyl (DPPH) was 90.30% at the concentration of 0.1 mg mL^?1 and that to hydroxyl radical was 65.18% at the concentration of 1.0 mg mL^?1. Moreover, it also showed strong antioxidant activity both in linoleic acid (LA) system and in chicken products. The molecular weight distribution of tea dregs hydrolysates was determined by nanofiltration tubular membrane, and the protein hydrolysates with molecular weight above 8000 Da had more effective antioxidant activity. The radical‐scavenging activities to DPPH and hydroxyl radical were 85.72% at 0.1 mg mL^?1 and 71.52% at 1.0 mg mL^?1, respectively. These findings suggest that the enzymatic hydrolysate of tea dregs protein probably possesses the specific peptides/amino acids which could stabilise or terminate the radicals through donating hydrogen. In addition, the hydrolysate could form a physical barrier around the fat droplets.     

克氏螯虾虾头Protamex蛋白酶水解产物的抗氧化活性和功能性质研究

使用Protamex蛋白酶对克氏螯虾虾头蛋白进行水解,以制备抗氧化活性肽。结果表明:虾头蛋白肽的还原力、Fe2+螯合力和DPPH自由基清除能力与蛋白肽的浓度之间存在量效关系,均随着肽浓度的增大而增大。通过对蛋白肽的分子量分布进行研究发现,95.14%的多肽的分子量小于1000Da。同时测定了酶解产物的功能特性,结果表明pH对虾头蛋白水解物的溶解性、起泡性和乳化性具有显著影响。     

Effects of physicochemical factors and in vitro gastrointestinal digestion on antioxidant activity of R‐phycoerythrin from red algae Bangia fusco‐purpurea

R‐phycoerythrin (R‐PE) was purified from the red algae Bangia fusco‐purpurea after 35–50% ammonium sulphate fractionation followed by ion‐exchange column chromatography on DEAE‐Sepharose, resulting in a purity (A₅₆₅/A₂₈₀) ratio of 5.1. The circular dichroism spectroscopy results suggested that the structure of R‐PE is predominately helical. The antioxidant activity of R‐PE was studied and revealed changes in conformation and antioxidant activity at different temperatures and pH values. After in vitro‐simulated gastrointestinal (GI) digestion of R‐PE, the scavenging activity of ABTS radical (EC₅₀, 769.9 μg mL^?1), DPPH radical (EC₅₀, 421.9 μg mL^?1), hydroxyl radical (EC₅₀, 32.4 μg mL^?1) and reducing power (A₇₀₀ = 0.5, 625.8 μg mL^?1) were measured. Gel filtration chromatography analysis showed that the molecular weight distribution of the final GI digest that still contained high antioxidant activity was <3 kDa. Our present results indicate that digestion‐resistant antioxidant peptides of R‐PE may be obtained by in vitro GI proteinases degradation.     

Effect of thermal treatment on the characteristic properties of loach peptide

The effects of thermal treatment on the changes in the colour, the molecular weight distribution, amino acid profiles and the overall antioxidant activities of loach peptide were investigated. When the thermal temperature was rising, the browning was enhanced. The contents of free amino acids Gly, Arg, Thr and Tyr were increased, whereas those of Asp, Ser, Met, Cys, Trp and Lys were decreased. The scavenging activities (1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) radical scavenging activity, the oxygen radical absorbance capacity (ORAC) and the reducing power of loach peptides after different thermal treatments were lower than those of the control, whereas the Cu²⁺ chelating activity was higher (except from the sample treated at 100 °C, 20 min). The heat treatment at 121 °C, 15 min was shown to be the best thermal processing, as it caused the least loss in radical scavenging activity and showed relatively higher Cu²⁺ chelating activity.     

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.     

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     

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.     

Effect of ultrasonic pretreatment on the antioxidant properties of porcine liver protein hydrolysates

In this study, the effect of ultrasonic pretreatment on the antioxidant activity of porcine liver protein hydrolysates (PLPHs) was investigated. The results showed that the degree of hydrolysis (DH) and peptide contents of the PLPHs increased as the time of ultrasonication increased. The hydrolysate pretreated with ultrasonication for 60 s exhibited the highest DH and peptide contents. The hydrolysate pretreated with ultrasonication for 45 s exhibited the highest ferrous ion chelating ability and reducing power. The hydrolysate pretreated with ultrasonication for 30 s exhibited the highest 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) radical scavenging activity and the higher inhibitory activity in the linoleic acid autoxidation system. The molecular weight of peptides in the hydrolysates was less than 6.2 kDa. The results clearly demonstrated that ultrasonic pretreatment enhances the antioxidant activities of the PLPHs in a short period of time (15–30 s).     

Effect of amino acids on Maillard reaction product formation and total antioxidant capacity in white pan bread

Maillard reaction products (MRPs) can be an important dietary antioxidant source. Bread is the most popular bakery product; however, limited information is available on the antioxidant activities of MRPs generated during bread-baking. This paper reports the effect of different amino acids on bread properties, melanoidin formation and antioxidant activities. Totally six amino acids at four different levels were evaluated. Increasing the amount of amino acid led to darker bread crust, higher melanoidin content and stronger antioxidant activity. Among the six amino acids, bread with glycine had the highest melanoidin content and ABTS radical scavenging activity up to 1079.77 ± 8.43 μmol TE mg⁻¹. Bread with lysine had the highest DPPH radical scavenging activity up to 281.97 ± 10.52 μmol TE mg⁻¹. Bread with alanine had the highest crust metal chelating activity compared with others. Adding certain dietary amino acids is a potential approach to enhance the antioxidant capacity of bread products.     

Isolation and identification of antioxidative peptides from rice endosperm protein enzymatic hydrolysate by consecutive chromatography and MALDI-TOF/TOF MS/MS

The defatted rice endosperm protein (REP) was, respectively, digested by five different protease treatments (Alcalase, Chymotrypsin, Neutrase, Papain and Flavorase), and Neutrase appears to be the most desirable for producing high quality antioxidant peptides from REP. Specially, the DPPH and hydroxyl radical scavenging activities of NHREP were higher than its superoxide radical scavenging activity, and the percentage inhibition of autooxidation of NHREP (80.09%) was similar to that of α-tocopherol (86.59%) on day 5. Furthermore, NHREP was purified consecutively, and the antioxidant peptides were identified to be Phe-Arg-Asp-Glu-His-Lys-Lys (FRDEHKK, 959.5 Da) and Lys-His-Asp-Arg-Gly-Asp-Glu-Phe (1002.5 Da) by MALDI-TOF/TOF MS/MS. Lastly, FRDEHKK was chemically synthesised. It significantly inhibited lipid peroxidation in an linoleic acid emulsion system more effectively than α-tocopherol, and enhanced the viability of t-BHP induced cytotoxicity up to 74.38% (for MRC-5) and 78.39% (for RAW264.7) at 80 μg/ml. Conclusively, it was feasible to produce natural antioxidants from REP.     

Phenolic Compounds and Antioxidant Capacities of 10 Common Edible Flowers from China

The free and bound phenolic compounds in 10 common Chinese edible flowers were investigated using reversed phase high‐performance liquid chromatography. Their antioxidant capacities were evaluated using 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) radical‐scavenging activity, 2,2'‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulphonic acid) (ABTS) radical‐scavenging activity, oxygen radical absorption capacity (ORAC), ferric reducing antioxidant power (FRAP), and cellular antioxidant activity (CAA). Free factions were more prominent in phenolic content and antioxidant capacity than bound fractions. Paeonia suffruticosa and Flos lonicerae showed the highest total phenolic content (TPC) 235.5 mg chlorogenic acid equivalents/g of dry weight and total flavonoid content 89.38 mg rutin equivalents/g of dry weight. The major phenolic compounds identified were gallic acid, chlorogenic acid, and rutin. P. suffruticosa had the highest antioxidant capacity in the DPPH, ABTS, and ORAC assays, which were 1028, 2065, 990 μmol Trolox equivalents/g of dry weight, respectively, whereas Rosa chinensis had the highest FRAP value (2645 μmol Fe²⁺ equivalents /g of dry weight). The P. suffruticosa soluble phenolics had the highest CAA, with the median effective dose (EC₅₀) 26.7 and 153 μmol quercetin equivalents/100 g of dry weight in the phosphate buffered saline (PBS) and no PBS wash protocol, respectively. TPC was strongly correlated with antioxidant capacity (R = 0.8443 to 0.9978, P < 0.01), which indicated that phenolics were the major contributors to the antioxidant activity of the selected edible flowers.     

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.