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.     

Effects of cationic property on the in vitro antioxidant activities of pea protein hydrolysate fractions

Yellow pea seed protein-derived peptides were produced through enzymatic hydrolysis of pea protein isolate, which was followed by ultrafiltration to isolate peptides with < 3 kDa sizes (PPH). The PPH was separated by cation-exchange chromatography into five peptide fractions (F1-F5) based on their affinity for the stationary phase. F1 contained peptides with the least amount (7.5%) of cationic amino acids while F5 contained peptides with more than 50% content of cationic amino acids. F1-F5 peptide fractions were evaluated for in vitro antioxidant activities in comparison to glutathione (GSH), an endogenous antioxidant peptide. Results showed that the peptide fraction with the least cationic property (F1) had significantly strongest (p < 0.05) scavenging activity against 1,1-diphenyl-2-picrylhydrazyl radical and hydrogen peroxide (H₂O₂) when compared to F2-F5. Generally, the scavenging of O₂?B⁻ and H₂O₂ was negatively related with the cationic property of the peptide fractions. Fractionation according to cationic property resulted in significantly increased (p < 0.05) O₂?B⁻ scavenging power of the peptide fractions (F1-F5) when compared to the unfractionated PPH. The peptide fractions showed weak or nil ferric reducing power and, regardless of cationic property, were significantly (p < 0.05) more effective at inhibiting lipid oxidation than GSH over a 7-day period. In contrast, with the exception of H₂O₂ scavenging, GSH was a more effective antioxidant than PPH and the peptide fractions. We concluded that the PPH and some of its fractions have the potential to be used as therapeutic agents against chronic diseases that develop from oxidative damage, and that the cationic property of amino acids contributed negatively to the antioxidant activity of the peptide fractions.     

Alcalase-generated proteolysates of stone fish (Actinopyga lecanora) flesh as a new source of antioxidant peptides

In the present study, the alcalase-generated proteolysates obtained after 8 h of proteolysis of stone fish flesh showed the most potent antioxidant activity in terms of DPPH^? radical scavenging activity (77.43%, IC₅₀ of 0.5 mg/mL), ABTS^? radical scavenging activity (92.73%, IC₅₀ of 0.33 mg/mL) and FRAP value (39.2 mmol/100 mL FeSO₄). These proteolysates profiled and characterized as antioxidative peptides. The proteolysates were initially subjected to ultrafiltration using MWCO Spin-X UF. Potent fractions were further characterized based on hydrophobicity using reversed-phase high-performance liquid chromatography (RP-HPLC) and isoelectric point using an OFFGEL isoelectric focusing fractionator. Results indicated that most of the antioxidative peptides found in fractions with a molecular weight (MW) of less than 2 kDa, hydrophilic (hydrophilicity >80%) and basic (pI = 9.7). The final purified fraction with the highest antioxidant activity was selected for peptide identification and sequencing using Q-TOF mass spectrometry. A total of four peptides were identified, from which Peptide 1 (GVSGLHID) showed the highest antioxidant activity and this has potential as a novel bioingredient of nutraceuticals and functional foods to promote human health.     

Phenolics from hull of Garcinia mangostana fruit and their antioxidant activities

The air-dried fruit hulls of Garcinia mangostana Linn. were extracted with 70% MeOH, and then partitioned into the n-BuOH fractions. Furthermore, three major phenolic components related to their antioxidant activities were purified by silica gel column chromatography and Sephadex LH-20 and then identified as P₁ (1,3,6,7-tetrahydroxy-2,8-(3-methyl-2-butenyl)), P₂ [1,3,6-trihydroxy-7-methoxy-2,8-(3-methyl-2-butenyl) xanthone] and P₃ (epicatechin) using UV–visible spectrophotometry, IR spectrophotometry and NMR spectroscopy, respectively. The antioxidant activities of three major phenolics were evaluated using different tests, including the free radical scavenging capability and total antioxidant activity in a linoleic acid peroxidation. These three phenolic compounds exhibited different antioxidant activities in these antioxidant tests. The hydroxyl radical and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging capabilities and the activity against linoleic acid peroxidation of P₁ were greater than those of P₂ and P₃, while the superoxide anion radical scavenging activity of P₃ was greater than that of P₁, but was close to that of P₂ or α-tocopherol. An activity-guided isolation and purification process was used to identify the components showing the strong DPPH radical scavenging activity from G. mangostana Linn.     

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.     

分子量对酪蛋白多肽抗氧化活性的影响

在对酪蛋白酶解产物制备工艺进行优化的基础上,对不同分子量抗氧化肽(>3ku,1～3ku,<1ku)的抗氧化活性进行了评价。首先对酶的种类、酶底物比及水解时间进行了单因素实验,最终确定采用碱性蛋白酶,在pH8.0,55℃,底物浓度5%,酶底物比0.192AU/g的条件下,酶解4h所得水解物抗氧化活性最高。经过超滤和凝胶过滤层析分离获得不同分子量的抗氧化肽,并采用2,2′-连氮基-双-(3-乙基苯并二氢噻唑啉-6-磺酸)二铵盐(ABTS+.)、羟自由基和超氧自由基清除活性评价其抗氧化性。结果表明,ABTS+.清除活性与分子量呈负相关(r=-0.898,p<0.01),分子量低于1ku组分活性最强(2mg/mL,Trolox当量为2.08±0.05mmol/L);分子量低于3ku的抗氧化肽羟自由基清除活性较高(IC50:1～3ku,4.43±0.03mg/mL;<1ku,4.35±0.06mg/mL);分子量高于3ku组分主要分布在3～5ku,超氧自由基清除活最强(10mg/mL,66.1%±1.0%)。     

Identification of potent antioxidant bioactive peptides from goat milk proteins

Goat milk proteins have gained increasing attention especially the bioactive peptides released from the parent proteins by digestive enzymes. Specifically, the interest in bioactives of goat milk is intensifying due to its reduced allergenicity compared to bovine milk. In this study, proteins of goat milk were fractionated into caseins (GCP) and whey proteins (GWP), hydrolyzed by pepsin and the generated peptides were examined for radical scavenging activities. The hydrolysates of whey (P-GWP) and casein (P-GCP) proteins exhibited potent superoxide anion (O₂^･−) scavenging activity in a dose-dependent manner, as investigated using the natural xanthine/xanthine oxidase (X/XOD) system. The P-GWP and P-GCP dramatically quenched the O₂^･− flux but had negligible effect on the catalytic function of the enzyme, indicating specificity to scavenge O₂^･− but not oxidase inhibition. Further, both P-GWP and P-GCP were able to remarkably quench the chemical DPPH radical. Fractionation of hydrolysates by size-exclusion chromatography produced four fractions (F1-F4) from both hydrolysates, with variable O₂^･− scavenging activities. However, the slow eluting fractions (F4) of both hydrolysates and fast eluting fraction (F2) of P-GCP contained peptides with the highest scavenging activities. Peptides in the active fractions of P-GWP and P-GCP, isolated by reversed phase-HPLC, exhibited significantly strong O₂^･− scavenging activities. MALDI-TOF-MS allowed the identification of several antioxidant peptides derived from both caseins and whey proteins, with β-casein and β-lactoglobulin being the major contributors, respectively. The results demonstrate that digestion with pepsin generates multiple soluble peptides from goat milk protein fractions with remarkable ability to scavenge superoxide radicals and thus providing a fascinating opportunity for their potential candidacy as antioxidant bioactive peptides.     

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 and Antiglycation Properties of Methanol Extract and Its Different Solvent Fractions of Musa paradisiaca L. (Cv. Nendran) Inflorescence

Methanolic extract of Musa paradisiaca inflorescence and its different solvent fractions, petroleum ether, ethyl acetate, butanol, and water were studied for their total phenolic and flavonoid content, free radical scavenging, and antiglycation activities, and these properties were compared with standard antioxidant compounds. Ethyl acetate fraction exhibited higher antioxidant and antiglycation properties than other fractions. IC₅₀ values of ethyl acetate fraction for 1,1-diphenyl-2-picrylhydrazyl method, 2,2-azinobis-3 ethylbenzothiazoline-6-sulphonic acid method, superoxide radical scavenging, hydroxyl radical scavenging, nitric oxide radical scavenging, and antiglycation activities were 9.80, 13.50, 26.40, 19.71, 25.73, and 31.00 μ g/ml, respectively. Total phenolic content of ethyl acetate (21.52 mg GAE/g) was significantly higher than other fractions. There was positive linear correlation between antioxidant activity and total phenolic content.      

Antioxidant and antibacterial activities of peptide fractions from flaxseed protein hydrolysed by protease from Bacillus altitudinis HK02

Flaxseed protein (FP) hydrolysates by crude protease of strain Bacillus altitudinis HK02 were further separated into five fractions by ultrafiltration membranes with a molecular weight cut‐off of 10, 5, 3 and 1 kDa for the analysis of antioxidant activities and antibacterial ability. The results demonstrated that the fraction of 1‐ to 3‐kDa peptides exhibited higher antioxidant activities on the free radical‐scavenging ability and the reducing power than those of Vit C, Vit E, BHA and other fractions. The fraction with a low molecular weight (<1 kDa) of peptides demonstrated the highest ferrous ion‐chelating ability and a higher inhibition of lipid peroxidation than BHA and other fractions. Moreover, it also exhibited the best growth inhibition of Pseudomonas aeruginosa and Escherichia coli. The results, including the concentration‐dependent effect of peptides fractions, demonstrated that it is feasible to derive functional ingredients of natural antioxidants along with antimicrobial activity from FPs hydrolysed by protease from B. altitudinis HK02.     

Neuronal cell protective effect of aerial parts of Chinese lizard’s tail (Saururus chinensis (Lour.) Baill.)

The antioxidant and neuronal cell protective effects of silica-gel open column chromatographic fractions, obtained from Chinese lizard’s tail (Saururus chinensis) methanolic extract, were investigated. The ABTS radical scavenging and ferric reducing antioxidant power (FRAP) assays indicated that the SC4 fraction (eluted with chloroform/methanol by 4:1, v/v) obtained from Chinese lizard’s tail was more potent radical scavengers and reducing agents than that of other 5 fractions. The SC4 fraction also presented protective effects against H₂O₂-induced neurotoxicity in a dose-dependent manner. Therefore, our study suggested that the SC4 fraction has strong antioxidant and neuronal protective effects which are correlated with its high level of phenolics, particularly quercitrin and quercetin.     

Isolation And Identification Of An Antioxidant Peptide Prepared From Fermented Peanut Meal Using Bacillus Subtilis Fermentation

In order to recover and utilize peanut protein resource, peanut meal was fermented using Bacillus subtilis to prepare antioxidant peptides in this study. The antioxidant activities of the peanut peptides were evaluated via six in vitro tests, namely, 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) scavenging activity, inhibition of linoleic acid autoxidation, reducing power, and metal chelating capacity. The peanut peptides showed 63.28% of scavenging effect on 1,1-diphenyl-2-picrylhydrazyl radical, 70.67% of scavenging effect on 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) and 39.32% of superoxide anion radical scavenging activity at 1.0 mg/ml, and 62.69% of inhibition on linoleic acid autooxidation at 0.8 mg/ml. Besides, the peanut peptides exhibited 32.8% of reducing power and 60.44% of Fe²⁺-chelating capacity at 2 mg/ml. Then the peanut peptides were desalted with macroporous resin; the fraction eluted with 75% ethanol showed the highest scavenging activity on 1,1-diphenyl-2-picrylhydrazyl radical. This fraction was subject to further purification using gel filtration chromatography and reverse phase-high performance liquid chromatography. At last, a peptide was gained, and it was identified to be Tyr-Pro with matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry. The results indicated that it is feasible to prepare natural antioxidants from peanut meal using B. subtilis fermentation.     

In vitro evaluation of the antioxidant activities in fruit extracts from citron and blood orange

Consumers are increasingly aware of diet related health problems and therefore demanding natural ingredients which are expected to be safe and health-promoting. Recently, number of studies on health benefits associated with citrus phytochemicals have been demonstrated. In the present study, an attempt has been made to isolate antioxidant fractions from two different citrus species such as Citron (Citrus medica) and blood orange (C. sinensis). Antioxidant fractions were extracted from mature, ripe fruits using five different solvents using a Soxhlet extractor. The total phenolic content of the extracts was determined by Folin–Ciocalteu method. MeOH:water (80:20) extract of citron and acetone extract of blood orange was found to contain maximum phenolics. The dried fractions were screened for their antioxidant activity potential using in vitro model systems such as 1, 1-diphenyl-2-picryl hydrazyl (DPPH), phosphomolybdenum method and as well as by the nitroblue tetrazolium (NBT) reduction test at different concentrations. The MeOH:water (80:20) fraction of citron showed highest radical scavenging activity 42.5%, 77.8% and 92.1% at 250, 500 and 1000 ppm, respectively, while MeOH:water (80:20) fraction of blood orange showed lowest DPPH radical scavenging activity at all the tested concentrations. Furthermore, all the fractions showed remarkable antioxidant capacity by the formation of phosphomolybdenum complex. In addition, superoxide radical scavenging activity was assayed using non-enzymatic (NADH/phenaxine methosulfate) superoxide generating system. All the extracts showed variable radical scavenging activity. The data obtained in the in vitro models clearly establish the antioxidant potency of citrus fruit extracts. However, comprehensive studies need to be conducted to ascertain the in vivo safety of such extracts in experimental animal models. To the best of our knowledge, this is the first report on antioxidant activity of citron and blood orange varieties of citrus fruits.     

Bioactivity of hydrolysates obtained from bovine casein using artichoke (Cynara scolymus L.) proteases

The objective of this work was to obtain casein hydrolysates with aspartic proteinases present in extracts from the artichoke flower (Cynara scolymus L.) and evaluate their antioxidant, antimicrobial, and angiotensin-I converting enzyme (ACE) inhibitory activity in vitro. The casein hydrolysates produced by the action of C. scolymus had elevated antihypertensive and antioxidant activity due to their high hydrophobic peptide content (93.84, 96.58, and 90.54% at 2, 4, and 16 h of hydrolysis, respectively). Hydrolysis time and molecular weight (<3 kDa) had a significant influence on the hypertensive and antioxidant activity of the hydrolysates, which were greater at hydrolysis times of 4 and 16 h and corresponding to the <3 kDa fractions. The <3 kDa fraction of the 16 h hydrolysate had an ACE inhibitory activity with a half-maximal inhibitory concentration (IC₅₀) of 71.77 µg peptides per mL; DPPH and ABTS^•+ radical scavenging activities of 6.27 µM and 6.21 mM Trolox equivalents per mg of peptides, respectively; and iron (II) chelation activity with an IC₅₀ of 221.49 µg of peptides per mL. Antimicrobial activity against Enterococcus faecalis was also observed in the hydrolysates. From the peptide sequences identified in the hydrolysates, we detected 22 peptides (from the BIOPEP database) that were already in their bioactive form (AMKPWIQPK, AMKPWIQPKTKVIPYVRYL, ARHPHPHLSFM, DAQSAPLRVY, FFVAPFPEVFGK, GPVRGPFPII, KVLPVPQK, LLYQEPVLGPVRGPFPIIV, MAIPPKKNQDK, NLHLPLPLL, PAAVRSPAQILQ, RELEELNVPGEIVESLSSSEESITR, RPKHPIKHQ, RPKHPIKHQGLPQEVLNENLLRF, SDIPNPIGSENSEK, TPVVVPPFLQP, VENLHLPLPLL, VKEAMAPK, VLNENLLR, VYPFPGPIH, VYQHQKAMKPWIQPKTKVIPYVRY, VYQHQKAMKPWIQPKTKVIPYVRYL) and are reported to display antioxidant, antimicrobial, and ACE inhibitory activity. We also identified 12,116, 14,513, and 25,169 peptide sequences in the hydrolysates at 2, 4, and 16 h, respectively, that were contained in the primary sequence, and these are reported to display ACE inhibitory, antioxidant, dipeptidyl peptidase IV inhibition, antithrombotic, opioid, immunomodulation, antiamnesic, anticancer, chelating, and hemolytic bioactivity.     

Assessment of the angiotensin-I-converting enzyme (ACE-I) inhibitory and antioxidant activities of hydrolysates of bovine brisket sarcoplasmic proteins produced by papain and characterisation of associated bioactive peptidic fractions

The main objective was to investigate the angiotensin-I-converting enzyme (ACE-I) inhibitory and antioxidant activities of sarcoplasmic proteins isolated from the brisket muscle (Pectoralis profundus) of 3 (Bos taurus) cattle and hydrolysed with papain for 24 h at 37 °C. Sarcoplasmic protein hydrolysates were ultra-filtered using molecular weight cut off (MWCO) membranes and 10-kDa and 3-kDa filtrates were obtained. The total sarcoplasmic protein extracts and the 3-kDa filtrates were tested for angiotensin I-converting enzyme inhibitory (ACE-I) activities. The total hydrolysates, 10-kDa and 3-kDa filtrates were also tested for their associated antioxidant activities using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity assay, the ferric ion reducing antioxidant power (FRAP) assay and the Fe²⁺ metal chelating ability assay. The peptidic content of the total hydrolysates, the 10-kDa and the 3-kDa filtrates were analysed using an ORBITRAP mass spectrometer, and mass spectral data obtained were analysed using TurboSEQUEST. Eleven peptides were characterised from the total hydrolysates, fifteen from the 10-kDa filtrate fractions, whilst nine peptides were characterised from the 3-kDa filtrate fractions. Similarities between the amino acid sequences of the peptides identified in this study and previously identified antioxidant and ACE-I inhibitory peptides detailed in the BIOPEP database were outlined.     

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.     

The antioxidant and free-radical scavenging activities of extract and fractions from corn silk (Zea mays L.) and related flavone glycosides

This study was designed to evaluate both the antioxidant and free-radical scavenging activities of extract and fractions from corn silk. N-butanol fraction (BF) demonstrated the highest total phenolic content (164.1 ± 9.7 μg GAE/g DCS) and total flavonoids content (69.4 ± 5.1 μg RE/g DCS), accompanied with the highest antioxidant activity compared to other fractions through all antioxidant assays. Two flavone glycosides showing potent antioxidant activity were isolated from BF and identified, by comparing spectral data (UV, FAB-MS and NMR) with literature values, to be isoorientin-2″-O-α-l-rhamnoside and 3′-methoxymaysin. The two isolated flavone glycosides, particularly isoorientin-2″-O-α-l-rhamnoside, demonstrated significant total antioxidant activity, DPPH radical scavenging activity, reducing power and iron-chelating capacity, with EC₅₀ values of 14.24 ± 1.49, 22.69 ± 2.33, 6.58 ± 1.07 and 30.25 ± 3.05 μg/ml, respectively. Results obtained indicated that corn silk extracts can be used potentially as a ready accessible and valuable bioactive source of natural antioxidants.     

Reducing,Radical Scavenging,and Chelation Properties of Fermented Soy Protein Meal Hydrolysate by Lactobacillus plantarum LP6

Fermented soybean protein meal hydrolysate (FSPMH) was fractionated by gel filtration on Sephadex G-15. The fractions obtained were subjected to various antioxidant assays, amino acid and molecular weight determinations. Among the seven fractions, the highest antioxidant activity was found in fraction F2, with significant differences (P < 0.01) 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, hydroxyl radicals (^.OH) scavenging and Cu²⁺ chelating activity. Fraction F2 exhibited scavenging of DPPH (59.43%), ^.OH (72.80%) and 44.47% Cu²⁺ chelating activity. All other fractions showed variable activities in different assays. Amino acid analyses of F2 fraction with the strongest antioxidant activity also had the highest percentage of related antioxidative amino acids content (Histidine 3.46, Serine 5.78, Valine 4.08 and Lysine 11.49 g/100 g protein) compared with other six fractions. The molecular weight distribution of F2 was found to vary from 170 to 1500 Da.