Functional Properties and Bioactivities of Pine Nut (Pinus gerardiana) Protein Isolates and Its Enzymatic Hydrolysates

The functional properties and bioactivities of the pine nut protein isolates (PPI) and its enzymatic hydrolysates (PPH) prepared with Alcalase at 5 %, 10 %, 15 % and 25 % degree of hydrolysis (DH) were studied. The solubility of PPH significantly increased (p?<?0.05) with the increase of the DH, while the foaming capacity of PPH was only improved at a low DH. However, enzymatic hydrolysis reduced the emulsifying capacity of PPH. The DPPH radical scavenging and inhibition of linoleic acid oxidation activities of PPH were significantly improved by a low DH (5 %) compared with those of PPH with a higher DH and the original PPI (p?<?0.05). The reducing power of PPH at all DH decreased in comparison to that of the original PPI. Potent angiotensin-converting enzyme (ACE) inhibitory peptides could be generated by hydrolysis with Alcalase, and the ACE inhibitory activity of PPH increased (p?<?0.05) with the DH. These results revealed that a low degree of enzymatic hydrolysis was appropriate to obtain PPH with improved functional properties and good antioxidant activities, while a high degree of hydrolysis was essential to obtain highly potent ACE inhibitory peptides from PPI. These results suggest that the control of the DH may be an effective strategy to modify specific functional and bioactive properties of PPH, and PPH has potential as a functional food ingredient for related functional and health benefits.     

Antioxidative activity and functional properties of protein hydrolysate of yellow stripe trevally (Selaroides leptolepis) as influenced by the degree of hydrolysis and enzyme type

Antioxidative activity and functional properties of protein hydrolysates from yellow stripe trevally (Selaroides leptolepis) meat, hydrolyzed by Alcalase 2.4L (HA) and Flavourzyme 500L (HF) with different degrees of hydrolysis (DH) were investigated. As the DH increased, DPPH radical-scavenging activity and reducing power of HA decreased (p < 0.05) but no differences were observed for HF (p > 0.05). Metal chelating activity of both HA and HF increased with increasing DH (p < 0.05). HF generally had a higher (p < 0.05) chelating activity than had HA at the same DH tested. At low DH (5%), HA exhibited a better DPPH radical-scavenging activity while, at high DH (25%), HF had a higher (p < 0.05) reducing power. For the functional properties, hydrolysis by both enzymes increased protein solubility to above 85% over a wide pH range (2–12). When the DH increased, the interfacial activities (emulsion activity index, emulsion stability index, foaming capacity, foam stability) of hydrolysates decreased (p < 0.05), possibly caused by the shorter peptide chain length. At the same DH, the functionalities of protein hydrolysate depended on the enzyme used. The results reveal that antioxidative activity and functionalities of protein hydrolysates from yellow stripe trevally meat were determined by the DH and by the enzyme type employed.     

ACE-inhibitory activity of tilapia protein hydrolysates

Fish processing wastes can be used for preparing bioactive peptides with various functionalities. Our objective was to evaluate the in vitro angiotensin converting enzyme (ACE) inhibitory activity of tilapia protein hydrolysates and its corresponding fractionates. Tilapia protein was alkali-solubilised at pH 11.0 and recovered at pH 5.5 to obtain a stable substrate. This substrate was hydrolysed using two enzymes, Cryotin-F or Flavourzyme, to 7.5% and 25% degree of hydrolysis (DH). The hydrolysates were ultra-filtered into three fractions: >30 kDa fraction, 10–30 kDa fraction, and <10 kDa fractions. Both hydrolysates and fractionates were tested for ACE inhibition. Results showed that both Cryotin and Flavourzyme hydrolysates with 25% DH gave maximum ACE inhibitory activity. Low MW peptides showed higher ACE inhibition than high MW peptides. The inhibitory activity of fractionates was lower than that of the corresponding hydrolysates, possibly due to separation and removal of synergistic peptides by ultrafiltration. Amongst fractionates, all the 7.5% DH Cryotin fractions and 25% DH Flavourzyme fractions exhibited optimum % ACE inhibition. The results of this research could be used for optimising enzyme parameters to obtain peptides from tilapia with optimum in vitro ACE inhibitory activity.     

Antioxidative and functional properties of protein hydrolysate from defatted skipjack (Katsuwonous pelamis) roe

Antioxidative and functional properties of protein hydrolysate from defatted skipjack (Katsuwonous pelamis) roe, hydrolysed by Alcalase 2.4 L (RPH) with different degrees of hydrolysis (DH) at various concentrations were examined. As DH increased, the reduction of DPPH, ABTS radicals scavenging activities and reducing power were noticeable (p < 0.05). The increases in metal chelating activity and superoxide scavenging activity were attained with increasing DH (p < 0.05). However, chelating activity gradually decreased at DH above 30%. All activities except superoxide anion radical scavenging activity increased as the concentration of hydrolysate increased (p < 0.05). Hydrolysis using Alcalase could increase protein solubility to above 80% over a wide pH range (2–10). The highest emulsion ability index (EAI) and foam stability (FS) of hydrolysates were observed at low DH (5%) (p < 0.05). Concentrations of hydrolysates determined interfacial properties differently, depending on DH. The molecular weight distribution of RPH with 5%DH (RPH5) was determined using Sephadex G-75 column. Two major peaks with the molecular weight of 57.8 and 5.5 kDa were obtained. Fraction with MW of 5.5 had the strongest metal chelating activity and ABTS radical scavenging activity. The results reveal that protein hydrolysates from defatted skipjack roe could be used as food additives possessing both antioxidant activity and functional properties.     

Angiotensin-I-converting enzyme inhibitory activity and bitterness of enzymatically-produced hydrolysates of shrimp (Pandalopsis dispar) processing byproducts investigated by Taguchi design

The importance of water-to-substrate ratio, protease type, percent enzyme and incubation time on hydrolysates produced from shrimp processing byproducts was investigated using Taguchi’s L₁₆ (4⁵) experimental design. Protease type significantly (p < 0.05) influenced soluble yield, degree of hydrolysis (DH), angiotensin-I-converting enzyme (ACE) inhibitory activity and bitterness of hydrolysates, while percent enzyme only affected the DH. Hydrolysates produced by Alcalase and Protamex possessed strong ACE inhibitory activity (IC₅₀ = 100–200 μg/ml and 70 μg/ml, respectively), accompanied by high yield, high DH and strong bitterness. Furthermore, ACE inhibition was positively correlated (r² = 0.87) with bitterness of the hydrolysates. Fractionation by size-exclusion chromatography revealed that the bitter substances, which also showed strong ACE inhibition, were <3 kDa in size and contained many hydrophobic residues, including Tyr, Phe, Leu, Ile, Val and Lys. Despite the bitterness, these hydrolysates may have potential health benefits, arising from their potent ACE inhibitory activity.     

Improvement of functional properties of peanut protein isolate by conjugation with dextran through Maillard reaction

Reaction mixtures containing peanut protein isolate (PPI) and dextran (1:1 weight ratio) were dry-heated at 60 °C and 79% relative humidity for 7 days. SDS-PAGE analysis indicated that PPI had become complexed with dextran to form conjugates of higher molecular weight. Arachin, accounting for 50% of peanut proteins, was hard to glycosylate with dextran, which might limit the extent of glycosylation of PPI. The thermal stability of PPI was remarkably improved by mixture/conjugation with dextran. Proteins in mixtures/conjugates might have a more compacted tertiary conformation than PPI. The protein solubility of conjugates at pH 4.5-6.0 was remarkably increased compared with the PPI/mixture. Mixture with dextran could significantly improve the emulsifying and foaming properties of PPI (p < 0.05). Conjugation with dextran could further enhance emulsifying and foaming properties of PPI.     

Free radical scavenging activity of porcine plasma protein hydrolysates determined by electron spin resonance spectrometer

A study was conducted to investigate the antioxidant capacity of porcine plasma protein before and after enzymatic hydrolysis. Porcine plasma protein was hydrolyzed by using Alcalase with degree of hydrolysis (DH) ranged from 0 to 17.8%. The free radical scavenging effects of porcine plasma protein hydrolysates (PPH) were evaluated by electron spin resonance (ESR) spectrometer. The reducing power of PPH increased with increasing of DH (P < 0.05). The 5-h PPH exhibited the strongest inhibition of lipid oxidation, as indicated by lowest thiobarbituric acid-reactive substance values in a liposome-oxidizing system, and the strongest free radical scavenging ability on 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH), hydroxyl (OH) and superoxide (O₂-) radicals. The increase of protein concentration enhanced (P < 0.05) free radical scavenging effect of PPH. Although non-hydrolyzed plasma protein displayed an antioxidative effect, it was far less potent than PPH. The results indicated that the antioxidant capacity of porcine plasma protein could be enhanced by enzymatic hydrolysis of Alcalase.     

Functional properties of fish protein hydrolysates from Pacific whiting(Merluccius productus) muscle produced by a commercial protease

Pacific whiting (Merluccius productus) muscle was used to produce hydrolysates with 10%, 15% and 20% degree of hydrolysis (DH) using the commercial protease Alcalase^® and were characterized at pH 4.0, 7.0 and 10 according their solubility, emulsifying and foaming properties. Protein recovered in soluble fractions increased proportionally with the hydrolytic process, yielded 48.6 ± 1.9, 58.6 ± 4.1 and 67.8 ± 1.4 of total protein after 10%, 15% and 20% DH, respectively. Freeze-dried hydrolysates presented almost 100% solubility (p > 0.05) at the different pHs evaluated. Emulsifying properties (EC, EAI and ESI) were not affected by DH as most samples showed similar (p > 0.05) results. Higher EC (p ? 0.05) than sodium caseinate, used as control, were obtained at pH 4 for most hydrolysates. Hydrolysates showed very low foaming capacity not affected by pH; but foam stability was equal or even better (p > 0.05) than bovine serum albumin (BSA), except at pH 4.0. Results suggest that hydrolysates from Pacific whiting muscle can be produced with similar or better functional properties than the food ingredients used as standards.     

Effects of partial hydrolysis and plasticizer content on the properties of film from cuttlefish (Sepia pharaonis) skin gelatin

Properties of film from cuttlefish (Sepia pharaonis) ventral skin gelatin with different degree of hydrolysis (DH: 0.40, 0.80 and 1.20%) added with glycerol as plasticizer at various levels (10, 15 and 20%, based on protein) were investigated. Films prepared from gelatin with all DH had the lower tensile strength (TS) and elongation at break (EAB) but higher water vapor permeability (WVP), compared with the control film (without hydrolysis) (p < 0.05). At the same glycerol content, both TS and EAB decreased, while WVP increased (p < 0.05) with increasing %DH. At the same DH, TS generally decreased as glycerol content increased (p < 0.05), however glycerol content had no effect on EAB when gelatins with 0.80 and 1.20% DH were used (p > 0.05). DH and glycerol content had no marked impact on color and the difference in color (ΔE^∗) of resulting films. Electrophoretic study revealed that degradation of gelatin and their corresponding films was more pronounced with increased %DH, resulting in the lower mechanical properties of films. Based on FTIR spectra, with the increasing %DH as well as glycerol content, higher amplitudes for amide-A and amide-B peaks were observed, compared with film from gelatin without hydrolysis (control film) due to the increased –NH₂ group caused by hydrolysis and the lower interaction of –NH₂ group in the presence of higher glycerol. Thermo-gravimetric analysis indicated that film prepared from gelatin with 1.20% DH exhibited the higher heat susceptibility and weight loss in the temperature range of 50–600 °C, compared with control film. Thus, both chain length of gelatin and glycerol content directly affected the properties of cuttlefish skin gelatin films.     

Compositions,functional properties and antioxidative activity of protein hydrolysates prepared from round scad (Decapterus maruadsi)

Composition, functional properties and antioxidative activity of a protein hydrolysate prepared from defatted round scad (Decapterus maruadsi) mince, using Flavourzyme, with a degree of hydrolysis (DH) of 60%, were determined. The protein hydrolysate had a high protein content (48.0%) and a high ash content (24.56%). It was brownish yellow in colour (L^∗ = 58.00, a^∗ = 8.38, b^∗ = 28.32). The protein hydrolysate contained a high amount of essential amino acids (48.04%) and had arginine and lysine as the dominant amino acids. Na⁺ was the predominant mineral in the hydrolysate. The protein hydrolysate had an excellent solubility (99%) and possessed interfacial properties, which were governed by their concentrations. The emulsifying activity index of the protein hydrolysate decreased with increasing concentration (p < 0.05). Conversely, the foaming abilities increased as the hydrolysate concentrations increased (p < 0.05). During storage at 25 °C and 4 °C for 6 weeks, the antioxidative activities and the solubility of round scad protein hydrolysate slightly decreased (p < 0.05). Yellowness (b^∗-value) of the protein hydrolysate became more intense as the storage time increased but the rate of increase was more pronounced at 25 °C than at 4 °C.     

High‐pressure microfluidisation pretreatment disaggregate peanut protein isolates to prepare antihypertensive peptide fractions

High‐pressure microfluidisation (HPM) pretreatment was applied to increase in vitro antihypertensive activity of peanut peptide fractions (PPF). The morphology of protein in aqueous dispersion revealed that peanut protein isolate (PPI) disaggregated at relatively low pressure (≤120 MPa) and re‐aggregated at relatively high pressures (150–210 MPa). The treated pressure of 120 MPa could lead to the most disaggregation of PPI. Small peptides contents, trichloroacetic acid‐nitrogen soluble index (TCA‐NSI) and degree of hydrolysis (DH) of peanut protein hydrolysates (PPH) all reached the highest at 120 MPa. Consequently, it possessed the highest angiotensin converting enzyme (ACE) and renin inhibitory activity. The highest surface hydrophobicity occurred at 120 MPa pretreatment samples. Thirty‐nine oligopeptides at 120 MPa pretreatment were identified by ultra‐performance liquid chromatography‐quadrupole time‐of‐flight (UPLC‐Q‐TOF) mass spectrometer combined with Progenesis QI for Proteomics software compared with 29 and 35 at control and 210 MPa, respectively. This meant that disaggregation of PPI at 120 MPa resulted in the release of new hydrophobic peptide.     

A novel approach of LED light radiation improves the antioxidant activity of pea seedlings

Influence of light-emitting diode (LED) light on antioxidant activity of radiated pea seedlings was first studied using red (625–630 nm) and blue (465–470 nm) LED lights as light sources in an attempt to determine and compare the changes in chlorophyll and β-carotene contents, and Trolox equivalent antioxidant capacity (TEAC, μM). After radiation for 96 h, comparing to white light group, red light radiated seedlings displayed significant (p < 0.05) increases in stem length and leaf area, while blue light radiation significantly (p < 0.05) increased the stem length and seedling weight. Chlorophyll in leaves increased rapidly when seedlings were radiated by blue light but no significant (p > 0.05) difference was observed among light radiated seedlings after 96-h cultivation. β-Carotene content of LED radiated leaves was significantly (p < 0.05) higher in red light (54.47 ± 2.35 μg/g) group than in the others. TEAC value of ethanol and acetone extracts (50 mg/mL) of 240 pieces of red light radiated seedlings cultured for 96 h reached 106.48 and 81.68 μM, respectively, were higher than the other treatments. In conclusion, the contribution of red light to significant β-carotene expression and antioxidant activity for nutrition and health benefits and blue light to seedling weight and chlorophyll induction of radiated pea seedlings are emphasized.     

Antioxidant activity and functional properties of porcine plasma protein hydrolysate as influenced by the degree of hydrolysis

Antioxidant activity and functional properties of porcine blood plasma protein hydrolysates (PPH) prepared with Alcalase at 6.2%, 12.7% and 17.6% of degree of hydrolysis (DH) were investigated. The PPH showed stronger radical-scavenging ability and possessed stronger Cu²⁺-chelation ability and a reducing power compared to non-hydrolysed plasma protein (P < 0.05). The antioxidant activity of PPH, indicated by thiobarbituric acid-reactive substance (TBARS) values in a liposome-oxidising system, increased with increasing DH (P < 0.05). The Alcalase hydrolysis increased protein solubility from its original 68.46–81.79% (non-hydrolysed) to 82.95–94.94% (hydrolysed) over a broad pH range (3.0–8.0). However, hydrolysis decreased surface hydrophobicity and suppressed emulsifying and foaming capacity of the plasma protein. To identify antioxidant peptide, PPH was subjected to ultrafiltration, ion-exchange chromatography and reverse-phase high performance liquid chromatography (RP-HPLC), and the amino acid sequences of isolated peptides were determined by liquid chromatography/tendem mass spectrometry (LC–MS/MS). The peptide with the strongest antioxidant activity had the amino acid sequence of His-Asn-Gly-Asn. The results indicated that PPH could be used as a novel antioxidant but may be of limited utility as an emulsifying or foaming agent.     

Antioxidant activity and phenolic content of raw and blanched Amaranthus species

The study was aimed to determine the antioxidant activity (total antioxidant and free radical-scavenging activities) and total phenolic content of Amaranthus sp. The effects of different blanching times (10 and 15 min) on antioxidant activity and phenolic content were also studied. Four types of Amaranthus species locally known as spinach, namely ‘bayam putih’ (Amaranthus paniculatus) (BP), ‘bayam merah’ (Amaranthus gangeticus) (BM), ‘bayam itik’ (Amaranthus blitum) (BI) and ‘bayam panjang’ (Amaranthus viridis) (BPG), were selected. Total antioxidant activity of water-soluble components in raw spinach was in the order of BI ≈ BM ≈ BPG > BP, whereas free radical-scavenging activity was in the order of BI > BPG > BM > BP. The total phenolic contents of BM and BP were significantly higher (p < 0.05) than other samples. All the studied spinach species possessed different antioxidant activities and phenolic contents. Antioxidant activities and phenolic contents of all the spinach were in the order of raw > blanched 10 min > blanched 15 min. Blanching up to 15 min may affect losses of antioxidant activity and phenolic content, depending on the species of spinach.     

Phenolic content and antioxidant activity of cantaloupe (cucumis melo) methanolic extracts

The objectives of this study were to determine phenolic content and antioxidant activity of methanolic extracts from different parts of cantaloupe (leaf, stem, skin, seed and flesh). The flesh extract afforded the highest yield (89.6 ± 0.3%) whilst the lowest yield was obtained from the seed (13.7 ± 0.5%) (p < 0.05). The leaf extract showed the highest total phenolic content (26.4 ± 0.3 mg GAE/g extract) and total flavonoid content (69.7 ± 3.37 μg RE/g extract) accompanied with best antioxidant activity through all antioxidant assays (p < 0.05). In addition, the stem extract also exhibited good antioxidant activity. Thus, these results suggest that methanolic extracts of cantaloupe leaf and stem may serve as a potential source of natural antioxidant for food and nutraceutical application.     

红衣组分对花生分离蛋白及其酶解产物理化和抗氧化性质的影响

以花生为原料,研究了花生红衣组分对花生分离蛋白及其酶解产物理化和抗氧化性质的影响。研究结果表明:含红衣的分离蛋白酶解速度低于不含红衣的;相同水解度条件下,含红衣的表面疏水性小于脱红衣的;GPC分布中,含红衣的峰值大于脱红衣的;红衣组分能够提高花生分离蛋白及其酶解产物的溶解性、乳化稳定性、乳化活性;在相同水解度条件下,含红衣的水解产物多酚含量显著高于脱红衣的,多酚含量的差异与花生分离蛋白及其水解产物的抗氧化性具有正相关性。     

Production of enzymatic hydrolysates with antioxidant and angiotensin-I converting enzyme inhibitory activity from pumpkin oil cake protein isolate

Protein isolate from pumpkin oil cake (PuOC PI) was hydrolysed by alcalase, flavourzyme and by sequential use of these enzymes, respectively, and the antioxidant properties and angiotensin-I converting enzyme (ACE) inhibitory activities of hydrolysates were evaluated. Under the same reaction conditions, alcalase hydrolysates showed a higher degree of hydrolysis (DH) than did flavourzyme hydrolysates. The highest DH’s by individual enzymes were 53.23 ± 0.7% and 37.17 ± 1.05%, respectively, both at 60 min. The increase of radical scavenging activity (RSA) in hydrolysates was positively correlated with the increase of DH, for both enzymes, though hydrolysates with flavourzyme showed two- or three-fold lower RSA than with alcalase. The highest bioactive potential was determined in the alcalase hydrolysate at 60 min, with RSA being 7.59 ± 0.081 mM TEAC/mg and ACE-inhibitory activity 71.05 ± 7.5% (IC₅₀ = 0.422 mg/ml). When this hydrolysate was further hydrolysed by flavourzyme, DH increased up to 69.29 ± 0.9%, but lower RSA (4.82 ± 0.21 mM TEAC/mg) and ACE-inhibitory activity (55.81 ± 6.196%) were determined in the final hydrolysate. This study suggested that the PuOC proteins could be converted into protein hydrolysates with antioxidant and ACE-inhibitory activities by enzymatic hydrolysis. Alcalase was shown as promising enzyme in further development of bioprocesses for the production of new bioactive food ingredients.     

Antioxidant activity of ethanolic extract of Cortex fraxini and use in peanut oil

Cortex fraxini was extracted with 95% ethanol to obtain a crude antioxidant extract. The antioxidant activity was evaluated using the linoleic acid peroxidation method and the free radical scavenging assays, namely 2,2′-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl radicals. Cortex fraxini extract (CFE) showed high inhibition of peroxidation of linoleic acid when compared with butylated hydroxytoluene (BHT). CFE also exhibited excellent scavenging activity on DPPH and hydroxyl radicals. Total antioxidant activity was measured by the reduction of Mo(VI) to Mo(V) by the extract, and subsequent formation of a green phosphate/Mo(V) complex at acid pH. CFE had significant total antioxidant activity and the effects were increased with increasing reaction time. The total phenolic content of the sample, analyzed by using Folin–Ciocalteu’s reagent, was 91.33 mg/g dry weight expressed as pyrocatechol equivalents. Then the suitability of CFE as an antioxidant was determined in peanut oil, and the decrease of lipid oxidation was monitored using thiobarbituric acid-reactive substances (TBARS) assay. CFE treatment significantly (P < 0.05) reduced lipid oxidation in peanut oil compared to the control. No significant differences (P = 0.05) in lipid oxidation were detected between CFE antioxidant and BHT antioxidant samples.     

Influence of the degree of hydrolysis (DH) on antioxidant properties and radical-scavenging activities of peanut peptides prepared from fermented peanut meal

To study the influence of degree of hydrolysis (DH) on antioxidant properties of peanut peptides, the peanut meal was fermented by Bacillus subtilis. The fermentation time was 48, 72, and 96 h, respectively, to prepare peanut peptides at different degree of hydrolysis. The peanut peptides (11.18, 16.20, and 21.41% of DH, respectively) were extracted from fermentation liquid. The antioxidant properties of these peanut peptides were evaluated based on DPPH^· radical (1,1-Diphenyl-2-picrylhydrazyl radical) scavenging activity, superoxide anion-scavenging activity, reducing power, metal-chelating activity, and inhibition of linoleic acid autooxidation. Peanut peptides (21.41% of DH) at 1 mg/mL exhibited 80.86 and 29.35% of scavenging concentration percent on DPPH^· and superoxide anion radical, respectively. In addition, the reducing power of peanut peptides was 0.368 at 2 mg/mL, and they possessed 76.32% of Fe²⁺-chelation ability at 2 mg/mL and 63.75% of inhibition of linoleic acid autooxidation at 0.8 mg/mL. The antioxidant activities of the peanut peptides (21.41% of DH) were stronger compared with others (11.18 and 16.20% of DH), and this indicated the antioxidant activities of peanut peptides increased with increasing DH (p < 0.05). To know much about the peanut peptides, they were subjected to amino acid analysis and determination of molecular weight distribution. Some acidic amino acids and essential amino acids were found, and the average molecular weight distributions were concentrated in <1,400 Da (86.96%). Combined with the results of the amino acid profiles, the peanut peptides were believed to have high nutritive value besides antioxidant activities.     

Phenolic profiles and antioxidant activities of commercial beers

The phenolic profiles and corresponding antioxidant activities of 34 commercial beers in Chinese markets were evaluated. Results found remarkable variations in total and individual phenolic contents as well as antioxidant activity across beer brands. Gallic and ferulic acids were the dominant phenolic compounds identified in the tested beer samples and both of them accounted for >50% of the total phenolic compounds. Results from Pearson correlation analysis suggested that five antioxidant activity assays positively correlated well (p < 0.01) with each other and showed significant positive correlations (p < 0.05) with (+)-catechin, protocatechuic, and caffeic acids contents. Stepwise linear regression further demonstrated that different phenolic components responsible for beer antioxidant activity were dependent on the method used, and that ferulic acid, syringic acid, (+)-catechin, caffeic acid, protocatechuic acid and (−)-epicatechin together made 55.0–88.1% of contribution to the antioxidant activity of beer.