Inhibition of polyphenol oxidase obtained from various sources by 2,3‐diaminopropionic acid

This paper reports for the first time the inhibition of the catecholase activities of mushroom, artichoke (Cynara scolymus L) and Ocimum basilicum L polyphenol oxidase by 2,3‐diaminopropionic acid. Polyphenol oxidases from artichoke and O basilicum L were purified by ammonium sulfate precipitation, dialysis and a Sepharose 4B‐L ‐tyrosine‐p‐aminobenzoic acid‐affinity column. In inhibition studies, 2,3‐diaminopropionic acid showed uncompetitive inhibition for mushroom PPO using catechol and pyrogallol as substrates, competitive inhibition for O basilicum L PPO using catechol as a substrate, and uncompetitive inhibition for artichoke PPO using catechol as a substrate. Furthermore, sodium azide, which is an inhibitor of PPO, was used as an inhibitor for comparison with the inhibition potency of 2,3‐diaminopropionic acid. The highest 2,3‐diaminopropionic acid inhibition observed with O basilicum L (K_i = 0.89 mM ), followed by artichoke (K_i = 1.42 mM ) and mushroom (K_i = 2.47 mM ), respectively. Copyright © 2005 Society of Chemical Industry     

Elucidation of the mechanism of enzymatic browning inhibition by sodium chlorite

Sodium chlorite (SC) is a well known anti-microbial agent and its strong inhibitory effect on enzymatic browning of fresh-cut produce has recently been identified. We investigated the effect of SC on polyphenol oxidase (PPO) and its substrate, chlorogenic acid (CA), as it relates to the mechanisms of browning inhibition by SC. Results indicate that the browning reaction of CA (1.0 mM) catalyzed by PPO (33 U/mL) was significantly inhibited by 1.0 mM SC at pH 4.6. Two PPO isoforms were identified by native polyacrylamide gel electrophoresis, and both were inactivated by SC (3.0 mM). This suggests that SC serves as a PPO inhibitor to prevent enzymatic browning. Furthermore, the effect of SC on the stability of CA in both acidic (pH 4.5) and basic conditions (pH 8.3) was studied by UV–Vis scan and LC–MS analysis. The results showed that at the presence of SC (3.0 mM), CA (0.1 mM) degraded to quinic acid and caffeic acid as well as other intermediates. Hence, the anti-browning property of SC can be attributed to the two modes of action: the inactivation of polyphenol oxidase directly and the oxidative degradation of phenolic substrates.     

Effect of Infrared Blanching on Enzyme Activity and Retention of β‐Carotene and Vitamin C in Dried Mango

The objectives of this work were to evaluate infrared (IR) dry blanching in comparison with conventional water blanching prior to hot air drying of mango to inactivate polyphenol oxidase (PPO) and ascorbic acid oxidase (AAO) enzymes, and to study its effect on color change and retention of vitamin C and β‐carotene. Mango cylinders were blanched under similar temperature–time conditions either by IR heating or by immersion in a water bath during 2 min at 90 °C (high‐temperature‐short‐time—HTST) or for 10 min at 65 °C (low‐temperature‐long‐time—LTLT). After blanching mango was hot air dried at 70 °C. PPO was completely inactivated during the blanching treatments, but AAO had a moderate remaining activity after LTLT treatment (～30%) and a low remaining activity after HTST treatment (9% to 15%). A higher retention of vitamin C was observed in mango subjected to IR dry blanching, 88.3 ± 1.0% (HTST) and 69.2 ± 2.9% (LTLT), compared with water blanching, 61.4 ± 5.3% (HTST) and 50.7 ± 9.6% (LTLT). All‐trans‐β‐carotene retention was significantly higher in water blanched dried mango, 93.2 ± 5.2% (LTLT) and 91.4 ± 5.1% (HTST), compared with IR dry blanched, 73.6 ± 3.6% (LTLT) and 76.9 ± 2.9% (HTST). Increased levels of 13‐cis‐β‐carotene isomer were detected only in IR dry blanched mango, and the corresponding dried mango also had a slightly darker color. IR blanching of mango prior to drying can improve the retention of vitamin C, but not the retention of carotenoids, which showed to be more dependent on the temperature than the blanching process. A reduction of drying time was observed in LTLT IR‐blanching mango.     

Polyphenol Oxidase Inhibitor from Blue Mussel (Mytilus edulis) Extract

Enzymatic browning remains a problem for the fruit and vegetable industry, especially new emerging markets like pre‐cuts. A crude inhibitor from blue mussel (Mytilus edulis) showed broad inhibition for apple (58%), mushroom (32%), and potato (44%) polyphenol oxidase (PPO) and was further characterized. Inhibition increased as the concentration of inhibitor increased in the reaction mixture eventually leveling off at a maximum inhibition of 92% for apple PPO. The inhibitor was capable of bleaching the brown color formed in the reaction mixture with apple PPO. Identification of the inhibitor by mass spectrometry and high‐performance liquid chromatography revealed it to be hypotaurine (C₂H₇NO₂S). Hypotaurine and other sulfinic acid analogs (methane and benzene sulfinic acids) showed very good inhibition for apple PPO at various concentrations with the highest inhibition occurring at 500 μM for hypotaurine (89%), methane sulfinic acid (100%), and benzene sulfinic acid (100%). Practical Application: An inhibitor found in the expressed liquid from blue mussel shows very good inhibition on enzymatic browning. Since this enzyme is responsible for losses to the fruit and vegetable industry, natural inhibitors that prevent browning would be valuable. Finding alternative chemistries that inhibit browning and understanding their mode of action would be beneficial to the fruit and vegetable industries and their segments such as pre‐cuts, juices, and so on. Inhibitors from products ingested by consumers are more acceptable as natural ingredients.     

Inhibition of loquat enzymatic browning by sulfhydryl compounds

The effectiveness of a series of sulfhydryl compounds in inhibiting polyphenoloxidase (PPO) activity in a model system (chlorogenic acid solution) and in loquat juices was evaluated. Application of different concentrations of sulfhydryl compounds to chlorogenic acid solution and fresh loquat juice showed that l-cysteine appeared to be an effective browning inhibitor. The required concentration of l-cysteine for 90% browning inhibition depended on loquat cultivars, and ranged from 0.6 mM for “Nagasaki” to 2.0 mM for “Yukawa” or “Toi”. The difference may be due to the concentration of endogenous phenolic compounds inherent in each cultivar. The results from oxidized product analysis by loquat PPO implied that the mechanism of browning inhibition by thiols involved the formation of a thiol-conjugated reaction product.     

Cyclodextrin and methacrylic acid octyl phenols poly(ethylene oxide) ester‐induced synergistic effect in a novel poly(AM‐co‐A‐β‐CD‐co‐AE) polymer

Owing to superior properties such as temperature resistance and salt tolerance etc., modified polyacrylamide (PAM) as one of the main injected polymers has been widely investigated to enhance oil production in reservoirs. Herein, a novel poly(AM‐co‐A‐β‐CD‐co‐AE) polymer was synthesized by utilizing β‐CD and AE to copolymerize with AM and characterized by FT‐IR and SEM. Furthermore, the temperature resistance and salt tolerance of poly(AM‐co‐A‐β‐CD‐co‐AE) polymer were explored. The results showed that the presence of the poly(AM‐co‐A‐β‐CD‐co‐AE) polymer better achieved temperature resistance and salt tolerance properties than is the case with PAM, which has potential application for enhancing oil recovery in the high‐temperature and high‐mineralization oilfield. On the other hand, the inhibition performance of poly(AM‐co‐A‐β‐CD‐co‐AE) polymer as a corrosion inhibitor was evaluated by SEM and electrochemical techniques. SEM observations of the carbon steel surface confirmed the protective role of the corrosion inhibitor. The results of potentiodynamic polarization and EIS measurements on the corrosion inhibition of carbon steel samples in 0.5 M sulfuric acid solutions revealed that the highest inhibition efficiency of it over 90% was obtained, indicating poly(AM‐co‐A‐β‐CD‐co‐AE) polymer acts as a more efficient corrosion inhibitor for carbon steel.     

Role of anthocyanins,polyphenol oxidase and phenols in lychee pericarp browning

Postharvest browning of lychee fruits is investigated in relation to anthocyanins, polyphenol oxidase (PPO) and phenols, using a purified PPO, anthocyanin extract and phenolic extract. Lychee PPO cannot oxidise the degradation of anthocyanins, but the presence of phenolic extract stimulates pigment degradation by PPO, leading to the development of brown pigments. The absorbance at 410 nm of the reaction solution is correlated well with the peel browning index. Pyrogallol, catechol and 4‐methylcatechol are good stimulators, but no action with chlorogenic acid, p‐cresol, resorcinol or tyrosine is observed. A decrease in the ascorbic acid content of lychee pericarp is associated with an increase in the peel browning index, and the addition of ascorbic acid to the reaction mixture exerts a preventive action on the anthocyanin degradation. Data here suggest that anthocyanins, PPO and phenols contribute to lychee pericarp browning involved in anthocyanin–PPO–phenol reactions. © 2000 Society of Chemical Industry     

Efficacy of various naturally occurring caffeic acid derivatives in preventing post‐harvest protein losses in forages

BACKGROUND: In red clover, oxidation of endogenous o‐diphenols by polyphenol oxidase (PPO) inhibits post‐harvest proteolyis. This system is transferable to alfalfa by providing PPO (via a transgene) and o‐diphenol PPO substrates (via exogenous application). To exploit the PPO system for protein protection, it would be advantageous to produce PPO substrates in alfalfa, which lacks them. We assessed the extent of PPO‐mediated proteolytic inhibition by phenolic compounds, especially those whose biosynthesis could be engineered into alfalfa. RESULTS: Tested compounds included o‐diphenols (caffeic acid, phaselic acid, chlorogenic acid, clovamide) and monophenols (p‐coumaric acid, p‐coumaroyl‐malic acid). In the presence of PPO, 2 mmol o‐diphenol g^?1 protein reduced 24 h proteolysis 68–87% (P < 0.001) and as little as 0.25 mmol g^?1 protein still decreased 24 h proteolysis 43–60% (P < 0.001). At high concentrations, clovamide inhibited 24 h proteolysis 50% (P < 0.001) in the absence of PPO, likely due to non‐PPO oxidation. Monophenol p‐coumaric acid did not inhibit 24 h proteolyis, although high levels of its malate ester did exhibit PPO‐ and oxygen‐independent inhibition (37%, P < 0.001). CONCLUSIONS: For PPO‐mediated proteolytic inhibition, pathways for both phaselic acid and chlorogenic acid may be good targets for engineering into alfalfa. Clovamide may be useful for inhibiting proteolysis without PPO. Published 2012 by John Wiley & Sons, Ltd.     

Effect of temperature on the drying characteristics,colour, antioxidant and beta‐carotene contents of two apricot varieties

In this study, the drying characteristics, colour, oxygen reactive antioxidant capacity (ORAC) and beta‐carotene contents of two apricot varieties dried at different temperatures were compared. The hot air drying of apricot slices for both varieties consisted of a constant rate period (CRP) and two of falling rate periods (FRP). The CRP drying rate and the first and second FRP drying coefficients increased with drying temperature for both apricot varieties. The first and second FRP of both apricot varieties gave activation energies of 23.5–28.7 and 25.6–29.3 kJ mole^?1, respectively. The colour values (L*, a* and b*) of both dried apricot varieties decreased with increasing temperature, while the total colour change of both dried apricot varieties increased with temperature. The chroma, hue angle and browning index values of both dried apricot varieties decreased with increasing temperature, and the hydrophilic ORAC and beta‐carotene contents increased with drying temperature.     

Flavan‐3‐ol C‐glycosides – Preparation and model experiments mimicking their human intestinal transit

In order to study the human intestinal transit of flavan‐3‐ol C‐glycosides, several C‐glycosyl derivatives were prepared by non‐enzymatic reaction of (+)‐catechin with α‐D ‐glucose, α‐D ‐galactose and α‐D ‐rhamnose, respectively. In contrast to literature data, we propose that the reaction mechanism proceeds in analogy to the rearrangement of flavan‐3‐ols during epimerization under alkaline conditions. Four of the 12 synthesized flavan‐3‐ol C‐glycosides were incubated under aerobic conditions at 37°C using saliva (2 min) and simulated gastric juice (3 h). To simulate human intestine, the C‐glycosides were also incubated under anaerobic conditions at 37°C both in human ileostomy fluid (10 h) and colostomy fluid (24 h), respectively. The flavan‐3‐ol C‐glycosides under study, i.e. (+)‐epicatechin 8‐C‐β‐D ‐glucopyranoside (1a), (+)‐epicatechin 6‐C‐β‐D ‐glucopyranoside (1d), (+)‐catechin 6‐C‐β‐D ‐galactopyranoside (2b), (+)‐catechin 6‐C‐β‐D ‐rhamnopyranoside (3b) were analyzed in the incubation samples by HPLC‐DAD and HPLC‐DAD‐MS/MS. They were found to be stable in the course of incubation in saliva, simulated gastric juice and ileostomy fluid and underwent degradation in colostomy fluid. While the 6‐C‐β‐D ‐glucopyranoside 1d was completely metabolized between 2 and 4 h, decomposition of the 6‐C‐β‐D ‐galactopyranoside 2b reached only 16±2% within 4 h of incubation. Linear degradation rates of 1d and 2b in colostomy fluid differed significantly. As microbial metabolism of flavan‐3‐ols is known not to be influenced by the stereochemistry of the aglycon, varying degradation rates are ascribed to the effect of the sugar moiety. Based on these results we assume that flavan‐3‐ol C‐glycosides pass through the upper gastrointestinal tract (oral cavity, stomach and small intestine) unmodified and are then metabolized by the colonic microflora.     

POLYPHENOLOXIDASE ACTIVITY OF MINIMALLY PROCESSED 'JONAGORED' APPLES (MALUS DOMESTICA)

The influence of three chemical dips using ascorbic acid (AA), citric acid (CA) and calcium chloride (CC) on the polyphenoloxidase (PPO) activity and on the total phenolic content of minimally processed (MP) apple (Malus domestica, cv. Jonagored) during cold storage was evaluated and a potential relationship with enzymatic browning was investigated. An ascorbic acid dip (42.6 mM) of 5 min duration was the most efficient chemical treatment in reducing the PPO activity of apple cubes. A 92% inhibition was achieved after 7 days of storage at 4C. All treatments were advantageous in comparison to the control in reducing color changes. Color changes, determined by absorbance at 420 nm (soluble pigments) and lightness (L) (insoluble pigments) of apple cubes treated with ascorbic acid were correlated with total phenolic content. No correlation was observed between PPO activity and tristimulus color parameters, browning index or total phenolic content of AA‐treated apple cubes.     

Fatty acids and all‐trans‐β‐carotene are correlated in differently colored rice landraces

A set of 54 rice landrace samples was compiled from various Asian countries, including six red/brownish and eight black/purple varieties. Brown rice samples were analyzed for lipid content and fatty acid profile, as well as all‐trans‐β‐carotene content. Black/purple varieties were found to be higher in crude lipid content than the red/brownish and colorless varieties. They also had a higher β‐carotene content than the other two color classes. The highest β‐carotene content determined was 0.22 mg kg⁻¹. Black/purple varieties tended to have a higher proportion of saturated fatty acids in their lipid fraction and a lower proportion of unsaturated fatty acids. The differences were statistically significant (P < 0.05) for oleic acid, which accounted for 42.1% of the lipid fraction in black/purple varieties and for 45.3% and 46.3% in red/brownish and colorless varieties, respectively. β‐Carotene content showed a significantly positive correlation with the crude lipid content (P < 0.001) and the content of saturated fatty acids (P < 0.001) on a dry matter basis. However, it was not correlated with the unsaturated fatty acids content on a dry matter basis. Within the total lipid extract, β‐carotene showed a significantly positive correlation with the proportion of saturated fatty acids (P < 0.01), especially palmitic acid (P < 0.01), and a significantly negative correlation with unsaturated fatty acids (P < 0.001), especially oleic acid (P < 0.01). Copyright © 2005 Society of Chemical Industry     

Effect of rice bran protein extract on enzymatic browning inhibition in potato puree

Effect of rice bran protein extract (RBPE) on enzymatic browning inhibition in potato puree was studied by colour measurement and polyphenol oxidase (PPO) inhibition. RBPE inhibited browning in potato puree and showed a higher per cent potato PPO inhibition at pH 4.0 and 5.0 than those at pH 3.0, 6.0 and 7.0 (P ≤ 0.05). RBPE heated to 40 and 60 °C had browning inhibition in potato puree and per cent potato PPO inhibition at a similar extent to unheated RBPE (P > 0.05). Browning inhibition and per cent potato PPO inhibition of RBPE were decreased when it was heated to 80 °C (P ≤ 0.05). RBPE inhibited browning in potato puree higher than 5, 10 and 20 mm ascorbic acid and 5 and 10 mm citric acid (P ≤ 0.05). Regarding the kinetics study, RBPE exhibited a mixed‐type inhibition for potato PPO. Therefore, RBPE has a potential to be used as a natural antibrowning agent in the potato industry.     

Prevention of Enzymatic Browning of Chinese Yam (Dioscorea spp.) Using Electrolyzed Oxidizing Water

In this study, the effects of electrolyzed oxidizing water (EOW) on the prevention of enzymatic browning of fresh‐cut “Jiu Jinhuang” Chinese yam were investigated. The yams were immersed in the inhibitors for 25 min at 20 °C. Compared with the tap water (TW) treatment, the chromatic attributes were significantly different after 72 h of storage (P < 0.05). The activities of polyphenol oxidase (PPO, EC 1.10.3.1), peroxidase (POD, EC 1.11.1.7), and _L‐phenylalanine ammonia lyase (PAL, EC 4.3.1.5) were inhibited when measured at 24 h. The contents of phenolic acids, including gallic and chlorogenic acid, in the group treated with the slightly acidic electrolyzed water (SAEW) were higher than those treated with TW and neutral electrolyzed water (NEW). The group treated with NEW had the highest total phenol content (P < 0.05, at 24 h), while the group treated with SAEW had the highest flavonoid content (P < 0.05) during storage. Without being treated with inhibitors, the K_m and V_max values of yam PPO were 0.0044 mol/L and 0.02627 U/min, respectively, and the K_i of samples treated with SAEW and citric acid (CA) were 15.6607 and 2.3969 μmol/L, respectively. These results indicate that EOW is beneficial as a browning inhibitor.     

Effects of argon enriched low-oxygen atmospheres and of high-oxygen atmospheres on the kinetics of polyphenoloxidase (PPO)

Abstract: The reported benefits of enrichment of air atmospheres with argon or oxygen for control of enzymatic browning were investigated by determining the effects of these atmospheres on PPO kinetics. Kinetics of purified apple PPO and a commercially available mushroom PPO were studied in an in vitro model system. Enrichment with argon produced greater inhibitory effects than the current industry practice of enrichment with nitrogen. Km^app values (mM) for apple PPO in 3%O₂/97%Ar, 3%O₂/97%N₂, and air, were 133, 87, and 48, respectively. The data indicate that inhibition by both gases is competitive, and also support the hypothesis that the greater inhibitory effect of argon was proportional to the size of the Van der Waals radius of argon against nitrogen (1.91Å against 1.54Å). Much smaller inhibitory effects were observed in the presence of 80% O₂ (Km^app 57mM), and the nature of this inhibition was less clear. The results suggest that the benefits of argon enrichment may be relatively small, and may require critical enzyme, substrate, and gas levels to be successful. However, these benefits may be exploitable commercially in some fresh‐cut products, and may allow less anoxic atmospheres to be used. Practical Application: Control of enzymatic browning without sulfites continues to be a challenge in some fresh‐cut products. While sporadic benefits of these atmospheres in control of enzymatic browning have been reported, results have been inconsistent in commercial practice. The results suggest that the benefits of argon enrichment may be relatively small, and may require critical enzyme, substrate, and gas levels to be successful. However, these benefits may be exploitable commercially in some fresh‐cut products, and allow less anoxic atmospheres to be used.     

Mechanism of Browning in Fresh Highbush Blueberry Fruit (Vaccinium corymbosum L). Role of Blueberry Polyphenol Oxidase,Chlorogenic Acid and Anthocyanins

Browning reactions which occurred after crushing fresh blueberries (Vaccinium corymbosum L) were followed by determination of chlorogenic acid colour density, polymer colour and the level of polymers. A polyphenol oxidase (PPO) activity plays a dominant role in enzymatic browning. Peroxidase (POD) does not contribute to brown colour formation. The loss of 29% of the colour in a model system containing PPO and blueberry anthocyanins indicated that PPO could act directly on these pigments. However, the rate of anthocyanin degradation was stimulated by the addition of chlorogenic acid. © 1997 SCI.     

Enzyme activity of α‐chymotrypsin after derivatization with phenolic compounds

α‐Chymotrypsin was allowed to react with selected phenolic and related compounds (chlorogenic acid, m‐, o‐, p‐dihydroxybenzene, p‐benzoquinone). The derivatized enzymes obtained were characterized in terms of their activity. In vitro experiments illustrated that the enzymatic activity of the derivatives was adversely affected. The kinetics of the enzymatic reactions showed that the hydrolysis of selected food proteins becomes slower and the affinity of the enzyme to these substrates declined as measured by Michaelis‐Menten constant and maximum velocity of the enzymatic reaction. This enzyme inhibition depended on the reactivity of the phenolic and related substances tested as well as on the degree of the derivatization. Further, influence of the enzyme‐substrate ratio was also demonstrated. The effects of the derivatization are more pronounced with increasing concentration of the substrates.     

Changes in browning‐related components of apple slices during different stages of instant controlled pressure drop‐assisted hot air drying (AD‐DIC)

The effect of instant controlled pressure drop‐assisted hot air drying (AD‐DIC) on browning‐related components in apple slices was studied. Results showed the AD temperature had no significant effect on colour index of final apple slices. The aspartic acid, glutamic acid, lysine, histidine, arginine, sucrose and dehydroascorbic acid (DHAA) in apple slices increased firstly during the AD process and then decreased during DIC process. The fructose, glucose, reduced ascorbic acid and major phenolic compounds reduced with the abrupt increase of 5‐hydroxymethylfurfural (5‐HMF) during AD‐DIC. There were significant correlations (P < 0.05) among moisture content (w.b.), water activity (a_w), water state, reduced ascorbic acid, DHAA, chlorogenic acid, procyanidin B₁, (+)‐catechin, (?)‐epicatechin and phlorizin during AD‐DIC. The colour difference showed that the major browning reaction of apple slices during the predrying process was enzymatic browning, while nonenzymatic browning played a crucial role on green‐red value (CIE a* value) during DIC.     

Identification and in vitro biological activities of flavonols in garlic leaf and shoot: inhibition of soybean lipoxygenase and hyaluronidase activities and scavenging of free radicals

We isolated four flavonols from garlic (Allium sativum L) leaf and shoot and measured the in vitro antioxidant activity of the isolated flavonols and their aglycones. The chemical structures of the compounds were shown to be quercetin 3‐O‐β‐D ‐glucopyranoside (isoquercitrin), quercetin 3‐O‐β‐D ‐xylopyranoside (reynoutrin), kaempferol 3‐O‐β‐D ‐glucopyranoside (astragalin) and isorhamnetin 3‐O‐β‐D ‐glucopyranoside based on FAB‐MS and NMR analyses. Assays of DPPH (1,1‐diphenyl‐β‐picryl hydrazyl) and hydroxyl radical scavenging, inhibition of linoleic acid peroxidation, and soybean lipoxygenase (LO) and hyaluronidase (HYA) inhibition were used to evaluate the antioxidant activity. Quercetin and its glycosides showed the highest antioxidant activity among the compounds. In the LO assay, the IC₅₀ values of quercetin, isoquercitrin and reynoutrin were 16.9, 40.1 and 32.9 µM respectively. Quercetin was the most effective among the flavonols. In the HYA assay the IC₅₀ values of quercetin, isoquercitrin and reynoutrin were 23.0, 20.9 and 22.1 mM respectively. Isoquercitrin had the most potent inhibitory activity on HYA. The inhibition patterns of the flavonols on LO and HYA were elucidated as mixed types of competitive and non‐competitive inhibition according to Lineweaver–Burk plot results. Although most garlic shoots and leaves are discarded and not used at present, our results suggest that ancillary garlic parts could be utilised as functional foods or ingredients. Copyright © 2004 Society of Chemical Industry     

Amino acid composition and anti‐polyphenol oxidase of peptide fractions from sericin hydrolysate

Sericin hydrolysate (SH), prepared from enzymatic hydrolysis of sericin, was investigated for its antipolyphenol oxidase (PPO) properties. SH decreased PPO activity from both purified mushroom PPO and extracts from apple and eggplant, and retarded browning in fresh‐cut apple and eggplant. SH was a competitive inhibitor using catechol as a substrate. SH exhibited copper ion‐chelating power and reducing power abilities. Fractionation of SH using size exclusion chromatography resulted in four fractions, designated as F1, F2, F3 and F4, with PPO inhibition of 35.75%, 3.89%, 24.52% and 14.75%, respectively. Ser and Asp were major amino acids found in F1. Amino acid sequences in F1, as investigated by LC‐MS/MS using de novo sequencing, contained a high ratio of amino acids with chelating ability. Moreover, amino acids with reducing power ability and with antityrosinase ability were also identified in the sequences.