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.     

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.     

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.     

Inhibitory effect of sericin on polyphenol oxidase and its application as edible coating

Sericin, a water‐soluble globular protein derived from silk industry wastewater, was investigated for food industrial applications. The results proved that sericin retarded polyphenol oxidase (PPO) activity. However, the degree of inhibition varied depending on the enzyme origins, the types of substrate, sericin content and sericin molecular size. Using catechol as a substrate, under the conditions studied, sericin lowered purified mushroom PPO and apple extract PPO activity by 40% and mango extract PPO by 75%. Kinetic studies on purified mushroom PPO indicated that the type of inhibition of sericin was dependent on the substrates used. Inhibitory effects of sericin increased as the sericin content increased. The reduction in sericin molecular size by enzymatic hydrolysis produced sericin hydrolysate with ability to decrease PPO activity approximately three times greater than that of sericin. Fresh‐cut Red Delicious apples coated with sericin showed significant reduction in weight loss and improvement in the colour and texture.     

Cloning and some properties of Japanese pear (Pyrus pyrifolia) polyphenol oxidase,and changes in browning potential during fruit maturation

A PCR‐amplified genomic DNA fragment encoding Japanese pear (Pyrus pyrifolia) polyphenol oxidase (PPO) was cloned and sequenced. The DNA appears to encode a 66 kDa precursor protein consisting of a 56 kDa mature protein and a 9.5 kDa N‐terminal transit peptide. The amino acid sequence showed high homology with apple PPO. The PPO mainly existed as a soluble fraction in cells and was limitedly proteolysed, while the mature form (56 kDa) was detected in plastids. Immature fruits showing high browning potential had high PPO activity and a high level of phenolics, while mature fruits showing little browning had high PPO activity but a low level of phenolics. Copyright © 2003 Society of Chemical Industry     

Maillard reaction products as "natural antibrowning" agents in fruit and vegetable technology

The effects of Maillard reaction products (MRPs), synthesized from a sugar (pentose, hexose, or disaccharide) and either a cysteine-related compound, an amino acid, or a sulfur compound, were investigated on polyphenoloxidase (PPO) activity from apple, mushroom, and eggplant. The optimal conditions for the production of inhibitory MRPs were performed using two-factor and five-level central experimental designs. It resulted that thiol-derived MRPs were highly prone to give rise to inhibitory compounds of PPO activity. Technological assays were also performed to test the efficiency of selected MRPs in the prevention of enzymatic browning in raw and minimally processed fruits and vegetables.     

Effect of sand roasting and microwave cooking on antioxidant activity of barley

Eight different barley cultivars were roasted in sand and cooked in a microwave oven and evaluated for antioxidant activity (AOA), total phenolic content (TPC), non-enzymatic browning, polyphenol oxidase activity (PPO), total flavonoid content (TFC), reducing power and metal chelating activity. Sand roasting resulted in higher puffing index. Greater increase in yellowness of the roasted flour was brought about by microwave cooking. A significant decrease in TPC (8.5 to 49.6%) and TFC (24.5 to 53.2%) was observed after roasting. The AOA significantly increased (16.8 to 108.2%) after roasting with sand roasted barley exhibiting higher AOA. The reducing power and metal chelating activity significantly increased by up to 77.5% and 78.9%, respectively after roasting with sand roasted samples showing greater effect. The non-enzymatic browning index increased (315 to 774%) and was higher for sand roasted barley. The microwave cooking brought about a greater reduction (45.1 to 76.8%) in PPO activity.     

Deodorization of Garlic Breath by Foods,and the Role of Polyphenol Oxidase and Phenolic Compounds

Garlic causes a strong garlic breath that may persist for almost a day. Therefore, it is important to study deodorization techniques for garlic breath. The volatiles responsible for garlic breath include diallyl disulfide, allyl mercaptan, allyl methyl disulfide, and allyl methyl sulfide. After eating garlic, water (control), raw, juiced or heated apple, raw or heated lettuce, raw or juiced mint leaves, or green tea were consumed immediately. The levels of the garlic volatiles on the breath were analyzed from 1 to 60 min by selected ion flow tube mass spectrometry (SIFT‐MS). Garlic was also blended with water (control), polyphenol oxidase (PPO), rosemarinic acid, quercetin or catechin, and the volatiles in the headspace analyzed from 3 to 40 min by SIFT‐MS. Raw apple, raw lettuce, and mint leaves significantly decreased all of the garlic breath volatiles in vivo. The proposed mechanism is enzymatic deodorization where volatiles react with phenolic compounds. Apple juice and mint juice also had a deodorizing effect on most of the garlic volatiles but were generally not as effective as the raw food, probably because the juice had enzymatic activity but the phenolic compounds had already polymerized. Both heated apple and heated lettuce produced a significant reduction of diallyl disulfide and allyl mercaptan. The presence of phenolic compounds that react with the volatile compounds even in the absence of enzymes is the most likely mechanism. Green tea had no deodorizing effect on the garlic volatile compounds. Rosmarinic acid, catechin, quercetin, and PPO significantly decreased all garlic breath volatiles in vitro. Rosmarinic acid was the most effective at deodorization.     

Antioxidant activities of the fractionated protein hydrolysates from heat stable defatted rice bran

The hydrolysates from heat stable rice bran (HSDRB) treated by Alcalase 2.4L and Protease 500G were desalted and fractionated by hydrophobicity using a nonpolar, styrene divinylbenzene resin and macroporous adsorption resin DA201‐C. The antioxidant activities of HSDRB hydrolysates (HSDRBH) eluted by 25%, 50%, 75% and 100% ethanol were investigated using 2, 2‐di (4‐tertoctylphenyl)‐1‐picrylhydrazyl free radical‐scavenging activity assay, reducing power assay, ferrous ion‐chelating activity assay and lipid peroxidation inhibition assay. The HSDRBH‐75 had the highest reducing power assay and inhibition ratio of linoleic acid autoxidation, which might be associated with reduced molecular weight, amino acid composition and hydrophobicity. The highest metal‐chelating activity of four different fractions (at the concentration of 4 mg mL^?1) had a positive correlation (r = 0.768, P = 0.116) with the total content of basic amino acid (Lys, Arg, and His) and a negative correlation (r = ?0.886, P = 0.057) with the range of molecular weight (Mw < 1000 Da). The HSDRBH‐75 had the highest antioxidant activities in many assays, which suggests that it may become a good natural antioxidant as a food additive.     

Ruminal micro‐organisms do not adapt to increase utilization of poly‐phenol oxidase protected red clover protein and glycerol‐based lipid

BACKGROUND: The enzyme polyphenol oxidase (PPO) reduces the extent of proteolysis and lipolysis within red clover fed to ruminants with subsequent increases in the efficiency of N utilization and the level of beneficial polyunsaturated fatty acids in their products (meat and milk). It has also been reported that red clover feeding alters the rumen microbial population compared to grass feeding. This study investigated whether the observed shifts in the microbial population of the rumen when ruminants are fed red clover silage (RC) as opposed to grass silage (G) represented an adaptation by the micro‐organisms to increase the utilization of PPO‐protected protein and glycerol‐based lipid. RESULTS: The experiment consisted of two periods where ruminally fistulated dairy cows were offered either RC or G for 2 weeks, followed by collection of rumen fluid, which was then used in in vitro incubations to investigate lipolysis and proteolysis over time in plant material derived from red clover plants with either wild type PPO expression (PPO+) or PPO expression reduced to undetectable levels by gene silencing (PPO?). Proteolysis and lipolysis (P < 0.05) were lower after 24 h of incubation in the PPO+ treatment than the PPO? treatment irrespective of rumen fluid. Biohydrogenation of C18 polyunsaturated fatty acids was also lower on the PPO+ treatment than the PPO? treatment, with no effect of rumen fluid. CONCLUSION: These results suggest that microbial changes to red clover feeding did not result in an increased ability of the micro‐organisms in the present study to utilize either PPO‐protected protein or glycerol‐based lipid. Copyright © 2008 Society of Chemical Industry     

蜂蜜对苹果酶促褐变的抑制及有效成分分析

作者以防止新鲜苹果切口的褐变为目标,比对不同食品配料组成的复配成分,发现蜂蜜作为涂膜材料可以有效延缓鲜切苹果片的褐变。将鲜切苹果片浸泡在质量分数0.1 g/g的椴树蜂蜜水溶液中,8 d后其仍能维持新鲜的色泽;探究了蜂蜜对苹果多酚氧化酶（polyphenol oxidase, PPO）抑制的动力学,发现蜂蜜对苹果PPO起非竞争性抑制作用,酶活抑制能力来自蜂蜜的抗氧化、清除自由基（相关系数r>0.6）能力;进一步分析蜂蜜成分与酶活抑制率的关系,发现蜂蜜中蛋白质与多酚（r值分别为0.79、0.63）是蜂蜜发挥抗褐变作用的重要成分。     

Inactivation of apple (Malus domestica Borkh) polyphenol oxidases by radio frequency combined with pulsed electric field treatment

Radio frequency (RF) preprocessing combined with pulsed electric field (PEF) processing was employed to inactivate polyphenol oxidase (PPO) in apple juice. PPO enzyme levels, loss of total phenolic content (TPC), colours and volatile components in the apple juice were subsequently determined and compared with conventional processing methods (60 °C for 10 min and 70 °C for 10 min). Results indicated that, when the apple tissue was preprocessed using RF for 10 min, the residual activity of PPO decreased to 13.57%; when the squeezed juice was processed by PEF, the residual activity decreased to about 5% at 15–35 kV cm^?1 for 400 μs. RF treatment caused no significant loss in TPC. Compared with the conventionally processed samples, the apple juice that was RF‐treated for 10 min and PEF‐treated at 15 kV cm^?1 for 400 μs increased its lightness and maintained its fresh‐like flavour.     

蛋白交联中多酚氧化酶的酶源筛选及酶学性质研究

从多种植物和真菌中筛选出多酚氧化酶(PPO)活力高的原料,并对其酶学性质进行研究,以利于其在蛋白交联中的应用。双孢蘑菇和茄子中PPO 酶活相对较高,达到11004U/g 和9376U/g。双孢蘑菇PPO 和茄子PPO 的最适温度和pH 值均分别为20℃和7.0;在温度较高时,茄子PPO 比双孢蘑菇PPO 稳定性更好。Zn2+、Mn2+、Ag+对双孢蘑菇PPO 和茄子PPO 活力表现明显的抑制作用,Cu2+ 对双孢蘑菇PPO 活力表现明显的促进作用,而对茄子PPO 却有很强的抑制作用。双孢蘑菇PPO 和茄子PPO 的Km 和Vmax 分别为5.5mmol/L、1666.7U 和8.75mmol/L、2500U。     

Polyphenol oxidase inactivation and vitamin C degradation kinetics of Fuji apple quarters by high humidity air impingement blanching

In this study, polyphenol oxidase (PPO) and vitamin C were used as the indicators of enzymes and nutrients to evaluate the apple quality during high humidity air impingement blanching (HHAIB) process. The PPO can be completely inactivated within 7 min at 90–120 °C and can retain relatively more vitamin C in the case of PPO fully inactivation. PPO inactivation followed zero‐order kinetics model at 90 and 100 °C, and followed first‐order fraction model at 110 and 120 °C. Activation energy (E_a) of PPO inactivation was between 11.61 and 13.66 kJ mol^?1 by Arrhenius equation. Vitamin C degradation under all processing temperatures was well described by first‐order model and its E_a value was 26.69 kJ mol^?1. Therefore, the HHAIB process was proved to be an effective pretreatment for Fuji apple quarters to inactivate PPO fast and meanwhile to maintain produce quality.     

Sweet potato protein hydrolysates: antioxidant activity and protective effects on oxidative DNA damage

In this study, sweet potato protein (SPP) hydrolysates were prepared by six enzymes (alcalase, proleather FG‐F, AS1.398, neutrase, papain and pepsin). The antioxidant activities and protective effect against oxidative DNA damage of SPP hydrolysates were investigated. Alcalase hydrolysates exhibited the highest hydroxyl radical‐scavenging activity (IC₅₀ 1.74 mg mL^?1) and Fe²⁺‐chelating ability (IC₅₀ 1.54 mg mL^?1) (P < 0.05). Compared with other five hydrolysates, the hydrolysates obtained by alcalase had the most abundant <3‐kDa fractions. In addition, below 3‐kDa fractions of alcalase hydrolysates showed the highest antioxidant activities and protective effects against DNA damage through both scavenging hydroxyl radicals and chelating Fe²⁺, which was probably because of the increase in several antioxidant amino acids, such as His, Met, Cys, Tyr and Phe, as well as the hydrophobic amino acids. The results suggested that enzymatic hydrolysis could be used as an effective technique to produce high value‐added peptides products from SPP.     

Polyphenol oxidase activity in grass and its effect on plant‐mediated lipolysis and proteolysis of Dactylis glomerata (cocksfoot) in a simulated rumen environment

Little is known about the level or activity of polyphenol oxidase (PPO) in grasses and its potential impact on proteolysis and lipolysis. Six grass species were initially screened for PPO activity (740.6, 291.9, 213.6, 119.0, 16.3 and 6.5 U g^?1 fresh weight (FW) for cocksfoot, hybrid ryegrass, Italian ryegrass, perennial ryegrass, timothy and tall fescue respectively). Cocksfoot, which expressed the highest activity, was then used to determine the effect of PPO on plant‐mediated proteolysis and lipolysis in a simulated rumen environment. Sourced cocksfoot was macerated and incubated in an antibiotic‐containing anaerobic medium with or without ascorbate to deactivate PPO in the dark at 39 °C over five time points. At each time point (0, 1, 2, 6 and 24 h), six replicate samples were destructively harvested; three of the replicates were used for lipid analysis and the other three for protein, free amino acid and bound phenol determination. Characterisation of the herbage showed PPO activities of 649.6 and 0 U g^?1 FW, which were reflected in the extent of phenol (derived from quinones) binding to protein after 24 h of incubation, namely 65.1 and 29.6 mg bound phenol g^?1 protein (P < 0.001) for cocksfoot and cocksfoot + ascorbate respectively. Proteolysis, measured as free amino acids released into the incubation buffer, was significantly reduced (P < 0.001) with increasing PPO activity, with values after the 24 h incubation of 0.03 and 0.07 mmol L^?1 g^?1 FW for cocksfoot and cocksfoot + ascorbate respectively. Lipolysis, measured as the proportional decline in the membrane lipid polar fraction, was likewise reduced (P < 0.001) with increasing PPO activity, with values after the 24 h incubation of 0.43 and 0.65 for cocksfoot and cocksfoot + ascorbate respectively. Changes that occurred in protein and the lipid fractions (polar fraction, monoacylglycerol + diacylglycerol, triacylglycerol and free fatty acids) during the incubations are also reported and discussed. These results support the selection of forages high in PPO activity to reduce protein and lipid losses in silo and potentially in the rumen. Copyright © 2006 Society of Chemical Industry     

Purification and identification of antioxidant peptide from black pomfret, <Emphasis Type="Italic">Parastromateus niger</Emphasis> (Bloch, 1975) viscera protein hydrolysate

To utilize fish waste, black pomfret, Parastromateus niger viscera was analysed for its proximate and amino acid composition followed by hydrolysis using various proteases to extract antioxidant peptide. Antioxidant activities of the crude hydrolysate was evaluated using DPPH (54%), metal chelating (78.6%) at a concentration of 1 mg/mL, whereas the reducing power assay was done with different concentration (0.5–2.5 mg/mL) and the activity also increased with increasing concentration (0.021–0.068). Furthermore, the hydrolysate was purified by diethylaminoethyl (DEAE) ion-exchange and Sephadex G-25 gel filtration chromatography. Finally, the purified peptide had a mass of 701.9 Da, and the amino acid sequence was identified as Ala-Met-Thr-Gly-Leu-Glu-Ala using electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Moreover, the protection ability of the peptide toward hydroxyl radical-induced oxidative DNA damage and inhibiting lipid peroxidation was evaluated and compared with natural antioxidant α-tocopherol.     

Convenient partial purification of polyphenol oxidase from apple skin by cationic reversed micellar extraction

Polyphenol oxidase (PPO) was selectively extracted from reconstituted freeze-dried apple skin using reverse micelles formed by a cationic surfactant, dodecyl trimethyl ammonium bromide (DTAB). An optimum forward extraction was achieved with sodium phosphate buffer (pH 6, 100 mM, no added KCl) and an organic phase (isooctane:hexanol at a ratio of 5:1) containing 100 mM DTAB. The solubilised PPO was efficiently recovered by a stripping solution (pH 6, 1 M KCl) containing 10% ethanol. Under the optimised conditions, the purification fold and recovered activity of PPO were 12.6% and 71%, respectively. This purification fold and recovery were maintained when the extraction volume increased from 10–200 ml. Overall, reversed micellar extraction can be used as an efficient first step for the purification of PPO from apple skin.     

Inhibition of enzymatic browning in actual food systems by the Maillard reaction products

BACKGROUND: The Maillard reaction occurring between amino acids and sugars produces neo‐formed compounds having certain levels of antioxidant activity depending on the reaction conditions and the type of reactants. The objective of this study was to investigate enzymatic browning inhibition capacity of Maillard reaction products (MRPs) formed from different amino acids including arginine (Arg), histidine (His), lysine (Lys) and proline (Pro). RESULTS: The inhibitory effects of the MRPs on polyphenol oxidase (PPO) were determined. The total antioxidant capacity (TAC) of MRPs derived from different amino acids were in the order Arg > His > Lys > Pro. The TAC and PPO inhibition of MRPs were evaluated as a function of temperature (80–120 °C), time (1–6 h) and pH (2–12). Arg‐Glc and His‐Glc MRPs exhibited strong TAC and PPO inhibition. Increasing temperature (up to 100 °C) and time also increased TAC and PPO inhibition. Kinetics analysis indicated a mixed type inhibition of PPO by MRPs. CONCLUSION: The results indicate that the MRPs derived from Arg and His under certain reaction conditions significantly prevent enzymatic browning in actual food systems. The intermediate compounds capable of preventing enzymatic browning are reductones and dehydroreductones, as confirmed by liquid chromatographic–mass spectrometric analyses. Copyright © 2010 Society of Chemical Industry