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.     

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     

Inhibition kinetics of polyphenol oxidase by glutamic acid

The inhibition of polyphenol oxidase (PPO) by glutamic acid was investigated. Application of different concentrations of glutamic acid to mushroom solution and Ocimum basilicum L. extract showed that glutamic acid appeared to be an effective browning inhibitor. Glutamic acid showed uncompetitive inhibition for mushroom and Ocimum basilicum L. polyphenol oxidases using 4-methylcatechol as a substrate, for mushroom PPO using catechol as a substrate and for Ocimum basilicum L. polyphenol oxidase using pyrogallol as a substrate; mixed-type inhibition for mushroom polyphenol oxidase using pyrogallol as a substrate; and non-competitive inhibition for Ocimum basilicum L. polyphenol oxidase using catechol as a substrate. Furthermore, sodium azide was used as an inhibitor for comparison with the inhibition potency of glutamic acid. It was found that glutamic acid was a more power inhibitor than sodium azide. The type of inhibition observed depended on the substrate, inhibitor and enzyme source used.     

Inhibition kinetic and mechanism of polyphenol oxidase from various sources by diethyldithiocarbamic acid

Inhibition kinetics and mechanism of polyphenol oxidases (PPO) partially purified from various sources such as Thymbra spicata L. var. spicata and Ocimum basilicum L., and of mushroom PPO bought from Sigma by diethyldithiocarbamic acid have been described using catechol, 4-methylcatechol and pyrogallol as substrates. The inhibition type was competitive for O. basilicum L. PPO using catechol and 4-methylcatechol as substrates, for mushroom PPO using catechol, 4-methylcatechol and pyrogallol as substrates, and for T. spicata L. var. spicata PPO using 4-methylcatechol as a substrate; uncompetitive inhibition for T. spicata L. var. spicata PPO using pyrogallol as a substrate; and non-competitive inhibition for O. basilicum L. and T. spicata L. var. spicata PPO using pyrogallol and catechol as substrates, respectively. The inhibition effect of diethyldithiocarbamic acid on enzymatic browning varied greatly from one phenol to another and from one enzyme to another. Hence, no general rule can easily be established with regard to the type of inhibition observed.     

PARTIAL PURIFICATION AND CHARACTERIZATION OF POLYPHENOL OXIDASE FROM FRESH-CUT CHINESE WATER CHESTNUT

The characteristics of polyphenol oxidase (PPO) from Chinese water chestnut (CWC) and its potential inhibitors for browning reactions were investigated. PPO was isolated from fresh‐cut CWC and was purified on a Sephadex G‐100 column, with a yield of total activity close to 10%. The molecular weight, Michaelis constant (K_m), substrate specificity, optimal pH and temperature of CWC PPO were examined. Kinetic studies indicated that the K_m and V_max values of CWC PPO for catechol were 10.32 mmol/L and 6.452 × 10⁴ U/min, respectively. The optimal pH and temperature for CWC PPO was 6.5 and 40C, respectively. Among the browning inhibitors tested, 4‐hexylresorcinol, at a concentration of 0.3 mmol/L, showed the strongest inhibition (70%) against the PPO activity of CWC, followed by 3.0 mmol/L N‐acetyl‐L‐cysteine with an inhibition of 53%.     

Synergistic effect of high-intensity ultrasound and β-cyclodextrin treatments on browning control in apple juice

This work evaluated the synergistic effects of combined high-intensity ultrasound (HIU) with β-cyclodextrin (β-CD) treatments on inhibiting browning of apple juice and explored the mechanism through simulation system. The combined treatment of 300 W HIU with 0.006 g mL⁻¹ β-CD had a synergistic impact on maintaining juice colour, resulting in a 39.06% reduction in browning degree, only a 36.64% decrease in total phenolic content, and a 17.82% reduction in PPO activity. The inhibition of enzymatic browning in simulated system revealed that HIU suppressed the enzyme (Polyphenol oxidase, PPO) and β-CD inhibited enzyme (PPO) and embedded substrate (polyphenol). The results of spectroscopic analysis showed that the particle-size distribution of PPO narrowed, the content of α-helix in the secondary structure increased, the fluorescence intensity increased, and the maximum wavelength was red-shifted after HIU and β-CD treatment. Changes in structure could further result in PPO activity loss. Hence, the combined treatment could synthetically alleviate the browning of apple juice.     

Purification of Polyphenoloxidase from the Purple‐fleshed Potato (Solanum tuberosum Jasim) and its Secondary Structure

ABSTRACT: Polyphenoloxidase (PPO) was purified from purple‐fleshed potatoes (Solanum tuberosum Jasim) using membrane concentration, ammonium sulfate fractionation, Resource Q ion exchange chromatography, and Sephacryl S‐200 HR gel permeation chromatography. PPO was purified 78‐fold from a crude extract. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis results showed that the purified enzyme has a major subunit molecular weight of 40 kDa. To elucidate the secondary structure of the purified PPO, circular dichroism (CD) was performed. The CD spectrum of the purified enzyme showed that PPO contains 35% α‐helix, 30% β‐turn, and 35% random coil structure.     

Antioxidant and Anti‐inflammatory Activities of Methanol Extracts of Tremella fuciformis and Its Major Phenolic Acids

Methanol extract subfractions of the edible white jelly mushroom (Tremella fuciformis), were assessed for the following antioxidant properties: ABTS⁺ radical scavenging activity, 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) radical scavenging activity, and inhibitory activity of human low‐density lipoprotein (LDL) oxidation. Among the subfractions tested, the chloroform subfraction exhibited the strongest antioxidant activity, with the highest total phenolic content (66.31 μg CAE/mg extract) and flavonoids content (5.12 μg QE/mg extract). The ABTS⁺ radical scavenging activity of the chloroform subfraction was 7.89 μmol trolox/mg extract, which was the highest among all subfractions. This subfraction also showed the highest DPPH radical scavenging activity and inhibitory activity of LDL oxidation. In addition, the chloroform subfraction demonstrated anti‐inflammatory activity through inhibition of nitric oxide production and inducible nitric oxide synthase expression in RAW 264.7 cells. Major phenolic acids from the mushroom extract were identified as 4‐hydroxybenzoic acid (323 mg/kg dry weight of mushroom), gentisic acid (174 mg/kg dry weight of mushroom), and 4‐coumaric acid (30 mg/kg dry weight of mushroom).     

Polyphenol Oxidase from Bean Sprouts (Glycine max L.)

ABSTRACT: Polyphenol oxidase (PPO) was purified and characterized from bean sprouts by ammonium sulfate precipitation, DEAE‐Toyopearl 650M, CM‐Toyopearl 650M, SuperQ‐Toyopearl 650S and QAE‐Toyopearl 550C column chromatographies. Substrate staining of the crude extract on electrophoresis showed the presence of 2 isozymic forms of this enzyme. The molecular weight of the purified enzyme was estimated to be about 54 kDa. The optimum pH was 9.0 and optimum temperature 40 °C. Heat inactivation occurred about 30 °C. PPO showed activity to catechol, pyrogallol and dopamine. These compounds such as ascorbic acid, L‐cysteine, 2‐mercaptoethanol, and glutathione used was the effective inhibitor. Enzyme activity was maintained for 7 d at 4 °C but suddenly decreased after 8 d.     

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.     

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.     

气调包装对平菇贮藏内在品质的影响

为了解气调包装(MAP)对平菇贮藏内在品质的影响,研究了经MAP处理后平菇多酚氧化酶(PPO)特性以及质构的变化。PPO活性是影响平菇货架期的内在因素之一。采用硫酸铵沉淀法得到平菇多酚氧化酶粗提液。试验结果表明,亚硫酸氢钠、柠檬酸均能有效地抑制平菇多酚氧化酶的活性,氯化钙对其影响不大。低温能有效地抑制平菇质构品质的下降,2.0% CaCl2处理效果比较明显,能达到延缓果实成熟和衰老的目的。     

Inhibitory effect of rice bran extract on polyphenol oxidase of potato and banana

Rice bran was extracted with water and its effects on potato and banana polyphenol oxidase (PPO) were investigated. Rice bran extract (RBE), conc. 0.3 g mL^?1, exhibited PPO inhibition in potato and banana PPO with % inhibition of 69.31% and 47.63%, respectively (P ≤ 0.05). RBE showed a concentration‐dependent inhibition on potato and banana PPO. RBE (conc. 0.3 g mL^?1) inhibited potato PPO higher than ascorbic acid, citric acid, NaCl and EDTA (final conc. 20 mg L^?1); and it also inhibited banana PPO higher than citric acid, NaCl and EDTA (final conc. 20 mg L^?1), respectively. The combination of RBE with citric acid or ascorbic acid appeared to be additive inhibitory effect on banana and potato PPO. Kinetic study of the inhibition on potato and banana PPO by RBE showed that RBE was a mixed‐type inhibitor; however, RBE appeared to be able to act directly on enzyme structure rather than substrate structure.     

PARTIAL PROPERTIES OF POLYPHENOL OXIDASE IN MANGO (MANGIFERA INDICA L. CV. "TAINONG") PULP

The properties of polyphenol oxidase (PPO, EC 1.14.18.1) from an extract of mango pulp were studied. PPO, with catechol as substrate, had an optimum pH at 7.0 and optimum temperature at 30C. PPO showed activity with dihydroxyphenols and trihydroxyphenols, but not with monohydroxyphenols. Kinetic parameters maximum velocity and Michaelis constant for PPO were 256.28 U/min and 6.30 mM with catechol, and 199.61 U/min and 47.81 mM with pyrogallol. The activity of PPO was well retained after heating the extract for 15 min at 50C, and 98% of the activity was lost after the extract was heated for 5 min at 80C. PPO was effectively inhibited by ascorbic acid as well as by β‐mercaptoethanol and L‐cysteine, and was enhanced by sodium dodecyl sulfate.     

Activation and conformational changes of mushroom polyphenoloxidase by high pressure microfluidization treatment

Activation and conformational changes of mushroom polyphenoloxidase (PPO) after high pressure microfluidization treatment were observed by means of UV spectrophotometer, far-UV circular dichroism, fluorescence emission spectra, UV absorption spectra and sulphydryl groups detection. The results indicated that, treated under pressures of 90 MPa, 110 MPa, 130 MPa and 150 MPa one pass, and 150 MPa 1, 2 and 3 passes, respectively, mushroom PPO exhibited an increase in activity. The circular dichroism (CD) analysis demonstrated that some of secondary structures such as α-helix were destroyed. There were some indices that the increase of relative activity was accompanied by a decrease in α-helix content. The fluorescence emission spectra analysis indicated that Trp and Tyr residues in mushroom PPO were more or less exposed to solvent, and the result was in good agreement with that of UV absorption spectra analysis. The sulphydryl groups detection showed that the sulphydryl groups content on the surface of mushroom PPO was increased.Industrial relevanceResults of the present study show that high pressure microfluidization can not be used to inactivate mushroom PPO. However, it provides a new way for enzyme preparation, such as enzyme modification, with higher activity. High pressure microfluidization can be easily operated, with shorter treatment time and higher food safety compared with chemical methods.     

INHIBITION STUDIES ON BURDOCK POLYPHENYL OXIDASE (PPO) ACTIVITY

The inhibitory effect of 4-hexylresorcinol (HR), ascorbic acid (AA), citric acid (CA), and bisulfites on polyphenol oxidase (PPO) of burdock roots was studied. HR was an effective inhibitor at low concentrations used in this study for crude PPO from root slices. A binary mixture of HR and AA at 1.0% concentration for 5 min achieved more than 90% inhibition of crude PPO. AA also showed high antibrowning activity for the purified PPO comparable to that of bisulfites, whereas its activity was much lower than that of HR in the crude extract. The inhibitory effect of the combined use of inhibitors was overall in the decreasing order of HR+AA, HR+CA and AA+CA, irrespective of the crude PPO or the purified PPO from burdock slices.     

Edible mushroom (Flammulina velutipes) extract inhibits melanosis in Kuruma shrimp (Marsupenaeus japonicus)

This study compared the potential of an aqueous extract of an edible mushroom (Flammulina velutipes) to prevent melanosis in cultured Kuruma shrimp (Marsupenaeus japonicus) with other antimelanosic compounds in vivo. The mushroom extract contained 9.1 mg/mL ergothioneine (ESH). Immersion of live full-grown shrimp in a 0.5% w/v solution of mushroom extract significantly reduced PPO activity in shrimp hemolymph. In addition, expression of the prophenoloxidase (proPO) gene decreased in hemocytes, suggesting that the extract blocked the activation of the proPO cascade. Consequently, the development of melanosis in the treated shrimp was significantly suppressed during ice storage. Treatment with a 0.05% w/v solution of sodium ascorbate and 4-hexyl-1,3-benzenediol had the same effect. In vitro experiments showed that ESH effectively inhibited PPO activity and activation of the proPO cascade in hemocyte lysate supernatant. This study suggests that in vivo application of F. velutipes mushroom extract is an effective natural alternative to synthetic antimelanosic agents to inhibit postmortem melanosis in shrimp. Practical Application: The extract of an edible mushroom (F. velutipes) containing ergothioneine can be a promising natural alternative to synthetic antimelanosic agents used to prevent postharvest melanosis in shrimp and other crustaceans. Furthermore, utilization of the mushroom trimmings could also help address the growing concerns on the disposal of such agricultural wastes and instead use it into a novel purpose as a source of antimelanosic and antioxidants for food and industrial application.     

IDENTIFICATION OF PROCYANIDIN A2 AS POLYPHENOL OXIDASE SUBSTRATE IN PERICARP TISSUES OF LITCHI FRUIT

Postharvest browning of litchi fruit results in short shelf life and reduced commercial value. Experiments were conducted to separate, purify and identify polyphenol oxidase (PPO ) substrates that cause litchi fruit to brown. PPO and its substrate were extracted from the pericarp tissues of litchi fruit. The litchi PPO substrate was purified using polyamide column, silica gel column and Sephadex LH‐20 column chromatography. The browning substrate was selected by a 0.5% FeCl₃ solution and then identified using a partially purified litchi PPO. Analyses of ultraviolet spectrometry, nuclear magnetic resonance and electrospray ionization mass spectrometry indicated that the PPO substrate was procyanidin A2. The substrate can be oxidized to ο‐quinones by litchi PPO and then form brown‐colored by‐products, resulting in pericarp browning of harvested litchi fruit.     

Effect of oyster mushroom (Pleurotus Ostreatus) and its ethanolic extract in diet on absorption and turnover of cholesterol in hypercholesterolemic rat

The effect of the diet containing 5% of powdered oyster mushroom (Pleurotus ostreatus) or an equivalent amount of mushroom ethanolic extract on cholesterol content in serum and liver, on its distribution in lipoproteins, absorption and turnover was studied in male Wistar rats (initial body weight about 70 g) fed a diet with 0.3% cholesterol. 12 weeks of feeding with whole oyster mushroom or mushroom extract reduced cholesterol level in serum by 52 and 33%, respectively. However, cholesterol content in liver was reduced only by whole oyster mushroom (by 20%). Diminished serum cholesterol level was mediated in 60% by reduction of cholesterol in very-low-density lipoproteins. Both whole oyster mushroom and mushroom extract increased the concentration of cholesterol in high-density lipoproteins. Consuming whole oyster mushroom decreased cholesterol absorption (estimated by dual-isotope plasma ratio method) by nearly 16% while no significant effect of mushroom extract could be demonstrated. Feeding the diet containing whole oyster mushroom or its extract reduced the half-times of decay curve of cholesterol-4-¹⁴C by 29 and 35%, respectively and reciprocally increased the fractional catabolic rate of plasma cholesterol.     

PURIFICATION AND KINETIC CHARACTERIZATION OF POLYPHENOL OXIDASE FROM TOMATO FRUITS (LYCOPERSICON ESCULENTUM CV. MUCHAMIEL)

Two different polyphenol oxidase (PPO) fractions, soluble and particulate, were purified from unripe tomato fruits (Lycopersicon esculentum M. cv. Muchamiel). The PPO present in the soluble fraction was purified fivefold with a 43.5% yield after ammonium sulfate fractionation. PPO in the particulate was purified 4.56‐fold with a 23% yield using the nonionic detergent Triton X‐114. A strong correlation between tomato fruit PPO activity and the physiological disorder blossom‐end rot (BER) was found, with a large increase of the PPO activity in the particulate fraction. Kinetic characterization, including kinetic parameters, pH and temperature profiles, substrate specificity and inhibitors showed similarities in both the soluble and the particulate enzyme(s). However, thermal stability of the particulate enzyme was significantly higher than stability of the soluble PPO, thus indicating possible structural differences. Cupric ions were activators, probably because of their ability to reactivate PPO partly denatured during purification.