Inhibition Of Polyphenol Oxidase Research Articles

Inhibition of polyphenoloxidase from apple by Maillard reaction products prepared from glucose or fructose with l-cysteine under various conditions of pH and temperature

The effects of Maillard reaction products (MRPs), synthesized from equimolar glucose or fructose with l-cysteine (1 mol l −1) aqueous model mixtures, by modulating pH and temperature of heating, according to a two-factor and five-level experimental design, were investigated on polyphenoloxidase (PPO) activity from apple. Final pH and absorbance measurements at 350 nm were also selected as indicators of the Maillard reaction development and checked. In general, inhibitory potency (IP) of the mixtures increased with the increase in temperature (80–120°C) and the decrease in pH (pH 2.0–12.0) of the reaction medium. A linear relationship between the IP and heating time (0–48 h) or Abs.350 nm (0–70 AU) was demonstrated for glucose/cysteine system heated from 80°C to 120°C. Polarographic and spectrophotometric data were used to calculate kinetic constants and activation energy ( Ea) values of inhibitory MRPs formation versus PPO activity and of those compounds absorbing at 350 nm. Ea values for these reactions were close, being 191 and 124 kJ mol −1, respectively. The experimental design allowed to conclude that linear effects of both factors as well as a quadratic effect of pH were significant, leading to optimum conditions for the production of glucose-derived MRPs inhibitors. In most cases, glucose produced MRPs with higher IP compared to counterpart fructose–cysteine MRPs.

Apple Polyphenol Oxidase Inhibitor(s) from Common Housefly (Musca Domestica L.)

ABSTRACT: A new natural apple polyphenol oxidase (PPO) inhibitor(s) from housefly (Musca domestica L.) was discovered. Crude inhibitor (s) isolated by buffer extraction, heat treatment, and dialysis from housefly pupae inhibited the activity of apple PPO up to 90% at pH values above 5.0. Inhibition was strictly pH‐dependent. The inhibitor(s) was further characterized by employing heat, freezing and thawing, irradiation, pH adjustment, and ultrafiltration studies. The potential PPO inhibitor(s) was stable to heating at 100 °C for 1 h, repeated freezing and thawing, and irradiation. The inhibitor(s) was most stable at pH around 5.0 and least stable at alkaline pH. The PPO inhibition profile of housefly during metamorphosis also was evaluated.

Kinetics of Inhibition of Polyphenol Oxidase Mediated Browning in Apple Juice by β-Cyclodextrin and L -Ascorbate-2-triphosphate

Polyphenol Oxidase (PPO) mediated browning in raw fruits and vegetables is a major cause of quality deterioration in fruits and vegetables and derived food products. Here the rate of browning reaction in apple juice treated individually and in combination(1:1) of β -Cyclodextrin (β -CD) and L -Ascorbate-2-triphosphate (L -AATP) is described. It was observed that the rate of quinone formation can be minimized using a combination of β -CD and L -AATP as compared to individual treatment with these agents. Kinetic experiments revealed that both compounds are non-competitive inhibitors of PPO.

Inhibitory effect of unheated and heated d-glucose, d-fructose and l-cysteine solutions and Maillard reaction product model systems on polyphenoloxidase from apple. II. Kinetic study and mechanism of inhibition

A previous study showed that Maillard reaction products (MRP) derived from equimolar glucose or fructose and l-cysteine model solutions (1 M) were potent inhibitors of polyphenoloxidase (PPO) from apple. In the present paper, a kinetic study was undertaken of the behaviour of a Michaelis–Menten PPO-catalyzed reaction in the presence of MRP heated at 90 °C for various times (2–39 h), using 4-methylcatechol as the substrate. Results obtained revealed that both MRP systems were mixed-type inhibitors, the glucose/cysteine model solution being the most effective one. Activity of preincubated PPO at 0 °C for different times with either heated l-cysteine or MRP was only slightly restored via exhaustive dialysis. Recovery of enzyme activity was more marked when cupric ions were added to the reaction medium. Possible mechanisms of inhibition of PPO activity by MRP are discussed, in relation to chelation of cupric ions.

INHIBITION OF SURFACE BROWNING, CUT AVOCADO

AbstractBrowning prevention of avocado halves using polyphenol oxidase (PPO) inhibitors along with sulfites was investigated. Browning inhibitiors were selected on the basis of the inhibition of a crude extract of avocado PPO. The most effective inhibitors were ascorbic acid and sodium sulfite, these compounds plus citric acid were mixed to get an inhibitory solution. In vivo experiments were performed on avocado halves by measuring Hunter color values after 15 days' storage to test the potential of solutions to prevent browning. Results showed that immersion in solutions along with refrigerated storage was needed to get an inhibitory effect. Color preservation was not due to oxygen exclusion nor to low pH and sulfite was unnecessary. The best treatment was the storage of avocado halves in a 1% ascorbic acid/1 % citric acid solution at pH 5 and 7C.

Polyphenol oxidases from latex of Hevea brasiliensis: purification and characterization

Polyphenol oxidase (PPO) was isolated from the B-serum obtained after repetitive freeze-thawing of the bottom fraction isolated from ultracentrifuged fresh latex. The B-serum was subjected to acetone precipitation and CM-Sepharose chromatography, affording two PPOs, PPO-I and PPO-II, which, upon SDS-PAGE, were 32 and 34 kDa, respectively. Both PPOs possessed the same pI (9.2), optimum pH (7) and optimum temperature (35–45 °C). They are stable up to 60 °C and active at broad pH ranges from 4–9. The K m values of PPO-I for dopamine, l-dopa and catechol as substrates are 2.08, 8.33 and 9.09 mM, while those for PPO-II are 2.12, 4.76 and 7.14 mM, respectively. Among various PPO inhibitors tested, 4-hexylresorcinol was the most potent. Anionic detergents were among the most effective activators of the enzymes, while cationic and nonionic detergents showed little and no effect on the PPO activities, respectively.

Substrate specificity, heat inactivation and inhibition of polyphenol oxidase from different aubergine cultivars

The effects of substrate specificity, heat inactivation, temperature, pH and inhibitors on polyphenol oxidase (PPO) activity obtained from three different aubergine cultivars were investigated to identify the most appropriate aubergine cultivar for dried preparations. PPO obtained from different aubergine cultivars (cultivars I, II and III) was partially purified by (NH4)2SO4 precipitation followed by dialysis. PPO showed activity with catechol and 4‐methylcatechol but not with L‐tyrosine. The best substrate for cultivar I (Vmax: 3333.3 EU min−1 mL−1, Km: 8.7 mM and Vmax/Km: 384.9 min−1) and cultivar III (Vmax: 1000 EU min−1 mL−1, Km: 9.3 mM and Vmax/Km: 107.5 min−1) was catechol, but 4‐methylcatechol was the best substrate for cultivar II (Vmax: 5000 EU min−1 mL−1, Km: 35.5 mM and Vmax/Km: 140.8 min−1). The optimum pH for aubergine PPO was 7.0 with catechol as a substrate and 6.0 with 4‐methylcatechol. Heat inactivation studies showed a decrease in enzyme activity at temperatures above 40 °C. For catechol and 4‐methylcatechol substrates, the optimum temperature for maximum PPO activity was 30 °C for all aubergine cultivars except cultivar I using catechol which was 20 °C. The effects of compounds such as tropolone, D,L‐dithiothreitrol and glutathione as inhibitors of the reactions catalysed by PPO were tested. Generally, tropolone was the most effective inhibitor.

Activity and Inhibition of Polyphenol Oxidase in Extracts of Bran and Other Milling Fractions from a Variety of Wheat Cultivars

ABSTRACTStudies were conducted to compare polyphenol oxidase (PPO) specific activities in various milling fractions of a variety of wheat cultivars and determine the levels of activities in a number of cultivars from different localities and harvesting seasons. Substrate specificities were also investigated. Bran was singled out as the richest source of PPO activity, which may also influence the activity in the other milling fractions that are known to have some proportion of bran content. We showed by gel electrophoresis and spectrophotometrically that the protein responsible for PPO activity apparently exists as a single isoform in bran and that the observed enzyme activity is likely to be a tyrosinase type, not a laccase or peroxidase. The specific activity was not significantly different between the reduction shorts and break shorts from the same cultivar, indicating a similar level of bran contamination in these fractions. Very low levels of PPO activity were recorded in the flour of all cultivars studied. Bran was used, therefore, to determine the varietal differences in the PPO activities in a number of cultivars from different localities and seasons of harvest. Results showed that the most significant determinant of PPO activity was the genotype, and this may be influenced by seasonality. We also determined that, apart from substrate preferences by the PPO enzyme, some phenolic acids actually inhibit PPO. Furthermore, we found that bran of some cultivars extracted with acidified methanol inhibited PPO activity substantially, whereas other extracts had less inhibitory properties. Thus, these unknown compounds in wheat may inhibit endogenous PPO activity.

PREVENTION OF BROWNING OF POTATO SLICES USING POLYPHENOLOXIDASE INHIBITORS AND ORGANIC ACIDS

ABSTRACTBrowning of minimally processed potato slices stored at low temperatures without active modification of the atmosphere in the storage container was inhibited by use of combinations of polyphenoloxidase inhibitors with organic acids. Slices of two cultivars‘Atlantic’and‘Russet Burbank’were dipped in solutions of various combinations of 4‐hexyl resorcinol, N‐acetyl‐cysteine and citric acid. Treated potato slices were kept in storage at 5C without browning or decrease in firmness for up to 21 days. The limiting factor in storage became visible microbial decay. Browning of slices from potatoes stored 1 month longer at 10C was also inhibited, but storage was limited to 14 days because of increased susceptibility to decay and decreased firmness with most treatments. However, no differences were found in inhibition of browning or retention of firmness of slices of the two cultivars after treatment with the combination of 0.25 M citric acid, 0.25 M N‐acetylcysteine and 0.01 M 4‐hexylresorcinol.

Identification of novel polyphenol oxidase inhibitors by enzymatic one-pot synthesis and deconvolution of combinatorial libraries.

The feasibility of enzymatic synthesis of combinatorial libraries using multifunctional starting materials [i.e., 2,4-dihydroxy-N-(2-hydroxyethyl)benzamide, 1; 4-hydroxyphenethyl alcohol, 2; 3,5-dihydroxybenzyl alcohol, 3; and 4-hydroxybenzyl alcohol, 4] with six vinyl esters, in a one-pot reaction, was investigated. Candida antarctica lipase was employed as a biocatalyst. The resulting 24-compound library contained all the expected species with no significant bias toward particular combinations of substrates. As expected, the library contained a substance(s) that showed significant inhibition of polyphenol oxidase, which was used as a model target. The deconvolution was accomplished via resynthesis of ten partial libraries, which were prepared with either an equimolar mixture of the four alcohols and a single vinyl ester, or a single alcohol and equimolar mixture of the activated esters. Analysis of the inhibition pattern observed with these partial libraries suggested that 4-hydroxybenzyl benzoate (4e) should be the most potent inhibitor. This conclusion was confirmed by the preparation and comparison of all 24 components of the initial library. Finally, it was shown that 4e was a competitive inhibitor of polyphenol oxidase, with a K(i) of 40 microM. This value compared favorably with a K(i) of 400 microM, which was determined for parent phenol 4.

PARTIAL CHARACTERIZATION OF A PEPTIDE FROM HONEY THAT INHIBITS MUSHROOM POLYPHENOL OXIDASE

Mushroom polyphenol oxidase (PPO) was inhibited by one type of honey in a model system. Honey was a noncompetitive inhibitor of PPO catalyzed oxidation of catechol. The Arrhenius activation energy (Ea) of thermal inactivation of mushroom PPO difered in the presence and absence of honey. The Ea value in the presence of honey was 2 7.71 W/mol; whereas, Ea of the control was 83.14 W/mol. Thus, the temperature coefficient of PPO inactivation was lower in the presence of honey. Characterization of the inhibitor was carried out by gel electrophoresis (SDS-PAGE and NativePAGE). An inhibitory peptide paction was obtained after native PAGE. The molecular weight was determined to be 0.6 kDa by Sephadex G15 column chromatography.

Characterization of polyphenol oxidase in coffee

Polyphenol oxidase (PPO) was characterized in partially purified extracts of leaves (PPO-L) and fruit endosperm (PPO-E) of coffee ( Coffea arabica L.). PPO activity was higher in early developmental stages of both leaves and endosperm of fruits. Wounding or exposure of coffee leaves to methyl jasmonate increased PPO activity 1.5–4-fold. PPO was not latent and was not activated by protease treatment. PPO activity was stimulated 10–15% with sodium dodecyl sulphate (SDS) at 0.35–1.75 mM, but at higher concentrations activities were similar to the control samples, without detergent. Prolonged incubation of extracts with trypsin or proteinase K inhibited PPO activity but pepsin had no effect. Inhibition of PPO with proteinase K was increased in the presence of SDS. PPO activity from both tissues was optimal at pH 6–7 and at an assay temperature of 30°C. Activity was highest with chlorogenic acid as substrate with a K m of 0.882 mM (PPO-L) and 2.27 mM (PPO-E). Hexadecyl trimethyl-ammonium bromide, polyvinylpyrrolidone 40, cinnamic acid and salicylhydroxamic acid inhibited PPO from both tissues. Both enzymes were inactivated by heat but the activity in endosperm extracts was more heat labile than that from leaves. The apparent M r determined by gel filtration was 46 (PPO-L) and 50 kDa (PPO-E). Activity-stained SDS–polyacrylamide gel electrophoresis (PAGE) gels and western blots probed with PPO antibodies suggested the existence of a 67 kDa PPO which is susceptible to proteolytic cleavage that generates a 45 kDa active form.

PHYSICO-CHEMICAL PROPERTIES of POLYPHENOL OXIDASE FROM d'ANJOU and BARTLETT PEARS (PYRUS COMMUNIS L.)

Polyphenol oxidase (PPO) and phenolics were studied in two pear cultivars, d'Anjou and Bartlett. PPO activity, total phenolics and chlorogenic acid concentration differed significantly with respect to cultivar. PPO activity, total phenolics and chlorogenic acid all decreased in overripe fruits. The pH optimum (d'Anjou, 4.7 and Bartlett, 5.5), and optimum temperature (d'Anjou 40C and Bartlett, 20C) for maximum PPO activity were determined. Among substrates, 4-methylcatechol followed by catechol and dopamine, was the most readily oxidized substrate of PPO from both pear cultivars. The enzyme from both cultivars did not show any activity with any of the monohydroxy substrates. K m of 13.33 and 10.92 mM were determined with 4-methylcatechol for d'Anjou and Bartlett pear PPO, respectively. Benzoic and cinnamic acid series compounds were poor inhibitors of PPO. However, L-cysteine, sodium metabisulfite, ascorbic acid and thiourea proved to be effective inhibitors of this enzyme. Heating at 65C for 30 min resulted in a loss of 60-72% of PPO activity.

Myricetin, an antioxidant flavonol, is a substrate of polyphenol oxidase

The present study demonstrates the antiradical efficiency of myricetin, a flavonol widely distributed in fruits and vegetables, by testing its ability to react with two different free radicals, ABTS+· and DPPH·. The polyphenolic nature of myricetin led us to consider the possibility of its oxidation by polyphenol oxidase (PPO). The results reported show that myricetin can be oxidised by PPO extracted and partially purified from broad bean seeds. The reaction was followed by recording spectral changes with time, maximal spectral changes being observed at 372 nm. The presence of two isosbestic points (at 274 and 314 nm) suggested that only one absorbing product was formed. The spectral changes were not observed in the absence of PPO. The oxidation rate varied with the pH, reaching its highest value at pH 5.5. The myricetin oxidation rate increased in the presence of SDS, an activing agent of polyphenol oxidase. Maximal activity was obtained at 1.3 mM SDS. The kinetic parameters were also determined: V m = 1.35 µM min−1, K m = 0.3,mM, V m/K m = 4.5 × 10−3 min−1. Flavonol oxidation was inhibited by a selective PPO inhibitor such as cinnamic acid (KI = 1 mM). The results reported show that myricetin oxidation was strictly dependent on the presence of polyphenol oxidase. © 1999 Society of Chemical Industry

Synthesis of crown ether compounds as potential inhibitors of enzymatic discolouration of foods

The synthesis of the following crown ether containing compounds as potential polyphenol oxidase inhibitors is described: 4'-N(benzo-crown-5)succinamic acid (I), 4'-N(benzo-15-crown-5)malemic acid (II), ester of 4'-N(benzo-15-crown-5)bis[4'-N(benzo-15-crown-5)]phthalamic acid (III) and complex of benzo-18-crown-6 with 2-propenyl-acrylamide (IV). The complexes of I, II and IV with potassium 2-propenyl-acrylamide are also described.

Production of a banana (Musa cavendishii) extract containing no polyphenol oxidase by ultrafiltration

The objective of this research was the production of a banana extract containing no polyphenol oxidase by ultrafiltration. Banana juice was extracted after inhibition of polyphenol oxidase using a solution containing 0.15 g per 100 ml of sodium metabisulphite and 1 g per 100 ml of ascorbic acid in distilled water to avoid initial browning. Polyphenol oxidase was then retained by ultrafiltration using polysulphone membranes with a cut-off of 20 kDaltons. Two transmembrane pressures, 600 and 800 kPa, were used but 600 kPa was preferred because of the more stable permeate flux which decreased less with time and concentration of extract. © 1999 Society of Chemical Industry

Cyclodextrins as activator and inhibitor of latent banana pulp polyphenol oxidase.

The effect of cyclodextrins (CDs) on o-diphenol oxidation catalyzed by banana polyphenol oxidase (PPO) was studied. The oxidation of dopamine, the natural substrate of banana, in the presence of cyclodextrins was unaffected, because this hydrophilic phenol does not form inclusion complexes with CDs. However, when a hydrophobic phenol such as tert-butylcatechol (TBC) was used, a marked inhibition was observed with beta-, hydroxypropyl-beta-, and maltosyl-beta- CDs. This inhibition was due to the complexation of TBC in the CD core, demonstrating that banana pulp PPO worked only toward free substrate and not toward the complex TBC-CDs. In addition, the effect of some inhibitors in the presence of CDs and dopamine as substrate was studied. Increasing concentrations of CDs, in the presence of two inhibitors (4-iodophenol and cinnamic acid) were able to activate the inhibited enzyme to reach the noninhibited level by complexing the inhibitors in the hydrophobic core of the CDs. This dual effect of CDs as activator and inhibitor was tested in crude banana pulp extracts, with surprising activation effects never before described being observed.

Oxidation of the flavonol quercetin by polyphenol oxidase.

Because direct oxidation of flavonols by polyphenol oxidase (PPO) has not previously been reported and, given the importance of flavonols, the ability of broad bean seed PPO to oxidize the flavonol quercetin was studied. The reaction was followed by recording spectral changes with time. Maximal spectral changes were observed at 291 nm (increase) and at 372 nm (decrease). The presence of two isosbectic points (at 272 and 342 nm) suggested the formation of only one absorbent product. These spectral changes were not observed in the absence of PPO. The oxidation rate, which varied with pH, was highest at pH 5.0. The following kinetic parameters were also determined: V(m) = 11 microM/min, K(m) = 646 microM, V(m)/K(m) = 17 x 10(-)(2) min(-)(1). Flavonol oxidation was efficiently inhibited (K(I) = 3.5 microM) by specific PPO inhibitors such as 4-hexylresorcinol. The results obtained showed that quercetin oxidation was strictly dependent on the presence of PPO.

Oxidation of the flavonol fisetin by polyphenol oxidase

The present study demonstrates the antiradical efficiency of fisetin, a flavonol widely distributed in fruits and vegetables, by its ability to react with two different free radicals, ABTS +⋅ and DPPH ⋅. The polyphenolic nature of fisetin led us to consider whether it might be oxidised by polyphenol oxidase (PPO), and the results reported show that it can be oxidised by PPO extracted and partially purified from broad bean seeds. The reaction was followed by recording spectral changes with time, with maximal spectral changes being observed at 282 nm (increase in absorbance) and at 362 nm (decrease). The presence of two isosbectic points (at 265 and 304 nm) suggested that only one absorbent product was formed. These spectral changes were not observed in the absence of PPO. The oxidation rate varied with the pH, reaching its highest value at pH 5.5. The fisetin oxidation rate increased in the presence of sodium dodecyl sulfate, an activator of polyphenol oxidase. Maximal activity was obtained at 0.87 mM sodium dodecyl sulfate. The following kinetic parameters were determined: V max=49 μM/min, K m=0.6 mM, V max/ K m=8.2×10 −2 min −1. Flavonol oxidation was inhibited by selective PPO inhibitors such as cinnamic acid (a classical competitive inhibitor, K i=1.4 mM) and 4-hexylresorcinol, which behaved as a slow-binding inhibitor. The results reported show that fisetin oxidation was strictly dependent on the presence of polyphenol oxidase.

Inhibition of polyphenol oxidase and the browning control of litchi fruit by glutathione and citric acid

Polyphenol oxidase (PPO, EC 1.10.3.2) from litchi peel was partially purified by ammonium sulfate fractionation and gel filtration, and a 16-fold purification of PPO achieved. The use of 10 mmol litre −1 glutathione and 100 mmol litre −1 citric acid was found to give good control of the browning of litchi fruit and 80–85% inhibition of PPO observed. Application of glutathione in combination with citric acid is recommended as a way of slowing the browning of litchi fruit.