Oxidation Of Polyphenols Research Articles

Identification and Characterisation of a Bryophyte Polyphenol Oxidase Encoding Gene from Physcomitrella patens

Polyphenol oxidases (PPO) are enzymes of secondary plant metabolism that catalyse the oxidation of polyphenols to quinones. Because of their ubiquitous appearance in the plant kingdom, an important role is assumed; however, the exact physiological function of PPOs remains unclear. In this work, the identification, cloning, and characterisation of a bryophyte PPO from the moss Physcomitrella patens is presented. PPO activity from protein extracts was determined polarographically after activation by SDS. Four Physcomitrella ESTs with homologies to known plant PPOs were selected from publicly accessible databases, and PCR experiments demonstrated that they belong to the same gene, named Pp_ppo1. The identified cDNA was found to be 2402 bp long, containing a single open reading frame of 1611 bp encoding for a 536 amino acid protein with a molecular mass of 60.1 kDa. Cloning and sequencing of a genomic part of Pp_ppo1 revealed the presence of a 94-bp intron. The time course of Pp_ppo1 gene expression in liquid culture was monitored by real time RT-PCR, revealing increasing transcription levels until the 4th day, a maximum between the 4th and the 8th day, and decreasing transcription until the 12th day. A comparison of the deduced amino acid sequence of Pp_ppo1 with seed plant PPOs revealed similarities such as the presence of two highly conserved copper-binding domains and a similar pattern of hydrophobic regions, but also differences such as a stronger membrane association and a shorter signal sequence, thus reflecting the phylogenetic distance of Physcomitrella from seed plants.

The kinetics of polyphenol degradation during the drying of Malaysian cocoa beans

SummaryThe main objective of the study was to determine the kinetics of the polyphenol oxidation reaction in cocoa beans during air drying at various air temperatures and humidities. The temperatures used were between 40 and 60 °C and the relative humidities were between 50 and 80%. The higher the temperature and relative humidity of the drying air, the lower the amount of polyphenol residue in the cocoa beans, because of enzymatic oxidation of polyphenols. At higher drying temperatures, non‐enzymatic oxidation of polyphenols could also occur. Computer simulation results gave rate constants for the polyphenol oxidation reaction (k1) and the condensation reaction (k2), at various air conditions, in the range of 0.055–0.200 and 0.136–0.155 h−1 respectively. The activation energies obtained for the polyphenol oxidation reaction were in the range of 27 800–30 312 J K−1 mol−1. The reaction kinetics of the enzymatic browning reaction fitted a pseudo first‐order reaction.

Browning development in white wines: dependence on compositional parameters and impact on antioxidant characteristics

Browning is a serious problem in relation to white wine quality and mostly affects the sensory attributes, whereas its impact on the dietary value of wines has never been investigated. Browning, however, is well known to be associated with polyphenol oxidation, and therefore it may be accompanied by changes in the antioxidant capacity. On the basis of this consideration, experimental white wines vinified and stored under identical conditions were subjected to accelerated browning, and efforts were made to distinguish compositional parameters that might be related to the extent of browning development. Further, the consequences of browning on the reducing power and antiradical activity were assessed. It was shown that none of the parameters examined (titratable acidity, pH, total SO2, total polyphenols and total flavanols) play an important role in browning onset, but SO2 appeared to exert a statistically significant influence (r2=0.6394, P<0.05) on the percentage change in the antiradical activity. Evidence also suggested that browning development might be prominently associated with decreases in both antiradical activity (r2=0.7891, P<0.01) and reducing power (r2=0.6925, P<0.05).

Maillard reaction and enzymatic browning affect the allergenicity of Pru av 1, the major allergen from cherry (Prunus avium).

The influence of thermal processing and nonenymatic as well as polyphenoloxidase-catalyzed browning reaction on the allergenicity of the major cherry allergen Pru av 1 was investigated. After thermal treatment of the recombinant protein rPru av 1 in the absence or presence of carbohydrates, SDS-PAGE, enzyme allergosorbent tests, and inhibition assays revealed that thermal treatment of rPru av 1 alone did not show any influence on the IgE-binding activity of the protein at least for 30 min, thus correlating well with the refolding of the allergen in buffer solution as demonstrated by CD spectroscopic experiments. Incubation of the protein with starch and maltose also showed no effect on IgE-binding activity, whereas reaction with glucose and ribose and, even more pronounced, with the carbohydrate breakdown products glyceraldehyde and glyoxal induced a strong decrease of the IgE-binding capacity of rPru av 1. In the second part of the study, the effect of polyphenoloxidase-catalyzed oxidation of polyphenols on food allergen activity was investigated. Incubation of rPru av 1 with epicatechin in the presence of tyrosinase led to a drastic decrease in IgE-binding activity of the protein. Variations of the phenolic compound revealed caffeic acid and epicatechin as the most active inhibitors of the IgE-binding activity of rPru av 1, followed by catechin and gallic acid, and, finally, by quercetin and rutin, showing significantly lower activity. On the basis of these data, reactive intermediates formed during thermal carbohydrate degradation as well as during enzymatic polyphenol oxidation are suggested as the active chemical species responsible for modifying nucleophilic amino acid side chains of proteins, thus inducing an irreversible change in the tertiary structure of the protein and resulting in a loss of conformational epitopes of the allergen.

Estimation of leaf transmittance in the near infrared region through reflectance measurements

A method, which allows one to compensate for the incomplete collection of transmitted light (T) by an integrating sphere, has recently been developed, and shown to be reliable provided that the absorptance (A) of the leaf in the NIR region (750–800 nm) can be neglected, allowing one to set R+T=1, where R denotes the reflectance; this implies that proper compensation can only be applied to healthy leaves, which do not absorb in the NIR region. To overcome this limitation, the feasibility of an alternative, requiring neither measurements of T nor an elaborate analysis of radiative transport through a leaf, is explored. Not surprisingly, this simplistic alternative provides results which (in general) do not agree with those found by using the compensation method, but the two approaches converge in the spectral regions where absorptance is low (that is, where R+T⩾0.9). The “T-through-R” method, as described here, thus provides an additional check on the correction factor used in conjunction with the integrating sphere, and extends the applicability of the compensation method to situations where NIR absorptance is not negligible, e.g., in the presence of `browning' pigments produced upon the oxidation of polyphenols during leaf senescence.

Estimation of leaf transmittance in the near infrared region through reflectance measurements

A method, which allows one to compensate for the incomplete collection of transmitted light ( T) by an integrating sphere, has recently been developed, and shown to be reliable provided that the absorptance ( A) of the leaf in the NIR region (750–800 nm) can be neglected, allowing one to set R+ T=1, where R denotes the reflectance; this implies that proper compensation can only be applied to healthy leaves, which do not absorb in the NIR region. To overcome this limitation, the feasibility of an alternative, requiring neither measurements of T nor an elaborate analysis of radiative transport through a leaf, is explored. Not surprisingly, this simplistic alternative provides results which (in general) do not agree with those found by using the compensation method, but the two approaches converge in the spectral regions where absorptance is low (that is, where R+ T⩾0.9). The “ T-through- R” method, as described here, thus provides an additional check on the correction factor used in conjunction with the integrating sphere, and extends the applicability of the compensation method to situations where NIR absorptance is not negligible, e.g., in the presence of `browning' pigments produced upon the oxidation of polyphenols during leaf senescence.

Reaction of iron(III) with theaflavin: complexation and oxidative products

Theaflavins are a family of compounds, whose chemistry has been sparsely investigated. They can comprise up to 40% the dry weight of black tea. They are known to chelate metals, however very little knowledge exists on the mechanisms involved. There is some correlation between both of these areas in that following degradation of the iron theaflavin complex, subsequent redox reactions may lead to the formation of similar products on both occasions. The interaction of iron(III) with theaflavin at pH <3.0 is investigated by means of liquid chromatography mass spectroscopy (LC-MS), stopped flow spectroscopy and multivariate data analysis. Iron theaflavin complexes are formed which subsequently decay to form a number of oxidative species. The difficulties involved in the elucidation of the structure of polymeric phenolic compounds from black tea has been highlighted by numerous authors. The intermediates and major low molecular weight oxidised theaflavin products from the reaction of excess iron with theaflavin have been detected and identified using multivariate data analysis of diode array spectroscopic data. It is not possible to characterise the extremely polar high molecular weight oxidation products obtained from polyphenol oxidation. High performance liquid chromatography (HPLC) and electrospray mass spectroscopy (ES-MS) detected the low molecular weight oxidised theaflavin species present in the system. Enzymatic oxidation of theaflavin using peroxidase (POD) resulted in the formation of one major low molecular weight species oxidative product, which was fully characterised using nuclear magnetic resonance spectroscopy (NMR), high performance liquid chromatography (HPLC), electrospray mass spectroscopy (ES-MS), UV–visible (UV–Vis) and Fourier transform infra-red spectroscopy (FT-IR). The major objective of this work is to investigate the reaction of iron(III) with theaflavin and to add some insight into the mechanistic interaction of iron(III) with this family of compounds.

機能性ポリフェノールの合成と酸化反応

機能性ポリフェノールの合成と酸化反応

Inhibition of apple polyphenol oxidase activity by procyanidins and polyphenol oxidation products.

The rate of consumption of dissolved oxygen by apple polyphenol oxidase in cider apple juices did not correlate with polyphenol oxidase activity in the fruits and decreased faster than could be explained by the decrease of its polyphenolic substrates. The kinetics parameters of a crude polyphenol oxidase extract, prepared from apple (Braeburn cultivar), were determined using caffeoylquinic acid as a substrate. Three apple procyanidin fractions of n 80, 10.5, and 4 were purified from the parenchyma of cider apples of various cultivars. Procyanidins, caffeoylquinic acid, (-)-epicatechin, and a mixture of caffeoylquinic acid and (-)-epicatechin were oxidized by reaction with caffeoylquinic acid o-quinone in order to form oxidation products. All the fractions were evaluated for their inhibitory effect on PPO activity. Native procyanidins inhibited polyphenol oxidase activity, the inhibition intensity increasing with n. The polyphenol oxidase activity decreased by 50% for 0.026 g/L of the fraction of n 80, 0.17 g/L of the fraction of n 10.5, and 1 g/L of the fraction of n 4. The inhibitory effect of oxidized procyanidins was twice that of native procyanidins. Oxidation products of caffeoylquinic acid and (-)-epicatechin also inhibited polyphenol oxidase.

A New Laccase Biosensor For Polyphenols Determination

The relevance of polyphenols in human health is a well known fact. Prompted by that, a very intensive research has been directed to get a method to detect them, wich will improve the current ones. Laccase (p-diphenol:dioxygen oxidoreductase EC 1.10.3.2) is a multi-copper oxidase, wich couples catalytic oxidation of phenolic substrates with four electron reduction of dioxygen to water [1]. A maximum catalytic response in oxigenated electrolyte was observed between 4.5 and 5.5 [2], while for pH &gt; 6.9 the laccase was found to be inactive [3]. We prepared a biosensor with laccase immobilised on a polyether sulphone membrane, at pH 4.5, wich was applied at Universal Sensors base electrode. Reduction of the product of oxidation of several polyphenols, catalysed by laccase, was done at a potential for wich the polyphenol of interest was found to respond. Reduction of catechol was found to occur at a potential of -200mV, wich is often referred to in the literature for polyphenolic biosensors. However other polyphenols did not respond at that potential. It was observed that (+)- catechin produced a very large cathodic current when +100mV were applied to the laccase biosensor, both in aqueous acetate and 12% ethanol acetate buffer, whereas caffeic acid responded at -50mV. Other polyphenols tested were gallic acid, malvidin, quercetin, rutin, trans-resveratrol

A PROPOSED MECHANISM BEHIND THE DEVELOPMENT OF INTERNAL BROWNING IN PEARS (PYRUS COMMUNIS CV CONFERENCE)

Storage of pears under low oxygen levels (0.5-1.0 kPa) leads to decreased ascorbic acid and ATP levels, a lower ATP-production, and to internal browning, a storage disorder in pears. Addition of 5 kPa carbon dioxide to the storage atmosphere increased the severity of this disorder. Experiments showed that anoxia can result in off-flavours, but not in internal browning. Internal browning is caused by brown pigments (melanins), which are formed due to oxidation of vacuolar polyphenols under the influence of tyrosinase (EC 1.14.18.1). We hypothesise that internal browning is initiated by a combination of oxygen radical action and a lack of maintenance energy for, amongst others, the regeneration of antioxidants. The two factors together lead to decompartmentation, bringing tyrosinase from the plastids and substrates from the vacuole together.

Laboratory-Scale Studies of the Impact of Oxygen on Mashing

An assessment of the impact of oxygen and hydrogen peroxide on mashing and wort parameters has been made on a laboratory scale. Oxygen has been stridently eliminated by using an anaerobic chamber during mash analysis. Additionally the relative importance of proanthocyanidin species has been assessed by comparing the behaviour of conventional malt and a malt produced from a low proanthocyanidin variety. It seems that oxygen and peroxide act independently in causing the oxidation of thiol-containing materials and polyphenols in mashes and that oxygen is not primarily exerting its impacts through the intermediacy of peroxide. The removal of thiols (presumably at least in part through the production of disulphide bridges between proteins) and of polyphenols (presumably via polymerisation) both contribute to increased wort turbidity and decreased rates of wort separation after mashing. Three inhibitors (nordihydroguaiaretic acid, ethylenediamenetetraacetate and potassium cyanide) have been employed in an attempt to differentiate between enzymic and non-enzymic events and also to identify whether lipoxygenase and peroxidase are catalysing key events. Whilst it seems that peroxidase has a key role in catalysing the oxidation of polyphenols by H 2 O 2 , it does not appear that either peroxidase or lipoxygenase is involved in the removal of measurable thiol. Nonetheless a significant proportion of the thiol elimination is likely enzyme-catalysed. We have been unable to demonstrate the production of hydroperoxides in mashes, but added hydroperoxide is undetectable, which suggests that these materials are either lost by onward conversion or by adsorption onto spent grains.

Multiple forms of phenol oxidase from Kolkhida tea leaves ( Camelia Sinensis L.) and Mycelia Sterilia IBR 35219/2 and their role in tea production

Molecular weights, (MW) pH optimum and substrate specificity of multiple forms of phenol oxidases from Kolkhida tea leaves ( Camelia sinensis L.) and microscopic fungus Mycelia sterilia IBR 35219/2 have been studied. It has been shown that Kolkhida tea leaves consist of phenol oxidases with MW 28,000, 41,000, 58,000, 118,000 and 250,000. Duration of M. sterilia IBR 35219/2 cultivation affects formation and activities of multiple forms of phenol oxidases (MW 240,000, 93,000, 58,000 and 41,000) in the culture filtrate. Maximum activity of phenol oxidases is revealed at 36-h duration period of cultivation. By this time Phenol oxidase, mainly, with MW 240,000 is produced. Both Kolkhida tea leaves phenol oxidase with MW 250,000 and M. sterilia IBR 35219/2 phenol oxidase with MW 240,000 are catechol oxidases with similar substrate specificities. Multiple forms of phenol oxidase from tea leaf with MW 118,000±3000 and 58,000±2000 do not reveal hydroxylase activity but they intensively catalyze oxidation of o-diphenols. High molecular weight forms of phenol oxidase of both Kolkhida tea leaves (MW 250,000, 118 00 and 58,000) and M. sterilia IBR 35219/2 (MW 240,000) catalyze the oxidation of catechins, mainly (−)epigallocatecin and (−)epigallocatechin gallate. Theaflavins and thearubugins produced at this time regulate phenol oxidase activity. High concentration of these products completely inhibits activity of phenol oxidase. Phenol oxidases of low molecular weights (MW 28,000 and 41,000) from tea leaves catalyze hydroxylation of monophenols, namely, p-coumaric acid, producing o-diphenols. Phenol oxidase of microscopic fungus M. sterilia IBR 35219/2 with MW 240,000 catalyzes oxidation of green tea extract polyphenols.

Higher radical scavenging activities of polyphenolic antioxidants can be ascribed to chemical reactions following their oxidation

Radical scavenging activities of 34 natural antioxidants were investigated from an electrochemical viewpoint. While the correlation of the oxidation potentials with their DPPH radical scavenging activities (represented by EC 50) was not high (the correlation coefficient, r=0.73), the number of electrons n required for oxidation of an antioxidant, being obtained by continuous flow-column electrolysis with a slower flow rate (0.05 ml min −1), did show a good correlation with EC 50 (1/EC 50=1.67 n+0.50 with r=0.94). The n values of most polyphenols were increased with a decrease in the flow rate, while those of nonpolyphenols were invariant. This suggests that a slower subsequent chemical reaction(s) should be involved in the oxidation of polyphenols, whose higher radical scavenging activities seem to be ascribed to the chemical reactions. In this study, we have proposed a possible mechanism for the oxidation of polyphenols, in which the oxidizable -OH moieties are reproduced through an oxidative dimerization (or more highly polymerization).

CFD simulation of heat transfer and polyphenol oxidation during tea fermentation

This paper describes a computational fluid dynamics (CFD) model developed to simulate airflow, heat transfer and enzymatic oxidation of polyphenols during black tea fermentation. The airflow through the packed bed of macerated tealeaves is modelled by the porous-medium flow equation. Spatial distribution of temperature within the packed bed of tealeaf is obtained by solving the heat transfer equation that incorporates the source heat of enzymatic oxidation and convective heat of airflow. Spatial distributions of polyphenolic compounds involved in enzymatic oxidation of tea fermentation are then predicted using species equations. The rates of depletion of reactants and formation of products of polyphenols are modelled by source/sink terms of the species equations with the rates of reactions related to the temperature. Solutions of the species equations then provide the basis for the calculation of source heat generated by the oxidative reactions. Some preliminary results are presented. The aim is to demonstrate that the CFD model provides a valuable tool to examine the effect of process conditions on the complex reaction kinetics and pathways that are responsible for the formation of polyphenolic compounds during tea fermentation.

Oxidation of polyphenols in unfermented and partly fermented cocoa beans by cocoa polyphenol oxidase and tyrosinase

AbstractUnfermented and partly fermented dried cocoa beans have an excessively astringent and bitter flavour owing to their high polyphenol content. Studies on the remaining polyphenol oxidase activity and the oxidation of polyphenols in these beans have been conducted in relation to two factors, ie incubation time (0, 1, 2, 4, 8 and 16 h) and pH of incubation (3.5, 4.5, 5.5 and 6.5). Owing to unsuccessful polyphenol oxidation during incubation of partly fermented beans, incubation–enrichment treatments were carried out by combining incubation time (0, 8, 16, 24 and 32 h) and addition of crude cocoa polyphenol oxidase and purified tyrosinase from mushroom at 88 and 8800 units g−1 and pH 5.5. Results showed that the remaining polyphenol oxidase activities of unfermented and partly fermented dried beans were 1 and 0.08% with specific activities of 9 and 1% respectively. The polyphenols of unfermented beans were effectively oxidised by incubation at 45 °C and pH 3.5–6.5 without enzyme enrichment; while those of partly fermented beans required enzyme enrichment. Both crude cocoa polyphenol oxidase and tyrosinase could be used for enzyme enrichment, but tyrosinase seemed to be more effective.© 2002 Society of Chemical Industry

Oxidation of Polyphenols and the Effect on In vitro Iron Accessibility in a Model Food System

ABSTRACT: Tannic acid and green tea extract were added to a model food system of wheat bread, before and after incubation with polyphenol oxidase (mushroom tyrosinase), and the effect on In vitro iron accessibility was studied. Tannic acid and green tea extract had a marked inhibitory effect on the In vitro accessibility of iron when added to the model food system. Incubation of the polyphenols with tyrosinase before addition to the model food system significantly increased the accessibility of iron. The results from the study therefore suggest that oxidation of polyphenols may be a promising way to increase the bioavailability of iron in polyphenol containing foods.

Allocation of extracellular enzymatic activity in relation to litter composition, N deposition, and mass loss

Decomposition of plant material is a complex process that requiresinteraction among a diversity of microorganisms whose presence and activity issubject to regulation by a wide range of environmental factors. Analysis ofextracellular enzyme activity (EEA) provides a way to relate the functionalorganization of microdecomposer communities to environmental variables. In thisstudy, we examined EEA in relation to litter composition and nitrogendeposition. Mesh bags containing senescent leaves of Quercusborealis (red oak), Acer rubrum (red maple) andCornus florida (flowering dogwood) were placed on forestfloor plots in southeastern New York. One-third of the plots were sprayedmonthly with distilled water. The other plots were sprayed monthly withNH4NO3 solution at dose rates equivalent to 2 or 8 g N m−2 y−1. Mass loss, litter composition, fungal mass, and the activities ofeight enzymes were measured on 13 dates for each litter type. Dogwood wasfollowed for one year, maple for two, oak for three. For each litter type andtreatment, enzymatic turnover activities were calculated from regressions of LN(%mass remaining) vs. cumulative activity. The decomposition of dogwood litterwas more efficient than that of maple and oak. Maple litter had the lowestfungal mass and required the most enzymatic work to decompose, even though itsmass loss rate was twice that of oak. Across litter types, N amendment reducedapparent enzymatic efficiencies and shifted EEA away from N acquisition andtoward P acquisition, and away from polyphenol oxidation and towardpolysaccharide hydrolysis. The effect of these shifts on decomposition ratevaried with litter composition: dogwood was stimulated, oak was inhibited andmaple showed mixed effects. The results show that relatively small shifts intheactivity of one or two critical enzymes can significantly alter decompositionrates.

Oxidation of polyphenols in phytate-reduced high-tannin cereals: effect on different phenolic groups and on in vitro accessible iron.

After reduction of phytate with phytase, water slurries of two high-tannin cereal flours were incubated with polyphenol oxidase (mushroom tyrosinase), and the effects on different phenolic groups and on in vitro accessible iron were studied. Enzyme incubation was also performed after cooking, soaking, and germination of the cereals. Phytase incubation significantly decreased the phytate content, and incubation with polyphenol oxidase had a reducing effect on the total phenol content, as well as on the amount of catechol and resorcinol groups. The in vitro accessible iron increased when the cereals were incubated with phytase and polyphenol oxidase, and the highest accessibility of iron was obtained when the germinated samples were incubated. The results from this study imply that oxidation of polyphenols in high-tannin cereals, after reduction of phytate, may be used to increase the bioavailability of iron in foods prepared from these cereals.

Isolation and characterization of melanic pigments derived from tea and tea polyphenols

The dark brown pigments derived from tea and tea polyphenols were studied. Physical and chemical properties revealed that pigments directly extracted from tea leaves and derived from tea polyphenols were similar to typical melanins. Further investigation showed that both melanic pigments possessed similar antioxidant capability, due to their chelating and scavenging properties. The inhibitory effect of melanic pigments, either from tea or tea polyphenols, was significantly stronger than that of non-treated tea polyphenols. According to these properties, we have extracted melanin from tea. In addition, oxidation of tea polyphenols also provided an alternative method to maximize the yields. The extracted melanin is an antioxidant, which interrupted free radical reactions at a step in the development chain by its scavenging properties and, at the step of initiation, by its ability to chelate metals.