Studies on the enzymic browning of apples. Inhibition of apple 0-diphenol oxidase by phenolic acids

The nature of the inhibition of a particulate preparation of apple o-diphenol oxidase by substituted cinnamic acids has been investigated for three substrates: 4-methylcatechol, chlorogenic acid and catechin. Solubilisation of the enzyme preparation altered the pattern of inhibitor/substrate relationships, and these results are discussed on the basis of the occurrence of separate catalytic and inhibitor sites on the enzyme.     

Purification and properties of polyphenol oxidase in head lettuce (Lactuca sativa)

Polyphenol oxidase (EC 1.10.3.1) in head lettuce (Lactuca sativa L) was purified by ammonium sulphate fractionation, ion exchange chromatography and gel filtration. The enzyme was found to be homogeneous by polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be about 56 000 amu by Sephadex G-100 gel filtration. The purified enzyme quickly oxidised chlorogenic acid (5-caffeoyl quinic acid) and (—)-epicatechin. The K^m values for the enzyme, using chlorogenic acid (pH 4·5, 30°C) and (—)-epicatechin (pH 7·0, 30°C) as substrate, were 0·67 mM and 0·91 mM, respectively. The optimal pH of chlorogenic acid oxidase and (—)-epicatechin oxidase activities were 4·5 and 7·8, respectively, and both activities were stable in the pH range 6–8 at 5°C for 20 h. Potassium cyanide and sodium diethyldithiocarbamate markedly inhibited both activities of the purified enzyme. The inhibitory effect of metallic ions such as Ca²⁺, Mn²⁺, Co²⁺ and Ni²⁺ for chlorogenic acid oxidase activity was stronger than that for (—)-epicatechin oxidase activity.     

Selected Properties of Polyphenol Oxidase Obtained from Ispir Sugar Bean

Polyphenol oxidase enzyme was isolated from Ispir sugar bean by ammonium sulphate precipitation and its biochemical properties were investigated. For this purpose, K_M and V_max values for optimum conditions of pH, temperature, and ionic strength were determined for catechol, catechin, and chlorogenic acid as substrates. Enzyme activities were measured spectrophotometrically at 420 nm using the same substrates at optimum conditions. K_M values were found to be 2.4875, 1.3154, and 2.2487 M for catechol, catechin, and chlorogenic acid, respectively. V_max values were 3.1480, 0.6130, and 0.5039 EU/ml.min for the same substrates, respectively. These results indicated that catechol was used as a subsrate for inhibition studies. For catechol substrate, dithiothreitol, glutathione, thiourea, and L-cysteine chlorid were inhibitors. For these inhibitors, K_i constants were calculated from Lineweaver-Burk plots and inhibiton types were estimated. Moreover, I₅₀ values were also determined. The most effective inhibitor was found to be glutathione.     

Some properties of polyphenol oxidase from lily

A study of crude polyphenol oxidase (PPO) from lily bulbs was carried out to provide information useful for guiding food processing operations. Optimum pH for the enzyme activity in the presence of catechol, were 4.0 and 7.0 at room temperature(approximately 20 °C) and the enzyme was stable in the pH range from 5.0 to 6.5 at 4 °C for 10 h. Its optimum temperature was 40 °C and the heat inactivation of the enzyme followed first‐order kinetics. Lily PPO possessed a diphenolase activity toward catechol, catechin and gallic acid; catechin was the best substrate for the enzyme considering the V_max/K_m ratio. The most effective enzyme inhibitor was sodium sulphite, although ascorbic acid, l ‐cysteine and thiourea were also effective inhibitors at high concentration. But NaCl and citric acid were poor inhibitors of the enzyme. Data generated by this study might help to better prevent lily bulbs browning.     

Anti-inflammatory effects of 4 medicinal plant extracts in lipopolysaccharide-induced RAW 264.7 cells

The anti-inflammatory activity of 4 plant extracts [guava (Psidium guajava) leaf, capillary wormwood (Artemisia capillaris Thunb.), Chinese goldthread (Coptis chinensis), and dandelion (Taraxacum platycarpum)] was investigated in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Six phenolic compounds (gallic acid, caffeic acid, chlorogenic acid, catechin, quercetin, and baicalin) were analyzed using LC-MS/MS. Guava leaf extracts showed the highest inhibitory effects on LPS-induced nitric oxide (NO, 52.58%) and prostaglandin E₂ (PGE₂, 43.45%) production. The total phenolic contents (TPC) in guava leaf, capillary wormwood, Chinese goldthread, and dandelion were 426.84, 154.42, 41.73, and 122.04 mg of gallic acid equivalent (GAE)/g of extract, respectively. TPC was positively correlated with the NO-inhibitory effect (r= 0.963, p<0.05) and the PGE₂-inhibitory effect (r=0.971, p<0.05) at 30 μg/mL of treatment. The guava leaf extracts contained the highest levels of gallic acid and catechin, while the capillary wormwood extracts contained the highest levels of chlorogenic acid and quercetin.

     

Effect of various inhibitors on enzymatic browning,antioxidant activity and total phenol content of fresh lettuce (Lactuca sativa)

In this study, polyphenol oxidase (PPO) was isolated from fresh lettuce. Its optimum temperature and pH were found to be 40 °C and 7.0, respectively. Lettuce PPO was shown to use catechin, catechol, chlorogenic acid, caffeic acid and gallic acid as substrates. Among the substrates used, the greatest substrate specificity was observed with chlorogenic acid. Lettuce PPO was sensitive to some inhibitors. Ascorbic acid, cysteine, oxalic acid and citric acid were tested as potential inhibitors of lettuce PPO. Cysteine was the most effective inhibitor. Total phenol and total antioxidant activity contents were also determined in the presence of these inhibitors at room and refrigerator temperatures. Ascorbic acid and cysteine increased the total antioxidant activity of lettuce while citric and oxalic acids had no effect on the total antioxidant activity. Lettuce phenolics were protected from oxidation by ascorbic acid and cysteine.     

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

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

Effect of whey protein concentrate on phenolic profile and browning of fresh-cut lettuce (Lactuca Sativa)

The in vitro kinetics of lettuce PPO with respect to dissolved oxygen using catechin, chlorogenic acid, caffeic acid and gallic acid has been examined. In-vitro lettuce polyphenol oxidase (PPO) activity was determined by measuring the consumption of oxygen during the oxidation reaction. The effect of whey protein concentrate (WPC) was tested on the inhibition of lettuce PPO comparing with ascorbic acid (AA) and cysteine. A competitive model that considered inhibitors was the most appropriate model to explain reaction kinetics. Browning of lettuce was also monitored during storage for 24 h. Addition of WPC prevented loss of lightness in lettuce. Loss of identified phenolic compounds in lettuce was measured during the enzymatic browning process by high-performance liquid chromatography. Degradation of identified phenolic compounds followed first order kinetics during storage. Combination of WPC with cysteine was proposed for the protection of phenolics compounds against PPO-catalysed oxidation.     

Mechanism of Browning in Fresh Highbush Blueberry Fruit (Vaccinium corymbosum L). Partial Purification and Characterisation of Blueberry Polyphenol Oxidase

The most efficient method for polyphenol oxidase (PPO) extraction from Highbush blueberry fruits was the preparation of an acetone powder. No activity was detected after direct extraction with phosphate buffer (pH 6·5) and detergents such as Triton X-100. PPO has been purified 19-fold, by ultrafiltration, ammonium sulphate precipitation and hydrophobic chromatography. Native-PAGE of the purified fraction revealed the presence of two isoforms. PPO has an observed optimum pH at 4·0, followed by a shoulder at pH 5·0. Caffeic acid is the best substrate (100%), followed by chlorogenic acid (60%) and pyrocatechol (32·5%). No activity was detected towards catechin, protocatechuic acid, resorcinol and monophenols. © 1997 SCI.     

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

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

Optimization of enzymatic oligomerization reaction conditions for three milk protein products via ceteris-paribus approach

Via a ceteris-paribus approach, optimum reaction conditions for an oxidoreductase- (lactoperoxidase; laccase; glucose oxidase) induced oligomerization of milk proteins were assessed for three different milk protein products (sodium caseinate; whey protein isolate; skim milk powder).Optimum protein monomer modification conditions were enzyme- and substrate-specifically identified, yielding protein monomer modification levels of, e.g., 58% in case of lactoperoxidase-induced (w/w = 5% protein; 1.8 μmol hydrogen peroxide/mg protein; 4.8 U lactoperoxidase/mg protein; 50 °C; 1 h; pH 7.0), 92% in case of laccase-induced (w/w = 5% protein; 0.02 μmol chlorogenic acid/mg protein; 0.01 U laccase/mg protein; 40 °C; 1 h; pH 7.0) and 86% in case of glucose oxidase-induced (w/w = 1% protein; 0.5 U glucose oxidase/mg protein; 40 °C; 16 h; pH 7.0) modification of total milk proteins from skim milk powder.The study for the first time provides a comprehensive overview over reaction conditions facilitating high degrees of milk protein monomer modification upon oxidoreductase-induced oligomerization in regard to food protein tailoring via application of less substrate- and reaction-specific enzymes than transglutaminase.     

Phenolic compounds and fatty acid composition of organic and conventional grown pecan kernels

BACKGROUND: In this study, differences in contents of phenolic compounds and fatty acids in pecan kernels of organically versus conventionally grown pecan cultivars (Cheyenne, Desirable, and Wichita) were evaluated. RESULTS: Although nine phenolic compounds (gallic acid, catechol, catechin, epicatechin, m‐coumaric acid, chlorogenic acid, ellagic acid, caffeic acid and an ellagic acid derivative) were identified in the methanol extract (80% methanol) of defatted kernels, only three compounds (gallic acid, catechin and ellagic acid) existed in sufficient amounts to accurately quantify levels in different cultivars and to study differences in organic versus conventional cultivation. Levels of ellagic acid and catechin found in organically grown ‘Desirable’ were fourfold and twofold higher than in conventional samples, respectively. Furthermore, significant differences in these two compounds were also observed when comparing values between cultivars. Oil content was also significantly greater only in organically grown ‘Desirable’. Oleic acid was the major fatty acid present and its content was significantly higher in organically versus conventionally grown ‘Desirable’ pecans, while there was no difference in levels of oleic acid in ‘Wichita’ and ‘Cheyenne’. On the other hand, linoleic acid content was significantly less in organically versus conventionally grown ‘Desirable’ pecans. CONCLUSION: Overall, these results showed that the effects of cultural differences (i.e. organic versus conventional cultivation) on kernel composition largely depend on the type of pecan cultivar. Copyright © 2009 Society of Chemical Industry     

Phenolic content and antioxidant capacity versus consumer acceptance of soaked and vacuum impregnated frozen nectarines

Nectarines (Prunus persica L. cv. Maria Laura) were manually selected, cut in slices and divided into four groups: fresh, untreated frozen, soaked in osmotic solution and subsequently frozen, and vacuum impregnated (VI) and subsequently frozen. This investigation was focused on evaluation of consumer acceptance with respect to treated versus untreated frozen nectarine slices. In a preliminary acceptance test of untreated frozen nectarine slices, fruits were generally rejected on the basis of a darkened appearance and “oxidized” taste. These negative attributes were probably linked to the activity of polyphenol oxidase (PPO) and depletion of phenols due to cell rupture during freeze–thaw procedures. For these reasons, in order to evaluate the tendency of fruit to oxidation, several analyses were performed: the antioxidant capacity of phenolic fraction and the o-diphenol content were estimated by spectrophotometric assays, whereas the hydroxycinnamic acid (chlorogenic and neochlorogenic acids) composition was evaluated by high performance liquid chromatography (HPLC). Phenolic content and antioxidant capacity were found to correlate well with the acceptance level of frozen nectarine slices. In this regard a higher phenolic content associated with a higher acceptance level of nectarine samples.     

Application of induced voltage in cloudy apple juice: enzymatic browning and bioactive and flavouring compounds

Controlling enzymatic browning and decreasing the loss of phenolic and flavour compounds by emerging technologies have drawn interest. In this study, magneto-induced voltages (900–1800 V, 20-45 kHz) with mild temperature (20–50 °C) were applied to inactivate polyphenol oxidase (PPO) and peroxidase (POD) in cloudy apple juice. Results suggested that PPO and POD activities could be reduced by 98.5% and 99.4%, respectively, following the condition: induced voltage 1800 V; frequency 45 kHz; temperature 50 °C and duration 3 min. And, phenolic compounds included gallic acid, catechin, chlorogenic acid, p-hydroxybenzoic acid, caffeic acid, rutin and p-coumaric acid, as well as major aroma compounds consisted of 22 volatile constituents which classified into three categories: esters, alcohols and aldehydes were also investigated. It suggested the phenolic compounds and major aroma compounds remained unchanged after the electrical treatment. This study suggests that the induced voltage produced by electromagnetic induction is an alternative to conventional heating methods.     

Comparison of phytochemical profiles,antioxidant and cellular antioxidant activities of seven cultivars of Aloe

A comparative assessment of the phytochemical profiles and antioxidant activities of seven cultivars of Aloe was conducted to evaluate the potential health benefits of Aloe. Aloe arborescens contained the highest levels of phenolic content, total antioxidant capacity by the oxygen radical scavenging capacity assay and cellular antioxidant activity assay. Aloe vera showed the highest levels of flavonoid content and antioxidant capacity by the peroxyl radical scavenging capacity assay. Aloe greenii had the highest CAA value with a PBS wash before adding ABAP. There were no significant differences observed between Aloe arborescens and Aloe greenii. Aloin, aloe‐emodin‐8‐O‐beta‐D‐glucopyranoside, catechin, epicatechin, sinapic acid and chlorogenic acid were identified in Aloe samples by the HPLC analysis. Aloin, aloe‐emodin‐8‐O‐beta‐D‐glucopyranoside and catechin showed strong relationships with antioxidant activity. Significant levels of aloin, aloe‐emodin‐8‐O‐beta‐D‐glucopyranoside and catechin were determined in Aloe greenii, Aloe vera and Aloe saponaria, respectively.     

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.     

Chemical Composition and Bioactivities of Two Common Chaenomeles Fruits in China: Chaenomeles speciosa and Chaenomeles sinensis

Contents of total flavonoids, total phenolics, total triterpenes, total condensed tannin and total saponins in peels, flesh and endocarps of Chaenomeles speciosa (CSP) and Chaenomeles sinensis (CSS) were determined by colorimetric method, while 5 phenolics (vanillic, gallic, chlorogenic, ferulic and p‐coumaric acids), 2 triterpenes (oleanolic and ursolic acids), and 3 flavonoids (rutin, catechin and epicatechin) were identified and quantified by high‐performance liquid chromatography‐mass spectrometry (HPLC‐MS) and HPLC, and antioxidant and α‐glucosidase inhibitory activities of them also were evaluated as well as their digestive characteristics. In the correlation analysis, total phenolics, vanillic acid, catechin, ursolic acid and oleanolic acid all contribute to DPPH^· scavenge capacity, gallic acid contributes to total ferric reducing antioxidant power, while total triterpenes, total saponins, chlorogenic acid and ferullic acid contribute to α‐glucosidase inhibitory activity. In the principal component analysis, endocarps of CSP and CSS both show better quality than their peels and flesh, respectively. In vitro digestion can increase contents of total flavonoids, total condensed tannin and total saponins, while contents of total phenolics and total triterpenes decreased greatly. Our study would contribute to the full use of discarded parts of the 2 Chaenomeles and be helpful to establish a good foundation for further research of CSP and CSS.     

Assembly of amine oxidase and D-amino acid oxidase in the cytosol of peroxisome-deficient mutants of the yeast Hansenula polymorpha during growth of cells on glucose in the presence of primary amines or D-alanine as the sole nitrogen source

We have studied growth of two peroxisome-deficient mutant strains of Hansenula polymorpha on glucose in the presence of different organic nitrogen sources (methylamine, ethylamine and D -alanine), the metabolism of which is mediated by peroxisome-borne oxidases in wild-type (WT) cells. Both strains grew well on each of these substrates with growth rates comparable to WT cells. Growth on both methylamine and ethylamine was associated with enhanced levels of catalase and amine oxidase in the cells; in D -alanine-grown cells D -amino acid oxidase activity and increased. In WT cells of H-polymorpha the activities of these enzymes were confined to the peroxisomal matrix; however, in both peroxisome-deficient strains their activities were localized in the cytosol. Electron microscopy indicated that, dependent on the stage of growth, the enzymes may form large protein aggregates. The molecular masses of both amine oxidase and D -amino acid oxidase in the mutant strains were identical to their respective counterparts in WT cells, indicating that both proteins were correctly assembled and active in the cytosol.     

Inhibition by carboxylic acids of an o-diphenol oxidase from Prunus avium fruits

Aromatic carboxylic acids strongly inhibited 4-methylcatechol oxidation by an o-diphenol oxidase extracted from sweet cherry fruits (Prunus avium). Esterification of the acids decreased their inhibitory strength. Inhibitors containing the benzene nucleus showed a greater effectiveness than the corresponding aliphatic and heterocyclic compounds (except 2-naphthalenecarboxylic acid). Similar inhibitory effects were observed by replacing the benzene ring with a highly unsaturated open chain. The inhibitory properties of benzoic acid, cinnamic acid, p-coumaric acid and 3,4-dihydroxybenzoic acid were investigated. It is proposed that the catalytic and inhibitory sites are close together in the enzyme molecule.     

Polyphenol oxidase from yali pear (Pyrus bretschneideri)

Polyphenol oxidase (PPO) was isolated from Yali pear (Pyrus bretschneideri R). At the end of purification by ion exchange chromatography on DEAE-cellulose, 10.8-fold purification was achieved. The enzyme showed activity to catechol, pyrogallol, chlorogenic acid and DL-DOPA; of these four, chlorogenic acid was the best substrate. The optimum pH for the PPO was 7.0. PPO activity was not destroyed by heating to 30° for 30 min. The effects of various compounds as inhibitors of the reaction catalysed by the enzyme were tested.