O-dihydroxyphenoloxidase action on natural polyhydric phenolics and enzymic browning of edible yams

Ten cultivars of five edible yam species were examined for o-dihydroxyphenoloxidase (o-DPOase) activity against yam extracts containing phenolic compounds, and for their propensity to brown when cut slices are exposed to air. (+)-Catechin levels in Dioscorea cayenensis (260 mg kg^?1) and D. bulbifera (240 mg kg^?1) were similar to those in D. dumetorum (270 mg kg^?1), but the latter contained less o-DPOase and showed less tendency to browning. D. alata contained more (+)-catechin than other yams, and one cultivar with a higher (+)-catechin content (660 mg kg^?1) showed more browning than another cultivar containing 430 mg kg^?1 (+)-catechin. Five cultivars of D. rotundata showed less tendency to brown, and had lower (+)-catechin content (90–190 mg kg^?1) than the other yams examined, but showed marked variation in o-DPOase activity. o-DPOase activity, assayed by recording the rate of oxygen consumption, varied between 75 units (1 unit=μmol O₂ consumed min^?1 100 g^?1 fresh yam tissue) in D. dumetorum and 2380 units in one D. rotundata cultivar when a crude extract of phenolic compounds diluted to contain 10 mM (+)-catechin was used as substrate and from 60 to 3480 units for these same two enzyme extracts when assayed using crude extracts of phenolic compounds (also adjusted to contain 10 mM (+)-catechin) prepared from the same yam as the enzyme extract. However, in general, the o-DPOase activities recorded using the D. alata extract showed little correlation with the activity when the phenolic extract was prepared from the same yam as the enzyme extract. Incubation of crude extracts containing phenolics with crude extracts of o-DPOase at 20°C for 24 h resulted in a decrease in the quantity of (+)-catechin estimated by h.p.l.c. Cyanidin released by hydrolysis with HCl under mild conditions was also measured. The level in D. alata was sufficiently high to account for ‘pinking’ when this species is boiled. It is concluded that the o-DPOase-catalysed oxidation of (+)-catechin is largely responsible for the browning of yams. The possible influence of other factors, including the reducing agent ascorbic acid in moderating the rate and extent of browning observed, is discussed.     

Partial characterization of peroxidase from the leaves of thymbra plant (Thymbra spicata L. var. spicata)

Peroxidase (EC 1.11.1.7; donor: hydrogen-peroxide oxidoreductase) catalyses the oxidation of various electron donor substrates (e.g. phenols, aromatic amines). In this study, the peroxidase was extracted from Thymbra spicata L. var. spicata and, then partially purified with (NH₄)₂SO₄ precipitation and dialysis. The substrate specificity of peroxidase was investigated using 2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS), o-dianisidine, o-phenylenediamine, catechol and guaiacol as substrates. Furthermore, the effects of buffer concentration, pH, temperature and thermal inactivation on enzyme activity were also studied. The results obtained have shown that (i) the best substrate is o-dianisidine, followed by ABTS, catechol, guaiacol and o-phenylenediamine, respectively; (ii) the best buffer concentration is 40 mM for o-dianisidine and catechol, 10 mM for ABTS and guaiacol, and 100 mM for o-phenylenediamine; (iii) optimum pH is 2.5 for ABTS and o-phenylenediamine, 6.0 for o-dianisidine, and 7.0 for catechol and guaiacol; (iv) optimum temperature is 20 °C for catechol, 40 °C for ABTS, guaiacol and o-dianisidine, and 50 °C for o-phenylenediamine; and (v) the enzyme activity in the thermal inactivation experiments was enhanced with increase in temperature with o-dianisidine as a substrate while its activity decreased with o-phenylenediamine.     

Polyphenol oxidase from bayberry (Myrica rubra Sieb. et Zucc.) and its role in anthocyanin degradation

Polyphenol oxidase (PPO) was extracted from bayberry (Myrica rubra Sieb. et Zucc. cv. Biqi), and its partial biochemical characteristics were studied. Stable and highly active PPO extracts were obtained using insoluble polyvinylpolypyrrolidone (PVPP) in sodium phosphate, pH 7.0, buffer. The highest PPO activity was observed in the ripe fruits. Optimum pH and temperature for bayberry PPO activity were pH 6.0 and T = 30 °C with 0.1 M catechol as substrate. PPO showed activity using the substrates of catechol, gallic acid and protocatechuic acid, but no activity with the substrates (+)-catechin, p-coumaric acid or caffeic acid. K_m and V_max values were 313 mM and 3.26ΔOD/min/g FW, respectively, with catechol as the substrate. Bayberry PPO did not act directly on cyanidin 3-glucoside but the rate of anthocyanin degradation was stimulated by the addition of gallic acid.     

Acid phosphatase in the tubers of Dioscorea species and its purification from the white yam (D. rotundata poir)

Acid phosphatase activity was determined in 15 cultivars from four species of yam. A 12-fold purification of the enzyme from Dioscorea rotundata (cv. chikakwondo) gave a homogeneous preparation as demonstrated by polyacrylamide gel electrophoresis. This enzyme preparation has an apparent molecular weight of 115 000 ±2000 and an optimum activity at a pH of 5·20 and a temperature of 50°C. The K_m of the enzyme is 3·81 mM with disodium p-nitrophenylphosphate (p-NNP) as a substrate. The energy of activation, heat of activation, energy of inactivation and heat of inactivation are 7·0, 6·4, 4·41 and 4·34 kcal M^?1, respectively. Although it has very little activity with most organic phosphoric acid esters, it is significantly inhibited by Ca²⁺, Hg²⁺ and EDTA and activated by Mg²⁺. The enzyme has a half-life of 50,17 or 13 days, respectively, when stored at 6-8°C, 0°C or room temperature (29±2°C).     

Glutathione and β-amylase,peroxidase and o-diphenolase activities during sprouting of minisett yams (Dioscorea sp)

Glutathione, β-amylase, o-diphenolase (EC 1.10.3.1) and peroxidase (EC 1.11.1.7) activities were assayed in minisetts derived from the head, middle and tail sections of sprouted Dioscorea alata (cv Sweet Yam) and D cayenensis (cv Round Leaf) yam tubers. In both tubers the head portions exhibited highest initial activities followed by the middle and tail, respectively. Minisetts obtained from the head, middle and tail sections of D alata sprouted uniformly whereas those obtained from D cayenensis tubers showed a much slower, sporadic sprouting pattern, with the head portions displaying dominant sprouting, The results revealed that sprouting initiation of the minisetts from both species correlated well with increased glutathione levels. High levels of peroxidase and reduced levels of o-diphenolase seemed to enhance the sprouting of the minisetts.     

Properties of starches of some West African yams

The tuber starches of several species and varieties of yam (Dioscorea) grown as food crops in West Africa were examined. The granule sizes were investigated, and the forms of the starch granules are illustrated in photographs. Amylose contents were also measured, together with ‘pasting temperatures’, viscous properties and gel strength. The granules of D. rotundata and D. alata were large (10–70 μ) while those of D. esculenta and D. dumetorum were much smaller (1–5 μ). The starch of D. rotundata gave the most viscous solutions, but that of D. alata gave the strongest gel. Pasting temperatures ranged between 76° and 85°.     

An infestation by Araecerus fasciculatus (Degeer) (Coleoptera: Anthribidae) and Decadarchis minuscula (Walsingham) (Lepidoptera: Tineidae) on stored fresh yam tubers in South-East Nigeria

In the course of storage trials in South-East Nigeria, four species of yam became infested with Araecerus fasciculatus (Degeer) and Decadarchis minuscula (Walsingham). Infestation was first observed in early February on Dioscorea dumetorum (Kunth) Pax. but quickly spread to D. rotundata Poir. and thence to D. alata L. and D. cayenensis Lam. The order of species attacked was probably related to their rate of drying. Both insects occurred together and attacked mainly the cut and damaged areas of the stored tubers.     

Improving the application of gibberellic acid to prolong dormancy of yam tubers (Dioscorea spp)

With the aim of reducing the cost and time needed to treat yam tubers with gibberellic acid (GA₃), this study compared several new methods of application with the established dipping procedure (150 mg kg^?1 for 1 h). Both GA₃‐containing soil paste (25 mg kg^?1) and gelatinized starch (860 mg kg^?1) were applied to tuber heads of Dioscorea alata and D cayenensis‐rotundata in the Ivory Coast. Soil paste, gelatinized starch and dipping consistently prolonged dormancy and reduced fresh matter losses by 23–39% in D cayenensis‐rotundata 3‐year means. Although dipping reduced the storage losses most efficiently, soil paste and gelatinized starch used considerably less GA₃. Both new treatments were easily prepared and quickly applied. Soil paste was most effective when the treatment was repeated before the end of dormancy. The third new method, spraying the tubers with a GA₃ solution (150 mg kg^?1), was not effective. In general, the optimal time of application was immediately post‐harvest. For D alata, treatment only 1 month after harvest was particularly ineffective, whereas D cayenensis‐rotundata tubers could be treated with some effect up to the end of dormancy. To achieve extended storage periods of healthy tubers of D cayenensis‐rotundata, GA₃ application may be recommended as post‐harvest practice. Copyright © 2003 Society of Chemical Industry     

Effect of short-term storage on phenolic content,o-diphenolase and peroxidase activities of cut yam tubers (Dioscorea sp)

The effect of short-term storage on the protein, phosphorus and phenolic content as well as peroxidase and o-diphenolase activities of cut, harvested Jamaican yam (Dioscorea sp) tubers (D rotundata. D alata and D cayenensis) was studied. There was an initial increase in the total phenolic content up to the third week of storage followed by a gradual decrease to the sixth week. Phenolic content was found to be highest in D cayenensis followed by D rotundata and D alata. The activities of peroxidase (EC 1. 11. 1. 7) and o-diphenolase (EC 1. 10.3.1) increased steadily up to the third week of storage and thereafter decreased to the fifth week. The intensity and rapidity of browning in tubers when cut, correlated very closely with the tuber o-diphenolase and phenolic content levels while the onset of rotting correlated with the peroxidase activity levels in the species studied.     

α-Anomer-selective glucosylation of (+)-catechin by the crude enzyme, showing glucosyl transfer activity, of Xanthomonas campestris WU-9701

α-Anomer-selective glucosylation of (+)-catechin was carried out using the crude enzyme, showing α-glucose transferring activity, of Xanthomonas campestris WU-9701 with maltose as a glucosyl donor. When 60 mg of (+)-catechin and 50 mg of the enzyme (5.25 units as maltose hydrolysing activity) were incubated in 10 ml of 10 mM citrate-Na₂HPO₄ buffer (pH 6.5) containing 1.2 M maltose at 45°C, only one (+)-catechin glucoside was selectively obtained as a product. The (+)-catechin glucoside was identified as (+)-catechin 3′-O-α- -glucopyranoside (α-C-G) by ¹³C-NMR, ¹H-NMR and two-dimensional HMBC analysis. The reaction at 45°C for 36 h under the optimum conditions gave 12 mM α-C-G, 5.4 mg/ml in the reaction mixture, and the maximum molar conversion yield based on the amount of (+)-catechin supplied reached 57.1%. At 20°C, the solubility in pure water of α-C-G, of 450 mg/ml, was approximately 100 fold higher than that of (+)-catechin, of 4.6 mg/ml. Since α-C-G has no bitter taste and a slight sweet taste compared with (+)-catechin which has a very bitter taste, α-C-G may be a desirable additive for foods, particularly sweet foods.     

Biochemical composition and storage of Jamaican yams (Dioscorea sp)

Dioscorea yam tubers of 11 cultivars from five common species grown in Jamaica were analysed for the following: protein content, total phenolics, vitamin C, total lipids, fatty acid composition and activity of the polyphenol oxidase enzyme. The results show that the yams grown in Jamaica are of good nutritional value with considerable amounts of protein, vitamin C, low lipids with only one cultivar ‘renta yam’ (D alata) possessing high levels of phenolic compounds. The fatty acids present in the total lipid extracts show that yam tubers generally possess high levels of saturated fatty acids mainly palmitic acid. However, the species D alata (cv white yam) and the species D trifida (cv yampie) have high levels of the unsaturated fatty acid, linolenic. All the polyphenol oxidases from the 11 cultivars show activities towards the diphenol substrates, catechol and DL-DOPA (DL-3,4-dihydroxyphenylalanine). However, no oxidation was observed with L-tyrosine, a monophenol substrate. All cultivars studied were found to have different lengths of dormancy which varied with storage conditions. When the harvested tubers were washed, sunned and stored at 20?C in a dark cupboard, it was possible to extend their lengths of dormancy by a further 11 weeks.     

DIRECT SYNTHESIS OF THE BARLEY PROANTHOCYANIDINS PRODELPHINIDIN B3, PRODELPHINIDIN C2 AND TWO TRIMERIC PROANTHOCYANIDINS WITH A MIXED PRODELPHINIDIN-PROCYANIDIN STEREOCHEMISTRY

Reduced (+)-dihydromyricetin and/or reduced (+)-dihydroquercetin have been condensed with (+)-catechin to give the following all-trans-products: (4,8)-(+)-gallocatechin-(+)-catechin (prodelphinidin B3); (4,8:4,8)-(+)-gallocatechin-(+)-gallocatechin-(+)-catechin (prodelphinidin C2); (4, 8:4,8)-(+)-gallocatechin-(+)-catechin-(+)-catechin and (4,8:4,8)-(+)-catechin-(+)-gallocatechin-(+)-catechin. ¹H N.m.r. spectra (360 MHz, CDCI₂, room temperature) of the acetates are presented in flavour of the above structures, the structural interpretation being simplified to a great extent by inspection of the ¹H n.m.r. data of the acetates of the well characterised all-trans compounds procyanidin B3, procyanidin B6, and procyanidin C2.     

PURIFICATION OF PEACH POLYPHENOL OXIDASE IN THE PRESENCE OF ADDED PROTEASE INHIBITORS1

Crude preparations of peach fruit (Prunus persica Batsch cv. Redskin) polyphenol oxidase (PPO) showed many apparent isoenzyme forms. Some of these forms were probably the result of proteolytic action of peach proteases while other forms were the result of association of PPO with carbohydrate materials. In the presence of protease inhibitors, Trasylol and phenylmethylsul-fonyl fluoride, three apparent isoenzyme forms of PPO were purified to homogeneity. The purification scheme included hydrophobic chromatography on phenyl sepharose CL-4B, hydroxylapatite chromatography, DEAE cellulose chromatography, and gel filtration on Ultrogel AcA 34. Minor contaminants remaining after these steps were separated from PPO by gel electrophoresis. The major PPO isoenzyme form (A) was purified 44 fold with an overall yield of 5.6% and contained no detectable carbohydrates. Isoenzyme forms A' and A' were purified 104 and 67 fold respectively, but still were associated with carbohydrate material. Cesium chloride centrifugation partially removed the carbohydrates associated with PPO A' and A'. Purified peach PPO A showed greater activity toward D-catechin (539%) and pyrogallol(l82%) than to catechol (100%). An apparent K₃ of 4, 0.3, and 2 mM was obtained with D-catechin, pyrogallol and catechol, respectively. The enzyme was severely inhibited by 10 μM 2,3-naphthalenediol (91%) and by 10 pM diethyl dithiocarbamate (100%).     

Biochemical properties and potential endogenous substrates of polyphenoloxidase from chufa (Eleocharis tuberosa) corms

Polyphenoloxidase (PPO) was partially purified from chufa corms through ammonium sulphate precipitation and dialysis. Biochemical properties of chufa PPO were analysed using exogenous substrate catechol. Optimal pH and temperature for PPO activity were 5 and 45 °C. Ethylenediaminetetraacetic acid disodium salt and l-cysteine could not inhibit the PPO activity. However, sodium thiosulphate pentahydrate exhibited the strongest inhibiting effect, followed by ascorbic acid and anhydrous sodium sulphite. Except for K⁺, other metal ions such as Zn²⁺, Cu²⁺, Fe³⁺, Ca²⁺, Fe²⁺ and Na⁺ accelerated the enzymatic reaction between catechol and PPO. Kinetic analysis showed that the apparent K_m and V_max values were around 10.77 mM and 82 units/ml min. In addition, (−)-gallocatechin gallate, (−)-epicatechin gallate and (+)-catechin gallate isolated and identified from chufa corms were supposed to be the potential endogenous PPO substrates due to their ortho-diphenolic or pyrogallolic structures. These polyphenols might be catalysed by PPO, resulting in the browning of chufa corms after fresh-cut processing.     

Purification and Characterization of Polyphenol Oxidase from Akko XIII Loquat (Eriobotrya japonica cv Akko XIII)

Akko XIII is an important loquat variety grown in Turkey. As with many fruits and vegetables, enzymatic browning catalyzed by polyphenol oxidase (PPO) also occurs in loquats. PPO from Akko XIII loquat was extracted and purified through (NH₄)₂SO₄ precipitation, dialysis and ion exchange chromatography. The enzyme showed several peaks with PPO activity on DEAE-Toyopearl 650 M column, of which only two (isoenzyme A and isoenzyme B) were characterized. Assay of activity of the isoenzymes between pH 3.04 and 7.80 using catechol as substrate showed two activity peaks, one at acidic pH and the other at neutral pH. pH optima of isoenzyme A and B were found to be at 7.4 and 4.98, respectively. The Km values of isoenzyme A and B using catechol as substrate were found to be 152.3 mM and 5.4 mM, respectively. They both displayed maximal activity at 30^oC. The two isoenzymes displayed different heat resistance and sensitivity towards various inhibitors.     

Changes in Grape Seed Phenols as Affected By Enzymic and Chemical Oxidation in vitro

An in vitro oxidation system containing all the phenolic compounds of an extract of grape seeds and phenol oxidases (catechol oxidase from grapes and laccase from grey mold of grape Botrytis cinerea) was used to determine the effects of enzymic oxidation on seven phenolic compounds: gallic acid, (+)-catechin; (-)-epicatechin and four procyanidins, dimers of (+)-catechin and (-)-epicatechin units. HPLC determinations showed that there was considerable depletion of phenolic compounds in the extract by both enzymes since the quantities of all the compounds fell during enzymic oxidation. When a pure chemical oxidation was compared, the decrease was very much slower.     

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     

Biochemical characterization and process stability of polyphenoloxidase extracted from Victoria grape (Vitis vinifera ssp. Sativa)

Polyphenol oxidase (PPO) was isolated from Victoria grapes (Vitis vinifera ssp. Sativa) grown in South Africa and its biochemical characteristics were studied. Optimum pH and temperature for grape PPO activity were pH 5.0 and T = 25 °C with 10 mM catechol in McIlvaine buffer as substrate. PPO showed activity using the following substances: catechol, 4 methyl catechol, d, l-DOPA, (+) catechin and chlorogenic acid. K_m and V_max values were 52.6 ± 0.00436 mM and 653 ± 24.0 OD_400 nm/min in the case of 10 mM catechol as a substrate. Eight inhibitors were tested in this study and the most effective inhibitors were found to be ascorbic acid, l-cysteine and sodium metabisulfite. Kinetic studies showed that the thermal inactivation of Victoria grape PPO followed first-order kinetics, with an activation energy, E_a = 225 ± 13.5 of kJ/mol. Both in semipurified extract and in grape juice, PPO showed a pronounced high pressure stability.     

Biochemical characteristics and thermal inhibition kinetics of polyphenol oxidase extracted from Thompson seedless grape

Polyphenol oxidase (PPO) was isolated from Thompson seedless grape (Vitis vinifera ‘Thompson Seedless’), and its biochemical characteristics were studied. The PPO showed activity to catechol and D, L-DOPA, but not towards monophenol l-Tyrosine, diphenols guaiacol and caffeic acid, and triphenols pyrogallic acid and gallic acid. Apparent Michaelis–Menten constant (K _m) and maximum velocity of the reaction (V _max) values were 45.0 ± 0.05 mM and 500.0 ± 15.3 OD_400 nm/min for catechol, and 34.6 ± 0.03 mM and 384.6 ± 11.7 OD_478 nm/min for D, L-DOPA, respectively. The obtained similar specificity values of V _max/K _m ratio of catechol and D, L-DOPA indicated their similar affinity to Thompson seedless PPO. The most effective inhibitor was l-cysteine, followed in decreasing order by ascorbic acid, sodium metabisulfite, EDTA, NaCl, and citric acid. It was discovered that metal ions of Mg²⁺ and Cu²⁺ increased, while Zn²⁺ and K⁺ reduced the PPO activity. Sugars showed inhibition on the PPO activity, with higher effect by sucrose and lower effect by fructose and glucose. Optimum pH and temperature for grape PPO activity were 6.0 and 25 °C with 10 mM catechol as substrate. The enzyme was heat stable between 10 and 25 °C, but showed significant activity loss at temperatures higher than 40 °C and completely inactivation at 70 °C for 10 min. Thermal inactivation of PPO showed a first-order kinetic with an activation energy (E _a) of 146.1 ± 10.8 kJ/mol at pH 6.0.     

Characterisation of starches from West African yams

Starches from four varieties of West African yams were extracted and characterised. The physicochemical properties investigated (granule size and morphology, amylose content, crystal form, gelatinisation and pasting behaviour) depended strongly on the yam variety. The starch granules extracted from water yam (Dioscorea alata), white yam (D rotundata) and yellow yam (D cayensis) varieties showed mononodal particle size distributions centred between 31 and 35 µm, while the bitter yam (D dumetorum) exhibited a binodal size distribution of starch granules centred at 4.5 and 9 µm. Light microscopy confirmed the variation in starch granule size and shape with yam variety. The X-ray diffractogram of yellow yam was of the B type, while bitter yam showed an A pattern. The starches extracted from the white and water yams were of the intermediate C-type patterns. The temperatures of onset of gelatinisation were derived from DSC and RVA measurements; values of 69.4 and 75.0 °C for the yellow yam, 71.5 and 78.2 °C for the white yam, 76.5 and 79.8 °C for the water yam and 78.1 and 83.1 °C for the bitter yam were obtained. © 1999 Society of Chemical Industry