Comparative characterization of monophenolase and diphenolase activities from a wild edible mushroom (Macrolepiota mastoidea)

In this study, Macrolepiota mastoidea, a wild edible mushroom, was evaluated for its polyphenol oxidase potential. Native electrophoresis, stained by l-dihydroxyphenylalanine, of the crude extracts from this species showed two bands having R_f values of 0.38 (minor) and 0.50 (major), supporting a polyphenol oxidase potential. The crude extracts showed monophenolase activity against 3-(4-hydroxyphenyl)-propionic acid and diphenolase activity against 4-methylcatechol as substrates. Monophenolase and diphenolase activities of enzyme extract prepared from M. mastoidea showed pH optimum values at pH 6.0 and pH 4.0, respectively. The extracts retained about 100% and 60% of their original monophenolase and diphenolase activities at their optimum pH values, respectively. It was estimated from thermodynamic data that M. mastoidea had a thermostable monophenolase activity. Thiourea and ascorbic acid were highly potential inhibitors for monophenolase, and ascorbic acid and sodium metabisulphite for diphenolase activity. It is clear from the present results that the enzyme extracts prepared from M. mastoidea possess polyphenol oxidase activities with interesting properties.     

Inactivation Kinetics of Polyphenol Oxidase in an Aqueous Model System under Stand-Alone and Combined Ultrasound and Ultraviolet Treatments

The objectives of this work were to study the ultrasound- and ultraviolet light-induced inactivation kinetics of polyphenol oxidase extracted from different sources in a model system. The polyphenol oxidase crude extract was obtained from bananas, apples, quince, eggplants, plums, dill, and cultured mushrooms, which exhibited high enzyme activity. The polyphenol oxidase crude extract was treated with ultrasound and ultraviolet light at 40°C temperature for 40 min. The study showed that the polyphenol oxidase enzyme was inactivated between 12 and 100% during ultrasound only treatment; between 4 and 29% during ultraviolet light only treatment; and between 80 and 100% during simultaneous ultrasound and ultraviolet light treatment. Based on the measurements, an exponential decay model for determining polyphenol oxidase inactivation kinetics was developed. The model provides high determination coefficients (R²): 0.968–0.999 with ultrasound only treatment, 0.881–0.990 with ultraviolet only treatment, and 0.975–1.000 with simultaneous ultrasound and ultraviolet treatment. The polyphenol oxidase kinetics evaluation showed that different treatments provided different inactivation times, or D-values. The D-values were 7.0–656.1 min for ultrasound only treatment (D_US), 251–1887 min for ultraviolet only treatment (D_UV), and 3.3–59.4 min for combined ultrasound and ultraviolet treatment (D_US+UV).     

Determination of kinetic properties of polyphenol oxidase from Thymus (Thymus longicaulis subsp. chaubardii var. chaubardii)

A partial characterization of polyphenol oxidase (PPO) activity of Thymus longicaulis subsp. chaubardii var. chaubardii is described. Polyphenol oxidase of Thymus was isolated by (NH₄)₂SO₄ precipitation and dialysis. The effects of substrate specificity, pH, temperature, heat-inactivation and glutathione inhibitor on polyphenol oxidase activity obtained from T. longicaulis subsp. chaubardii var. chaubardii were investigated. Polyphenol oxidase showed activity toward catechol, 4-methylcatechol and pyrogallol. Pyrogallol was the most suitable substrate, due to the lowest K_M (5.5 mM) and the biggest V_max/K_M (1260/min) values. It was found that the optimum pH values did not change with temperature, and were 6.5 for catechol and pyrogallol and 5.5 for 4-methycatechol at all temperatures. Optimum temperatures were 25 °C for catechol and 4-methylcatechol, and 35 °C for pyrogallol. Again, it was found that optimum temperature did not change with pH. Activation energy values were calculated from the Arrhenius equation and found to be in the range −1.72 and −7.48 kcal/mol for catechol, −3.56 and −9.17 kcal/mol for 4-methylcatechol, and −1.60 and −3.98 kcal/mol for pyrogallol as substrates, respectively. From heat-inactivation studies, the required times for 50% inactivation, using catechol, 4-methylcatechol and pyrogallol substrates, were 68.9, 66.4 and 96.3 min at 45 °C, 19.9, 17.9 and 34.3 min at 65 °C, and 4.1, 2.1 and 11.9 min at 85 °C, respectively. I₅₀ and K_i values for glutathione inhibitor, using catechol, 4-methylcatechol and pyrogallol substrates, were calculated, and it was found that the type of inhibition was competitive.     

Biochemical studies of cocoa bean polyphenol oxidase

Polyphenol oxidase (EC 1.14.18.1) was isolated and partially purified from cocoa beans. The properties of the enzyme were studied. The Michaelis constant K_m for catechol was 1 × 10^?2 M . The pH optimum of polyphenol oxidase activity assayed with catechol as substrate occurred at pH 6.8 and was characterised by a relatively high thermal stability, 50% of its activity was lost after heating for 40, 25 and 5 min at 60, 69 and 80°C respectively. The optimum temperature for the enzyme activity with catechol as substrate was around 45°C. The enzyme was reactive towards 3-(3,4-dihydroxy phenyl)-DL -alanine, 3-hydroxytyramine hydrochloride and 4-methyl catechol but showed no activity towards tyrosine, p-cresol, and 4-hydroxy-phenol. A rapid deactivation of the enzyme was observed when catechol of concentration > 40 mM was used as substrate. The enzyme activity was inhibited by ascorbic acid, L -cysteine, sodium bisulphite and thiourea.     

Purification and Characterization of Polyphenol Oxidase From Agaricus bisporus

A polyphenol oxidase was isolated from Agaricus bisporus using ammonium sulfate precipitation, combined with DEAE–Sepharose Fast Flow chromatography and Phenyl Sepharose CL-4B chromatography. The molecular weight of the purified polyphenol oxidase was determined to be about 43 kDa with N-terminal sequence Ala-Thr-Asn-Ser-Gly-Thr-Leu-Ile-Ile-Phe by Edman degradation. The optimum temperature and pH for the polyphenol oxidase activity was 20°C and a pH of 6.5–7.0. Moreover, it was stable at 30 and 40°C over a 60 min pre-incubation time period. The K_m and V_max values of this polyphenol oxidase for catechol were 0.67 mM and 3333 U/ml min^?1, respectively. In addition, following the cross linking with polyphenol oxidase, the emulsifying activity index, foam capacity, and foam stability of the whey proteins were evaluated to be modified. This work was useful to better understand polyphenol oxidase from A. bisporus, and may lead to its practical use in the future.     

Studies of the physico-chemical properties and polyphenoloxidase activity in seeds from hybrid sunflower (Helianthus annuus) varieties grown in India

Physico-chemical properties and polyphenol oxidase (PPO) activities of seven hybrid sunflower varieties commonly grown in Punjab state of India were studied. Seeds were studied for variations in physico-chemical properties, 100 kernel weight, density, bulk density, moisture, hull, oil, protein and ash content; expelled cake was studied for moisture, oil, crude fibre, ash and protein content and expelled-defatted cake for acid detergent fibre, lignin, moisture, ash and protein contents. PPO activity in all varieties, determined using pyrogallol as substrate at pH 6.5, varied between 0.212 and 0.294 Ab/min/mg protein. The PPO enzyme activity for MSFH-8 and Mega-363 was observed to be maximum at 55 and 60°C, respectively. The enzyme showed a K_m value between 1.01 and 1.968 mM for pyrogallol and V_max value between 0.21 and 2.0 Ab/min. ©     

Effects of Grilling on Total Polyphenol Content and Antioxidant Capacity of Eggplant (Solanum melongena L.)

Cooking can change the polyphenol contents of eggplant. This study elucidated the effects of grilling on total polyphenol content (TPC), antioxidant capacity, and the inner structures of eggplant. After identical hollowing, cylindrical eggplant samples were prepared and were then grilled until their center temperatures (CT) respectively reached 50, 65, 75, 85, and 95 °C. Chemical assays and observations of the inner structures clarified that TPC and 1, 1‐diphenyl‐2‐picrylhydrazyl (DPPH) radical scavenging activity decreased as CT increased when CT was below 65 °C. Results also showed that TPC and DPPH radical scavenging activity increased as CT increased when CT was between 65 °C and 95 °C. For CT 65 °C, the samples retained polyphenol oxidase (PPO) activity up to 40% of the raw state activity. The 3 grilled eggplant models, chlorogenic acid, chlorogenic acid—sugar and chlorogenic acid—amino acid model, yielded results showing that phenol functional groups on chlorogenic acid were thermally stable and that phenol functional groups on chlorogenic acid reacted neither with sugar nor with amino acids. Results show that PPO activity is a primary reason for the decrease of the 2 indices. Optical microscopic and scanning electron microscopic observations revealed collapsed cells and inter‐tissue cracks around the surface area for CT 85 and 95 °C. Scanning electron microscopic observations clarified that intercellular bonds for CT 85 and 95 °C became thinner than those for CT 75 °C around the middle area. The phenomena explained above are reasons for the increase of TPC and DPPH radical scavenging activity.     

Protective Effect of Melanoidins from Fructose–Glutamic Acid on Polyphenol Oxidase Inactivation by Ultraviolet–Visible Irradiation

Ultraviolet irradiation has been proved to be effective for inactivating polyphenol oxidase in some fruit derivatives. However, some compounds that may be found in these products, such as melanoidins, can protect the enzyme during the irradiation process. In this piece of work, the protective effect of melanoidins synthesized from fructose and glutamic acid on Agaricus bisporus polyphenol oxidase inactivation by ultraviolet–visible irradiation has been assessed. The polymers with molecular mass lower than 150 kDa had a greater protective effect than those molecules higher than 150 kDa. If the obtained melanoidins are not fractioned by their molecular mass, the protective effect that they exert is lower. It was found that the most effective radiation to inactivate this polyphenol oxidase is that between 260 and 310 nm.     

Polyphenol oxidase properties,anti-urease,and anti-acetylcholinesterase activity of Diospyros lotus L. (Plum Persimmon)

Diospyros lotus fruit polyphenol oxidase was purified using affinity chromatography, resulting in a 15-fold enrichment in specific activity. The purified enzyme, having 16.5 kDa molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, exhibited the highest activity toward 4-methylcatechol. Maximum diphenolase activity was reached at pH 7.0 and 60°C in the presence of 4-methylcatechol. K_m and V_max values were calculated as 3.8 mM and 1250 U/mg protein, respectively. Ascorbic acid was a promising inhibitor with an IC₅₀ value of 0.121 µM. The activity of the purified enzyme was stimulated by Fe²⁺, Sr²⁺, Zn²⁺, and K⁺ and deeply inhibited by Hg²⁺, at 1 mM final concentration. Aqueous extract of Diospyros lotus L. fruit showed strong substantial urease and acetylcholinesterase inhibition, with IC₅₀ values of 1.55 ± 0.05 and 16.75 ± 0.11 mg/mL, respectively.     

Control of Enzymatic Browning in Processed Mushrooms (Agaricus bisporus)

Control of polyphenol oxidase (E.C. 1.14.18) activity by the use of citric acid was investigated. The enzyme was inactivated at pH 4.0 and was stable to 10 min exposures at 25°C in the pH range 4.0–8.0. At pH 6.5 the enzyme was active at 45°C but not at 70°C and thermal inactivation followed pseudo first-order kientics. At pH 6.5 the activation energy (E_a) for enzyme inactivation was 41.1 Kcal/mole while at pH 3.5 two rate constants and hence two values for E_a were observed. Between 0–5 min E_a for inactivation of polyphenol oxidase was 8.7 Kcal/mole and >5 min Ea was 21.8 Kcal/mole.     

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.     

Polyphenol oxidase potentials of three wild mushroom species harvested from Lişer High Plateau,Trabzon

Crude enzyme extracts were prepared from Armillaria mellea (A. mellea), Lepista nuda (L. nuda) and Hypholoma fasciculare (H. fasciculare), which were harvested from the Li?er High Plateau-Maçka (Trabzon, Turkey). The crude polyphenol oxidase (PPO) extracts from each mushroom were highly active against 4-methylcatechol. Native polyacrylamide gel electrophoresis, stained by L-3,4-dihydroxyphenylalanine, showed the polyphenol oxidase potentials. The optimum pH value, for each enzyme, was 7.0. When enzyme extracts were incubated at pH 7.0 for 24 h at 4 °C, it was observed that L. nuda and H. fasciculare enzyme activities decreased by about 26% and 18%, respectively, but, A. mellea enzyme activity increased by about 11%. The temperature optima of A. mellea, L. nuda and H. fasciculare were, respectively, 30, 30 and 20 °C. Cr³⁺ and Cu²⁺ ions inhibited each activity. Also, sodium metabisulphite and ascorbic acid were strong inhibitors of the enzyme activities.     

Biochemical and Structural Changes of Taro (Colocasia esculenta) Tubers During Simple Thermal Treatments (Low Temperature) or in Combination with Chemicals

The present study set out to study the structural and biochemical modification of taro (Colocasia esculenta) grown in Cameroon, through simple heating or in association with chemicals. Both techniques are known to inactivate the reactions of polyphenol oxidase. The textural analysis was performed on tubers heated in water, 0.5% NaCl and 1% NaCl solution at various times from 0 to 105?min. The result showed optimum pH and temperature for taro polyphenol oxidase (PPO) activity at pH?6.0 and t?=?40?°C with 10?mM catechol in 0.1?M phosphate buffer as substrate. K _m and V _max values were about 7.317?±?0.012?mM and 0.148?±?0.0003 OD_{430 nm}/minute. Seven inhibitors were tested in this study, and the most effective inhibitors were found to be NaCl, CaCl2 and KCl. Kinetic studies showed that the thermal inactivation of taro PPO followed first-order kinetics, with an activation energy of E _a?=?422.79?±?0.52?kJ/mol. The textural modification of taro tubers during heating follows the kinetic of the fractional model. It was noticed that the activation energy increased with the concentration of NaCl.     

Inactivation of polyphenol oxidase in muscadine grape juice by dense phase-CO2 processing

The effect of dense phase-CO₂ processing (DP-CO₂) on polyphenol oxidase (PPO) activity, polyphenolic and antioxidant changes in muscadine grape juice under different processing pressures (27.6, 38.3, and 48.3 MPa) and CO₂ concentrations (0, 7.5, and 15%) were measured. Subsequently two DP-CO₂ conditions (48.3 MPa at 0 or 15% CO₂) were evaluated for polyphenolic and antioxidant changes during storage (4 °C, four weeks). Pressure alone was responsible for a 40% decrease in PPO activity that resulted in 16–40% polyphenolic and antioxidant losses. Increasing CO₂ from 0 to 7.5% was responsible for an additional 35% decrease in enzyme activity and a two-fold greater polyphenolic retention. However, insignificant changes in PPO activity or polyphenolic retention were observed when CO₂ was increased to 15%. During storage, juices with residual PPO activity following processing resulted in greater polyphenolic (8- to 10-fold) and antioxidant capacity (four-fold) degradation compared to control juices with no PPO activity. This study demonstrated that partial PPO inactivation can be obtained by DP-CO₂ and that CO₂ level was the primary variable influencing PPO activity and polyphenolics and antioxidant capacity retention in muscadine grape juice.     

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

Unfermented 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     

Purification and some properties of polyphenol oxidase of longan fruit

Polyphenol oxidase (PPO) was isolated from longan (Dimocarpus longan Lour.) fruit peel, with a 46-fold purification of PPO by ammonium sulfate, Sephadex G-200 and Phenyl Sepharose being achieved. Pyrogallol, 4-methylcatechol, and catechol were good substrates for the enzyme, and activity with chlorogenic acid, p-cresol, resorcinol, or tyrosine was not observed. The optimal pH for PPO activity was 6.5 with 4-methylcatechol. The enzyme had a remarkably temperature optimum (35°C) and was relatively stable, requiring a little more than 20 min at 50°C for 50% loss of activity. Reduced glutathione, l-cysteine, thiourea, FeSO₄ and SnCl₂ markedly inhibited PPO activity, whereas MnSO₄ and CaCl₂ enhanced PPO activity. Data obtained in this study might help to better understand longan fruit peel browning.     

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 (K_I = 1 mM ). The results reported show that myricetin oxidation was strictly dependent on the presence of polyphenol oxidase. © 1999 Society of Chemical Industry     

Effect of high hydrostatic pressure and thermal processing on the nutritional quality and enzyme activity of fruit smoothies

Fruit smoothie samples were thermally (P₇₀ > 10 min) or high hydrostatic pressure (HHP) processed (450 MPa/20 °C/5 min or 600 MPa/20 °C/10 min) and the total antioxidant capacity (TAC), levels of antioxidant groups [total phenols (TP), anthocyanins and ascorbic acid], instrumental colour, polyphenol oxidase (PPO) enzyme activity and dissolved oxygen were examined over a storage period of 10 h at 4 °C. Thermal processing of smoothies reduced (p < 0.001) TAC and TP values, ascorbic acid and L and a colour attributes (lightness and redness respectively) compared to fresh and HHP-450 processed samples. Conversely, it did result in complete inactivation of PPO enzyme, with no activity detected. Of the HHP treatments, HHP-450 samples had higher (p < 0.001) levels of total antioxidant, phenols and anthocyanin content than HHP-600 samples. However, the latter was more effective in reducing (p < 0.001) the endogenous enzyme activity of the smoothies. .Ascorbic acid content degraded over the storage for all smoothies. HHP-600 samples had high initial values, which declined slowly over storage, while thermal samples had the lowest initial value (0.5 h) that fell below detectable limits by 10 h. Despite these data, less pronounced effects were observed for storage. No significant effects were observed for total anthocyanin and phenolic contents as well as L and colour change (ΔE) variables. Overall, HHP processing of smoothies at moderate temperatures may be a suitable alternative to traditional thermal processing.     

Inactivation of polyphenol oxidase by ultraviolet irradiation: Protective effect of melanins

Most of the studies about UV irradiation of fruit and vegetable derivatives have been carried out in order to assess its effect on microbial inactivation. Nevertheless, there are few references about UV influence on some enzyme activities that are important in this kind of food, especially polyphenol oxidase, which is responsible for enzymatic browning in fruit and vegetable tissues containing phenolic or polyphenolic compounds. In this work, the effect of UV–Vis irradiation on polyphenol oxidase from Agaricus bisporus was investigated. A reduction of 58.7% in enzyme activity was achieved in the first 90 s, and it was completely inactivated after 35 min of treatment. In addition, the protective effect of melanins synthesized by the action of the same polyphenol oxidase was assessed. These pigments absorbed some of the radiant energy and led to a slower inactivation of polyphenol oxidase.     

Effect of carbon dioxide on the inactivation of florida spiny lobster polyphenol oxidase

Florida spiny lobster (Panulirus argus) polyphenol oxidase (PPO) exposed to CO₂ (1 atm) at 33, 38, and 43°C showed a decrease in enzyme activity with increased heating time. Enzyme inactivation by CO₂ was faster for PPO heated at 43 than at 33 or 38°C. Inactivation kinetics revealed PPO was more labile to CO₂ and heat in the range 33–43°C than to heat alone. Kinetic data also revealed that CO₂, in addition to pH changes, was involved in the loss of PPO activity. Studies using get electrophoresis showed no differences in protein patterns and isoelectric point between CO₂-treated and non-treated control. The pH of CO₂-treated PPO stock solution was brought back from 5.4 to 8.0 after 6 weeks of frozen-storage and no enzyme reactivation was observed.