Heat inactivation and kinetics of polyphenoloxidase from palmito (Euterpe edulis)

Polyphenoloxidase (PPO, EC 1.14.18.1)was extracted from palmito (Euterpe edulis Mart) using 0.1 M phosphate buffer, pH 7.5. Partial purification of the enzyme was achieved by a combination of (NH₄)₂SO₄precipitation (35–90% saturation) and Sephadex G-25 and DEAE-cellulose chromatography. The purified preparation gave five protein bands on polyacrylamide gel electrophoresis, three of them with PPO activity. The K_mvalues for chlorogenic acid, caffeic acid, catechol, 4-methylcatechol and catechin were 0.57, 0.59, 1.1, 2.0 and 6.25 mM , respectively. PPO has a molecular weight of 51 000 Da, maximum activity at pH 5.6 with chlorogenic acid as substrate, and was stable between pH 5.0 and 8.0. The enzyme was heat stable at 50–60°C and inactivated at 75°C. The heat stability of palmito PPO was found to be pH dependent; at 50°C and pH 4.0 the enzyme was fully inactivated after 30 min. The pH/activity studies showed two groups with pK values c 4.6 and 6.7 involved in PPO catalysis.     

Preservation of raw milk with CO2

The effect of CO₂ on the growth of psychrotrophic milk spoilage organisms was studied, both in raw fresh milk and in pure cultures of three species ofPseudomonas growing in sterilised milk. Changes of sensory properties of CO₂-treated samples after heat treatment were also analysed. Inhibition of psychrotrophic growth at 7 °C in milk treated with CO₂ to a pH 6.2 or 6.0 was impaired by a gradual reduction of the CO₂ content during storage. Growth inhibition was considerably improved by pH adjustment at 24-h intervals. Sensory analysis showed significant differences between non-acidified and acidified samples after heat treatment at 75 °C for 20 s or 110 °C for 5 min. No sensory differences were found between non-acidified and acidified milks degassed before heat treatment.     

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.     

Characterization of Polyphenoloxidase from Stanley Plums

Five plum cultivars were investigated for polyphenoloxidase. Stanley cultivar, which showed highest PPO activity, was selected for characterization of this enzyme. The activity of crude enzyme was 3.5 times greater in the flesh than in the skin of the fruit. The enzyme showed a K_m of 20 raM of catechol and V_max of 5.41 × 10^?1 O.D./ min at pH 6.0. Its pH and temperature optima were 6.0 and 20°C respectively. The enzyme lost activity below pH 4.5, but was stable even at 70°C. Among substrates, 4-methylcatechol was oxidized more rapidy, although catechol, dopamine, pyrogallol and caffeic acid were also good substrates. The enzyme was strongly inhibited by sodium metabisulfite (Na₂S₂O₅), L-cysteine and ascorbic acid.     

Properties of polyphenol oxidase in mango (Mangifera indica) kernel

Polyphenol oxidase (PPO) activity of filtered extract of ground mango kernel suspension (400 g litre⁻¹) was studied spectrophotometrically at 420 nm using catechol as substrate. The enzyme was most active at pH 6·0 and 25°C. Activity was reduced by 50% at pH values of 5·0 and 7·1, and also at temperatures of 14°C and 30°C. The calculated activation energy and the Michaelis constant (K_m) were 21·4 kcal mol⁻¹ °C⁻¹ and 24·6 mM , respectively. The V_max value was 2·14 units g⁻¹ mango kernel. The time to heat inactivate PPO decreased rapidly to < 10 min with increasing temperature of ⩾ 70°C at 50% activity. © 1998 SCI.     

Heat Inactivation Kinetics of Apple Polyphenoloxidase and Activation of its Latent Form

Heat inactivation kinetics of crude polyphenoloxidase (PPO) from six apple cultivars (Golden Delicious, Starking Delicious, Granny Smith; Gloster, Starcrimson and Amasya) were studied at three temperatures (68°, 73° and 78°C). PPO activity initially increased and then decreased with heat, following a first order kinetic model. Increase in activity indicated presence of latent PPO. Regression coefficients for the linear portions of inactivation curves were computed to determine inactivation parameters. Reaction data at 78°C revealed that PPO in Amasya was the least and Starking Delicious the most heat-stable. Rate constants for heat inactivation at 78°C ranged from 15.99–28.27. 10^?2 min^?1. Activation energies varied between 54.7–77.2 kcal. mol^?1 with z values of 7.1–10.0C°.PPO in apples was generally more heat-stable than PPO in most fruits.     

Polyphenoloxidase activity from strawberry fruit (Fragaria ananassa,Duch., cv Selva): characterisation and partial purification

In this work, polyphenoloxidase (PPO) from Selva strawberry fruit (Fragaria × ananassa, Duch) was extracted, characterised and partially purified. The activity of PPO was analysed in crude extracts obtained from either fresh fruits or acetone powder. The presence of NaCl and Triton X‐100 in the extraction buffer caused a marked increase in enzyme extractability. The enzyme showed an apparent K_m value of 11.2 mM with pyrocatechol as substrate. The maximum enzyme activity was observed at 50 °C and pH 5.3–6.0 without SDS and pH 7.2 in the presence of SDS. The presence of SDS increased PPO activity at pH 7.2 but diminished it at pH 6.0. The enzyme showed high thermal stability and maintained activities equal to or greater than 50% of its maximum activity in the 2.6–9.3 pH range. One polyphenoloxidase isoenzyme was detected in crude extracts of all ripening stages, showing an isoelectric point of 7.3. The specific activity of PPO decreased continuously through fruit ripening. Maximum specific activities were found at the ‘small green’ and ‘large green’ ripening stages. A total enzyme extract was partially purified by means of (NH₄)₂SO₄ precipitation and cationic exchange chromatography in an FPLC system. The purification grade achieved was near 25. The partially purified enzyme showed an isoelectric point equal to 7.3 and a molecular mass of 135 ± 4 kDa for the native protein. © 2000 Society of Chemical Industry     

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.     

Influence of hot‐air treatment,superatmospheric O2 and elevated CO2 on bioactive compounds and storage properties of fresh‐cut pomegranate arils

The impact of heat treatment using hot air (HT 45 °C and 55 °C for 1 h) and two active modified atmosphere packaging (MAP) conditions of high oxygen atmosphere (HOA: 80 kPa O₂, 20 kPa N₂) and high CO₂ atmosphere (HCA: 20 kPa CO₂, 80 kPa N₂), individually or combined, on the antioxidant capacity, polyphenols, vitamin C content, total anthocyanins, polyphenoloxydase (PPO) activity and shelf life of fresh‐cut (FC) pomegranate arils stored for 14 days at 4 °C was studied. The results indicate that HT 45 °C along with HOA inhibited PPO activity and prevented loss of antioxidant capacity, vitamin C and phenolic compounds in arils, in comparison with control and HT 55 °C. All treatments reduced the accumulation of anthocyanins, but HCA‐treated arils lost more anthocyanins besides having worse a* colour parameter values. No significant differences in titrable acidity (TA) and total soluble solids (TSS) were observed between treatments. The combination of HOA and HT 45 °C enhanced the benefits of applying each treatment separately and could be useful to improve and extend postharvest life of pomegranate FC arils.     

Enhanced inactivation of pectin methyl esterase in orange juice using modified supercritical carbon dioxide treatment

The aim of the study is to quantify the effect of ethanol addition and exposure surface on the inactivation of pectin methyl esterase (PME), a juice clarifying enzyme, in orange juice using supercritical carbon dioxide (SC‐CO₂). Addition of ethanol to the SC‐CO₂ at 2% (v/v) caused greater inactivation than SC‐CO₂ alone, with a maximum reduction of PME activity of 97% at 30 MPa and 40 °C for 60 min. As the surface area to volume ratio was increased, the rate of inactivation of PME increased. Analysis of first‐order reaction kinetic data revealed that D values were greatly influenced by ethanol addition and agitation. With the addition of 2% ethanol, the D value reduced by half, that is, 56 min from 109 min. With impeller agitation of the sample at 1100 ± 100 rpm, the D value for PME was further reduced to 43 and 30 min without and with ethanol, respectively. The activity of PME treated with SC‐CO₂ remained unchanged after 14 days of storage at 4 °C. Treatment did not significantly change pH or colour, but did significantly increase the cloud values of the juice, resulting in a cloud stabilised juice with similar qualities to fresh juice.     

Characterization of Sultaniye grape (Vitis vinifera L. cv. Sultana) polyphenol oxidase

Polyphenol oxidase (PPO) was extracted from Sultaniye grapes grown in Turkey, and its characteristics in terms of pH and temperature optima, thermal inactivation, kinetic parameters and potency of some PPO inhibitors were studied. Optimum pH and temperature for grape PPO were found to be 3.4 and 30 °C, using catechol as substrate. K_m and V_max values were found to be 44.5 ± 5.47 mm and 0.695 ± 0.0353 OD₄₁₀ min^?1, respectively. Four inhibitors were tested in this study and the most potent inhibitor was sodium metabisulphite, followed by ascorbic acid. From the thermal inactivation studies in the range of 65–80 °C, the half‐life values of the enzyme ranged between 2.6 and 49.5 min. Activation energy (E_a) and Z values were calculated to be 208.5 kJ mol^?1 (r² = 0.9544) and 10.95 °C (r² = 0.9517), respectively.     

Muscadine Grape Polyphenoloxidase: Partial Purification by High Pressure Liquid Chromatography and Some Properties

A rapid method of isolating grape polyphenoloxidase (PPO) was developed, using two muscadine cultivars, “Noble” and “Welder”. PPO extracts from acetone powders were partially purified followed by filtration through 0.2μ membrane filter by HPLC. Reactivity of the active fraction per unit protein, increased 10-fold for Noble and 33-fold for Welder cultivars over values for the corresponding crude extracts. Electrophoretic analysis and activity stain showed that the enzyme displayed a single band in Welder and two bands for Noble. Optimum pH for PPO activity in Welder was pH 4.5 while that for Noble was pH 4.5–5.5. The enzyme in the Noble cultivar was relatively more active at alkaline pH. Activites of the enzymes were stable at temperatures up to 30°C for both cultivars.     

Properties of Trypsin from the Pyloric Ceca of Atlantic Cod (Gadus morhua)

Trypsin (EC 3.4.21.4) was isolated from the pyloric ceca of Atlantic cod and purified to homogeneity by affinity chromatography. The enzyme catalyzed the hydrolysis of benzoyl arginine p-nitroanilide (BAPA, pH 8.2 and 25°C) such that V_max was 250 BAPA units per micromole trypsin and K_m was 1.48 mM. For the hydrolysis of tosyl arginine methyl ester (TAME, pH 8.1 and 25°C), V_max was 18.2 × 10³ TAME units/micromole trypsin, and K_m 0.22 mM. The pH and temperature optima with BAPA substrate were 7.5 and 40°C, respectively. Atlantic cod trypsin was most active and stable at alkaline pH. The enzyme was heat labile, losing more than 50% of its activity after incubation at 50°C for 30 min. Amino acid analysis of Atlantic cod trypsin revealed that the enzyme was rich in residues such as serine, glycine, glutamate and aspartate, but poor in basic amino acid residues compared to trypsins from warm blooded animals.     

The effect of thermal treatment on β‐glucosidase inactivation in vanilla bean (Vanilla planifolia Andrews)

The conditions for enzyme activity (pH and temperature) and kinetic parameters for the thermal inactivation of β‐glucosidase enzyme in vanilla beans have been investigated. The maximum enzyme activity was detected at pH 6.5 and 38 °C. The values obtained for V_max and K_m were 62.05 units and 2.07 mm, respectively. When hot water treatment (the most practical method of vanilla bean killing) was applied, β‐glucosidase treated at pH 6.0 and 60 °C for 3 min lost 51% of activity, while at 70 °C for 90 s the enzyme lost 60% of activity and at 80 °C for 30 s the enzyme lost 48% of its activity. When vanilla beans were cured in an oven at 60 °C for 36 to 48 h all β‐glucosidase activity was lost.     

Changes in selected quality characteristics of minimally processed carambola (Averrhoa carambola L.) when treated with ascorbic acid

‘B 10’ carambola of ripening stage (RS) 3 and 4 were minimally processed (MP) and then dipped in 0, 15 and 30 mg L^?1 ascorbic acid (AA). The 1‐cm‐thick slices were then dried, packed into cling‐wrapped‐foam tray and stored at 7 °C for 0, 3 and 5 days. Skin colour (L*, C* and h°), flesh firmness, soluble solids concentration, vitamin C content, titratable acidity, pH, degree of browning, polyphenoloxidase (PPO) activity and sensory attributes of MP carambola treated with AA were determined. AA treatment had significant effect in decreasing cut surface browning degree but no significant effect on all the selected quality characteristics of the MP carambola. In the sensory evaluation, flesh colour, sweetness, flavour and overall taste were significantly affected by AA treatment especially at 15 mg L^?1. The RS of fruit significantly affected skin colour (C* and h°), pH and sensory attributes of colour and flavour of the MP carambola. As storage day (SD) progressed, skin colour (C* and h°), flesh firmness and vitamin C content, cut surface browning, PPO activity and all the sensory attributes of MP carambola decreased significantly. Flesh firmness of the MP carambola was affected by the interaction between AA × SD. Sensory attributes of MP carambola were affected significantly by AA × RS. All the sensory attributes of MP carambola positively correlated to each other but negatively correlated with browning degree. PPO activity positively correlated with browning degree. Copyright © 2007 Society of Chemical Industry     

Thermal inactivation kinetics parameters of browning enzymes in starfruit (Averrhoa carambola L.) juice

This study aimed to evaluate the thermal inactivation kinetics of polyphenol oxidase (PPO) and peroxidase (POD) in starfruit juice. It followed the Malaysia Food Regulations 1985 and CODEX STAN 247-2005. Glucose, fructose and sucrose were the main sugars in starfruit juice. The total soluble solids, pH, titratable acidity, and total phenolics content of the starfruit juice produced were 8.13 ± 0.25 °Brix, 3.80 ± 0.05, 0.43% ± 0.02% malic acid, and 93.67 ± 4.96 mg GAEL⁻¹, respectively. Thermal inactivation kinetics of PPO and POD followed the first-order kinetic model. The decimal reduction time at 83.6 °C (D_83.6) of PPO and POD was 198.48 and 98.4 s, respectively, while the thermal resistance constant (z value) of PPO and POD was 12.8 and 5.4 °C, respectively. In conclusion, PPO might be a suitable signal for thermal processing on starfruit juice since it has higher heat resistance than POD.     

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.     

Thermal inactivation of polyphenoloxidase and peroxidase in green coconut (Cocos nucifera) water

Coconut water is an isotonic beverage naturally obtained from the green coconut. After extracted and exposed to air, it is rapidly degraded by enzymes peroxidase (POD) and polyphenoloxidase (PPO). To study the effect of thermal processing on coconut water enzymatic activity, batch process was conducted at three different temperatures, and at eight holding times. The residual activity values suggest the presence of two isoenzymes with different thermal resistances, at least, and a two‐component first‐order model was considered to model the enzymatic inactivation parameters. The decimal reduction time at 86.9 ^°C (D_{86.9 °C}) determined were 6.0 s and 11.3 min for PPO heat labile and heat resistant fractions, respectively, with average z‐value = 5.6 °C (temperature difference required for tenfold change in D). For POD, D_{86.9 °C} = 8.6 s (z = 3.4 °C) for the heat labile fraction was obtained and D_{86.9 °C} = 26.3 min (z = 6.7 °C) for the heat resistant one.     

Characterization and Inhibitors of Polyphenol Oxidase from Chinese Toon

Chinese toon is the unique and traditional woody vegetable in China. Enzymatic browning catalyzed by polyphenol oxidase (PPO) is prone to happen during the harvest, storage, and processing of Chinese toon so that the sensory quality and the nutritional content of Chinese toon products are seriously influenced. In order to prolong shelf life and storage period, the characterization of Chinese toon PPO (CtPPO) has been analyzed in this study. The PPO was extracted and fractionated by 25–70% (NH₄)₂SO₄, and the biochemical characteristics were analyzed. Based on the V_max /K_m ratio, pyrogallol was the most suitable substrate, followed by catechol and gallic acid. CtPPO exhibited no affinity with methyl gallate. The molecular mass of CtPPO was approximately 84.55 kDa estimated by SDS-PAGE. The native PAGE showed four prominent bands. Optimal pH and temperature were 6.2, 40°C and 8.5, 80°C for catechol and pyrogallol, respectively. CtPPO showed high and stable activity at pH ranging from 5.0 to 7.2 for catechol and pH 7.4 to 9.5 for pyrogallol. Activation energy (Ea) values were 263.79 KJ/mol for catechol and 103.91 KJ/mol for pyrogallol. In addition, CtPPO showed stronger heat resistance above 70°C for pyrogallol than catechol in the half-life values, D-values and other thermodynamic parameters. Ascorbic acid was the most effective inhibitor, followed by L-cysteine and citric acid. Purple onion peel extract and pomegranate rind extract were effective natural inhibitors, but the former was more valid.     

Carbon Dioxide Inhibition of Escherichia coli and Staphylococcus aureus on a pH-adjusted Surface in a Model System

Growth of Escherichia coli and Staphylococcus aureuson the surface of Trypticase Soy Agar (TSA) packaged with various CO₂ partial pressures (0, 20, 40, 60, 80, 100%, balance N₂) was compared to the control (N₂ 100%) on TSA in which the pH was adjusted to equal that in CO₂ atmospheres at 15°C and 30°C. At 15°C, the biostatic effect was noted with all CO₂ partial pressures for both species. At 30°C, the biostatic effect of CO₂ was almost completely nullified for E. coli, but that for S. aureus was still effective. S. aureus was more sensitive to the inhibitory effects of CO₂ than E. coli at both the temperatures.