PARTIAL PURIFICATION AND CHARACTERIZATION OF POLYPHENOL OXIDASE FROM FRESH-CUT CHINESE WATER CHESTNUT

The characteristics of polyphenol oxidase (PPO) from Chinese water chestnut (CWC) and its potential inhibitors for browning reactions were investigated. PPO was isolated from fresh‐cut CWC and was purified on a Sephadex G‐100 column, with a yield of total activity close to 10%. The molecular weight, Michaelis constant (K_m), substrate specificity, optimal pH and temperature of CWC PPO were examined. Kinetic studies indicated that the K_m and V_max values of CWC PPO for catechol were 10.32 mmol/L and 6.452 × 10⁴ U/min, respectively. The optimal pH and temperature for CWC PPO was 6.5 and 40C, respectively. Among the browning inhibitors tested, 4‐hexylresorcinol, at a concentration of 0.3 mmol/L, showed the strongest inhibition (70%) against the PPO activity of CWC, followed by 3.0 mmol/L N‐acetyl‐L‐cysteine with an inhibition of 53%.     

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.     

Determination of some biochemical properties of polyphenol oxidase from Emir grape (Vitis vinifera L. cv. Emir)

Polyphenol oxidase (PPO) was extracted from Emir 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. The optimum pH and temperature for grape PPO were found to be 4.2 and 25 °C respectively using catechol as substrate. K_m and V_max values were found to be 25.1 ± 2.72 mmol L⁻¹ and 0.925 ± 0.04 OD₄₁₀ min⁻¹ respectively. Of the inhibitors tested, the most potent was sodium metabisulfite, followed by ascorbic acid. The thermal inactivation curve was biphasic. Activation energy (E_a) and Z values were calculated as 251.4 kJ mol⁻¹ (r² = 0.996) and 8.92 °C (r² = 0.993) respectively. Copyright © 2006 Society of Chemical Industry     

Respiratory parameters of onion bulbs (Allium cepa) during storage. Effects of ionising radiation and temperature

The O₂ and CO₂ respiration rates of untreated and irradiated onion bulbs (Allium cepa) at 0.15 and 0.30 kGy were measured at 4, 10 and 20 °C. The O₂ respiration rate increased for 24 h after treatment from 0.19 mmole kg⁻¹ h⁻¹ at 20 °C for control samples up to 0.26 and 0.39 mmole kg⁻¹ h⁻¹ for 0.15 and 0.3 kGy irradiated onions respectively. Respiratory quotient (RQ) increased with temperature. The Q₁₀ of the respiration of the control samples (1.61) was lower than that of any other plant tissue, but it increased with storage duration and irradiation dose. The respiration rate of control onions increased steadily over 25 weeks of storage at 4 °C, while that of the irradiated samples decreased during the same period after a peak observed after irradiation treatment. The apparent K_m for the Menten–Michaelis equation was determined on a new respirometer and averaged 1.6 kPa at 10 °C and 6.3 kPa at 20 °C. However, at this higher temperature (20 °C) apparent K_m varied with O₂ partial pressure, proving that the respiration of onion bulbs does not follow a Menten–Michaelis‐like process. The Fermentative Index (FI) of onions was measured under anoxic conditions as CO₂ production rates in mmole kg⁻¹ h⁻¹ at 4, 10 and 20 °C. © 2000 Society of Chemical Industry     

Characterization of polyphenol oxidase from Uapaca kirkiana fruit

Polyphenol oxidase (PPO), the enzyme responsible for the postharvest spoilage of fruits, was extracted and purified from Uapaca kirkiana peel and pulp by ammonium sulfate precipitation and dialysis. Further purification of peel PPO was carried out by gel filtration chromatography. Optimum pH values were 7 and 8 for peel and pulp PPO, respectively. The optimum temperatures for peel and pulp PPO were 45 and 35 °C, respectively. Inhibition studies of the PPO enzyme were performed using citric acid, sodium azide, sodium metabisulfite and thiourea. The most effective inhibitors were sodium azide and citric acid for both peel and pulp PPO. V_max and K_m values were 13.63 units min^?1 and 4.923 mmol L^?1, respectively, for peel PPO and 14.03 units min^?1 and 5.43 mmol L^?1, respectively, for pulp PPO. Three isoenzymes of Uapaca kirkiana PPO were detected by polyacrylamide gel electrophoresis. One of the isoenzymes could be identified as having a molecular weight of 26 625 Da. Copyright © 2005 Society of Chemical Industry     

A preliminary study of monosaccharide composition and α‐glucosidase inhibitory effect of polysaccharides from pumpkin (Cucurbita moschata) fruit

In this work, crude polysaccharide extracts were extracted from pumpkin (Cucurbita moschata) fruit by hot water. After removal of proteins and purification, polysaccharides of pumpkin fruit (PP1‐1) were subjected to structural identification. Gas chromatography analysis indicated that PP1‐1 comprised of galactose (86.4%), and glucose (13.6%). The molecular weight of PP1‐1 was measured to be 0.87 × 10⁴ Da by gel permeation chromatography. The inhibitory kinetic evaluation showed that it was non‐competitive inhibition of PP1‐1 on the α‐glucosidase‐catalysed hydrolysis of PNPG. The Michaelis–Menten constant (K_m) was 0.106 m , and the inhibitory constants (K_i), 0.435 mg.     

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     

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     

APPLICATION OF A NEW CONTINUOUS FLOW SPECTROPHOTOMETRIC METHOD FOR THE CHARACTERIZATION OF POLYPHENOL OXIDASE NATURALLY IMMOBILIZED ON COCONUT FIBER

The association of continuous flow injection and spectrophotometry affords a simple, novel and rapid way of monitoring continuously the activity of naturally immobilized enzymes in their natural environment, thus eliminating cumbersome purification. The method was applied to determine the activity of polyphenol oxidase (PPO) enzymes naturally immobilized on coconut (Cocus nucifera, L.) fiber tissues. Maximum enzyme activity occurred at a temperature of 25C and at pH 6.0 using catechol as substrate. Thermal stability was assayed in a temperature range of 20 to 75C. The PPO exhibited excellent thermal stability, with only 50% loss in its activity at 75C after 4.3 min exposure. For catechol apparent Michaelis‐Menten constant (apparent Km), apparent Vmax and the apparent activation energy were 9.1 × 10^?3 mol L^?1, 0.20 abs min^?1 and 10.5 kcal mol^?1, respectively. The immobilized PPO showed high activity for o‐diphenols. The reactivity order was caffeic acid > pyrogallol > catechol. Complete inhibition of the enzyme was observed with 1 × 10^?3 mol L^?1 concentration of cyanide, thiourea, L‐cysteine, ascorbic acid, sodium sulfite, nitrates of cadmium, zinc and mercury, individually. Benzoic acid, 3‐hydroxy‐benzoic acid, 4‐acetamidephenol, sodium azide, resorcinol, L‐cystine and EDTA at equal concentrations inhibited PPO partially.     

Enzymolysis reaction kinetics and thermodynamics of rapeseed protein with sequential dual‐frequency ultrasound pretreatment

We evaluated the effect of sequential dual‐frequency ultrasound (SDFU) pretreatments on rapeseed protein enzymolysis, using alcalase as a model enzyme. Hydrolysed protein concentrations, enzymolysis kinetics and thermodynamic parameters were investigated. The results showed that the hydrolysed rapeseed protein concentration following SDFU pretreatments was higher compared to that of the control for up to 75 min of enzymolysis at various substrate concentrations of 5–25 g L^?1; both control and SDFU pretreatment groups showed first‐order reaction kinetics. Compared to the control, the Michaelis–Menten constant (K_M) value decreased remarkably by 17.61%, while an increase in the binding frequency between enzyme and substrate (K_A) by 10.47% was observed. The thermodynamic parameters, enthalpy, entropy and activation energy were reduced in the SDFU pretreatment group compared to the control by 31.78%, 18.0% and 29.56%, respectively. SDFU pretreatment showed little effect on Gibbs free energy at the various temperatures studied.     

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.     

Latent polyphenol oxidase from quince fruit pulp (Cydonia oblonga): purification,activation and some properties

Quince fruit polyphenol oxidase (PPO) was partially purified using a combination of phase partitioning in Triton X‐114 and PEG 8000/phosphate with a final ammonium sulfate fractionation between 30% and 75%, to avoid the deep browning of the enzyme due to the high amount of oxidizing substances present in the quince pulp. The clean and stable enzyme was partially purified in a latent form and could be optimally activated by the presence of 0.5 g dm^?3 sodium dodecyl sulfate (SDS) with an optimum pH of 5.0. In the absence of SDS, the enzyme showed maximum activity at acid pH. The apparent kinetic parameters of the latent enzyme were determined at pH 5.0, the V_m value being 15 times higher in the presence of SDS than in its absence, whereas the K_M was the same in both cases, with a value of 1.2 mmol L^?1. The effect of several inhibitors was studied, tropolone being the most active with a K_i value of 4.7 µmol L^?1. In addition, the effect of cyclodextrins was studied, and the complexation constant (K_c) between 4‐tert‐butylcatechol and cyclodextrins was calculated using an enzymatic method. The value obtained for K_c was 15 310 mol L^?1. Copyright © 2006 Society of Chemical Industry     

Inhibition of polyphenol oxidase and the browning control of litchi fruit by glutathione and citric acid

Polyphenol oxidase (PPO, EC 1.10.3.2) from litchi peel was partially purified by ammonium sulfate fractionation and gel filtration, and a 16-fold purification of PPO achieved. The use of 10 mmol litre⁻¹ glutathione and 100 mmol litre⁻¹ citric acid was found to give good control of the browning of litchi fruit and 80–85% inhibition of PPO observed. Application of glutathione in combination with citric acid is recommended as a way of slowing the browning of litchi fruit.     

Purification of polyphenol oxidase and the browning control of litchi fruit by glutathione and citric acid

Polyphenol oxidase (PPO, EC 1.10.3.2) was purified to homogeneity from litchi peel yielding a single protein with a molecular weight of about 75.6 kD by Sephadex G‐100 gel filtration, and a 108‐fold purification of PPO achieved. The enzyme was determined to be composed of two similar subunits. Glutathione, L ‐cysteine and citric acid suppressed PPO activity markedly, whereas ascorbic acid and n‐propyl gallate showed a little inhibition. Moreover, the effect was enhanced by the addition of citric acid. On the basis of the inhibition of PPO activity in vitro, the use of 10 mmol l ⁻¹ glutathione and 100 mmol⁻¹ l citric acid was found to give good control of the browning of litchi fruit, and an 80–85% inhibition of PPO activity was observed. It is suggested that application of glutathione in combination with citric acid may slow down the browning of litchi fruit. © 1999 Society of Chemical Industry     

Microwave-assisted digestion of β-lactoglobulin by pronase, α-chymotrypsin and pepsin

The effects of microwave irradiation (MWI) on kinetic parameters for pronase, α-chymotrypsin and pepsin hydrolysis of bovine β-lactoglobulin (β-Lg) were evaluated. The experiments were performed under MWI or conventional heat (CH) at 40 °C. The initial velocity (V₀) of peptide bonds cleavage was measured by o-phthaldialdehyde method; the peptide profile was analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE). Higher catalytic effectiveness (K_cat K_m⁻¹) values were obtained in the pronase and α-chymotrypsin digestions performed under MWI (7793 and 2073 min⁻¹ mM⁻¹, respectively) in comparison with the values in the respective CH digestions (1802 and 941 min⁻¹ mM⁻¹, respectively). The Michaelis–Menten constant (K_m) for either enzyme was reduced under MWI. Pepsin showed very low activity on β-Lg at pH 4.0 regardless of the heating procedure used. For two enzymes, pronase and α-chymotrypsin, differences in SDS–PAGE profiles were obtained due to the MWI applied during the enzymatic hydrolysis. The combined enzyme/MWI treatments could have a relevant application in the development of β-Lg hydrolysates.     

Antioxidant activity of anacardic acids

Anacardic acids, 6-pentadec(en)ylsalicylic acids isolated from the cashew Anacardium occidentale L. (Anacardiaceae) nut and apple, were found to possess preventive antioxidant activity while salicylic acid did not show this activity. These anacardic acids prevent generation of superoxide radicals by inhibiting xanthine oxidase (EC 1.1.3.22, Grade IV) without radical-scavenging activity. Notably, the inhibition kinetics of anacardic acids do not follow hyperbolic dependence of enzyme inhibition on inhibitor contents (Michaelis–Menten equation) but follow the Hill equation instead. Anacardic acid (C_15:1) inhibited the soybean lipoxygenase-1 (EC 1.13.11.12, Type 1) catalyzed oxidation of linoleic acid with an IC₅₀ of 6.8 μM. The inhibition is a slow and reversible reaction without residual enzyme activity. The inhibition kinetics indicate that anacardic acid (C_15:1) is a competitive inhibitor and the inhibition constant, K_I, was 2.8 μM. Anacardic acids act as antioxidants in a variety ways, including inhibition of various prooxidant enzymes involved in the production of the reactive oxygen species and chelate divalent metal ions such as Fe²⁺ or Cu²⁺, but do not quench reactive oxygen species. The C₁₅-alkenyl side chain is largely associated with the activity.     

Effects of α-, β- and maltosyl-β-cyclodextrins use on the glucoraphanin–sulforaphane system of broccoli juice

Cyclodextrins (CDs) are macromolecules with several industrial applications, being particularly used in the food industry as health-promoting compounds protection agents, as flavour stabilizers, or to eliminate undesired tastes and browning reactions, among others. This study shows the effects of α- (10, 30 and 40 mmol L⁻¹), β- (3, 6 and 10 mmol L⁻¹) and maltosyl-β-CDs (30, 60 and 90 mmol L⁻¹) use on the health-promoting glucoraphanin–sulforaphane system of a broccoli juice up to 24 h at 22 °C. Maltosyl-β-CD (90 mmol L⁻¹) highly retained glucoraphanin content after 24 h at 22 °C, showing better effectiveness than β-CD (10 mmol L⁻¹). Sulforaphane was efficiently encapsulated with β-CD at just 3 mmol L⁻¹, and the sulforaphane formed was stable during 3 h at 22 °C. On the other hand, 40 mmol L⁻¹ α-CD retained a high glucoraphanin content in broccoli juice. In contrast, glucoraphanin levels in juice without CDs decreased by 71% after 24 h. Consequently, CDs addition may potentially preserve glucoraphanin in this broccoli juice during industrial processing with the possibility to be later transformed by endogenous myrosinase after ingestion to the health-promoting sulforaphane. © 2018 Society of Chemical Industry     

Batch kinetics and modelling of ethanolic fermentation of whey

The fermentation of whey by Kluyveromyces marxianus strain MTCC 1288 was studied using varying lactose concentrations at constant temperature and pH. The increase in substrate concentration up to a certain limit was accompanied by an increase in ethanol formation, for example, at a substrate concentration of 10 g L^?1, the production of ethanol was 0.618 g L^?1 whereas at 50 g L^?1 it was 3.98 g L^?1. However, an increase in lactose concentration to 100 g L^?1 led to a drastic decrease in product formation and substrate utilization. The maximum ethanol yield was obtained with an initial lactose concentration of 50 g L^?1. A method of batch kinetics was utilized to formulate a mathematical model using substrate and product inhibition constants. The model successfully simulated the batch kinetics observed at S₀ = 10 and 50 g L^?1 but failed in case of S₀ = 100 g L^?1 because of strong substrate inhibition.     

Evaluation of Models for Spinach Respiratory Metabolism Under Low Oxygen Atmospheres

Anaerobic respiration is a major problem that causes the deterioration of fresh produce packaged under low O₂ atmospheres. The problem becomes more severe and causes high losses in the packages handling at ambient conditions, especially in developing countries. In designing modified atmosphere packaging, the risk of anaerobic development greatly depends upon the accuracy of respiration rate prediction; therefore, the respiration rate model for a particular produce has to be identified. In this study, different atmospheric storage conditions in a closed system were realized to examine the adaptability of respiration rate models for spinach storage under low O₂ at an expected ambient temperature of 25 °C. Six models were applied and it was found that, for aerobic conditions, the respiration rate could be described with a constant respiratory quotient by three models, viz., (a) Michaelis–Menten model without inhibition, (b) Michaelis–Menten model with uncompetitive inhibition, and (c) Langmuir adsorption model, whereas three other models, viz. (d) Michaelis–Menten model with competitive inhibition, (e) Michaelis–Menten model with noncompetitive inhibition, and (f) Michaelis–Menten with mixed inhibition could not be fitted. Among the three successful models, the Michaelis–Menten with uncompetitive inhibition was found to be the most suitable model for practical applications in developing countries where cold-chain systems are lacking. This model can be applied for the prediction of gas composition and optimize the packages, particularly to ensure the aerobic respiration.