Extraction Of Polyphenol Oxidase Research Articles

Evaluation of blue textile dye decolorization by immobilized polyphenol oxidase using pumice stone under optimum conditions

Industrial dyes are major pollutants in wastewater and river water with an initial visible concentration of 1 mg/L. Recent studies have shown the possibility of using polyphenol oxidase in catalytic biological treatment due to its ability to oxidize a large number of dyes and pollutants in wastewater and the flexibility to work in wide ranges of temperature, pH and salinity. It is easy availability as well as the low economic cost resulting from its use in biological treatments, this enzyme polyphenol oxidase was used. The findings in this study showed that the extraction of polyphenol oxidase (PPO) from potato peel was homogenized with potassium phosphate buffer (0.1 M, pH 7) at a ratio of 1:10 (weight: volume) for two min. The result of enzyme purification by ion exchange chromatography showed that the yield of this step was 64 % and the specific activity was 6541.67 U/mg. The polyphenol oxidase was immobilized covalently on the functionalized pumice stone (25.2 U/gm) compared with other methods. The characterization results demonstrated that the optimum pH for immobilized and free enzyme activity was 6.0 while the pH range of free and immobilized polyphenol oxidase stability was from 4.5 -7.0 and 3.5 - 8.5 respectively. The better temperature for free and immobilized polyphenol oxidase activity was 25 ºC, whereas the free and immobilized polyphenol oxidase was stable at 15-35 and 15-50 °C respectively. The outcomes showed that the decolorization efficiency of blue textile dye employing immobilized polyphenol oxidase reached 99.85 % after 24 hr. for 100 mg/L concentration while for other concentrations 200, 300, 400, 500 and 1000 mg/L the decolorization efficiencies were 85.96, 76.15, 72.54, 66.94 and 63.5 % respectively. Based on the results, the immobilized polyphenol oxidase on pumice stone is highly efficient in removing textile dyes at large concentrations and in different environmental conditions.

Preparation of rodenticide for (Sprague Dawley Rats Rattus rattus) from the residue of tin adsorption arriving to Zero Residue Level (ZRL)

This paper is deal with study the potential of Banana Peel Residue (after extraction of Polyphenol Oxidase (PPO) enzyme) to remove tin (Sn+2) ions from simulated synthesis aqueous solutions (SSAS) through various operating parameters using adsorption technique in sorption unit. Results show that the removal efficiency were 94.55% for tin ions from (SSAS) and the removal efficiency decreased with increasing of initial concentration, pH and SSAS flow rate while the removal efficiency increased with increasing packing height of adsorbent material and treatment time. The waste of banana peel residue (remaining after adsorbed tin) was predestined to investigate the utilization of it in useful method. It can prepare a cheap rodenticide from these BPR waste without any further treatment. The results explain that the BPR adsorb chromium ions showed a good behaviour as rodenticide for sprague dawley rats Rattus rattus. By this way it can perform different benefits which are: remove the chromium ions polluted the water, get rid of agricultural waste banana peel residue, in the same time, prepare a cheap and active rodenticide. It can discard more type of waste in a non-cost and eco-friendly method accessing to zero residue level (ZRL).

Effect of Ultrasound on the Activity of Mushroom (Agaricus bisporous) Polyphenol Oxidase and Observation of Structural Changes Using Time-resolved Fluorescence

Polyphenol oxidase (PPO) is an enzyme associated with the browning process that can occur from mechanical injury and postharvest storage. Thus, its inactivation to inhibit this process is of great interest to the food industry. Recently a nonthermal technology, high frequency ultrasound (100–1 MHz), has found usage in this aspect. In this work, the application of high-frequency (378 kHz, 583 kHz, 1144 kHz, and 1175 kHz) and low frequency (20 kHz) treatment on a PPO extract (from mushrooms) by monitoring the residual enzymatic activity is described. A control thermal treatment at 40 °C was also performed for comparison purposes. High-frequency inactivation data fitted well using the Weibull model, whereas those obtained upon low frequency followed first-order kinetics. The inactivation rate constant obtained ranged from 0.0054 (20 kHz) to 0.028 min−1 (at 583 kHz). To elucidate changes in the enzyme structure time-resolved spectroscopy of a commercial PPO enzyme model was employed. Results indicated that ultrasound-induced structural changes in PPO, in keeping with the activity behaviour upon sonication.

Modification of Cu2+ in polyphenol oxidase extract from purple eggplant for phenol degradation in coal wastewater treatment

Cracking coal-forming organic compounds during the gasification process produces liquid waste containing phenolic compounds that require special handling based on their toxicity. As one of the components, there is liquid waste resulting from the coal gasification process. The purpose of this research was to study the activity of polyphenol oxidase (PPO) on phenol after the addition of Cu2+ to purple eggplant (Solanum melongena L.) extract and its potential to work more effectively in phenol biodegradation for coal wastewater containing phenol. Enzyme activity and phenol determination were carried out spectrophotometrically. The results showed PPO activity of 25.90-38.10 U/mL; 4.0 mM phenol and the activity of PPO-Cu2+ was 21.58-46.32 U/mL; 2-10 mM CuSO4; 2.0-4.0 mM phenol. Based on Michaelis Menten’s graph, the initial rate of PPO-Cu2+ was 0.015 mM/min and the initial rate of PPO was 0.15 mM/min using 2 mM phenol as a substrate. Lineweaver-Burk’s graph shows the KM of PPO-Cu2+ = 6.92 mM, which is lower than KM of PPO = 13.05 mM. Its means that the phenol response has a higher affinity for PPO-Cu2+ than PPO. The application of PPO-Cu2+ in purple eggplant extract works effectively as much as 46.7% for artificial coal liquid waste containing phenol.

Extraction and Characterization of Polyphenol Oxidase from Plant Materials: A Review

Polyphenol oxidase (PPO) is a copper-containing enzyme that can be used for different applications including wastewater treatment and biosensing. Given these wide arrays of application, the reaction and biochemical characteristics of the enzyme must be known to further determine its other applications and to control the essential factors during processing. The purpose of this research was to review the different factors that influence the effective extraction and characterization of PPO from plant materials. The pH of the extraction mixture, extraction temperature, type of buffer, mass to solvent ratio, extraction time, and additives are the factors that influence the effective extraction of PPO from plant materials. Since PPOs taken from different plant sources have varied protein structures, these factors have different effects during extraction. The isolated PPO from the extraction process can be characterized based on its activity as a function of pH, temperature, and type of substrate, and on the values of its kinetic parameters (Km and Vmax). PPO isolated from different plant sources shows varied optimum pH, optimum temperature, substrate affinity, and kinetic parameter values.

CONTROLLING OF POLYPHENOL OXIDASE-BASED BROWNING IN SELECTED FRUITS AND VEGETABLES

The activity of polyphenol oxidase (PPO) lowers the nutritive qualities, and hence lower the cost of the fruits and vegetables. The present study was designed to use different physical and chemical treatments in fruits and vegetables for inactivation of PPO. The crude PPO was extracted from selected fruits (Apple, Banana, Mango, Grapes, Peach) and vegetables (Potato, Eggplant, and Lettuce) using standard protocols and PPO activity was recorded with spectrophotometer using catechol as substrate. Physical treatments were performed at 4C, 45C and 5C for 20 minutes. The chemical treatments were performed using EDTA (1mg/L), ascorbic acid (0.5%), sodium chloride (1g/L), citric acid (0.75%) and honey (10% and 50%). Results indicated that PPO inhibition was maximum in banana (20.699%) at 55C for 20 minutes. The PPO extract from mango showed maximum inhibition (75.930%) with 50% honey. In vegetables, the PPO extract of potato showed (38.731%) inhibition at 4�C for 20 minutes and (72.874%) with 50% honey. The results indicated that storage at 4�C and treatment with citric acid (0.75%), ascorbic acid (0.5%), and honey (10% and 50%) were most effective for reduction of enzymatic browning in plant products. Results of this study provide bases for the development of dual control strategies to prevent browning of fruits, vegetables and other food products.

Inactivation of Potato Polyphenol Oxidase Using Microwave Cold Plasma Treatment.

This study was conducted to examine the effects of microwave cold plasma (CP) treatment on inactivation of polyphenol oxidase (PPO) of potato. The PPO activity and treatment variables were fit to first-order kinetics, the Weibull model, and the second-order model. The optimum CP-generation power and treatment time for inactivating PPO in the PPO extract were found to be 900 W and 40 min, respectively, which resulted in the highest inactivation of PPO (49.5%). PPO activity after CP treatment of potato slices decreased from 72.4% to 59.0% as the sample surface-to-volume ratio increased from 7.1 to 9.0. CP treatment delayed the browning of potato slices. Microwave CP treatment effectively inactivated PPO in potatoes, demonstrating the potential of CP treatment for controlling PPO activity in foods. PRACTICAL APPLICATION: This study demonstrated that microwave CP treatment, a nonthermal food processing technology, inactivates PPO activity in potatoes. The results showed that the inactivation effect of CP treatment on PPO corresponded to the surface-to-volume ratio of potato slices. Furthermore, this study proposed an enzyme inactivation model that is suitable for predicting the inactivation of PPO activity and confirmed that CP treatment delayed browning in potatoes.

Effect of High Pressure Carbon Dioxide on polyphenoloxidase from Litopenaeus vannamei

The extraction of polyphenoloxidase (PPO) from Litopenaeus vannamei was studied at different temperatures, pH and ionic strength conditions. Optimum extracts at pH = 7.2 were treated by hiph pressure carbon dioxide (HPCD) technology at 20 MPa and in a temperature range from 35 to 50 °C. Inactivation kinetics for PPO extracts were determined and fitted to the Weibull model. The effect of HPCD on PPO activity was also evaluated in shrimps in vivo, finding that PPO could be easier inactivated than in the crude PPO extract. Color of Litopenaeus vannamei was visually followed right after HPCD treatment and thermal treatment at the same temperature and during storage at 4 °C during a week, showing good results when shrimps were treated with HPCD in the absence of water.

Purification and characterization of polyphenol oxidase (PPO) from water yam (Dioscorea alata)

ABSTRACTPolyphenol oxidase (PPO) is responsible for the enzymatic browning of fruits and vegetables, and thus has negative effects on the quality of food. In this study, extraction and purification of PPO from water yam was performed successfully, and the characteristics of PPO and inhibitors of enzyme activity were analysed. The results showed that PPO activity improved by 4.58-fold through the DEAE-Sepharose and Superdex G-75 purification technique, which reached electrophoretic purity. The enzyme molecular weight was estimated to be 32 kDa. The optimum pH and temperature values of PPO were 6.0 and 35°C, respectively, and it was not thermostable at higher temperatures. PPO had the highest substrate affinity towards catechol (Km = 27.82 mmol/L and Vmax = 1.464 △A/min). The most effective inhibitor was L-cysteine followed by ascorbic acid and sodium sulfite.

Protection of emulsified polyunsaturated fatty acids against in vitro ruminal biohydrogenation by polyphenol oxidase: Characterization of the cross-linked emulsion

In the current study, linseed oil emulsions were cross-linked by polyphenol oxidase to study the effect of emulsion characteristics on the protection of polyunsaturated fatty acids against biohydrogenation by rumen bacteria in vitro. Polyphenol oxidase (PPO) catalyzes the oxidation of diphenolic substrate to o-quinones which are reactive towards amino- and thiol- groups on proteins, forming a complex of covalently bound protein cross-linked by protein-bound phenols (PBP). Such cross-linking of protein-stabilized emulsions was shown to yield high in vitro ruminal protection of polyunsaturated fatty acids before, by addition of PPO extract and diphenolic substrate to protein-stabilized emulsions. The current study examined the relationship between characteristics of cross-linked emulsions and in vitro ruminal protection by evaluation of two hypotheses: protection efficiency would be impaired when 1) the amount of PPO in the emulsion decreased or 2) the amount of non-adsorbed protein in the emulsion increased. Hence, in a first experiment, emulsions containing the same protein concentration and varying amounts of PPO extract (0,2-0,8 g/g) were analyzed, while in experiment 2, emulsions containing varying amounts of non-adsorbed protein and a similar amount of PPO extract (0,8 g/g) were investigated. In the latter experiment, variation in non-adsorbed protein was obtained by altering the emulsifier concentration (10 or 0 mg/g whey protein isolate) and/or removal of the majority (>90%) of the non-adsorbed protein after emulsion preparation, i.e. washing of the emulsion, before addition of PPO extract. In vitro ruminal biohydrogenation was evaluated after 24 h in a rumen-simulated environment containing rumen inoculum. Results of experiment 1 showed protection efficiency decreased for decreasing amounts of PPO extract. Additionally, in vitro ruminal protection was found to be positively correlated to mean droplet size, protein load and proportion of PBP in the free protein fraction of the emulsions after cross-linking, while proportion of PBP in the adsorbed protein layer did not differ between treatments. In experiment 2, an increase in emulsifier concentration from 10 to 50 g/g WPI lowered the amount of PBP in both protein fractions. Washing of 50 mg/g WPI emulsions, enhanced in vitro ruminal protection of 18:3n-3 and 18:2n-6 and increased the proportion of PBP in both free and adsorbed protein. Moreover, protein load and the amount of total adsorbed PBP were positively affected by washing. Comparison of emulsions with similar protein load between experiments showed in vitro ruminal protection efficiency increased for higher proportions of PBP in the adsorbed protein layer. Moreover, an excess of PBP on the possible binding positions on the adsorbed protein seemed to promote in vitro ruminal protection efficiency.

Extraction and purification of polyphenol oxidase: A review

Abstract Polyphenol oxidase (PPO) is a copper containing enzyme widely occurred in many plants, animals, bacteria and some fungi. Polyphenol oxidase has wide applications in the field of biosensor formation, food industry, medicine and waste water treatment. Extraction and purification of PPO was studied by many researchers from wide variety of plants and fermentation process using various methods such as salt precipitation, three phase partitioning (TPP), aqueous two phase extraction (ATPE), reverse micellar extraction (RME) and chromatographic techniques. Physio-chemical characterization of the same was performed by the researchers using different techniques. Diversity in the resources of the enzyme also made scientist to study its substrate specificity, optimized activity conditions and inhibitors in detail. Aim of this review is to summarize the detail study regarding extraction and purification of polyphenol oxidase performed by researchers from wide variety of sources.

Enzymatic synthesis, purification and in vitro antioxidant capacity of polyphenolic oxidation products from apple juice

5-O-Caffeoylquinic acid (CQA) and (−)-epicatechin (EC) commonly present in apple were oxidized with a polyphenoloxidase extract (PPO) from apple to produce oxidation products formed in the course of apple processing into juice. The oxidation products were characterized by RP-HPLC-ESI-MS. CQA homodimers and EC–CQA heterodimers were the main compounds produced as shown on the extracted ion LC-MS chromatograms of the corresponding deprotonated molecules at m/z 705 and 641, respectively. Thirteen fractions purified by semi preparative HPLC were obtained as pure compounds according to the MS spectrum. In vitro DPPH˙ radical scavenging activity of those purified oxidation products was evaluated and compared to the native parent molecules and Trolox. The results were discussed on the basis of structure–activity relationship. On a molar basis, heterodimers exhibited higher antioxidant potential than homodimers. However, they showed a lower activity than an equimolar mixture of the parent molecules. These results provide new information about antioxidant activities of polyphenolic oxidation products which have an impact on the nutritional value of apple juices and other apple-derived foods.

Purification and Characterization of Polyphenol Oxidase From Waste Potato Peel by Aqueous Two-Phase Extraction

Potato peel from food industrial waste is a good source of polyphenol oxidase (PPO). This work illustrates the application of an aqueous two-phase system (ATPS) for the extraction and purification of PPO from potato peel. ATPS was composed of polyethylene glycol (PEG) and potassium phosphate buffer. Effect of different process parameters, namely, PEG, potassium phosphate buffer, NaCl concentration, and pH of the system, on partition coefficient, purification factor, and yield of PPO enzyme were evaluated. Response surface methodology (RSM) was utilized as a statistical tool for the optimization of ATPS. Optimized experimental conditions were found to be PEG1500 17.62% (w/w), potassium phosphate buffer 15.11% (w/w), and NaCl 2.08 mM at pH 7. At optimized condition, maximum partition coefficient, purification factor, and yield were found to be 3.7, 4.5, and 77.8%, respectively. After partial purification of PPO from ATPS, further purification was done by gel chromatography where its purity was increased up to 12.6-fold. The purified PPO enzyme was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by Km value 3.3 mM, and Vmax value 3333 U/mL, and enzyme stable ranges for temperature and pH of PPO were determined. These results revealed that ATPS would be an attractive option for obtaining purified PPO from waste potato peel.

First extraction of polyphenol oxidase from edible desert truffle (Terfezia leonis Tul.) and its thermal behavior

Polyphenol oxidase (PPO) is a common enzyme with large applications in food processing and analysis, especially based on their monophenolase activity. In this context, extraction and surfactant-mediated activation of PPO from desert truffle Terfezia leonis Tul. were successfully achieved. In the presence of l-tyrosine, the cresolase activity was optimal in the pH 5–6 domain and in the 35–45 °C temperature range. In the presence of pyrocatechol, the catecholase activity was optimal at neutral pH and 30 °C. Kinetics studies revealed higher affinity of PPO for l-tyrosine than for pyrocatechol. Both enzyme–substrate complexes were structurally robust, and their thermosensitivity was mainly related to entropy changes. These properties may reflect the adaptation to desert conditions where T. leonis grows and should be useful for the development of enzymatic catalysts and sensors.

English

  Polyphenol oxidase (PPO) has been shown to be responsible for browning reactions and discolouration in many fruit and vegetable products. Crude polyphenol oxidase (PPO) of ripe and unripe plantain (Musa paradisiaca cultivars – Agbagba, Cardaba and plantain hybrid) was isolated and some of the characteristics of the PPO extracts investigated. The activity of the enzyme was evaluated using spectrophotomeric method. Plantain PPO (ripe and unripe) catalyzes oxidation of both various substrates with catechol being the most readily oxidized substrate. The optimum pH of the enzyme was between pH 6 and 7 for the three cultivars. Thermal inactivation data showed that activity of the enzyme was lost after heating for 15 and 4 min at 50 and 80°C, respectively. Browning reactions of plantain PPO were inhibited by sodium metabisulphite (0.1%), ascorbic acid (0.2%), malic acid (1.0%) and the least sodium chloride (>1.0%) which is similar to banana PPO. Plantain polyphenol oxidase was active towards catechol but not with cresol.   Key words: plantain (Musa paradisiaca) cultivars, polyphenol oxidase, catechol, enzyme activity

An Improved Method of Optimizing the Extraction of Polyphenol Oxidase from Potato (Solanum tuberosum L.) Peel

The present study has an objective to optimize the extraction of Polyphenol Oxidase (PPO) from potato ( Solanum tuberosum L.) peel. Response surface methodology (RSM) was used to design experiments and study the effect of six influential extraction parameters: extraction buffer concentration (100-500 mM), pH of extraction buffer (4.5-8.5), time (1-12 hours), temperature (4-40°C), concentration of PMSF (1-5 mM) and volume of extraction buffer (200-1000 ml) on the extraction of PPO. The dependent variable was considered as response function which was specific activity (SA) of extracted PPO. ANOVA was performed to obtain the regression equation that could predict the responses within given range. From RSM generated model, the optimum conditions for the maximum extraction of PPO were phosphate buffer concentration of 100 mm, buffer pH of 4.5, extraction time of 1 hour, 40°C temperature, PMSF concentration of 5 mM and buffer volume of 200 ml. Finally, this study illustrates a cost effective and less time consuming method to maximize the extraction of PPO from a vegetable waste.

Inactivation of polyphenol oxidase from Pacific white shrimp by dense phase carbon dioxide

The inactivation of polyphenol oxidase (PPO) from Pacific white shrimp exposed to dense phase carbon dioxide (DPCD) treatment was investigated. PPO activity showed a dramatic loss at 4–25 MPa and 37 °C. At the lower pressure (4–15 MPa), the experimental data of inactivation followed the first-order reaction kinetic model, the pressure sensitivity (ZP) of the kinetic parameters was 49.02 MPa and the activation volume (△ V≠) was − 120.88 cm3/mol. At the higher pressure (20 and 25 MPa), the experimental data of inactivation followed the biexponential kinetic model. The kinetic rate constant kF and kS of fast and stable fractions were 2.45 and 0.08 min− 1, respectively. The decimal reduction time DF and DS were 0.94 and 29.43 min at 25 MPa and 37 °C, respectively. After DPCD treatment, the loss activity of PPO had no restoration storing for 6 days at 4 °C. The results of SDS-PAGE and activity staining also showed that DPCD treatment had the obvious inhibitory effect on PPO from Pacific white shrimp. The PPO activity in vivo was easier to be inactivated than that in crude PPO extracts under the same DPCD treatment conditions.

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.

Use of Experimental Design Methodology To Prepare Maillard Reaction Products from Glucose and Cysteine Inhibitors of Polyphenol Oxidase from Eggplant (Solanum melongena)

Polyphenol oxidase (PPO) from eggplant was extracted and partially purified by a two-step fractionation-precipitation using ammonium sulfate and phenylsepharose hydrophobic interaction chromatography. The eggplant PPO extract was characterized concerning its kinetic properties. Optimal conditions to obtain Maillard reaction products (MRPs) with a maximal inhibitory potency (IP) toward PPO activity were determined using the surface response methodology and a four-factor and five-level experimental design. The MRPs were prepared from cysteine (0.25 M) and glucose (0-1 M), at several initial pH values (2-6) and at differing heating times (3-19 h) and temperatures (95-115 degrees C). The maximal IP was obtained after heating a model system of glucose/cysteine (1/0.25 M) at pH 2 for 3 h 20 min at 115 degrees C. The soluble part of this MRP, called MRP(IPmax), was a noncompetitive inhibitor toward eggplant PPO. The IP of MRP(IPmax) on PPO activity was very potent as compared to that displayed by benzoic, p-coumaric, and t-cinnamic acids, as well as sorbic acid and 4-hexylresorcinol. The activity of preincubated PPO at 0 degrees C with MRP(IPmax) was only slightly restored after dialysis or gel filtration.

Partial characterization of peroxidase and polyphenol oxidase activities in blackberry fruits.

A partial characterization of peroxidase (POD) and polyphenol oxidase (PPO) activities in blackberry fruits is described. Two cultivars of blackberry (Wild and Thornless) were analyzed for POD and PPO activities. Stable and highly active POD and PPO extracts were obtained using insoluble poly(vinylpyrrolidone) and Triton X-100 in 0.05 M sodium phosphate, pH 7.5, buffer. Blackberry POD and PPO activities have a pH optimum of 6.5, in a reaction mixture of 0.2 M sodium phosphate. Optimal POD activity was found with 3% o-dianisidine. Maximum PPO activity was found with catechol (catecholase activity) followed by 4-methylcatechol. Polyacrylamide gel electrophoresis of blackberry extracts under non-denaturing conditions resolved in various bands. In the POD extracts of Wild fruits, there was only one band with a mobility of 0.12. In the Thornless POD extracts there were three well-resolved bands, with R(f) values of 0.63, 0.36, and 0.09. Both the Wild and Thornless blackberry cultivars produced a single band of PPO, with R(f) values of 0.1 for Wild and 0.06 for Thornless.