Browning inhibition in fresh-cut Chinese water chestnut under high pressure CO2 treatment: Regulation of reactive oxygen species and membrane lipid metabolism.

Fresh-cut Chinese water chestnut (CWC) was treated with high pressure CO2 (HPCD) to inhibit the browning reactions, and the underlying mechanism was investigated in this study. Results showed that HPCD at 2 MPa pressure significantly inhibited lipoxygenase activity and enhanced superoxide dismutase activity, leading to decreased malondialdehyde and H2O2 contents in surface tissue. Moreover, HPCD could reduce total phenols/flavonoids content of surface tissue. Compare with control, homoeriodictyol, hesperetin, and isorhamnetin contents of 2 MPa HPCD-treated samples on day 10 were reduced by 95.72%, 94.31%, and 94.02%, respectively. Furthermore, HPCD treatment enhanced antioxidant enzyme activities, and improved the O2- scavenging ability and reducing power of inner tissue. In conclusion, by regulating ROS and membrane lipid metabolism, HPCD treatment with appropriate pressure could retard the biosynthesis of flavonoids and enzymatic oxidation of phenolic compounds in surface tissue, and enhance antioxidant activity of inner tissue, thereby, delaying the quality deterioration of fresh-cut CWC.

Eugenol treatment delays the flesh browning of fresh-cut water chestnut (Eleocharis tuberosa) through regulating the metabolisms of phenolics and reactive oxygen species.

The potential mechanism behind the browning inhibition in fresh-cut water chestnuts (FWC) after eugenol (EUG) treatment was investigated by comparing the difference in browning behavior between surface and inner tissues. EUG treatment was found to inactivate browning-related enzymes and reduce phenolic contents in surface tissue. Molecular docking further confirmed the hydrophobic interactions and hydrogen bonding between EUG and phenylalanine ammonia-lyase (PAL). Moreover, EUG also enhanced reactive oxygen species (ROS)-scavenging enzyme activities, ultimately decreasing the O2 - generation rates. Regarding inner tissue, EUG induced the accumulation of colorless phenolic compounds and increased the antioxidant capacity. In conclusion, 1.5 % EUG exhibited the best inhibitory effect on FWC browning, which partly attribute to the direct inhibitory effects on PAL activity. Furthermore, EUG could also enhance the enzymatic/non-enzymatic antioxidant capacity and alleviate the ROS damage to membranes, thereby, preventing the contact between oxidative enzymes and phenols and indirectly inhibiting the enzymatic browning in FWC.

Changes in Browning Degree and Reducibility of Polyphenols during Autoxidation and Enzymatic Oxidation.

In the present study, the browning degree and reducing power of browning products of catechin (CT), epicatechin (EC), caffeic acid (CA), and chlorogenic acid (CGA) in autoxidation and enzymatic oxidation were investigated. Influencing factors were considered, such as pH, substrate species and composition, and eugenol. Results show that polyphenols' autoxidation was intensified in an alkaline environment, but the reducing power was not improved. Products of enzymatic oxidation at a neutral pH have higher reducing power than autoxidation. In enzymatic oxidation, the browning degree of mixed substrates was higher than that of a single polyphenol. The reducing power of flavonoid mixed solution (CT and EC) was higher than those of phenolic acids' (CA and CGA) in autoxidation and enzymatic oxidation. Eugenol activity studies have shown that eugenol could increase autoxidation browning but inhibit enzymatic browning. Activity test and molecular docking results show that eugenol could inhibit tyrosinase.

Enzymatic, Phyto-, and Physicochemical Evaluation of Apple Juice under High-Pressure Carbon Dioxide and Thermal Processing.

In this study, the changes in enzyme activities, total polyphenols, phenolic profile, and physicochemical properties from thermally (25-75 °C) and high-pressure carbon dioxide (HP-CO2) (25-65 °C/20 MPa)-treated apple juice were investigated. The HP-CO2 exhibited complete inactivation of polyphenol oxidase (PPO) at 65 °C, whereas PPO was still active at 75 °C under thermal processing (TP). Similarly, the relative activity of peroxidase (POD) significantly decreased by 71% at 65 °C under HP-CO2 processing, whereas TP was less effective. HP-CO2 and TP treatments at 65 °C reduced the browning degree (BD) value to 0.47 and 0.89, respectively. Thus, HP-CO2 inhibits the browning reactions caused by PPO and POD enzymes at each operating temperature. The concentration of epicatechin and catechin increased significantly with increasing temperature above 45 °C in TP-treated juices. HP-CO2 treatment increased the same phenolic compounds at 35 °C and 9 MPa, whereas high-temperature and -pressure conditions caused insignificant changes in concentration of epicatechin and catechin. Changes in others phenolic compounds were insignificant under TP and HP-CO2 treatment. Overall, HP-CO2 is a promising technology to get high-quality juices with lower enzyme activity.

Inactivation and structural changes of polyphenol oxidase in quince (Cydonia oblonga Miller) juice subjected to ultrasonic treatment.

BACKGROUND: Polyphenol oxidase (PPO) is considered a problem in the food industry because it starts browning reactions during fruit and vegetable processing. Ultrasonic treatment is a technology used to inactivate the enzyme; however, the mechanism behind PPO inactivation is still unclear. For this reason, the inactivation, aggregation, and structural changes in PPO from quince juice subjected to ultrasonic treatments were investigated. Different intensities and times of ultrasonic treatment were used. Changes in the activity, aggregation, conformation, and structure of PPO were investigated through different structural analyses. RESULTS: Compared to untreated juice, the PPO activity in treated juice was reduced to 35% at a high ultrasonic intensity of 400 W for 20 min. The structure of PPO determined from particle size distribution (PSD) analysis showed that ultrasound treatment caused initial dissociation and subsequent aggregation leading to structural modification. The spectra of circular dichroism (CD) analysis of ultrasonic treated PPO protein showed a significant loss of α-helix, and reorganization of secondary structure. Fluorescence analysis showed a significant increase in fluorescence intensity of PPO after ultrasound treatment with evident blue shift, revealing disruption in the tertiary structure. CONCLUSION: In summary, ultrasonic treatment triggered protein aggregation, distortion of tertiary structure, and loss of α-helix conformation of secondary structure causing inactivation of the PPO enzyme. Hence, ultrasound processing at high intensity and duration could cause the inactivation of the PPO enzyme by inducing aggregation and structural modifications. © 2019 Society of Chemical Industry.

Ultrasonic Processing Induced Activity and Structural Changes of Polyphenol Oxidase in Orange (Citrus sinensis Osbeck).

Apart from non-enzymatic browning, polyphenol oxidase (PPO) also plays a role in the browning reaction of orange (Citrus sinensis Osbeck) juice, and needs to be inactivated during the processing. In this study, the protein with high PPO activity was purified from orange (Citrus sinensis Osbeck) and inactivated by ultrasonic processing. Fluorescence spectroscopy, circular dichroism (CD) and Dynamic light scattering (DLS) were used to investigate the ultrasonic effect on PPO activity and structural changes on purified PPO. DLS analysis illustrated that ultrasonic processing leads to initial dissociation and final aggregation of the protein. Fluorescence spectroscopy analysis showed the decrease in fluorescence intensity leading to the exposure of Trp residues to the polar environment, thereby causing the disruption of the tertiary structure after ultrasonic processing. Loss of α-helix conformation leading to the reorganization of secondary structure was triggered after the ultrasonic processing, according to CD analysis. Ultrasonic processing could induce aggregation and modification in the tertiary and secondary structure of a protein containing high PPO activity in orange (Citrus sinensis Osbeck), thereby causing inactivation of the enzyme.

Catalytic and Structural Characterization of a Browning-Related Protein in Oriental Sweet Melon (Cucumis Melo var. Makuwa Makino).

Polyphenol oxidase (PPO) in plants plays an important role in browning reactions and may affect the quality of sweet melon products. In this study, a browning-related protein (BRP) with PPO activity was partially purified from oriental sweet melon (Cucumis melo var. makuwa Makino) by salt precipitation and column chromatography. The BRP possessed a high degree of identity with several chitinase proteins, particularly defense-related proteins, by MS identification. Pyrogallol was determined as the most appropriate substrate for BRP (Km = 0.04278 M). BRP exhibited extreme resistance under alkaline and high temperature conditions when pyrogallol was used as substrate. Polyacrylamide gel electrophoresis (PAGE) analysis indicated that BRP was a homo-dimer of two subunits and had a molecular weight of 37 kDa. Structural analysis indicated that the α-helix was the dominant conformation of BRP. The active site of the protein might be buried deeply in the protein, and BRP might be monodispersed in an aqueous system.

Aggregation and Conformational Changes in Native and Thermally Treated Polyphenol Oxidase From Apple Juice (Malus domestica).

This study investigated the effects of heat treatment after purification on dissociation, aggregation, and structural modification of polyphenol oxidase (PPO) activity from apple (Malus domestica) juice. PPO activity at the 70°C for 10 min was still activated and drastically decreased since 20-60 min with catechol and pyrogallol as substrate. Moreover, spectral results of fluorescence and circular dichroism (CD) indicated that increasing temperature for shorter and longer durations can cause reorganization of the secondary structure of PPO and demolished the native configuration of PPO respectively. Compared with native PPO, all thermally treated PPO showed reduced activity with gradually increasing particle size shift toward section III of some fully assembled proteins treated at 70°C for 10 min (2,670 nm). Polyacrylamide gel electrophoresis (PAGE) analysis also exhibited the increase in protein content at the 70°C for 10 min with molecular size 35 kDa (7.7 ± 0.016c). Hence, thermally treated juice subjected to purification at high temperature for a short time could induce the aggregation of protein and is not really effective for PPO inactivation. For PPO, higher degree of long duration can induce the inactivation of the enzyme after processing.

Effect of ultrasonic processing on the changes in activity, aggregation and the secondary and tertiary structure of polyphenol oxidase in oriental sweet melon (Cucumis melo var. makuwa Makino).

BACKGROUND: Polyphenol oxidase (PPO) mainly contributes to the browning reaction of fruits and vegetables and causes serious damage to the quality of sweet melon products. However, traditional methods to inactivate browning may induce more unexpected risks than ultrasonic processing. Meanwhile, there are no reports on the effect of ultrasound on PPO directly purified from sweet melon. RESULTS: The PPO in the original juice was less inactivated than the purified form when treated with ultrasound. As for purified PPO, superior to thermal treatment, less heat was needed to inactivate the PPO with ultrasonic treatment. At intensity lower than 200 W, ultrasound did not significantly affect the structure and activity of PPO (P > 0.05), and latent PPO was activated. At intensity higher than 200 W, ultrasound inactivated PPO, induced aggregation and dissociation of PPO particles and significantly decreased the α-helix structure content. CONCLUSION: Low-frequency high-intensity ultrasound caused an inactivation effect and conformational changes of purified PPO from oriental sweet melons. Changes in the PPO structure induced by ultrasound eventually inactivated the enzyme. Ultrasound may be a potential method to inactivate PPO in oriental sweet melons. © 2016 Society of Chemical Industry.

Inactivation, aggregation, secondary and tertiary structural changes of germin-like protein in Satsuma mandarine with high polyphenol oxidase activity induced by ultrasonic processing.

The inhibition of Polyphenol oxidase (PPO) in plants has been widely researched for their important roles in browning reaction. A newly found germin-like protein (GLP) with high PPO activity in Satsuma mandarine was inactivated by low-frequency high-intensity ultrasonic (20 kHz) processing. The effects of ultrasound on PPO activity and structure of GLP were investigated using dynamic light scattering (DLS) analysis, transmission electron microscopy (TEM), circular dichroism (CD) spectral measurement and fluorescence spectral measurement. The lowest PPO activity achieved was 27.4% following ultrasonication for 30 min at 400 W. DLS analysis showed ultrasound caused both aggregation and dissociation of GLP particles. TEM images also demonstrated protein aggregation phenomena. CD spectra exhibited a certain number of loss in α-helix structure content. Fluorescence spectra showed remarkable increase in fluorescence intensity with tiny blue-shift following ultrasonication. In conclusion, ultrasound applied in this study induced structural changes of GLP and eventually inactivated PPO activity.

Characterization of germin-like protein with polyphenol oxidase activity from Satsuma mandarine.

Polyphenol oxidases (PPOs) catalyzing the oxygen dependent oxidation of phenols to quinones are ubiquitously distributed in plants and are assumed to be involved in plant defense against pests and pathogens. A protein with high PPO activity was identified in Satsuma mandarine, extracted with Tris-HCl buffer, purified by salt precipitation and column chromatography, and characterized by mass spectrometry as germin-like protein (GLP), which belongs to pathogenesis related protein (PR) family. In the present study, the structure and enzymatic properties of GLP were characterized using spectroscopy methods. Based on native PAGE analysis, the molecular weight of GLP was estimated to be 108 kDa and GLP was identified as a pentamer containing five subunits of 22 kDa. The optimum pH and temperature for PPO catalyzing activity of GLP was 6.5 and 65°C, respectively. Kinetic constants were 0.0365 M and 0.0196 M with the substrates catechol and pyrogallol, respectively. The structural characterization of GLP provided better insights into the regions responsible for its PPO activity.

Enzyme inactivation in food processing using high pressure carbon dioxide technology.

High pressure carbon dioxide (HPCD) is an effective non-thermal processing technique for inactivating deleterious enzymes in liquid and solid food systems. This processing method avoids high temperatures and exerts a minimal impact on the nutritional and sensory properties of foods, but extends shelf life by inhibiting or killing microorganisms and enzymes. Indigenous enzymes in food such as polyphenol oxidase (PPO), pectin methylesterase (PME), and lypoxygenase (LOX) may cause undesirable chemical changes in food attributes, showing the loss in color, texture, and flavor. For more than two decades, HPCD has proved its effectiveness in inactivating these enzymes. The HPCD-induced inactivation of some microbial enzymes responsible for microbial metabolism is also included. This review presents a survey of the published knowledge regarding the use of HPCD for the inactivation of these enzymes, and analyzes the factors controlling the efficiency of HPCD and speculates on the underlying mechanism that leads to enzyme inactivation.

Aggregation and homogenization, surface charge and structural change, and inactivation of mushroom tyrosinase in an aqueous system by subcritical/supercritical carbon dioxide.

The subcritical/supercritical carbon dioxide (SS CO(2)) has gained considerable attention in green chemistry industry for its advantage as nontoxic, nonflammable, and inexpensive. The effects of SS CO(2) treatments on aggregation and homogenization, surface charge, secondary and tertiary structure, and activity of mushroom tyrosinase in an aqueous system were investigated using a number of methods including dynamic light scattering (DLS), zeta potential measurement, circular dichroism (CD) spectropolarimeter, and spectrofluorometer. With a treatment time of 20 min, three treatment temperatures (35, 45, and 55 °C) and four pressures (5, 8, 12, and 15 MPa) had been selected. The aggregation and homogenization of the globular protein particles was induced by SS CO(2) as suggested by the particle size distribution (PSD) patterns that were closely related to the pressure and temperature. The surface charge of the tyrosinase decreased following the SS CO(2) treatments, and its variation tendency shows a favorable consistency with that of its PSD patterns. The α-helix conformation in secondary structure and fluorescence intensity reflecting tertiary structure also decreased, together with the λ(max) red-shifted with the increasing pressure. The results also indicated that SS CO(2) could enhance inactivation effect of the temperature on the tyrosinase with its lowest residual activity being about 60% under the condition of 8 MPa, 55 °C, and 20 min treatment time. The loss in the activity of the tyrosinase was correlated to its aggregation and homogenization effect induced by SS CO(2), which led to the change of surface charge as well as secondary and tertiary structure.