Affinity gel synthesis from the p-aminobenzoic acid derivative 4-amino-2-methylbenzoic acid and purification of polyphenol oxidase from various plant sources.

The polyphenol oxidase (PPO) enzyme, which causes enzymatic browning, has been repeatedly purified from fruit and vegetables by affinity chromatography. In the present research, Sepharose 4B-l-tyrosine-4-amino-2-methylbenzoic acid, a novel affinity gel for the purification of the PPO enzyme with high efficiency, was synthesized. Additionally, Sepharose 4B-l-tyrosine-p-aminobenzoic acid affinity gel, known in the literature, was also synthesized, and 9.02, 16.57, and 28.13 purification folds were obtained for the PPO enzymes of potato, mushroom, and eggplant by the reference gel. The PPO enzymes of potato, mushroom, and eggplant were purified 41.17, 64.47, and 56.78-fold from the new 4-amino-2-methylbenzoic acid gel. Following their isolation from the new affinity column, the assessment of PPO enzyme purity involved the utilization of SDS-PAGE. According to the results from SDS-PAGE and native PAGE, the molecular weight of each enzyme was 50 kDa. Then, the inhibition effects of naringin, morin hydrate, esculin hydrate, homovanillic acid, vanillic acid, phloridzin dihydrate, and p-coumaric acid phenolic compounds on purified potato, mushroom, and eggplant PPO enzyme were investigated. Among the tested phenolic compounds, morin hydrate was determined to be the most potent inhibitor on the potato (Ki: 0.07 ± 0.03 µM), mushroom (Ki: 0.7 ± 0.3 µM), and eggplant (Ki: 4.8 ± 1.2 µM) PPO enzymes. The studies found that the weakest inhibitor was homovanillic acid for the potato (Ki: 1112 ± 324 µM), mushroom (Ki: 567 ± 81 µM), and eggplant (Ki: 2016.7 ± 805.6 µM) PPO enzymes. Kinetic assays indicated that morin hydrate was a remarkable inhibitor on PPO.

Anti-browning effect of Rosa roxburghii on apple juice and identification of polyphenol oxidase inhibitors.

Enzymatic browning control of cloudy fruit juice with natural substances has received much attention for improving its nutritional and commercial value. This study explored the anti-browning potential of Rosa roxburghii in apple juice. The anti-browning effects and mechanisms were evaluated by serial measurements of appearance, browning index, polyphenol oxidase (PPO) activity, UPLC-QE-Orbitrap-MS identification, inhibition kinetics and molecular docking. The results showed that Rosa roxburghii juice (0.25%-1.25% w/w) could effectively inhibit browning and PPO activity of apple juice. Ascorbic acid (1.67 g/100 g) as a reducing agent was a main anti-browning factor. Furthermore, seven phenolic compounds in Rosa roxburghii were screened as PPO inhibitors. Representative phenolic inhibitors induced mixed or competitive inhibition of PPO, mainly driven by hydrophobic forces and hydrogen bonds. This work demonstrates that Rosa roxburghii is a promising natural anti-browning ingredient to control the browning of cloudy apple juice due to abundant ascorbic acid and PPO inhibitors.

Anti-polyphenol oxidase properties of total flavonoids from young loquat fruits: inhibitory activity and mechanism.

This study investigated anti-polyphenol oxidase activity and mechanism of purified total flavonoids (PTF) from young loquat fruits. PTF remarkably inhibited the activity of polyphenol oxidase (PPO) with an IC50 value of 21.03 ± 2.37 µg/mL. Based on enzyme kinetics, PTF was found to be a potent, mixed-type, and reversible inhibitor of PPO. The fluorescence intensity of PPO was quenched by PTF through forming a PTF-PPO complex in a static procedure. Therefore, this study authenticated PTF as an efficient PPO inhibitor, which would contribute to their utilization in food industry.

Inhibitory mechanism of salicylic acid on polyphenol oxidase: A cooperation between acidification and binding effects.

Salicylic acid is generally considered to combine with polyphenol oxidase (PPO) to inhibit activity and enzymatic browning, while its acidification effect on PPO activity was usually neglected. In this study, the inhibitory mechanism of salicylic acid on PPO was examined from acidification and binding effects by altering the buffer conditions. As the buffer concentration increased, contribution of acidification decreased while the binding effect became more predominant. Salicylic acid exhibited competitive inhibition on PPO, inducing the changes in secondary structure with a reduction in α-helix. Molecular docking results showed that salicylic acid interacted with residues HIS61, HIS85, HIS259, HIS263 and VAL283 through hydrogen bond and hydrophobic interaction. Furthermore, acidic pH enhanced the binding of salicylic acid to PPO with lower binding energy, additional hydrogen bond and electrostatic interactions. Therefore, both acidification and binding effects were important for salicylic acid on PPO inhibition and enzymatic browning control in fruit and vegetables.

Kinetic, spectroscopic, and molecular docking studies on the inhibition of membrane-bound polyphenol oxidase from Granny Smith apples (Malus domestica Borkh.).

We investigated the inhibitory effect and binding mechanism of four selected compounds (ascorbic acid, l-cysteine, glutathione, and citric acid) on membrane-bound polyphenol oxidases (mPPO) using spectroscopic and molecular docking techniques. Kinetic analysis demonstrated that these inhibitors reversibly inhibited the mPPO activity. Fluorescence spectroscopy revealed that the intrinsic fluorescence intensity of mPPO was quenched by inhibitors with a single class of the inhibition site on mPPO. Amino acid residues His 180, His 201, His 366, Cys 184, Glu 328, and Asn 333 were the important binding sites in the active center. These sites were identified using molecular docking techniques. Our findings suggested that the inhibitors were allosterically bound to the active center of mPPO through hydrogen bonds and ion contacts. This study provides new insights into the active site residues responsible for catalyzing mPPO and provides applicable information about the design of mPPO inhibitors.

The effect of ultrasonic on reducing anti-browning minimum effective concentration of purslane extract on fresh-cut potato slices during storage.

Enzymatic browning is one of the major difficulties for the preservation and commercial value of fresh-cut products. To research more healthy and inexpensive anti-browning methods, we investigated the effect of ultrasonic coupling purslane extract on the browning resistance of fresh-cut potato during 8d storage at 4 °C. Firstly, the optimal ultrasonic time (10 min) was obtained. Then, the results showed that the combined application with lower purslane extract concentration (0.02%, w/w) could achieve a better anti-browning effect than the optimal concentration of alone purslane extract (0.05%, w/w). The combined application not only significantly inhibited the key enzyme activities of polyphenol oxidase (PPO) and peroxidase (POD), but also effectively reduced the damage to cell membrane, maintained its integrity and permeability. Meanwhile, it also improved antioxidant capacity during storage. Overall, the ultrasonic cavitation combined with purslane extract would be a promising method for fresh-cut industry.

Peptides based on the reactive center loop of Manduca sexta serpin-3 block its protease inhibitory function.

One innate immune response in insects is the proteolytic activation of hemolymph prophenoloxidase (proPO), regulated by protease inhibitors called serpins. In the inhibition reaction of serpins, a protease cleaves a peptide bond in a solvent-exposed reactive center loop (RCL) of the serpin, and the serpin undergoes a conformational change, incorporating the amino-terminal segment of the RCL into serpin ß-sheet A as a new strand. This results in an irreversible inhibitory complex of the serpin with the protease. We synthesized four peptides with sequences from the hinge region in the RCL of Manduca sexta serpin-3 and found they were able to block serpin-3 inhibitory activity, resulting in suppression of inhibitory protease-serpin complex formation. An RCL-derived peptide with the sequence Ser-Val-Ala-Phe-Ser (SVAFS) displayed robust blocking activity against serpin-3. Addition of acetyl-SVAFS-amide to hemolymph led to unregulated proPO activation. Serpin-3 associated with Ac-SVAFS-COO- had an altered circular dichroism spectrum and enhanced thermal resistance to change in secondary structure, indicating that these two molecules formed a binary complex, most likely by insertion of the peptide into ß-sheet A. The interference of RCL-derived peptides with serpin activity may lead to new possibilities of "silencing" arthropod serpins with unknown functions for investigation of their physiological roles.

Inhibitory Effect of Azamacrocyclic Ligands on Polyphenol Oxidase in Model and Food Systems.

Enzymatic browning is one of the main problems faced by the food industry due to the enzyme polyphenol oxidase (PPO) provoking an undesirable color change in the presence of oxygen. Here, we report the evaluation of 10 different azamacrocyclic compounds with diverse morphologies as potential inhibitors against the activity of PPO, both in model and real systems. An initial screening of 10 ligands shows that all azamacrocyclic compounds inhibit to some extent the enzymatic browning, but the molecular structure plays a crucial role on the power of inhibition. Kinetic studies of the most active ligand (L2) reveal a S-parabolic I-parabolic noncompetitive inhibition mechanism and a remarkable inhibition at micromolar concentration (IC50 = 10 µM). Furthermore, L2 action has been proven on apple juice to significantly reduce the enzymatic browning.

Recent Trends in Controlling the Enzymatic Browning of Fruit and Vegetable Products.

Enzymatic browning because of polyphenol oxidases (PPOs) contributes to the color quality of fruit and vegetable (FV) products. Physical and chemical methods have been developed to inhibit the activity of PPOs, and several synthetic chemical compounds are commonly being used as PPO inhibitors in FV products. Recently, there has been an emphasis on consumer-oriented innovations in the food industry. Consumers tend to urge the use of natural and environment-friendly PPO inhibitors. The purpose of this review is to summarize the mechanisms underlying the anti-browning action of chemical PPO inhibitors and current trends in the research on these inhibitors. Based on their mechanisms of action, chemical inhibitors can be categorized as antioxidants, reducing agents, chelating agents, acidulants, and/or mixed-type PPO inhibitors. Here, we focused on the food ingredients, dietary components, food by-products, and waste associated with anti-browning activity.

Tyrosinase Inhibition and Kinetic Details of Puerol A Having But-2-Enolide Structure from Amorpha fruticosa.

Puerol A (1) from Amorpha fruticosa showed highly potent inhibition against both monophenolase (IC50 = 2.2 µM) and diphenolase (IC50 = 3.8 µM) of tyrosinase. We tried to obtain a full story of enzyme inhibitory behavior for inhibitor 1 because the butenolide skeleton has never been reported as a tyrosinase inhibitor. Puerol A was proved as a reversible, competitive, simple slow-binding inhibitor, according to the respective parameters; k3 = 0.0279 µM-1 min-1 and k4 = 0.003 min-1. A longer lag-phase and a reduced static-state activity of the enzyme explained that puerol A had a tight formation of the complex with Emet. Dose-dependent inhibition was also confirmed by high-performance liquid chromatography (HPLC) analysis using N-acetyl-l-tyrosine as a substrate, which was completely inhibited at 20 µM. A high binding affinity of 1 to tyrosinase was confirmed by fluorescence quenching analysis. Moreover, puerol A decreased melanin content in the B16 melanoma cell dose-dependently with an IC50 of 11.4 µM.

Optimization of extraction of loquat flowers polyphenolics and its antioxidant and anti-polyphenol oxidase properties.

In this study, the conditions of extraction of loquat flowers polyphenolics were optimized through response surface methodology (RSM). Proper extraction conditions were: solid to liquid ratio 1 g per 50 mL and ethanol concentration 50% at 61°C for 9 min. Furthermore, the antioxidant and anti-polyphenol oxidase (PPO) activity of purified total polyphenolics (PTP) were investigated. PTP displayed strong antioxidant activity with IC50 values of 126.3 ± 8.9, 162.4 ± 6.3 and 94.97 mg ascorbic acid equivalent/g dry weight (mg AAE/d.w.) for ABTS, DPPH, and FRAP assays. In addition, PTP has a substantial inhibitory activity on PPO (IC50 = 115 ± 9.2 µg/mL). From the kinetics analysis, it was proved to be a reversible and mixed-type inhibitor of PPO with KI and KIS values of 76.77 µg/mL and 227.86 µg/mL, respectively. Further, the molecular mechanism underlying the inhibition of PPO by PTP was investigated by molecular docking techniques. The results showed that PTP units could form interaction with the catalytic pocket of PPO through the interaction with amino acid residues in the enzyme active center. The antioxidant activities of PTP together with its effect on PPO activity provide a strong starting point for their practical usage in the food industry.

A new insight into purification of polyphenol oxidase and inhibition effect of curcumin and quercetin on potato polyphenol oxidase.

In the literature, the polyphenol oxidase (PPO) enzyme has been purified a many times via Sepharose 4B-l-tyrosine-p-aminobenzoic acid affinity column. In order to study PPO purification efficiency, 2-aminophenol and 4-aminophenol were applied as a spacer arm to CNBr-activated Sepharose 4B. The effects of the spacer arm on specific activity, purification fold, and electrophoretic properties were investigated. The best performance with 11.7-fold purification and 23951 U/mg protein specific activity was achieved with the 4-aminophenol extension arm. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with was done to check the purity of the potato PPO enzyme obtained from affinity columns. According to the results of SDS-PAGE and native PAGE, the molecular weight of the enzyme is 50 kDa. Furthermore, the inhibition effects of curcumin and quercetin on the enzyme activity were examined, and the IC50 and Ki values were computed for the mentioned substances. IC50 values were determined to be 0.018 and 0.029 mM for potato PPO with curcumin and quercetin inhibitors with catechol as a substrate, respectively. IC50 value was also determined to be 0.0086 mM for quercetin inhibitor with 4-methylcatechol as a substrate. Ki constant was 0.0753 ± 0.0085 mM for curcumin using catechol as a substrate. No inhibition effect was observed for curcumin with the 4-methylcatechol substrate. The Ki constant for quercetin was 0.0398 ± 0.00743 mM with the 4-methylcatechol substrate and 0.0109 ± 0.0021 mM with the catechol substrate.

Cysteine Protease Inhibitors Reduce Enzymatic Browning of Potato by Lowering the Accumulation of Free Amino Acids.

Enzymatic browning is a major issue affecting the quality of processed potato (Solanum tuberosum L.). To understand the molecular mechanism of browning, transcriptional analyses were performed by employing potatoes that differed in browning. Coexpression analysis indicated that 9 out of 15 upregulated genes in browning-less groups encoded for potato protease inhibitors (StPIs). In addition, gene otology analysis showed that the enriched terms were mainly involved in protease inhibitors. Overexpression of cysteine StPI 143 and StPI 146 individually reduced browning and lowered protease activities and tyrosine and total free amino acid (FAA) contents, but they could not decrease polyphenol oxidase activity. Moreover, supplementing exogenous tyrosine or total FAAs into transgenic potato mash to wild-type amounts promoted mash browning, browning with total FAAs, more than with tyrosine, resembling wild-type levels. These results implied that cysteine StPIs reduced browning via lowering the accumulation of FAAs in addition to tyrosine. Our findings have enriched the knowledge about the roles and mechanisms of protease inhibitors in regulating enzymatic browning of potato, which provide new ways for controlling potato browning.

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.

Effect of phlorotannins on melanosis and quality changes of Pacific white shrimp (Litopenaeus vannamei) during iced storage.

Effect of phlorotannins (PT) treatments on the ployhenoloxidase (PPO) activity and quality changes of Pacific white shrimp during a 16-day period of storage in ice were studied. Among seaweeds, Sargassum tenerimum had the highest amount of PT (10.00 mg phloroglucinol/g), PPO inhibitory activity (71.94%) and therefore selected for PT extraction. The shrimp treated with 5% PT (w/v) showed the least melanosis score, pH, TVB-N values and lipid oxidation among all treatments throughout the iced storage. Lower counts of mesophilic and psychrotrophic bacteria (1-2 log CFU/g) were obtained with 5% PT treatment compared to the control at the last day of storage (P < 0.05). Sensory evaluation proved that 5% PT treatment could cause a 4-days increase in the shelf-life of shrimp compared to the control, PT1% and PT2% treatments. Therefore, 5% phlorotannins from S. tenerimum could be used as a safe melanosis inhibitor for the treatment of shrimp.

The nature of ß-cyclodextrin inhibition of potato polyphenol oxidase-catalyzed reactions.

There is general interest in strategies to control polyphenol oxidase (PPO)-initiated enzymatic browning because it is often associated with declining food quality. Cyclodextrins are cyclic glucan oligosaccharides that form inclusion complexes with a number of PPO substrates. This study focuses on the effect of ß-cyclodextrins (ßCyD) on PPO-catalyzed reactions. Potato enzyme extracts and semi-purified potato PPO served as enzyme sources. Substrates included phenolics endogenous to potatoes. Reaction time-courses were followed spectrophotometrically; rates were compared by analysis of variance. Extents of ßCyD inhibition of PPO-catalyzed reactions are shown to be substrate specific and can be quantitatively accounted for based on degrees of ßCyD substrate sequestration. There was no evidence for direct irreversible ßCyD inactivation of potato PPO. An apparent "direct PPO inactivation" by ßCyD is shown to result from a sequence of sequestration-dependent reactions that occur in commonly employed assay systems for the quantification of PPO in fruits and vegetables.

Effect of mild heat treatment on browning-related parameters in fresh-cut Iceberg lettuce.

Enzymatic browning of Iceberg lettuce was studied by subjecting midrib tissues to a series of mild heat treatments. The effects of wounding and subsequent application of a mild heat treatment were examined by monitoring the browning potential (BP) and the activity of three browning-related enzymes (i.e., phenylalanine ammonia lyase [PAL], polyphenol oxidase [PPO], and peroxidase [POD]) during refrigerated storage up to 10 days. Efficient inhibition of browning was achieved by treatment at 50°C for 60 s. The wound-induced increase of the BP and the activity of PAL and POD was effectively suppressed, maintaining their values at initial levels up to 7 days of storage. PPO activity, on the contrary, remained unchanged after wounding, whether or not followed by heat treatment. BP, PAL activity and POD were found to be strongly correlated, whereas meaningful associations for PPO with the other parameters could not be established. PRACTICAL APPLICATIONS: In an attempt to answer to the growing demand in the fresh-cut produce industry to control browning, heat treatment was investigated as interesting alternative to chemical preservation methods. Efficient control of enzymatic browning in fresh-cut Iceberg lettuce could be achieved by heat treatment at 50°C for 60 s. Experimental data are provided showing the effects of wounding and subsequent heat treatment on visual browning, the BP and the activity of PAL, PPO, and POD during refrigerated storage up to 10 days. Using this data, correlations were found for BP, PAL activity, and POD activity, but not for PPO. Although undesired side effects of heat treatment (e.g., tissue softening) cannot be excluded, the obtained information might be useful for further research, serving as a baseline for wound-induced effects on browning-related parameters in fresh-cut lettuce and possible mechanisms of action of inhibitory treatments.

Exogenous proline has favorable effects on growth and browning suppression in rice but not in tobacco.

Proline is one of the amino acids that compose proteins and has various roles under non-stress and stress conditions. In this study, we investigated the effect of proline on the growth and browning of two plants, tobacco and rice, by exogenous application and endogenous increase of proline. Exogenous proline had a different effect on the growth and browning between tobacco and rice: proline affected negatively the growth of tobacco seedlings and favorably that of rice seedlings. In addition, proline prevented browning only in rice cultured cells, consistent with the increase of proline contents, but not in tobacco BY-2 cells. These results might be due to the difference of exogenous proline uptake activity in these cells. From the Lineweaver-Burk plots, proline inhibited polyphenol oxidase activity in vitro, which is a major factor of enzymatic browning in plants, by affecting the enzyme-substrate complex. Proline could suppress the browning of the plant callus by inhibition of PPO activity.

Acid Inhibition on Polyphenol Oxidase and Peroxidase in Processing of Anthocyanin-Rich Juice and Co-product Recovery from Purple-Fleshed Sweetpotatoes.

With high phytochemical and starch contents, purple-fleshed sweetpotatoes (PFSP) have been processed into various functional ingredients and food products including juices and natural colorants. For juice processing, PFSP are usually subjected to heat treatment for inactivation of pigment-degrading enzymes. However, heating of sweetpotatoes gelatinizes starch and produces thick slurry with cooked flavor, which are the drawbacks. Development of alternative processes to overcome the stated problems will be beneficial to sweetpotato processors. This study demonstrated that acidified water (≥3% w/v citric acid) was effective in inhibiting polyphenol oxidase and peroxidase in raw PFSP resulting in an attractive reddish juice. About 93% total phenolics (TP) and 83% total monomeric anthocyanins (TMA) in PFSP were extracted by two repeated extractions. The combined PFSP juice (3.2 L/kg PFSP) had high levels of TP (1,850 mg/L) and TMA (475 mg/L). With the developed process, 167 g dried starch, and 140 g dried high-fiber pomace were obtained for each kg raw PFSP, besides the highly pigmented juice. Pasteurization of the PFSP juice samples (pH 3.2) at 80 °C for 12 s resulted in 15% loss in TMA and had no effect on TP. The results indicated an efficient process to produce sweetpotato juice with high bioactive compounds and recovery of starch and high dietary fiber pomace as co-products. PRACTICAL APPLICATION: Purple-fleshed sweetpotatoes (PFSP) are rich in polyphenolics and antioxidant activities. In PFSP juice extraction, heat treatment to inactivate the pigment-degrading enzymes results in starch gelatinization and cooked flavor. A nonthermal process using acidified water was developed for producing anthocyanin-rich juice from PFSP and concurrently recovering native starch and dried pomace, which would increase the economic feasibility of the developed process. The results demonstrate an efficient process for the sweetpotato industry in producing PFSP pigmented juice and co-products for various food applications.

Effect of purslane (Portulaca oleracea L.) extract on anti-browning of fresh-cut potato slices during storage.

Enzymatic browning is a crucial reaction affecting the quality of fresh-cut fruit and vegetables. Purslane is an edible Chinese folk medicine with extensive distribution and containing a lot of polyphenols and alkaloids. However, little research on its' anti-browning effect on fresh-cut food was reported. In this study, the effectiveness of 0.05% (w/w) purslane aqueous extract treatment efficiently inhibited the activities of polyphenol oxidase (PPO), peroxidase (POD) and phenylalanine ammonia-lyase (PAL), the membrane integrity was effectively maintained, and malondialdehyde (MDA) content increases was retarded during whole storage period at 4 °C. Oddly, the higher purslane extract concentration, the lower endogenesis phenolic content. Additionally, thirty polyphenols and fifty-six alkaloids were found in purslane aqueous extract by LC-MS/MS. All results suggest that purslane aqueous extract is a promising nutritive anti-browning agent for fresh-cut potato.