Characterization and Inhibition of Plant Polyphenol Oxidase for Quality Preservation in Post-Harvest Processing

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.

Extraction and purification of polyphenol oxidase: A review

The purification of the polyphenol oxidase (PPO) enzyme from Kirmizi Kismis grape (Vitis vinifera L.) was performed 61.23 times using affinity chromatography. The molecular weight of the enzyme was found to be about 38.1kDa by SDS-PAGE as a single band. The optimum pH and temperature values were revealed to be 5.0 and 30°C, respectively, in the presence of 4-methyl catechol substrate. The thermal stability of PPO was examined and it was observed to maintain its activity at 20°C for 1hr. Km and Vmax values were determined to be 4.8mM and 2000,0EU/ml for 4-methyl catechol as a substrate. IC50 and Ki values and inhibition types were found for various browning agents and ascorbic acid had the strongest inhibitory impact on PPO. The inhibitory impact of Na+ , K+ , Mg2+ , Cu2+ , and Al3+ metal ions on the enzyme activity at final concentrations of 1mM and 10mM was examined. PRACTICAL APPLICATIONS: Grapes grown and processed take a significant place in our life. The grape has antioxidant, anticarcinogenic, antidiabetic and protective properties against bacteria and viruses. Furthermore, it takes an important position in the country's economy and social life due to providing raw materials to the food industry and having high export potential. Polyphenol oxidase, which is the leading actor of enzymatic browning reactions causing serious economic losses every year, was purified and characterized from Kirmizi Kismis grape (Vitis vinifera L.). This ancient grape variety has industrial processing and export potential due to its long storage life and resistance to oxidation. Therefore, the purification and biochemical characterization of polyphenol oxidase from Kirmizi Kismis grape are of great importance.

Polyphenol oxidase (PPO) was firstly purified from damson plum as a high antioxidant source. PPO was treated by 0–80% ammonium sulfate precipitation and dialysis. Characterization results were determined for catechol, 4-methyl catechol, pyrogallol and caffeic acid as 0.05 M/pH: 7.2/25 °C; 0.2 M/pH: 4.5/10 °C; 0.01 M/pH: 6.8/5 °C, and 0.2 M/pH: 8.5/10 °C, respectively. V max and KM values were calculated for same substrates as 17,219.97 U/(mL*min) and 11.67 mM; 7309.72 U/(mL*min) and 5 mM; 12,580.12 U/(mL*min) and 3.74 mM; 12,100.41 U/(mL*min) and 6.25 mM, respectively. Catechol gave the highest V max value among substrates. Affinity purification was performed by using Sepharose 4B-L-Tyrosine-p-aminobenzoic acid and Sepharose 6B-L-Tyrosine-p-aminobenzoic acid. Single bands were approximately observed at 50 kDa for each affinity sample in SDS-PAGE and Native-PAGE. 93.88 and 10.46 purification-folds were obtained for PPO by reference Sepharose-4B and original Sepharose-6B gels. Metal effects upon PPO activity were also investigated due to the importance of enzymatic browning in foods. Cu+2 activation and Fe+2 inhibition were observed with a final metal concentration of 1 mM at 219.66 and 43.18%, respectively. PPO purification from damson plum by affinity chromatography, its characterization, stability evaluation by statistically, and effects of metal ions on damson plum PPO have not been investigated in the literature.

Honey, pineapple, chilli pepper, and onion extracts were investigated for their ability to inhibit enzymatic browning in sweet potatoes. Honey had the highest inhibition (41.39%–48.0%), comparable with l-ascorbic acid. Heated onion extract increased polyphenol oxidase (PPO) inhibition by 2.5-fold when 4-methylcatechol was used as substrate. However, percentage inhibition decreased drastically (7.42%–19.67%) when heated pineapple extract was used. Fresh chilli pepper extract showed a higher PPO inhibition (45.97%) than that of heated extract. These natural inhibitors were also compared to synthetic inhibitors that ranked: l-cysteine > l-ascorbic acid > sodium chloride. l-cysteine was the most effective chemical inhibitor which inhibited up to 70.4% PPO activity. It was found that natural inhibitors extracted from common food items are able to control the enzymatic browning of sweet potato. These natural inhibitors extracts are safe to human and would also be potentially replaced chemical inhibitors for the preservation of other fruits as well as vegetables. 1 Practical applications Fruits and vegetables are prone to enzymatic browning. This is caused by the oxidation reaction of endogenous phenolic compounds, catalyzed by PPO. PPO oxidizes o-diphenols to o-quinones, which will turn into brown pigments as the o-quinones polymerize immediately. Enzymatic browning of fruits and vegetables always take places after harvest, during the post-harvest processing as well as storage. Thus, identification of suitable and acceptable inhibitors is important in order to prevent food browning and to increase the food marketability for food industry. This study indicated that sweet potato PPO was successfully inhibited by extracts from common food items such as honey, chilli pepper, pineapple, and onion which are safe for human consumption. Thus, these natural inhibitors could also be applied to similar food materials such as apples, star-fruit, and normal potatoes. We expect that the inhibitors examined in this study can be isolated and identified as lead molecules for the development of natural anti-browning food additives.

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

Polyphenol oxidase (PPO) catalyzes in the presence of oxygen, the oxidation of mono- and di- phenols to o-quinones. The browning in fruits, vegetables and their processed products occurs as a result of this oxidation reaction. PPO in this study was isolated and characterized from two species of garden egg; Solanum mellongena depressum and Solanum gilo. Extracts were purified using a combination of ammonium sulphate precipitation, ion exchange chromatography on DEAE sephadex and gel filtration on Sephadex G-200 column. The effect of pH and temperature were carried out, pH and thermal stability, kinetic and substrate specificity, the effect of inhibitors and activator on the enzyme activity was investigated. About 6% protein yield for both species and a purification fold of 30.0 and 41.0 for S. depressum and S. gilo respectively was achieved. The optima pH of activity were found to be 4.0 – 4.5 and 7.0 for S. depressum and 4.0 and 8.0 for S. gilo while 4.5 and 8.0 were obtain in presence of SDS, the activity of the PPO increased in the presence of small concentration of SDS in all the pH investigated. The temperature optimum for both species was observed at 30°C. The PPO were stable at 30°C retaining about 88% and 87% of initial activity after 60minutes for S. depressum and S. gilo respectively while PPO from S. depressum was inactivated after 40min and 80°C and 70°C. A minimal remaining activity of 5% was observed at 80°C after 60min incubation time. PPO was fairly stable at pH 6.0 – 8.0 retaining a percentage remaining activity of 40.3% to 50% for S depressum and 45.5% to 52.1% for S. gilo. The PPO exhibited a marked activity towards o-diphenol and lower activities for monophenols. Ascorbic acid, EDTA and SDS inhibited enzymatic activity while the Km values using catechol, DOPA and catechin as substrate were 0.3mM, 0.095mM and 1.09mM for S. depressum and 1.9mM, 0.414mM and 0.56mM for S. gilo. PPO from S. depressum exhibited a higher enzymatic activity compared to S. gilo while S. gilo retained a fairly more stable activity.

Polyphenol oxidase (PPO) is a critical molecule because of its participation in enzymatic browning in fruits and vegetables during ripening, handling, storage, and processing. In this contribution, PPO was extracted, purified, and characterized from Golden Delicious apples, PPO activity was increased 319-fold with a yield of 1.6%. A monomer of 58 kDa was obtained by gel filtration and SDS-PAGE; the optimum temperature and pH were 35°C and 6.0, respectively; the best catalytic efficiencies (Vmax/Km ratios [U/(mg·mM)]) were 15,402 for 4-methylcatechol and 5,941 for pyrogallol. The most efficient inhibitors were sodium metabisulfite (≥1 mM) and tropolone (≥10 mM). PPO activity decreased as the temperature increased and thermal inactivation was achieved at 80°C in 5 min. An Ea of 69.2 kJ/mol was estimated from the kinetic inactivation at different temperatures. Microwave irradiation inactivates PPO after 120 s in crude extract and after 60 s in fresh apple juice. Practical applications According to FAO (Food and Agricultural Organization) data, Mexico produced about 858,608 tons of apple in 2013, Mexico is in 30th place in the world overall apple production. It is important to characterize the Polyphenol Oxidase (PPO) from Golden Delicious apple and provide data on its inactivation. PPO has been considered a crucial molecule because of its participation in enzymatic browning in several fruit and vegetables during ripening, handling, storage, and processing, being responsible for economical loses for producers and industry. In this contribution PPO was extracted, purified, and partially characterized from Mexican Golden Delicious apples by fast protein liquid chromatography. Also some inactivation methods like the use of several inhibitors and antibrowning agents, thermal and microwave inactivation where tested. The information obtained in this research is important for better understanding of PPO from Golden Delicious apple and gives information on ways to optimize inactivation process of the purified enzyme or in apple juice.

Enzymatic browning is a colour reaction occurring in plants, including cereals, fruit and horticultural crops, due to oxidation during postharvest processing and storage. This has a negative impact on the colour, flavour, nutritional properties and shelf life of food products. Browning is usually caused by polyphenol oxidases (PPOs), following cell damage caused by senescence, wounding and the attack of pests and pathogens. Several studies indicated that PPOs play a role in plant immunity, and emerging evidence suggested that PPOs might also be involved in other physiological processes. Genomic investigations ultimately led to the isolation of PPO homologs in several crops, which will be possibly characterized at the functional level in the near future. Here, focusing on the botanic families of Poaceae and Solanaceae, we provide an overview on available scientific literature on PPOs, resulting in useful information on biochemical, physiological and genetic aspects.

A comparative study of polyphenol oxidase (PPO) from two Turkish varieties of quince (Eşme and Kalecik) was conducted to determine some of the characteristics of the enzymes in terms of temperature and pH optima, substrate specificity, thermal inactivation and potency of some PPO inhibitors. PPO was partially purified by ammonium sulfate precipitation followed by dialysis. The optimum pH and temperature of the two PPOs were found to be similar. The apparent substrate specificity was established from Vmax/Km as 4-methylcatechol > catechol > pyrogallol for PPO from Eşme variety (PPOEşme). The substrate specificity for PPO from Kalecik variety (PPOKalecik) was catechol > 4-methylcatechol > pyrogallol. Heat stabilities and substrate specificities of the two enzymes differed. PRACTICAL APPLICATION Turkey is the main quince producer in the world. There are many quince types and cultivars grown in different parts of Turkey. It is consumed as fresh fruit, as well as processed into different products. As with many fruits and vegetables, enzymatic browning catalyzed by polyphenol oxidase (PPO) (EC 1.14.18.1) also occurs in quince. A successful prevention of these reactions requires characterization of PPO. The biochemical properties of PPOs determined in this study are important for controlling enzymatic browning which occurs during handling and processing of quince fruits.

Development of computer vision system to predict peroxidase and polyphenol oxidase enzymes to evaluate the process of banana peel browning using genetic programming modeling

Purpose of review: Enzymatic browning of fruits and vegetables during postharvest handling and processing degrades the sensory properties and nutritional value and discourages consumer purchase of fresh-cut products. Consequently, enzymatic browning results in significant economic losses for the fresh produce industry. This paper discusses the biochemistry of enzymatic browning, and focuses on technologies that can be used to prevent browning of fresh-cut fruits and vegetables and maintain good product quality and safety for consumers. Main findings: Enzymatic browning results from oxidation of phenolic compounds catalysed by polyphenol oxidase (PPO) followed by non-enzymatic formation of pigments. PPOs exhibit either mono- or di-phenol oxidase activity, or both types of activities. Peroxidase (POD) and phenylalanine ammonia lyase (PAL) are also found to be closely associated with the browning of fresh-cut fruits and vegetables. A range of physical and chemical treatments that have the potential to be adopted by fresh-cut industry for browning inhibition were reviewed. The effective treatments can be divided into three methods: 1) dipping in anti-browning solutions; 2) modified atmosphere packaging; and 3) heat shock and refrigerated storage. The importance of balancing browning inhibition and pathogen inactivation in fresh-cut produce was particularly emphasised. Directions for future research: Understanding the details of enzymatic browning, which occurs during the processing of fresh-cut products, is necessary for improving browning control. The activities of PPO, POD and PAL, as well as their interactions during browning reactions in fresh-cut produce need more investigation. The relationship between enzymatic browning and the content of total phenolic compounds (or specific phenolic composition) also requires further research. In addition, the development of dual controls to prevent both browning and pathogen contamination in fresh-cut produce is critical to maintaining the quality and safety of fresh-cut produce.

Enzymatic browning of sprouts during storage is a serious problem negatively influencing their consumer quality. Identifying and understanding the mechanism of inhibition of polyphenol oxidases (PPOs) in lentil sprouts may offer inexpensive alternatives to prevent browning. This study focused on the biochemical characteristics of PPOs from stored lentil sprouts, providing data that may be directly implemented in improving the consumer quality of sprouts. The purification resulted in approximately 25-fold enrichment of two PPO isoenzymes (PPO I and PPO II). The optimum pH for total PPOs, as well as for PPO I and PPO II isoenzymes, was 4.5–5.5, 4.5–5.0, and 5.5, respectively. The optimal temperature for PPOs was 35 °C. Total PPOs and the PPO I and PPO II isoenzymes had the greatest affinity for catechol (Km = 1.32, 1.76, and 0.94 mM, respectively). Ascorbic acid was the most effective in the inhibition of dark color formation by total PPOs, and showed ca. 62%, 43%, and 24% inhibition at 20-, 2-, and 0.2-mM concentrations. Ascorbic acid, l-cysteine, and sodium metabisulfite (20 mM) significantly inhibited color development in the reactions catalyzed by both isoenzymes of PPO. Ba2+, Fe3+, and Mn2+ (10 mM) completely inhibited PPO activity. This study of the effect of antibrowning compounds and cations on PPO activity provides data that can be used to protect lentil sprouts against enzymatic browning during storage and processing.

Polyphenol oxidases (PPOs) oxidize o-diphenols to o-quinones, which cause browning reactions in many wounded fruits, vegetables, and plants including the forage crop red clover (Trifolium pratense L.). Production of o-quinones in red clover inhibits postharvest proteolysis during the ensiling process. The cDNAs encoding three red clover PPOs were expressed individually in alfalfa (Medicago sativa L.), which lacks detectable endogenous foliar PPO activity and o-diphenols. Several physical and biochemical characteristics of the red clover PPOs in alfalfa extracts were determined. In transgenic alfalfa extracts, red clover PPOs exist in a latent state and are activated (10-40-fold increase in activity) by long incubations (>2 days) at ambient temperature or short incubations (<10 min) at > or =65 degrees C. PPO1 appears to be more stable at high temperatures than PPO2 or PPO3. During incubation at ambient temperature, the molecular masses of the PPO enzymes were reduced by approximately 20 kDa. The apparent pH optima of latent PPO1, PPO2, and PPO3 are 5.5, 6.9, and 5.1, respectively, and latent PPO1 is slightly activated (~5-fold) by low pH. Activation of the PPOs shifts the pH optima to approximately 7, and the activated PPOs retain substantial levels of activity as the pH increases above their optima. The latent and activated PPOs were surveyed for ability to oxidize various o-diphenols, and activation of the PPOs had little effect on substrate specificity. Activation increases the V max but not the affinity of the PPO enzymes for caffeic acid. Results indicate red clover PPOs undergo structural and kinetic changes during activation and provide new insights to their effects in postharvest physiology.

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