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.     

IDENTIFICATION OF PROCYANIDIN A2 AS POLYPHENOL OXIDASE SUBSTRATE IN PERICARP TISSUES OF LITCHI FRUIT

Postharvest browning of litchi fruit results in short shelf life and reduced commercial value. Experiments were conducted to separate, purify and identify polyphenol oxidase (PPO ) substrates that cause litchi fruit to brown. PPO and its substrate were extracted from the pericarp tissues of litchi fruit. The litchi PPO substrate was purified using polyamide column, silica gel column and Sephadex LH‐20 column chromatography. The browning substrate was selected by a 0.5% FeCl₃ solution and then identified using a partially purified litchi PPO. Analyses of ultraviolet spectrometry, nuclear magnetic resonance and electrospray ionization mass spectrometry indicated that the PPO substrate was procyanidin A2. The substrate can be oxidized to ο‐quinones by litchi PPO and then form brown‐colored by‐products, resulting in pericarp browning of harvested litchi fruit.     

Identification of (−)-epicatechin as the direct substrate for polyphenol oxidase isolated from litchi pericarp

Postharvest browning of litchi fruit results in a short life and a reduced commercial value. The experiments were conducted to separate, purify and identify the polyphenol oxidase (PPO) substrates that cause litchi fruit to brown. PPO and its substrates were, respectively, extracted from fruit pericarp tissues. The substrates for litchi PPO were separated and purified using polyamide column chromatography, silica gel column chromatography and preparative thin layer chromatography. The substrate was further identified by 0.5% FeCl₃ solution and enzymatic reaction with litchi PPO. On the basis of UV, ¹H NMR, ¹³C NMR, and ESI-MS data, the direct substrate for the PPO from litchi fruit pericarp tissues was identified to be (−)-epicatechin.     

Role of endogenous and exogenous phenolics in litchi anthocyanin degradation caused by polyphenol oxidase

The degradation of anthocyanins and/or the oxidation of phenolics caused by polyphenol oxidase (PPO) results in an enzymatic browning reaction of fruits and vegetables. This work was conducted with a view to explaining the unexpected observation that litchi (Litchi chinensis Sonn.) PPO did not directly oxidise litchi anthocyanins. PPO and anthocyanin from litchi fruit pericarp were extracted and purified, respectively, and then the anthocyanin degradation by PPO in the presence of (−)-epicatechin (endogenous PPO substrate), and catechol and gallic acid (exogenous PPO substrates) were analysed comparatively. The results showed that catechol was the most effective in litchi anthocyanin degradation, followed by (−)-epicatechin and gallic acid, but no significant differences existed between catechol and (−)-epicatechin. The study suggested that litchi PPO directly oxidised (−)-epicatechin; then oxidative products of (−)-epicatechin in turn catalysed litchi anthocyanin degradation, and finally resulted in the browning reaction, which can account for pericarp browning of postharvest litchi fruit.     

Polyphenol Oxidase Inhibitor from Blue Mussel (Mytilus edulis) Extract

Enzymatic browning remains a problem for the fruit and vegetable industry, especially new emerging markets like pre‐cuts. A crude inhibitor from blue mussel (Mytilus edulis) showed broad inhibition for apple (58%), mushroom (32%), and potato (44%) polyphenol oxidase (PPO) and was further characterized. Inhibition increased as the concentration of inhibitor increased in the reaction mixture eventually leveling off at a maximum inhibition of 92% for apple PPO. The inhibitor was capable of bleaching the brown color formed in the reaction mixture with apple PPO. Identification of the inhibitor by mass spectrometry and high‐performance liquid chromatography revealed it to be hypotaurine (C₂H₇NO₂S). Hypotaurine and other sulfinic acid analogs (methane and benzene sulfinic acids) showed very good inhibition for apple PPO at various concentrations with the highest inhibition occurring at 500 μM for hypotaurine (89%), methane sulfinic acid (100%), and benzene sulfinic acid (100%). Practical Application: An inhibitor found in the expressed liquid from blue mussel shows very good inhibition on enzymatic browning. Since this enzyme is responsible for losses to the fruit and vegetable industry, natural inhibitors that prevent browning would be valuable. Finding alternative chemistries that inhibit browning and understanding their mode of action would be beneficial to the fruit and vegetable industries and their segments such as pre‐cuts, juices, and so on. Inhibitors from products ingested by consumers are more acceptable as natural ingredients.     

Inhibitory effect of rice bran extract on polyphenol oxidase of potato and banana

Rice bran was extracted with water and its effects on potato and banana polyphenol oxidase (PPO) were investigated. Rice bran extract (RBE), conc. 0.3 g mL^?1, exhibited PPO inhibition in potato and banana PPO with % inhibition of 69.31% and 47.63%, respectively (P ≤ 0.05). RBE showed a concentration‐dependent inhibition on potato and banana PPO. RBE (conc. 0.3 g mL^?1) inhibited potato PPO higher than ascorbic acid, citric acid, NaCl and EDTA (final conc. 20 mg L^?1); and it also inhibited banana PPO higher than citric acid, NaCl and EDTA (final conc. 20 mg L^?1), respectively. The combination of RBE with citric acid or ascorbic acid appeared to be additive inhibitory effect on banana and potato PPO. Kinetic study of the inhibition on potato and banana PPO by RBE showed that RBE was a mixed‐type inhibitor; however, RBE appeared to be able to act directly on enzyme structure rather than substrate structure.     

Effect of rice bran protein extract on enzymatic browning inhibition in potato puree

Effect of rice bran protein extract (RBPE) on enzymatic browning inhibition in potato puree was studied by colour measurement and polyphenol oxidase (PPO) inhibition. RBPE inhibited browning in potato puree and showed a higher per cent potato PPO inhibition at pH 4.0 and 5.0 than those at pH 3.0, 6.0 and 7.0 (P ≤ 0.05). RBPE heated to 40 and 60 °C had browning inhibition in potato puree and per cent potato PPO inhibition at a similar extent to unheated RBPE (P > 0.05). Browning inhibition and per cent potato PPO inhibition of RBPE were decreased when it was heated to 80 °C (P ≤ 0.05). RBPE inhibited browning in potato puree higher than 5, 10 and 20 mm ascorbic acid and 5 and 10 mm citric acid (P ≤ 0.05). Regarding the kinetics study, RBPE exhibited a mixed‐type inhibition for potato PPO. Therefore, RBPE has a potential to be used as a natural antibrowning agent in the potato industry.     

The effectiveness of ozone and acidulant treatments in extending the refrigerated shelf life of fresh-cut potatoes

Abstract: The objective of the study was to determine the effectiveness of acidulant dip treatments (with or without aqueous ozone) to reduce enzymatic browning and to extend the shelf life of fresh‐cut potato slices during refrigerated storage (4 °C) for 28 d. Potato slices subjected to aqueous ozone (2 ppm) had significantly (P≤ 0.05) higher L‐values and lower a‐values, but ozone did not appear to have any effect on aerobic plate counts (APCs) or polyphenol oxidase (PPO) activity. NatureSeal (NS) and sodium acid sulfate (SAS) were the most effective acidulant treatments in reducing browning (significantly [P≤ 0.05] higher L‐values, lower a‐values, and browning index values) regardless of ozone treatment. NS and SAS also had lower PPO activity compared to other treatments on days 0 and 28, and significantly (P≤ 0.05) lower APCs (≤2.00 log CFU/g) over refrigerated storage. Therefore, the SAS treatment was comparable to NS, a commercially available product, and showed promise as an effective antibrowning dip to reduce browning and spoilage in fresh‐cut potato products. Practical Application: A 1% SAS dip treatment which included 1% citric and 1% ascorbic acid was found to be an effective antibrowning dip for fresh‐cut potatoes along with NatureSeal®'s PS‐10, compared to other treatments. They were both effective in maintaining low microbial counts over refrigerated storage. Additionally, aqueous ozone washes (2 ppm) showed significant benefits to reduce browning; however, ozone did not affect microbial counts or PPO enzyme activity. Therefore, the SAS treatment could have potential use in the fruit and vegetable industry to reduce browning and spoilage in fresh‐cut potato products.     

POLYPHENOLOXIDASE ACTIVITY OF MINIMALLY PROCESSED 'JONAGORED' APPLES (MALUS DOMESTICA)

The influence of three chemical dips using ascorbic acid (AA), citric acid (CA) and calcium chloride (CC) on the polyphenoloxidase (PPO) activity and on the total phenolic content of minimally processed (MP) apple (Malus domestica, cv. Jonagored) during cold storage was evaluated and a potential relationship with enzymatic browning was investigated. An ascorbic acid dip (42.6 mM) of 5 min duration was the most efficient chemical treatment in reducing the PPO activity of apple cubes. A 92% inhibition was achieved after 7 days of storage at 4C. All treatments were advantageous in comparison to the control in reducing color changes. Color changes, determined by absorbance at 420 nm (soluble pigments) and lightness (L) (insoluble pigments) of apple cubes treated with ascorbic acid were correlated with total phenolic content. No correlation was observed between PPO activity and tristimulus color parameters, browning index or total phenolic content of AA‐treated apple cubes.     

Modelling the effect of superatmospheric oxygen concentrations on in vitro mushroom PPO activity

The kinetics of polyphenol oxidase (PPO, EC 1.14.18.1) with respect to oxygen concentrations from 5 to 100% using chlorogenic acid (CGA) as substrate was examined. In vitro mushroom PPO activity was determined by measuring the consumption of oxygen during the oxidation reaction. A differential Michaelis–Menten model was fitted to the obtained total depletion curves. The product concentration as well as the concentration of oxygen had a clear inhibitory effect on the reaction rate. However, the inhibitory effect of oxygen was more evident at low product concentration. A linear mixed inhibition model that considered both the product (oxidised CGA) and oxygen as inhibitors was developed. A model with the product as a competitive inhibitor and oxygen as an uncompetitive inhibitor was the most appropriate to explain the reaction kinetics. The values of the inhibition constants calculated from the model were 0.0032 mmol L⁻¹ for K_m (Michaelis–Menten constant related to oxygen), 0.023 mmol L⁻¹ for K_mc (constant for competitive inhibition due to the product), 1.630 mmol L⁻¹ for K_mu (constant for uncompetitive inhibition due to oxygen) and 1.77 × 10⁻⁴ mmol L⁻¹ s⁻¹ for V_max (maximum reaction rate). The results indicate that superatmospheric oxygen concentrations could be effective in preventing enzymatic browning by PPO. Copyright © 2006 Society of Chemical Industry     

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%.     

Polyphenol oxidase activity,phenolic acid composition and browning in cashew apple (Anacardium occidentale, L.) after processing

This study describes the extraction and characterisation of cashew apple polyphenol oxidase (PPO) and the effect of wounding on cashew apple phenolic acid composition, PPO activity and fruit browning. Purification factor was 59 at 95% (NH₄)₂SO₄ saturation. For PPO activity, the optimal substrate was catechol and the optimum pH was 6.5. PPO K_m and V_max values were 18.8 mM and 13.6 U min⁻¹ ml⁻¹, respectively. Ascorbic acid, citric acid, sodium sulphite and sodium metabisulphite decreased PPO activity, while sodium chloride increased PPO activity. Wounding at 2 °C and 27 °C for 24 h increased PPO activity but storage at 40 °C reduced PPO activity. Gallic acid, protocatechuic acid and cinnamic acid (free and conjugate) were identified in cashew apple juice. Cutting and subsequent storage at 40 °C hydrolysed cinnamic acid. 5-Hydroxymethylfurfural content in cashew apple juice increased after injury and storage at higher temperatures, indicating non-enzymatic browning.     

荔枝与荔枝酒褐变控制

以荔枝及荔枝酒为试料,研究荔枝多酚氧化酶(PPO)的酶学特性及荔枝酒发酵过程中的褐变机理及控制措施。结果表明：荔枝PPO存在同功酶,荔枝PPO最适底物为没食子酸;以没食子酸为底物,荔枝PPO最适反应pH值为8.0,最适反应温度50℃,荔枝PPO的动力学参数为：米氏参数(Km)为26.033mmol/L,最大反应速度(Vmax)为34.816g/(L·min);荔枝PPO对酸碱、对热稳定性较差;荔枝酒发酵过程中褐变的主要原因是其PPO引起的酶促褐变。因此,低温贮藏和高温热处理可有效抑制荔枝及荔枝酒在贮藏和加工中酶促褐变反应。     

Effect of integrated application of chitosan coating and modified atmosphere packaging on overall quality retention in litchi cultivars

BACKGROUND: The storage life of litchi is limited due to pericarp browning and decay. Modified atmosphere packaging (MAP) showed promising results for ensuring quality retention. However, to improve the efficiency of MAP the integrated treatment of a chitosan coating and MAP was investigated. RESULTS: The effect of chitosan (1.0 g L⁻¹) + MAP was compared with MAP (control), and was effective in preventing decay, browning and retaining the pericarp colour in the cultivar McLean's Red. Chitosan (1.0 g L⁻¹) + MAP significantly reduced polyphenol oxidase (PPO) and peroxidase (POD) activity, retained membrane integrity, anthocyanin content and prevented the decline of pericarp colour values during storage. The POD activity was greater than the PPO activity in the cultivars McLean's Red and Mauritius. The two cultivars differed in anthocyanin content and the activity of oxidation enzymes. The gas compositions within the packages were compared between chitosan at 1.0 g L⁻¹ and 20.0 g L⁻¹ concentration for both cultivars. Chitosan (20.0 g L⁻¹) + MAP lowered the respiration during storage in both cultivars compared to 1.0 g L⁻¹ + MAP. CONCLUSION: The McLean's Red cultivar is better suited for chitosan (1.0 g L⁻¹) + MAP integrated treatment than is Mauritius in retaining overall quality. Copyright © 2009 Society of Chemical Industry     

INHIBITORY EFFECT OF ANTHOCYANIN EXTRACT FROM SEED COAT OF BLACK BEAN ON PERICARP BROWNING AND LIPID PEROXIDATION OF LITCHI FRUIT DURING STORAGE

ABSTRACT

Anthocyanins were extracted from seed coats of black beans (Glycine max[L.]) and the inhibitory effects of anthocyanin extract on pericarp browning and lipid peroxidation of litchi fruit were investigated. Litchi fruit were infiltrated for 3 min with 0 (control) or 50 mg/L of anthocyanin extract at a reduced pressure of 53 kPa, then packed in 0.03 mm thick polyethylene bags, and finally stored at 28C for 6 days. Changes in browning index, contents of anthocyanins and total phenol, peroxidase (POD) activity, levels of relative leakage rate and lipid peroxidation, α,α‐diphenyl‐β‐picrylhydrazy (DPPH) radical scavenging activity and reducing power were evaluated. Application of anthocyanin extract from black bean delayed pericarp browning of litchi fruit during storage, which was associated with reduced POD activity and higher contents of anthocyanins and total phenol. Moreover, the anthocyanin extract was found to have a direct inhibition on the POD activity in vitro. Furthermore, application of the anthocyanin extract apparently reduced lipid peroxidation and relatively maintained membrane integrity, which may account for browning inhibition to an extent. Finally, higher DPPH radical scavenging activity and reducing power of the fruits treated with the anthocyanin extract than control fruits possibly benefited in scavenging free radicals and reducing lipid peroxidation. It is, therefore, suggested that inhibited POD activity and reduced lipid peroxidation by the anthocyanin extract from seed coats of black beans were responsible for the inhibition of pericarp browning of litchi fruits.

PRACTICAL APPLICATIONS

Lipid peroxidation is a major cause of quality deterioration of postharvest fruits and vegetables. Some synthetic antioxidants are beneficial in inhibiting lipid peroxidation. However, considering that synthetic antioxidants, such as butylhydroxyanisole and butylated hydroxytoluene (BHT), have potential toxicity, the use of natural extracts to extend the shelf life of postharvest fruits and vegetables is a tendency. In our previous study, it has been found that anthocyanin extract from litchi pericarp has stronger antioxidant activities or free radical scavenging activities than BHT and ascorbic acid ( Duan et al. 2007 ). In this study, the application of anthocyanin extract from seed coat of black soybean showed reduced lipid peroxidation and pericarp browning. It could be used potentially as a postharvest technology for reducing or replacing the use of other chemicals, but it requires further investigation.     

Effect of washing with citric acid and antioxidants on the colour and microbiological quality of whole mushrooms (Agaricus bisporus L.)

The effect of washing with citric acid followed by anti‐browning treatment involving rinsing with sodium d ‐isoascorbate or sodium l ‐ascorbate on the colour, microbiological quality and bacterial blotch of whole mushrooms when stored at 5 °C for up to 13 days at 80% RH was evaluated. Washing with 1% citric acid reduced Pseudomonas counts avoiding bacterial blotch, but caused an important deterioration of colour. When washing with citric acid was followed by an anti‐browning treatment, mushrooms colour was improved, at the same time as bacterial blotch was reduced, although the decrease of Pseudomonas counts was smaller than in mushrooms washed only with citric acid. The most effective treatment was washing with 1% citric acid followed by anti‐browning treatment with 1.5% sodium l ‐ascorbate.     

Changes in selected quality characteristics of minimally processed carambola (Averrhoa carambola L.) when treated with ascorbic acid

‘B 10’ carambola of ripening stage (RS) 3 and 4 were minimally processed (MP) and then dipped in 0, 15 and 30 mg L^?1 ascorbic acid (AA). The 1‐cm‐thick slices were then dried, packed into cling‐wrapped‐foam tray and stored at 7 °C for 0, 3 and 5 days. Skin colour (L*, C* and h°), flesh firmness, soluble solids concentration, vitamin C content, titratable acidity, pH, degree of browning, polyphenoloxidase (PPO) activity and sensory attributes of MP carambola treated with AA were determined. AA treatment had significant effect in decreasing cut surface browning degree but no significant effect on all the selected quality characteristics of the MP carambola. In the sensory evaluation, flesh colour, sweetness, flavour and overall taste were significantly affected by AA treatment especially at 15 mg L^?1. The RS of fruit significantly affected skin colour (C* and h°), pH and sensory attributes of colour and flavour of the MP carambola. As storage day (SD) progressed, skin colour (C* and h°), flesh firmness and vitamin C content, cut surface browning, PPO activity and all the sensory attributes of MP carambola decreased significantly. Flesh firmness of the MP carambola was affected by the interaction between AA × SD. Sensory attributes of MP carambola were affected significantly by AA × RS. All the sensory attributes of MP carambola positively correlated to each other but negatively correlated with browning degree. PPO activity positively correlated with browning degree. Copyright © 2007 Society of Chemical Industry     

EFFECT OF OXYGEN CONCENTRATION ON THE BIOCHEMICAL AND CHEMICAL CHANGES OF STORED LONGAN FRUIT

ABSTRACT

Longan fruits were stored for 6 days in atmosphere of 5, 21 (air) or 60% O₂ (balance N₂) at 28C and 90–95% relative humidity to examine effects of low and high O₂ concentration on enzymatic browning and quality attributes of the fruit. Changes in pericarp browning, pulp breakdown, disease development, total phenol content, activities of phenol metabolism‐associated enzymes, relative leakage rate, α,α‐diphenyl‐β‐picrylhydrazy (DPPH) radical scavenging activity, and contents of total soluble solids, titratable acidity and ascorbic acid were evaluated. Storage of fruit in a 5% O₂ atmosphere markedly delayed pericarp browning in association with maintenance of high total phenolic content and reduced activities of polyphenol oxidase (PPO), peroxidase (POD) and phenylalanine ammonia lyase. Moreover, the fruit stored in a 5% O₂ atmosphere exhibited a lower relative leakage rate and higher DPPH radical scavenging activity than fruit stored in air. This presumably was beneficial in maintaining compartmentation of enzymes and substrates, and thus, reducing pericarp browning. Pulp breakdown and disease development were also reduced by exposure to a 5% oxygenatmosphere. On the contrary, exposure of longan fruit to a 60% O₂ atmosphere accelerated pericarp browning, pulp breakdown and decay development. PPO and POD activities and relative leakage rate were similar for control and 60% O₂‐treated fruit after 4 and 6 days of storage. Furthermore, treatment with 60% O₂ significantly decreased the phenolic content and DPPH scavenging activity of fruit. In addition, exposure to 5 or 60% O₂ resulted in a higher level of total soluble solids, but a lower level of ascorbic acid of longan fruit flesh. In conclusion, exposure to a 5% O₂ atmosphere showed great potential to reduce pericarp browning and extend shelf life of longan fruit.

PRACTICAL APPLICATIONS

Pericarp browning and pulp breakdown are the major causes of deterioration in postharvest longan. Conventional controlled atmosphere with low O₂ and high CO₂ is effective in maintaining quality and extending shelf life of fruits and vegetables, including inhibition of tissue browning. In this study, 5%‐controlled atmosphere reduced significantly pericarp browning, pulp breakdown and rot development. It could potentially be useful as a postharvest technology of longan fruit for reducing or replacing the use of chemicals such as SO₂ and fungicides, but it requires further investigation.     

Purification and enzymatic characteristics of a novel polyphenol oxidase from lotus seed (Nelumbo nucifera Gaertn.)

A polyphenol oxidase (PPO) from lotus seed was purified by the procedures including ammonium sulphate precipitation and affinity chromatography. The apparent molecular mass was 38.6 kDa by SDS‐PAGE. Kinetic studies showed that the K_m and V_max values for catechol were 6.04 mm and 416.67 U, respectively. The PPO performed optimal activity in 20 °C and pH 7.0. The enzymatic activity could be mainly maintained up to 50 °C and pH 4.0–7.0. The activity could be inhibited by various inhibitors including thiourea, urea, sodium hydrogen sulphite, EDTA·2Na, SDS, citric acid, guanidine hydrochloride, ascorbic acid, sodium sulphite and sodium thiosulphate. The metal ions Ba²⁺, Mg²⁺, Ca²⁺, Mn²⁺, Co²⁺ and Zn²⁺ could inhibit the activity of PPO, while Cu²⁺ performed obvious enhancement. The enzymatic properties of PPO could probably provide practical application in inhibiting the PPO activity and preventing enzymatic browning in the process of picking, transportation, processing and storage of fresh lotus seeds.     

宣木瓜多酚氧化酶酶学特性与抑制剂研究

研究宣木瓜不同部位的多酚氧化酶(PPO)的活性及其温度、pH值、底物专一性等特性和抑制方法。结果表明：果心中的PPO活性明显高于果皮和果肉,宣木瓜组织的褐变主要是由多酚氧化酶引起;PPO最适底物为邻苯二酚;最适温度20℃,在80℃处理5min即可钝化PPO的活性;最适pH5.5,在酸性条件下稳定性优于碱性;抗坏血酸、L-半胱氨酸、亚硫酸氢钠、柠檬酸和氯化钠均能有效的抑制PPO的活性。