Inhibition kinetics of polyphenol oxidase by glutamic acid

The inhibition of polyphenol oxidase (PPO) by glutamic acid was investigated. Application of different concentrations of glutamic acid to mushroom solution and Ocimum basilicum L. extract showed that glutamic acid appeared to be an effective browning inhibitor. Glutamic acid showed uncompetitive inhibition for mushroom and Ocimum basilicum L. polyphenol oxidases using 4-methylcatechol as a substrate, for mushroom PPO using catechol as a substrate and for Ocimum basilicum L. polyphenol oxidase using pyrogallol as a substrate; mixed-type inhibition for mushroom polyphenol oxidase using pyrogallol as a substrate; and non-competitive inhibition for Ocimum basilicum L. polyphenol oxidase using catechol as a substrate. Furthermore, sodium azide was used as an inhibitor for comparison with the inhibition potency of glutamic acid. It was found that glutamic acid was a more power inhibitor than sodium azide. The type of inhibition observed depended on the substrate, inhibitor and enzyme source used.     

The role of polyphenol oxidase and peroxidase in the browning of water caltrop pericarp during heat treatment

The mechanism of browning involving enzymatic browning was investigated in the pericarp of water caltrop, an Asian vegetable popular for its taste and medicinal properties. Polyphenol oxidase (PPO) and peroxidase (POD) activities were determined in pericarp at various times and temperatures. Water caltrop consisted of 44.22% moisture content, 37.23% crude fibre, and 2.63% crude protein. PPO and POD activities dropped from 62 and 38 units/g sample, respectively, as water temperature was increased from 30 to 80 °C. Optimum pH and temperature for PPO activity was at pH 5.0, 25–45 °C, and POD activity peaked at 60 °C. High PPO and POD activities at 40–50 °C resulted in degradation of phenolic compounds, which led to increased aggregation of browning pigments and discolouration (lower L-values) of the pericarp. Enzymatic browning was determined as the major factor in the browning discolouration of heat-treated water caltrop pericarp.     

Peroxidase and polyphenol oxidase thermal inactivation by microwaves in green coconut water simulated solutions

Enzymes from coconut water such as peroxidase (POD) and polyphenol oxidase (PPO) when in contact with oxygen begin reactions causing nutritional and color losses. Solutions simulating the chemical constituents of coconut water were submitted to a batch process in a microwave oven. PPO and POD inactivation data could be characterized by: PPO/water D_93 °C=16.5 s (z=35.5 °C); PPO/sugars D_91 °C=18 s (z=33°C); POD/water D_91.5 °C=44 s (z=24 °C) and POD/sugars D_92 °C=20.5 s (z=19.5 °C). The contact between salts and enzymes promoted a drastic reduction of the initial activity. After the incidence of microwave energy at temperatures above 90 °C, enzymes activity was not detected. These results can indicate an adequate choice of temperature conditions to inactivate coconut water enzymes. The knowledge of how green coconut water constituents influence POD and PPO activity will supply useful information about microwave processing of coconut water.     

冻结速率对苹果片多酚氧化酶和过氧化物酶活性影响的研究

低温断裂是果蔬超速冻过程中常见的问题。本文初步研究了速冻过程对苹果片生化特性的影响,发现不同的冻结速率会引起可溶性过氧化物酶(POD)的活性较大幅度变化,而可溶性多酚氧化酶(PPO)活性变化较小。通过对实验结果的初步分析,指出速冻过程与冻藏食品的保存质量有密切关系     

Role of polyphenol oxidase and peroxidase in shaping the phenolic profile of virgin olive oil

The main objective of this study was to assess the ability of polyphenol oxidase (PPO) and peroxidase (POX) enzymatic activities present in olive fruit tissues to oxidize main phenolic compounds related to the process to obtain virgin olive oil (VOO) in order to ascertain their involvement in shaping the phenolic profile of this product. In the two olive cultivars under study, Arbequina and Picual, olive PPO activity was found to be largely present in the fruit mesocarp whereas most olive POX activity is in the seed. Moreover, both enzymatic activities display relatively constant values after the onset of fruit ripening when the fruit is harvested for VOO production. Results showed that both PPO and POX activities present in olive fruit at ripening stages are able to oxidize main phenolic glycosides present in the fruit as well as those phenolic compounds arising during the industrial process to obtain the oil, especially secoiridoid compounds derived from oleuropein that mainly determine VOO nutritional and sensorial properties. Experimental data suggest a key role for endogenous olive PPO and POX enzymatic activities in determining the phenolic profile of VOO.     

Effect of ascorbic acid on the odours of cloudy apple juice

Ascorbic acid is used in apple juice as an antibrowning agent. This study investigated the effect of ascorbic acid (0.0–0.2% w/v) on the odours of cloudy apple juice using sensory evaluation and gas chromatography (GC). The increase in ascorbic acid concentration in the apple juice resulted in increases in green and unnatural odours and decreases in fresh, fruity and apple-like odours. In the GC determination, 23 volatile compounds were detected in apple juice. Aroma value, which showed the relative importance of volatile compounds, was used to elucidate the changes in odours of apple juice due to the addition of ascorbic acid. The aroma values of hexanal and trans-2-hexenal in the apple juice with 0.2% w/v ascorbic acid increased about 4 and 5-fold from those in the ascorbic acid-free apple juice, respectively. On the other hand, the aroma values of esters insignificantly changed in the apple juice with ascorbic acid. The increases in aroma values of aldehydes corresponded well with the increase in green odour in the apple juice with ascorbic acid.     

Improvement of frozen banana (Musa cavendishii, cv. Enana) colour by blanching: relationship between browning, phenols and polyphenol oxidase and peroxidase activities

 Browning in banana (Musa cavendishii, cv. Enana) processed products is a result of phenol oxidation catalysed by polyphenol oxidase (PPO) and peroxidase (POD) or of other non-enzymatic reactions (Maillard and Strecker mechanisms). Microwave and steam blanching significantly reduced PPO and POD activities and phenol levels in banana flesh, steam blanching being the most effective method for enzyme inactivation. Freezing/thawing processes produced a significant increase in phenol levels in all samples, due to cellular breakdown. After microwave heating browning processes occurred while steam-treated samples did not exhibit a significant colour change. Extractable PPO and POD activities in all banana samples increased as a consequence of freezing/thawing: steam-blanched slices exhibited lower residual activities. High correlations occurred between phenols and browning (r=0.86) in control samples. Blanched samples (microwave or steam) only exhibited correlations between PPO (r=0.80) and POD (r=0.80) activities and browning. Received: 22 February 1996     

Improvement of frozen banana (Musa cavendishii, cv. Enana) colour by blanching: relationship between browning, phenols and polyphenol oxidase and peroxidase activities

 Browning in banana (Musa cavendishii, cv. Enana) processed products is a result of phenol oxidation catalysed by polyphenol oxidase (PPO) and peroxidase (POD) or of other non-enzymatic reactions (Maillard and Strecker mechanisms). Microwave and steam blanching significantly reduced PPO and POD activities and phenol levels in banana flesh, steam blanching being the most effective method for enzyme inactivation. Freezing/thawing processes produced a significant increase in phenol levels in all samples, due to cellular breakdown. After microwave heating browning processes occurred while steam-treated samples did not exhibit a significant colour change. Extractable PPO and POD activities in all banana samples increased as a consequence of freezing/thawing: steam-blanched slices exhibited lower residual activities. High correlations occurred between phenols and browning (r=0.86) in control samples. Blanched samples (microwave or steam) only exhibited correlations between PPO (r=0.80) and POD (r=0.80) activities and browning. Received: 22 February 1996     

Kinetic characterisation and thermal inactivation study of polyphenol oxidase and peroxidase from table grape (Crimson Seedless)

Polyphenol oxidase (PPO) and peroxidase (POD) were extracted from a table grape (Crimson Seedless) using Triton X-114 and characterized using spectrophotometric methods. Both PPO and POD were activated by acid shock. However, in the presence of the anionic detergent sodium dodecil sulphate (SDS), PPO was activated whereas POD was inactivated. The enzymes were kinetically characterized and both followed Michaelis–Menten kinetics, although with different values of their kinetic parameters. The V_m/K_m ratio showed that Crimson Seedless grape PPO presents a similar affinity for 4-tert-butyl-catechol (TBC) whether activated by acid shock (0.018 min⁻¹) or SDS (0.023 min⁻¹). With regards to POD, the K_m and V_m values for 2,2′-azinobis(3-ethylbenzothiazolinesulphonic acid) (ABTS) were 0.79 mM and 1.20 μM/min, respectively. In the case of H₂O₂, the K_m and V_m value were 0.4 mM and 0.93 μM/min, respectively. PPO and POD showed similar thermostability, losing >90% of relative activity after only 5 min of incubation at 78 °C and 75 °C, respectively. In addition, PPO´s activation energy was similar to that obtained for POD (295.5 kJ/mol and 271.9 kJ/mol, respectively).     

Convenient partial purification of polyphenol oxidase from apple skin by cationic reversed micellar extraction

Polyphenol oxidase (PPO) was selectively extracted from reconstituted freeze-dried apple skin using reverse micelles formed by a cationic surfactant, dodecyl trimethyl ammonium bromide (DTAB). An optimum forward extraction was achieved with sodium phosphate buffer (pH 6, 100 mM, no added KCl) and an organic phase (isooctane:hexanol at a ratio of 5:1) containing 100 mM DTAB. The solubilised PPO was efficiently recovered by a stripping solution (pH 6, 1 M KCl) containing 10% ethanol. Under the optimised conditions, the purification fold and recovered activity of PPO were 12.6% and 71%, respectively. This purification fold and recovery were maintained when the extraction volume increased from 10–200 ml. Overall, reversed micellar extraction can be used as an efficient first step for the purification of PPO from apple skin.     

不同杂交后代苹果与富士苹果的多酚氧化酶特性比较

不同杂交后代苹果54号、45号及富士苹果多酚氧化酶特性比较结果表明：不同苹果的最适pH值不同，45号和富士为5．5，54号为6．0；45号、54号和富士最适温度分别为30，35，40℃；富士热稳定性最强，其次是45号，最次为54号；不同苹果PPO的最适底物依次为绿原酸、咖啡酸、儿茶酚；Na2S203对PPO活性抑制效果最佳，L-半胱氨酸与抗坏血酸的抑制效果较好，EDTA-2Na的抑制效果较差；不同苹果PPO同工酶酶带不同，染色深浅不同。富士有四条酶带，45号和54号只有一条共有酶带，富士酶带数最多，染色最深。     

Inactivation of peroxidase and polyphenol oxidase in red beet (Beta vulgaris L.) extract with continuous high pressure carbon dioxide

The inactivation of peroxidase (POD) and polyphenol oxidase (PPO) in red beet extract (RBE) with continuous high pressure carbon dioxide (HPCD) was investigated. HPCD treatment at 7.5 MPa (55 °C, 30 min) resulted in a reduction of their activities by approximately 73% and 93%, respectively. Compared with thermal treatment, continuous HPCD treatment reduced the decimal reduction time (D) of POD and PPO from 555.6 min to 55.9 min and 161.3 min to 32.1 min, respectively. The inactivation process could be described by first-order kinetics (r² > 0.70, p < 0.05); D values declined when temperature increased and continuous HPCD at 7.5 MPa and 55 °C resulted in the highest reaction rate constant (k value; smallest D value). The activation energy of the inactivation was reduced by HPCD treatment from 92.5 kJ/mol to 69.8 kJ/mol and 57.1 kJ/mol to 49.5 kJ/mol for POD and PPO, respectively. Continuous HPCD treatment had little effect on the antioxidant capacities of RBE samples.     

Inactivation kinetics of apple polyphenol oxidase in different pressure–temperature domains

The impact of high hydrostatic pressure and temperature on the stability of polyphenol oxidase (PPO) was studied in cloudy apple juice. Application of 200–500 MPa near room temperature or heat treatment at 45–55 °C at ambient pressure caused an increase of PPO activity of up to 65% in freshly squeezed apple juice. Combined pressure–temperature inactivation experiments with fully activated PPO (5 min treatment at 400 MPa and 20 °C) were carried out in the range of 0.1–700 MPa and 20–80 °C. Enzyme inactivation kinetics followed a 2.2 order reaction scheme at all pressure–temperature conditions tested. A polynomial model was successfully applied to describe the rate of PPO inactivation as a function of pressure and temperature and was used to construct a pressure–temperature isokinetic diagram. This diagram clearly showed synergistic effects of pressure and temperature on the inactivation of apple PPO at pressures above 300 MPa and antagonistic effects at lower pressures. Compared to ambient pressure conditions, temperatures required to inactivate PPO in apple juice were increased 10–15 °C at 100–300 MPa.

Industrial relevance

High pressure processing of fresh fruits is gaining popularity in the food industry because of its ability to inactivate microorganisms and some enzymes near room temperature with little impact on flavour or nutritional attributes of the food. However, quantitative data regarding the impact of process parameters on the target reaction are required to economically utilise this technology. This paper provides a mathematical model describing the combined effect of pressure, temperature and treatment time on the inactivation of PPO in cloudy apple juice.     

Quantitative modelling approaches for ascorbic acid degradation and non-enzymatic browning of orange juice during ultrasound processing

The objective of this study was to develop a deterministic modelling approach for non-enzymatic browning (NEB) and ascorbic acid (AA) degradation in orange juice during ultrasound processing. Freshly squeezed orange juice was sonicated using a 1500 W ultrasonic processor at a constant frequency of 20 kHz and processing variables of amplitude level (24.4–61.0 μm), temperature (5–30 °C) and time (0–10 min). The rate constants of the NEB and AA were estimated by a primary model (zero and first order) while their relationship with respect to the processing factors was tested for a number of models, i.e., second order polynomial, different types of Ratkowsky-type model, and an Arrhenius-type model. The non-monotonic behaviour of NEB has been described more accurately by the use of a polynomial model. The rate constants of AA were described by a similar type of model having a monotonic behaviour. A synergistic effect of temperature for different amplitudes on the rate constant of both NEB and AA was observed, while an antagonistic effect of amplitude on the rate of NEB was evident. The models with the best fit were integrated to produce contour plots for the combined amplitude and temperature. The constructed contour plots illustrate that low temperatures and intermediate amplitudes, i.e., 42.7 μm, result in lower NEB and AA deterioration and consequently better quality orange juice. The overall developed modelling approaches exploit quality data in order to identify the optimal processing regions for eliminating quality deterioration of orange juice during ultrasound processing which is of high importance to the food industry.     

Inhibition kinetic and mechanism of polyphenol oxidase from various sources by diethyldithiocarbamic acid

Inhibition kinetics and mechanism of polyphenol oxidases (PPO) partially purified from various sources such as Thymbra spicata L. var. spicata and Ocimum basilicum L., and of mushroom PPO bought from Sigma by diethyldithiocarbamic acid have been described using catechol, 4-methylcatechol and pyrogallol as substrates. The inhibition type was competitive for O. basilicum L. PPO using catechol and 4-methylcatechol as substrates, for mushroom PPO using catechol, 4-methylcatechol and pyrogallol as substrates, and for T. spicata L. var. spicata PPO using 4-methylcatechol as a substrate; uncompetitive inhibition for T. spicata L. var. spicata PPO using pyrogallol as a substrate; and non-competitive inhibition for O. basilicum L. and T. spicata L. var. spicata PPO using pyrogallol and catechol as substrates, respectively. The inhibition effect of diethyldithiocarbamic acid on enzymatic browning varied greatly from one phenol to another and from one enzyme to another. Hence, no general rule can easily be established with regard to the type of inhibition observed.     

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.     

苹果多酚氧化酶特性及无硫护色脱水研究

脱水苹果片在加工过程中极易发生褐变。在对苹果多酚氧化酶特性进行分析的基础上,通过设计正交实验得到最佳无硫护色剂组合,并初步确定真空干燥的条件。结果表明,苹果多酚氧化酶的最适pH为6.0,最适温度为36℃,100℃热烫60s可使其完全失活。苹果片的最佳无硫护色剂组合为0.6%柠檬酸、0.06%L-半胱氨酸、1.5%氯化钠,在真空度为0.09MPa,温度为50℃的条件下,可得到色泽较好的苹果片。     

Inactivation of polyphenol oxidases in cloudy apple juice exposed to supercritical carbon dioxide

The inactivation of polyphenol oxidase (PPO) in cloudy apple juice exposed to supercritical carbon dioxide (SCCO₂) treatment was investigated. Higher pressure, higher temperature, and longer treatment time caused more inactivation of PPO. The maximum reduction of PPO activity reached more than 60% at 30 MPa and 55 °C for 60 min. The experimental data followed first-order reaction kinetics; the kinetic rate constant k and the decimal reduction time D were closely related to the pressure and temperature of SCCO₂ treatment. Higher pressures or higher temperatures resulted in lower D values (higher k), the D value of PPO was minimized to 145 min treated by the combination of 30 MPa and 55 °C. Activation energy of 18.00 kJ /mol, was significantly reduced by SCCO₂ treatment at 30 MPa, as compared to activation energy of 72.0 kJ/mol for identical treatment at atmospheric pressure. Pressure and temperature sensitivity of kinetic parameters were studied. Z_P at 55 °C was 66.7 MPa and Z_T at 30 MPa was 108 °C.     

山药多酚氧化酶酶学特性及褐变控制研究

以鲜山药为试材，应用分光光度法对山药多酚氧化酶（PPO）酶学特性进行研究，以单一抑制荆对山药的褐变控制效果研究为依据，采用正交试验，确定复合抑制剂在山药褐变控制过程中的最佳组合。研究结果发现：山药PPO最适温度为30℃，最适pH为5．0和7．0。米氏方程显示：PPO与邻苯二酚有极强的亲和力。单一抑制剂在对山药的褐变抑制效果依次为VC＞异VC钠〉柠檬酸＞NaCl。复合抑制剂对山药的褐变最佳抑制效果的配比为：NaCl 40mmol／L、VC 3mmol／L、柠檬酸12．5mmol／L。     

蓝莓多酚氧化酶的部分纯化及其酶学特性研究

目的：明确蓝莓多酚氧化酶(PPO)的酶学特性。方法：分别采用硫酸铵分级沉淀法和疏水层析法分离纯化蓝莓PPO,比较两者的纯化效果,并对蓝莓PPO的酶促反应动力学、最适反应温度和pH以及稳定性进行研究。结果：硫酸铵分级沉淀法与疏水层析法的纯化倍数相近,但两者的得率分别为61%和84%。以绿原酸和邻苯二酚为底物时,蓝莓PPO的米氏常数(K_m)分别为23.38mM和6.13mM。该酶的最适pH值为6.0,最适反应温度为25℃。该酶在40℃以上不稳定,而在70℃时,其酶活在60s后残余酶活仅为7.23%。随着pH值的不断降低,其稳定性会不断下降。结论：疏水层析法对蓝莓PPO的分离纯化效果更好。蓝莓PPO对邻苯二酚的亲和力更高,该酶对温度和pH都较为敏感。