Purification of polyphenol oxidase and the browning control of litchi fruit by glutathione and citric acid

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     

Effect of oxalic acid on control of postharvest browning of litchi fruit

Litchi (Litchi chinensis Sonn.) fruit, cv. Huaizhi, was treated with 2 and 4 mM oxalic acid and stored at room temperature to investigate the effect of oxalic acid on pericarp browning. The results showed that the pericarp browning indices of the fruit, treated with both oxalic acid concentrations, were significantly lower than that of the control, due to increase of membrane integrity, inhibition of anthocyanin degradation, decline of oxidation, and maintanance of relatively low peroxidase activity in the fruit during storage. It appears that application of oxalic acid can effectively control the pericarp browning of litchi fruit during postharvest storage.     

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.     

Antioxidant activities and contents of polyphenol oxidase substrates from pericarp tissues of litchi fruit

The experiments were performed to extract and purify substrates for polyphenol oxidase (PPO) from pericarp tissue of postharvest litchi fruit. Two purified PPO substrates were identified as (−)-epicatechin and procyanidin A2. The antioxidant properties of two PPO substrates were further evaluated in the present study. Variation in the content of the major substrate (−)-epicatechin of litchi fruit during storage at 25 °C was analysed using the HPLC-UV method. The results showed that (−)-epicatechin exhibited stronger antioxidant capability than procyanidin A2, in terms of reducing power and scavenging activities of DPPH radical, hydroxyl radical and superoxide radical. Furthermore, (−)-epicatechin content in pericarp tissue tended to decrease with increasing skin browning index of litchi fruit during storage at 25 °C. Thus, these two compounds can be used as potential antioxidants in litchi waste and the fresh pericarp tissue of litchi fruit exhibited a better utilisation value.     

红枣多酚氧化酶酶促褐变控制研究

确定了红枣多酚氧化酶最适pH为6.0～6.5,最适温度为27℃;其对pH和温度较为敏感,通过调节pH和适当的热处理即可有效抑制其活性。同时在上述最适条件下,研究了以柠檬酸、抗坏血酸、亚硫酸钠作为抑制剂对红枣多酚氧化酶的抑制作用,并在单因素实验的基础上,采用响应面分析法对复合抑制剂的复配进行了优化,获得最佳添加配比为:柠檬酸0.24%,抗坏血酸0.14%,亚硫酸钠0.08%;此条件下红枣多酚氧化酶相对残余酶活为0.51%。     

荔枝果肉多酚氧化酶提取与初步纯化

酶促褐变是影响荔枝果肉加工和贮藏的重要问题,多酚氧化酶(PPO)是引起酶促褐变的重要酶类.用磷酸缓冲液匀浆提取荔枝果肉中的多酚氧化酶,提取液最佳离子浓度为0.1mol/L、最适pH为7.5、最佳提取时间为2h,最适料液比为1:3.并通过硫酸铵沉淀和Phenyl Sepharowe CL-4B(苯基琼脂糖CL-4B)疏水柱层析进行初步纯化,该酶被纯化了9.4倍,得率为48%.     

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.     

低温结合气调包装对荔枝保鲜作用

本文探究了常温条件下不同浓度的嘧菌酯和甲基硫菌灵对采后荔枝果实保鲜效果的影响,在贮藏期间通过对荔枝生理指标进行分析从而筛选出较优的杀菌剂配方,并对筛选后的杀菌剂配方进行验证以及膳食暴露评估,最终获得可以在常温贮藏条件下延长荔枝货架期的保鲜方法。结果发现,在25 ℃贮藏条件下,采用25%嘧菌酯1600倍稀释液（M1600）、25%嘧菌酯1600倍稀释液复配70%甲基硫菌灵700倍稀释液（M1600+J700）处理荔枝可以显著抑制果实的呼吸强度（P<0.05）,降低果皮的相对电导率（P<0.05）和果实的腐烂率（P<0.05）,且在一定程度上抑制了果肉中可溶性固形物TSS和维生素C含量的降低,较好地维持了荔枝果实的感官品质和食用价值。经对比发现,两种杀菌剂对荔枝的保鲜效果相当,但单独使用M1600在抑制荔枝果皮褐变方面效果较优。通过对贮藏期间荔枝全果和果肉中杀菌剂残留量的检测以及膳食暴露评估发现,单独使用嘧菌酯处理荔枝后整果中嘧菌酯的风险熵RQ远小于1,可以同时达到满足提高荔枝贮藏品质、安全食用以及降低经济成本的目的。该研究为荔枝等非呼吸跃变型果实采后品质提升的研究提供了技术参考。

     

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.     

Characterization of polyphenol oxidase from Uapaca kirkiana fruit

Polyphenol oxidase (PPO), the enzyme responsible for the postharvest spoilage of fruits, was extracted and purified from Uapaca kirkiana peel and pulp by ammonium sulfate precipitation and dialysis. Further purification of peel PPO was carried out by gel filtration chromatography. Optimum pH values were 7 and 8 for peel and pulp PPO, respectively. The optimum temperatures for peel and pulp PPO were 45 and 35 °C, respectively. Inhibition studies of the PPO enzyme were performed using citric acid, sodium azide, sodium metabisulfite and thiourea. The most effective inhibitors were sodium azide and citric acid for both peel and pulp PPO. V_max and K_m values were 13.63 units min^?1 and 4.923 mmol L^?1, respectively, for peel PPO and 14.03 units min^?1 and 5.43 mmol L^?1, respectively, for pulp PPO. Three isoenzymes of Uapaca kirkiana PPO were detected by polyacrylamide gel electrophoresis. One of the isoenzymes could be identified as having a molecular weight of 26 625 Da. Copyright © 2005 Society of Chemical Industry     

模型溶液研究荔枝干制过程中的非酶褐变反应

建立荔枝干制过程的模型溶液,通过测定其在荔枝干制条件下,反应体系在280、420nm处的吸光值变化,研究四种非酶褐变反应对颜色的贡献作用。结果显示:美拉德反应和焦糖化是主要的非酶褐变反应,VC、多酚类物质对颜色形成不起主要的贡献作用;果糖和蔗糖在非酶褐变反应中起重要的作用,葡萄糖的作用几乎可忽略不计。     

Role of anthocyanins,polyphenol oxidase and phenols in lychee pericarp browning

Postharvest browning of lychee fruits is investigated in relation to anthocyanins, polyphenol oxidase (PPO) and phenols, using a purified PPO, anthocyanin extract and phenolic extract. Lychee PPO cannot oxidise the degradation of anthocyanins, but the presence of phenolic extract stimulates pigment degradation by PPO, leading to the development of brown pigments. The absorbance at 410 nm of the reaction solution is correlated well with the peel browning index. Pyrogallol, catechol and 4‐methylcatechol are good stimulators, but no action with chlorogenic acid, p‐cresol, resorcinol or tyrosine is observed. A decrease in the ascorbic acid content of lychee pericarp is associated with an increase in the peel browning index, and the addition of ascorbic acid to the reaction mixture exerts a preventive action on the anthocyanin degradation. Data here suggest that anthocyanins, PPO and phenols contribute to lychee pericarp browning involved in anthocyanin–PPO–phenol reactions. © 2000 Society of Chemical Industry     

板栗品种褐变度差异性及其多酚氧化酶活性的相关性研究

以37个板栗品种为试材,研究了板栗果实褐变差异性及其与多酚氧化酶（PPO）活性之间的关系,并根据褐变差异性对各品种进行聚类分析。结果表明:板栗不同品种间褐变差异显著（p<0.05）,根据褐变差异性聚类分析,可将37个品种可以分为5个等级;不同品种间PPO活性差异显著（p<0.05）;通过探讨不同品种间褐变度与PPO活性的关系,发现随着板栗品种间PPO活性的升高,褐变度也开始升高,当PPO活性达到90 U·min-1·g-1时,PPO活性对褐变影响开始降低;通过褐变聚类分析,将板栗PPO活性分为5类,在优选或改良品种时,可将品种的PPO活性在第一或第二类活性范围以下作为选择依据之一。      

Inhibition of polyphenol oxidase obtained from various sources by 2,3‐diaminopropionic acid

This paper reports for the first time the inhibition of the catecholase activities of mushroom, artichoke (Cynara scolymus L) and Ocimum basilicum L polyphenol oxidase by 2,3‐diaminopropionic acid. Polyphenol oxidases from artichoke and O basilicum L were purified by ammonium sulfate precipitation, dialysis and a Sepharose 4B‐L ‐tyrosine‐p‐aminobenzoic acid‐affinity column. In inhibition studies, 2,3‐diaminopropionic acid showed uncompetitive inhibition for mushroom PPO using catechol and pyrogallol as substrates, competitive inhibition for O basilicum L PPO using catechol as a substrate, and uncompetitive inhibition for artichoke PPO using catechol as a substrate. Furthermore, sodium azide, which is an inhibitor of PPO, was used as an inhibitor for comparison with the inhibition potency of 2,3‐diaminopropionic acid. The highest 2,3‐diaminopropionic acid inhibition observed with O basilicum L (K_i = 0.89 mM ), followed by artichoke (K_i = 1.42 mM ) and mushroom (K_i = 2.47 mM ), respectively. Copyright © 2005 Society of Chemical Industry     

龙眼菠萝复合果酒褐变抑制的研究

研究了龙眼菠萝复合果酒加工过程中原料配比、原料成熟度、抑制剂处理、杀菌条件对果酒褐变的抑制效果.结果表明:龙眼的配比越大、成熟度越大,果酒的褐变程度越大;杀菌温度90℃、杀菌时间20min、柠檬酸添加量0.3％时,对果酒有较好的褐变抑制效果,但抗坏血酸、半胱氨酸、CaCl2对该果酒的褐变抑制效果较差,不适宜用作龙眼菠萝复合果酒的褐变抑制剂.     

Screening legume forages for soluble phenols,polyphenol oxidase and extract browning

Red clover (Trifolium pralense) is a legume that will brown quickly upon harvesting. This browning of red clover has been correlated to a reduction in proteolysis and requires the presence of a polyphenol oxidase and soluble phenol substrates. A method was developed to screen forages for soluble phenols, soluble polyphenol oxidase activity and extract browning. Fourteen different legumes and 24 red clovers (22 plant introductions and two cultivars), field grown in replicated plots, were screened. Of the different legumes, only red clover had measurable soluble polyphenol oxidase activity (7.70 abs g^?1leaf) and underwent browning (0.245 abs g^?1 leaf). This extract browning and polyphenol oxidase activity were present in all red clovers tested and ranged from 0.153 to 0.458 and 6.00 to 9.00 abs g^?1 leaf, respectively. All of the legumes, including all red clovers, gave a positive response for soluble phenols.     

草酸与柠檬酸抑制鲜切香蕉酶褐变的比较研究

对草酸和柠檬酸抑制鲜切香蕉片酶褐变的效果进行了研究。鲜切香蕉片分别用0、5、10、20、40、60、80、100mmol/L的草酸和柠檬酸溶液浸泡5min,然后置于白纸上常温存放8h,每2h抽样测定香蕉片的L*值;鲜切香蕉片分别用60mmol/L草酸、60mmol/L柠檬酸、60mmol/L草酸+0.5%异抗坏血酸钠和60mmol/L柠檬酸+0.5%异抗坏血酸钠溶液浸泡5min,然后装在塑料托盘里,用PE保鲜膜包扎,在10℃下贮藏5d,每天抽样测定香蕉片的L*值。结果表明,无论常温还是低温下,草酸和柠檬酸均可有效抑制鲜切香蕉褐变,而且草酸抑制鲜切香蕉褐变的效果均好于柠檬酸。     

Purification and some properties of polyphenol oxidase of longan fruit

Polyphenol oxidase (PPO) was isolated from longan (Dimocarpus longan Lour.) fruit peel, with a 46-fold purification of PPO by ammonium sulfate, Sephadex G-200 and Phenyl Sepharose being achieved. Pyrogallol, 4-methylcatechol, and catechol were good substrates for the enzyme, and activity with chlorogenic acid, p-cresol, resorcinol, or tyrosine was not observed. The optimal pH for PPO activity was 6.5 with 4-methylcatechol. The enzyme had a remarkably temperature optimum (35°C) and was relatively stable, requiring a little more than 20 min at 50°C for 50% loss of activity. Reduced glutathione, l-cysteine, thiourea, FeSO₄ and SnCl₂ markedly inhibited PPO activity, whereas MnSO₄ and CaCl₂ enhanced PPO activity. Data obtained in this study might help to better understand longan fruit peel browning.     

Inhibition of polyphenol oxidase and peroxidase activities on fresh-cut apple by simultaneous treatment of ultrasound and ascorbic acid

The effects of ultrasound and ascorbic acid on activity changes of polyphenol oxidase and peroxidase, of fresh-cut apple during storage, were investigated. The combined treatment of ultrasound and ascorbic acid inactivated monophenolase, diphenolase, and peroxidase, whilst the individual treatment of ultrasound or ascorbic acid had inverse and limited inhibitory effect on the enzymes. The main protein bands had a molecular weight of approximately 63 kDa. A diffuse band, lacking the electrophoretic mobility of proteins, was observed after combined treatment. This investigation revealed that simultaneous treatment with ultrasound and ascorbic acid had synergistic inhibitory effects on several enzymes related to enzymatic browning.     

马铃薯贮藏期间褐变强度与多酚氧化酶活性变化的研究

测定了十个马铃薯品种在六个月窖藏期间褐变强度和多酚氧化酶活性的变化。结果表明:马铃薯的褐变强度和多酚氧化酶活性在品种间的差异达极显著水平,除渭薯8号外,其它品种在贮藏期间的变化也达到极显著水平。在贮藏期间,不同品种的褐变强度和多酚氧化酶活性的变化趋势不同,但同一品种褐变强度与多酚氧化酶活性之间存在中度的正相关,相关系数r为0.736。