Polyphenol Oxidase, Peroxidase and PhenylalanineAmmonium Lyase Induced in Postharvest Peach Fruitsby Inoculation with Pichia membranefaciensor Rhizopus stolonifer

Rhizopus rot of peach fruits could be significantly suppressed by Pichia membranefaciens. Polyphenol oxidase (PPO), peroxidase (POD) and phenylalanine ammonium-lyase (PAL) activities induced by inoculation with P. membrane faciens or R. stolonifer were studied in postharvest peach fruits. The activities of PPO and PAL in peaches increased significantly after being inoculated with P. membrane faciens + R. stolonifer by 24 h, the activities maintained at a high level throughout the experiment. Under the condition of infected with R. stolonifer alone, activity of PPO and PAL could also increased, but the levels were lower than those treated with P. membrane faciens+ R. stolonifer. However, fruits inoculaed with P. membrane-faciens + R. stolonifer or R. stolonifer alone did not stimulated POD activity. The results suggest that the activation of these defense enzymes is involved in the action of P. membrane faciens against R. stolonifer.

Inhibitory effect of tartary buckwheat seedling extracts and associated flavonoid compounds on the polyphenol oxidase activity in potatoes(Solanum tuberosum L.)

To improve the processing quality of potatoes,phosphate buffer extract(PBE),50%ethanol(E50),and aqueous extract(AE)of tartary buckwheat seedlings were evaluated for their ability to inhibit the enzymatic browning of potatoes.The results suggest that all extracts of tartary buckwheat seedlings exert significant inhibitory effects on the polyphenol oxidase(PPO)activity in potatoes.The relative concentrations required for a 50%reduction in the PPO activity(IC50)were 0.21,0.28 and 0.41 mg mL^-1,for PBE,E50 and AE,respectively.The strongest inhibitory activity was observed for PBE,followed by E50 and AE.Four flavone compounds in the PBE of tartary buckwheat seedlings(i.e.,rutin,kaempferol-3-O-rutinoside,quercetin,and kaempferol)were identified by high-performance liquid chromatography.These compounds were subsequently evaluated for their roles in the inhibition of PPO from potatoes using a model system.The results indicated that rutin exhibited the highest inhibition rate on the PPO of potato.A synergistic inhibitory effect was observed by mixing rutin,kaempferol-3-Orutinoside,quercetin,and proteins.The inhibitory patterns of rutin,kaempferol-3-O-rutinoside,and quercetin on the enzyme were noncompetitive and reversible,with inhibitory constants of 0.12,0.31,and 0.40 mg mL^-1,respectively.Flavonoids from tartary buckwheat seedlings may exhibit a common mechanism with phenolic compounds,involving the blockage of the reaction of oxygen with PPO leading to the inhibition of the enzymes involved in browning.Based on these results,extracts of tartary buckwheat seedlings can be used as potent natural inhibitors.

Potential of plant polyphenol oxidases in the decolorization and removal of textile and non-textile dyes

In this study an effort has been made to use plant polyphenol oxidases; potato （Solanum tuberosum） and brinjal （Solanum melongena）, for the treatment of various important dyes used in textile and other industries. The ammonium sulphate fractionated enzyme preparations were used to treat a number of dyes under various experimental conditions. Majority of the treated dyes were maximally decolorized at pH 3.0. Some of the dyes were quickly decolorized whereas others were marginally decolorized. The initial first hour was sufficient for the maximum decolorization of dyes. The rate of decolorization was quite slow on long treatment of dyes. Enhancement in the dye decolorization was noticed on increasing the concentration of enzymes. The complex mixtures of dyes were treated with both preparations of polyphenol oxidases in the buffers of varying pH values. Potato polyphenol oxidase was significantly more effective in decolorizing the dyes to higher extent as compared to the enzyme obtained from brinjal polyphenol oxidase. Decolorization of dyes and their mixtures, followed by the formation of an insoluble precipitate, which could be easily removed simply by centrifugation.

Effects of Terpinen-4-ol on Four Metabolic Enzymes and Polyphenol Oxidase (PPO) in Mythimna separta Walker

To study insecticidal mechanism of terpinen-4-ol, a main insecticidal composition in the essential oil of Sabina vulgaris, the 5th instar larvae of Mythimna separta, were investigated with terpinen-4-ol by topical application. The activities of phosphatase, glutathione S-transferase （GSTs）, cytochrome P450 （P450）, and polyphenol oxidase （PPO） of tested insects were determined in all poisoning stages, including exciting stage, convulsing stage, paralysis stage, and recover stage. The result showed that the activities of both acid phosphatase （ACP） and alkaline phosphatase （AKP） in treated insects were induced by terpinen-4-ol, but ACP was inhibited in paralysis stage. The activities of GSTs were inhibited in exciting stage, convulsing stage, and paralysis stage, but gradually recovered in recover stage. O-demethylase activity of cytochrome P450 was inhibited by terpinen-4-ol, and the inhibition rate in all poisoning stages were 26.27, 46.03, 80.24, and 90.22%, respectively. PPO activities were strongly inhibited by terpinen-4-ol both in vitro and in vivo. In conclusion, the activities of P450, GSTs, and PPO could have relation with toxicity of terpinen-4-ol against larvae of the Mythimna separta, but recover stage of the poisoning insects might be related to GSTs induced. As a new insecticide or synergist, terpinen- 4-ol has a potential value in field of insecticide resistance management.

Study on Enzymatic Kinetics of Nephelium lappaceum Polyphenol Oxidase

[Objective] This study aimed to investigate the enzymatic kinetics of polyphenol oxidase in peel of Nephelium lappaceum to explore the environmental factors affecting its catalytic activity. [ Method ] With N. lappaceum peel as the experimental material and catechol as substrate, effects of temperature, pH, four inhibitors （EDTA, Vc, sodium bisulfite and citric acid） and substrate concentration on the activity of polyphenol oxidase were investigated. [ Result] The optimal temperature for polyphenol oxidase in N. lappaceum peel was 40 %, and the optimal pH was 6.8. EDTA, Vc, sodium bisulfite and citric acid all showed ideal in- hibitory effects on the activity of polyphenol oxidase; specifically, EDTA had the strongest inhibitory effect. [ Conclusion] At low temperature, EDTA, Vc, sodi- um bisulflte and citric acid with certain concentrations can inhibit the catalytic activity of polyphenol oxidase in peel of N. lappaceum, extend the storage and trans- portation time of N. lappaceum fruit, and inhibit the browning. This study provides theoretical basis for the development and utilization of polyphenol oxidase in peel of N. lappaceum.

Study on Polyphenol Oxidase Activity and Total Phenol Content of Docynia indica

[Objectives] To study the polyphenol oxidase (PPO) activity and total phenol content of Docynia indica .[Methods] The tender branches and petioles of the medicinal and edible plant D. indica were used as experimental materials, and the annual changes of total phenol content and polyphenol oxidase (PPO) activity were analyzed.[Results] pH 7.0 was the optimum value of polyphenol oxidase activity in D. indica plants;the best substrate was catechol with the concentration of 0.15 mol/L;the optimum temperature was 25 ℃.[Conclusions] The total phenol content of D. indica plants was the lowest in April, May and September of each year. The polyphenol oxidase activity of D. indica plants at flowering and fruiting stage was significantly higher than that at pre-flowering stage.

Purification and Partial Characterization of Polyphenol Oxidase from Sapodilla Plum (<i>Achras sapota</i>)

The browning of fruits can be considered as an enzymatic oxidation which is believed to be one of the main causes of quality loss during post-harvest handling. The enzymes responsible for this are the oxidoreductases;the polyphenol oxidase (PPO) (monophenol, o-diphenol, oxygen oxidoreductase;EC 1.14.18.1) belongs to this group. This enzyme, which is found in the sapodilla plum (Achras sapota), was purified using a phenylsepharose and a SephacrylS-200 columns. The molecular weight of the purified enzyme was estimated to be about 66 kDa by gel filtration and 29 kDa by SDS-PAGE. A single protein band was found using the latter system (SDS-PAGE), which shows that the PPO of the pulp of the sapodilla plum may be composed of two protein subunits with similar molecular weight. The optimum pH was 7.0 and the optimum temperature 60℃. The most effective inhibitors tested were ascorbic acid, sodium metabisulfite and acetic acid.

Polyphenol Oxidase Inactivation by Microwave Oven and Its Effect on Phenolic Profile of Loquat (<i>Eriobotrya japonica</i>) Fruit

The objective of this research was investigated the effect of polyphenol oxidase microwave treatment on phenolic composition, antioxidant activity and microstructure of loquat fruit. Phenolic profile of methanolic extracts prepared from fresh, and microwave-treated samples were analyzed. Antioxidant activity was also evaluated by 2,2’-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS?+) and 1,1-diphenyl-2-picrylhydrazyl (DPPH+) methods. In addition, polyphenol oxidase inactivation was carried out using a response surface methodology to establish the optimal conditions of treatment. The phenolic content of fresh mesocarp was 311 ± 0.60 mg gallic acid equivalents (GAE)/100g dry weight (DW) and that of microwave-treated mesocarp was 1230 ± 0.36 mg GAE/100g DW. Total phenolic content of water/ methanol extract significantly increases after microwave treatment rather than methanolic extract of fresh loquat. Five glycoside phenolics were identified by HPLC-DAD-MS as 3-caffeoylquinic acid, 3-p-coumaroylquinic acid, 5-caffeoylquinic acid and quercetin-3-O-sambubioside. Methanolic extract of microwave-treated mesocarp showed higher antioxidant activity than that of fresh mesocarp. Thus, polyphenol oxidase inactivation by microwave energy preserved the integrity of phenolic compounds as well as antioxidant activity in mesocarp extracts prepared from loquat fruit. It was also noted that phenolics were more abundant in the microwaved samples than in the fresh samples.

Polyphenol Oxidase Activity and Colour Changes of ‘Starking’ Apple Cubes Coated with Alginate and Dehydrated with Air

The objective was to study the effect of alginate coating on polyphenol oxidase (PPO) activity and colour of ‘Starking’ apple cubes during dehydration with hot air. Apple cubes were dehydrated at 20oC, 35oC, or 40oC, with a parallel airflow. Analysis of PPO activity, colour (L*, a*, b*) and dry matter were performed along the dehydration process at each temperature. All samples presented a peak in relative PPO activity in the beginning of the drying. Exponential models fitted well the experimental data after the peak. Cubes without coating presented lower PPO activity, suggesting lower browning, than coated samples throughout the dehydration process, for all temperatures. Better results for coated samples were obtained with a perpendicular airflow drying at 40oC, after dipping the whole apple in water at 60oC for 10 min. In order to prevent coated samples from browning, drying by perpendicular airflow preceded by a thermal treatment of the whole apple is required.

Immobilization and Properties of Polyphenol Oxidase

[Objectives]This study was conducted to investigate the effects of embedding conditions on activity and catalytic properties of immobilized polyphenol oxidase.[Methods]Polyphenol oxidase was immobilized in a polymer material by the embedding method,and the optimal immobilization conditions were obtained by single factor tests:CaCl_(2) concentration 2.0%,sodium alginate concentration 2.0%,immobilization time 2 h and mass ratio of enzyme to carrier 10 mg/100 g,under which the immobilized enzyme activity was 93.33 U/g.Under the above conditions,the properties of polyphenol oxidase immobilized by sodium alginate(A-PPO)and free polyphenol oxidase were studied.[Results]The thermostability of A-PPO was better than that of the free enzyme,but the pH stability of A-PPO was inhibited.The Michaelis constant K_(m) values of free polyphenol oxidase and A-PPO were 0.37 and 0.48 mmol/L,respectively,and the maximum reaction rate V_(max) values were 0.38 and 0.51 mmol/(L·g),respectively.[Conclusions]This study provides a theoretical basis for the study of the properties of polyphenol oxidase.

<i>In Silico</i>and <i>in Vitro</i>Approach for the Understanding of the Xanthine Oxidase Inhibitory Activity of Uruguayan Tannat Grape Pomace and Propolis Poliphenols

The use of food additives with xanthine oxidase (XO) inhibitory activity offers an alternative approach to hyperuricemic and gout disease treatment, and provides an example of antioxidant nutraceutics. The in vitro and in silico XO inhibitory activity of polyphenols from Uruguayan Tannat grape pomaces and propolis extracts was evaluated as well as the scavenging capacity of said compounds. When comparing propolis and grape pomace samples, the in vitro studies demonstrated that polyphenols extracted from propolis are more active as free radical scavengers than those from Tannat grape pomace. Both natural products effectively inhibited XO but the capacity of phenols present in GP is higher than the one present in P. The high content of anthocyanins in GP, absent in P, could account for this observation. In silico assays allowed us to determine relevant ligand-receptor interactions between polyphenols, from a database built with previously reported polyphenols from both natural products, and the active site of XO. The in silico results showed that compound (E)-isoprenylcaffeate from propolis was the best potential XO inhibitor displaying hydrophobic aromatic interaction between the conjugated ring of the caffeate moiety and polar interactions between hydroxyl groups from caffeate with the active site polar residues. Among grape pomaces, the Cyanidin-3-O-(6-(E)-p-coumaroyl)-glucoside was the best XO inhibitor;its moiety oxychromenyl being relevant to the docking stabilization. All these results lead us to propose Uruguayan propolis and Tannat grape pomace extracts as food additives as well as phytopharmaceuticals to decrease the uric acid levels in gout disease and to act against oxidative stress.

采前氨基乙氧基乙烯甘氨酸处理对‘黄冠’梨长期冷藏后果实品质和果心褐变的影响

为延长‘黄冠’梨贮藏期,减轻长期贮藏后货架期间果实品质劣变,采用不同浓度氨基乙氧基乙烯甘氨酸(aminoethoxyvinylglycine,AVG)在采前喷施‘黄冠’梨果实,在冷藏180 d及货架7 d((180+7)d)后检测相关指标。结果显示,采前喷施AVG可延缓‘黄冠’梨果实硬度下降,维持可溶性固形物含量以及可滴定酸含量,有效抑制果面褐斑和果心褐变的发生,其中以200 mg/L AVG效果最好。‘黄冠’梨果实在长期冷藏时果心发生褐变,同时熊果苷、绿原酸以及总酚含量增加,多酚氧化酶活性升高;PbPAL1、PbPAL2、PbPPO1、PbPPO5、PbLOX1以及PbLOX5在冷藏期间表达量升高,货架期表达量随之下降。采前200 mg/L AVG处理‘黄冠’梨,可显著促进熊果苷、绿原酸以及总酚在整个贮藏过程中含量的增加,抑制多酚氧化酶活性升高,抑制PbPAL1、PbPAL2、PbPPO1、PbPPO5以及PbLOX5的表达,进而有效减少果心褐变的发生。

大肠杆菌多酚氧化酶的分子克隆及异源高效表达

菜籽粕、棉籽粕等非粮饲料资源存在的酚类化合物芥子碱、棉酚等抗营养因子,严重影响了其饲喂价值。本课题组筛选出的一株大肠杆菌属芥子碱降解菌SDB2已被证实能通过分泌多酚氧化酶来降解芥子碱和棉酚,本试验旨在运用基因工程技术克隆SDB2多酚氧化酶基因,实现其高效表达,为SDB2多酚氧化酶在饲料工业上的规模化应用奠定基础。从大肠杆菌SDB2中克隆出多酚氧化酶基因,将其与质粒pET28a连接后转化至大肠杆菌BL21感受态细胞中培养,通过PCR鉴定及质粒酶切验证方法构建重组菌株,同时对重组克隆基因进行生物信息学分析。采用“2×2”交叉试验设计,以温度和诱导剂IPTG浓度两个影响因素诱导SDB2多酚氧化酶基因在重组菌株中高效表达,经SDS-PAGE和WB双重验证后,确立最佳诱导条件,最终纯化出目标酶蛋白。结果表明:(1)成功克隆SDB2多酚氧化酶基因,构建出异源表达重组菌株。(2)克隆出的SDB2重组多酚氧化酶基因含目标核苷酸741个,总编码氨基酸263个(含标签氨基酸20个),氨基酸组成的蛋白相对分子质量为28499.0,理论等电点为6.94,据此预测出SDB2重组多酚氧化酶三维结构模型。(3)确立SDB2多酚氧化酶异源高效表达的最佳诱导条件为温度37℃,IPTG浓度1 mmol,该条件下表达出大量可溶性酶蛋白,有利于工业化生产。本试验条件下,大肠杆菌SDB2多酚氧化酶成功实现了分子克隆及异源高效表达,为我国非粮饲料资源实现高效利用提供了技术参考。

灰霉病早期胁迫下花椰菜多酚氧化酶活性的高光谱研究

利用高光谱技术实现灰霉病早期胁迫下花椰菜多酚氧化酶(PPO)活性的快速无损检测。为了使预测效果更好,在900~1700 nm光谱范围内采集253个健康花椰菜样本及257个染病花椰菜样本的光谱信息,并使用分光光度计法对样本中多酚氧化酶活性进行测定。对健康及染病花椰菜样本PPO活性均值进行分析,发现健康花椰菜PPO活性均值(10.257 U·g^(-1))小于染病花椰菜PPO活性均值(12.324 U·g^(-1))。利用光谱-理化值共生距离(SPXY)算法对样本进行校正集(健康样本193个,染病样本197个)和预测集(健康样本60个,染病样本60个)的划分,对划分后的样本集进行六种单一预处理(卷积平滑算法SG、去趋势算法DT、中值滤波MF、归一化处理NOR、标准正态变量变换SNV、基线校正Baseline)。利用相关系数(R)和均方根误差(RMSE)作为模型评价指标,发现预处理能够有效提高模型的精度和稳定性,其中健康样本经NOR预处理后的预测集建模效果最好;染病样本经DT预处理后的预测集建模效果最好。采用连续投影算法(SPA)与回归系数法(RC)提取特征波长,建立PPO活性值的偏最小二乘回归(PLSR)、最小二乘支持向量机(LS-SVM)及BP神经网络三种预测模型,探究不同特征波长提取方法对模型精度的影响,比较不同建模方式对花椰菜PPO活性预测的准确度。结果说明提取特征波长可以优化光谱信息,SPA和RC对两种样本提取的波长个数分别为9,12,7和11,其中SPA优化后的光谱数据对健康样本PPO活性预测效果较好,RC优化后的光谱数据对染病样本PPO活性预测效果较好。对比分析模型效果,发现LS-SVM模型对两种样本和其对应的酶活性产生了很好的拟合效果。最终发现,SPA-LS-SVM模型对于健康花椰菜PPO活性预测效果较好,其预测相关系数(Rp)为0.832,预测均方根误差(RMSEP)为1.676;RC-LS-SVM模型对于染病花椰菜PPO活性预测效果较好,其Rp为0.848,RMSEP为1.156。研究结果表明高光谱技术可以实现灰霉病早期胁迫下花椰菜多酚氧化酶活性的测定,为快速检测花椰菜多酚氧化酶活性及便携式仪器开发提供了科学依据。

月柿果片不同部位酚类组成及褐变酶活力分布特征

为研究月柿果片不同部位酚类组成与其在微波干燥过程中色泽变化不均匀的关系,将月柿果片由顶到底分为A、B、C 3个区域,由外到内分成1、2两个区域,从酶促褐变视角探究新鲜月柿果片不同部位水分、总酚、可溶性单宁、总黄酮含量及多酚氧化酶(polyphenol oxidase,PPO)、过氧化物酶(peroxidase enzyme,POD)活力的分布特征。结果表明,在纵向方向上,水分含量由顶到底依次减少,POD活力则呈现相反的趋势;总酚和总黄酮含量分布为A区>C区>B区;可溶性单宁含量呈现A1区<B1区<C1区、B2区<C2区<A2区的变化趋势;PPO活力呈现B区>C区>A区的分布特征。在横向方向上,水分含量和PPO、POD活力分布表现为1区<2区;总酚、可溶性单宁和总黄酮含量呈现A1区<A2区、B1区>B2区、C1区>C2区的变化趋势。此外,主成分分析表明月柿果片不同部位活性成分组成有明显差异。

蜂蜜对苹果酶促褐变的抑制及有效成分分析

作者以防止新鲜苹果切口的褐变为目标,比对不同食品配料组成的复配成分,发现蜂蜜作为涂膜材料可以有效延缓鲜切苹果片的褐变。将鲜切苹果片浸泡在质量分数0.1 g/g的椴树蜂蜜水溶液中,8 d后其仍能维持新鲜的色泽;探究了蜂蜜对苹果多酚氧化酶(polyphenol oxidase,PPO)抑制的动力学,发现蜂蜜对苹果PPO起非竞争性抑制作用,酶活抑制能力来自蜂蜜的抗氧化、清除自由基(相关系数r>0.6)能力;进一步分析蜂蜜成分与酶活抑制率的关系,发现蜂蜜中蛋白质与多酚(r值分别为0.79、0.63)是蜂蜜发挥抗褐变作用的重要成分。

马铃薯抗褐变品种的鉴定与筛选

褐变是马铃薯贮藏和加工过程中常见现象,影响其营养、感官和食味品质,降低其商品价值。以24个马铃薯品种为试验材料,通过测定多酚氧化酶(Polyphenol oxidase,PPO)活性、褐变强度、维生素C含量和褐化指数4个相关指标,对不同马铃薯品种抗褐变能力进行综合鉴定和评价。不同马铃薯品种PPO活性、褐变强度、维生素C含量、褐化指数均存在明显差异。选择PPO活性在15 U以内的11个品种为预选褐变材料;选择维生素C含量在8 mg/100 g以上的7个品种为预选褐变材料;选择初始褐变强度小于0.2,变化值在0.5以内的7个品种为预选褐变材料;选择24 h总褐化指数在Ⅱ级以内,且开始发生褐变时间大于1 h的11个品种为预选褐变材料。对4个褐变相关指标进行相关性和聚类分析,将测试的24个马铃薯品种聚为3类。类型Ⅰ为易褐变类型,类型Ⅱ是中间水平类型,类型Ⅲ是抗褐变类型。筛选出6个抗褐变马铃薯品种,即‘东农314’‘东农324’‘东农325’‘东农328’‘延薯13号’和‘中薯5号’。研究可为马铃薯种质资源创新和抗褐变育种提供基础材料。

褐变抑制剂对果蔬多酚氧化酶的影响

果蔬贮藏过程中,褐变问题严重影响其经济价值及营养价值,由多酚氧化酶引起的酶促褐变备受关注,寻找合适的抑制方法是国内外研究的热点。一些化学抑制剂对于酶促褐变具有良好的抑制效果,本文综述了果蔬褐变的机理,抗坏血酸、半胱氨酸、柠檬酸抑制剂的作用原理及具体应用,旨在为解决由酶促褐变引起的果蔬生产加工问题提供参考,促进果蔬品质的提升。

不同湿度条件对雪茄烟叶晾制过程中酶促棕色化反应及品质的影响

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