中华管鞭虾多酚氧化酶的分离制备

对中华管鞭虾中多酚氧化酶(PPO)的提取分离及其影响因素进行研究,讨论了新鲜管鞭虾的保藏温度、提取方法、浸提时间、缓冲液pH值、料液比和超声波提取等单因素影响,在此基础上采用正交法对各因素进一步优化。结果表明:在室温状态下,使用溶液浸提法,最佳时间为8h,最佳料液比为1:2,缓冲液最适pH值为8,超声时间为20min。     

Evidence of an active laccase-like enzyme in deepwater pink shrimp (Parapenaeus longirostris)

This paper demonstrates the presence of an active laccase-like enzyme from deepwater pink shrimp (Parapenaeus longirostris) using polyacrylamide gel electrophoresis. This enzyme was found in all anatomical parts of the deepwater pink shrimp, but particularly in the cephalothorax, and became active during the course of storage. Gel staining with laccase-specific substrates such as ADA, DMP and DAB was used to characterize a protein of around 44 kDa as containing laccase activity. The enzyme was inhibited by a specific inhibitor, CTAB. 4-Hexylresorcinol, a specific inhibitor of polyphenoloxidase (PPO), did not inhibit the laccase-like enzyme. Low concentrations of antioxidants ascorbic acid or sodium metabisulphite were sufficient to inhibit the laccase-like enzyme. ABTS and DMP were subsequently used to characterize the enzyme. Given the evidence of this enzyme in deepwater pink shrimp, new melanosis-inhibiting compounds that are suitable for consumption need to be found to complement specific inhibitors of PPO activity.     

Characterisation and tissue distribution of polyphenol oxidase of deepwater pink shrimp (Parapenaeus longirostris)

Characterisation and tissue distribution of polyphenol oxidase (PPO) was studied in deepwater pink shrimp (Parapenaeus longirostris) post mortem. PPO activity was the highest in the carapace, followed by that in the abdomen exoskeleton, cephalotorax, pleopods and telson. No PPO activity was found in the abdomen muscle and in the pereopods and maxillipeds using the enzymatic assay. Storage of whole shrimps and of the different organs showed that melanosis (blackening) required the presence of the cephalotorax to be initiated, indicating that its development depends on other factors in addition to the PPO levels. Further characterisation was carried out in extracts partly purified using 40–70% ammonium sulfate fractionation. The enzyme had the highest activity at pH 4.5 and was most stable at pH 4.5 and 9.0. No clear maximum was observed in the 15–60 °C range but the higher stability was achieved at 30–35 °C. Apparent kinetic constants in the partly purified PPO from carapace were K_M = 1.85 mM and V_max = 38.5 U/mg of protein, pointing to a high affinity and reactivity of the enzyme when assayed with DOPA. Electrophoretic mobility was studied in native PAGE and non-reducing SDS–PAGE followed by staining with DOPA. Approximate MW of 500 kDa and 200 kDa were observed, respectively. These two forms could correspond to aggregates of minor PPO subunits that could not be resolved in these electrophoretic systems. The peptide mass fingerprinting obtained by MALDI-TOF analysis showed some peptides whose homology with hemocyanins and different PPO subunit precursors has already been demonstrated in the same species.     

Comparison of two treatment methods on the purification of shrimp polyphenol oxidase

The effects of different stirring time and two treatment methods (salt and organic solvent) on the recovery of shrimp polyphenol oxidase (PPO) were investigated. Stirring for 30 min yielded maximal PPO recovery. With respect to PPO specific activity, yield and purification fold enhancement, the use of butanol treatment followed by Phenyl Sepharose CL-4B chromatography was shown to be better than ammonium sulphate fractionation and then Phenyl Sepharose chromatography. White shrimp PPO was more susceptible than pink shrimp PPO to inactivation during purification. © 1997 SCI.     

贝莱斯芽孢杆菌处理对厚皮甜瓜保鲜效果及保护酶活性的影响

为探究生防菌处理对厚皮甜瓜保鲜效果和保护酶活性的影响,本研究选用西州蜜25号厚皮甜瓜作为研究对象,通过在25 ℃和4 ℃条件下在厚皮甜瓜表面接种贝莱斯芽孢杆菌BG-2,测定贮藏期间拮抗菌在厚皮甜瓜表面定殖特性、保鲜效果和厚皮甜瓜过氧化物酶（Peroxidase,POD）、多酚氧化酶（Polyphenol oxidase,PPO）和苯丙氨酸解氨酶（Phenylalnine ammonialyase,PAL）活性的变化。结果表明,在25 ℃和4 ℃条件下,拮抗菌在厚皮甜瓜表面定殖数量均呈现先上升后下降的趋势且能稳定定殖。接种拮抗菌的厚皮甜瓜腐烂效果、抑制pH和可溶性固形物含量的下降显著优于对照组（P<0.05）。POD和PAL活性呈现先上升后下降的趋势,PPO活性在室温下呈现先上升后下降趋势,在低温下呈现缓慢上升趋势。25 ℃下,拮抗菌处理组POD在第20 d时酶活最高,PAL和PPO在第10 d时酶活最高;4 ℃下,拮抗菌处理组POD和PAL在第20 d时酶活最高。PPO处理在第40 d时酶活最高。且拮抗菌处理过的厚皮甜瓜酶活始终高于病原菌处理组。在25 ℃和4 ℃贝莱斯芽孢杆菌BG-2均能在厚皮甜瓜表皮上稳定定殖,抑制贮藏期厚皮甜瓜腐烂,延缓pH、可溶性固形物、POD、PPO和PAL酶活性下降,且BG-2和低温相结合对上述效果更好。本研究为延长新疆厚皮甜瓜贮藏期提供理论依据,为生防菌作用在厚皮甜瓜采后保鲜提供技术支持。     

六堡茶渥堆过程中产多酚氧化酶菌株的筛选及多酚氧化酶的分离纯化

本研究采用传统分离法从渥堆六堡茶中共分离出42株菌,然后利用平板初筛和液体发酵进行复筛,从42株微生物中筛选出10株产PPO的微生物并测定其酶活.用盐析及透析法对产酶活性最高的菌株所产的多酚氧化酶进行纯化,纯化后的酶液用SDS-PAGE及Native-PAGE电泳方法确定PPO的分子量范围和PPO种类.结果表明,渥堆六...     

Effect of cinnamaldehyde on melanosis and spoilage of Pacific white shrimp (Litopenaeus vannamei) during storage

BACKGROUND: Shrimp is a very perishable product and postmortem changes occur rapidly. Sulfiting agents were once and are still widely used as a preservative in the shrimp industry. However, the application of sulfite in shrimp may pose a risk to human health. Thus development of a natural preservative as a sulfite alternative to extend the shelf life of Pacific white shrimp is urgently needed. RESULTS: The effects of cinnamaldehyde essential oil (1 and 5 g kg^?1) on the shelf life of Pacific white shrimp stored at 4 °C were investigated. As the concentration of cinnamaldehyde increased, residual polyphenoloxidase (PPO) enzyme activity decreased. Kinetic analysis showed that cinnamaldehyde was a noncompetitive inhibitor for the oxidation of L ‐DOPA (L ‐3,4‐dihydroxyphenylalanine) by PPO of Pacific white shrimp. Based on this study, shrimp treated with 5 g kg^?1 cinnamaldehyde possessed the lowest aerobic plate count, total volatile basic nitrogen, and pH values in all treatments after 10 days of storage. According to the results of L*, cinnamaldehyde showed inhibitory activity toward the formation of melanosis. CONCLUSION: Treatment with cinnamaldehyde could improve the sensory properties and extend the shelf life of Pacific white shrimp to 8 days. Therefore, cinnamaldehyde could be used as a promising natural preservative for inhibiting melanosis and preventing the growth of microbes during the chilled storage of Pacific white shrimp. Copyright © 2012 Society of Chemical Industry     

Polyphenoloxidase Activity in Germinated Legume Seeds

Polyphenoloxidase (PPO) activity was estimated in the whole seed and its morphological components of germinated chickpea, pigeon-pea, greengram and blackgram. Increase in enzyme activity on germination was highest in blackgram and lowest in chickpea. Pyrogallol oxidizing activity was more predominant in the legume seeds and the seed fractions. Embryo was richest in PPO activity but the cotyledons were the major contributor to the whole PPO activity. Except in black-gram, the seed coat fraction was devoid of any PPO activity. The seed coat of pigeonpea and chickpea contained a water soluble and heat stable PPO inhibitor. Induction of the enzyme on germination did not fully explain the reduction in tannin content of the legume seeds. Role of polyphenol oxidase in seed germination is briefly discussed.     

4-HR与抗坏血酸对中华管鞭虾多酚氧化酶的抑制动力学模拟分析

研究无硫复合抑制剂主要作用成分4-己基间苯二酚（4-hexylresorcinol,4-HR）、抗坏血酸对中华管鞭虾多酚氧化酶（polyphenoloxidase,PPO）活性的抑制作用,并从抑制剂化学结构及动力学模型出发分析推断其抑制机理。结果表明,4-HR、抗坏血酸均能抑制PPO活力,且对酶促反应有延滞作用,两者半抑制浓度分别为0.072、2.02?mmol/L,对中华管鞭虾PPO的抑制作用都表现为竞争性抑制;化学动力学模拟分析显示4-HR、抗坏血酸均可与PPO双铜活性中心结合,其中4-HR可生成一种高稳定的多酚氧化酶络合物,持续有效地消耗PPO。4-HR、抗坏血酸均能有效抑制中华管鞭虾PPO酶促反应,其中4-HR抑制效果更显著。本实验为无硫复合抑制剂在海水虾捕捞与加工过程中的应用推广提供理论依据。     

The effect of heating conditions on polyphenol oxidase, proteases and melanosis in pre-cooked Pacific white shrimp during refrigerated storage

Pre-cooked Pacific white shrimp (Litopenaeus vannamei) is an important shrimp product. However melanosis, especially in the cephalothorax including carapace and internal organs, is more likely caused by the remaining polyphenol oxidase (PPO) after pre-cooking. Thus, PPO from carapace and proteases from hepatopancreas of Pacific white shrimp were characterised and the remaining activities of both enzymes were monitored in pre-cooked shrimp during storage at 4 °C. Based on activity staining using L-β-(3,4 dihydroxylphenyl) alanine as a substrate, PPO consisted of two isoforms with apparent molecular weight of 210 and 220 kDa. No difference in activity band was observed when analysed under reducing and non-reducing condition. Proteases from hepatopancreas were able to activate PPO to some degree. For the in vitro study, both enzymes were quite stable when heated at temperature up to 70 °C but the loss in activities increased with increasing heating time (0-120 s). When Pacific white shrimp were pre-cooked to obtain different core temperatures (50-90 °C), different PPO and protease activities were retained. Higher core temperatures were associated with lower PPO and protease activities, but higher cooking loss. When the shrimp were pre-cooked at 80 °C, the residual PPO and protease activities were 3.9% and 5.4%, respectively and cooking yield of 95.6% was obtained. The resulting pre-cooked shrimp possessed lower melanosis score during 7 days of storage at 4 °C. Thus, pre-cooking of shrimp to obtain a core temperature of 80 °C, with a holding time of 30 s, could prevent the severe cooking loss and lower melanosis during subsequent storage.     

Inactivation of polyphenol oxidase from Pacific white shrimp by dense phase carbon dioxide

The inactivation of polyphenol oxidase (PPO) from Pacific white shrimp exposed to dense phase carbon dioxide (DPCD) treatment was investigated. PPO activity showed a dramatic loss at 4–25 MPa and 37 °C. At the lower pressure (4–15 MPa), the experimental data of inactivation followed the first-order reaction kinetic model, the pressure sensitivity (Z_P) of the kinetic parameters was 49.02 MPa and the activation volume (△ V^≠) was − 120.88 cm³/mol. At the higher pressure (20 and 25 MPa), the experimental data of inactivation followed the biexponential kinetic model. The kinetic rate constant k_F and k_S of fast and stable fractions were 2.45 and 0.08 min^{− 1}, respectively. The decimal reduction time D_F and D_S were 0.94 and 29.43 min at 25 MPa and 37 °C, respectively. After DPCD treatment, the loss activity of PPO had no restoration storing for 6 days at 4 °C. The results of SDS-PAGE and activity staining also showed that DPCD treatment had the obvious inhibitory effect on PPO from Pacific white shrimp. The PPO activity in vivo was easier to be inactivated than that in crude PPO extracts under the same DPCD treatment conditions.

Industrial relevance

There is a growing interest in non-thermal pasteurization methods, which could retain food's freshlike physical, nutritional, and sensory qualities. Pacific white shrimp accounts for 90% of the global aquaculture shrimp production, they are becoming increasingly popular. However, enzymatic browning of shrimp has been of great concern to food scientists and food processors. Dense phase carbon dioxide (DPCD) may be an adequate tool to obtain high quality since PPO activity could not be inactivated totally by high pressure under 400 MPa yet. The present work deals with the inactivation of PPO from Pacific white shrimp exposed to DPCD treatment in order to explore the feasibility of shrimp by DPCD process.     

蛋白交联中多酚氧化酶的酶源筛选及酶学性质研究

从多种植物和真菌中筛选出多酚氧化酶(PPO)活力高的原料,并对其酶学性质进行研究,以利于其在蛋白交联中的应用。双孢蘑菇和茄子中PPO 酶活相对较高,达到11004U/g 和9376U/g。双孢蘑菇PPO 和茄子PPO 的最适温度和pH 值均分别为20℃和7.0;在温度较高时,茄子PPO 比双孢蘑菇PPO 稳定性更好。Zn2+、Mn2+、Ag+对双孢蘑菇PPO 和茄子PPO 活力表现明显的抑制作用,Cu2+ 对双孢蘑菇PPO 活力表现明显的促进作用,而对茄子PPO 却有很强的抑制作用。双孢蘑菇PPO 和茄子PPO 的Km 和Vmax 分别为5.5mmol/L、1666.7U 和8.75mmol/L、2500U。     

Polyphenol oxidase activity and browning of three avocado varieties

A comparison between the in vitro total and specific activity of polyphenol oxidase of three avocado varieties, Fuerte, Horeshim and Lerman, showed clear differences that were correlated with the in vivo rate of browning of the corresponding freshly cut mature fruit; Fuerte had the highest values, followed by Horeshim and then Lerman. A good correlation existed between the PPO activity in the crude homogenate of the fresh fruit and the crude enzyme extracted from the acetone powder prepared from each variety. The possibility that the relatively low PPO activity in the crude enzyme of the Lerman avocado was due to the presence of an inhibitor and to factor(s) degrading the enzyme, or that the relatively high activity of the Fuerte enzyme was due to an activator, was tested in various ways and ruled out. It was therefore concluded that the differences in the rate of browning of the three avocado varieties studied were directly related to the PPO activities as expressed in the crude enzyme fraction.     

浸渍冻结对凡纳滨对虾冻藏过程中品质的影响

为了研究浸渍冻结对凡纳滨对虾冻藏品质的影响,以挥发性盐基氮含量、脂肪氧化值、pH值、盐溶性蛋白含量、Ca2+-ATP酶活力、多酚氧化酶活力、感官评价为指标,考察浸渍冻结和静止空气冻结对凡纳滨对虾品质特性的影响。结果表明：冻藏期间静止空气冻结对虾的挥发性盐基氮含量、脂肪氧化值、pH值等腐败指标均高于浸渍冻结对虾,并且随着冻藏时间延长而显著性增大（P＜0.05）;盐溶性蛋白含量、Ca2+-ATP酶活力和多酚氧化酶活力随冻藏时间延长而下降,蛋白质逐渐变性,品质下降,但浸渍冻结对虾的下降程度小于静止空气冻结的。感官评价进一步显示,两种冻结方式处理对虾的组织、气味、色泽和外观4 方面有不同程度下降,品质不断下降,但浸渍冻结对虾的感官评分高于静止空气冻结的对虾。综合以上结果,浸渍冻结更有利于对虾冻藏过程中品质的保持。     

Changes in semen quality and morphology of the reproductive tract of the male tammar wallaby parallel seasonal breeding activity in the female

Changes in semen quality and morphology of the male reproductive tract were studied throughout the year in the highly promiscuous tammar wallaby. Body size, semen quality and gross morphology of the reproductive organs were assessed in adult males each month from January to November. The mean weight of males was similar in most periods sampled, but males were slightly heavier in the minor (P < 0.05) than the non-breeding season. Since body weight was correlated with weights of the testes, epididymides and accessory sex glands, organ weights were adjusted for body weight in subsequent analyses. In the major breeding season (late January/early February), when most females go through a brief, highly synchronized oestrus, the testes, prostate, Cowper's glands, crus penis and urethral bulb were heaviest, volume and coagulation of ejaculates were greatest, and sperm motility had increased. Semen samples collected by electroejaculation at this time contained low numbers of spermatozoa, possibly as a result of dilution and entrapment by the seminal coagulum or depletion of epididymal stores during intense multiple mating activity. In the non-breeding season (late May-July), when mating does not normally occur in the wild, there was a significant decrease in the relative weight of nearly all male reproductive organs and a decline in most semen parameters. In the minor breeding season (September-November), when pubertal females undergo their first oestrus and mating, the weights of testes, epididymides and most accessory sex glands had significantly increased similar to those of males in the major breeding season. The total number and motility of ejaculated spermatozoa were highest during this period, but the volume and coagulation of ejaculates and weight of the prostate had only increased to levels that were intermediate between the major and non-breeding seasons. Ejaculate volume was strongly correlated with prostate weight, and % motile spermatozoa was strongly correlated with epididymis weight. Semen quality thus varied seasonally with changes in androgen-dependent reproductive organs in the male tammar wallaby and appeared to be influenced by the seasonal timing of oestrus in females. Semen quality may also improve in response to an increase in the number of available oestrous females.     

Study of the quality changes and myofibrillar proteins of white shrimp (Litopenaeus vannamei) under modified atmosphere packaging with varying CO2 levels

This paper studied the effect of modified atmosphere packaging (MAP) with 40–80 % CO₂, 5 % O₂ and 15–55 % N₂ on the quality of Pacific white shrimp (Litopenaeus vannamei) during 10-day storage at (4 ± 1) °C. The effect of CO₂ on the shrimp quality was compared by analyzing the quality indices such as total viable counts (TVC), total volatile basic nitrogen (TVB-N), pH and polyphenoloxidase (PPO) activity, whiteness and sensory attributes. Changes in myofibril length of shrimp flesh were also studied, which correspond to flesh softening. The findings suggest that the shrimp packaged in modified atmosphere had better quality indices than the control. Compared the MAP-batches, 80 % CO₂/15 % N₂/5 % O₂ led to the lowest level of TVC, TVB-N values, PPO activity and the highest sensory scores. Therefore, high-CO₂ packaging should be recommended to delay quality loss of shrimp during transportation and storage.     

鲜切芦蒿PPO特性的研究

芦蒿去除不可食部分、冰水清洗、切分后用研钵碾成匀浆状于低温(4℃)离心机中离心(10000r/min,30min),获得的粗酶液用于多酚氧化酶(PPO)活力的测定和酶特性的研究。结果表明:鲜切芦蒿PPO适宜的pH值为6.5,温度为40℃。鲜切芦蒿PPO对60℃以下的温度不敏感,而在80℃以上温度下很易失活。鲜切芦蒿PPO对酚底物的亲和力依次为:绿原酸、愈创木酚>焦性没食子酸苯酚>酪氨酸>儿茶酚>间苯二酚>苯酚,且与儿茶酚结合时出现最大活力。抑制鲜切芦蒿PPO活力以焦亚硫酸钠效果最好,当其浓度达到0.33mmol/L(即0.06%)时能使PPO完全失活;亚硫酸氢钠和L-Cys的抑制效果也十分强;而EDTA-2Na浓度达3.0mmol/L对鲜切芦蒿PPO的抑制也仅为29.55%,作用很微弱;植酸、AA、醋酸锌、CA和4-HR的抑制作用介于L-Cys和EDTA-2Na之间。     

Inhibitory effect of sericin on polyphenol oxidase and its application as edible coating

Sericin, a water‐soluble globular protein derived from silk industry wastewater, was investigated for food industrial applications. The results proved that sericin retarded polyphenol oxidase (PPO) activity. However, the degree of inhibition varied depending on the enzyme origins, the types of substrate, sericin content and sericin molecular size. Using catechol as a substrate, under the conditions studied, sericin lowered purified mushroom PPO and apple extract PPO activity by 40% and mango extract PPO by 75%. Kinetic studies on purified mushroom PPO indicated that the type of inhibition of sericin was dependent on the substrates used. Inhibitory effects of sericin increased as the sericin content increased. The reduction in sericin molecular size by enzymatic hydrolysis produced sericin hydrolysate with ability to decrease PPO activity approximately three times greater than that of sericin. Fresh‐cut Red Delicious apples coated with sericin showed significant reduction in weight loss and improvement in the colour and texture.     

Characterization of Polyphenol Oxidase from Airen Grapes

Polyphenol oxidase (PPO) was isolated from grapes grown in Spain and its characteristics were studied. The partially purified enzyme had both cresolase and catecholase activities. Catecholase activity had a pH optimum in a range 3.5–4.5 and was characterized by a relatively high stability to heat. The apparent K_M for 4-methylcatechol was 9.5 mM. Cresolase activity presents a lag period which is modulated by different factors: enzyme concentration, substrate concentration, temperature or pH. The presence of o-diphenols in the reaction medium abolishes the lag period, these acting as co-substrates. The apparent K_M towards p-cresol and the activation constant for o-diphenol for cresolase activity were 0.35 mM and 1.75 μM, respectively.     

茭白中多酚氧化酶活性的测定及护色效果研究

茭白在加工过程中会发生酶褐变,影响茭白的质量。文中研究了温度、pH值对茭白中PPO(多酚氧化酶)活性的影响,设计了不同的护色方案,以确定茭白的无硫护色条件。结果表明,茭白中多酚氧化酶活性最适温度36℃,最适pH值为5.6。用质量分数为0.1%L-半胱氨酸、0.7%柠檬酸、0.07%抗坏血酸、0.02%CaCl2配成的复合护色液护色20min后,茭白在整个实验期间内,色白,无褐变现象产生。