Cholesterol oxidation in meat products and its regulation by supplementation of sodium nitrite and apple polyphenol before processing

The levels of cholesterol oxidation derivatives (OxChol) in eight commercial species of meat products were examined. These products contained more than 1 mg/100 g of OxChol, and 7beta-hydroxycholesterol + 5beta-epoxycholesterol (111-1092 microg/100 g), 5alpha-epoxycholesterol (80-712 microg/100 g), cholestanetriol (0-368 microg/100 g), and 7-ketocholesterol (708-1204 microg/100 g) were detected. To know the interaction of sodium nitrite supplementation against cholesterol oxidation in meat products, sausage was produced with or without varying levels of sodium nitrite and stored in the refrigerator for 15 days. As a result, cholesterol oxidation in sausage was inhibited by addition of sodium nitrite in a dose-dependent manner. This observation may be associated with inactivation of O(2)(-) radical and stabilization of polyunsaturated fatty acids (PUFAs). In fact, the levels of OxChol in sausage increased, accompanying the decrease of coexisting linoleic acid when sodium nitrite was not added to sausage meat. Thus, cholesterol oxidation in meat products seems to be considarably promoted by the oxidation of coexisting PUFAs. On the other hand, additive apple polyphenol also inhibited linoleic acid oxidation in sausage and then suppressed cholesterol oxidation through its radical scavenging effects. Therefore, apple polyphenol, having a large amount of an oligomer of catechin, may interfere with cholesterol oxidation in meat processing or storage of meat products through its antioxidative action and be useful as a new antioxitant for meat products when it is added to the original meat before processing.     

Inhibition of polyphenol oxidases activity by various dipeptides

In an effort to develop natural and nontoxic inhibitors on the activity of mushroom polyphenol oxidase (PPO) the effect of various glycyl-dipeptides (GlyAsp, GlyGly, GlyHis, GlyLeu, GlyLys, GlyPhe, GlyPro, GlyTyr) was investigated. The inhibition study with dihydroxyphenylalanine (DOPA) as substrate is based on separation of the enzymatic reaction components by reversed phase HPLC and the UV detection of the dopachrome formed. The results have evidenced that several of tested dipeptides inhibited PPO activity in the range of 20-40% while GlyPro and GlyLeu had no effect. The study has also permitted the characterization of the following kinetic pattern: a linear-mixed-type mechanism for GlyAsp, GlyGly, GlyLys, and GlyPhe and a hyperbolic-mixed-type for GlyTyr. It was not possible to identify the inhibition mechanism for GlyHis, although it affects PPO activity. In addition the effects of GlyAsp, GlyLys and GlyHis were evaluated for lessening the browning of fresh Golden Delicious apple and Irish White Skinned potato. The effectiveness of such inhibitors was determined by the difference between the colors observed in the dipeptide-treated sample and the controls using the color space CIE-Lab system. The % browning inhibition on potato (20-50%) was greater than of apple (20-30%) by the all tested dipeptides. Only GlyLys presented the significant value of 50%.     

A kinetic study of p-cresol oxidation by quince fruit polyphenol oxidase

The monophenolase activity of quince pulp polyphenol oxidase was characterized by extracting samples using a combination of a two-phase partition step in Triton X-114, followed by a PEG 8000/phosphate partition step, and a final ammonium sulfate fractionation between 30 and 75%. The purification method avoids the loss of cresolase activity described in another quince pulp polyphenol oxidase. The activity was characterized by a lag period, whose duration depended on the substrate concentration, the pH, and the presence of catalytic amounts of o-diphenol. By increasing the concentration of o-diphenols, it was possible to evaluate the enzyme activation constant, K(act), which showed a value of 4.5 microM for 4-methylcatechol. A general kinetic mechanism for this enzyme is used to explain the loss of activity that normally occurs during quince pulp polyphenol oxidase purification.     

Oligomeric procyanidins in apple polyphenol are main active components for inhibition of pancreatic lipase and triglyceride absorption

Inhibitory effects of apple polyphenol extract (AP) and procyanidin contained in AP on in vitro pancreatic lipase activity and in vivo triglyceride absorption in mice and humans were examined. AP and procyanidin considerably inhibited in vitro pancreatic lipase activity. However, polyphenols, except for procyanidin, in AP (i.e., catechins, chalcones, and phenol carboxylic acids) showed weak inhibitory activities on pancreatic lipase. Procyanidins separated by normal-phase chromatography according to the degree of polymerization were also examined. Inhibitory effects of procyanidins increased according to the degree of polymerization from dimer to pentamer. On the other hand, pentamer or greater procyanidins showed maximal inhibitory effects on pancreatic lipase. These results suggested that with respect to in vitro pancreatic lipase inhibition, the degree of polymerization was an important factor and oligomeric procyanidin mainly contributed. Next, we performed a triglyceride tolerance test in mice and humans. Simultaneous ingestion of AP and triglyceride significantly inhibited an increase of plasma triglyceride levels in both models. These results suggested that the oligomeric procyanidins contained in AP inhibited triglyceride absorption by inhibiting pancreatic lipase activity in mice and humans.     

Degradation of pentachlorophenol by potato polyphenol oxidase

In this study, polyphenol oxidase (PPO) was extracted from commercial potatoes. Degradation of pentachlorophenol by potato PPO was investigated. The experimental results show that potato PPO is more active in weak acid than in basic condition and that the optimum pH for the reaction is 5.0. The degradation of pentachlorophenol by potato PPO reaches a maximum at 298 K. After reaction for 1 h, the removal of both pentachlorophenol and total organic carbon is >70% with 6.0 units/mL potato PPO at pH 5.0 and 298 K. Pentachlorophenol can be degraded through dechlorination and ring-opening by potato PPO. The work demonstrates that pentachlorophenol can be effectively eliminated by crude potato PPO.     

Characterization of monophenolase activity of polyphenol oxidase from iceberg lettuce

Polyphenol oxidase (EC 1.14.18.1), a thylakoid membrane-bound enzyme, was isolated by sonication of osmotically shocked chloroplasts from iceberg lettuce (Lactuca sativa). The enzyme showed monophenolase activity when assayed on (p-hydroxyphenyl)propionic acid with 3-methyl-2-benzothiazolinone hydrazone in a reliable continuous spectrophotometric method, with high sensitivity, accuracy, and precision. The monophenolase activity showed a lag period before the steady-state rate (V(ss)) was reached. Both kinetic parameters, the lag period and the steady-state rate, depended on the pH, the enzyme and substrate concentrations, and the presence of catalytic amounts of o-diphenol. This activity shows inhibition by high substrate concentration. The experimental results correspond with the mechanism previously described for PPO from other sources. Kinetic constants K(m), V(max), and K(i) were determined.     

Partial characterization of polyphenol oxidase activity in raspberry fruits.

A partial characterization of polyphenol oxidase (PPO) activity in raspberry fruits is described. Two early cultivars harvested in May/June (Heritage and Autumm Bliss) and two late cultivars harvested in October-November (Ceva and Rubi) were analyzed for PPO activity. Stable and highly active PPO extracts were obtained using insoluble poly(vinylpyrrolidone) (PVP) and Triton X-100 in sodium phosphate, pH 7.0 buffer. Polyacrylamide gel electrophoresis of raspberry extracts under nondenaturing conditions resolved in one band (R(f)()(1) = 0.25). Raspberry PPO activity has pH optima of 8.0 and 5.5, both with catechol (0.1 M). Maximum activity was with D-catechin (catecholase activity), followed by p-coumaric acid (cresolase activity). Heritage raspberry also showed PPO activity toward 4-methylcatechol. Ceva and Autumm Bliss raspberries showed the higher PPO activity using catechol as substrate.     

Kinetic characterization of the oxidation of chlorogenic acid by polyphenol oxidase and peroxidase. Characteristics of the o-quinone

Chlorogenic acid is the major diphenol of many fruits, where it is oxidized enzymatically by polyphenol oxidase (PPO) or peroxidase (POD) to its o-quinone. In spectrophotometric studies of chlorogenic acid oxidation with a periodate ratio of [CGA]0/[IO4-]0 < 1 and [CGA]0/[IO4-]0 > 1, the o-quinone was characterized as follows: lambda(max) at 400 nm and epsilon = 2000 and 2200 M-1 cm-1 at pH 4.5 and 7.0, respectively. In studies of o-quinone generated by the oxidation of chlorogenic acid using a periodate at ratio of [CGA]0/[IO4-]0 > 1, a reaction with the remaining substrate was detected, showing rate constants of k = 2.73 +/- 0.17 M-1 s-1 and k' = 0.05 +/- 0.01 M-1 s-1 at the above pH values. A chronometric spectrophotometric method is proposed to kinetically characterize the action of the PPO or POD on the basis of measuring the time it takes for a given amount of ascorbic acid to be consumed in the reaction with the o-quinone. The kinetic constants of mushroom PPO and horseradish POD are determined.     

Oxidation of the flavonol quercetin by polyphenol oxidase

Because direct oxidation of flavonols by polyphenol oxidase (PPO) has not previously been reported and, given the importance of flavonols, the ability of broad bean seed PPO to oxidize the flavonol quercetin was studied. The reaction was followed by recording spectral changes with time. Maximal spectral changes were observed at 291 nm (increase) and at 372 nm (decrease). The presence of two isosbectic points (at 272 and 342 nm) suggested the formation of only one absorbent product. These spectral changes were not observed in the absence of PPO. The oxidation rate, which varied with pH, was highest at pH 5.0. The following kinetic parameters were also determined: V(m) = 11 microM/min, K(m) = 646 microM, V(m)/K(m) = 17 x 10(-)(2) min(-)(1). Flavonol oxidation was efficiently inhibited (K(I) = 3.5 microM) by specific PPO inhibitors such as 4-hexylresorcinol. The results obtained showed that quercetin oxidation was strictly dependent on the presence of PPO.     

Fast apple (Malus x domestica) and tobacco (Nicotiana tobacum) leaf polyphenol oxidase activity assay for screening transgenic plants

A spectrophotometric assay method for the analysis of polyphenol oxidase (PPO), in apple and tobacco leaves, has been optimized to increase efficiency in the screening of large numbers of transgenic plants. Crude protein extracts from leaf punches were prepared in a FastPrep homogenizer. The addition of Triton X-100 during extraction resulted in 44 and 74% increases in the PPO activity recovered, from apple and tobacco, respectively. The enzyme kinetics differed markedly between apple and tobacco. Apple leaf PPO was isolated in a latent state and was activated by the addition of SDS. In contrast, tobacco PPO activity was inhibited by SDS, particularly at acidic pH. Apple PPO showed a pronounced pH optimum around pH 6, whereas the pH profile for tobacco PPO was much flatter, with a broad optimum around pH 4. The calculated Km' value for apple PPO, using 4-methylcatechol as substrate, was 8.1, and for tobacco the Km was 4.3. The PPO reaction was strongly inhibited by tropolone, a Cu competitor, and restored by the addition of Cu2+. Several factors affecting variability in leaf PPO activity levels in plants are discussed.     

Inhibition of key digestive enzymes by cocoa extracts and procyanidins

This study determined the in vitro inhibitory effects of cocoa extracts and procyanidins against pancreatic α-amylase (PA), pancreatic lipase (PL), and secreted phospholipase A(2) (PLA(2)) and characterized the kinetics of such inhibition. Lavado, regular, and Dutch-processed cocoa extracts as well as cocoa procyanidins (degree of polymerization (DP) = 2-10) were examined. Cocoa extracts and procyanidins dose-dependently inhibited PA, PL, and PLA(2). Lavado cocoa extract was the most potent inhibitor (IC(50) = 8.5-47 μg/mL). An inverse correlation between log IC(50) and DP (R(2) > 0.93) was observed. Kinetic analysis suggested that regular cocoa extract, the pentamer, and decamer inhibited PL activity in a mixed mode. The pentamer and decamer noncompetitively inhibited PLA(2) activity, whereas regular cocoa extract inhibited PLA(2) competitively. This study demonstrates that cocoa polyphenols can inhibit digestive enzymes in vitro and may, in conjunction with a low-calorie diet, play a role in body weight management.     

Low-density lipoprotein antioxidant activity of phenolic compounds and polyphenol oxidase activity in selected clingstone peach cultivars

The antioxidant potential of eight clingstone peach cultivars was investigated by determining phenolic compounds and inhibition of low-density lipoprotein (LDL) oxidation. Cultivars low in polyphenol oxidase (PPO) were also selected to minimize enzymatic browning. Inhibition of LDL oxidation varied from 17.0 to 37.1% in peach flesh extract, from 15.2 to 49.8% in whole peach extract, and from 18.2 to 48.1% in peel extract. Total phenols were 432.8-768.1 mg/kg in flesh extract, 483.3-803.0 mg/kg in whole extract, and 910.9-1922.9 mg/kg in peel extract. The correlation coefficient between relative LDL antioxidant activity and concentration of total phenols was 0.76. Peel PPO activity was higher than flesh activity in most cultivars. The lowest PPO and specific activities were found in the Walgant cultivar, followed by Kakamas and 18-8-23. These three cultivars combine the desirable characteristics of strong antioxidant activity, low PPO activity, and lower susceptibility to browning reactions.     

Isolation and structural elucidation of some procyanidins from apple by low-temperature nuclear magnetic resonance

Procyanidin fractions from apple were separated according to the degree of polymerization using normal phase chromatography. Evaluation of physiological functionalities of procyanidins requires individual structural determination. However, it is difficult to elucidate the structure of procyanidins, in particular those with (+)-epicatechin (1) or (-)-catechin (2) units, and determine whether the interflavanoid bonds are 4beta-->8 or 4beta-->6 without cleavage and acetylation. Structural determination used LC-MS and low-temperature NMR. Nine procyanidins were separated by preparative HPLC consisting of three well-known procyanidins [procyanidin B1 (3), procyanidin B2 (4), and procyanidin C1 (5)] and six new procyanidins [epicatechin-(4beta-->8)-epicatechin-(4beta-->8)-catechin (6); epicatechin-(4beta-->6)-epicatechin-(4beta-->8)-catechin (7); epicatechin-(4beta-->6)-epicatechin-(4beta-->8)-epicatechin (8); epicatechin-(4beta-->8)-epicatechin-(4beta-->6)-catechin (9); epicatechin-(4beta-->8)-epicatechin-(4beta-->6)-epicatechin (10); and epicatechin-(4beta-->8)-epicatechin-(4beta-->8)-epicatechin-(4beta-->8)-epicatechin (11)]. Compounds 6-11 were detected for the first time as apple constituents.     

多酚氧化酶高强度脉冲磁场灭活及动力学模型

为了找到一种有效控制果蔬中多酚氧化酶（PPO）活性的方法,该文研究了高强度脉冲磁场（PMF）对PPO活性的影响,并进行了灭酶动力学模型的研究。结果表明,当PPO于磁场强度2.5、3.5和4.5特斯拉（T）分别处理5至40个脉冲时,酶的残余活性随着磁场强度和脉冲数的增加而逐渐降低。在4.5 T处理40个脉冲时,酶的灭活率最高达到93.10%。对灭活动力学曲线分别用Bigelow模型、Weibull模型和Hülsheger模型进行拟合,发现Weibull模型对PMF下PPO的灭活的拟合度最好。可见,高强度脉冲磁场可以作为一种有效杀灭果蔬中多酚氧化酶的非热技术,且酶的灭活过程符合Weibull模型,该模型可以为实际应用提供参考。     

Oxygenation of bisphenol A to quinones by polyphenol oxidase in vegetables

To understand conversion of bisphenol A and its related compounds under some chemical and biological environments, oxidation of these compounds was performed. Bisphenol A was oxidized to monoquinone and bisquinone derivatives by Fremy's salt, a radical oxidant; but salcomine and alkali did not catalyze the oxidation by molecular oxygen. Bisphenol A, bisphenol B, and 3,4'-(1-methylethylidene)bisphenol were converted to their monoquinone derivatives in the presence of oxygen and polyphenol oxidase from mushroom at 25 degrees C at pH 6.5. Among crude enzyme solutions of fruits and vegetables, potato, mushroom, eggplant, edible burdock, and yacon showed remarkable oxidative activity on bisphenol A. The highest activity was observed in potato, and the main product obtained by the enzymatic oxygenation was the monoquinone derivative of bisphenol A, accompanied by a small amount of the bisquinone derivative. The oxidation reactions found here will be useful for developing techniques for elimination of phenolic endocrine disrupters from the environment.     

Inhibition of Df-protease associated with allergic diseases by polyphenol.

It was reported that Df-protease from house dust mite (Dermatophagoides farinae) catalyzes the activation of the kallikrein-kinin system in human plasma and is closely associated with mite-induced allergy. Therefore, to prevent the release of kinin by Df-protease, the inhibitory activity of polyphenols including catechins and flavonols was tested in vitro and in vivo. Among them, myricetin and epigallocatechin gallate (EGCg) effectively inhibited the amidase activity of Df-protease with K(i) values of 1 x 10(-)(8) and 6 x 10(-)(4) M, respectively. The kinin release in human plasma was extensively inhibited by the addition of EGCg in comparison with myricetin. Enhancement of vascular permeability in guinea pigs caused by Df-protease was markedly suppressed by EGCg.     

Purification and characterization of polyphenol oxidase from rape flower

The purification and partial enzymology characteristics of polyphenol oxidase (PPO) from rape flower were studied. After preliminary treatments, the crude enzyme solution was in turn purified with ammonium sulfate, dialysis, and Sephadex G-75 gel chromatography. The optimal conditions and stability of PPO were examined at different pH values and temperatures. Subsequently, PPO was also characterized by substrate (catechol) concentrations, inhibitors, kinetic parameters, and molecular weight. Results showed that the optimal pH for PPO activity was 5.5 in the presence of catechol and that PPO was relatively stable at pH 3.5-5.5. PPO was moderately stable at temperatures from 60 to 70 °C, whereas it was easily denatured at 80-90 °C. Ethylenediaminetetraacetic acid, sodium chloride, and calcium chloride had little inhibitive effects on PPO, whereas citric acid, sodium sulfite, and ascorbic acid had strongly inhibitive effects. The Michaelis-Menten constant (K(m)) and maximal reaction velocity (V(max)) of PPO were 0.767 mol/L and 0.519 Ab/min/mL of the crude PPO solution, respectively. PPO was finally purified to homogeneity with a purification factor of 4.41-fold and a recovery of 12.41%. Its molecular weight was 60.4 kDa, indicating that the PPO is a dimer. The data obtained in this research may help to prevent the enzymatic browning of rape flower during its storage and processing.     

Effect of reddening-ripening on the antioxidant activity of polyphenol extracts from cv. 'Annurca' apple fruits

Apple is among the most consumed fruits worldwide, and several studies suggest that apple polyphenols could play a role in the prevention of degenerative diseases. 'Annurca' apple fruit undergoes, after harvest, a typical reddening treatment to turn the apples' skin red, and it is noted for its high firmness. This paper reports the effect of reddening-ripening treatment on polyphenol concentration and antioxidant activity of both peel and flesh extracts. The in vitro antioxidant properties have been compared with the protective effect against the cytotoxic effects of reactive oxygen species using Caco-2 cells as model system. Pretreatment of cells with different polyphenolic apple extracts provides a remarkable protection against oxidative damage. This effect seems to be associated with the antioxidant activity of 'Annurca' apple polyphenolic compounds. The flesh has antioxidant properties comparable to those possessed by the peel. Neither the reddening nor the fruit conservation causes changes in the antioxidant properties possessed by this apple variety. The data indicate that polyphenolic compounds in 'Annurca' apples are relatively stable in the peel and also in the flesh; therefore, the health benefits of polyphenols should be maintained during long-term storage. Finally, a diet rich in apple antioxidants could exert a beneficial effect in the prevention of intestinal pathologies related to the production of reactive oxygen species.     

The effects of triacontanol on seedling growth and polyphenol oxidase activity in dark and light growth lettuce

The present study was designed to study the effects of various concentrations (0.00; 0.01, 0.10 and 1.00‐mg/ml) of triacontanol on root, stem and leaf growth and polyphenol oxidase activity in each respective tissue; in Grand Rapids and Great Lakes varieties of eleven‐day‐old dark and light grown lettuce seedlings.

Root, stem and leaf growth was less than in non‐triacontanol‐treated Grand Rapids seedlings in both TC‐treated dark and light exposed seedlings. With respect to Great Lakes seedlings, all dark‐grown roots exhibited greater growth than the corresponding untreated control. Light‐grown Great Lakes roots treated with 0.01‐mg/ml and 1.0‐mg/ml or triacontanol respectively, grew more than control or 0.1‐mg/ml triacontanol‐treated seedlings. Both dark and light‐grown triacontanol‐treated stem and leaf tissues of Great Lakes seedlings all produced less growth than the untreated controls.

The Grand Rapids variety had less polyphenol oxidase activity in both dark and light‐grown root and stem tissues than in untreated controls; however, both dark and light‐grown leaf tissue, treat ed with 0.01‐mg/ml and 1.0‐mg/ml of triacontanol respectively exhibited more polyphenol oxidase activity than 1.0‐mg/ml triacontanol ‐treated or untreated control tissues.

TC treatment of 0.1‐mg/ml caused no enhancement of PPO activity in dark or light‐grown root, stem and leaf tissues of Great Lakes tissue, however, seedlings treated with a concentration of 0.01‐mg/ml TC exhibited more PPO activity than non‐TC‐treated controls.     

Partial purification of latent persimmon fruit polyphenol oxidase

Persimmon fruit polyphenol oxidase (PPO) was partially purified using a combination of phase partitioning with Triton X-114 and ammonium sulfate fractionation between 50 and 75%. The enzyme, which showed both monophenolase and diphenolase activities, was partially purified in a latent form and could be optimally activated by the presence of 1 mM sodium dodecyl sulfate (SDS) with an optimum pH of 5.5. In the absence of SDS, the enzyme showed maximum activity at acid pH. SDS-PAGE showed the presence of a single band when L-DOPA was used as substrate. The apparent kinetic parameters of the latent enzyme were determined at pH 5.5, the V(m) value being 15 times higher in the presence of SDS than in its absence, whereas the K(M) was the same in both cases, with a value of 0.68 mM. The effect of several inhibitors was studied, tropolone being the most active with a K(i) value of 0.45 microM. In addition, the effect of cyclodextrins (CDs) was studied, and the complexation constant (K(c)) between 4-tert-butylcatechol (TBC) and CDs was calculated using an enzymatic method. The value obtained for K(c) was 15580 M(-1).