Contribution of pyrrole formation and polymerization to the nonenzymatic browning produced by amino-carbonyl reactions

Recent studies have hypothesized that pyrrole formation and polymerization may be contribute to the nonenzymatic browning produced in both oxidized lipid/protein reactions and the Maillard reaction. To develop a methodology that would allow investigation of the contribution of this browning mechanism, the kinetics of formation of color, fluorescence, and pyrrolization in 4, 5(E)-epoxy-2(E)-heptenal/lysine and linolenic acid/lysine model systems were studied. In both cases similar kinetics for the three measurements were observed at the two temperatures assayed (37 and 60 degrees C), and there was a high correlation among color, fluorescence, and pyrrolization measurements obtained as a function of incubation time. Because the color and fluorescence production in the 4,5(E)-epoxy-2(E)-heptenal/lysine system is a consequence of pyrrole formation and polymerization, the high correlations observed with the unsaturated fatty acid also suggest a contribution of the pyrrole formation and polymerization to the development of color and fluorescence observed in the fatty acid/lysine system. Although the contribution of other mechanisms cannot be discarded, all of these results suggest that when the pyrrole formation and polymerization mechanism contributes to the nonenzymatic browning of foods, a high correlation among color, fluorescence, and pyrrolization measurements should be expected.     

Effect of pH and temperature on comparative nonenzymatic browning of proteins produced by oxidized lipids and carbohydrates

Bovine serum albumin (BSA) was incubated for 24 h in the presence of 10 mM ribose (RI), methyl linoleate hydroperoxides, or the secondary products of methyl linoleate oxidation (SP), at five temperatures (25, 37, 50, 80, and 120 degrees C) and different pHs (4, 7, and 10), to study the influence of these variables in the browning, fluorescence, amino acid losses, and pyrrolization of the modified proteins. All treated proteins exhibited similar colors and fluorescence spectra, and the spectra of their Ehrlich adducts were also analogous. However, at 25-50 degrees C the proteins treated with oxidized lipids exhibited higher color changes, amino acid losses, and pyrrolization than the BSA treated with RI, and these effects were much higher in proteins treated with RI at 80-120 degrees C. The effect of pH was similar in proteins treated with RI or SP. These results suggested a similarity for browned proteins obtained from both carbohydrates and oxidized lipids. In addition, both reactions seem to be complementary, because melanoidin formation derived from oxidized lipids can be produced under conditions different from those carbohydrate/protein reactions.     

Characterization of key chromophores formed by nonenzymatic browning of hexoses and L-alanine by using the color activity concept

Thermal treatment of an aqueous solution of D-glucose and L-alanine in the presence of the carbohydrate degradation product furan-2-aldehyde resulted in the formation of a variety of colored compounds, among which (Z)-2-[(2-furyl)methylidene]-5, 6-di(2-furyl)-6H-pyran-3-one (I), [E]- and [Z]-1, 2-bis(2-furyl)-1-pentene-3,4-dione (IIa/IIb), 4, 5-bis(2-furyl)-2-methyl-3H-furan-2-one (III), and (S,S)- and (S, R)-2-[4, 5-bis(2-furyl)-2-hydroxy-2-methyl-3(2H)-pyrrol-1-yl]propionic acid (IVa/IVb) as well as 2-[(2-furyl)methylidene]-4-hydroxy-5-[(E)-(2-furyl)methylidene]methyl -2H-furan-3-one (V) were successfully identified as the most intense by application of the color dilution analysis. To measure the contribution of these colorants to the overall color of the browned Maillard mixture, color activity values were calculated as the ratio of the concentration to the visual detection threshold of each colorant. By application of this color activity concept, 16.0% of the overall color of the Maillard mixture accounted for these five types of colorants, thus confirming them as key chromophores. On the basis of synthetic model experiments, the formation pathways leading to the chromophores IIa/IIb, III, and IVa/IVb were proposed.     

Degradation of oligosaccharides in nonenzymatic browning by formation of alpha-dicarbonyl compounds via a "peeling off" mechanism

The formation of alpha-dicarbonyl-containing substances and Amadori rearrangement products was studied in the glycine-catalyzed (Maillard reaction) and uncatalyzed thermal degradation of glucose, maltose, and maltotriose using o-phenylenediamine as trapping agent. Various degradation products, especially alpha-dicarbonyl compounds, are formed from carbohydrates with differing degrees of polymerization during nonenzymatic browning. The different Amadori rearrangement products, isomerization products, and alpha-dicarbonyls produced by the used carbohydrates were quantified throughout the observed reaction time, and the relevance of the different degradation pathways is discussed. In the Maillard reaction (MR) the amino-catalyzed rearrangement with subsequent elimination of water predominated, giving rise to hexosuloses with alpha-dicarbonyl structure, whereas under caramelization conditions more sugar fragments with an alpha-dicarbonyl moiety were formed. For the MR of oligosaccharides a mechanism is proposed in which 1,4-dideoxyosone is formed as the predominating alpha-dicarbonyl in the quasi-water-free thermolysis of di- and trisaccharides in the presence of glycine.     

Manothermosonication of foods and food-resembling systems: effect on nutrient content and nonenzymatic browning

The effect of manothermosonication (MTS), an emergent technology for food preservation, on thiamin, riboflavin, carotenoids, and ascorbic acid was evaluated in milk and orange juice. The effect of both heat treatment and MTS on several compounds produced in nonenzymatic browning in model systems was also studied. MTS does not affect significantly the nutrient content studied. However, it changes the behavior of nonenzymatic browning. No formation of 5-(hydroxymethyl)-2-furfuraldehyde (HMF) was detected in fruit juice model systems after heat and MTS treatments at the experimental conditions used. In a milk-resembling system, free HMF formation by MTS is higher compared to that by heat treatment. As the MTS temperature increases, free HMF production by both treatments equaled on another. For bound HMF the production rate is lower by MTS than by heat treatment under the experimental conditions used. Formation kinetics of brown pigments and that of fluorescent compounds are different for both treatments. Fluorescence and brown pigment production are faster in MTS.     

alpha-Dicarbonyl compounds formed by nonenzymatic browning during the dry heating of caseinate and lactose

A method using high-performance liquid chromatography with UV and electrospray ionization mass spectrometry detection was developed for monitoring the alpha-dicarbonyl compound profiles generated from nonenzymatic browning using o-phenylenediamine (OPD) as a trapping agent. The alpha-dicarbonyl compounds were generated by the "dry" reaction of sodium caseinate and lactose heated at various relative humidities (RHs). The proportions of alpha-dicarbonyls formed were different for samples heated at low, intermediate, and high RHs. This study shows that relatively large amounts of 3-deoxypentosulose and galactosyl 2-pentosulose are produced under high RHs, while galactosyl hexosulose and 1,4-dideoxyhexosulose are elevated under low RH conditions. Both caramelization and Maillard reaction pathways contributed to the generation of alpha-dicarbonyls.     

Studies on radical intermediates in the early stage of the nonenzymatic browning reaction of carbohydrates and amino acids

Determination of the color intensity of heated mixtures of L-alanine and carbohydrate degradation products revealed furan-2-carboxaldehyde and glycolaldehyde as by far the most effective color precursors. EPR studies demonstrated that furan-2-carboxaldehyde generated colored compounds exclusively via ionic mechanisms, whereas glycolaldehyde led to color development accompanied by intense radical formation. In agreement with literature data, these radicals were also detected in heated mixtures of L-alanine and pentoses or hexoses, respectively, and were identified as 1,4-dialkylpyrazinium radical cations by EPR as well as LC/MS measurements. Studies on the mechanisms of radical formation revealed that under the reaction conditions applied, glyoxal is formed as an early product in hexose/L-alanine mixtures prior to radical formation. Reductones then initiate radical formation upon reduction of glyoxal and/or glyoxal imines, formed upon reaction with the amino acid, into glycolaldehyde, which was found as the most effective radical precursor. LC/MS measurements gave evidence that these pyrazinium radicals cations are not stable but are easily transformed into hydroxylated 1,4-dialkyl-1, 4-dihydropyrazines upon oxidation and hydrolysis of intermediate diquarternary pyrazinium ions. Besides other types of color precursors, these intermediates might be involved in the formation of colored compounds in the Maillard reaction.     

3-deoxypentosulose: an alpha-dicarbonyl compound predominating in nonenzymatic browning of oligosaccharides in aqueous solution

The thermal degradation of D-glucose, maltose, and maltotriose in aqueous solution was investigated under caramelization (no glycine) and Maillard (with glycine) conditions. Degradation of the sugar and alpha-dicarbonyls product was monitored. Under both caramelization and Maillard reaction conditions, 3-deoxypentosulose was the predominating alpha-dicarbonyl compound formed from maltose and maltotriose. In the absence of an amino compound, however, 3-deoxypentosulose is formed in much lower concentration. It was concluded that 3-deoxypentosulose is formed by a pathway specific for oligo- and polysaccharides since this alpha-dicarbonyl is formed from the alpha-1-->4 glucans such as maltose and maltotriose but not from glucose. For its formation, a retro Claisen reaction of an enolization product of 1-amino-1,4-dideoxyhexosulose is proposed as the route to its formation. 1-Amino-1,4-dideoxyhexosulose could be formed by vinylogous alpha-elimination from the 2,3-enediol structure after Amadori rearrangement, favored by planar alignment of the bonds between C1 and C4. Subsequent rearrangement by keto-enoltautomerization leads to a 1-imino-3-keto structure. In this structure, attack of a hydroxyl anion, provided by water at neutral pH, could cause a splitting off of the C1. This reaction gives rise to formic acid or formamide and a pentose derivative, which reacts further to give 3-deoxypentosulose.     

Effect of tocopherols in the antioxidative activity of oxidized lipid-amine reaction products

Phosphatidylethanolamine (PE), phosphatidylcholine (PC), lysine (Lys), and mixtures of them were tested for antioxidative activity in a tocopherol-stripped olive oil (TSO) and the same oil after addition of 250 microg of alpha-tocopherol g of oil/(tocopherol-added olive oil, TAO) to evaluate the role of tocopherol in the antioxidant activity of oxidized lipid-amine products. Neither PE nor PC nor Lys protected TSO when tested alone, but both PE and Lys increased the induction period (IP) of TAO. On the contrary, PE/Lys and PC/Lys mixtures, but not PC/PE mixtures, protected both TSO and TAO. These results were a consequence of both the formation of oxidized lipid-amine products, which were determined by gas chromatography-mass spectrometry after their conversion into volatile derivatives, and a synergism between alpha-tocopherol and the produced compounds. These results were confirmed by analyzing the antioxidative activity of two of the produced carbonyl-amine products: 6-amino-2-(1H-pyrrol-1-yl)hexanoic acid (1) and 2,3-dipalmitoylpropyl 2-(1H-pyrrol-1-yl)ethyl phosphate (2). The hydrophilic compound 1 was more antioxidant than the analogous lipophilic compound 2, and this antioxidative activity was observed in TAO and not in TSO. All these results suggested that antioxidative activity of carbonyl-amine products may be greatly increased with the addition of tocopherols, and those products derived from Lys are more antioxidant in bulk oils than those derived from PE.     

Impact of browning reactions and bran pigments on color of parboiled rice

Rice color changes from white to amber during parboiling (soaking and steaming). Color parameters indicated that, during soaking, yellow bran pigments leached out in the water. The levels of the Maillard precursors (i.e., reducing sugars (RS) and free alpha-amino nitrogen (FAN)) depended on soaking temperature and time: leaching of RS was compensated by enzymic formation for long soaking times (>60 min), while proteolytic activity was too low to compensate for FAN leaching. Rice soaking under nitrogen, oxygen, or ambient conditions and determination of polyphenol oxidase activity allowed us to conclude that the effect of enzymic color changes on the soaked rice color was rather small. Color measurements of brown and milled mildly, intermediately, and severely parboiled rice samples showed that both brown and milled rice samples were darker and more red and yellow after parboiling and that the effect depended on the severity of parboiling conditions. Furthermore, steaming affected the rice color more and in a way opposite to that observed in soaking. The changes in RS and the loss of FAN during parboiling suggested that Maillard type reactions occur during brown rice steaming. Analyses of furosine levels confirmed Maillard browning of outer bran layers and endosperm during steaming. The level of this Maillard indicator increased with the severity of parboiling conditions in both brown and milled parboiled rice. Measurements of the levels of bran pigments indicated that bran pigments diffuse into the endosperm during parboiling and contribute to the parboiled rice color.     

Progress of browning reactions during storage of liquid infant milks.

Changes in furfural compounds and reactive lysine were monitorized in three commercial liquid infant milks during 9 months of storage at 20, 30, and 37 degrees C. Samples consisted of two ultrahigh temperature (UHT)-treated milks and one conventionally sterilized milk. Reactive lysine remained constant throughout storage at the three temperatures, whereas, in general an increase in furfural compounds was observed. The heat treatment used in the manufacture of milk is an important factor that influences the levels of furfural compounds although the composition of the milk is also a critical factor. Finally, a study was conducted to find the kinetic equations describing furfural compounds changes and allowing for the prediction of the influence of time and temperature of storage on those changes.     

玉米苞叶中总黄酮提取及其体外抗氧化性能的研究

运用超声波技术提取玉米苞叶中的总黄酮,用亚硝酸钠-硝酸铝比色法测定总黄酮含量,得出玉米苞叶中总黄酮提取的最佳工艺条件:超声波功率700W,温度60℃,料液比(g/mL)1∶50,乙醇浓度为60%,提取时间为35min,玉米苞叶中总黄酮最大提取率为1.225%。体外抗氧化试验表明:玉米苞叶中的总黄酮对超氧阴离子自由基、羟基自由基清除能力较好,其IC50分别为1.5μg/mL左右和0.14mg/mL,清除能力均高于Vc;对DPPH自由基体系的清除率达到50%时,玉米苞叶中总黄酮的IC50为0.2mg/mL左右,Vc的IC50为0.075mg/mL,说明玉米苞叶中的总黄酮清除DPPH自由基的能力低于Vc,但在所选浓度剂量范围内,其最大清除率高于Vc。试验表明,玉米苞叶中的总黄酮是一种较好的天然自由基清除剂。     

Two types of radicals in whole milk powder. Effect of lactose crystallization, lipid oxidation, and browning reactions

Whole milk powder was stored in closed vials at 60 degrees C to induce crystallization of lactose within a short time scale. After an induction period of 3-4 days simultaneous crystallization of lactose, increase of water activity, formation of browning products, and increase of radical content took place. Radicals detected before lactose crystallization were characterized by a narrow ESR spectrum (g = 2.006) and could be depleted by removal of oxygen and therefore were assigned to oxidation processes. Late-stage radicals present after crystallization of lactose gave much wider spectra (g = 2.0048) and were independent of oxygen availability and assigned to late-stage Maillard reaction products. The study indicates that the processes of lactose crystallization, browning, and formation of radical species (g = 2.0048) are strongly coupled, while lipid oxidation is less dependent on the other processes.     

Effect of pepper lipoxygenase activity and its linked reactions on pigments of the pepper fruit.

The products formed during the enzymatic reaction catalyzed by the lipoxygenase of pepper (variety Agridulce) have in vitro a strong destructive action on the carotenoid pigments of the fruit. When conditions and proportions of enzyme and pigments are similar to those found in the fruit, and at a reaction temperature of 20 degrees C, almost 30% of the pigments are destroyed after 24 h of reaction. Of this amount, 2.5% is due to autoxidation of pigments, 4. 5% to oxidation induced by the presence of linoleic under saturating conditions, and the remaining 22% to the presence in the medium of reaction products of the lipoxygenase-catalyzed reaction. When the enzyme acts under substrate-saturating conditions, the rate of pigment destruction by lipoxygenase can be considered maximal at the experimental temperature. The fact that in vitro pepper lipoxygenase induces a heavy destruction of pigments and that, in vivo, its activity remains almost constant during over-ripening could explain why up to 40% of the pigment content in some varieties is lost during the postharvest period.     

Comparative evaluation of cytotoxicity and antioxidative activity of 20 flavonoids

Flavonoids are common dietary components with many health benefits shown through epidemiological studies. However, the fact that flavonoids also act as pro-oxidants and mutagens makes the safety of flavonoids uncertain when used at higher doses. To give a preliminary evaluation on the correlation between beneficial and harmful effects of flavonoids, the antioxidative activity and cytotoxicity of 20 flavonoids from food and herbs were investigated in vitro. The results indicated that luteolin, hydroxygenkwanin, and kaempferol possessed significant dual properties, whereas flavokawain B, flavokawain C, cardamonin, and uvangoletin showed a marked cytotoxicity. The relationships between structure and antioxidant and cytotoxic activity are intensively discussed. In view of the toxicity, the intake of flavonoids in large amounts should not yet be encouraged.     

黑莓原花青素超声波辅助提取优化及抗氧化性研究

论文采用响应曲面法研究了超声波辅助提取时温度、时间、料液比、超声波功率及其交互作用对黑莓原花青素提取效果的影响,同时研究了黑莓原花青素对二苯代苦味酰自由基(DPPH)、·OH及·O2-的清除效果.用SAS软件确定了黑莓原花青素超声波提取的工艺参数为:提取温度为70.9℃,料液比为1∶9.14,时间为30.5 min,超声波功率为526.9 W.黑莓原花青素对DPPH、·O2-及·OH 3种自由基具有显著的清除效果,且明显优于茶多酚,当浓度为3.0 μg/mL时,对DPPH、·O2-及·OH 3种自由基的最大清除率分别可达到82.54%、79.90%和65.90%,且黑莓原花青素对3种自由基的清除效果与其浓度之间存在明显的量效关系.     

Changes produced in the antioxidative activity of phospholipids as a consequence of their oxidation

The antioxidative activities of native and oxidized soybean phosphatidylcholine (PC), phosphatidylthanolamine (PE), and phosphatidylinositol (PI) in the protection of soybean oil heated in the dark under air at 60 degrees C were studied in an attempt to clarify the consequences that phospholipid oxidation has on antioxidative activities. The three native phospholipids protected the oil when assayed at 200 ppm, and phospholipid oxidation decreased the antioxidative activity of both PC and PI. However, slightly oxidized PE was more antioxidative than native PE, most likely as a consequence of the formation by amino-carbonyl reactions of pyrrolized phospholipids, which were determined and for which antioxidative properties are known. Nevertheless, further increases in PE oxidation produced a decrease in its antioxidative activity. These results suggest that two opposite reactions are competing in the antioxidative activity of amino phospholipids upon oxidation: fatty acid chain oxidation, which decreases phospholipid antioxidative activity, and amino-carbonyl reactions, which produce derivatives with antioxidant properties. This last property may be useful to increase the antioxidative activity of commercial lecithins containing amino phospholipids.     

Tyrosinase inhibitory activity of cucumber compounds: enzymes responsible for browning in cucumber

The inhibition of mushroom tyrosinase by cucumber extracts was evaluated. The inhibitory effect was measured by both polarographic and spectrophotometric methods. The commercial aldehyde, trans,cis-2,6-nonadienal, described as a major volatile compound of cucumber, was characterized as a noncompetitive inhibitor against 4-tert-butylcatechol oxidation by mushroom tyrosinase. The K(I) obtained was 3.4 mM. Polyphenol oxidase (PPO) activity was not detected in cucumber skin extracts. However, the presence of PPO was revealed by Western blot; a single band was found with a M(r) of 53 kDa. These results support the assumption that the enzyme PPO is present in the cucumber skin, but its activity is inhibited. Peroxidase (PO) was also found in cucumber skin extracts. This enzyme was detected in the soluble fraction but not in the membrane fraction. The kinetic characterization of PO was carried out. Native isoelectric focusing revealed several acidic PO isoenzymes with a pI in the range between 5 and 6, a basic isoenzyme, and one principal neutral isoenzyme of pI = 7.2.     

gamma-Radiation influences browning, antioxidant activity, and malondialdehyde level of apple juice

Apple juice was gamma-irradiated at 5 degrees C at doses ranging from 0 to 8.9 kGy and then stored at 5 degrees C for 15 days. Ionizing radiation reduced the browning of apple juice and increased antioxidant activity measured by the ferric-reducing antioxidant power (FRAP) assay. The magnitude of changes increased with radiation dose. The level of malondialdehyde (MDA) measured using the thiobarbituric acid reactive substrates assay increased at radiation doses above 2.67 kGy. The browning of irradiated juices increased during storage at 5 degrees C, but the irradiated juices were still lighter than controls at the end of storage. Differences in FRAP values disappeared during early periods of storage while higher MDA levels were observed in irradiated samples during most of the storage period. Elimination of suspended matter from apple juice did not alter irradiation-induced changes in browning, FRAP, or MDA formation. As compared to irradiation conducted at 5 and 20 degrees C, treatment at -15 degrees C was less effective in reducing browning and in increasing MDA formation but elevated FRAP values. The exclusion of oxygen from juices did not affect the reduction in browning due to irradiation but promoted the increase in FRAP values and decreased the irradiation-induced MDA formation.     

环境湿度对高温胁迫下苹果果皮组织抗氧化特性的影响

苹果果实日灼是一种由高温和强光引起的生理伤害,同时还与其他气候因子有关,如空气相对湿度和风速等.张建光等观察表明,果实日灼一般出现在一天中空气相对湿度比较低的时段.李长存等在研究山楂果实日灼时发现,气温达到33℃时,在73%相对湿度条件下,平均果实日灼率为16.7%；而在56%的相对湿度下,果实日灼率高达83.7%.这表明,空气相对湿度对果实日灼的发生有重要影响.然而,迄今为止在相对湿度对高温胁迫下果实抗氧化能力影响方面的研究甚少。本试验利用离体果实,在控制条件下研究了不同湿度对果实表皮组织超氧阴离子（O-2）和丙二醛（MDA）含量以及超氧化物歧化酶（SOD）和过氧化物酶（POD）活性变化的影响,从而为揭示湿度与高温伤害的关系提供理论依据。