Antioxidative activity of amino phospholipids and phospholipid/amino Acid mixtures in edible oils as determined by the Rancimat method

Phosphatidylethanolamine (PE), phosphatidylcholine (PC), lysine (Lys), and mixtures of them were tested for antioxidative activity in refined olive oil by the Rancimat method to investigate the role of the chemical reactions produced in the Rancimat vessel on the induction periods (IPs) obtained. PE and Lys, but not PC, increased the IPs of the oil when tested alone. In addition, PE/Lys and PC/Lys mixtures, but not PC/PE mixtures, exhibited a synergistic effect. All these results can be understood considering the in situ formation of oxidized lipid/amino compound reaction products with antioxidative activities. Thus, the formation of pyrroles could be detected after derivatization with p-(dimethylamino)benzaldehyde, and some of these compounds could be unambiguously identified by GC-MS after their conversion into volatile derivatives. In addition, the formed products contributed to the color developed, and a correlation was observed between the Rancimat IPs obtained and the yellowness index of the oxidized oils recovered from the Rancimat. Furthermore, the differences observed in the antioxidative activities of PE, PC, Lys, and their mixtures could be explained according to the lipophility and hydrophility of the oxidized lipid/amino compound reaction products formed. All these results suggest that chemical reactions are being produced in the Rancimat vessel and the Rancimat IPs obtained are a consequence of the antioxidative activities of the products formed in these reactions. Furthermore, Rancimat may be a valuable tool for testing antioxidative activities of antioxidants produced during food processing if favorable conditions for antioxidant formation are employed.     

Effect of pH and temperature on comparative antioxidant activity of nonenzymatically browned proteins produced by reaction with oxidized lipids and carbohydrates

The antioxidative activity of nonenzymatically browned bovine serum albumin (BSA) produced by reaction with ribose (RI), hydroperoxides of methyl linoleate oxidation (HP), and secondary products of methyl linoleate oxidation (SP), at different pHs (4, 7, and 10) and temperatures (25, 37, 50, 80, and 120 degrees C), was studied to compare the antioxidative effects of carbohydrate- and oxidized lipids-modified proteins. The modified proteins (RIBSA, HPBSA, and SPBSA) were tested for antioxidative activity (at 100 ppm) in soybean oil using the thiobarbituric acid-reactive substances (TBARS) assay. All of them decreased significantly (p < 0.05) the TBARS formation in the oil and exhibited different effectiveness as a function of the temperature and the pH of the medium. In addition, there was a good correlation between the antioxidative activity of the protein and the amino acid losses produced during the nonenzymatic browning. These results are in agreement with an analogous and complimentary contribution of both Maillard and oxidized lipid/protein reactions to the antioxidative activity produced in foods during processing and storage.     

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.     

A new insight into the formation of odor active carbonyls by thermally-induced degradation of phospholipids in self-assembly structures

The role of molecular organization in heated aqueous dispersions of egg phosphatidylcholine (PC) and egg phosphatidylethanolamine (PE) was studied with respect to the formation of key odorants. Evidence was found for the crucial role of self-assembly structures adopted by phospholipid molecules on the quantitative composition of volatile constituents. The concentrations of seven aldehydes and one vinyl ketone were determined by isotope dilution assay in heated aqueous dispersions of PC and PE present in various ratios. Addition of PE to PC drastically decreased the amount of (E,E)-2,4-decadienal formed, which cannot be explained by the differences in the fatty acid composition of PC and PE. The free amino group in PE does not explain this phenomenon either, as replacing PE by phosphatidic acid distearylester also reduced the amounts of (E,E)-2,4-decadienal. We suggest that the type of self-assembly structure adopted by phospholipids in water significantly influences the reaction yields. However, the mechanisms leading to the preferred formation of phospholipid-derived odorants in a lamellar phase, as compared to the reversed hexagonal phase, remain unknown.     

Tracking phospholipid profiling of muscle from Ctennopharyngodon idellus during storage by shotgun lipidomics

This paper aims to study phospholipid (PL) profiling of muscle from Ctenopharyngodon idellus during room-temperature storage for 72 h by direct-infusion electrospray ionization tandem mass spectrometry (ESI-MS/MS). Five classes of PLs, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), and sphingomyelin (SM), were analyzed. At least 110 molecular species of PLs were identified, including 32 species of PC, 34 species of PE, 24 species of PS, 18 species of PI, and 2 species of SM. The result showed that oxidation and hydrolysis are the two main causes for the deterioration of PLs in fish muscle during storage. Most content of PL molecular species increased and then decreased gradually. However, some special PE molecular species with former low abundance, such as PE 32:1, PE 34:2, and PE 34:1, emerged during the storage in quantity. It indicated that those PE molecular species may come from the microbe bred in the muscle. This phenomenon was found and discussed for the first time. The possible relevance between the emergence of these special PE molecular species and the freshness of the fish muscle during storage will be investigated in further studies.     

Comparative evaluation of the emulsifying properties of phosphatidylcholine after enzymatic acyl modification

The ability of enzymatically synthesized structured phosphatidylcholine (PC) containing caprylic acid to form and stabilize oil-in-water emulsions prepared with different triglycerides [medium chain triglycerides (MCT), soybean oil, and enzymatically synthesized structured lipids] was examined and compared with natural soybean PC and deoiled lecithin. Emulsions were prepared with varying oil and emulsifier concentrations. The particle size distribution, creaming stability, and viscosity were measured for the evaluation of the emulsifying properties. With an increase in the oil concentration, there was an increase in particle size, viscosity, and creaming layer. With an increase in the phospholipid (PL) concentration, there was usually a decrease in particle size and an increase in viscosity, where the emulsion stability was increased. General emulsions prepared with structured lipids resulted in smaller particle sizes as compared to MCT and soybean oil. Deoiled lecithin was able to increase the viscosity more significantly and give smaller particle sizes as compared to the other emulsifiers, thus producing more stable emulsions. However, in certain cases, structured PC was superior to deoiled lecithin and soybean PC. This observation was made for emulsions prepared with soybean oil or structured lipid at an oil/water ratio of 10:90. At an oil/water ratio of 30:70, the deoiled lecithin performed better as compared to the other PLs with all oil types. However, structured PC produced more stable emulsions as compared to natural soybean PC in MCT and soybean oil.     

Antioxidative properties of tripeptide libraries prepared by the combinatorial chemistry

Two series of combinatorial tripeptide libraries were constructed, based on an antioxidative peptide isolated from a soybean protein hydrolysate. One was a library of 108 peptides containing either His or Tyr residues. Another was a library of 114 peptides related to Pro-His-His, which had been identified as an active core of the antioxidative peptide. The antioxidative properties of these libraries were examined by several methods, such as the antioxidative activity against the peroxidation of linoleic acid, the reducing activity, the radical scavenging activity, and the peroxynitrite scavenging activity. Two Tyr-containg tripeptides showed higher activities than those of two His-containing tripeptides in the peroxidation of linoleic acid. Tyr-His-Tyr showed a strong synergistic effects with phenolic antioxidants. However, the tripeptide had only marginal reducing activity and a moderate peroxynitrite scavenging activity. Cysteine-containing tripeptides showed the strong peroxynitrite scavenging activity. Change of either the N-terminus or C-terminus of Pro-His-His to other amino acid residues did not significantly alter their antioxidative activity. Tripeptides containing Trp or Tyr residues at the C-terminus had strong radical scavenging activities, but very weak peroxynitrite scavenging activity. The present results allow us to understand why protein digests have such a variety of antioxidative properties.     

Antiatherogenic effects of structured lipid containing conjugated linoleic acid in C57BL/6J mice

Conjugated linoleic acids (CLA) were enzymatically acidolyzed with olive oil to produce structured lipids (SL), and their antiatherosclerotic properties were investigated in C57BL/6J mice. Twenty-eight mice were divided into four groups and fed control diet or atherogenic diets supplemented with high cholesterol and high fat (HCHF) containing 5% of lard, olive oil, or SL based on control diet for 4 weeks. The supplementation of SL diet (0.6% CLA) significantly reduced the levels of serum total cholesterol and total triglyceride and increased high-density lipoprotein cholesterol level as compared to lard and olive oil diet groups (p < 0.05). The activity of liver acyl CoA:cholesterol acyltransferase (ACAT) of mice fed the SL diet was significantly lower than that of mice fed the lard or olive oil diet. A reduced formation of aortic fatty streak was observed in SL group. The extent of CLA incorporation depended on tissues or types of phospholipids. More CLA was incorporated in adipose tissue (1.85 mol %) than in the liver (0.33 mol %). Besides, more CLA was found in phosphatidylethanolamine (PE) (0.47 mol %) than in phosphatidylcholine (PC) (0.05 mol %) of hepatic phospholipids. Hepatic phospholipids (PC and PE) of mice fed the SL diet contained reduced contents of arachidonic and linoleic acid compared with mice fed the olive oil or lard diet. The present study suggests that SL could be considered as a functional oil for preventing risks of atheroscelerosis.     

Determination of antioxidant properties of aroma extracts from various beans

Aroma extracts from fresh soybeans, mung beans, kidney beans, and azuki beans were prepared using simultaneous steam distillation and solvent extraction (SDE) under mild conditions (55 degrees C and 95 mmHg). Extracts were examined for antioxidative activities in two different assays. The aroma extracts isolated from all beans inhibited the oxidation of hexanal for nearly one month at a level of 250 microL/mL. Mung bean and soybean extracts inhibited malonaldehyde (MA) formation from cod-liver oil by 86% and 88%, respectively, at the 250 microL/mL level. Azuki and kidney bean extracts inhibited MA formation from cod-liver oil by 76% and 53%, respectively, at the 250 microL/mL level. The antioxidative activities of mung bean and soybean extracts were comparable with that of the natural antioxidant, alpha-tocopherol (vitamin E).     

Maillard reaction products inhibit oxidation of human low-density lipoproteins in vitro

Dietary intake of antioxidants has been associated with a reduced risk of cardiovascular diseases, which is very likely caused by their capability of prevent oxidation of low-density lipoproteins (LDL). During food processing and storage, substances with antioxidative properties are formed by Maillard reactions. In this study, the activity of Maillard products to inhibit copper-induced oxidation of human LDL in vitro was investigated. d-Glucose was heated with an equimolar amount of glycine, l-lysine, or l-arginine, for 1 h under reflux. The increase of the antioxidative activity (AOA) of the Maillard mixtures was highly significant compared to the control mixtures. Additionally, two defined Maillard products with amino reductone structure were tested. 3-Hydroxy-4-(morpholino)-3-buten-2-one (1) and amino hexose reductone (2) showed a significant and dose dependent AOA. Compound 1 was about half as active as ascorbic acid, which served as positive control. Thus, it can be concluded that Maillard products, particularly those with amino reductone structure, have the strong potential to inhibit LDL oxidation.     

Effects of phenolic propyl esters on the oxidative stability of refined sunflower oil

The oxidative stability of refined sunflower oil in the presence and in the absence of propyl caffeate (PC), propyl hydrocaffeate (PHC), propyl ferulate (PF), and propyl isoferulate (PI) has been evaluated according to the Rancimat method. The antioxidant activity of the phenolic derivatives was compared with that obtained with native [alpha-tocopherol (alpha-TOH)] and synthetic [propyl gallate (PG)] antioxidants. The results allow the establishment of a decreasing order of antioxidant power: PG > PHC > PC > alpha-TOH > PI > PF. The oxidative stability was improved neither by the addition of PF nor by a supplement of alpha-TOH. Moreover, a positive antioxidant effect was obtained for PC that was placed between those of alpha-TOH and PG. The antioxidant activity of PHC was higher than that of its analogue (PC). A dose-dependent effect was observed for PG, PHC, and PC. A chain-breaking mechanism was proposed for the antioxidant activity of propyl phenolic esters because the same ranking order of efficacy was obtained for their antiradical activities evaluated by using the 2,2-diphenyl-1-picrylhydrazyl radical method.     

Effect of the pyrrole polymerization mechanism on the antioxidative activity of nonenzymatic browning reactions

The present investigation was undertaken to study how the antioxidative activity (AA) of nonenzymatic browning reactions is changing at the same time that the browning (by the pyrrole polymerization mechanism) is being produced. The antioxidative activities of eight model pyrroles (pyrrole, 1-methylpyrrole, 2,5-dimethylpyrrole, 1,2,5-trimethylpyrrole, 2-acetylpyrrole, 2-acetyl-1-methylpyrrole, pyrrole-2-carboxaldehyde, and 1-methyl-2-pyrrolecarboxaldehyde) as well as the browning reaction of 2-(1-hydroxyethyl)-1-methylpyrrole (HMP) and the dimer (DIM) produced during HMP browning were determined. The results obtained suggest that the AAs observed in nonenzymatic browning reactions are the result of the AAs of the different oxidized lipid/amino acid reaction products formed. Thus, the different pyrrole derivatives produced in these reactions had different AAs, and the highest AAs were observed for alkyl-substituted pyrroles without free alpha-positions. Because some of these pyrrole derivatives are implicated in nonenzymatic browning production and this browning production implies the loss of hydroxyl groups and the transformation of some pyrroles with one type of substitution into others, changes in AA during browning production were observed, and the resulting DIM derivative was more antioxidant than HMP or higher polymers. These results explain the AA observed in fatty acid/protein mixtures after slight oxidation and suggest that, when the pyrrole polymerization mechanism is predominant, slightly browned samples may be more antioxidant than samples in which nonenzymatic browning has been highly developed.     

Development of yellow pigmentation in squid (Loligo peali) as a result of lipid oxidation

The impact of lipid oxidation on yellow pigment formation in squid lipids and proteins was studied. When the squid microsomes were oxidized with iron and ascorbate, thiobarbituric acid reactive substance were observed to increase simultaneously with b values (yellowness) and pyrrole compounds concomitantly with a decrease in free amines. Oxidized microsomes were not able to change the solubility, sulfhydryl content, or color of salt-soluble squid myofibrillar proteins. Aldehydic lipid oxidation products were able to decrease solubility and sulfhydryl content of salt-soluble squid myofibrillar proteins but had no impact on color. Aldehydic lipid oxidation products increased b values (yellowness) and pyrrole compounds and decreased free amines in both squid phospholipid and egg yolk lecithin liposomes. The ability of aldehydic lipid oxidation products to change the physical and chemical properties of egg yolk lecithin liposomes increased with increasing level of unsaturation and when the carbon number was increased from 6 to 7. These data suggest that off-color formation in squid muscle could be due to nonenzymatic browning reactions occurring between aldehydic lipid oxidation products and the amines on phospholipids headgroups.     

Effect of heating oxymyoglobin and metmyoglobin on the oxidation of muscle microsomes

Myoglobin (Mb) and its iron have been proposed to be major prooxidants in cooked meats. To understand the mechanisms and differentiate between the prooxidant and antioxidant potential of oxymyoglobin (OxyMb) and metmyoglobin (MetMb), their prooxidant activity, iron content, solubility, free radical scavenging activity, and iron binding capacity were determined as a function of thermal processing. The ability of native and heat denatured OxyMb and MetMb to promote the oxidation of muscle microsomes was different. MetMb promoted lipid oxidation in both its native and denatured states. Conversely, OxyMb became antioxidative when the protein was heated to temperatures >or=75 degrees C. The increased antioxidant activity of heat denatured OxyMb was likely due to a decrease in its prooxidative activity due to its loss of solubility. These data show that the impact on oxidative reactions of Mb is the result of the balance between its antioxidant and prooxidant activities.     

Odorants generated by thermally induced degradation of phospholipids

The qualitative and quantitative aroma composition of heated aqueous dispersions of egg phosphatidylcholine (PC) and egg phosphatidylethanolamine (PE) were characterized by aroma extract dilution analysis and isotope dilution assay. On the basis of FD-factors and odor activity values, trans-4,5-epoxy-(E)-2-decenal was found to be the most potent odorant followed by (E,E)-2,4-decadienal, 1-octen-3-one, and hexanal. The amount of (E,E)-2,4-decadienal in PC was about 20-fold higher compared to PE, while hexanal was the major odor-active compound in the PE sample. (E,Z,Z)-2,4,7-Tridecatrienal was identified for the first time as an odor-active volatile constituent of heated phospholipids exhibiting a characteristic egg white-like note. Further odorants first reported in thermally treated phospholipids were (Z)-2-decenal, (E)-2-decenal, and (E)-2-undecenal. Differences in the fatty acid composition of PC and PE can only partially explain the quantitative results found in this study, thus suggesting that further parameters may influence the formation of carbonyls from heated aqueous dispersions of phospholipids.     

Contribution of phospholipid pyrrolization to the color reversion produced during deodorization of poorly degummed vegetable oils

The Ehrlich reaction was optimized to determine the formation of pyrrolized phospholipids in edible oils in an attempt to understand the color reversion produced during the deodorization of poorly degummed edible oils. The procedure consisted of the treatment of the oil with p-(dimethylamino)benzaldehyde in tetrahydrofuran/2-propanol at a controlled acidity and temperature and the spectrophotometric determination of adducts produced. The extinction coefficient of Ehrlich adducts was calculated by using 1-[1-(2-hydroxyethyl)-1H-pyrrol-2-yl]propan-1-ol (1) as a standard and was 15 300 M(-)(1) cm(-)(1). The response was linear and reproducible within the range of 0.334-48.6 microM of compound 1. When the assay was applied to a soybean oil treated with 100-1000 ppm of phosphatidylethanolamine and submitted to deodorization, the formation of pyrrolized phospholipids was observed at the same time that the disappearance of the phospholipid and the oil darkening were produced. The main changes were observed during the first steps of the deodorization process, when the oil was heated between 80 and 160 degrees C. During the initial heating of the oil until achieving 200 degrees C, oil darkening, phosphatidylethanolamine disappearance, and pyrrolized phospholipid formation were correlated, therefore suggesting a contribution of phospholipid pyrrolization to the oil darkening produced.     

Antioxidant properties of aroma compounds isolated from soybeans and mung beans

Aroma compounds contained in the extracts of soybean and mung bean that possess antioxidant activity were identified by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). The major aroma constituents of soybeans were 1-octen-3-ol (13.699 ppm), maltol (1.662 ppm), phenylethyl alcohol (1.474 ppm), hexanol (1.430 ppm), and gamma-butyrolactone (1.370 ppm). The major aroma constituents of mung beans were hexanol (3.234 ppm), benzyl alcohol (2.060 ppm), gamma-butyrolactone (1.857 ppm), 2-methyl-2-propenal (1. 633 ppm), and pentanol (1.363 ppm). The major aroma chemicals of soybeans and mung beans were examined for antioxidative activities in two different assays. Eugenol, maltol, benzyl alcohol, and 1-octen-3-ol showed potent antioxidative activities in two different assays. Eugenol, maltol, benzyl alcohol, and 1-octen-3-ol inhibited the oxidation of hexanal by 100%, 93%, 84%, and 32%, respectively, for a period of 40 days at the 500 microg/mL level. Eugenol, maltol, benzyl alcohol, and 1-octen-3-ol inhibited malonaldehyde (MA) formation from cod liver oil by 91%, 78%, 78%, and 78%, respectively, at the 160 microg/mL level. The antioxidative activity of eugenol was comparable to that of the natural antioxidant alpha-tocopherol (vitamin E).     

Antioxidant activity of cysteine, tryptophan, and methionine residues in continuous phase beta-lactoglobulin in oil-in-water emulsions

Proteins dispersed in the continuous phase of oil-in-water emulsions are capable of inhibiting lipid oxidation reactions. The antioxidant activity of these proteins is thought to encompass both free radical scavenging by amino acid residues and chelation of prooxidative transition metals; however, the precise mechanism by which this occurs remains unclear. In this study, the oxidative stability of cysteine, tryptophan, and methionine residues in continuous phase beta-lactoglobulin (beta-Lg) in a Brij-stabilized menhaden oil-in-water emulsion was determined. The presence of low concentrations of continuous phase beta-Lg (250 and 750 microg/mL) significantly inhibited lipid oxidation as determined by lipid hydroperoxides and thiobarbituric acid reactive substances analysis. It was observed that cysteine oxidized before tryptophan in beta-Lg, and both residues oxidized before lipid oxidation could be detected. No oxidation of the methionine residues of beta-Lg was observed despite its reported high oxidative susceptibility. It is conceivable that surface exposure of amino acid residues greatly affects their oxidation kinetics, which may explain why some residues are preferentially oxidized relative to others. Further elucidation of the mechanisms governing free radical scavenging of amino acids could lead to more effective applications of proteins as antioxidants within oil-in-water food emulsions.     

Antioxidant mechanisms of enzymatic hydrolysates of beta-lactoglobulin in food lipid dispersions

The antioxidant activities of aqueous phase beta-lactoglobulin (beta-Lg) and its chymotryptic hydrolysates (CTH) were compared in this study. Proteins and peptides have been shown to inhibit lipid oxidation reactions in oil-in-water emulsions; however, a more fundamental understanding of the antioxidant activity of these compounds in dispersed food lipid systems is lacking. CTH was more effective than an equivalent concentration of beta-Lg in retarding lipid oxidation reactions when dispersed in the continuous phase of Brij-stabilized oil-in-water emulsions (pH 7). Furthermore, it was observed that CTH had higher peroxyl radical scavenging and iron-binding values than beta-Lg. Liquid chromatography-mass spectrometry (LC-MS) was used to measure the rate of oxidation of three oxidatively labile amino acid residues (Tyr, Met, and Phe) in certain CTH peptide fragments. Significant oxidation of specific Tyr and Met residues present in two separate 12 amino acid peptide fragments was observed in the days preceding lipid oxidation (39 and 55% of Tyr and Met were oxidized, respectively, by day 4 of the study); however, no significant oxidation of the Phe residue present in a specific 14 amino acid peptide fragment could be observed during the same time period. These data could suggest that Met and Tyr residues are capable of scavenging radical species and have the potential to improve the oxidative stability dispersed food lipids.     

Jojoba seed meal proteins associated with proteolytic and protease inhibitory activities

The jojoba, Simmondsia chinensis, is a characteristic desert plant native to the Sonoran desert. The jojoba meal after oil extraction is rich in protein. The major jojoba proteins were albumins (79%) and globulins (21%), which have similar amino acid compositions and also showed a labile thrombin-inhibitory activity. SDS-PAGE showed two major proteins at 50 kDa and 25 kDa both in the albumins and in the globulins. The 25 kDa protein has trypsin- and chymotrypsin-inhibitory activities. In vitro digestibility of the globulins and albumins resembled that of casein and soybean protein concentrates and was increased after heat treatment. The increased digestibility achieved by boiling may be attributed to inactivation of the protease inhibitors and denaturation of proteins.