Catalase Expression Is Modulated by Vancomycin and Ciprofloxacin and Influences the Formation of Free Radicals in Staphylococcus aureus Cultures.

Detection of free radicals in biological systems is challenging due to their short half-lives. We have applied electron spin resonance (ESR) spectroscopy combined with spin traps using the probes PBN (N-tert-butyl-α-phenylnitrone) and DMPO (5,5-dimethyl-1-pyrroline N-oxide) to assess free radical formation in the human pathogen Staphylococcus aureus treated with a bactericidal antibiotic, vancomycin or ciprofloxacin. While we were unable to detect ESR signals in bacterial cells, hydroxyl radicals were observed in the supernatant of bacterial cell cultures. Surprisingly, the strongest signal was detected in broth medium without bacterial cells present and it was mitigated by iron chelation or by addition of catalase, which catalyzes the decomposition of hydrogen peroxide to water and oxygen. This suggests that the signal originates from hydroxyl radicals formed by the Fenton reaction, in which iron is oxidized by hydrogen peroxide. Previously, hydroxyl radicals have been proposed to be generated within bacterial cells in response to bactericidal antibiotics. We found that when S. aureus was exposed to vancomycin or ciprofloxacin, hydroxyl radical formation in the broth was indeed increased compared to the level seen with untreated bacterial cells. However, S. aureus cells express catalase, and the antibiotic-mediated increase in hydroxyl radical formation was correlated with reduced katA expression and catalase activity in the presence of either antibiotic. Therefore, our results show that in S. aureus, bactericidal antibiotics modulate catalase expression, which in turn influences the formation of free radicals in the surrounding broth medium. If similar regulation is found in other bacterial species, it might explain why bactericidal antibiotics are perceived as inducing formation of free radicals.

Quantification of protein thiols using ThioGlo 1 fluorescent derivatives and HPLC separation.

A method for quantification of total soluble protein-derived thiols in beer was developed based on the formation of fluorescent adducts with the maleimide compound ThioGlo 1. The problem of interference from fluorescent adducts of sulfite and ThioGlo 1 was solved by HPLC separation of the adducts followed by fluorescence detection. Using standard addition of GSH, a detection limit of 0.028 µM thiols was achieved. The application and validation of the method was demonstrated for beers with different color intensities, and the application range is in principle for any biological system containing thiols. However, the quantification of cysteine was complicated by a lower fluorescence response of its ThioGlo 1 adducts. Based on the studies of the responses of a series of cysteine-derived thiols and (1)H NMR studies of the structures of ThioGlo 1 adducts with GSH and cysteine, it was concluded that thiols with a neighboring free amino group yield ThioGlo 1 adducts with a reduced fluorescence intensity.

Temperature affects the kinetics but not the products of the reaction between 4-methylbenzoquinone and lysine.

Quinones are electrophilic compounds that can undergo Michael addition or Schiff base reaction with nucleophilic amines, but the effect of temperature has not been systematically studied. The aim of this study was to characterize how temperature affects the reaction mechanism and kinetics of 4-methylbenzoquinone (4MBQ) with lysine (Lys), Nα-acetyl Lys or Nε-acetyl Lys. The products were identified and characterized by LC-MS/MS, which revealed formation of Michael addition products, Schiff base, and a di-adduct in Lys and Nα-acetyl Lys-containing reaction mixtures. The product profiles were not affected by temperature in the range of 15-100 °C. NMR analysis proved that Michael addition of Nα-acetyl Lys occurred on the C5 position of 4MBQ. Rate constants for the reactions studied by stopped-flow UV-vis spectrophotometry under pseudo-first-order conditions where the amines were present in excess in the range 15 °C to 45 °C showed the α-amino groups of Lys are more reactive than the ε-groups. The kinetics results revealed that the temperature dependence of reaction rates followed the Arrhenius law, with activation energies in the order: Lys < Nε-acetyl Lys < Nα-acetyl Lys. Our results provide detailed knowledge about the temperature dependence of the reaction between Lys residues and quinones under conditions relevant for storage of foods.

Textural and biochemical changes during ripening of old-fashioned salted herrings.

BACKGROUND: Understanding of the biochemical reactions taking place during ripening of salted herring is still rather limited. Therefore, salted herrings were traditionally produced and the impact of the brine composition was evaluated in relation to the development of the characteristic texture of salted herrings. The aim of this study was to measure the texture changes during ripening using two different methods and to correlate the texture changes with brine composition and with biochemical modifications at the molecular level. RESULTS: During ripening (up to 151 days), hardness was higher in salted herrings compared to raw herrings, irrespective of the brine composition. However, the increase in hardness of herring prepared with extra brine occurred later. After prolonged storage (371 days), hardness was found for both batches to decrease to the level of raw herring. The increase in hardness during the ripening period could be explained by free-radical-induced cross-linking of myosin and the formation of aggregates. In addition, degradation of these aggregates correlated with the decrease in hardness observed at 371 days. CONCLUSIONS: Texture changes during ripening of salted herrings can be explained by oxidative reactions inducing myosin cross-linking followed by subsequent degradation of these myosin aggregates. The brine composition might play a role in the development of herring texture but this need to be investigated in more details.

ESR spin trapping for in situ detection of radicals involved in the early stages of lipid oxidation of dried microencapsulated oils.

Different studies have shown that detection of free radicals by ESR spin trapping provides useful information on the susceptibility to oxidation of bulk oils and accordingly on the oxidative stability of different samples for comparative purposes. With the same goal, ESR spin trapping was evaluated in this work for in situ detection of radicals in dried microencapsulated oils (DMOs). By testing different oils, encapsulation matrices and oxidation conditions, results showed that ESR spin trapping can be useful to evaluate the oxidative susceptibility of DMOs, but ESR data should be interpreted cautiously, as the great complexity of the reactions involved may lead to data misinterpretations. Conditions for oxygen availability can have important impacts on the rates of both spin trapping and spin-adduct quenching affecting the levels of radicals observed. The kinetics of oxidation, spin trapping and spin-adduct decay should be known first in bulk oils for correct data interpretation in DMOs.

Measurement of hydrogen peroxide vapor in powders with potassium titanium oxide oxalate loaded cellulose pellets as probes.

An image-based method for determining H2O2 vapor pressures in powder systems was developed based on cellulose pellets loaded with potassium titanium oxide oxalate (PTO Pellets) as probe particles. Solid titanyl salts change color after exposure to hydrogen peroxide vapor and the quantitative response of PTO pellets to H2O2 has been established by comparing reactions with H2O2 in liquid and solid states. Analysis of pictures of the color changes of PTO Pellets mixed into powders can be used to monitor the presence of ppm levels of H2O2 vapor inside powder systems such as bleach containing dry detergent powders.•H2O2 vapor quantification in dry systems with titanyl loaded cellulose particles.•Simple fabrication of H2O2 probe particles.•High sensitivity with LOD 0.190 ppm H2O2 .

Copper Binding in Sweet Worts Made from Specialty Malts.

Trace levels of copper can impact the flavor stability of beer. The main source of copper is malt, and the wort copper levels are established during mashing and lautering. This study focuses on sweet worts made from experimental roasted and caramel malts. Potentiometric titrations using ion-selective electrodes combined with electron paramagnetic resonance spectroscopy have been used to investigate Cu(II) binding in worts as well as the impact of Cu(II) ions on the wort oxidative stability. High-temperature treatment during malting decreased Cu(II) binding affinities in the worts, with roasted malt worts having lower affinities than caramel malt worts of similar color and pH. Electron paramagnetic resonance spectra indicated dipeptides as the main Cu(II) chelators. A positive correlation between Cu and free amino nitrogen levels in worts is demonstrated. In dark worts with high rates of radical formation, Cu(II) had pronounced antioxidative effects. In contrast, moderate prooxidative effects were observed when adding Cu(II) to pale worts with inherently low rates of oxidation.

The effect of molecular structure of polyphenols on the kinetics of the trapping reactions with methylglyoxal.

Reaction kinetics for the reactions between four polyphenols (trans-resveratrol, apigenin, kaempferol and fisetin) and methylglyoxal (MGO) were determined at pH 7.4 and 37 °C, and the effect of functional groups of polyphenols on MGO trapping ability was examined. Reaction products of polyphenols with MGO were characterized by HPLC-MS/MS. The apparent second order rate constants for the reaction of MGO with trans-resveratrol, apigenin, kaempferol and fisetin were (2.7 ± 0.2) × 10-2 M-1 s-1, (4.4 ± 0.4) × 10-3 M-1 s-1, (6.3 ± 0.2) × 10-2 M-1 s-1, and (3.6 ± 0.1) × 10-3 M-1 s-1, respectively. The rate constants were used to evaluate the possible competition between MGO with nucleophilic sites on proteins and polyphenols at concentrations relevant to UHT milk. Combining this information with solubility and flavour of the polyphenols, it was concluded that kaempferol could effectively trap MGO and hereby inhibit Maillard reactions in UHT milk.

Multiresponse Kinetic Modeling of Heat-Induced Equilibrium of ß-Carotene cis-trans Isomerization in Medium-Chain Triglyceride Oil.

The kinetics and mechanism of the stepwise cis-trans isomerization reactions of all-trans-ß-carotene dissolved in MCT (medium-chain triglyceride) oil at temperatures in the range of 80-160 °C have been analyzed using multiresponse modeling. Quantitation of the cis-isomers was performed using HPLC-DAD and quantitation at the reaction isosbestic point at 421 nm. Multiresponse kinetic modeling using the Bayesian criterion was initially performed at 120 °C to determine the best model. Subsequently, the reparametrized Arrhenius equation was used to calculate the activation energies of all reactions. The equilibrium constants for the individual isomerization reactions were determined from the rate constants and the final product distributions. The enthalpies and entropies of the isomerization reactions were determined from the temperature dependence of the equilibrium constants. The 13-cis and 13,13'-di-cis isomers were found to be the fastest formed isomers followed by the 9-cis, 9,13-di-cis, and 13,15-di-cis isomers, where the latter was found to be formed from 13-cis and not the 15-cis isomer. The relative free energies of the ß-carotene isomers were determined as all-trans < 13-cis < 9-cis < 13,13'-di-cis < 9,13-di-cis ≈ 15-cis < 13,15-di-cis. The entropic contribution of each reaction was found to play an important role in the ordering. It is concluded that the ß-carotene system is quite labile at temperatures ranging from 80 to 160 °C and resulting in equilibrium distributions of the cis-trans isomers.

Trapping of Carbonyl Compounds by Epicatechin: Reaction Kinetics and Identification of Epicatechin Adducts in Stored UHT Milk.

The kinetics of the reaction between epicatechin and various carbonyl compounds typically formed in cooked and stored foods were evaluated in model systems at pH 7.4 and 37 °C, and the corresponding reaction products in stored ultrahigh temperature (UHT) milk-added epicatechin were identified by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The rate constants for the reactions of carbonyl compounds with epicatechin decreased in the following the order: methylglyoxal; 1.6 ± 0.2 M-1 s-1 > glyoxal; (5.9 ± 0.3) × 10-2 M-1 s-1 ≥ 5-(hydroxymethyl)furfural; (4.0 ± 0.2) × 10-2 M-1 s-1 ≥ acetaldehyde; (2.6 ± 0.3) × 10-2 M-1 s-1 ≥ phenylacetaldehyde; (2.1 ± 0.2) × 10-2 M-1 s-1 ≥ furfural; (4.3 ± 0.1) × 10-3 M-1 s-1 > 2-methylbutanal and 3-methylbutanal; ∼0 M-1 s-1. Reaction products generated by epicatechin and methylglyoxal, glyoxal, 5-(hydroxymethyl)furfural, and acetaldehyde were detected in UHT milk samples by incubating milk samples with epicatechin at 37 °C for 24 h. The lack of reaction between epicatechin and phenylacetaldehyde, furfural, 2-methylbutanal, and 3-methylbutanal in stored UHT milk may be due to their slow reaction rates or low concentration in stored UHT milk. It is demonstrated that epicatechin traps 5-(hydroxymethyl)furfural, acetaldehyde, glyoxal, and methylglyoxal and may thereby reduce off-flavor formation in UHT milk during storage both by trapping of precursors (methylglyoxal and glyoxal) for off-flavor formation and by direct trapping of off-flavors.

Quantitation of Protein Cysteine-Phenol Adducts in Minced Beef Containing 4-Methyl Catechol.

Thiol groups of cysteine (Cys) residues in proteins react with quinones, oxidation products of polyphenols, to form protein-polyphenol adducts. The aim of the present work was to quantify the amount of adduct formed between Cys residues and 4-methylcatechol (4MC) in minced beef. A Cys-4MC adduct standard was electrochemically synthesized and characterized by liquid chromatography-mass spectrometry (LC-MS) as well as NMR spectroscopy. Cys-4MC adducts were quantified after acidic hydrolysis of myofibrillar protein isolates (MPIs) and LC-MS/MS analysis of meat containing either 500 or 1500 ppm 4MC and stored at 4 °C for 7 days under a nitrogen or oxygen atmosphere. The concentrations of Cys-4MC were found to be 2.2 ± 0.3 nmol/mg MPI and 8.1 ± 0.9 nmol/mg MPI in meat containing 500 and 1500 ppm 4MC, respectively, and stored for 7 days under oxygen. The formation of the Cys-4MC adduct resulted in protein thiol loss, and ca. 62% of the thiol loss was estimated to account for the formation of the Cys-4MC adduct for meat containing 1500 ppm 4MC. Furthermore, protein polymerization increased in samples containing 4MC as evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the polymerization was found to originate from protein-polyphenol interactions as evaluated by a blotting assay with staining by nitroblue tetrazolium.

Identification of emulsifier potato peptides by bioinformatics: application to omega-3 delivery emulsions and release from potato industry side streams.

In this work, we developed a novel approach combining bioinformatics, testing of functionality and bottom-up proteomics to obtain peptide emulsifiers from potato side-streams. This is a significant advancement in the process to obtain emulsifier peptides and it is applicable to any type of protein. Our results indicated that structure at the interface is the major determining factor of the emulsifying activity of peptide emulsifiers. Fish oil-in-water emulsions with high physical stability were stabilized with peptides to be predicted to have facial amphiphilicity: (i) peptides with predominantly α-helix conformation at the interface and having 18-29 amino acids, and (ii) peptides with predominantly ß-strand conformation at the interface and having 13-15 amino acids. In addition, high physically stable emulsions were obtained with peptides that were predicted to have axial hydrophobic/hydrophilic regions. Peptides containing the sequence FCLKVGV showed high in vitro antioxidant activity and led to emulsions with high oxidative stability. Peptide-level proteomics data and sequence analysis revealed the feasibility to obtain the potent emulsifier peptides found in this study (e.g. γ-1) by trypsin-based hydrolysis of different side streams in the potato industry.

Effect of pH on the reaction between naringenin and methylglyoxal: A kinetic study.

Methylglyoxal (MGO) is a highly reactive ɑ-dicarbonyl compound that may adversely impact food quality and human health by modifying proteins. The kinetics of the reaction of naringenin with MGO was studied at pH 6-8 and 37 °C by UV-Vis spectrophotometry and reaction products were characterized by liquid chromatography-mass spectrometry (LC-MS/MS). The apparent second order rate constant (k2) increased at pH above the lowest pKa value of naringenin, indicating deprotonated naringenin as the main reactant. A Lederer-Manasse type reaction mechanism is suggested, with dehydration of the MGO-dihydrate as a rate determining step. The quantitative data obtained in the present study was used to simulate the competitive reaction between MGO and nucleophilic amino acid residues (Lys, Arg and Cys) and naringenin in milk. It is predicted that naringenin will be able to efficiently trap MGO during storage of milk, although the reversible trapping of MGO by Cys residues is initially kinetically favourable.

Oxygen permeability and oxidative stability of fish oil-loaded electrosprayed capsules measured by Electron Spin Resonance: Effect of dextran and glucose syrup as main encapsulating materials.

The oxygen permeability and oxidative stability of fish oil-loaded electrosprayed capsules were studied by Electron Spin Resonance (ESR). Electrosprayed capsules with dextran as main biopolymer showed a significantly faster broadening (ΔHpp) of 16-doxyl-stearate ESR spectrum when compared to glucose syrup capsules. This finding indicates a higher oxygen permeability of dextran capsules than glucose syrup capsules, which is explained by a reduced average free volume in the glucose syrup matrix than in the dextran shell. Moreover, glucose syrup capsules showed a significantly lower increase in the peak-to-peak amplitude of N-tert-butyl-α-phenylnitrone (PBN) ESR spectrum during storage when compared to dextran capsules. This implies a higher oxidative stability of glucose syrup capsules than dextran capsules, which correlated well with the lower oxygen permeability of the former. These results indicated the importance of the oxygen barrier properties of the wall materials when encapsulating long chain omega-3 polyunsaturated fatty acids by electrospraying.

Oxidation of porcine Myosin by hypervalent myoglobin: the role of thiol groups.

Oxidation of the myofibrillar muscle protein myosin from pork by hypervalent myoglobin species (MbFe(III)/H 2O2 radical generating system) was investigated in aqueous solution in the pH range of 5.0-7.8 by electron spin resonance (ESR) spectroscopy using N- tert-butyl-alpha-phenylnitrone (PBN) as spin trap and indirectly by determination of the rate of reduction of hypervalent myoglobin species by UV spectroscopy. Cross-linking of myosin was examined by SDS-PAGE. The target for oxidative modification of myosin was studied by thiol blocking by N-acetylmaleimide (NEM) and by determining oxidative modification of myosin thiols. The reaction between myosin and hypervalent myoglobin was fast and showed little dependence on pH. The myosin radicals formed were observed to be short-lived. Myosin thiols are suggested to be the main target for oxidative modification, as NEM-treated myosin did not form radicals in the presence of hypervalent myoglobin. A significant decrease in thiol content was already demonstrated 25 s after initiation of oxidation of myosin. The majority of myosin heavy chain (MHC) was demonstrated to be cross-linked through intermolecular disulfide bonding 1 h after initiation of oxidation. This demonstrates that thiols are important for radical formation and cross-linking of myosin during oxidation with hypervalent myoglobin at the pH of meat products.

Characterization of oxidative changes in salted herring (Clupea harengus) during ripening.

Salted herring were prepared in barrels according to a traditional recipe. The biochemical changes in the fish and in the brine were monitored during a prolonged ripening period (12 months). The process was followed by measuring pH, protein, salt, dry matter, free fatty acids, and lipid content in the brine and in the fish according to standard protocols. The results showed that most of the biochemical changes occurred at an early stage in the ripening process. Lipid oxidation was followed in the fish muscle using spectroscopic determination for lipid hydroperoxide (PV) and by GC-MS for determination of secondary oxidation products. Protein oxidation was determined using spectrophotometric determination of protein carbonyl groups. To follow protein degradation (proteolysis) and protein oxidation SDS-PAGE and immunoblotting for protein carbonyl were performed on both brine and fish during the ripening period. Results revealed that no lipid oxidation occurred in fish muscle during ripening but a significant level of protein oxidation was detected. Finally, iron alpha-tocopherol, and 3-methylbutanal levels were also measured. Alpha-tocopherol levels decreased during ripening, further supporting that oxidative reactions took place. Peroxidase activity was demonstrated in the brine, suggesting that hemoglobin might be a crucial parameter, which might trigger protein oxidation. This indicates that protein oxidation might be important for the development of the characteristic organoleptic properties of salted herring.

Efficiency of natural phenolic compounds regenerating alpha-tocopherol from alpha-tocopheroxyl radical.

Benzoic acid-derived phenolics (p-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, vanillic acid, syringic acid, and gallic acid) and the polyphenols epicatechin and epigallocatechin gallate (EGCG) were evaluated for their efficiency in regenerating alpha-tocopherol from alpha-tocopheroxyl radical in comparison with ascorbyl palmitate, which is known to regenerate alpha-tocopherol. Ethanolic solutions of phenolic compounds were added to a homogeneous hexane medium containing alpha-tocopheroxyl radical generated by reaction of alpha-tocopherol in molar excess with 1,1-diphenyl-2-picrylhydrazyl radical, and the alpha-tocopheroxyl radical was monitored by electron spin resonance spectroscopy. p-Hydroxybenzoic acid, vanillic acid, and syringic acid (400 microM) did not exhibit a significant effect on alpha-tocopheroxyl radical concentration (0.6-0.7 microM). In contrast, 3,4-dihydroxybenzoic acid and gallic acid were able to reduce the concentration of alpha-tocopheroxyl radical by 16 and 64%, respectively. Epicatechin showed a reduction of alpha-tocopheroxyl radical similar to gallic acid, and EGCG and ascorbyl palmitate were the most effective, reducing alpha-tocopheroxyl radical completely at a much lower phenolic concentration (66.7 microM). The moles of alpha-tocopheroxyl radical reduced per mole of ascorbyl palmitate (0.93), EGCG (0.066), gallic acid (4.3 x 10(-4)), and epicatechin (4.5 x 10(-4)) were determined, and the logarithm of these stoichoimetric ratios showed a negative linear relationship with the bond dissociation enthalpies of the O-H bond of the phenolics. The relative capacity to reduce alpha-tocopheroxyl radical was found to be ascorbyl palmitate (2142) > EGCG (151) > gallic acid (1) approximately epicatechin (1).

Puerarin and conjugate bases as radical scavengers and antioxidants: molecular mechanism and synergism with beta-carotene.

The 4'-hydroxyl group of puerarin, a C-glycoside of the isoflavonoid daidzein, was shown, using 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical cation and stopped-flow spectroscopy and by comparison with the 7-propylpuerarin (A ring derivative) and 4'-propylpuerarin (B ring derivative), to be a more efficient radical scavenger as compared to the 7-hydroxyl group by a factor of 2, a difference increasing upon deprotonation. The difference in radical scavenging agreed with the oxidation potentials (cyclic voltammetry in acetonitrile, 0.1 M Bu4NBF4 at 25 degrees C): E/mV=862+/-3 for puerarin, 905+/-10 for 7-propylpuerarin, and 1064+/-2 for 4'-propylpuerarin relative to ferrocene/ferricenium. In aqueous solution, the reduction potential was shown to decrease for increasing pH, and deprotonation of the 4'-hydroxyl group increased radical scavenging more than deprotonation of the 7-hydroxyl group. The 7-hydroxyl was found to be more acidic (pKa1=7.20+/-0.01 in puerarin and pKa=7.23+/-0.01 in 4'-propylpuerarin) than the 4'-hydroxyl group (pKa2=9.84+/-0.08 in puerarin and pKa=9.51+/-0.02 in 7-propylpuerarin); aqueous solution, ionic strength of 0.1, and 25 degrees C. In phosphatidyl choline liposome of pH 7.4, puerarin and beta-carotene each showed a modest antioxidant activity measured as prolongation of the lag phase for formation of conjugate dienes and using the water-soluble radical initiator APPH with effects of puerarin and beta-carotene being additive. For the lipophilic initiator AMVN, the antioxidative effect decreased for puerarin and increased for beta-carotene as compared to APPH and showed a clear synergism. A regeneration of beta-carotene, effective in the liposome lipid phase as antioxidant, from the cation radical by deprotonated forms of puerarin was demonstrated in 9:1 chloroform/methanol using laser flash photolysis with k2=2.7x10(4) L mol-1 s-1 for the bimolecular process between the cation radical and the puerarin dianion.

Kinetic Models for the Role of Protein Thiols during Oxidation in Beer.

Thiol-containing proteins have been suggested to have antioxidative properties in beer. A kinetic model has been setup for the reactivity of thiols during early stages of oxidative degradation of beer. Kinetic analysis based on the proposed reaction mechanism allowed evaluation of the relative reactivity of beer components, such as bitter acids from hops and polyphenols. The rate constants for the reaction of 1-hydroxyethyl radicals, which are generated during radical mediated oxidation of ethanol in beer, with hop bitter acids and thiols were very similar, and the concentration of these compounds in beer is therefore essential for the relative reactivity. For a standard pilsner beer with 35 international bitter units with typical concentrations of thiols and hop bitter acids, thiols were found to react with ca. 9% of 1-hydroxyethyl radicals, while bitter acids from hops accounted for ca. 88% of the reaction with 1-hydroxyethyl radicals. Polyphenols were not found to account for any major part of the reaction with 1-hydroxyethyl radicals due to low reaction rates and low concentrations in pilsner beer compared to the other components. The kinetic model suggests that the concentration of thiols has to be increased in order to contribute with any significant antioxidative protection and that the fate of thiols during oxidation must be considered since some thiol oxidation products may induce further damage.

Interactions between macromolecule-bound antioxidants and Trolox during liposome autoxidation: A multivariate approach.

The interactions between free and macromolecule-bound antioxidants were investigated in order to evaluate their combined effects on the antioxidant environment. Dietary fiber (DF), protein and lipid-bound antioxidants, obtained from whole wheat, soybean and olive oil products, respectively and Trolox were used for this purpose. Experimental studies were carried out in autoxidizing liposome medium by monitoring the development of fluorescent products formed by lipid oxidation. Chemometric methods were used both at experimental design and multivariate data analysis stages. Comparison of the simple addition effects of Trolox and bound antioxidants with measured values on lipid oxidation revealed synergetic interactions for DF and refined olive oil-bound antioxidants, and antagonistic interactions for protein and extra virgin olive oil-bound antioxidants with Trolox. A generalized version of logistic function was successfully used for modelling the oxidation curve of liposomes. Principal component analysis revealed two separate phases of liposome autoxidation.