Selected spin probes for the electron spin resonance study of the dynamics of water and lipids in doughs.

New water-soluble and lipid-soluble spin probes suitable for the ESR investigation of the physical states and interactions of components of dough have been developed. This paper reports some preliminary findings on the suitability of these probes for this type of investigation. Rotational correlation times have been measured for the spin probes in water, dough, oil, and a starch/water mixture. An increase in rotational correlation time of the spin probe corresponds to an increase in microviscosity of the medium. Changes observed in correlation times of the water-soluble spin probes in doughs and in water/starch mixtures clearly correspond to a gelatinization process when the mixtures are heated above 60 degrees C. These irreversible changes, clearly important in the baking process, were monitored by following the change of mobility of a spin probe in doughs over a wide temperature range. The similarity of the results from the two sets of experiments suggests that the phenomenon of the increase of correlation times with temperature in doughs is attributable to the starch component. The lipid-soluble spin probe was found to be located preferentially in the lipid phase of the dough.     

ESR/spin probe study of ice cream

Spin probes based on the 1,1,3,3-tetramethylisoindolin-2-yl structure have been used, in conjunction with electron spin resonance spectroscopy (ESR), to study the physical changes occurring in ice cream during freezing and melting. The ESR measurements allowed the rotational correlation times, tau(B), of the spin probes to be determined. Two probes were used together in a given sample of ice cream, namely, 1,1,3,3-tetramethylisoindolin-2-yl (TMIO), which samples the fat phase, and the sodium salt of 1,1,3,3-tetramethylisoindolin-2-yloxyl-5-sulfonate (NaTMIOS), which samples the aqueous phase. Data from the TMIO probe showed that when ice cream is cooled, the fat phase is a mixture of solid and liquid fat until a temperature of approximately -60 degrees C is reached. The water-soluble probe NaTMIOS showed that the aqueous phase changes completely from liquid to solid within 1 degrees C of -18 degrees C. On cooling further to -24.7 degrees C and then allowing it to warm to +25.0 degrees C, the rotational correlation times of the NaTMIOS were slow to recover to their previous values. For the lipid phase, tau(B)(298) was found to be 65.7 +/- 2.0 ps and the corresponding activation enthalpy, DeltaH, was 32.5 +/- 0.9 kJ mol(-)(1): These values are typical of those expected to be found in the type of fat used to make ice cream. The water phase gave corresponding values of 32.2 +/- 0.5 ps and 24.5 +/- 0.4 kJ mol(-)(1) values, which are those expected for a sucrose concentration of 24%.     

Effect of Moisture Content on Thermomechanical Behavior of Concentrated Wheat Starch-Water Preparations

The rheological behavior of wheat starch preparations at intermediate moisture contents (25–60%, w/w) was studied by dynamic mechanical thermal analysis (DMTA). Differential scanning calorimetry (DSC) and electron spin resonance (ESR) experiments were also performed in parallel. Upon heating wheat starch preparations from 25 to 85°C, DMTA showed first a slight decrease in storage modulus (G′) to 45–60°C, then an increase of the shear modulus (predominant effect of swelling) to 68–74°C, followed by a decrease (predominant effect of melting-softening) to 85°C. In this 25–85°C temperature range, the initial swelling and subsequent softening were less pronounced with decreasing moisture content. The 45% moisture content level appeared critical, since there was a radical change in the thermomechanical behavior below this concentration. DSC showed that gelatinization did not appear as a single endotherm but as two endotherms. Whatever the moisture content, the melting started within a quite narrow temperature range, while the end of melting shifted progressively to higher temperatures as moisture content was decreased. ESR showed first a slight decrease in the water-soluble probe (Tempol) mobility as temperature was increased to 47–50°C, followed by a pronounced decrease to 57–60°C. Then, a progressive increase in probe mobility was observed to 85°C. These changes in probe mobility suggest some modifications of the kinetic and thermodynamic properties of the aqueous phase associated with changes in the starch physical state. For the lowest moisture contents, the probe mobility was quite stable during heating.     

Nonenzymatic browning in food models in the vicinity of the glass transition: effects of fructose,glucose, and xylose as reducing sugar

Effects of a reducing sugar, fructose, glucose, or xylose, and glass transition on the nonenzymatic browning (NEB) rate in maltodextrin (MD), poly(vinylpyrrolidone) (PVP), and water systems were studied. Glass transition temperatures (T(g)) were determined using DSC. Water contents were determined gravimetrically, and NEB rates were followed at several temperatures spectrophotometrically at 280 and 420 nm. Reducing sugar did not affect water contents, but xylose reduced the T(g) of the solid models. Sugars showed decreasing NEB reactivity in the order xylose > fructose > glucose in every matrix material. The NEB reactivity and temperature dependence of the single sugars varied in different matrices. The NEB rates of the solid models increased at temperatures 10-20 degrees C above the T(g), and nonlinearity was observed in Arrhenius plots in the vicinity of T(g). The temperature dependence of nonenzymatic browning could also be modeled using the WLF equation.     

Maillard reaction kinetics in model preservation systems in the vicinity of the glass transition: experiment and theory

Rates of reactant consumption for the Maillard reaction between lysine and glucose were measured for a noncrystallizing trehalose-sucrose-water matrix in the glass transition region. At temperatures above the glass transition temperature (T(g)), the consumption rates showed Arrhenius temperature dependence with activation energies of 135 and 140 kJ mol(-1) for lysine and glucose, respectively. Finite reaction rates were observed for glassy samples that were faster than that of one of the nonglassy samples. A comparison of experimental results with predicted diffusion-controlled reaction rate constants indicated that the reaction was reaction-controlled at temperatures above T(g) and approached the diffusion-influenced regime in the glassy state. The needs for further research on reactant diffusivity, the theory of the orientation dependence of reactivity, and a detailed understanding of the reaction mechanism and kinetics were identified.     

Heat-induced aggregation of Phaseolus vulgaris L. Proteins: an electron spin resonance study

The mechanism of heat-induced aggregation of Phaseolus vulgaris L. proteins and of subunit interactions of importance for susceptibility of proteins to proteolysis was studied by electron spin resonance (ESR) spectroscopy. The mobility of a spin label bound to lysine residues was monitored at two different pH-induced (neutral and alkaline) association states of proteins extracted from raw and cooked common bean. The molecular weight of the protein complexes was assessed by size exclusion-high performance liquid chromatography (SE-HPLC) of labeled proteins. Upon alkaline dissociation, both native and denatured protein subunits underwent a reassociation process to form soluble complexes of molecular weight higher than the species originally present at neutral pH. However, unlike native proteins, impaired mobility of the spin label was observed in the aggregates that are formed after dissociation of subunits of denatured proteins, indicating a reduced accessibility of lysine residues. Trapping of lysine residues inside protein aggregates may explain limited digestibility in the small intestine of proteins in cooked legumes.     

5,5-Dimethyl-2-pyrrolidone-N-oxyl formation in electron spin resonance studies of electrolyzed NaCl solution using 5,5-dimethyl-1-pyrroline-N-oxide as a spin trapping agent

Electrolyzed oxidizing (EO) water has recently generated much interest as a disinfectant in the food industry. 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) is a spin trapping agent widely used in the electron spin resonance (ESR) characterization of oxygen-centered free radicals. The reaction between electrolyzed water, collected from the anode side of a two-chamber electrolyzer, and DMPO was investigated by ESR spectroscopy. Addition of DMPO to EO water generated an ESR spectrum identical to that of 5,5-dimethyl-2-pyrrolidone-N-oxyl (DMPOX), suggesting that a compound from EO water oxidized DMPO with the formation of DMPOX. To further investigate the electrolytically generated compound that oxidized DMPO, aqueous solutions of different sodium salts (sodium chloride, sodium citrate, and sodium iodide) with similar conductivities were electrolyzed. The DMPOX signal was not detected in the electrolyzed sodium citrate sample, suggesting that DMPOX formation in the electrolyzed NaCl sample might be due to an electrolytically generated chlorine species. A low DMPOX signal was also observed from the electrolyzed NaI sample, suggesting that a similar species obtained through the electrolysis of I- can also oxidize DMPO. Hypochlorous acid is proposed to oxidize the spin trap DMPO with the formation of DMPOX. In a neutral pH environment, electrolyzed water also oxidized DMPO to DMPOX. This is consistent with the DMPOX formation in the reaction of chlorine water (containing HOCl and Cl2) or sodium hypochlorite with DMPO.     

Structure and dynamics of high molecular weight glutenin subunits of durum wheat (Triticum durum) in water and alcohol solutions studied by electron paramagnetic resonance and circular dichroism spectroscopies

Complementary information on the structure and dynamics of high molecular weight glutenin subunits (HMW-GS) of durum wheat (Triticum durum) was obtained by means of two spectroscopic techniques. Electron paramagnetic resonance was used to investigate the dynamics of the HMW-GS hydrated with two 2-propanol/water mixtures at temperatures between 268 and 308 K by specific spin labeling of their cysteine residues. Spectra were of a composite type, resulting from two populations of spin labels differing in molecular mobility, both undergoing isotropic rotational diffusion. Diffusional coefficients and populations of the fast- and slow-moving spin labels, determined by an accurate spectral line shape analysis, are discussed as a function of temperature and water content in the solvent systems. Far-UV circular dichroism was employed to provide information on the secondary structure of the HMW-GS in three different solvents [aqueous 50% (v/v) 2-propanol, aqueous 0.1% (v/v) trifluoroacetic acid, and trifluoroethanol]. For the first one, the influence of temperature on HMW-GS structure was also investigated.     

Electron spin resonance estimation of hydroxyl radical scavenging capacity for lipophilic antioxidants

Hydroxyl radical scavenging capacity estimation for lipophilic antioxidants is a challenge due to their poor solubility in aqueous radical generating and measuring systems. In this study, an electron spin resonance (ESR) method was developed and validated for its application in estimating the relative hydroxyl radical (HO*) scavenging capacity for lipophilic antioxidants under physiological pH using a Fenton Fe2+/H2O2 system for radical generation and acetonitrile as a solvent. The Fenton Fe2+/H2O2 system generates a constant flux of pure HO* under the assay conditions. The method was validated by linearity, precision, and reproducibility using selected known lipophilic antioxidants including alpha-tocopherol, lutein, beta-carotene, and BHT. The potential effects of commonly used water-miscible and water-immiscible organic solvents on the Fenton Fe2+/H2O2 HO* generating system as well as their possible interactions with the fluorescent and spectroscopic probes were also reported. In addition, the limitation of the ESR assay was described.     

Modeling bleaching of tomato derivatives at subzero temperatures

This work was addressed to obtain a predictive model of the rate of bleaching in tomato derivatives at subzero temperatures. To this aim, a tomato puree was freeze-dried and equilibrated at increasing solid fractions. The bleaching rate was assessed by measuring tomato color during storage for up to 18 months at temperatures from -30 to 0 degrees C. The temperature dependence of the tomato-bleaching rate was neither predictable using the Arrhenius equation nor simply related to tomato physical state. The lack of a clear Arrhenius relation was attributed to the occurrence of temperature-dependent phenomena, such as ice crystallization and oxygen solubility modifications, which strongly changed the local concentration of reactants. A modified Arrhenius equation predicting the tomato-bleaching rate in the entire temperature range was proposed. Tomato concentration, and hence its physical state, affected the temperature dependence of bleaching, modifying apparent activation energy and frequency factor of the modified Arrhenius equation. In light of these considerations, a mathematical model was set up and validated to accurately predict the tomato-bleaching rate on the basis of only its concentration and storage temperature.     

Comparison of three methods based on electron spin resonance spectrometry for evaluation of oxidative stability of processed cheese.

Electron spin resonance (ESR) spectrometry has been adapted to accelerated (light and temperature) storage experiments with processed cheese comparing the spin trapping technique with 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) as spin trap added to the cheese and the spin labeling technique with 2,2,6, 6-tetramethylpiperidine-1-oxyl radical (TEMPO) as spin label added to the cheese. Both methods showed that light was the more important factor compared to temperature for early stages (up to 11 days) in the formation of radicals in processed cheese. For the spin labeling technique other, unidentified reactions interfered during the early stages of the accelerated storage, indicating that longer reaction times are required for the evaluation, and the spin trapping technique is recommended. As a third method, direct measurement of free radicals formed in processed cheese stored for 15 months and subsequently freeze-dried showed that for longer storage, temperature is more important for the steady-state concentration of radicals than light exposure. In agreement with this result, formation of secondary lipid oxidation products determined as thiobarbituric acid reactive substances was found to correlate with the direct ESR measurement.     

The question of high- or low-temperature glass transition in frozen fish. Construction of the supplemented state diagram for tuna muscle by differential scanning calorimetry

The thermal behavior of fresh tuna muscle, rehydrated freeze-dried tuna muscle, and tuna sarcoplasmic protein fraction was studied by three types of differential scanning calorimetry (DSC): conventional DSC, alternating DSC, and sensitive micro-DSC. The relationship between glass transition temperature, T(g), and water content was established. Only a low-temperature glass transition was detected for fresh tuna and freeze-dried tuna rehydrated to high water contents, whereas for sarcoplasmic protein fraction both a low-temperature and an apparent high-temperature glass transition were detected for samples of high water content. Construction of the supplemented state diagrams for whole tuna muscle and for tuna sarcoplasmic protein fraction confirmed the low-temperature transition to be glass transition of the maximally freeze-dehydrated phase. The apparent upper transition of sarcoplasmic protein fraction was shown not to be a glass transition but rather to originate from the onset of melting of ice, and the temperature of this event should be denoted T(m)'. The glass transition temperature and the concentration of the maximally freeze dehydrated tuna muscle are -74 degrees C and 79% (w/w), respectively.     

Electron spin resonance studies of free radicals in gamma-irradiated soybean paste.

Free radicals in gamma-irradiated soybean paste were investigated by electron spin resonance (ESR) spectroscopy to determine the effect of temperature (77-296 K) and moisture content (1-54%) of samples irradiated at high dose (1-40 kGy). The samples were kept in liquid nitrogen (77 K) during irradiation and subsequent ESR measurements. The spectra shown at 77 K consisted of the hydrogen atom lines at low and high field and complicated symmetric spectrum. By increasing the microwave power, the line shape of ESR spectra altered, which indicated the detection of different paramagnetic centers at different microwave powers. In saturation curves, it was possible to select four types of spectra components which were different in their relaxation times. By the different irradiation doses, the change in free radical concentration showed a curvilinearly increasing relationship with irradiation dose in wet samples, whereas a proportional relationship was observed with dried samples. This might indicate that the indirect process of free radical formation was involved with the existence of free water radicals in the wet samples.     

In vivo ESR spin trapping detection of carbon-centered alpha-Farnesene radicals

Lyophilisates of the ascomycetes Penicillium solitum and Aspergillus niger converted alpha-farnesene to 7-hydroxyfarnesene as the major product. The radical mechanism of this bioconversion was proven by electron spin resonance (ESR) and GC-MS using the spin trapping technique. Intermediate carbon-centered radicals of alpha-farnesene were captured using two spin traps, 2-methyl-2-nitrosopropane and alpha-(4-pyridyl-1-oxide)- N- tert-butylnitrone, respectively. The evaluation of the coupling constants and hyperfine couplings of the ESR spectra showed that tertiary carbon radicals were trapped. The radical position at C7 of alpha-farnesene was derived from EI and CI mass spectra of the corresponding MNP spin adduct. The present study demonstrates that the complementary application of ESR and MS spectrometric data allows the detailed evaluation of a radical mechanism of a fungal terpene transformation reaction.     

Temperature effect on lactose crystallization, maillard reactions, and lipid oxidation in whole milk powder

Whole milk powder with an initial water content of 4.4% (w/w) and a water activity of 0.23 stored in hermetically sealed vials for up to 147 days below (37 and 45 degrees C) and above (55 degrees C) the glass transition temperature (T(g) determined to have the value 48 degrees C) showed a strong temperature dependence for quality deterioration corresponding to energies of activation close to 200 kJ/mol for most deteriorative processes. The glass transition was found not to cause any deviation from Arrhenius temperature dependence. Lactose crystallization, which occurred as a gradual process as monitored by isothermal calorimetry, is concluded to liberate bound water (a(w) increase to 0.46) with a modest time delay (approximately 2 days at 55 degrees C) and with concomitant surface browning as evidenced by an increasing Hunter b-value. Browning and formation of bound hydroxymethyl-furfural determined by HPLC seem to be coupled, while formation of another Maillard reaction product, furosine, occurred gradually and was initiated prior to crystallization. Initiation of lipid oxidation, as detected by lipid-derived radicals (high g-value ESR spectra), and progression of lipid oxidation, as detected by headspace GC, seem not to be affected by lactose crystallization and browning, and no indication of browning products acting as antioxidants could be determined.     

Electron spin resonance spin trapping for analysis of lipid oxidation in oils: inhibiting effect of the spin trap alpha-phenyl-N-tert-butylnitrone on lipid oxidation

The electron spin resonance (ESR) spin trapping technique was investigated as an analytical approach to follow lipid oxidation of rapeseed oil, sunflower oil, and fish oil during storage at 40 degrees C. Unlike previous investigations, alpha-phenyl-N-tert-butylnitrone (PBN), used as spin trap, was added to the fresh oils and formation of radicals was monitored during storage. Results were compared with the development in peroxide value (PV) and the thiobarbituric acid index (TBA). Increasing radical development was detected during the initial stages of oxidation, during which no significant changes in PV and TBA were observed. Evidence of spin adduct depletion was found during prolonged storage, suggesting that although spin trapping of radicals may be used to follow early events in lipid oxidation, it is not a suitable parameter for long periods of time. Addition of the spin trap after sequential samplings is recommended for getting an insight of oxidative changes during storage. Further, the influence of the spin trap (PBN) on lipid oxidation was studied in detail by application of PV and TBA and by following the depletion of naturally occurring tocopherol. PBN was found to possess a profound inhibiting effect on lipid oxidation. Such an effect was found to be dependent on the nature of the oil, and it was observed that the lower the oxidative stability, the larger the effect of PBN on lipid oxidation. This effect was interpreted in terms of the capability of PBN to react with peroxyl radicals, which in turn depends on the initial tocopherol content of the oils.     

Oxidative reactions during early stages of beer brewing studied by electron spin resonance and spin trapping

An electron spin resonance (ESR)-based method was used for evaluating the levels of radical formation during mashing and in sweet wort. The method included the addition of 5% (v/v) ethanol together with the spin trap alpha-4-pyridyl(1-oxide)- N- tert-butylnitrone (POBN) to wort, followed by monitoring the rate of formation of POBN spin adducts during aerobic heating of the wort. The presence of ethanol makes the spin trapping method more selective and sensitive for the detection of highly reactive radicals such as hydroxyl and alkoxyl radicals. Samples of wort that were collected during the early stages of the mashing process gave higher rates of spin adduct formation than wort samples collected during the later stages. The lower oxidative stability of the early wort samples was confirmed by measuring the rate of oxygen consumption during heating of the wort. The addition of Fe(II) to the wort samples increased the rate of spin adduct formation, whereas the addition of Fe(II) during the mashing had no effect on the oxidative stability of the wort samples. Analysis of the iron content in the sweet wort samples demonstrated that iron added during the mashing had no effect on the iron level in the wort. The moderate temperatures during the early steps of mashing allow the endogenous malt enzymes to be active. The potential antioxidative effects of different redox-active enzymes during mashing were tested by measuring the rate of spin adduct formation in samples of wort. Surprisingly, a high catalase dosage caused a significant, 20% reduction of the initial rate of radical formation, whereas superoxide dismutase had no effect on the oxidation rates. This suggests that hydrogen peroxide and superoxide are not the only intermediates that play a role in the oxidative reactions occurring during aerobic oxidation of sweet wort.     

Air-liquid partition coefficients of aroma volatiles in frozen sugar solutions

Static headspace gas chromatography was used to measure air-solution partition coefficients of homologous series of methyl ketones and ethyl esters in aqueous sugar solutions. The measurements were performed in a temperature range from 25 to -25 degrees C. A rather unexpected temperature dependence of the partition coefficients was observed at subzero temperatures; that is, partition coefficients were found to increase in the temperature interval from 0 to -10 degrees C. A simple model, based on the freeze concentration effect, is proposed to explain the observed temperature dependence.     

Detection of free radical transfer in lipoxygenase I-B-catalyzed linoleic acid-soybean protein interaction by electron spin resonance spectroscopy (ESR)

Effects of lipoxygenase I-B (LOX)-catalyzed oxidation of linoleic acid on soybean proteins was evaluated by electron spin resonance (ESR) and fluorescence spectroscopy in different model systems in the presence or absence of antioxidants. A strong central singlet signal was detected by ESR spectroscopy and identified as the carbon radical (g value range 2.0041-2.0054). A downfield shoulder attributed to the sulfur radical (g value 2.019-2.028) was also observed. The changes in soybean proteins were accompanied by an increase in fluorescence, indicating the formation of cross-links. Natural antioxidants such as ascorbic acid and alpha-tocopherol as well as synthetic antioxidants butyl hydroxytoluene (BHT) inhibited the development of both the free radical signal and the fluorescence when added to soybean proteins prior to incubation with linoleic acid and lipoxygenase I-B; the central singlet signal attributed to the carbon radical was reduced by 35-65%. This paper clearly indicates direct free radical transfer from oxidizing linoleic acid catalyzed by LOX to soybean proteins.     

Application of electron spin resonance spectroscopy and spin probes to investigate the effect of ingredients on changes in wheat dough during heating

The change in microviscosity of the aqueous and lipid phases of wheat flour dough, during heating and subsequent cooling, has been measured using novel spin probes based on the isoindolin-yloxyl structure. The spin probes, water and/or lipid soluble, were used with combinations of dough ingredients: diacetyl tartaric acid ester of monoglycerides (DATEM), salt, yeast, and sodium ascorbate. The lipid soluble probe showed that DATEM does not produce a homogeneous phase with endogenous lipids but is found in a separate, less mobile phase. Also, the lipids were shown not to be involved in the baking process, although DATEM may be incorporated into the gelled starch matrix. The water soluble probe enabled starch gelatinization to be investigated in detail and showed that gelatinization produces a reduction of dielectric constant. The technique is appropriate for the detailed examination of the behavior of different ingredients during baking and also potentially to examine interactions between ingredients and flour components in dough.