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.     

Kinetics and mechanism of the primary steps of degradation of carotenoids by acid in homogeneous solution

The kinetics of reaction between trifluoroacetic acid as an acid of medium strength and the carotenoids beta-carotene, zeaxanthin, canthaxanthin, and astaxanthin has been examined in detail including the effects of dioxygen, acid concentration, and carotenoid structure. Reaction between acid and carotenoid leads to species absorbing in the red and near-infrared (NIR) spectral regions, intermediates that subsequently disappear. ESR experiments clearly show that these species are not carotenoid radicals, although their NIR absorption is similar to the absorption of carotenoid radical cations. Under most reaction conditions, the disappearance of carotenoids follows pseudo-zero-order kinetics, whereas the reaction order is >1 with respect to acid, and the long-lived (hours) intermediates are suggested to be mono- (700 nm) and diprotonated carotenoid ( approximately 950 nm). Acid induces cis/trans-isomerization via the protonated intermediates, which also decay to nonradical species with shorter conjugated systems-most probably carotenoid esters. Slow protonization of the methine carbon is the primary step in the degradation, but dioxygen increases the rate as a result of formation of a charge-transfer complex with the carotenoids as indicated by a red-shift of the NIR absorption bands. Carotenoids with carbonyl groups (astaxanthin and canthaxanthin) have slower rates of degradation than beta-carotene and zeaxanthin, indicating preferential nondegradative protonation of the carbonyl groups.     

Effect of temperature and glassy states on the molecular mobility of solutes in frozen tuna muscle as studied by electron spin resonance spectroscopy with spin probe detection

The mobility of solutes in frozen food systems (tuna muscle, sarcoplasmic protein fraction of tuna muscle, and carbohydrate-water) has been studied using the temperature dependence of the shape of electron spin resonance (ESR) spectra of the spin probe 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL). The spin probe was incorporated into the tuna meat from an aqueous solution of TEMPOL or by contact with a layer of TEMPOL crystals. The melting/freezing of freeze-concentrated solutes in frozen tuna meat was observed to take place over a range of temperatures from -25 to -10 degrees C. Lower temperatures gave ESR powder spectra due to the decreased mobility of the spin probe, and the temperature dependence of the mobility of the spin probe did not show abrupt changes at the glass transition temperatures of the systems. The mobility of nonglass forming solutes is concluded to be decoupled from the glass forming components. Similar behavior was also observed for TEMPOL in frozen, aqueous carbohydrate systems. The temperature dependence of the mobility of TEMPOL in the frozen systems was analyzed using the Arrhenius equation, and the logarithm of the Arrhenius preexponential factor tau(a) was found to be linearly correlated with the activation energy for all of the tuna and carbohydrate samples, indicating a common molecular mechanism for the observed mobility of TEMPOL in all of the systems. The linear correlation also suggests that the observed mobility of TEMPOL in the frozen aqueous systems is dominated by enthalpy-entropy compensation effects, where the mobility of TEMPOL is thermodynamically strongly coupled to the closest surrounding molecules.     

Kinetics and mechanism of lactosylation of alpha-lactalbumin

The lactosylation of alpha-lactalbumin in aqueous solution was followed at pH(c) = 6.0, 6.3, 7.0, 7.3, and 7.9 and constant ionic strength (I = 0.080) at 50-60 degrees C by reversed-phase high-performance liquid chromatography (RP-HPLC) and electrospray mass spectrometry (MS). The rate of the lactosylation reaction increased with increasing pH and with temperature most significantly at lower pH. The rate of lactosylation could be described by an acid dissociation curve corresponding to pK(a) of the epsilon-amino group of lysine in alpha-lactalbumin. From initial rates for conditions of excess of lactose, pseudo-first-order rate constants were calculated and further transferred into second-order rate constants by dividing with the lactose concentration. Second-order rate constants for protonated and unprotonated lysine in alpha-lactalbumin both showed Arrhenius behavior, and using transition-state theory, DeltaH# = 31 +/- 2 kJ/mol and DeltaS# = -266 +/- 48 J/(mol . K) were determined for the unprotonated form and DeltaH# = 158 +/- 49 kJ/mol and DeltaS# = 80 +/- 150 J/(mol . K) for the protonated form, respectively. On the basis of the marked differences in activation parameters, initial formation of a lactosylamine is suggested as rate-determining for reaction between lactose and a protonated lysine in alpha-lactalbumin, while subsequent water elimination to form a Schiff base becomes rate-determining for the unprotonated form.     

Deactivation of triplet-excited riboflavin by purine derivatives: important role of uric acid in light-induced oxidation of milk sensitized by riboflavin

The reactivity of purine derivatives (uric acid, xanthine, hypoxanthine, and purine) toward triplet-excited riboflavin in aqueous solution at pH 6.4 is described on the basis of kinetic (laser flash photolysis), electrochemical (square-wave voltammetry), and theoretical data (density functional theory, DFT). Direct deactivation of triplet-excited riboflavin in aqueous solution, pH 6.4 at 24 degrees C, in the presence of uric acid, xanthine, and hypoxanthine strongly suggests a direct electron transfer from the purine to the triplet-excited riboflavin with k = 2.9 x 10(9) M(-1) s(-1) (DeltaH(++) = 14.7 kJ mol(-1), DeltaS(++) = -15.6 J mol(-1) K(-1)), 1.2 x 10(9) M(-1) s(-1) (DeltaH(++) = 34.3 kJ mol(-1), DeltaS(++) = +45.3 J mol(-1) K(-1)), and 1.7 x10(8) M(-1) s(-1) (DeltaH(++) = 122 kJ mol(-1), DeltaS(++) = +319 J mol(-1) K(-1)), respectively. From the respective one-electron oxidation potentials collected in aqueous solution at pH 6.4 for uric acid (E = +0.686 vs normal hydrogen electrode, NHE), xanthine (E = +1.106 vs NHE), and hypoxanthine (E = +1.654 vs NHE), the overall free energy changes for electron transfer from the quencher to the triplet-excited riboflavin are as follows: uric acid (DeltaG(o) = -114 kJ mol(-1)), xanthine (DeltaG(o) = -73.5 kJ mol(-1)), hypoxanthine (DeltaG(o) = -20.6 kJ mol(-1)), and purine (DeltaG(o) > 0). The inertness observed for purine toward triplet-excited riboflavin corroborates with its electrochemical inactivity in the potential range from 0 up to 2 V vs NHE. These data are in agreement with the DFT results, which show that the energy of the purine highest occupied molecular orbital (HOMO) (-0.2685 arbitrary unit) is lower than the energy of the semioccupied molecular orbital (SOMO) (-0.2557 a.u.) of triplet-excited riboflavin, indicating an endergonic process for the electron-transfer process. The rate-determining step for deactivation by purine derivatives can be assigned to an electron transfer from the purine derivative to the SOMO orbital of the triplet-excited riboflavin. The results show that uric acid may compete with oxygen and other antioxidants to deactivate triplet-excited riboflavin in milk serum and other biological fluids leading to a free radical process.     

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.     

Mechanism of oxymyoglobin oxidation in the presence of oxidizing lipids in bovine muscle

Lipid oxidation and oxymyoglobin oxidation were measured in bovine muscle homogenates. M. longissimus dorsi homogenates (25% w/w, pH 5.7) were prepared, held at 4 degrees C, and subjected to one of the following treatments: (i) stirred and bubbled with oxygen, (ii) stirred with no oxygen, or (iii) neither stirred nor bubbled with oxygen (control). Lipid oxidation was initiated with 45 microM ferric chloride/sodium ascorbate. Lipid oxidation was highest and oxymyoglobin oxidation lowest in the homogenate bubbled with oxygen while lipid oxidation was lowest and oxymyoglobin oxidation highest in the control homogenate. Dissolved oxygen became depleted over time in the control homogenate, remained high in the homogenate bubbled with oxygen, but decreased and then increased in the homogenate stirred with no oxygen. Free radical formation was lower in the control homogenate than in the stirred homogenates as determined by spin trapping and electron spin resonance detection. The data indicated that lipid oxidation-induced oxygen depletion, as opposed to primary or secondary lipid oxidation products, is a likely cause of oxymyoglobin oxidation in muscle systems.     

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.     

β-carotene radical cation addition to green tea polyphenols. Mechanism of antioxidant antagonism in peroxidizing liposomes

Green tea polyphenols, (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), and (-)-epigallocatechin gallate (EGCG), all showed antioxidative effect in liposomes for lipid oxidation initiated in the lipid phase (antioxidant efficiency EC > EGCG > ECG > EGC) or in the aqueous phase (EC ? EGC > EGCG > ECG) as monitored by the formation of conjugated dienes. For initiation in the lipid phase, β-carotene, itself active as an antioxidant, showed antagonism with the polyphenols (EC > ECG > EGCG > EGC). The Trolox equivalent antioxidant capacity (TEAC EGC > EGCG > ECG > EC) correlates with the lowest phenol O-H bond dissociation enthalpy (BDE) as calculated by density functional theory (DFT). Surface-enhanced Raman spectroscopy (SERS) was used to assess the reducing power of the phenolic hydroxyls in corroboration with DFT calculations. For homogeneous (1:9 v/v methanol/chloroform) solution, the β-carotene radical cation reacted readily with each of the polyphenol monoanions (but not with the neutral polyphenols) with a rate approaching the diffusion limit for EC as studied by laser flash photolysis at 25 °C monitoring the radical cation at 950 nm. The rate constant did not correlate with polyphenol HOMO/LUMO energy gap (DFT calculations), and β-carotene was not regenerated by an electron transfer reaction (monitored at 500 nm). It is suggested that the β-carotene radical cation is rather reacting with the tea polyphenols through addition, as further evidenced by steady-state absorption spectroscopy and liquid chromatography-mass spectroscopy (LC-MS), in effect preventing regeneration of β-carotene as an active lipid phase antioxidant and leading to the observed antagonism.