Degradation kinetics of C-Phycocyanin under isothermal and dynamic thermal treatments.

C-Phycocyanin (C-PC) represents an alternative to artificial blue/green dyes in food products. This study characterized and gained insights into C-PC thermal stability mechanisms and provided a model to estimate its thermal degradation. Aqueous solutions of C-PC (0.3 µM, pH:6.1) were isothermally heated at 45-80 °C. C-PC degradation was monitored based on the photophysical properties of its lumiphores (phycocyanobilins and aromatic aminoacids-AAs). While C-PC was stable at 45 °C, less than 10 min at 80 °C sufficed to degrade most of it. The thermal degradation curves were characterized using the Weibull model, which was validated with data obtained under non-isothermal conditions. Deviations between estimated and experimental values were lower than 8%. Hypsochromic shifts of the AAs' spectra (from 340 to 315 nm) and increase (>30%) in anisotropy at λexc = 280 and 520 nm suggest that colour losses are not solely associated with alterations of the chromophore but also with conformational changes and possible aggregation of the protein subunits.

Temperature-dependence of riboflavin phosphorescence in cryosolvents.

The molecular mobility of amorphous excipients is important for the stability of biomaterials during preservation, facilitating matrix formulation and product design. Phosphorescence spectroscopy is a sensitive optical method to study molecular mobility. However, there is a need to expand the pool of probes available for analysis since molecules differ in sensitivity. This research explored the feasibility and limitations of using riboflavin as a phosphorescent probe for monitoring matrix molecular mobility. Phosphorescence decays of riboflavin in four amorphous cryosolvents (aqueous solutions of glycerol, ethanol, sucrose, and dextran) were collected at 77 K to capture its natural phosphorescence lifetime (estimated at 170 ms). Decays were also collected during ballistic heating to assess the sensitivity of riboflavin towards changes in matrix molecular mobility. Riboflavin exhibited good sensitivity towards matrix secondary relaxations in the glass, indicating that riboflavin has excellent potential as an edible phosphorescent probe for molecular mobility in food and pharmaceutical products.

Potential applications of luminescent molecular rotors in food science and engineering.

Fluorescent molecular rotors (MRs) are compounds whose emission is modulated by segmental mobility; photoexcitation generates a locally excited (LE), planar state that can relax either by radiative decay (emission of a photon) or by formation of a twisted intramolecular charge transfer (TICT) state that can relax nonradiatively due to internal rotation. If the local environment around the probe allows for rapid internal rotation in the excited state, fast non-radiative decay can either effectively quench the fluorescence or generate a second, red-shifted emission band. Conversely, any environmental restriction to twisting in the excited state due to free volume, crowding or viscosity, slows rotational relaxation and promotes fluorescence emission from the LE state. The environmental sensitivity of MRs has been exploited extensively in biological applications to sense microviscosity in biofluids, the stability and physical state of biomembranes, and conformational changes in macromolecules. The application of MRs in food research, however, has been only marginally explored. In this review, we summarize the main characteristics of fluorescent MRs, their current applications in biological research and their current and potential applications as sensors of physical properties in food science and engineering.

Tryptophan Fluorescence Yields and Lifetimes as a Probe of Conformational Changes in Human Glucokinase.

Five variants of glucokinase (ATP-D-hexose-6-phosphotransferase, EC 2.7.1.1) including wild type and single Trp mutants with the Trp residue at positions 65, 99, 167 and 257 were prepared. The fluorescence of Trp in all locations studied showed intensity changes when glucose bound, indicating that conformational change occurs globally over the entire protein. While the fluorescence quantum yield changes upon glucose binding, the enzyme's absorption spectra, emission spectra and fluorescence lifetimes change very little. These results are consistent with the existence of a dark complex for excited state Trp. Addition of glycerol, L-glucose, sucrose, or trehalose increases the binding affinity of glucose to the enzyme and increases fluorescence intensity. The effect of these osmolytes is thought to shift the protein conformation to a condensed, high affinity form. Based upon these results, we consider the nature of quenching of the Trp excited state. Amide groups are known to quench indole fluorescence and amides of the polypeptide chain make interact with excited state Trp in the relatively unstructured, glucose-free enzyme. Also, removal of water around the aromatic ring by addition of glucose substrate or osmolyte may reduce the quenching.

Solvent-Slaved Dynamic Processes Observed by Tryptophan Phosphorescence of Human Serum Albumin.

Despite extensive experimental and computational efforts to understand the nature of the hierarchy of protein fluctuations and the modulating role of the protein hydration shell, a detailed microscopic description of the dynamics of the protein-solvent system has yet to be achieved. By using single tryptophan protein phosphorescence, we follow site-specific internal protein dynamics over a broad temperature range and demonstrate three independent dynamic processes. Process I is seen at temperatures below the bulk solvent Tg, has low activation energy, and is likely due to fast vibrations that may be enabled by water mobility on the protein surface. Process II is observed above 170 K, with activation energy typical of ß relaxations in a glass; it has the same temperature dependence as fluctuations of hydration shell waters. Process III is observed at T > 200 K; it has super-Arrhenius temperature dependence and closely follows the primary relaxation of the bulk. The fluorescence of pyranine bound to the protein reports on the mobility of water in the hydration shell; it reveals a shift in emission spectra with increasing temperature, indicative of a changing H-bond network at the surface of the protein. These results support a model of solvent-slaved protein dynamics.

Preparation and characterization of zein thermo-modified starch films.

A new method of preparing films from zein thermo-modified starch was presented. According to data of micro-visco-amylography and rheometer, dry heating with zein significantly decreased the pasting properties of waxy corn starch and distarch phosphate, the values of G' and G″ of starches dry heated with zein decreased, while the loss tangent increased as a function of heating time. Scanning electron microscopy images showed that the surfaces of starch granules became rough after dry heating with zein, suggesting interactions between zein and the granule surface. In films made from mixtures, the starch/zein compatibility was improved through dry heating. Compared with films cast from mixtures of separately heat-treated starch and zein, films made from the starch/zein mixtures heated together possessed higher water contact angle and tensile strength. Simple dry heating with zein could thus be used as a promising modification method for improving the functionality of edible films from starches.

Revisiting time-resolved protein phosphorescence.

Analysis of time-resolved phosphorescence data from proteins presents certain problems. Care must be taken in establishing that the analysis is not confounded in the early part of the decay by other emitting species. These species may include tyrosine, impurities found in the solvent, or impurities bound to the protein. In this paper, analysis of the phosphorescence of simple mixtures of tryptophan, tyrosine, and tryptophan + tyrosine in glycerol-water solvent has demonstrated the necessity of accounting for tyrosine emission in the analysis of protein phosphorescence. The tyrosine emission is especially strong at cold temperatures and becomes negligible above approximately 185 K in this solvent. Two fitting procedures have been developed to describe the bimodal emission that results from a single-tryptophan protein that contains a significant number of tyrosine residues. The methods utilize either a maximum entropy method-derived lifetime distribution or the stretched exponential function. In both cases some prior information regarding the expected decay characteristics of the tryptophan residue is applied to guide the separation of the tryptophan component from the tyrosine component. This prior information is obtained by comparing the tail of the protein decay to decays of free-tryptophan in solvent at a variety of temperatures until a match is found having close overlap on a log-intensity decay plot.

Fluorescence quenching study of resveratrol binding to zein and gliadin: Towards a more rational approach to resveratrol encapsulation using water-insoluble proteins.

Several health benefits have been ascribed to consumption of resveratrol, a polyphenol that can be extracted from grape skins. However, its use as a nutraceutical ingredient is compromised by its low water solubility, chemical stability, and bioavailability. Encapsulation of resveratrol in protein nanoparticles can be used to overcome these issues. Fluorescence quenching experiments were used to study the interaction of resveratrol with gliadin and zein. Resveratrol interacted with both proteins, but the binding constant was higher for zein than for gliadin at 35 °C. Furthermore, binding between resveratrol and gliadin increased at higher temperatures, which was not observed for zein. Analysis of the thermodynamic parameters suggested that resveratrol-gliadin binding mainly occurs through hydrophobic interactions while the binding with zein is predominantly mediated through hydrogen bonds. These results help rationalise ingredient selection and production of protein nanoparticles and microparticles for encapsulation, protection and release of resveratrol and potentially other bioactive compounds.

Effects of glycerol on the molecular mobility and hydrogen bond network in starch matrix.

The effects of glycerol on molecular mobility and hydrogen bonding network in an amorphous glassy starch matrix were studied using phosphorescence and IR spectroscopy. Amorphous potato starch films containing varying amounts of glycerol (0, 5, 10, 20 and 30 wt.%) were formulated by rapidly dehydrating aqueous potato starch gel (5%, w/v) with a corresponding content of glycerol; X-ray diffraction data confirm that the films contained negligible content of crystalline starch. Erythrosin B (Ery B) phosphorescence was used to monitor the molecular mobility of these matrices over the temperature range from 0 to 100°C. Analysis of Ery B emission peak frequency, band width and intensity decay provided information about thermally-activated modes of molecular mobility in the matrix. Dipolar relaxation around the triplet state of Ery B was enhanced by addition of glycerol and the extent of relaxation increased at low and intermediate but decreased at higher temperature. The glycerol content-dependent onset temperature for this transition was 70°C for pure starch and decreased to 40°C for a matrix with 30% glycerol. Measurements of the rate of non-radiative decay from the Ery B triplet state indicated that glycerol plasticized the starch matrix above ∼10 wt.% while acting as an antiplastizer to increase the matrix molecular mobility at lower content. These matrix properties were related to glycerol-dependent increases in hydrogen bond strength as measured by IR.

Effect of additives on physicochemical properties in amorphous starch matrices.

The effect of the addition of non-reducing sugars or methylcellulose on the matrix physical properties and rate of non-enzymatic browning (NBR) between exogenous glucose+lysine in a starch-based glassy matrix were studied, using the methods of luminescence and FTIR. Amorphous starch-based matrices were formulated by rapidly dehydrating potato starch gel mixed with additives at weight ratios of 7:93 (additive:starch). Data on the phosphorescence emission energy and lifetime from erythrosin B dispersed in the matrices indicated that sugars decreased starch matrix mobility in a Tg-dependent manner, except for trehalose that interacted with starch in a unique mode, while methylcellulose, the additive with the highest Tg, increased the molecular mobility. Using FTIR, we found that methylcellulose decreased the strength of hydrogen bond network and sugars enhanced the hydrogen bond strength in the order: trehalose>maltitol>sucrose. Comparing those changes with the rate of NBR between exogenous glucose+lysine, we suggest that NBR rates are primarily influenced by matrix mobility, which is modulated by the hydrogen bond network, and interactions among components.

Influence of antioxidant structure on local molecular mobility in amorphous sucrose.

The effect of the antioxidants gallic acid and methyl, propyl, and octyl gallate on the molecular mobility and hydrogen bond network in amorphous sucrose was studied. Solid amorphous sucrose films with and without the addition of antioxidants at a mole ratio of 1:5 (antioxidant/sucrose) were cast from solution onto quartz slides. Local molecular mobility from 0 to 70°C was measured using tryptophan amino acid as a luminescent probe dispersed in the films. Phosphorescence from the tryptophan probe provides spectroscopic characteristics-emission spectrum and lifetime-that are sensitive to changes in molecular mobility induced by the addition of antioxidants. Local molecular mobility detected by tryptophan increased in the following order: sucrosesucrose-gallic acid>sucrose-propyl gallate>sucrose>sucrose-octyl gallate) that was nearly the reverse of that seen in matrix mobility. Analysis of the differential effects of the antioxidants suggests that the presence of the hydroxyl benzoyl head group increased matrix molecular mobility and hydrogen bond strength while the saturated carbon chain decreased mobility and bond strength. The influence of the carboxyl group on matrix properties was comparable to that of the formyloxy group. These results indicate that the addition of specific functional ingredients such as antioxidants may significantly affect the physical properties and consequently functional properties of amorphous edible films in ways that might condition their use. The observed changes are closely related to the chemical structure of the added species.

Influence of glycerol on molecular mobility and hydrogen bond network in amorphous glucose matrix.

The effect of glycerol on molecular mobility and hydrogen bonding in amorphous glucose matrix was studied. Phosphorescence from erythrosin B (Ery B) was used to characterize the temperature dependence of mobility in glucose/glycerol films over the temperature range from 100 °C down to -10 °C. Analysis of emission energy and excited state decay kinetics from Ery B provided information about thermally activated modes of matrix dipolar relaxation around and collisions with the excited triplet state of the probe. Both the average rate of matrix mobility and the width of the distribution of matrix mobility rates largely scaled with the effect of glycerol on the glass transition temperature of the glucose/glycerol mixture. The IR hydrogen bond bandwidth increased at higher glycerol content, suggesting that the strength of the bond became more widely distributed with the added glycerol. An increase with temperature in the hydrogen bond peak frequency indicated the transformation of associated hydroxyl to free hydroxyl. These results support a model in which glycerol plasticizes the glucose matrix at mole ratios of 0.1 and above while providing no evidence for the antiplasticization seen in other sugar matrices.

Antioxidants modulate molecular mobility, oxygen permeability, and microstructure in zein films.

The effect of octyl gallate and propyl gallate on the molecular mobility, oxygen permeability, and microstructure of zein/glycerol films was studied. Films were cast from 70% ethanol/water containing 20% (w/w) glycerol and different amounts of the antioxidants propyl gallate or octyl gallate. The oxygen permeability and local mobility of these films were measured using phosphorescence from the dispersed triplet probe erythrosin B. Although both antioxidants increased the local mobility of the zein matrix to about the same extent, octyl gallate and to a lesser extent propyl gallate dramatically increased the permeability of the film to oxygen. Atomic force microscopy imaging indicated that propyl gallate induced aggregation of zein complexes, which could lead to a more condensed film. These results indicate that the addition of specific functional ingredients, such as antioxidants, to edible films may significantly affect the physical properties and structure and, thus, functional properties in ways that influence their eventual use.

Effect of starch on the molecular mobility of amorphous sucrose.

Molecular mobility in amorphous solid biomaterials is modulated by the composition and environment (primarily temperature). Phosphorescence of the triplet probe erythrosin B was used to generate a mobility map within amorphous sucrose films doped with starch ranging from 0.001 to 0.1 g starch/g sucrose. Data on the emission energy and lifetime of erythrosin B in sucrose and sucrose-starch films over the temperature range from 5 to 100 °C indicates that starch influences the molecular mobility as well as dynamic site heterogeneity of amorphous sucrose in a dose-dependent manner. At a starch/sucrose weight (wt) ratio below 0.005, both emission energy and lifetime decreased, and both the dipolar relaxation rate and nonradiative quenching rate k(TS0) increased, indicating that starch increased the matrix molecular mobility. At a ratio above 0.005, both emission energy and lifetime increased, and the dipolar relaxation rate and nonradiative quenching rate decreased, indicating that starch decreased the matrix mobility both in the glass and in the melt. The mobility showed a minimum value at a ratio of 0.01. The interactions existing in the sucrose-starch matrix are considered as the determining factor to influence the molecular mobility of sucrose-starch mixtures. Changes in the distribution of emission energies (emission bandwidth) and lifetimes indicated that starch increased the spectral heterogeneity at high contents while showing insignificant change or a slight decrease in the heterogeneity at low starch contents. These data illustrate the complex effects of a polymer with mainly linear structure and flexible conformation on the mobility of an amorphous, hydrogen bonded sugar matrix.

Vanillin phosphorescence as a probe of molecular mobility in amorphous sucrose.

Changes in molecular mobility are important in defining the stability and quality of amorphous solid foods, pharmaceuticals, and other solid biomaterials. Predictions of stability must consider matrix mobility below and above T(g) (the glass transition temperature); measurement of molecular mobility in amorphous solids over time scales ranging from <10(-9) s to >10(8) s requires specialized methods. This research investigated how the steady-state and time-resolved emission and intensity of phosphorescence from vanillin (4-hydroxy-3-methoxy benzaldehyde), a common flavor compound, can be used to probe molecular mobility when dispersed within amorphous pure sucrose films. Phosphorescence emission spectra and time-resolved intensity decays, measured in sucrose as a function of temperature in the absence of oxygen, were strongly modulated by matrix molecular mobility. Temperature had a significant effect on vanillin phosphorescence peak frequency and bandwidth, intensity, and lifetime both in the glass and in the melt. Time-resolved phosphorescence intensity decays from vanillin were multiexponential both below and above the glass transition temperature, indicating that the pure (single component) amorphous matrix was dynamically heterogeneous on the molecular level. These data show that vanillin is a promising intrinsic probe of molecular mobility and dynamic heterogeneity in amorphous solid foods and perhaps pharmaceuticals.

Effect of xanthan on the molecular mobility of amorphous sucrose detected by erythrosin B phosphorescence.

Molecular mobility in amorphous solids is modulated by composition and environmental conditions such as temperature. Phosphorescence of erythrosin B was used to generate a mobility map of amorphous sucrose film doped with xanthan gum at weight ratios of xanthan/sucrose ranging from 0.0001 to 0.01. On the basis of analysis of the emission energy and lifetime of erythrosin B in pure sucrose and sucrose-xanthan films over the temperature range from 5 to 100 degrees C, we conclude that xanthan influences the molecular mobility as well as the dynamic site heterogeneity of amorphous sucrose in a dose-dependent fashion. At xanthan/sucrose weight ratios below approximately 0.0005, both emission energy and lifetime decreased and k(TS0) (the nonradiative decay rate of the triplet state) increased, indicating that xanthan increased the matrix molecular mobility. At weight ratios above 0.001, both emission energy and lifetime increased and k(TS0) decreased, indicating that xanthan decreased matrix mobility, reaching a plateau at weight ratios between 0.005 and 0.01. The concentration at which the effect of xanthan switched from increasing to decreasing mobility was similar to the concentration at which polymer chains overlapped in solution, suggesting that the dynamic changeover reflected the onset of chain overlap in the amorphous solid. Systematic trends in the emission bandwidth and lifetime heterogeneity and variations in the emission lifetime vs wavelength indicated that xanthan reduced the matrix dynamic site heterogeneity except at a weight ratio of 0.01. These data illustrate the complex effects of a polymer with a rigid structure and large side chains on the mobility of an amorphous, hydrogen-bonded sugar matrix.

Lactocin 160, a Bacteriocin Produced by Vaginal Lactobacillus rhamnosus, Targets Cytoplasmic Membranes of the Vaginal Pathogen, Gardnerella vaginalis.

Bacterial vaginosis (BV) is a commonly occurring vaginal infection that is associated with a variety of serious risks related to the reproductive health of women. Conventional antibiotic treatment for this condition is frequently ineffective because the antibiotics tend to inhibit healthy vaginal microflora along with the pathogens. Lactocin 160, a bacteriocin produced by healthy vaginal lactobacilli, is a promising alternative to antibiotics; this compound specifically inhibits the BV-associated vaginal pathogens such as Gardnerella vaginalis and Prevotella bivia without affecting the healthy microflora. This study investigates the molecular mechanism of action for lactocin 160 and reveals that this compound targets the cytoplasmic membrane of G. vaginalis, causing the efflux of ATP molecules and dissipation of the proton motive force.

Processing stability of squalene in amaranth and antioxidant potential of amaranth extract.

The processing stability of squalene in amaranth and the antioxidant capacity of the oil-rich fraction of amaranth were studied. The processes investigated were continuous puffing and roasting. Puffing was carried out using a single screw extruder, while roasting was carried out in a convection oven. High-performance liquid chromatography was used to quantify squalene content before and after processing. The L-ORAC method was used to study the antioxidant activity of pure squalene and lipophilic amaranth extract containing squalene. It was found that squalene was stable during all of the processing operations with a maximum loss of 12% during roasting (150 degrees C, 20 min) and no loss during puffing. The L-ORAC test showed pure squalene to be a weak antioxidant, whereas the lipophilic extract of amaranth showed higher antioxidant activity as compared to pure squalene at the same concentration, suggesting that tocotrienols and other minor ingredients also played a role as antioxidants.

Effect of gelatin on molecular mobility in amorphous sucrose detected by erythrosin B phosphorescence.

We have used phosphorescence from the triplet probe erythrosin B (Ery B) to evaluate the effect of gelatin on the molecular mobility of the amorphous sucrose matrix as a function of temperature. Ery B was dispersed in amorphous sucrose and sucrose-gelatin films at ratios of approximately 1:10(4) (probe/sucrose), and delayed emission spectra and emission decay transients were measured over the temperature range from 5 to 100 degrees C. Analysis of spectra using a lognormal function provided the peak energy and bandwidth of the emission. The emission peak frequency decreased at low (0.00022-0.0007) gelatin concentrations and increased at high (above 0.0022) gelatin concentrations, indicating that gelatin increased the extent, and thus the rate, of dipolar relaxation at low gelatin content and decreased the extent at higher gelatin content. Decay transients were well fit to a stretched exponential function at all gelatin contents and temperatures. Analysis of the emission lifetimes provided a measure of the rate of non-radiative decay to the ground state, an indicator of matrix molecular mobility. This rate increased at low (0.00022-0.0022) and decreased at high (>0.0073) gelatin wt ratios. Analysis of the effect of gelatin on the emission bandwidth, the stretching exponent beta, and the variation of lifetime across the emission band indicated that matrix dynamic site heterogeneity increased at low and decreased at high gelatin wt ratios. These results provide a novel insight into the complex dynamic effects of the gelatin polymer on the molecular mobility of the amorphous sucrose matrix.

The effect of salts on molecular mobility in amorphous sucrose monitored by erythrosin B phosphorescence.

Salts are present in most amorphous biomaterials such as dried or frozen solid foods, plant seeds, and bacterial spores, and in some pharmaceutical formulations. However, knowledge of how salts modulate the physical properties of amorphous solid sugars, a major component in these systems, is lacking. We have used phosphorescence of the triplet probe erythrosin B (Ery B) to monitor molecular mobility in amorphous sucrose films (dried against P(2)O(5)) containing the salts NaCl, MgCl(2), CaCl(2), NaAcetate, Na(3)Citrate, NaH(2)PO(4), or Na(2)HPO(4) at a mole ratio of 0.2:1 (salt/sucrose). All the salts examined, except NaH(2)PO(4), significantly increased the phosphorescence lifetime of Ery B over the temperature range from 5 to 100 degrees C. This increase is due to a reduction in the rate of collisional quenching of the triplet state due to interactions with the matrix, indicating that these salts decreased the matrix molecular mobility. NaAcetate, Na(3)Citrate, and Na(2)HPO(4) decreased mobility more than NaCl, CaCl(2), or MgCl(2), perhaps due to specific hydrogen bonding interactions between the anion and sucrose. Systematic variations in the probe emission lifetime across the excitation and emission bands at 25 degrees C indicate that there are sites of different mobilities within amorphous solid sucrose; this dynamic site heterogeneity was enhanced in the presence of the divalent cationic salts MgCl(2) and CaCl(2). These results suggest that salts may play a significant role in modulating the mobility, and thus the long-term stability, of amorphous biological matrixes.