Citrate increases glass transition temperature of vitrified sucrose preparations

The aim of this study was to investigate the effect of sodium citrate on the properties of dried amorphous sucrose glasses. Addition of sodium citrate to a sucrose solution followed by freeze-drying or convective drying resulted in a glass transition temperature (Tg) that was higher than the well-studied sucrose Tg. This result was obtained either at reduced water content of the analysed sample or by removal of water during Modulated DSC analysis. After removal of the remaining water ( < 3.5% w/w), a Tg of approximately 105 degrees C was obtained at a mass ratio of sodium citrate to sucrose of 0.3. FTIR analysis showed a similar increase in Tg as was found with Modulated DSC analysis. The Tg values were derived from breaks in the vibrational frequency vs. temperature plots in the OH stretching and bending regions. Elevated average strength of hydrogen bonding in the sucrose/citrate glass was concluded from the downshift of the OH stretching band of 25 cm(-1) and from the reduced wavenumber temperature coefficient (WTC). The antisymmetric carboxylate stretch of citrate sensed the glass transition of the mixture, from which we conclude that citrate interacts with the sucrose OH via its carboxylate groups.     

Impact of water activity, temperature, and physical state on the storage stability of Lactobacillus paracasei ssp. paracasei freeze-dried in a lactose matrix

The aim of this study was to determine whether the combined effect of water activity and temperature on inactivation rates of freeze-dried microorganisms in a lactose matrix could be explained in terms of the glass transition theory. The stabilized glass transition temperature, Tg, of the freeze-dried products was determined by differential scanning calorimetry at two different temperatures, T (20 and 37 degrees C), and different water activities (0.07-0.48). This information served as a basis for defining conditions of T and water activity, which led to storage of the bacteria in the glassy (T < Tg) and nonglassy (T > Tg) states. The rates of inactivation of the dry microorganisms subjected to different storage conditions were determined by plate counts and could be described by first-order kinetics. Rates were analyzed as a function of water activity, storage temperature, and the difference between Tg and T. Inactivation below Tg was low; however, Tg could not be regarded as an absolute threshold of bacteria stability during storage. When the cells were stored in the nonglassy state (T > Tg), inactivation proceeded faster, however, not as rapid as suggested by the temperature dependence of the viscosity above the glass transition temperature. Furthermore, the first-order rate constant, k, was dependent on the storage temperature per se rather than on the temperature difference between the glass transition temperature and the storage temperature (T - Tg).     

A fundamental approach for the estimation of the mechanical glass transition temperature in gelatin

The paper constitutes an attempt to overcome the empiricism prevalent in the estimation of the glass transition temperature (Tg) of gelatin networks using rheological techniques. In doing so, it presents a study of the viscoelastic properties of a well-characterised gelatin sample covering the structural properties from the rubbery region to the glassy state. The pattern of oscillatory behaviour on shear is given by a master curve produced by shifting data obtained at different temperatures along the logarithmic time scale. Data reduction does not hold for all temperatures thus giving rise to thermorheological complexity. Within the temperature range at which molecular processes are represented by a simple distribution of relaxation times, a fundamental argument is developed to pinpoint the mechanical Tg. This should improve confidence in measured glassy properties over the empirical indicators found in the literature. As a demonstration, the glass transition temperature of gelatin at "zero moisture" obtained using the proposed framework of analysis is contrasted with earlier attempts to identify the mechanical Tg of gelatin solids.     

Effect of water as a diluent on the glass transition behaviour of malto-oligosaccharides, amylose and amylopectin

The glass transition behaviour of amorphous malto-oligomers from dimer to hexamer was investigated as a function of diluent (water) concentration using differential scanning calorimetry. The glass transition temperatures of the pure compounds ranged from 364 K for maltose to 448 K for maltohexaose. At low diluent concentrations the addition of water strongly depressed Tg. From the measurement of Tg and the heat capacity increment, delta Cp, of the transition for the pure compounds it was possible to predict the Tg of the malto-oligomer/water mixtures using a thermodynamic approach developed by Couchman. From the measurements on the malto-oligomers it was possible to obtain, by extrapolation, the high DP limits of delta Cp and Tg, which are appropriate to amylose and amylopectin. The predicted variation of Tg with diluent concentration for these materials was compared with the experimentally observed behaviour.     

Characterization of molecular mobility in seed tissues: an electron paramagnetic resonance spin probe study.

The relationship between molecular mobility (tauR) of the polar spin probe 3-carboxy-proxyl and water content and temperature was established in pea axes by electron paramagnetic resonance (EPR) and saturation transfer EPR. At room temperature, tauR increased during drying from 10(-11) s at 2.0 g water/g dry weight to 10(-4) s in the dry state. At water contents below 0.07 g water/g dry weight, tauR remained constant upon further drying. At the glass transition temperature, tauR was constant at approximately 10(-4) s for all water contents studied. Above Tg, isomobility lines were found that were approximately parallel to the Tg curve. The temperature dependence of tauR at all water contents studied followed Arrhenius behavior, with a break at Tg. Above Tg the activation energy for rotational motion was approximately 25 kJ/mol compared to 10 kJ/mol below Tg. The temperature dependence of tauR could also be described by the WLF equation, using constants deviating considerably from the universal constants. The temperature effect on tauR above Tg was much smaller in pea axes, as found previously for sugar and polymer glasses. Thus, although glasses are present in seeds, the melting of the glass by raising the temperature will cause only a moderate increase in molecular mobility in the cytoplasm as compared to a huge increase in amorphous sugars.     

Stability of dry liposomes in sugar glasses.

Sugars, particularly trehalose and sucrose, are used to stabilize liposomes during hydration (freeze-drying and air-drying). As a result, dry liposomes are trapped in a sugar glass, a supersaturated and thermodynamically unstable solid solution. We investigated the effects of the glassy state on liposome fusion and solute retention in the dry state. Solute leakage from dry liposomes was extremely slow at temperatures below the glass transition temperature (Tg); however, it increased exponentially as temperature increased to near or above the Tg, indicating that the glassy state had to be maintained for dry liposomes to retain trapped solutes. The leakage of solutes from dry liposomes followed the law of first-order kinetics and was correlated linearly with liposome fusion. The kinetics of solute leakage showed an excellent fit with the Arrhenius equation at temperatures both above and below the Tg, with a transitional break near the Tg. The activation energy of solute leakage was 1320 kJ/mol at temperatures above the Tg, but increased to 1991 kJ/mol at temperatures below the Tg. The stabilization effect of sugar glass on dry liposomes may be associated with the elevated energy barrier for liposome fusion and the physical separation of dry liposomes in the glassy state. The half-life of solute retention in dry liposomes may be prolonged by storing dry liposomes at temperatures below the Tg and by increasing the Tg of the dry liposome preparation.     

Glass transition of gluten. 2: The effect of lipids and emulsifiers.

The effects of lipids and emulsifiers on the glass transition temperature (Tg) of gluten have been studied as a function of water content, using differential scanning calorimetry, dynamic mechanical thermal analysis, a three point bend test and pulsed nuclear magnetic resonance. Lipids and emulsifiers were found to have little or no effect on Tg, with the exception of the lower molecular weight caproic and hydroxycaproic acids, which had a plasticizing effect. In the case of hydroxycaproic acid this was found to be in accordance with the Couchman-Karasz equation. The magnitude of the drop in modulus at Tg was increased in the presence of these additives at low water contents. The effect of lipids and emulsifiers on the Tg of gluten and their compatibility with gluten appears to depend both on their hydrophobicity and on their molecular weight.     

Molecular weight effects on the glass transition of gelatin/cosolute mixtures

The structural properties of four gelatin fractions in mixture with sucrose and glucose syrup have been investigated extensively using small deformation dynamic oscillation. The total level of solids was 80%, the number average molecular weight of the protein ranged from 29.2 to 68 kD, and the temperatures were between 60 and -60 degrees C. Remarkably, the nature of the time and temperature dependence on the viscoelastic functions of all samples could be reduced to master curves using horizontal shift factors. The construction of master curves indicates a common mechanism of structure formation, which, in accordance with the synthetic polymer literature, comprises the rubbery zone, glass transition region, and glassy state. Application of Ferry's free-volume formalism and Rouse theory suggests that there is no change in the thermodynamic state of materials during vitrification, with changes in molecular weight simply introducing shifts in the time scale and temperature range of contributions to viscoelasticity. The thermorheological simplicity allowed development of the concept of "rheological" Tg. This was defined as the point between free-volume phenomena of the polymeric backbone occurring in the glass transition region and an energetic barrier to rotation required for local chain rearrangements in the glassy state. Mechanical relaxation and retardation distribution functions were calculated, thus obtaining values for the effective friction coefficient per monomer unit of the protein. It appears that the local friction coefficient is governed by a linear relationship between fractional free volume and the decreasing molecular weight of the protein, which introduces additional voids due to molecular ends.     

Interactions between anesthetics and lipid mixtures. Normal alcohols.

The effects of normal alcohols up to 1-dodecanol on phase transitions in phosphatidylcholines and phosphatidylethanolamines have been studied using chlorophyll a as fluorescent probe. With the phosphatidylcholines, alcohols up to octanol cause a lowering of the transition temperature, and a broadening of the transition, whereas for dipalmitoylphosphatidylethanolamine, only a lowering of the transition is observed. The lowering of the phase transition temperature in dipalmitoylphosphatidylcholine by butanol and hexanol is close to that expected for ideal behavior, but the behavior of the longer chain alcohols becomes less ideal. The effects of these alcohols on mixtures of lipids have been studied, and they illustrate the care necessary if these plots of temperatures of onset and completion of gel phase formation are to be called "phase diagrams". The effect of 1 -octanol on mixtures of lipids is to increase the proportion of lipid present in the lipid-crystalline state. In contrast, 1-decanol causes an increase in the phase transition temperature for dimyristoylphosphatidylcholine, although it lowers the transition temperature for dipalmitoylphosphatidylcholine, and 1 -dodecanol raises the transition temperature for both of these phosphatidylcholines, although it lowers that for dipalmitoylphosphatidylethanolamine. Dodecanol appears to behave in these lipid bilayer membranes as a lipid with a phase transition temperature of ca. 55 degrees C. Anesthesia is discussed as a phenomenon of liquidus extension: alcohols up to 1 -octanol increase the proportion of lipid in the liquidus state and result in anesthesia, whereas the longer alcohols do not, and result in catalepsy.     

Solubility and diffusion of nitrogen in maltodextrin/protein tablets

The gas transport properties of compacted tablets consisting of an amorphous mixture of maltodextrin and sodium caseinate were studied by dissolving nitrogen gas in the tablets and then determining the gas release over time as a function of temperature and water activity. Gas was dissolved in the tablet matrix by heating the tablets under pressure, generally to temperatures above the glass transition temperature of the matrix, holding them at these conditions for a specified time and then rapidly cooling them while maintaining the external pressure. The solubility of nitrogen was found to be largely determined by the free volume of the matrix, which in turn can be influenced to some degree by thermal and pressure treatments during gas loading. At the levels of free volume studied, the dissolved nitrogen is densely packed in the free volume, the packing density being virtually independent of the externally applied pressure. Release of gas from the tablets at temperatures below the glass transition temperature is generally well described by Fickian diffusion. The effective diffusion coefficient of gas release is strongly dependent on the microstructure and porosity of the tablet matrix, and an approximate model describing the relationship between tablet structure and rate of gas release is formulated. The model is in semiquantitative agreement with the rates of gas diffusion obtained for tablets and dense granules. Owing to the structural heterogeneity and variability of the tablets and the history-dependent properties of the tablet matrix, the effective diffusion coefficients of gas release from the tablets showed a relatively large spread. The temperature dependence of diffusional release follows an Arrhenius relation below the glass transition temperature. This allows the prediction of the nitrogen retention in the tablets as function of time, temperature and pressure.     

Glass transition investigations on highly crosslinked epoxy resins by molecular dynamics simulations

Molecular dynamics simulations at the atomistic level were performed to investigate the glass transition of a highly crosslinked thermoset epoxy resin system composed of diglycidyl ether bisphenol A and isophorone diamine. The crosslinked model was first constructed using a cyclic dynamic method, and extended by investigating the effect of conversion degree on the static properties of local structure, internal energy and volume shrinkage. Based on this model, a systematic investigation on volume, energy and dynamic properties against temperature was made, which determined the glass transition temperature (T_g). The T_gs obtained from various volumetric and energy properties agree well with the differential scanning calorimetry experimental data available, yet a dynamic T_g obtained from the diffusion coefficient is relatively higher. Moreover, the investigation on epoxy segmental dynamics confirmed that the glass transition of the highly crosslinked epoxy resin has a strong dependence on the backbone bond torsional kinetics.     

Thermodynamic reversibility of phase transitions. Specific effects of alcohols on phosphatidylcholines

The gel-to-fluid phase transitions of several phosphatidylethanolamines (PE's) and phosphatidylcholines (PC's) have been investigated in the presence of three short-chain alcohols. The effects of the alcohols on the thermodynamic reversibility of these transitions was studied and it was found that the transitions for PC's are not thermodynamically reversible at relatively high alcohol concentrations. The PE transitions are thermodynamically reversible for all alcohol concentrations, and the PE's do not exhibit the biphasic effects of alcohol on the transition temperature previously reported for the PC's (Rowe, E.S. (1983) Biochemistry 22, 3299-3305). The biphasic transition temperature effects and the thermodynamic irreversibility of PC transitions at high alcohol concentrations appear to be correlated with the induction of a fully interdigitated gel phase recently reported in the literature (Simon, S.A. and McIntosh, T.J. (1984) Biochim. Biophys. Acta 773, 169-172). The biological significance of these findings is discussed.     

Calorimetric study of the glass transition of denatured collagen

Temperature dependence of heat capacity of native and denatured collagen samples with different content of bound water (6 divided by 27%) has been studied by DSC method in the temperature range from -50 to 150 degrees C. Heat capacity of denatured samples demonstrates a jump of 0.50 J/g.grad. at temperature Tg, which depends on humidity of the sample. It has been shown that Tg value also depends on the heating rate and thermal history. Annealing at the temperature below Tg produces an additional maximum in the temperature dependence on heat capacity. The magnitude of this maximum, as well as the Tg value increase with the annealing time. It is concluded that these properties of heat capacity reflect glass transition in the denatured collagen.     

Non-bioengineered silk gland fibroin protein: characterization and evaluation of matrices for potential tissue engineering applications

The possibility of using wild non-mulberry silk protein as a biopolymer remains unexplored compared to domesticated mulberry silk protein. One of the main reasons for this was for not having any suitable method of extraction of silk protein fibroin from cocoons and silk glands. In this study non-bioengineered non-mulberry silk gland fibroin protein from tropical tasar silkworm Antheraea mylitta, is regenerated and characterized using 1% (w/v) sodium dodecyl sulfate (SDS). The new technique is important and unique because it uses a mild surfactant for fibroin dissolution and is advantageous over other previous reported techniques using chaotropic salts. Fabricated fibroin films are smooth as confirmed by atomic force microscopy. Circular dichroism spectrometry along with Fourier transformed infrared spectroscopy and X-ray diffraction reveal random coil/alpha-helix conformations in regenerated fibroin which transform to beta-sheets, resulting in crystalline structure and protein insolubility through ethanol treatment. Differential scanning calorimetry shows an increase in glass transition (Tg) temperature and enhanced degradation temperature on alcohol treatment. Enhanced cell attachment and viability of AH927 feline fibroblasts were observed on fibroin matrices. Higher mechanical strength along with controllable water stability of regenerated gland fibroin films make non-mulberry Indian tropical tasar silk gland fibroin protein a promising biomaterial for tissue engineering applications.     

Bone-tissue-engineering material poly(propylene fumarate): correlation between molecular weight, chain dimensions, and physical properties

Poly(propylene fumarate) (PPF) is an important biodegradable and cross-linkable polymer designed for bone-tissue-engineering applications. For the first time we report the extensive characterization of this biomaterial including molecular weight dependences of physical properties such as glass transition temperature Tg, thermal degradation temperature Td, density rho, melt viscosity eta0, hydrodynamic radius RH, and intrinsic viscosity [eta]. The temperature dependence of eta0 changes progressively with molecular weight, whereas it can be unified when the temperature is normalized to Tg. The plateau modulus and entanglement molecular weight Me have been obtained from the rheological master curves. A variety of chain microstructure parameters such as the Mark-Houwink-Sakurada constants K and alpha, characteristic ratio Cinfinity, unperturbed chain dimension r0(2)/M, packing lengthp, Kuhn length b, and tube diameter a have been deduced. Further correlation between the microstructure and macroscopic physical properties has been discussed in light of recent progress in polymer dynamics to supply a better understanding about this unsaturated polyester to advance its biomedical uses. The molecular weight dependence of Tg for six polymer species including PPF has been summarized to support that Me is irrelevant for the finite length effect on the glass transition, whereas surprisingly these polymers can be divided into two groups when their normalized Tg is plotted simply against Mw to indicate the deciding roles of inherent chain properties such as chain fragility, intermolecular cooperativity, and chain end mobility.     

Influence of alcohols, temperature, and region on the mobility of lipids in neuronal membrane

Fluorescence recovery after photobleaching was used to examine lipid diffusibility in different regions of Aplysia neurons. Differences in diffusion of 1-acyl-2-(6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4- yl)]aminohexanoyl)phosphatidylcholine (NBD-C6-PC) in the cell body, axon hillock, and axon were not apparent. Lipid diffusibility during temperature variations and exposure to alcohols was also examined by photobleaching techniques. For these studies, all measurements were made on the cell body. Alcohols were found to be selective in their effects upon the diffusibility of lipid probes. Neither ethanol nor butanol affected the diffusibility of NBD-PC. However, at the same concentrations, both of these alcohols caused a significant increase in the diffusion coefficient (D) for rhodamine-phosphatidylethanolamine (Rho-PE). The diffusion coefficient for NBD-PC in the cell body plasma membrane did not increase with warming, between 4 degrees C and 25 degrees C. The fraction of lipid probe free to diffuse (per cent recovery; %R) however, increased as temperature increased, within this range. The nonconventional relationship between temperature and D was even more pronounced for Rho-PE. As temperature increased, D became smaller for this probe, concurrent with an increase in %R. These results suggest that immobile viscous lipid is recruited into a mobile fraction as temperature increases, resulting in the maintenance of constant diffusibility. The effects of temperature on D and %R, and the selective effects of alcohols on lipid diffusibility suggest that the membrane is heterogeneously organized, on a submicroscopic scale, into domains. The implications of this organization for nerve function and responses of nervous systems to temperature and anesthetics are discussed.     

Glass transitions in soybean seed : relevance to anhydrous biology

We have investigated the mechanism by which anhydrobiotic organisms can survive severe dehydration. The method used was measurement of the rotational diffusion coefficient of a hydrophilic spin probe, inserted in the cytoplasm of soybean (Glycine max L.) axes, as a function of temperature and sample water content. Results indicate the existence of a hydration-dependent glass-like transition at physiological temperatures. No glass transitions have been observed in desiccation-intolerant samples, suggesting that the ability to withstand dehydration is associated with glass formation.     

Dual effect of ethanol and other aliphatic alcohols on the effectiveness of the Ca-pump and Ca-ATPase activity of the sarcoplasmic reticulum of skeletal muscles

The effect of ethanol and other low molecular weight alcohols having an anaesthetic action, on the activity of Ca-ATPase (EC 3.6.1.38) as well as on the Ca2+ uptake and efflux and the functional efficiency of Ca-pump in rabbit skeletal muscle sarcoplasmic reticulum membranes was studied. It was found that some alcohols, especially when taken at low concentrations, specifically stimulate the activity of the Ca-pump and Ca-ATPase. The concentration (C) of the alcohol at which the maximum value of the Ca/ATP ratio is achieved, is well correlated with the value of the partition coefficient (P) for this alcohol in a two-phase water/octanol system. As the concentration of an alcohol rises, it primarily affects the release of Ca2+, but the Ca-pump still functions well and is able to compensate for the Ca2+ leakage up to a certain moment, after which the phospholipid bilayer structure changes crucially, and is beginning the denaturation of Ca-ATPase. Finally the increase in the concentration of either of the alcohols results in a complete loss of the Ca-ATPase activity. The specific effect of alcohols cannot be explained in terms of an unitary mechanism based on fluidity changes in the membrane. It is assumed that at low concentrations certain alcohols (or groups of related alcohols) are able to promote the specific transition of membrane proteins into the active state, whereas at higher concentrations all alcohols provide for the non-functional state of the proteins.     

Temperature dependency of molecular mobility in preserved seeds

Although cryogenic storage is presumed to provide nearly infinite longevity to cells, the actual timescale for changes in viability has not been addressed theoretically or empirically. Molecular mobility within preserved biological materials provides a first approximation of the rate of deteriorative reactions that ultimately affect shelf-life. Here, temperature effects on molecular mobility in partially dried seeds are calculated from heat capacities, measured using differential scanning calorimetry, and models for relaxation of glasses based on configurational entropy. Based on these analyses, glassy behavior in seeds containing 0.07 g H(2)O/g dm followed strict Vogel-Tamman-Fulcher (VTF) behavior at temperatures above and just below the glass transition temperature (Tg) at 28 degrees C. Temperature dependency of relaxation times followed Arrhenius kinetics as temperatures decreased well below Tg. The transition from VTF to Arrhenius kinetics occurred between approximately 5 and -10 degrees C. Overall, relaxation times calculated for seeds containing 0.07 g H(2)O/g dm decreased by approximately eight orders of magnitude when seeds were cooled from 60 to -60 degrees C, comparable to the magnitude of change in aging kinetics reported for seeds and pollen stored at a similar temperature range. The Kauzmann temperature (T(K)), often considered the point at which molecular mobility of glasses is practically nil, was calculated as -42 degrees C. Calculated relaxation times, temperature coefficients lower than expected from VTF kinetics, and T(K) that is 70 degrees C below Tg suggest there is molecular mobility, albeit limited, at cryogenic temperatures.     

Effect of sugars on headgroup mobility in freeze-dried dipalmitoylphosphatidylcholine bilayers: solid-state 31P NMR and FTIR studies.

The effect of the carbohydrates trehalose, glucose, and hydroxyethyl starch (HES) on the motional properties of the phosphate headgroup of freeze-dried dipalmitoylphosphatidylcholine (DPPC) liposomes was studied by means of 31P NMR, Fourier transform infrared spectroscopy (FTIR), and differential scanning calorimetry (DSC). The results show that trehalose, which is a strong glass former (Tg = 115 degreesC), elevates the onset of the lipid headgroup rotations and preserves some rotational mobility of the phosphate headgroups after cooling from the liquid-crystalline state. Glucose (Tg = 30 degreesC), a very effective depressant of the phase transition temperature of freeze-dried DPPC, markedly elevates the initiation of the temperature of headgroup rotations. On the other hand, the monosaccharide does not preserve the headgroup disordering when cooled from the liquid-crystalline state. These effects are consistent with formation of hydrogen bonds between the OH groups of the sugar and the polar headgroups of DPPC. They show, however, that hydrogen bonding is not sufficient for preservation of the dynamic properties of freeze-dried DPPC. HES, although a very good glass former (Tg > 110 degreesC), does not depress the phase transition temperature and affects only slightly the rotational properties of freeze-dried DPPC. This lack of effect of HES is associated with the absence of direct interactions with the lipid phosphates, as evidenced by the FTIR results. These data show that vitrification of the additive is not sufficient to affect the dynamic properties of dried DPPC.