Anti-plasticization of cassava starch by complexing fatty acids

The effect of adding 1–8% amylose complexing fatty acids (CFA), such as linoleic and oleic acids, on the glass transition temperature (T_g) of cassava starch (CS) with moisture content varying from 5 to 35% (dry basis) was studied. The main relaxation temperature (T_α), associated with the glass transition temperature of the samples (T_g), was determined by dynamic-mechanical-thermal analysis. The plasticizing behavior of water in the blends was evidenced by a decrease of T_α values with moisture content. The effect of CFA on CS was found to be a function of moisture content. At low moisture (<11%) it caused an anti-plasticization effect, while at higher moisture contents it produced plasticization. The anti-plasticizing effect of CFA on CS was attributed to amylose–lipid complex formation.     

Practical Considerations for Determination of Glass Transition Temperature of a Maximally Freeze Concentrated Solution

Glass transition temperature is a unique thermal characteristic of amorphous systems and is associated with changes in physical properties such as heat capacity, viscosity, electrical resistance, and molecular mobility. Glass transition temperature for amorphous solids is referred as (T _g), whereas for maximally freeze concentrated solution, the notation is (T _g′). This article is focused on the factors affecting determination of T _g′ for application to lyophilization process design and frozen storage stability. Also, this review provides a perspective on use of various types of solutes in protein formulation and their effect on T _g′. Although various analytical techniques are used for determination of T _g′ based on the changes in physical properties associated with glass transition, the differential scanning calorimetry (DSC) is the most commonly used technique. In this article, an overview of DSC technique is provided along with brief discussion on the alternate analytical techniques for T _g′ determination. Additionally, challenges associated with T _g′ determination, using DSC for protein formulations, are discussed. The purpose of this review is to provide a practical industry perspective on determination of T _g′ for protein formulations as it relates to design and development of lyophilization process and/or for frozen storage; however, a comprehensive review of glass transition temperature (T _g, T _g′), in general, is outside the scope of this work.     

A broad glass transition in hydrated proteins

We performed Raman and Brillouin scattering measurements to estimate glass transition temperature, T_g, of hydrated protein. The measurements reveal very broad glass transition in hydrated lysozyme with approximate T_g ∼ 180 ± 15 K. This result agrees with a broad range of T_g ∼ 160–200 K reported in literature for hydrated globular proteins and stresses the difference between behavior of hydrated biomolecules and simple glass-forming systems. Moreover, the main structural relaxation of the hydrated protein system that freezes at T_g ∼ 180 K remains unknown. We emphasize the difference between the “dynamic transition”, known as a sharp rise in mean-squared atomic displacement <r²> at temperatures around T_D ∼ 200–230 K, and the glass transition. They have different physical origin and should not be confused.     

The protein glass transition as measured by dielectric spectroscopy and differential scanning calorimetry

The glass transition and its related dynamics of myoglobin in water and in a water–glycerol mixture have been investigated by dielectric spectroscopy and differential scanning calorimetry (DSC). For all samples, the DSC measurements display a glass transition that extends over a large temperature range. Both the temperature of the transition and its broadness decrease rapidly with increasing amount of solvent in the system. The dielectric measurements show several dynamical processes, due to both protein and solvent relaxations, and in the case of pure water as solvent the main protein process (which most likely is due to conformational changes of the protein structure) exhibits a dynamic glass transition (i.e. reaches a relaxation time of 100 s) at about the same temperature as the calorimetric glass transition temperature T_g is found. This glass transition is most likely caused by the dynamic crossover and the associated vanishing of the α-relaxation of the main water relaxation, although it does not contribute to the calorimetric T_g. This is in contrast to myoglobin in water–glycerol, where the main solvent relaxation makes the strongest contribution to the calorimetric glass transition. For all samples it is clear that several proteins processes are involved in the calorimetric glass transition and the broadness of the transition depends on how much these different relaxations are separated in time.     

Refinement of a coarse-grained model of poly(2,6-dimethyl-1,4-phenylene ether) and its application to blends of PPE and PS

A coarse-grained (CG) molecular simulation model has been refined for poly(2,6-dimethyl-1,4-phenylene ether) (PPE). This was successfully validated against atomistic simulation and experimental data. Particularly, the glass transition temperature (T_g) of PPE was studied using both atomistic and CG models and compared favourably to experimental data. In addition, we used the CG model together with an existing Martini CG model of polystyrene (PS) to study the blending behaviour of these two polymers. We solved the problem to mix the different potentials and molecular dynamics of high-molecular-weight blends of PPE/PS was performed in detail.     

Adverse effect of cake collapse on the functional integrity of freeze-dried bull spermatozoa

Under optimal freeze-drying conditions, solutions exhibit a cake-like porous structure. However, if the solution temperature is higher than the glass transition temperature of the maximally freeze-concentrated phase (T_g′) during drying phase, the glassy matrix undergoes viscous flow, resulting in cake collapse. The purpose of the present study was to investigate the effect of cake collapse on the integrity of freeze-dried bull spermatozoa. In a preliminary experiment, factors affecting the T_g′ of conventional EGTA buffer (consisting of Tris–HCl, EGTA and NaCl) were investigated in order to establish the main experimental protocol because EGTA buffer T_g′ was too low (−45.0 °C) to suppress collapse. Modification of the EGTA buffer composition by complete removal of NaCl and addition of trehalose (mEGTA buffer) resulted in an increase of T_g′ up to −27.7 °C. In the main experiment, blastocyst yields after ooplasmic injection of freeze-dried sperm preserved in collapsed cakes (drying temperature: 0 or −15 °C) were significantly lower than those of sperm preserved in non-collapsed cake (drying temperature: −30 °C). In conclusion, freeze-dried cake collapse may be undesirable for maintaining sperm functions to support embryonic development, and can be inhibited by controlling both T_g′ of freeze-drying buffer and temperature during the drying phase.     

Storage of Yarrowia lipolytica lipase after spray-drying in the presence of additives

Lipase from Yarrowia lipolytica is an enzyme that presents numerous potentialities for biotechnological applications. This work describes the development of powders obtained by atomization of supernatants lipase from Y. lipolytica LGx6481. Two formulations were studied: one formulation with skim milk powder and gum arabic, and the other with maltodextrin, calcium chloride and gum arabic. After drying, powders were stored at 4 and 20 °C in aluminium hermetic bags to evaluate their stability over a period of one year. The influence of water activity and glass transition temperature (T_g) on the powder's storage were also studied.     

A novel method to predict the glass transition of 70% glycerol aqueous solution

Vitrification has been used to successfully cryopreserve cells and tissues for over 60 years. Glass transition temperature (T _g) of the vitrification is a critical parameter, which has been investigated experimentally. In this study, an isothermal–isobaric molecular simulation (NPT-MD) is proposed to investigate the glass transition and T _g of such vitrification solution. The cohesive energy density, solubility parameter (δ) and bulk modulus of the solution during the process of the glass transition are investigated as well. The results indicate that these properties as functions of temperature can give a definite inflexion; thus, these properties can be used to predict T _g more accurately than the heat capacity (C _p ), density (ρ), volume (V) and radial distribution function (rdf). At the same time, the predicted values of T _g agree well with the experimental results. Therefore, molecular dynamics simulation is a potential method for investigating the glass transition and T _g of the vitrification solutions.     

Glass transition behavior of octyl β-D-glucoside and octyl β-D-thioglucoside/water binary mixtures

The lyotropic behavior and glass-forming properties of octyl β-d-glucoside (C8Glu) and octyl β-d-thioglucoside (C8SGlu)/water binary mixtures were evaluated using differential scanning calorimetry (DSC) and polarizing optical microscopy (POM). The results clearly indicate that the mixture forms a glass in the supercooling state of liquid crystalline phases such as cubic, lamellar, and smectic. The glass transition temperature (T_g) of the mixture was strongly dependent on solute concentration, with a higher concentration correlating with a higher T_g. The experimental T_g was consistent with the predicted value calculated using the Couchman-Karasz equation in both the C8Glu and C8SGlu/water mixtures. The change of heat capacity at T_g showed the two bending points under variation of concentrations. And the highest temperature of phase transition from lamellar to isotropic solution was observed at around 50% molar concentration. It was expected that non-percolated state of water existed in extremely higher concentration ranges.     

Glassy State and Seed Storage Stability: The WLF Kinetics of Seed Viability Loss at T>Tgand the Plasticization Effect of Water on Storage Stability

The relationship between the glassy state in seeds and storage stability was examined, using the glass transition curve and a seed viability database from previous experiments. Storage data for seeds at various water contents were studied by Williams–Landel–Ferry (WLF) kinetics, whereas the glass transition curves of seeds with different storage stability were analysed by the Gordon–Taylor equation in terms of the plasticization effect of water on seed storage stability. It was found that the critical temperatures (T_c) for long-term storage of three orthodox seeds were near or below their glass transition temperatures (T_g), indicating the requirement for the presence of the glassy state for long-term seed storage. The rate of seed viability loss was a function of T-T_gat T>T_g, which fitted the WLF equation well, suggesting that storage stability was associated with the glass transition, and that the effect of water content on seed storage was correlated with the plasticization effect of water on intracellular glasses. A preliminary examination suggested a possible link between the glass transition curve and seed storage stability. According to the determined WLF constants, intracellular glasses in seeds fell into the second class of amorphous systems as defined by Slade and Levine (Critical Reviews in Food Science and Nutrition30: 115–360, 1991). These results support the interpretation that the glassy state plays an important role in storage stability and should be a major consideration in optimizing storage conditions.     

Water Content�CWater Activity�CGlass Transition Temperature Relationships of Spray-Dried Boroj�� as Related to Changes in Color and Mechanical Properties

The water content–water activity–glass transition temperature relationships of commercial spray-dried borojó powder, with and without maltodextrin, have been studied as related to changes in color and mechanical properties. The GAB and Gordon and Taylor models were well fitted to the sorption and glass transition data, respectively. The Boltzman equation adequately described the evolution of the mechanical parameter characterized in the samples with the difference between the experimental temperature and the glass transition temperature (T _g) of the sample. The color of the samples showed a sigmoid change with water activity. The changes in the mechanical properties of borojó powder related to collapse development started when the sample moved to the rubbery state and began to be significant at about 10 °C above T _g. The increase in the molecular mobility from this point on also favors browning reactions. Maltodextrin presence slows the caking kinetics but induces color changes to spray-dried borojó powder.     

Interactions between soluble sugars and POPC (1-palmitoyl-2-oleoylphosphatidylcholine) during dehydration: vitrification of sugars alters the phase behavior of the phospholipid

The phase behavior of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) was characterized as a function of hydration in the presence of combinations of sugars representative of sugars found in seed embryos having differing degrees of desiccation tolerance. The tendency of the sugar mixes to vitrify was also monitored as a function of hydration. Using differential scanning calorimetry, it was found that all sugars diminished the increase in the gel-to-fluid phase transition temperature (T_m) of POPC that occurred upon dehydration of the pure lipid. These results are analyzed in terms of the osmotic and volumetric properties of sugars. Also, it was found that in those samples for which the glass transition temperature (T_g) was greater than the (T_m) of POPC, T_m was lowered by approx. 20 C° from the value for the fully hydrated lipid. X-ray diffraction data confirmed that acyl chain freezing was deferred to a lower temperature during cooling of vitrified samples. The significance of these results is discussed in terms of the ability of many organisms to tolerate desiccation.     

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.     

Protein stability in the amorphous carbohydrate matrix: relevance to anhydrobiosis

The formation of intracellular glass is proposed to be relevant to protein stabilization and survival of anhydrobiotic organisms in the dry state. The stability of proteins in the amorphous carbohydrate matrix and its relevance to seed survival have been investigated in the present study. Glucose-6-phosphate dehydrogenase (G6PDH) was preserved in the amorphous glucose/sucrose (1:10, w/w) matrix by freeze-drying. The stability of freeze-dried G6PDH was examined at temperatures above and below the glass transition temperature (T_g). The rate of G6PDH inactivation in the amorphous carbohydrate matrix deviated significantly from the Arrhenius kinetics, and conformed to the Williams-Landel-Ferry (WLF) relationship. The temperature dependence of G6PDH inactivation in two sets of samples with different T_g values was compared. Identical temperature dependence of G6PDH inactivation was observed after temperature normalization by (T?T_g). Seed survival of Vigna radiata Wilczek (mung bean) showed a similar WLF kinetics at storage temperatures T≥T_g. In situ protein stability in mung bean embryonic axes was studied using differential scanning calorimetry (DSC). Thermal stability of seed proteins exhibited a strong dependence on the T_g of intracellular glass. These results indicate an important role of the glassy state in protein stabilization. Our data suggest an association between protein stability in intracellular glass and seed survival during storage.     

Simultaneous Determination of Glass Transition Temperatures of Several Polymers

Aims

A simple and easy optical method is proposed for the determination of glass transition temperature (T_g) of polymers.

Methods & Results

T_g was determined using the technique of microsphere imaging to monitor the variation of the refractive index of polymer microsphere as a function of temperature. It was demonstrated that the method can eliminate most thermal lag and has sensitivity about six fold higher than the conventional method in T_g determination. So the determined T_g is more accurate and varies less with cooling/heating rate than that obtained by conventional methods. The most attractive character of the method is that it can simultaneously determine the T_g of several polymers in a single experiment, so it can greatly save experimental time and heating energy.

Conclusion

The method is not only applicable for polymer microspheres, but also for the materials with arbitrary shapes. Therefore, it is expected to be broadly applied to different fundamental researches and practical applications of polymers.     

Evaluation of the relevance of the glassy state as stability criterion for freeze-dried bacteria by application of the Arrhenius and WLF model

The aim of this work was to describe the temperature dependence of microbial inactivation for several storage conditions and protective systems (lactose, trehalose and dextran) in relation to the physical state of the sample, i.e. the glassy or non-glassy state. The resulting inactivation rates k were described by applying two models, Arrhenius and Williams–Landel–Ferry (WLF), in order to evaluate the relevance of diffusional limitation as a protective mechanism. The application of the Arrhenius model revealed a significant decrease in activation energy E_a for storage conditions close to T_g. This finding is an indication that the protective effect of a surrounding glassy matrix can, at least, partly be ascribed to its inherent restricted diffusion and mobility. The application of the WLF model revealed that the temperature dependence of microbial inactivation above T_g is significantly weaker than predicted by the universal coefficients. Thus, it can be concluded that microbial inactivation is not directly linked with the mechanical relaxation behavior of the surrounding matrix as it was reported for viscosity and crystallization phenomena in case of disaccharide systems.     

Predict the glass transition temperature of glycerol-water binary cryoprotectant by molecular dynamic simulation

Vitrification is proposed to be the best way for the cryopreservation of organs. The glass transition temperature (T_g) of vitrification solutions is a critical parameter of fundamental importance for cryopreservation by vitrification. The instruments that can detect the thermodynamic, mechanical and dielectric changes of a substance may be used to determine the glass transition temperature. T_g is usually measured by using differential scanning calorimetry (DSC). In this study, the T_g of the glycerol-aqueous solution (60%, wt/%) was determined by isothermal-isobaric molecular dynamic simulation (NPT-MD). The software package Discover in Material Studio with the Polymer Consortium Force Field (PCFF) was used for the simulation. The state parameters of heat capacity at constant pressure (C_p), density (ρ), amorphous cell volume (V_cell) and specific volume (V_specific) and radial distribution function (rdf) were obtained by NPT-MD in the temperature range of 90–270 K. These parameters showed a discontinuity at a specific temperature in the plot of state parameter versus temperature. The temperature at the discontinuity is taken as the simulated T_g value for glycerol–water binary solution. The T_g values determined by simulation method were compared with the values in the literatures. The simulation values of T_g (160.06–167.51 K) agree well with the DSC results (163.60–167.10 K) and the DMA results (159.00 K). We drew the conclusion that molecular dynamic simulation (MDS) is a potential method for investigating the glass transition temperature (T_g) of glycerol–water binary cryoprotectants and may be used for other vitrification solutions.     

Effect of Water Content on the Glass Transition Temperature of Calcium Maltobionate and its Application to the Characterization of Non-Arrhenius Viscosity Behavior

To understand the fundamental physical properties of calcium maltobionate (MBCa), its water sorption isotherm, glass transition temperature (T _g), and viscosity (η) were investigated and compared with those of maltobionic acid (MBH) and maltose. Although amorphous maltose crystalized at water activity (a _w) higher than 0.43, MBCa and MBH maintained an amorphous state over the whole a _w range. In addition, MBCa had a higher T _g and greater resistance to water plasticizing than MBH and maltose. These properties of MBCa likely originate from the strong interaction between MBCa and water induced by electrostatic interactions. Moreover, the effects of temperature and water content on η of an aqueous MBCa solution were evaluated, and its behavior was described using a semi-empirical approach based on a combination of T _g extrapolated by the Gordon-Taylor equation and a non-Arrhenius formula known as the Vogel–Fulcher–Tammann equation. This result will be useful for understating the effect of MBCa addition on the solution’s properties.     

Dielectric properties of two diastereoisomers of the arabinose and their equimolar mixture

Dielectric relaxation measurements were performed on two enantiomers, d- and l-arabinose and their equimolar mixture, and compared to dielectric data obtained for d-ribose. d-Arabinose differs from d-ribose by having the opposite configuration at C2. This study reveals that both d- and l- of arabinose exhibit α-relaxation peaks with the same shape for the same α-relaxation time τ_α, and the same steepness index for the T_g-scale T-dependence of τ_α. However, the two isomers have slightly different glass transition temperatures T_g’s, and their secondary γ-relaxation times also differ slightly from the previously observed γ-relaxation in d-ribose at the same temperature. However, when samples of both investigated monosaccharides are annealed at higher temperatures, their glass transition temperatures become nearly identical. This is an effect of the mutarotation process, which leads to the formation of pairs of the enantiomers and accordingly they should have the same physical properties. The width of the α-relaxation of d- and l-arabinose is broader than that of d-ribose, as reflected by the smaller stretch exponent in the Kohlrausch-Williams-Watts function used to fit the data of the former (β_KWW = 0.46 ± 0.01) than the latter (β_KWW = 0.55 ± 0.01). The width of the α-relaxation of racemic mixture of the d- and l-arabinose is slightly broader than that of the pure isomers. While the dielectric loss data of d-ribose in the glassy state at ambient and elevated pressures show an inflexion indicating the presence of the JG β-relaxation, the data of d- and l-arabinose show no such feature for identification of the supposedly universal JG β-relaxation. Nevertheless, on comparing the loss spectra of d-arabinose with that of d-ribose, the presence of the JG β-relaxation in d-arabinose has been rationalized.