Increasing Trehalose Concentration Research Articles

Promotion of Thermal Inactivation Treatment of Apple Polyphenol Oxidase in the Presence of Trehalose.

Trehalose is known to protect enzymes from denaturation. In the present study, we observed promotion of apple polyphenol oxidase (PPO) inactivation in a trehalose solution with thermal treatment. Crude PPO from Fuji apple was mixed with either sucrose or trehalose solutions, then the samples treated at 25 or 65 °C. In the presence of trehalose, PPO activities were markedly decreased upon treatment at 65 °C with increasing trehalose concentration. Furthermore, the reduction in PPO activity in the presence of trehalose was proportional to storage time after thermal treatment and thermal treatment time. Comparing PPO activities between treatment time 0 and 90 min at 65 °C, activities decreased 89 % for trehalose concentration of 0.2 M. These results indicates that trehalose acts not only as inhibitor but as promoter of inactivation of PPO. The Lineweaver-Burk plot indicated that trehalose acts on PPO as a non-competitive inhibitor during the 65 °C treatment. Two mechanisms of PPO inactivation in the presence of trehalose were suggested; one is the suppression of PPO activation cause by a thermal treatment, and another is the conformational change to inactivation form of PPO in conjunction with trehalose and a thermal treatment. Additionally, apple juice including 0.2 or 0.5 M trehalose with 65 °C treatment indicated slow browning than the juice with 0.2 or 0.5 M sucrose or without sugars. This result demonstrates that the preventing of browning with trehalose is a viable industrial food process.

Structural transitions in TPGS micelles induced by trehalose as a model cryoprotectant

Cryoprotectants are included to protect colloidal dispersions from mechanical stress of ice crystals formed at sub-zero temperature. Trehalose (THL) remains a popular choice among sugar-based cryoprotectants. In the present study, we illustrate the effect of THL concentration (0.01–1 M) upon microstructural characteristics of nonionic micelles which were formulated with alpha tocopherol polyethylene glycol 1000 succinate (TPGS). The effect of THL incorporation in drug-loaded micelles was studied by using capsaicin (CAP), a hydrophobic alkaloid. Our interpretations are based on the results of cloud point, dynamic light scattering (DLS), small angle neutron scattering (SANS), nuclear magnetic resonance (NMR) and Fourier-transformed infrared (FTIR) spectroscopic measurements. Cloud point and spectral data reveal that the micelles underwent progressive dehydration at increasing THL concentration. Together with scattering data, it is discernible that THL molecules intercalated in the micelle corona and expelled water from the polar headgroups. In spite of this, micelle size did not change until 1 M THL. Freeze-thawed micelles also displayed identical size distribution. Structural transitions occurred upon solubilization of CAP; micelles changed from sphere to ellipsoids. We suggest that CAP molecules occupied the hydrophobic core and triggered the formation of ellipsoids which were retained in the presence of THL. These observations lead us to a conclusion that, by virtue of its selective action (removal of freezable water) in headgroup region, THL protects the morphology of micelles over a wide concentration range.

Phase behavior of poloxamer 188 in frozen aqueous solutions – Influence of processing conditions and cosolutes

The aim of the study is to investigate the thermal behavior of poloxamer 188 (P188) in binary (P188-water) and ternary (P188-trehalose-water) solutions during freezing and thawing. The thermal behavior of P188 in frozen (binary and ternary) systems was characterized by differential scanning calorimetry (DSC) and low-temperature X-ray powder diffractometry (XPRD) as a complementary technique. The influence of processing conditions (cooling rate, annealing) and a noncrystallizing co-solute (addition of trehalose) on the behavior of P188 was evaluated during freezing as well as thawing. In rapidly cooled (10 °C/min) aqueous binary solutions, P188 (10% w/v) was retained in the amorphous state. At slower cooling rates (0.5–5 °C/min), the extent of crystallization depended on the cooling rate. In ternary P188-trehalose-water systems (P188 4% w/v, trehalose 0–10% w/v), a concentration dependent inhibition of P188 crystallization was observed with increasing trehalose concentration. However, irrespective of trehalose concentration, annealing resulted in P188 crystallization. The presence of trehalose as well as the processing conditions (cooling rate and annealing) influenced the physical state of P188 at different stages of freezing and thawing. As the cooling rate decreased, the extent of P188 crystallization progressively increased. In presence of trehalose (≥4.0% w/v) crystallization of P188 (4.0% w/v) was inhibited and this effect could be reversed by annealing. Depending on the intended application, the physical form of P188 could be modulated, by annealing even in presence of a noncrystallizing solute.

Inhibition of GNNQQNY prion peptide aggregation by trehalose: a mechanistic view.

Deposition of amyloid fibrils is the seminal event in the pathogenesis of numerous neurodegenerative diseases. The formation of this amyloid assembly is the manifestation of a cascade of structural transitions including toxic oligomer formation in the early stages of aggregation. Thus a viable therapeutic strategy involves the use of small molecular ligands to interfere with this assembly. In this perspective, we have explored the kinetics of aggregate formation of the fibril forming GNNQQNY peptide fragment from the yeast prion protein SUP35 using multiple all atom MD simulations with explicit solvent and provided mechanistic insights into the way trehalose, an experimentally known aggregation inhibitor, modulates the aggregation pathway. The results suggest that the assimilation process is impeded by different barriers at smaller and larger oligomeric sizes: the initial one being easily surpassed at higher temperatures and peptide concentrations. The kinetic profile demonstrates that trehalose delays the aggregation process by increasing both these activation barriers, specifically the latter one. It increases the sampling of small-sized aggregates that lack the beta sheet conformation. Analysis reveals that the barrier in the growth of larger stable oligomers causes the formation of multiple stable small oligomers which then fuse together bimolecularly. The PCA of 26 properties was carried out to deconvolute the events within the temporary lag phases, which suggested dynamism in lags involving an increase in interchain contacts and burial of SASA. The predominant growth route is monomer addition, which changes to condensation on account of a large number of depolymerisation events in the presence of trehalose. The favourable interaction of trehalose specifically with the sidechain of the peptide promotes crowding of trehalose molecules in its vicinity - the combination of both these factors imparts the observed behaviour. Furthermore, increasing trehalose concentration leads to faster expulsion of water molecules than interpeptide interactions. These expelled water molecules have larger translational movement, suggesting an entropy factor to favor the assembly process. Different conformations observed under this condition suggest the role of water molecules in guiding the morphology of the aggregates as well. A similar scenario exists on increasing peptide concentration.

Chitosan-platelet-rich plasma implants for tissue repair - in vitro and in vivo characteristics

Event Abstract Back to Event Chitosan-platelet-rich plasma implants for tissue repair - in vitro and in vivo characteristics Gabrielle Depres-Tremblay1, Anik Chevrier2, Nicolas Tran-Khan2, Monica Nelea2 and Michael Buschmann1, 2 1 Polytechnique de Montreal, Biomedical engineering institute, Canada 2 Polytechnique de Montreal, Chemical engineering department, Canada Introduction: Freeze-dried chitosan formulations can be solubilised in platelet-rich plasma (PRP) to produce injectable mixtures that coagulate to form implants that resist platelet-mediated clot retraction and are resident for at least 2 weeks in vivo[1],[2]. The purpose of this study was to 1) investigate the mechanisms by which clot retraction is inhibited in chitosan-PRP hybrid clots, 2) to investigate release of platelet-derived growth factors from chitosan-PRP hybrid clots in culture medium, and 3) to inject freeze-dried chitosan-PRP formulations subcutaneously in rabbits to assess long-term implant retention and biodegradability. Materials and Methods: Freeze-dried chitosan cakes were prepared using 0.56% or 1% w/v chitosan (80% DDA and Mn 40kDa), 1% or 6% w/v trehalose concentration as lyoprotectant and 42.2mM calcium chloride (CaCl2) as clot activator. Cakes were solubilised in PRP obtained from a human donor and dispensed in either 1) Glass tubes for confocal and scanning electron microscopy imaging or 2) 24-well plates to monitor release of PDGF-AB over 7 days. Formulations containing 1% (w/v) chitosan of Mn 40kDa and different DDA (80%, 85%, 90%), 1% (w/v) trehalose and 42.2mM CaCl2 were solubilised with autologous PRP and injected subcutaneously in the back of five NZW rabbits (150µL/implant) to investigate implant retention and biodegradability at 2 weeks; 4 weeks and 6 weeks. Results: Platelet aggregates were smaller in chitosan-PRP hybrid clots compared to PRP only controls and increasing trehalose concentration further decreased the size of platelet aggregates (Fig 1 left). Platelets, cells and fibrin fibers were coated with chitosan in chitosan-PRP hybrid clots, while fibrin fibers were readily visible in PRP only controls (Fig 1 right). PDGF-AB release profiles were similar for the different chitosan-PRP hybrid clots and for PRP controls, but all were higher than whole blood alone (Fig 2 left). In vivo, PRP controls were completely degraded by 2 weeks, while all chitosan-PRP formulations tested (80%, 85% & 90% DDA) were retained up to 6 weeks post-implantation, where they induced cell recruitment of host-derived cells (Fig 2 right). Discussion: Chitosan inhibited platelet aggregation, partially through the physical mechanism of coating the cells and fibrin fibers in the hybrid clots. Increasing trehalose concentration further decreased the size of platelet aggregates through a currently unknown mechanism. Strong platelet aggregation is required for platelet-mediated clot retraction, so this is consistent with our previous results (1). PDGF-AB release was similar in chitosan-PRP hybrid clots and in PRP controls, which is unsurprising considering that PDGF-AB has an isoelectric point of 9.8 and solubilised chitosan-PRP formulations have a pH between 6.8-7.1. Chitosan-PRP hybrids resided up to 6 weeks post-implantation in vivo, while PRP controls were quickly degraded, suggesting that prolonged residency of chitosan itself leads to sustained recruitment of host cells. Conclusion: Chitosan and trehalose decreased the size of platelet aggregates, which is consistent with their inhibition of clot retraction. Chitosan-PRP does not provide sustained release of PDGF-AB but may do so for other growth factors with different charge properties. Prolonged residency of chitosan-PRP in vivo leads to increased biological activity compared to PRP alone. We acknowledge the technical contributions of Geneviève Picard, the funding sources (CIHR, CFI, GRSTB, NSERC) and the assistance of Ortho Regenerative Technologies Inc.

Molecular insights into the role of aqueous trehalose solution on temperature-induced protein denaturation.

To investigate the underlying mechanism by which trehalose acts as a bioprotectant against thermal denaturation of protein in aqueous solution, we carry out classical molecular dynamics simulations at two different temperatures. Though it is widely accepted that trehalose acts as an antidote against such protein structural destabilization and numerous hypotheses have been proposed in regard to its mechanism of stabilization, there is still no definitive generally accepted answer to this question and it remains a subject of active research. In view of this, in this article we report the thermal denaturation process of a 15-residue S-peptide analogue at 360 K temperature and the counteracting ability of trehalose of varying concentrations at that temperature. In order to verify the conformational stability of the peptide at ambient temperature condition, we also carry out a separate simulation of peptide-water binary system at 300 K temperature. The goal is to provide a molecular level understanding of how trehalose protects protein at elevated temperature. The Cα-rmsd calculation shows that in pure water, the peptide is stable at 300 K temperature and its unfolding is observed at 360 K. However, in peptide-water-trehalose ternary system, the value of Cα-rmsd decreases as trehalose concentration is increased. Remarkably, at the highest trehalose concentration considered in this study, the value of Cα-rmsd at 360 K is similar to that of water-peptide binary system at 300 K temperature. Further, the calculations of radius of gyration of Cα-atoms and helical percentage of the peptide residues support the above observations. The total number of hydrogen bonds formed by the peptide with solution species (trehalose and water) remains constant, though the peptide water hydrogen bond decreases and peptide trehalose hydrogen bond increases with increasing trehalose concentration. This finding suggests replacement of water molecules by trehalose molecules and supports water replacement hypothesis. The calculations of preferential interaction parameter show that at the peptide surface, trehalose molecules are slightly more preferred over water and for the most concentrated solutions, a prominent exclusion of water and enrichment of trehalose molecules is observed. Also observed are (i) trehalose-induced second shell collapse of water structure, (ii) the growth of trehalose cluster as concentration is increased, and (iii) trehalose-induced slowing down of the translational motion of both water and trehalose, the effect being more pronounced for the latter. Implications of these results for counteracting mechanism of trehalose are discussed.

Counteraction of trehalose on urea-induced protein unfolding: Thermodynamic and kinetic studies

Urea-induced protein denaturation can be effectively inhibited by trehalose, but the thermodynamic and kinetic behaviors are still unclear. Herein, the counteraction of trehalose on urea-induced unfolding of ferricytochrome c was studied. Thermodynamic parameters for the counteraction of trehalose were derived based on fluorescence spectroscopic data. Then the kinetics was emphatically investigated by stopped-flow fluorescence spectroscopy. Urea-induced unfolding of ferricytochrome c in 8.00mol/L urea solution reveals two observable phases, including fast and slow phases following a burst phase. Trehalose has little influence on the burst phase amplitude. Nevertheless, the observable unfolding pathway is significantly affected by trehalose. At lower trehalose concentrations (<0.20mol/L) in 8.00mol/L urea, the unfolding pathways still keep to show two phases. However, the rate constant and amplitude for the fast phase diminish with increasing trehalose concentration. In contrast, the rate constant for the slow phase shows only a slight change with a significant increase of the amplitude. At higher trehalose concentrations (>0.30mol/L), the unfolding pathway is transformed into a single slow phase. The rate constant and amplitude for the single phase also decrease with increasing trehalose concentration. The studies are expected to help our understanding of trehalose effects on protein stability.

The influence of trehalose on hydrophobic interactions of small nonpolar solute: A molecular dynamics simulation study

Molecular dynamics simulations were carried out to investigate the influences of aqueous trehalose solution on the hydrophobic interactions between neopentane molecules. In this study, we consider six different trehalose concentrations ranging from 0% to 56%. We observe that with increasing trehalose concentration the dispersion of solute neopentane takes place. The neopentane-neopentane association constant value decreases with addition of trehalose. Our preferential interaction calculations suggest that with increasing trehalose concentration neopentane interacts preferentially with water over trehalose. Site-site neopentane-trehalose rdfs indicate that trehalose molecules are expelled out from the neopentane surface. Also observed are (i) trehalose induced second shell collapse of water network (ii) decrease in average number of water-water and water-trehalose hydrogen bonds with increasing trehalose concentration. We also find that addition of trehalose decreases the translational motion of all the solution species. The decrease in diffusion coefficient value is more pronounced for trehalose. We, further, observe that the ratio of the diffusion coefficient values of water and trehalose increases with increasing trehalose concentration.

The effect of complex solvents on the structure and dynamics of protein solutions: The case of Lysozyme in trehalose/water mixtures

We present a Molecular Dynamics simulation study of the effect of trehalose concentration on the structure and dynamics of individual proteins immersed in trehalose/water mixtures. Hen egg-white Lysozyme is used in this study and trehalose concentrations of 0%, 10%, 20%, 30% and 100% by weight are explored. Surprisingly, we have found that changes in trehalose concentration do not change the global structural characteristics of the protein as measured by standard quantities like the mean square deviation, radius of gyration, solvent accessible surface area, inertia tensor and asphericity. Only in the limit of pure trehalose these metrics change significantly. Specifically, we found that the protein is compressed by 2% when immersed in pure trehalose. At the amino acid level there is noticeable rearrangement of the surface residues due to the change in polarity of the surrounding environment with the addition of trehalose. From a dynamic perspective, our computation of the Incoherent Intermediate Scattering Function shows that the protein slows down with increasing trehalose concentration; however, this slowdown is not monotonic. Finally, we also report in-depth results for the hydration layer around the protein including its structure, hydrogen-bonding characteristics and dynamic behavior at different length scales.

Equilibrium and kinetic studies of the counteraction of trehalose on acid-induced protein unfolding

It has been known that trehalose can counteract acid-induced protein denaturation and induce the formation of molten globule (MG) state, but the equilibrium and kinetic behaviors are still unclear. Herein, the counteraction of trehalose on the acid-induced unfolding of ferricytochrome c was studied at pH 2.0. Far-UV circular dichroism, Soret absorption and fluorescence spectra indicate that trehalose inhibits acid-induced protein denaturation and induces the formation of MG state. The kinetics of the counteracting effects of trehalose was investigated by stopped-flow fluorescence spectroscopy. It is found that the unfolding of ferricytochrome c displays three phases (i.e., fast, intermediate and slow phases) in the absence of trehalose. At trehalose <0.8M, the unfolding transition still occurs in three phases, but at trehalose >0.8M, it is transformed into a biphasic process without the fast phase. The rate constants for all the unfolding phases decrease linearly with increasing trehalose concentration. Meanwhile, the increase in the fluorescence intensity for the fast and intermediate phases diminishes with the increment of trehalose concentration. Nevertheless, there is little influence of trehalose on the fluorescence intensity change in the slow phase. The studies have provided new insight into the effects of trehalose on the stability of proteins.

Sugar feedingviatrehalose haemolymph concentration affects sex pheromone production in matedHeliothis virescensmoths

Long-distance, female-produced sex pheromones are widespread among moths. Larval feeding provides most of the nutrients for development of these insects but is not thought to influence the de novo production of the fatty-acid derived compounds used as pheromones by most species. Feeding on plant nectar (sugar) by adult moths is important for increasing female fitness and also for the pollination of many plant species. In this paper, I show that feeding on sucrose solution, as opposed to water, increases sex pheromone titre in mated, but not virgin, female Heliothis virescens. Mating caused a rapid decrease in haemolymph trehalose concentration, which was restored to near-virgin levels by sugar ingestion. When isolated mated female abdomens were cultured with different concentrations of trehalose, pheromone titre increased with increasing trehalose concentration. This effect was not observed when abdomens were cultured on saline containing the sugar rhamnose, which insects cannot metabolise to glucose. Virgins injected with the juvenile hormone (JH) analogue, methoprene, showed the same effects as mated females with respect to pheromone titre and haemolymph trehalose concentration. Thus, following mating increases in JH titre increase demand for, and lowering of, blood sugar to develop oocytes, which can be compensated for by sugar ingestion. Haemolymph trehalose concentration probably influences glycolysis in gland cells and, consequently, levels of cytosolic citrate and acetyl-CoA for pheromone biosynthesis. This increase in pheromone titre in sugar-fed, mated females may facilitate further mating and increased fecundity. Thus, exogenous sugar feeding is behaviourally and physiologically integrated with endogenous JH titre to maximise female fitness.

Trehalose Bioprotective Effects in Lysozyme Aqueous Solution Studied by Brillouin Scattering and Calorimetric Measurements

The bioprotective effect of trehalose in lysozyme aqueous solutions has been investigated by Brillouin scattering and modulated-temperature differential scanning calorimetry (MDSC). MDSC experiments show that the isothermal kinetics of thermally irreversible denaturation can be described by the Arrhenius equation. By the addition of trehalose, the irreversible denaturation of lysozyme is suppressed, and its activation energy is half that of the denaturation without trehalose. The sound velocity of lysozyme–trehalose–water ternary solutions obviously depends on the trehalose concentration. With increasing trehalose concentration, the sound velocity becomes higher because the hydration of trehalose reduces the hydrogen bonds between water molecules. Moreover, hydration around lysozyme molecules increases the sound velocity further. Trehalose molecules tend to aggregate with lysozyme molecules at high trehalose concentrations. The bioprotective effect of trehalose probably originates from the mechanical suppression of conformational fluctuations of lysozyme molecules.

Effect of trehalose on the glass transition and ice crystal growth in ice cream

SummaryThe effects of trehalose and sucrose on the rate of ice crystal growth in ice cream during accelerated shelf‐life were compared. Experimental and theoretical freezing curves were shown to be in good agreement. Glass transition temperatures (Tg) of maximally freeze concentrated trehalose and sucrose solutions (40% w/w) were found to be −39.5 °C and −47 °C respectively. For ice cream mixes, the Tg value increased from −46.4 °C for the 100% sucrose‐based mix to −42.0 °C for the 100% trehalose sweetened ice cream. However, no differences in viscosity, nucleation rate or inhibition of ice crystal growth were observed with increasing trehalose concentration in ice cream.

Concentration−Temperature Dependencies of Structural Relaxation Time in Trehalose−Water Solutions by Brillouin Inelastic UV Scattering

A Brillouin scattering investigation has been carried out on trehalose-water solutions in a wide range of concentrations (0<phi<0.74) in the ultraviolet regime. A complete set of data as a function of temperature (-10 degrees C<or=T<or=100 degrees C) has been obtained for each concentration. The T-phi evolution of the system has been analyzed in terms of energy position and line width of inelastic peaks. These results have been used to derive the structural relaxation time, tau, of the system. This was found to be in the tens of picoseconds time scale, and its T dependence can be described with an activation (Arrhenius) law. Most importantly, a significant slowing down of the relaxation dynamics has been observed as trehalose concentration was increased. At low phi, the activation energy of the relaxation has been found to be consistent with literature data for pure water and comparable with intermolecular hydrogen bond (HB) energy. This evidence strongly supports the hypothesis that the main microscopic mechanism responsible for the relaxation process in trehalose solutions lies in the continuous rearrangement of the HB network. Finally, the results are discussed in terms of the evolution of the system upon increasing trehalose concentration, in order to provide a complete description of the viscoelastic stiffening in real biological conditions.

How Homogeneous Are the Trehalose, Maltose, and Sucrose Water Solutions? An Insight from Molecular Dynamics Simulations

The structural properties resulting from the reciprocal influence between water and three well-known homologous disaccharides, namely, trehalose, maltose, and sucrose, in aqueous solutions have been investigated in the 4-66 wt % concentration range by means of molecular dynamics computer simulations. Hydration numbers clearly show that trehalose binds to a larger number of water molecules than do maltose or sucrose, thus affecting the water structure to a deeper extent. Two-dimensional radial distribution functions of trehalose solutions definitely reveal that water is preferentially localized at the hydration sites found in the trehalose dihydrate crystal, this tendency being enhanced when increasing trehalose concentration. Over a rather wide concentration range (4-49 wt %), the fluctuations of the radius of gyration and of the glycosidic dihedral angles of trehalose indicate a higher flexibility with respect to maltose and sucrose. At sugar concentrations between 33 and 66 wt %, the mean sugar cluster size and the number of sugar-sugar hydrogen bonds formed within sugar clusters reveal that trehalose is able to form larger clusters than sucrose but smaller than maltose. These features suggest that trehalose-water mixtures would be more homogeneous than the two others, thus reducing both desiccation stresses and ice formation.

Infrared spectroscopic studies on interactions of water and carbohydrates with a biological membrane

Infrared spectroscopy was used to investigate the changes in bands assigned to phospholipids and proteins in dehydrated and rehydrated sarcoplasmic reticulum. The changes in CH 2 and CH 3 stretching bands, amide bands, and phosphate stretching bands are similar to shifts in frequency seen for those bands in phospholipid and protein preparations during thermotropic phase transitions and hydration. IR studies on dry trehalose-sarcoplasmic reticulum mixtures show similar results; with increasing trehalose concentration in the dry mixtures, amide and phosphate bands shift to frequencies characteristic of hydrated samples. Changes in bands assigned to OH deformations in the trehalose suggest that the interaction between the carbohydrate and membrane is by means of hydrogen bonding between these OH groups and membrane components.