Combined effects of trehalose and cations on the thermal resistance of beta-galactosidase in freeze-dried systems

The purpose of this study was to investigate the combined effects of trehalose and cations on the preservation of beta-galactosidase in freeze-dried systems and their relationship to physical properties. Differential scanning calorimetry was employed to measure the glass transition temperature (T(g)) and the endothermal peak area, related to the amount of crystalline trehalose dihydrate present in the samples. In systems in which the trehalose matrix was humidified to conditions which allowed a high proportion of trehalose to crystallize, the enzyme was rapidly inactivated upon heating at 70 degrees C. In these conditions the addition of CsCl, NaCl and particularly KCl or MgCl(2), improved the enzyme stability with respect to that observed in matrices containing only trehalose. For a given moisture content, addition of salts produced very little change on the glass transition temperature; therefore the protective effect could not be attributed to a higher T(g) value. The crystallization of trehalose dihydrate in the humidified samples was delayed in the trehalose/salt systems (principally in the presence of Mg(2+)) and a parallel improvement of enzyme stability was observed.     

The glass transition temperature of mixtures of trehalose and hydroxyethyl starch

Although mixtures of HES and sugars are used to preserve cells during freezing or drying, little is known about the glass transition of HES, or how mixtures of HES and sugars vitrify. These difficulties may be due to the polydispersity between HES samples or differences in preparation techniques, as well as problems in measuring the glass transition temperature (T(g)) using differential scanning calorimetry (DSC). In this report, we examine the T(g) of mixtures of HES and trehalose sugar with <1% moisture content using DSC measurements. By extrapolating these measurements to pure HES using the Gordon-Taylor and Fox equations, we were able to estimate the T(g) of our HES sample at 44 degrees C. These results were additionally confirmed by using mixtures of glucose-HES which yielded a similar extrapolated T(g) value. Our approach to estimating the glass transition temperature of HES may be useful in other cases where glass transitions are not easily identified.     

Thermal properties of agave fructans (Agave tequilana Weber var. Azul)

Thermal properties of agave (A. tequilana Weber var. Azul) at different water contents were investigated. HP-TLC results showed a complex mixture of mono-, di-, oligo, and polysaccharides in agave fructans samples. The thermal decomposition temperatures were observed below to 200 °C. Modulated-differential scanning calorimetry studies showed a glass transition and a relaxation enthalpy processes in agave fructans. Samples with the highest moieties of monosaccharides showed the lower glass transition temperatures (Tg). The moisture sorption isotherm of agave fructans was determined at 20 °C and fitted to the GAB model. Gordon-Taylor equation was used to fit the Tg experimental data as a function of water content. Agave fructans was found to be an amorphous material. At low water activity (a_w) values (<0.4), agave fructans remained in a powdered amorphous state; and at intermediate a_w (0.4-0.75) collapsed and caked; and at high a_w (>0.75) changed in a highly viscous liquid-like solution.     

Trehalose transporter from African chironomid larvae improves desiccation tolerance of Chinese hamster ovary cells

Dry preservation has been explored as an energy-efficient alternative to cryopreservation, but the high sensitivity of mammalian cells to desiccation stress has been one of the major hurdles in storing cells in the desiccated state. An important strategy to reduce desiccation sensitivity involves use of the disaccharide trehalose. Trehalose is known to improve desiccation tolerance in mammalian cells when present on both sides of the cell membrane. Because trehalose is membrane impermeant the development of desiccation strategies involving this promising sugar is hindered. We explored the potential of using a high-capacity trehalose transporter (TRET1) from the African chironomid Polypedilum vanderplanki[21] to introduce trehalose into the cytoplasm of mammalian cells and thereby increase desiccation tolerance. When Chinese hamster ovary cells (CHO) were stably transfected with TRET1 (CHO-TRET1 cells) and incubated with 0.4M trehalose for 4h at 37°C, a sevenfold increase in trehalose uptake was observed compared to the wild-type CHO cells. Following trehalose loading, desiccation tolerance was investigated by evaporative drying of cells at 14% relative humidity. After desiccation to 2.60g of water per gram dry weight, a 170% increase in viability and a 400% increase in growth (after 7days) was observed for CHO-TRET1 relative to control CHO cells. Our results demonstrate the beneficial effect of intracellular trehalose for imparting tolerance to partial desiccation.     

The Physical and Morphological Characteristics of Mango Powder (<Emphasis Type="Italic">Mangifera indica</Emphasis> L. cv Tommy Atkins) Produced by Foam Mat Drying

The thermal behavior, moisture adsorption properties and structural and morphological characteristics of mango powders were evaluated. The powders were obtained by foam mat drying methodology using albumin (ALB), mixture (EB) of monoglycerides of fatty acids, sorbitan monostearate and polyoxyethylene sorbitan monostearate and a combination of the two (EB-ALB) as foaming agents. The evaluation was done by differential scanning calorimetry (DSC), X-ray diffraction (XRD) and scanning electron microscopy (SEM), and the moisture adsorption isotherms were also determined. The powder with EB had a glass transition temperature (T_g) of ?4.2 °C. The denaturation temperature of pure albumin (82.2 °C) increased in the powders with ALB to 117 °C and in those with EB-ALB to 102 °C, due to the thermal stability provided by the pulp sugars. The moisture sorption isotherm of the EB-powder showed a higher water equilibrium content than the other powders. All the powders were in the amorphous state. The morphology of the powder with EB showed corrugated particles, whereas those with ALB and EB-ALB showed particles with a less porous aspect and more compact surfaces than the powders with EB.     

A Raman Microspectroscopy Study of Water and Trehalose in Spin-Dried Cells

Long-term storage of desiccated nucleated mammalian cells at ambient temperature may be accomplished in a stable glassy state, which can be achieved by removal of water from the biological sample in the presence of glass-forming agents including trehalose. The stability of the glass may be compromised due to a nonuniform distribution of residual water and trehalose within and around the desiccated cells. Thus, quantification of water and trehalose contents at the single-cell level is critical for predicting the glass formation and stability for dry storage. Using Raman microspectroscopy, we estimated the trehalose and residual water contents in the microenvironment of spin-dried cells. Individual cells with or without intracellular trehalose were embedded in a solid thin layer of extracellular trehalose after spin-drying. We found strong evidence suggesting that the residual water was bound at a 2:1 water/trehalose molar ratio in both the extracellular and intracellular milieus. Other than the water associated with trehalose, we did not find any more residual water in the spin-dried sample, intra- or extracellularly. The extracellular trehalose film exhibited characteristics of an amorphous state with a glass transition temperature of ∼22°C. The intracellular milieu also dried to levels suitable for glass formation at room temperature. These findings demonstrate a method for quantification of water and trehalose in desiccated specimens using confocal Raman microspectroscopy. This approach has broad use in desiccation studies to carefully investigate the relationship of water and trehalose content and distribution with the tolerance to drying in mammalian cells.     

Literature review: supplemented phase diagram of the trehalose-water binary mixture

Trehalose is of great interest in many fields, including freeze-drying, cryoprotection, and anhydrobiosis. Although data for the trehalose-water supplemented phase diagram have previously appeared in the literature, the data have been widely scattered and reported in several units. In this study, literature data for the binary trehalose-water system were collected and analyzed. The literature data were found to be reasonably consistent, with substantial agreement on the melting points for water, trehalose, and trehalose dihydrate and the glass transition temperature of water. There was also good agreement for the solubility, freezing, and glass transition curves. However, there was no general agreement on the glass transition temperature of pure trehalose. Additionally, the trehalose-water glass transition curve was modeled using the Gordon-Taylor equation, with a value for k of 5.2. The collected data in this report will be of much use in further studies of the protective abilities of trehalose.     

Sorption of tartrate, citrate, and EDTA onto chitosan and its regeneration applying electrolysis

The equilibrium isotherm data obtained by the sorption of tartrate, citrate, and EDTA onto chitosan were analyzed using Langmuir and Freundlich equations. The process fits best the Langmuir equation. Kinetic investigations showed that the sorption process obeys the pseudo-second-order kinetic equation. Sorption and desorption peculiarities, FTIR investigations, and measurements of molecular weight enable one to hypothesize that sorption proceeds along with the electrostatic interaction between the positively charged -NH3+ groups of chitosan and the negatively charged -COO(-) of carboxylic acids in the formation of amide bonds between the -NH(2) groups of chitosan and the -COOH groups of the carboxylic acid. Electrolysis under galvanostatic conditions in a mixture of chitosan with a 0.1 mol L(-1) Na(2)SO(4) solution enables one to destroy the amide bonds in the cathode compartment of the electrochemical cell and to anodize organics in the anodic compartment. The choosing of relevant conditions of electrolysis enables one to obtain chitosan with properties (deacetylation degree, molecular weight, and sorption ability) similar to those of initial chitosan. After electrolysis the regenerated chitosan possesses the same or even higher ability for sorption of the carboxylic acids as the initial chitosan.     

The glass transition temperatures of sugar mixtures

We measured the glass transition temperatures of mono-, di-, and trisaccharide mixtures using differential scanning calorimeter (DSC) and analyzed these temperatures using the Gordon-Taylor equation. We found that the glass transition temperatures of monosaccharide-monosaccharide and disaccharide-disaccharide mixtures could be described by the conventional Gordon-Taylor equation. However, the glass transition temperatures of monosaccharide-disaccharide and monosaccharide-trisaccharide mixtures deviated from the conventional Gordon-Taylor equation and the amount of deviation in the monosaccharide-trisaccharide mixtures was larger than those in the monosaccharide-disaccharide mixtures. From these results, we conclude that the size and shape of the sugars play an important role in the glass transition temperature of the mixtures.     

Accumulation of trehalose in response to desiccation and salt stress in the terrestrial cyanobacterium Nostoc commune

Changes in photosynthetic activity and trehalose levels in field‐isolated, natural colonies of the terrestrial cyanobacterium Nostoc commune responding to desiccation and salt stress were investigated. As the water content decreased in N. commune colonies during desiccation, photosynthetic O₂‐evolving activity decreased and no activity was detected in desiccated colonies. A high level of O₂ evolution was restored in the colonies as they absorbed atmospheric moisture, indicating that only a small amount of water is required for reactivation of photosynthesis. No detectable trehalose was found in fully hydrated N. commune colonies; however, trehalose accumulation occurred in response to water loss during desiccation and high levels of trehalose were detected in the air‐dried colonies. Moreover, a 0.2 M NaCl treatment also induced trehalose accumulation to a level equivalent to that by desiccation. Photosynthetic O₂ evolution was inhibited by 0.2 M NaCl, indicating that N. commune can tolerate only low levels of salt. These results suggest that cessation of photosynthesis and trehalose accumulation occur in response to both matric water stress (desiccation) and osmotic water stress (high salt concentration), and that while trehalose may be a less effective osmoprotective compound than others, it is important for the extreme tolerance to desiccation observed in terrestrial cyanobacterium.     

Trehalose loading through the mitochondrial permeability transition pore enhances desiccation tolerance in rat liver mitochondria

Trehalose has extensively been used to improve the desiccation tolerance of mammalian cells. To test whether trehalose improves desiccation tolerance of mammalian mitochondria, we introduced trehalose into the matrix of isolated rat liver mitochondria by reversibly permeabilizing the inner membrane using the mitochondrial permeability transition pore (MPTP). Measurement of the trehalose concentration inside mitochondria using high performance liquid chromatography showed that the sugar permeated rapidly into the matrix upon opening the MPTP. The concentration of intra-matrix trehalose reached 0.29 mmol/mg protein (∼190 mM) in 5 min. Mitochondria, with and without trehalose loaded into the matrix, were desiccated in a buffer containing 0.25 M trehalose by diffusive drying. After re-hydration, the inner membrane integrity was assessed by measurement of mitochondrial membrane potential with the fluorescent probe JC-1. The results showed that following drying to similar water contents, the mitochondria loaded with trehalose had significantly higher inner membrane integrity than those without trehalose loading. These findings suggest the presence of trehalose in the mitochondrial matrix affords improved desiccation tolerance to the isolated mitochondria.     

A simple model for estimating Ni availability and leaf Ni accumulation for the Ni-hyperaccumulator Leptoplax emarginata

Aims

The aim of the present study was to predict kinetics of both Ni concentration in soil solution and leaf Ni mass for the Ni-hyperaccumulator Leptoplax emarginata cultivated on a fertilized and Ni-contaminated sandy topsoil.

Methods

The 0-D (independent of space) one-site rate-limited desorption model proposed by Ingwersen et al. (J Environ Qual 35:2055–2065, 2006) was modified. The plant sink term of the model was approximated by the biophysical equation which assumes that the leaf nickel mass is equal to the triple product of the Intact Plant Transpiration Stream Concentration Factor for Ni IPTSCF_Ni (xylem:solution Ni concentration ratio), Ni concentration in solution and the volume of transpired water. The model input variables were the constant mean IPTSCF_Ni value, determined from independent leaf Ni accumulation kinetics, and the exponential law fitting the transpiration rate kinetics. Using the best calibration, the model was validated and a sensitivity analysis was carried out thereafter. Models were formulated as sets of ordinary differential equation systems which were solved using the fourth-order Runge–Kutta method.

Results

The best model calibration was the joint parameter optimization: the two parameters of the Freundlich Ni adsorption isotherm and of the Ni desorption rate coefficient are obtained using the kinetics of Ni concentrations in the soil solutions for the reference unplanted Ni-contaminated topsoils. The model was validated reasonably well for both Ni concentration in soil solution and leaf Ni mass.

Conclusions

The joint parameter optimization of the two parameters of the Freundlich nickel sorption isotherm and of the Ni desorption rate was successful whereas the Freundlich batch Ni sorption isotherm dramatically overestimated Ni sorption. This joint approach is therefore recommended for any plant metal uptake model. The 0-D one-site rate-limited desorption model linked to a biophysical coupled Ni and water uptake model reasonably validated the kinetics of both Ni concentration in solution and leaf Ni mass. This promising simplified model for predicting both metal concentration in solution and leaf metal mass for metal needs further validations in culture chambers and further improvements in order to use it in the field as a one-dimensional model, taking into account soil moisture dynamics.     

Structural Relaxation During Drying and Rehydration of Food Materials��the Water Effect and the Origin of Hysteresis

The state of water in foodstuffs is a guiding principle in food design, and the equilibrium concept of water activity (Aw) is ubiquitous. It is regarded as a primary variable or “hurdle” in preservation technology, and a key variable influencing chemical reaction during storage. However, the amount of water in any system differs as function of water activity depending whether it is determined by water sorption or desorption. Even though this hysteresis behaviour has already been described in the literature, no physical interpretation of its origin has yet been proposed with respect to detailed molecular organisation. This work shows, for two different food powders, gluten and a milk-based product that the hysteresis disappears when either go through their glass transition. A more complete DSC analysis for gluten during different sorption/desorption cycles demonstrates that the hysteresis is dependent on the ageing of the material, which evolves in the glassy state and is induced by structural relaxation.     

Trehalose loading through the mitochondrial permeability transition pore enhances desiccation tolerance in rat liver mitochondria

Trehalose has extensively been used to improve the desiccation tolerance of mammalian cells. To test whether trehalose improves desiccation tolerance of mammalian mitochondria, we introduced trehalose into the matrix of isolated rat liver mitochondria by reversibly permeabilizing the inner membrane using the mitochondrial permeability transition pore (MPTP). Measurement of the trehalose concentration inside mitochondria using high performance liquid chromatography showed that the sugar permeated rapidly into the matrix upon opening the MPTP. The concentration of intra-matrix trehalose reached 0.29 mmol/mg protein (approximately 190 mM) in 5 min. Mitochondria, with and without trehalose loaded into the matrix, were desiccated in a buffer containing 0.25 M trehalose by diffusive drying. After re-hydration, the inner membrane integrity was assessed by measurement of mitochondrial membrane potential with the fluorescent probe JC-1. The results showed that following drying to similar water contents, the mitochondria loaded with trehalose had significantly higher inner membrane integrity than those without trehalose loading. These findings suggest the presence of trehalose in the mitochondrial matrix affords improved desiccation tolerance to the isolated mitochondria.     

An examination of the moisture sorption characteristics of commercial magnesium stearate

The objective of this study was to characterize the moisture sorption of magnesium stearate and the morphological changes, if any, resulting from moisture sorption. Six samples of commercial magnesium stearate USP were examined. Moisture sorption isotherms were obtained at 25°C and 5% to 98% relative humidity (RH) using a moisture balance. Changes in crystal form resulting from moisture sorption were determined by x-ray diffraction. There were differences in the shape of the isotherm, reversibility of moisture uptake, and shape of the hysteresis loop in the isotherms of crystalline and amorphous magnesium stearates. The isotherm of crystalline magnesium stearate was almost parallel to the pressure axis until and RH of ∼80%. The isotherm of the amorphous sample was characterized by continuous uptake of water over the entire range of RH. Exposure of amorphous magnesium stearate to RH greater than 70% resulted in the formation of the trihydrate. The trihydrate was converted into the anhydrous form when heated to a temperature of 100°C to 105°C. The trihydrate could be generated by exposing the anhydrate to RH higher than 70%.     

Continuously Distributed Glass Transition of Maca (Lepidium meyenii Walpers) Powder and Impact on Caking Properties

Food Biophysics - Water sorption isotherm, glass transition temperature (Tg), and caking properties of maca (Lepidium meyenii Walpers) powder were investigated. A broad endothermic shift reflecting...     

Effect of viscoelastic relaxation on moisture transport in foods. Part II: Sorption and drying of soybeans

The general fluid transport equation presented in Part-I of this paper is used for predicting moisture transport and viscoelastic stresses during sorption and drying of soybeans. Predicted drying curves were validated using experimental data obtained from literature (average absolute difference 6-13%). For drying temperatures used in the soybean processing industry (70–93 °C), smooth moisture profiles were obtained, which indicated Fickian (Darcian) transport. As the drying temperature approached the glass transition temperature (25 °C at 10% moisture content), the moisture profiles became sharper, which indicated non-Fickian (non-Darcian) transport. The viscoelastic stress profiles clearly exhibited the role of the force terms during imbibition and drying. Increase in drying temperature tends to decrease the stress relaxation function but reduction in moisture content during drying tends to increase it. The increase in stress due to the reduction in moisture content below 10% was not compensated by an increase in drying temperature. Drying of soybeans below 10% moisture content should be avoided in the industry because this will lead to thicker flakes that reduce the amount of oil recovery. During imbibition of soybeans, a high magnitude of stresses was obtained in the rubbery regions, which may cause critical regions prone to fissuring. The role of glass transition on stress development and critical region development was clearly observed during drying and imbibition of soybeans.Revised version: 5 October 2003     

海藻糖对脂肪酶的保护机理及酶失活动力学

采用自制的磁性固定化酶(MIE),考察了高温下二糖类对酶的保护作用。结果显示:海藻糖对悬浮于水溶液中的MIE没有保护作用;而在高温干燥后,对酶的保护作用效果依次为:海藻糖>乳糖>蔗糖,支持‘玻璃态学说’;此外,采用两步失活动力学模型能够较好的拟合酶的失活过程,并且得到酶的失活速率常数k和半衰期t1/2,加入海藻糖和乳糖之后,MIE的半衰期分别增长了31和23倍。     

Effect of the desiccation tolerance and cryopreservation methods on the viability of Citrus limon L. Burm cv. Eureka seeds

Cryopreservation of the germplasm for long-term periods is of great importance to maintain the genetic resource. Argentina is one of the world's highest lemon producing country. The performance of different cooling/warming rates in the cryopreservation method of Citrus limon L. Burm cv. Eureka seeds and their influence on the interval of optimal moisture content in the desiccation stage were analyzed. Water sorption isotherm was determined and modeled using D'Arcy & Watt equation; it provided important information concerning the amounts of water associated to strong, weak and multimolecular binding sites along the sorption isotherm. Seeds tolerated a wide range of desiccation conditions (0.1<a_w<0.85) showing a high viability (>80%), however desiccation to 0.0526 g H₂O g⁻¹ d.b. (a_w = 0.0901) produced a significant loss of viability. Differential Scanning Calorimetry was used to identify the thermal transitions of lipids and water in the seed; enthalpies were used to calculate the unfrozen water fraction (0.19 g H₂O g⁻¹ d.b. corresponding to a_w = 0.64). Two cooling/warming rates were tested on desiccated seeds (0.11<a_w<0.85): i) 200 °C min⁻¹ (reached with seeds placed inside a closed cryogenic vial); ii) 1000 °C min⁻¹ (reached with aluminum-foiled seeds placed in a perforated cryogenic vial). For both methods, viability was maximum (83.3%) at a_w = 0.64. Lethal ice formation was responsible for the loss of viability at a_w>0.64 corresponding to the unfrozen water fraction. The use of higher cooling/warming rates enables a wider range of desiccation conditions (0.33<a_w<0.76) in cryopreservation procedures. This work contributes to the optimization of cryopreservation methods of economically important germplasm.