Effect of nonionic detergents on the activity of a thermostable lipase from Bacillus stearothermophilus MC7

The influence of nonionic surfactants on the activity of a novel thermostable lipase from Bacillus stearothermophilus MC7 was investigated with a view to its potential for synthesis of structured lipids. A large number of modifiers within a broad concentration range were applied. The activity of the enzyme was measured at a relatively high reaction temperature. Highest degree of activation was observed when PEG₆₀₀₀ was applied (up to 2.3-fold increase). Modification essentially changed the performance of the lyophilised preparations—they keep up to 80% of the activity of the native enzyme in the presence of a detergent against 30% in its absence. The effect of sorbitan esters (spans) and polyoxyethylene derivatives of sorbitan esters (tweens) on lipase MC7 was estimated, their HLB value varying within the interval 2.1–16.7. Tweens were strong inhibitors at higher concentrations. For all spans, excepting span 60, an increase of enzyme activity with concentration was observed. All studied additives slow down the process of thermal denaturation. Lipase preparations preserve more than 60% of their activity after 30-min incubation at 75 °C in the presence of tween 60 or PEG₄₀₀₀.     

Freeze-dried egg yolk based extenders containing various antioxidants improve post-thawing quality and incubation resilience of goat spermatozoa

The aim of this study was to evaluate different antioxidants-supplemented freeze-dried egg yolk based extenders for the post-thawing quality and incubation resilience of goat spermatozoa. Pooled semen were diluted in a two-step dilution method to a final concentration of 1/5 (semen/extender) in control and antoxidant supplemented freeze-dried extenders (methionine, cysteamine and butylated hydroxytoluene). Semen samples were assessed for sperm motility, plasma membrane functional integrity using hypoosmotic swelling test (HOST), damaged acrosome using FITC-Pisum sativum agglutinin (PSA-FITC) and DNA integrity using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Membrane lipid peroxidation status was also analyzed using the malondialdehyde (MDA) concentration. In the study, antioxidant supplemented freeze-dried egg yolk based extenders have beneficial effect on goat sperm parameters. In addition, we achieved a higher quality in post thawed goat semen even after 6 h incubation when the extender was supplemented by 5 mM BHT or cysteamine.     

Influence of oligosaccharides on the viability and membrane properties of Lactobacillus reuteri TMW1.106 during freeze-drying

Freeze-drying is a process commonly used in starter culture preparation. To improve the survival rate of bacteria during the process, cryoprotectives are usually added before freezing. This study investigated the influence of the addition of sucrose, fructo-oligosaccharides (FOS), inulin and skim milk on the viability and membrane integrity of Lactobacillus reuteri TMW1.106 during freezing, freeze-drying and storage. The effect of drying adjuncts on survival was correlated to their interaction with bacterial membrane by determination of the parameters membrane fluidity and membrane lateral pressure. Sucrose, FOS and skim milk significantly enhanced survival of exponential-phase cells of L. reuteri during freeze-drying. Cellular viability during storage of exponential-phase cells remained highest for cells dried in the presence of skim milk and inulin. Membranes of these cells were completely permeabilized after freeze-drying. The application of FOS significantly improved survival of stationary phase cells of L. reuteri TMW1.106 after freeze-drying and storage. This increased viability of L. reuteri TMW1.106 in the presence of FOS correlated to improved membrane integrity. Fructo-oligosaccharides and fructans, but not gluco-oligosaccharides interacted with membrane vesicles prepared from L. reuteri TMW1.106 as indicated by increased membrane lateral pressure in the presence of FOS and fructans. Increased membrane integrity of stationary phase L. reuteri TMW1.106 was attributed to direct interactions between FOS and the membrane which leads to increased membrane fluidity and thus improved stability of the membrane during and rehydration.     

Recovery of tubulin functions after freeze-drying in the presence of trehalose

Microtubules represent cytoplasmic structures that are indispensable for the maintenance of cell morphology and motility generation. Due to their regular structural organization, microtubules have become of great interest for preparation of in vitro nanotransport systems. However, tubulin, the major building protein of microtubules, is a thermolabile protein and is usually stored at −80 °C to preserve its conformation and polymerization properties. Here we describe a novel method for freeze-drying of assembly-competent tubulin in the presence of a nonreducing sugar trehalose. Even after prolonged storage at ambient temperature, rehydrated tubulin is capable of binding antimitotic drugs and assembling to microtubules that bind microtubule-associated proteins in the usual way. Electron microscopy confirmed that rehydrated tubulin assembles into normal microtubules that are able to generate motility by interaction with the motor protein kinesin in a cell-free environment. Freeze-drying also preserved preformed microtubules. Rehydrated tubulin and microtubules can be used for preparation of diverse in vitro and in vivo assays as well as for preparation of bionanodevices.     

Mammalian freeze-dried sperm can maintain their calcium oscillation-inducing ability when microinjected into mouse eggs

Mammalian freeze-dried sperm can maintain their genetic integrity and event support full development to term when microinjected into mature oocytes. However, it is unknown whether freeze-dried sperm can still maintain their calcium oscillation-inducing capability. Here, we microinjected mouse and bovine freeze-dried sperm into mouse MII oocytes and examined their calcium oscillation-inducing ability following intracytoplasmic sperm injection (ICSI). Two pieces of information are revealed. First, nearly all oocytes injected with a freeze-dried mouse sperm head or a bovine sperm showed fertilization-like calcium oscillations, indicating that freeze-drying treatment does not affect the activity of the sperm factor responsible for calcium oscillations. Second, freeze-dried sperm exhibited high resistance to external temperature increase. This is shown by the finding that the freeze-dried sperm can maintain their calcium oscillation-inducing capacity even following exposure to 100 degrees C for 3 h. We therefore conclude that mammalian sperm can maintain their calcium oscillation-inducing capability following freeze-drying, rehydration, and ICSI treatments.     

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.     

A new approach for freezing of aqueous solutions under active control of the nucleation temperature

An experimental setup for controlled freezing of aqueous solutions is introduced. The special feature is a mechanism to actively control the nucleation temperature via electrofreezing: an ice nucleus generated at a platinum electrode by the application of an electric high voltage pulse initiates the crystallization of the sample. Using electrofreezing, the nucleation temperature in pure water can be precisely adjusted to a desired value over the whole temperature range between a maximum temperature Tn(max) close to the melting point and the temperature of spontaneous nucleation. However, the presence of additives can inhibit the nucleus formation. The influence of hydroxyethylstarch (HES), glucose, glycerol, additives commonly used in cryobiology, and NaCl on Tn(max) were investigated. While the decrease showed to be moderate for the non-ionic additives, the hindrance of nucleation by ionic NaCl makes the direct application of electrofreezing in solutions with physiological salt concentrations impossible. Therefore, in the multi-sample freezing device presented in this paper, the ice nucleus is produced in a separate volume of pure water inside an electrode cap. This way, the nucleus formation becomes independent of the sample composition. Using electrofreezing rather than conventional seeding methods allows automated freezing of many samples under equal conditions. Experiments performed with model solutions show the reliability and repeatability of this method to start crystallization in the test samples at different specified temperatures. The setup was designed to freeze samples of small volume for basic investigations in the field of cryopreservation and freeze-drying, but the mode of operation might be interesting for many other applications where a controlled nucleation of aqueous solutions is of importance.     

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.     

菌种冷冻干燥保藏的影响因素

菌种资源保藏是微生物学及相关学科研究的基础.冷冻干燥保藏法是菌种保藏最有效的方法之一,为进一步提高菌种保藏质量人们进行了大量的研究.本文介绍了菌种冷冻干燥保藏方法的原理和优点,同时详细介绍了菌种冷冻干燥保藏方法的影响因素.     

Trehalose–Water–Salt Interactions Related to the Stability of β-Galactosidase in Supercooled Media

The conservation of desirable properties in foods and ingredients is often based on the maintenance of the amorphous metastable properties of the systems. Enzymes may be stabilized by drying in saccharide matrices, but a second excipient is generally required to improve sugar protective effects. The effect of electrolytes on the thermophysical properties of sugar systems is of special interest because of their major influence on water structure and their possible interactions with biomolecules. Salts affect the kinetics of very important changes in sugar systems such as crystallization. The purpose of the present work was to analyze fungal β-galactosidase stability in supercooled systems of trehalose-containing electrolytes (water soluble acetates, citrates, and chlorides of magnesium and potassium). The degree of sugar crystallization was also related to enzyme stability. Potassium citrate and acetate improved enzyme stability during freeze-drying and thermal treatment of samples at water activity (a _w) of 0.22. However, trehalose crystallization inhibition at a _w = 0.43 (which was about 50–60%, related to the system without salt) impaired enzyme protection. Certain salts may act retarding sugar crystallization, but in the presence of salts, trehalose crystallization is even more critical because the enzyme is confined in a highly salt-concentrated region. Thus, crystallization inhibition by sugar–salt combinations should be carefully conducted. Santagapita, Research Fellow, CONICET, Argentina. Buera, Member of CONICET, Argentina. An erratum to this article can be found at