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).     

Glass transition temperatures of cassava starch-whey protein concentrate systems at low and intermediate water content

Glass transition temperatures of cassava starch (CS)-whey protein concentrate (WPC) blends were determined by means of differential scanning calorimetry (DSC) in a water content range of 8-20% (dry basis, d.b.). Water equilibration in the samples was carried out by storing them at room temperature (25 °C) during four weeks. Physical aging and phase segregation were observed in some samples after this storage period depending on the water content. Both, first DSC heating scans and tan δ curves of CS-WPC blends with intermediate water content (10-18%), showed two endothermic thermal events. The first one appeared at around 60 °C and was independent of water content. The second one was detected at higher temperatures and moved towards the low-temperature peak as the water content increased. The results can be explained by a phase segregation process that can take place when the samples are conditioned below their glass transition temperatures. The Gordon-Taylor equation described well the plasticizing effect of water on the blends. WPC was also found to decrease the glass transition temperature, at constant water content, an effect attributed to additional water produced during browning reactions in the blends.     

Low-temperature glass transition in proteins

The viscoelastic properties of solid samples (crystals, amorphous films) of hen egg white lysozyme, bovine serum albumin, and sperm whale myoglobin were studied in the temperature range of 100–300 K at different hydration levels. Decreasing the temperature was shown to cause a steplike increase in the Young's modulus of highly hydrated protein samples (with water content exceeding 0.3 g/g dry weight of protein) in the temperature range of 237–251 K, followed by a large increase in the modulus in the broad temperature interval of 240–130 K, which we refer to as a mechanical glass transition. Soaking the samples in 50% glycerol solution completely removed the steplike transition without significantly affecting the glass transition. The apparent activation energy determined from the frequency dependence of the glass-transition temperature was found to be 18 kcal/mol for wet lysozyme crystals. Lowering the humidity causes both the change of the Young's modulus in response to the transition and the activation energy to decrease. The thermal expansion coefficient of amorphous protein films also indicates the glass transition at 150–170 K. The data presented suggest that the glass transition in hydrated samples is located in the surface layer of proteins and related to the immobilization of the protein groups and strongly bound water.     

Physiological responses of pressed baker’s yeast cells pre-treated with citric, malic and succinic acids

Summary The dough-leavening power of baker’s yeast, Saccharomyces cerevisiae, is strongly influenced by conditions under which the pressed yeast is maintained prior to bread dough preparation. In this study, the influence of the yeast cell’s pre-treatment with organic acids (malic, succinic, and citric acids) was investigated at a wide range of pH values when the pressed yeast samples were exposed to 30 °C. Increased fermentative activity was observed immediately after pre-treatment of the cells with organic acids. When the pH of the pressed yeast containing added citric acid was raised from 3.5 to 7.5, increases in both fermentative and maltase activities were obtained. Improvements in viability and levels of total protein were also observed during storage in the presence of citric acid, notably at pH 7.5. Glycerol-3-phosphate dehydrogenase activity and levels of internal glycerol also increased in the presence of citrate. On the other hand, pressed yeast samples containing succinic acid at pH 7.5 showed decreased viability during storage despite the maintenance of high levels of fermentative activity, similar to pressed yeast containing malic acid at pH 4.5 and 7.5. Decreases in intracellular levels of trehalose were observed during storage in all cases. Overall, the results of this study revealed the potential benefits of adding organic acids to pressed yeast preparations for baking purposes.     

Collapse temperature of freeze-dried Lactobacillus bulgaricus suspensions and protective media

Optimization of the freeze-drying process needs to characterize the physical state of frozen and dried products. A protocol to measure the collapse temperature of complex biological media such as concentrated lactic acid bacteria using freeze-drying microscopy was first elaborated. Afterward, aqueous solutions of one or several components as well as concentrated lactic acid bacterial suspensions were analyzed in order to study how the structure of these materials is degraded during freeze-drying. A similar behavior toward collapse was observed for all aqueous solutions, which was characterized by two temperatures: the "microcollapse" temperature (T(microc), beginning of a local loss of structure) and the "collapse" temperature (T(c), beginning of an overall loss of structure). For aqueous solutions, these two temperatures were close, differing by less than 3 degrees C. Nevertheless, when lactic acid bacteria were added to aqueous solutions, the collapse temperatures increased. Moreover, the interval between microcollapse and collapse temperatures became larger. Lactic acid bacterial cells gave a kind of "robustness" to the freeze-dried product. Finally, comparing glass transition, measured by differential scanning calorimetry (DSC) and collapse temperature for aqueous solutions with noncrystallizable solutes, showed that these values belonged to the same temperature range (differing by less than 5 degrees C). As suggested in the literature, the glass transition temperature can thus be used as a first approximation of the collapse temperature of these media. However, for lactic acid bacterial suspensions, because the difference between collapse and glass transition temperatures was about 10 degrees C, this approximation was not justified. An elegant physical appearance of the dried cakes and an acceptable acidification activity recovery were obtained, when applying operating conditions during freeze-drying in vials that allowed the product temperature to be maintained during primary drying at a level lower than the collapse temperature of lactic acid bacterial suspensions. Consequently, the collapse temperature T(c) was proposed as the maximal product temperature preserving the structure from macroscopic collapse and an acceptable biological activity of cells.     

The preservation of liposomes by raffinose family oligosaccharides during drying is mediated by effects on fusion and lipid phase transitions

Raffinose family oligosaccharides (RFO) have been implicated as protective agents in the cellular dehydration tolerance, especially of many plant seeds. However, their efficacy in stabilizing membranes during dehydration has never been systematically investigated. We have analyzed the effects of sucrose, raffinose, stachyose, and verbascose on liposome stability during air-drying. With increasing degree of polymerization (DP), the RFO were progressively better able to stabilize liposomes against leakage of aqueous content and against membrane fusion after rehydration. Indeed, there was a very tight linear correlation between fusion and leakage for all RFO. These data indicate that increased protection of liposomes against leakage with increasing DP is due to better protection against fusion. This is in accord with the higher glass transition temperature of the longer chain oligosaccharides. Further evidence for the influence of glass transitions on membrane stability in the dry state was provided by experiments testing the temperature dependence of membrane fusion. During incubation at temperatures up to 95 degrees C for 2 h, fusion increased less with temperature in the presence of higher DP sugars. This indicates that RFO with a higher glass transition temperature are better able to protect dry membranes at elevated temperatures. In addition, Fourier-transform infrared (FTIR) spectroscopy showed a reduction of the gel to liquid-crystalline phase transition temperature of dry liposomes in the presence of all investigated sugars. However, the RFO became slightly less effective with increasing chain length, again pointing to a decisive role for preventing fusion. A direct interaction of the RFO with the lipids was indicated by a strong effect of the sugars on the phosphate asymmetric stretch region of the infrared spectrum.     

The preservation of liposomes by raffinose family oligosaccharides during drying is mediated by effects on fusion and lipid phase transitions

Raffinose family oligosaccharides (RFO) have been implicated as protective agents in the cellular dehydration tolerance, especially of many plant seeds. However, their efficacy in stabilizing membranes during dehydration has never been systematically investigated. We have analyzed the effects of sucrose, raffinose, stachyose, and verbascose on liposome stability during air-drying. With increasing degree of polymerization (DP), the RFO were progressively better able to stabilize liposomes against leakage of aqueous content and against membrane fusion after rehydration. Indeed, there was a very tight linear correlation between fusion and leakage for all RFO. These data indicate that increased protection of liposomes against leakage with increasing DP is due to better protection against fusion. This is in accord with the higher glass transition temperature of the longer chain oligosaccharides. Further evidence for the influence of glass transitions on membrane stability in the dry state was provided by experiments testing the temperature dependence of membrane fusion. During incubation at temperatures up to 95 °C for 2 h, fusion increased less with temperature in the presence of higher DP sugars. This indicates that RFO with a higher glass transition temperature are better able to protect dry membranes at elevated temperatures. In addition, Fourier-transform infrared (FTIR) spectroscopy showed a reduction of the gel to liquid-crystalline phase transition temperature of dry liposomes in the presence of all investigated sugars. However, the RFO became slightly less effective with increasing chain length, again pointing to a decisive role for preventing fusion. A direct interaction of the RFO with the lipids was indicated by a strong effect of the sugars on the phosphate asymmetric stretch region of the infrared spectrum.     

Enthalpy relaxation of freeze concentrated sucrose-water glass

The enthalpy relaxation of freeze concentrated sucrose-water glass was investigated using 40% sucrose, differential scanning calorimetry (DSC) with isothermal ageing for 1-6 days at various temperatures (-70, -65, -60, and -55 degrees C). The enthalpy relaxation was observed as an endothermic peak superimposed on the endothermic step-wise change due to the glass transition around -47 degrees C. The enthalpy relaxation was found to increase with ageing time and temperature. An 80% sucrose glass was also investigated at ageing temperatures of -60 and -65 degrees C, and this material exhibited a similar glass transition and enthalpy relaxation to that observed with the frozen 40% sucrose solution. The calculated activation energy of the enthalpy relaxation of the sucrose-water glass was smaller than that reported for pure sucrose. These results suggest that the freeze concentrated sucrose-water glass could have a higher molecular mobility and less stability than pure sucrose glass.     

Dynamical transition in a large globular protein: Macroscopic properties and glass transition

Hydrated soy-proteins display different macroscopic properties below and above approximately 25% moisture. This is relevant to the food industry in terms of processing and handling. Quasi-elastic neutron spectroscopy of a large globular soy-protein, glycinin, reveals that a similar moisture-content dependence exists for the microscopic dynamics as well. We find evidence of a transition analogous to those found in smaller proteins, when investigated as a function of temperature, at the so-called dynamical transition. In contrast, the glass transition seems to be unrelated. Small proteins are good model systems for the much larger proteins because the relaxation characteristics are rather similar despite the change in scale. For dry samples, which do not show the dynamical transition, the dynamics of the methyl group is probably the most important contribution to the QENS spectra, however a simple rotational model is not able to explain the data. Our results indicate that the dynamics that occurs above the transition temperature is unrelated to that at lower temperatures and that the transition is not simply related to the relaxation rate falling within the spectral window of the spectrometer.     

Mean square fluctuations of hydrogen atoms and water-biopolymer interactions in hydrated saccharides.

We have used the elastic neutron scattering technique to investigate the dynamics of the two main saccharidic components of starch: amylose and amylopectin. The measurements were carried out in the temperature range of 20 to 320 K and at different hydration levels from the dry state up to 0.47 g saccharide/g D(2)O. In the dry samples, the atomic dynamics is harmonic up to approximately 300 K. In the hydrated samples a "glass-like" transition leading to an anharmonic dynamics is observed. The onset of the anharmonicity occurs at temperatures that increase from approximately 180 K to 260 K upon decreasing hydration from 0.5 to 0.1 g saccharide/g D(2)O. This behavior is qualitatively similar to that observed in hydrated globular proteins, but quantitative differences are present. Assuming a simple asymmetric double-well potential model, the temperature and hydration dependence of the transition have been described in terms of few physical parameters.     

Is trehalose special for preserving dry biomaterials?

Simple sugars, especially disaccharides, stabilize biomaterials of various composition during air-drying or freeze-drying. We and others have provided evidence that direct interaction, an interaction that we believe is essential for the stabilization, between the sugar and polar groups in, for example, proteins and phospholipids occurs in the dry state. Some researchers, however, have suggested that the ability of the sugar to form a glass is the only requirement for stabilization. More recently, we have shown that both glass formation and direct interaction of the sugar and headgroup are often required for stabilization. In the present study, we present a state diagram for trehalose glass and suggest that the efficacy of this sugar for stabilization may be related to its higher glass transition temperatures at all water contents. We also show that trehalose and trehalose:liposome preparations form trehalose dihydrate as well as trehalose glass when rehydrated with water vapor. Formation of the dihydrate sequesters water, which might otherwise participate in lowering the glass transition temperature to below ambient. Because samples remain in the glassy state at ambient temperatures, viscosity is high and fusion between liposomes is prevented.     

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.     

面包酵母细胞出芽率与产品质量关系研究

速用压榨酵母(Compressed yeast,CY)和活性干酵母(Active dry yeast,ADY)是用于面点制作的主要两类酵母产品。发酵力则是其共同质量指标。目前,国产压榨酵母的发酵力波动很大,活性干酵母不仅发酵力低,而且保质期短。面包酵母的发酵力定义为由一定数量的酵母样品,面粉和水制成的面团在恒温下产生CO_2的能力。本文采用Burrows法测量面包酵母发酵力,面团由0.15克干酵母,20克面粉和15mol脱离子水混合而成,并以该面团在30℃、180min内产生的CO₂总量作为发酵力的表征,文献^[2]表明,面包酵母的发酵力与终细胞群体中的带芽细胞分率(Fraction of budding cells,FBC)有密切的关系,传统的概念是,为提高酵母产品质量,FBC越低愈好。经研究发现,压榨酵母的耐贮存力及活性干酵发酵力与FBC成反比,但对速用压榨酵母而言,其发酵力FBC的正相关关系甚为明显,这与传统有着根本的差异。     

Thermally induced changes in the structure and activity of yeast hexokinase B

Yeast hexokinase has been poorly characterized in regard with its stability. In the present study, various spectroscopic techniques were employed to investigate thermal stability of the monomeric form of yeast hexokinase B (YHB). The enzyme underwent a conformational transition with a T(m) of about 41.9 degrees C. The structural transition proved to be significantly reversible below 55 degrees C and irreversible at higher temperatures. Thermoinactivation studies revealed that enzymatic activity diminished significantly at high temperatures, with greater loss of activity observed above 55 degrees C. Release of ammonia upon deamidation of YHB obeyed a similar temperature-dependence pattern. Dynamic light scattering and size exclusion-HPLC indicated formation of stable aggregates. Taking various findings on the influence of osmolytes and chaperone-like agents on YHB thermal denaturation together, it is proposed that the purely conformational transition of YHB is reversible, and irreversibility is due to aggregation, as a major cause. Deamidation of a critical Asn or Gln residue(s) may also play an important role.     

Viscoelastic properties of pectin--co-solute mixtures at iso-free-volume states

Small-deformation oscillatory measurements were performed on pectin-sucrose-glucose syrup systems at a total level of solids of 81%, with the polysaccharide content being fixed at levels of industrial use (1%). The experimental temperature range was between 50 and - 50 degrees C. Analysis of the temperature dependence of viscoelastic processes by the equation of Williams, Landel, and Ferry provides values of fractional free volume for the temperatures covering the glass transition region. The shift factors used in the conversion of mechanical spectra into master curves were normalised at suitably different temperatures so that their temperature dependence becomes coincident. The treatment implies an iso-free-volume state and relates to changes in the monomeric friction coefficient with increasing levels of intermolecular interactions in the mixture. A free-volume related glass transition temperature was defined and manipulated markedly by introducing pectin of variable degrees of esterification to the sucrose-glucose syrup system.     

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.     

Glass transition and enthalpy relaxation of amorphous lactose glass

The glass transition temperature, T(g), and enthalpy relaxation of amorphous lactose glass were investigated by differential scanning calorimetry (DSC) for isothermal aging periods at various temperatures (25, 60, 75, and 90 degrees C) below T(g). Both T(g) and enthalpy relaxation were found to increase with increasing aging time and temperature. The enthalpy relaxation increased approximately exponentially with aging time at a temperature (90 degrees C) close to T(g) (102 degrees C). There was no significant change observed in the enthalpy relaxation around room temperature (25 degrees C) over an aging period of 1month. The Kohlrausch-Williams-Watts (KWW) model was able to fit the experimental enthalpy relaxation data well. The relaxation distribution parameter (beta) was determined to be in the range 0.81-0.89. The enthalpy relaxation time constant (tau) increased with decreasing aging temperature. The observed enthalpy relaxation data showed that molecular mobility in amorphous lactose glass was higher at temperatures closer to T(g). Lactose glass was stable for a long time at 25 degrees C. These findings should be helpful for improving the processing and storage stability of amorphous lactose and lactose containing food and pharmaceutical products.     

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.     

Influence of growth temperature on the acid phosphatase activity in the yeast Yarrowia lipolytica

Abstract The adaptation of the yeast Yarrowia lipolytica 76-18 to growth temperature was studied by measuring the levels of secreted and intracellular acid phosphatase activities during growth at five temperatures from 8 to 36 °C. The intracellular acid phosphatase activity is maximal at a growth temperature of 20 °C. The level of the secreted phosphatase activity decreases as growth temperature increases from 15 to 36 °C. It is the growth temperature itself and not the growth rate that regulates these activities. The observed dependence of the acid phosphatase activity on the growth temperature indicates a possible participation of acid phosphatases in the temperature adaptation of yeast cells.     

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.