Carolina Schebor

Glassy state and thermal inactivation of invertase and lactase in dried amorphous matrices

The thermal stability of enzymes lactase and invertase in dried, amorphous matrices of sugars (trehalose, maltose, lactose, sucrose, raffinose) and some other selected systems (casein, PVP, milk) was studied. The glass transition temperature (Tg) was limited as a threshold parameter for predicting enzyme inactivation because (a) enzyme inactivation was observed in glassy matrices, (b) a specific effect of enzyme stabilization by certain matrices particularly trehalose was observed, and (c) enzyme stability appeared to depend on heating temperature (T) “per se” rather than (T − Tg) . For these reasons, a protective mechanism by sugars related to the maintenance of the tertiary structure of the enzyme was favored. A rapid loss of enzyme (lactase) activity was observed in heated sucrose systems at T > Tg, and this was attributed to sucrose crystallization since it is known that upon crystallization the protective effect of sugars is lost. Thus, the stabilizing effect could be indirectly affected by the Tg of the matrix, since crystallization of sugars only occurs above Tg. Trehalose model systems (with added invertase) showed an exceptional stability toward “darkening” (e.g., non‐enzymatic browning) when heated in the dried state to elevated temperatures and for long periods of time.

Effect of pH, Counter Ion, and Phosphate Concentration on the Glass Transition Temperature of Freeze-Dried Sugar-Phosphate Mixtures

AbstractPurpose. The aim of the present work is to study the interaction of phosphate salts with trehalose and sucrose in freeze-dried matrices, particularly the effect of the salts on the glass transition temperature (Tg) of the sugars. Methods. Freeze-dried trehalose and sucrose systems containing different amounts of sodium or potassium phosphate were analyzed by differential scanning calorimetry to determine the Tg and by Fourier-transform infrared spectroscopy (FTIR) analysis to evaluate the strength of the interaction between sugars and phosphate ions. Results. Sucrose-phosphate mixtures show an increase in Tg up to 40°C in a broad pH range (4-9) compared to that of pure sucrose. Sucrose-phosphate mixtures exhibit a higher Tg than pure sucrose while retaining higher water contents. Trehalose-phosphate mixtures (having a Tg of 135°C at a pH of 8.8) are a better option than pure trehalose for preservation of labile materials. The -OH stretching of the sugars in the presence of phosphates decreases with increase in pH, indicating an increase in the sugar-phosphate interaction. Conclusions. Sugar-phosphate mixtures exhibit several interesting features that make them useful for lyophilization of labile molecules; Tg values much higher than those observed for the pure sugars can be obtained upon the addition of phosphate.

Glassy state in relation to the thermal inactivation of the enzyme invertase in amorphous dried matrices of trehalose, maltodextrin and PVP

Abstract The stabilization of invertase by its incorporation in aqueous trehalose and polymer solutions, followed by freeze-drying and desiccation to ‘zero’ moisture content, was studied. The dried amorphous preparations of trehalose, maltodextrin (MD; DE = 10.9), and poly(vinyl)pyrrolidone (PVP), molecular weights 360000, 40000 and 10000, greatly protected invertase—as compared with its behavior in liquid solution—from heat inactivation at elevated temperatures. Significant invertase inactivation was observed in heated PVP and MD matrices kept well below their glass-transition temperature. Under glassy conditions the extent of enzyme protection by MD and PVP systems was related to their glass-transition temperature (Tg) since systems of higher Tg afforded better protection. However, the data for trehalose deviated from this behavior since invertase stabilization was higher than expected on the basis of the results obtained with polymer matrices. Present results suggest that invertase inactivation in dried amorphous systems cannot be adequately explained by the glass-transition theory and this is particularly true for trehalose, for which some additional mechanism of enzyme protection is likely to operate.

Sucrose hydrolysis in a glassy starch matrix

The purpose of present work was to investigate whether the glassy state may prevent sucrose inversion in an acid-containing amorphous starch powder (native or pre-gelatinized). It was found that sucrose hydrolysis occurred to a significant extent in the glassy state (in both native and gelatinized starch). Moisture content of the system affected the extent of sucrose hydrolysis but its effect was not attributable to the plasticizing effect of water. Little reaction occurred at moisture contents below the so-called B.E.T. monolayer. Temperature was a critical factor controlling sucrose inversion.

Effect of physical properties on the stability of Lactobacillus bulgaricus in a freeze-dried galacto-oligosaccharides matrix

The ability of galacto-oligosaccharides (GOS) to protect Lactobacillus delbrueckii subsp. bulgaricus upon freeze drying was analyzed on the basis of their capacity to form glassy structures. Glass transition temperatures (T(g)) of a GOS matrix at various relative humidities (RH) were determined by DSC. Survival of L. bulgaricus in a glassy GOS matrix was investigated after freezing, freeze drying, equilibration at different RHs and storage at different temperatures. At 32 °C, a drastic viability loss was observed. At 20 °C, the survival was affected by the water content, having the samples stored at lower RHs, the highest survival percentages. At 4°C, no decay in the cells count was observed after 45 days of storage. The correlation between molecular mobility [as measured by Proton nuclear magnetic resonance (¹H NMR)] and loss of viability explained the efficiency of GOS as cryoprotectants. The preservation of microorganisms was improved at low molecular mobility and this condition was obtained at low water contents and low storage temperatures. These results are important in the developing of new functional foods containing pre and probiotics.

Non-enzymatic browning kinetics analysed through water-solids interactions and water mobility in dehydrated potato.

Non-enzymatic browning (NEB) development was studied in dehydrated potato at 70°C. It was related to the macroscopic and molecular properties and to water-solid interactions over a wide range of water activities. Time resolved (1)H NMR, thermal transitions and water sorption isotherms were evaluated. Although non-enzymatic browning could be detected in the glassy state; colour development was higher in the supercooled state. The reaction rate increased up to a water content of 26g/100g of solids (aw=0.84) and then decreased at higher water contents, concomitantly with the increase of water proton mobility. The joint analyses of NEB kinetics, water sorption isotherm and proton relaxation behaviour made it evident that the point at which the reaction rate decreased, after a maximum value, could be related to the appearance of highly mobile water. The results obtained in this work indicate that the prediction of chemical reaction kinetics can be performed through the integrated analysis of water sorption, water and solids mobility and the physical state of the matrix.

Glass transition temperature of regular and lactose hydrolyzed milk powders

Abstract The glass transition temperatures ( T g ) of lactose hydrolyzed milk powder (HMP) was determined and compared to that of regular milk powders (MP). Some physical and chemical changes (loss of flowing ability, browning development) were also evaluated during storage of the different milk powders. Sugars of HMP (glucose, galactose, lactose) influenced, but did not define, the T g values of the product, on the contrary lactose governed the observed T g values of MP samples. Changes in the flowing characteristics of milk powder and color development, stored at different water activity values, were much more evident in HMP than in regular milk powder. Results suggested that the T g value is not the unique parameter governing the flowability and color development in stored milk powders.

Physical and Functional Properties of Blackberry Freeze- and Spray-Dried Powders

The aim of the present work was to develop two products from blackberry juice by freeze and spray drying with potential use as food colorants or healthy ingredients. A characterization of the physical and functional properties of the powdered juices was done. Maltodextrin or a mixture of trehalose and maltodextrin were assessed as carrier matrices. Freeze-dried, maltodextrin-containing powders presented the best retention of bioactive compounds and antiradical activity; however, they showed a narrow relative humidity range for storage in the glassy state. Spray-dried powders showed better physical properties, bearing higher glass transition temperature and lower molecular mobility than freeze-dried formulations.

Glass transition temperatures and fermentative activity of heat-treated commercial active dry yeasts.

Differential scanning calorimetry thermograms of various samples of commercial instant active dry yeasts revealed a clear glass transition typical of amorphous carbohydrates and sugars. The resulting glass transition temperatures were found to decrease with increasing moisture content. The observed glass curve was similar to that of pure trehalose, which is known to accumulate in large amounts in bakers yeast. The effect of heat treatment at various temperatures on the fermentative activity (as measured by the metabolic production of CO2) of dry yeast was studied. First‐order plots were obtained representing the loss of fermentative activity as a function of heating time at the various temperatures assayed. Significant losses of fermentative activity were observed in vitrified yeast samples. The dependence of rate constants with temperature was found to follow Arrhenius behavior. The relationship between the loss of fermentative activity and glass transition was not verified, and the glass transition was not reflected on the temperature dependence of fermentative activity loss.

Effect of trehalose on the interaction of Alzheimer's Aβ-peptide and anionic lipid monolayers.

The interaction of amyloid β-peptide (Aβ) with cell membranes is believed to play a central role in the pathogenesis of Alzheimers disease. In particular, recent experimental evidence indicates that bilayer and monolayer membranes accelerate the aggregation and amyloid fibril formation rate of Aβ. Understanding that interaction could help develop therapeutic strategies for treatment of the disease. Trehalose, a disaccharide of glucose, has been shown to be effective in preventing the aggregation of numerous proteins. It has also been shown to delay the onset of certain amyloid-related diseases in a mouse model. Using Langmuir monolayers and molecular simulations of the corresponding system, we study several thermodynamic and kinetic aspects of the insertion of Aβ peptide into DPPG monolayers in water and trehalose subphases. In the water subphase, the insertion of the Aβ peptide into the monolayer exhibits a lag time which decreases with increasing temperature of the subphase. In the presence of trehalose, the lag time is completely eliminated and peptide insertion is completed within a shorter time period compared to that observed in pure water. Molecular simulations show that more peptide is inserted into the monolayer in the water subphase, and that such insertion is deeper. The peptide at the monolayer interface orients itself parallel to the monolayer, while it inserts with an angle of 50° in the trehalose subphase. Simulations also show that trehalose reduces the conformational change that the peptide undergoes when it inserts into the monolayer. This observation helps explain the experimentally observed elimination of the lag time by trehalose and the temperature dependence of the lag time in the water subphase.