Crystallization and X-ray diffraction of spray-dried and freeze-dried amorphous lactose

Crystallization of spray-dried and freeze-dried amorphous lactose over different relative vapor pressures (RVP) and storage times was studied. Crystallization was observed from increasing peak intensities in X-ray diffraction patterns. Lactose was crystallized in the samples stored at RVP of 44.1% and above in both types of dehydrated powders. The rate of crystallization increased with increasing RVP and storage time. Similar crystallization behavior of both spray-dried and freeze-dried lactose was observed. Lactose crystallized as alpha-lactose monohydrate, anhydrous beta-lactose, and the anhydrous form of alpha- and beta-lactose in a molar ratio of 5:3 and 4:1 in both spray-dried and freeze-dried forms. Peak intensities of X-ray diffraction patterns for anhydrous beta-lactose were decreased, and for alpha-lactose monohydrate increased with increasing storage RVP and time. The crystallization data were successfully modeled using Avrami equation at RVP of 54.5% and above. The crystallization data obtained is helpful in understanding and predicting storage stability of lactose-containing food and pharmaceutical products.     

Crystallization kinetics of amorphous lactose, whey-permeate and whey powders

Amorphous lactose, whey-permeate and whey powders have been converted to their crystalline forms by exposure to air at various temperatures and relative humidities. The total time required for sorption, induction and crystallization of these powders was observed by following the time-dependent mass change of the powders during treatment. These experiments have shown that higher temperatures and relative humidities lead to shorter crystallization times. Lactose crystallizes within 1 min at an air temperature of 100 degrees C and relative air humidity of 80%, whereas whey-permeate and whey powders requires up to 5 min at the same set of conditions. Thus, as previously described, the presence of proteins and salts in the whey-permeate and whey powders reduces the crystallization rate. The rate constants and activation energies have been determined over a range of temperatures and humidities to enable the calculation of crystallization times for the design of an industrial process that crystallizes whey and whey-permeate powders. Finally, the crystallization rates found in this work are sufficiently fast to be applicable in an industrial process that crystallizes whey and whey-permeate powders.     

Modification of the Solid-State Nature of Sulfathiazole and Sulfathiazole Sodium by Spray Drying

Solid-state characterisation of a drug following pharmaceutical processing and upon storage is fundamental to successful dosage form development. The aim of the study was to investigate the effects of using different solvents, feed concentrations and spray drier configuration on the solid-state nature of the highly polymorphic model drug, sulfathiazole (ST) and its sodium salt (STNa). The drugs were spray-dried from ethanol, acetone and mixtures of these organic solvents with water. Additionally, STNa was spray-dried from pure water. The physicochemical properties including the physical stability of the spray-dried powders were compared to the unprocessed materials. Spray drying of ST from either acetonic or ethanolic solutions with the spray drier operating in a closed cycle mode yielded crystalline powders. In contrast, the powders obtained from ethanolic solutions with the spray drier operating in an open cycle mode were amorphous. Amorphous ST crystallised to pure form I at ≤35 % relative humidity (RH) or to polymorphic mixtures at higher RH values. The usual crystal habit of form I is needle-like, but spherical particles of this polymorph were generated by spray drying. STNa solutions resulted in an amorphous material upon processing, regardless of the solvent and the spray drier configuration employed. Moisture induced crystallisation of amorphous STNa to a sesquihydrate, whilst crystallisation upon heating gave rise to a new anhydrous polymorph. This study indicated that control of processing and storage parameters can be exploited to produce drugs with a specific/desired solid-state nature.KEY WORDS: amorphous state, dynamic vapour sorption, particle habit, physical stability, polymorphism, sulfathiazole     

Thermally reversible hydration of beta-chitin

Thermally induced transition between anhydrous and hydrated forms of highly crystalline beta-chitin was studied by differential thermal calorimetry (DSC) and X-ray diffraction. DSC of wet beta-chitin in a sealed pan gave two well-defined endothermic peaks at 85.2 and 104.7 degrees C on heating and one broad exothermic peak at between 60 and 0 degrees C on cooling. These peaks were highly reproducible and became more distinct after repeated heating-cooling cycles. The X-ray diffraction pattern of wet beta-chitin at elevated temperature showed corresponding changes in d-spacing between the sheets formed by stacking of chitin molecules. These phenomena clearly show that water is reversibly incorporated into the beta-chitin crystal and that the temperature change induces transitions between anhydrous, monohydrate, and dihydrate forms. The DSC behavior in heating-cooling cycles, including reversion between the two endothermic peaks, indicated that the transition between monohydrate and dihydrate was a fast and narrow-temperature process, whereas the one between the anhydrous and the monohydrate form was a slow and wide-temperature process.     

The phase behavior of hydrated cholesterol.

The thermotropic phase behavior of cholesterol monohydrate in water was investigated by differential scanning calorimetry, polarizing light microscopy, and x-ray diffraction. In contrast to anhydrous cholesterol which undergoes a polymorphic crystalline transition at 39 degrees C and a crystalline to liquid transition at 151 degrees C, the closed system of cholesterol monohydrate and water exhibited three reversible endothermic transitions at 86, 123, and 157 degrees C. At 86 degrees C, cholesterol monohydrate loses its water of hydration, forming the high temperature polymorph of anhydrous cholesterol. At least 24 hours were required for re-hydration of cholesterol and the rate of hydration was dependent on the polymorphic crystalline form of anhydrous cholesterol. At 123 degrees C, anhydrous crystalline cholesterol in the presence of excess water undergoes a sharp transition to a birefringent liquid crystalline phase of smectic texture. The x-ray diffraction pattern obtained from this phase contained two sharp low-angle reflections at 37.4 and 18.7 A and a diffuse wide-angle reflection centered at 5.7 A, indicating a layered smectic type of liquid crystalline structure with each layer being two cholesterol molecules thick. The liquid crystalline phase is stable over the temperature range of 123 to 157 degrees C before melting to a liquid dispersed in water. The observation of a smectic liquid crystalline phase for hydrated cholesterol correlates with its high surface activity and helps to explain its ability to exist in high concentrations in biological membranes.     

Effects of temperature on the hydrolysis of lactose by immobilized beta-galactosidase in a capillary bed reactor

The effects of temperature on the hydrolysis of lactose by immobilized beta-galactosidase were studied in a continuous flow capillary bed reactor. Temperature affects the rates of enzymatic reactions in two ways. Higher temperatures increase the rate of the hydrolysis reaction, but also increase the rate of thermal deactivation of the enzyme. The effect of temperature on the kinetic parameters was studied by performing lactose hydrolysis experiments at 15, 20, 25, 30, and 40 degrees C. The kinetic parameters were observed to follow an Arrhenius-type temperature dependence. Galactose mutarotation has a significant impact on the overall rate of lactose hydrolysis. The temperature dependence of the mutarotation of galactose was effectively modelled by first-order reversible kinetics. The thermal deactivation characteristics of the immobilized enzyme reactor were investigated by performing lactose hydrolysis experiments at 52, 56, 60, and 64 degrees C. The thermal deactivation was modelled effectively as a first order decay process. Based on the estimated thermal deactivation rate constants, at an operating temperature of 40 degrees C, 10% of the enzyme activity would be lost in one year.     

Alpha-lactose monohydrate single crystals as hosts for matrix isolation of guest biopolymers.

Single crystals of alpha-lactose monohydrate show a remarkable tendency to include biopolymers, such as proteins, oligonucleotides and dextrans, within the growing lattice. Glycosylation increased the amount of protein contained within the crystals. The guest molecules were found only within the (010) growth sector of the hatchet shaped crystals, thereby binding preferentially to one of the seven developed crystal faces. The topographical features of the active surface are described.     

Effect of Carbonation of Supersaturated Lactose Solution on Crystallisation Behaviour of Alpha-Lactose Monohydrate

The effect of dissolution of carbon dioxide (CO₂) in lactose solution on the particle size, morphology, yield and densities of crystallized alpha-lactose monohydrate (α-LM) was investigated. Dry ice was added into a 50% w/w aqueous lactose solution (relative supersaturation S_R = 4.55) at 0, 500 and 1000 ppm in batch mode and closed system at 40°C. Agitation and sonication then were applied to the carbonated aqueous lactose solution for creating CO₂ bubbles to assist the nucleation process. For ultrasound (US) treatment, the aqueous lactose solution was sonicated at 50% amplitude for 1 min. All samples (CO₂ + agitation and CO₂ + US + agitation) were then agitated for 3 h at 25°C upon crystallisation process. Regardless of mechanical treatment, prior addition of CO₂ at higher concentration tended to induce formation of smaller crystals with triangular and trapezoidal-shaped morphologies whilst non-carbonated lactose favoured generation of bigger tomahawk-shaped crystals. A remarkable increase in α-LM yield was found in 1000 ppm carbonated sample prior to sonication as against non-carbonated counterpart. The crystal size d(0.5) was also found significantly lower (~32 μm) for the carbonated sample as compared to those of non-carbonated sample (~56 μm). The bulk and true densities of α-LM powder obtained were independent on CO₂ concentration added but addition of 1000 ppm CO₂ affected the packed densities of α-LM. Carbonation of lactose solution can be considered as an additional tool to manipulate crystal size, shape and yield of α-LM that may suitable for pharmaceutical and food applications.     

Transformation of lactulose trihydrate into anhydrous lactulose by fluidized bed drying and its characterization

Stable anhydrous lactulose was produced from lactulose trihydrate by stepwise heating on a fluidized bed. The processes were performed on stable powder forms. The anhydrous lactulose was characterized by an opaque white appearance, a coarse surface structure with random cracks and indentations, a high degree of crystallization, stability under humid conditions, and by X-ray powder diffraction, differential thermal analysis, and differential thermogravimetry. Those characteristics were different from those of the original trihydrate, which was transparent, had a smooth surface and a higher degree of crystallization, was stable under humid conditions and had different X-ray powder diffraction, differential thermal analysis, and thermogravimetric characteristics. The transformation was enhanced when the inlet temperature was 45-55 degrees C or when the temperature of the fluidized bed was over 40 degrees C. At these cutoff temperatures, both crystalline forms were observed.     

Lipoprotein lipase, a key role in atherosclerosis?

Enzymes from hyperthermophiles can be efficiently purified after expression in mesophilic hosts and are well-suited for crystallisation attempts. Two enzymes of histidine biosynthesis from Thermotoga maritima, N'-((5'-phosphoribosyl)-formimino)-5-aminoimidazol-4-carb oxamid ribonucleotide isomerase and the cyclase moiety of imidazoleglycerol phosphate synthase, were overexpressed in Escherichia coli, both in their native and seleno-methionine-labelled forms, purified by heat precipitation of host proteins and crystallised. N'-((5'-phosphoribosyl)-formimino)-5-aminoimidazol-4-carb oxamid ribonucleotide isomerase crystallised in four different forms, all suitable for X-ray structure solution, and the cyclase moiety of imidazoleglycerol phosphate synthase yielded one crystal form that diffracted to atomic resolution. The obtained crystals will enable the determination of the first three-dimensional structures of enzymes from the histidine biosynthetic pathway.     

The kinetic of lactose hydrolysis for the beta-galactosidase from Aspergillus niger

The kinetics of glucose liberation from lactose by means of the beta-glactosidase from Aspergillus niger has been studied in a wide range of the main variables. The analysis shows that the kinetic models proposed so far are not adequate. The main finding is that the reaction rate is not linearly correlated to the enzyme concentration-it increase more than proportionally. This nonlinear relationship results because this lactase can distinguish between alpha-and beta-galactose alpha-Galactose acts as competitive and anticompetitive inhibitor while beta-galactose is a competitive one. The competitive inhibition of the alpha-anomer is approximately 12 times more sever than that of the beta-anomer. The kinetics, including a simplified model for the mutarotation of galactose is given for a temperature of 50 degrees C at a pH of 3.5-the most likely conditions for the application of this lactase in acid whey treatment.     

FTIR study of state and phase transitions of low moisture sucrose and lactose

Mid-infrared spectra of freeze-dried sucrose and lactose systems were acquired over a range of temperatures (30-200 degrees C) and water contents (0-6.3%). Starting from the glassy state, the experimental conditions were selected to cover the main thermal transitions: the glass-rubber transition, the crystallisation and, for some samples, the subsequent melting. The FTIR spectra were very sensitive to the physical state. While subtle but systematic spectral differences between the glassy and rubbery states were detectable throughout the spectrum, a very pronounced increase in spectral resolution was observed as crystallisation occurred and was followed by the expected spectral broadening during melting. The temperatures at which these changes occurred were in satisfactory agreement with the transition temperatures measured by differential scanning calorimetry (DSC). The increase in molecular mobility as a result of increasing temperature or plasticisation by water led to a significant shift of the O-H stretching band to higher wavenumbers indicating a weakening of hydrogen bonding. This shift reached a maximum as the DSC measured crystallisation temperature range was approached. As expected, the crystallisation led to a highly effective hydrogen bonding network. This was more significant for lactose than for sucrose. No significant step change in hydrogen bonding was observed at Tg. As anticipated, the temperature at which these transitions occurred decreased with increasing water content but overlapped when observed in the context of the shifted temperature (T-Tg).     

A new graphical method for determining parameters in Michaelis-Menten-type kinetics for enzymatic lactose hydrolysis

A new graphical method was developed to determine the kinetic parameters in the Michaelis-Menten-type equation. This method was then applied to studying the kinetics of lactose hydrolysis by Aspergillus niger beta-galactosidase. In this study, the reaction temperature ranged between 8 and 60 degrees C, and the initial lactose concentration ranged between 2.5 and 20%. A kinetic model similar to the conventional Michaelis-Menten equation with competitive product inhibition by galactose was tested using this graphical method as well as a nonlinear computer regression method. The experimental data and the model fit together fairly well at 50 degrees C. However, a relative large disparity was found for reactions at 30 degrees C. A three-parameter integrated model derived from the reversible reaction mechanism simulates the experimental data very well at all temperatures studied. However, this reversible reaction model does not follow the Arrhenius temperature dependence. Nevertheless, reaction rate constants for the proposed model involving the enzyme-galactose complex (in addition to the Michaelis complex) as an intermediate in lactose hydrolysis follow the Arrhenius temperature dependence fairly well, suggesting that this model can be best used for describing the enzymatic lactose hydrolysis. The lack of fit between the model predictions and data may be largely attributed to the effects of galactose mutarotation and oligosaccharide formation during lactose hydrolysis.     

Measurement of borate in occupational environments

The hydration stability for inhalable borate particles has been characterized as a function of temperature and relative humidity when collected by a field personnel monitor. The rate of hydration was measured for boric acid (B[OH]3); Neobor borax 5 mol (Na2O x 2B2O3 x 5H2O); borax 10 mol (Na2O x 2B2O3 x 10H2O); anhydrous boric acid (B2O3); and anhydrous borax (Na2O x 2B2O3). The particle size is large in bulk commercial products, such that they can be handled and stored without problems. However, inhalable dust particles, in the range of 20 microm (MMD), undergo hydration/dehydration rapidly owing to their high surface-to-volume ratio. The hydration state of a collected air sample was found to be strongly dependent on the conditions of relative humidity and temperature during its collection. As a consequence, the actual chemical species of dust being inspired cannot be identified accurately. Inhalable particles of borax 10 mol placed in a field personal monitoring cartridge and exposed to dry air at 2.0 L/min at 70 degrees F for 7 h undergo rapid dehydration, producing a sodium borate residue having significantly less than four waters of hydration. Likewise, inhalable particles of anhydrous boric acid and anhydrous borax were found to hydrate under normal atmospheric conditions. Borax 5 mol and boric acid were found to be stable to dehydration. In most cases, the specific borate species or borate compounds collected in a field monitor cannot be accurately characterized other than by their boron (B) content.     

Use of novel immobilized beta-galactosidase reactor to hydrolyze the lactose constituent of skim milk

beta-galactosidase from Aspergillus oryzae immobilized in an axial-annular flow reactor was used to effect the hydrolysis of the lactose component of skim milk. Nonlinear regression methods were employed to determine the kinetic parameters of four rate expressions derived from a proposed enzymatic mechanism. Data taken at three different temperatures (30 degrees C, 40 degrees C, and 50 degrees C) were fit via nonlinear regression methods assuming an Arrhenius temperature model for each of the parameters. For the reaction conditions used in this research, a three-parameter rate expression which includes the separate competitive inhibition effects of alpha- and beta-galactose (and the associated mutarotation reaction) is sufficient to model the hydrolysis of lactose in skim milk. The effects of temperature on the individual kinetic parameters are small. The most significant effect appears in the term for inhibition by the beta anomer of galactose (E(A) = 10.3 kcal/mol). At 40 degrees C and a space time of 10 min, 70% of the lactose present in skim milk can be hydrolyzed with the axial-annular flow reactor. This reactor can be used to hydrolyze the lactose in skim milk without the problems observed with other reactor configurations, namely, plugging due to particulates, microbial contamination, and large pressure drop.     

De- and rehydration behavior of alpha,alpha-trehalose dihydrate under humidity-controlled atmospheres

Effects of humidity were investigated on de- and rehydration behavior of alpha,alpha-trehalose dihydrate (T(h)) throughout simultaneous measurements of differential scanning calorimetry and X-ray diffractometry (DSC-XRD) and simultaneous thermogravimetry and differential thermal analysis (TG-DTA). When T(h) was heated from room temperature under dry nitrogen atmosphere, a metastable anhydrous crystal (T(alpha)) was formed at 105 degrees C after dehydration of T(h). The resulting T(alpha) melted at 125 degrees C and became amorphous, followed by cold crystallization from 150 degrees C giving rise to a stable anhydrous crystal T(beta). Under a highly humid atmosphere, on the other hand, T(beta) was formed at 90 degrees C directly as a result of T(h) dehydration. T(alpha) was readily rehydrated and turned back to T(h) when nitrogen gas with low water vapor pressure of 2.1kPa was admitted, whereas high water vapor pressure up to 7.4kPa was required for rehydration of T(beta) into T(h). This study provided a picture of pathways that link various solid forms of trehalose, taking into account the effects of a humid environment.     

Solvation of the left-handed hexamer d(5BrC-G-5BrC-G-5 BrC-G) in crystals grown at two temperatures

Crystals of the hexadeoxyoligomer d(5BrC-G-5BrC-G-5BrC-G) were grown at different temperatures (5 degrees C, 18 degrees C and 37 degrees C) in the absence of divalent cations. The crystals grown at 5 degrees C did not diffract X-rays, while those grown at 18 degrees C and 37 degrees C did. The oligomer adopts the left-handed ZI conformation in both crystals. The main difference resides in a more extensive hydration shell in the crystal grown at high temperature than in the crystal grown at low temperature. The high-temperature crystal displays a spine of hydration running deep in the minor groove and linking exocyclic O-2 atoms of the pyrimidine rings. In both crystalline forms, a hydrated sodium ion bound to the N-7 of a guanine ring was found. Strings of water molecules bridging phosphate anionic oxygen atoms are found along the backbone. The absolute values of the propeller-twist are also different in both structures although the values of the twist are very similar. The results point to the importance of the crystallization conditions when analysing fine structural details like solvation properties of oligomers.     

Amorphization of sugar hydrates upon milling

The possibility to amorphize anhydrous crystalline sugars, like lactose, trehalose and glucose, by mechanical milling was previously reported. We test here the possibility to amorphize the corresponding crystalline hydrates: lactose monohydrate, trehalose dihydrate and glucose monohydrate using fully identical milling procedures. The results show that only the first hydrate amorphizes while the other two remain structurally invariant. These different behaviours are attributed to the plasticizing effect of the structural water molecules which can decrease the glass transition temperature below the milling temperature. The results reveal clearly the fundamental role of the glass transition in the solid-state amorphization process induced by milling, and they also explain why crystalline hydrates are systematically more difficult to amorphize by milling than their anhydrous counterpart. The investigations have been performed by differential scanning calorimetry and powder X-ray diffraction.     

Development of a digital video-microscopy technique to study lactose crystallisation kinetics in situ

Polarised light microscopy was employed non-invasively to monitor lactose crystallisation from non-seeded supersaturated solutions in real time. Images were continuously recorded, processed and characterised by image analysis, and the results were compared with those obtained by refractometry. Three crystallisation temperatures (10, 20 and 30 degrees C) and three different levels of initial relative supersaturation (C/C(s)=1.95; 2.34; 3.15) were investigated. Induction times using the imaging technique proved to be substantially lower than those determined using refractive index. Lactose crystals were isolated digitally to determine geometrical parameters of interest, such as perimeter, diameter, area, roundness and Feret mean, and to derive crystal growth rates. Mean growth rates obtained for single crystals were fitted to a combined mass transfer model (R(2)=0.9766). The model allowed the effects of temperature and supersaturation on crystallisation rate to be clearly identified. It also suggested that, in this set of experiments, surface integration seemed to be the rate controlling step. It is believed that a similar experimental set-up could be implemented in a real food system to characterise a particular process where crystallisation control is of interest and where traditional techniques are difficult to implement.     

Survival of the biocontrol yeast Pichia anomala after long-term storage in liquid formulations at different temperatures, assessed by flow cytometry

AIMS: Investigate the survival of liquid formulations of the biocontrol yeast Pichia anomala J121 at different temperatures, and develop a system for comparative studies of different storage conditions and formulations. METHODS AND RESULTS: The survival of P. anomala in liquid formulations with lactose, starch and trehalose amendments was measured during prolonged storage at temperatures ranging from -20 to +30 degrees C. The relative survival of the stored cells was rapidly estimated by flow cytometry. After 4 weeks incubation at 4 and 10 degrees C, 75-90% of the cells were viable, with no significant differences between the various formulations. Supplementing the storage buffer with lactose or trehalose increased the survival after longer incubations (8 and 12 weeks) at all temperatures (-20 to 30 degrees C). Trehalose was the most effective protectant at 20 and 30 degrees C (>20% viable cells after 12 weeks at 20 degrees C). The biocontrol activity was maintained after formulation and prolonged storage of P. anomala. CONCLUSIONS: The storage potential of liquid formulated P. anomala cells can be increased by supplementation with lactose or trehalose. The combination of a custom made incubation chamber and flow cytometry was suitable to evaluate stability of P. anomala formulations. SIGNIFICANCE AND IMPACT OF THE STUDY: Liquid formulated P. anomala have a long shelf life. The developed test system can be used to study different formulations of other biocontrol agents.