The Concept of Microwave Foam Drying Under Vacuum: A Gentle Preservation Method for Sensitive Biological Material.

Microwave vacuum drying as compared to conventional vacuum drying has evinced advantages regarding drying time, while comparable product characteristics were achieved when drying sensitive biological material. Due to the volumetric microwave input, a time reduction of up to 90% is possible. When drying viscous liquids, a foamed structure that remains stable during drying exhibits further advantages as the diffusion-limited third drying step is enhanced by the porous structure. As foams not only have to be thermally resistant during microwave vacuum processing, but also withstand the vacuum, a specific process for foam drying by microwaves under low pressure conditions was developed. Foam formation and stabilization was achieved by using a synergistic mixture of proteins and carbohydrates; Lactobacillus paracasei ssp. paracasei F19 (L. paracasei) served as a model sensitive substance. Investigation of surface activity and foaming properties as a function of L. paracasei concentration revealed a significant positive contribution of the bacterial cells. It was shown that L. paracasei directly adsorbed at the air-water interface. Besides, a structuring of the liquid lamellae was assumed. Moreover, drying time was reduced to at least 50% compared to microwave vacuum drying without foaming. It was further observed that the slight loss in survival was mainly due to the relatively high moisture content and high vacuum levels at the beginning of the process. However, foaming, vacuum application, and final drying, respectively, did not affect viability of the bacterial cells. Thus, by incorporation of lactic acid bacteria into foam structures, drying can be carried out in a fraction of time, and further results in high-product quality. PRACTICAL APPLICATION: The application of continuous foam drying offers an efficient and energy-saving alternative to the currently applied techniques for the processing of sensitive material. The process could be applied for the preservation of starter cultures and probiotics as well as in the pharmaceutical industry, when sensitive material such as therapeutic proteins is dried. This process is especially suitable for freezing-sensitive and thermolabile substances.

Rheological properties of fresh and reconstituted milk protein concentrates under standard and processing conditions.

As the processability of fresh and reconstituted milk protein concentrates crucially depends on their rheological properties, a considerable amount of studies focuses on this topic. By means of a direct comparison, we are the first to clearly show that distinct rheological differences can exist between fresh and reconstituted milk protein concentrates under standard and processing conditions. We show that reconstituted milk protein concentrates made from commercial milk protein powders exhibit higher viscosities than fresh ones. Furthermore, we found that during intense shearing, the reconstituted milk protein concentrates undergo a loss of structure, which manifests itself in a significant viscosity decrease. The inverse effect can be observed for fresh milk protein concentrates. Besides these differences, the reconstituted milk protein concentrates exhibit gel-like properties above a certain protein content. We attribute these observations to protein-protein interactions in the milk protein powder, which are induced by manufacturing and/or storing conditions. Our results demonstrate that rheological properties of fresh and reconstituted milk protein concentrates are quantitatively not invariably interchangeable. Thus, the purpose of this article is to emphasize the necessity for researchers and engineers to take into account the rheological particularities of different milk protein concentrates prior to usage.

Protective effect of sugars on storage stability of microwave freeze-dried and freeze-dried Lactobacillus paracasei F19.

AIMS: Microwave freeze drying (MWFD) in comparison with conventional freeze drying allows for intensification of the preservation process of lactic acid bacteria without imposing additional processing stress. Viability as a function of storage time of microwave freeze-dried Lactobacillus paracasei ssp. paracasei F19 was investigated in comparison to conventionally lyophilized bacteria of the same strain. Furthermore, the impact of the protectants, sorbitol, trehalose and maltodextrin, on shelf life was analysed. METHODS AND RESULTS: The highest inactivation rates of 0·035 and 0·045 day-1 , respectively, were found for cultures without protectants. Thus, all additives were found to exhibit a protective effect during storage with inactivation rates between 0·015 and 0·040 day-1 . Although trehalose and maltodextrin samples were in the glassy state during storage, in contrast to samples containing sorbitol as protectant, the best protective effect could be found for sorbitol with the lowest inactivation rate of 0·015 day-1 . CONCLUSIONS: Due to its low molecular weight, it might protect cells owing to better adsorption to the cytoplasma membrane. Sorbitol additionally shows antioxidative properties. Storage behaviour of microwave freeze-dried cultures follows the typical behaviour of a product dried by conventional lyophilization. No significant influence of the drying technique on storage behaviour was detected. SIGNIFICANCE AND IMPACT OF THE STUDY: General findings concerning storage behaviour in freeze drying are likely to be applicable in MWFD with only slight adjustments.

Influence of spore and carrier material surface hydrophobicity on decontamination efficacy with condensing hydrogen peroxide vapour.

AIMS: To investigate the influence of surface hydrophobicity of carrier material (CM) and bacterial spores of Bacillus subtilis SA 22 and Bacillus atrophaeus (DSM 675) on spore inactivation with condensing hydrogen peroxide (H2 O2 ) gas. METHODS AND RESULTS: Surface hydrophobicity of bacterial spores and CM was determined by means of contact angle measurement. Spores of B. subtilis showed water contact angles of 90°, spores of B. atrophaeus showed water contact angles of 42°. Above that, a resistance test against liquid H2 O2 at room temperature was conducted with resulting DH2O2 values of 101 s (B. subtilis) and 906 s (B. atrophaeus). The spores were deposited on CMs of different wettability (water contact angles of 115-30°). The spores were applied either individually or as an 1 : 1 mixture of both spore species. Exposure of biological indicators (BI) with 5200 ppm of gaseous H2 O2 at 70°C treatment temperature for defined times up to 10 s led to inactivation kinetics. Surfaces with high hydrophobicity showed faster spore inactivation than surfaces with lower hydrophobicity for single-spore species. Regarding the mixed BI, better survival of hydrophobic spores was expected. However, this effect can only be seen as a slight trend and is not significant after 10 s. CONCLUSIONS: Surface hydrophobicity of CMs does influence the decontamination with gaseous, condensing H2 O2 . However, surface hydrophobicity of spores in a mixed population does only have a small influence on inactivation results. SIGNIFICANCE AND IMPACT OF THE STUDY: The intensity of spore inactivation depends more on other factors than on the wettability of the bacterial spores. However, hydrophobic surfaces lead to faster inactivation effects and should thus be preferred for aseptic packaging technology.

Water ingress into a casein film quantified using time-resolved neutron imaging.

The migration of water into a casein film was probed with neutron radiography. From the neutron transmission images, the evolution of the water saturation profiles was extracted. The results indicate that the water influx is dominated by imbibition but also contains a diffusional component. The time dependence of the water ingress was quantified using a diffusion-like equation previously also applied to imbibition. A water transport coefficient D = 0.9 × 10(-9) m(2) s(-1) was found. This value and direct observation of the images indicate that the time taken for a typical adhesive casein-based layer to become saturated with water is of the order of hours.

Structural changes of deposited casein micelles induced by membrane filtration.

Casein micelles undergo shape changes when subjected to frontal filtration forces. Grazing incidence small angle X-ray scattering (GISAXS) and atomic force microscopy (AFM) allow a quantification of such structural changes on filtration cakes deposited on smooth silicon micro-sieves. A trans-membrane pressure of deltap = 400 mbar across the micro-sieve leads to an immediate film formation after deposition of casein solution. We observe significant changes in the GISAXS pattern depending on how many layers are stacked on top of each other. Compared to a deposit formed by one layer, GISAXS on a deposit formed by three layers of casein micelles leads to less scattering in the vertical and more scattering in the horizontal direction. Simulations show that the experimental results can be interpreted by a structural transformation from an originally spherical micelle shape to an ellipsoidal-deformed shape. The results are supported by AFM measurements showing a reduced lateral size of casein micelles deposited on top of a membrane pore. The observed shape changes could be due to filtration forces acting on densely packed deposits confining the micelles into ellipsoidal shapes.

Effect of membrane length, membrane resistance, and filtration conditions on the fractionation of milk proteins by microfiltration.

We investigated the fractionation of casein micelles and the whey protein ß-lactoglobulin (ß-LG) of skim milk by crossflow microfiltration (0.1 µm) for the first time by a novel approach as a function of membrane length and membrane resistance. A special module was constructed with 4 sections and used to assess the effects of membrane length by measuring flux and ß-LG permeation (or transmission) as a function of transmembrane pressure and membrane length. Depending on the position, the membranes were partly controlled by a deposit layer. A maximum for ß-LG mass flow through the various membrane sections was found, depending on the position along the membrane. To study the effect of convective flow toward the membrane, membranes with 4 different intrinsic permeation resistances were assessed in terms of the permeation and fouling effects along the flow channel. From these findings, we derived a ratio between transmembrane pressure and membrane resistance, which was useful in reducing the effect of deposit formation and, thus, to optimize the protein permeation. In addition, the fouling effect was investigated in terms of reversible and irreversible fouling and, in addition, by differentiation between pressure-induced fouling and adsorption-induced (pressure-independent) fouling, again as a function of membrane length.

Combined influence of fermentation and drying conditions on survival and metabolic activity of starter and probiotic cultures after low-temperature vacuum drying.

The influence of low temperature vacuum drying process parameters on the survival, metabolic activity and residual water content of three different bacterial strains (Lactobacillus paracasei ssp. paracasei, Lactobacillus delbrueckii ssp. bulgaricus and Bifidobacterium lactis) was investigated. Shelf temperature and chamber pressure were varied and optimized by response surface methodology with regard to survival and residual water content. It is shown that the survival rate after low temperature vacuum drying is comparable to that of freeze drying. Based on the optimization experiments the combined influence of fermentation pH and drying process parameters was studied for the most detrimental and the best process condition, respectively. The results show that interactions between process and fermentation conditions have to be taken in account and that these influences are highly strain specific.

Influence of the surface temperature of packaging specimens on the inactivation of Bacillus spores by means of gaseous H(2) O(2).

AIMS: To determine the influence of condensation as a function of the surface temperature of aseptic packaging, on the inactivation of Bacillus spores [Bacillus subtilis (DSM 347), B. subtilis SA22, Bacillus atrophaeus] having different surface properties by means of vaporized H(2) O(2). METHODS AND RESULTS: The packaging specimens inoculated with Bacillus spores were tempered and subsequently exposed to H(2) O(2) -vapour. During the exposure, surface temperature curves were measured and the spore survival was determined. Results showed that decreasing the initial surface temperature of the packaging specimens had a positive effect on the sporicidal activity of H(2) O(2) -vapour, where the effect was less pronounced for less hydrophilic spores. The surfaces of spores were characterized by means of the water contact angle. CONCLUSIONS: For starting surface temperatures below the dew point temperature of the sterilant gas, the condensation of highly concentrated liquid H(2) O(2) on the packaging surface accelerates the killing of the spores, while the inferior wettability of more hydrophobic spores compared to more hydrophilic ones diminishes the effect. SIGNIFICANCE AND IMPACT OF THE STUDY: Regarding industrial packaging sterilization, a mixed microflora has to be inactivated. Promoting the condensation of H(2) O(2) improves in general the killing of different species of spores, however, at various degrees depending on the wettability of spores.

Structural characterization of casein micelles: shape changes during film formation.

The objective of this study was to determine the effect of size-fractionation by centrifugation on the film structure of casein micelles. Fractionated casein micelles in solution were asymmetrically distributed with a small distribution width as measured by dynamic light scattering. Films prepared from the size-fractionated samples showed a smooth surface in optical microscopy images and a homogeneous microstructure in atomic force micrographs. The nano- and microstructure of casein films was probed by micro-beam grazing incidence small angle x-ray scattering (µGISAXS). Compared to the solution measurements, the sizes determined in the film were larger and broadly distributed. The measured GISAXS patterns clearly deviate from those simulated for a sphere and suggest a deformation of the casein micelles in the film.

Kinetics of formation and physicochemical characterization of thermally-induced beta-lactoglobulin aggregates.

The kinetics of heat denaturation and aggregation for beta-lactoglobulin dispersions (5% w/v) were studied at 3 pHs (6, 6.4, and 6.8) and at a heating temperature of 80 degrees C. Protein aggregates were characterized for hydrodynamic diameter, microstructure, and molecular weight by means of dynamic light scattering, transmission electron microscopy, and polyacrylamide gel electrophoresis, respectively. Concentration of native beta-lactoglobulin decreased with holding time and with a decrease in the pH. Apparent rate constants were calculated for beta-lactoglobulin denaturation applying the general kinetic equation solved for a reaction order of 1.5. Values of the apparent reaction rate constant k = 7.5, 6.3 and 5.6 x 10(-3) s(-1) were found for pH 6, 6.4, and 6.8, respectively. Decreasing the pH of the dispersions produced higher aggregate sizes. After a holding time of 900 s, average hydrodynamic diameters for beta-lactoglobulin aggregates at pH 6, 6.4, and 6.8 were 96, 49, and 42 nm, respectively. These results were confirmed by transmission electron microscopy images, where a shift in the size and morphology of aggregates was found, from large and spherical at pH 6 to smaller and linear aggregates at pH 6.8. beta-Lactoglobulin formed disulfide-linked intermediates (dimers, trimers, tetramers) and so on) which then formed high molecular weight aggregates. From the results obtained by DLS, TEM, and SDS-PAGE a mechanism for beta-lactoglobulin aggregation was proposed. This study shows that heat treatment can be used to produce protein aggregates with different sizes and morphologies to be utilized as ingredients in foods.

1H NMR investigation on the role of sorbitol for the survival of Lactobacillus paracasei ssp. paracasei in vacuum-dried preparations.

AIMS: The aim of this work was to study the effect of sorbitol as protective agent on the survival of the probiotic strain Lactobacillus paracasei ssp. paracasei (F19) after vacuum drying. METHODS AND RESULTS: The survival was studied after different drying times and for various concentrations of sorbitol by plate count method. Furthermore, time domain 1H NMR studies on dehydrated suspensions of Lact. paracasei ssp. paracasei were performed to study the proton mobility in the dried samples. From the obtained signal, T2 relaxation times of single components and fractions with different proton mobility were determined. It was found out that the survival is increased by the presence of a minimum amount of sorbitol that is dependent on drying time. Furthermore, it is shown that the protective effect can only be observed below a critical water content of c. 20%. Nuclear magnetic resonance (NMR) results indicate a transition of sorbitol from liquid to solid like behaviour during drying. The onset of the transition coincides with the critical water content found for a protective effect. CONCLUSIONS: The data suggest that sorbitol protons are incorporated into the dried cells of Lact. paracasei ssp. paracasei (F19) below the critical water content and therefore leading to an enhanced survival. SIGNIFICANCE AND IMPACT OF THE STUDY: The results help to better understand the underlying mechanism of protection of Lact. paracasei ssp. paracasei using sorbitol and to establish vacuum drying as potential alternative drying technique to standard freeze drying.

Changes in membrane fatty acids of Lactobacillus helveticus during vacuum drying with sorbitol.

AIMS: To examine changes in membrane fatty acid profile attributed to the physiological adaptation of Lactobacillus helveticus during vacuum drying. METHODS AND RESULTS: The viability and membrane integrity of the cells after vacuum drying were measured by plate counts and DNA fluorescence dyes. The physiological adaptation of cells dried in the presence of sorbitol was observed by determining changes in membrane fatty acid composition using gas chromatography. Results showed that viability and membrane integrity of Lact. helveticus cells increased when drying in the presence of sorbitol. The occurrence of the very low melting point polyunsaturated fatty acids linoleic and arachidonic acid was observed in cells dried in the presence of sorbitol. CONCLUSIONS: The physiological adaptation of cells occurred with cell membrane of Lact. helveticus during vacuum drying of cells in the presence of sorbitol. SIGNIFICANCE AND IMPACT OF THE STUDY: The study showed that physiological adaptation with membrane of the cells occurred during the drying process. The insight implies that instead of viability improvement of dried cells by the conventional stress induction during cultivation, the induction may be exercised thereafter without compromising growth of the cells.

Hydration behavior of casein micelles in thin film geometry: a GISANS study?

The water content of casein micelle films in water vapor atmosphere is investigated using time-resolved grazing incidence small-angle neutron scattering (GISANS). Initial dry casein films are prepared with a spin-coating method. At 30 degrees C, the formation of a water-equilibrated casein protein film is reached after 11 min with a total content of 0.36 g of water/g of protein. With increasing water vapor temperature up to 70 degrees C, an increase in the water content is found. With GISANS, lateral structures on the nanometer scale are resolved during the swelling experiment at different temperatures and modeled using two types of spheres: micelles and mini-micelles. Upon water uptake, molecular assemblies in the size range of 15 nm (mini-micelles) are attributed to the formation of a high-contrast D2O outer shell on the small objects that already exist in the protein film. For large objects (>100 nm), the mean size increases at high D2O vapor temperature because of possible aggregation between hydrated micelles. These results are discussed and compared with various proposed models for casein micelle structures.

Inactivation mechanisms of lactic acid starter cultures preserved by drying processes.

The preservation of lactic acid starter cultures by drying are of increased interest. A further improvement of cell viability is, however, still needed, and the insight into inactivation mechanisms of the cells is a prerequisite. In this present work, we review the inactivation mechanisms of lactic acid starter cultures during drying which are not yet completely understood. Inactivation is not only induced by dehydration inactivation but also by thermal- and cryo-injuries depending on the drying processes employed. The cell membrane has been reported as a major site of damage during drying or rehydration where transitions of membrane phases occur. Some drying processes, such as freeze drying or spray drying, involve subzero or very high temperatures. These physical conditions pose additional stresses to cells during the drying processes. Injuries of cells subjected to freezing temperatures may be due to the high electrolyte concentration (solution effect) or intracellular ice formation, depending on the cooling rate. High temperatures affect most essential cellular components. It is difficult to identify a critical component, although ribosomal functionality is speculated as the primary reason. The activation during storage is mainly due to membrane lipid oxidation, while the storage conditions such as temperature moisture content of the dried starter cultures are important factors.

Damage of cell envelope of Lactobacillus helveticus during vacuum drying.

AIMS: The aim of this study was to gain insight into the inactivation mechanisms of Lactobacillus helveticus during vacuum drying. METHODS AND RESULTS: Early stationary phase cells of L. helveticus were dried in a vacuum drier. Viability, cell integrity and metabolic activity of cells were assessed over time by plate counts on de Man Rogosa and Sharpe broth agar medium and cytological methods employing fluorescent reagents and nucleic acid stains. The cell envelope damage was visualized by atomic force microscopy (AFM). Fourier transform infrared spectroscopy (FT-IR) was used to indirectly observe changes in cell components during drying. Viability, metabolic activity and cell integrity decreased during vacuum drying, and different inactivation curves, characterized by the loss of ability to resume growth, and cell injuries were found. AFM images showed cracks on the surface of dried cells. Main changes in FT-IR spectra were attributed to the damage in cell envelope. CONCLUSION: The cell envelope was the main site of damage in L. helveticus during vacuum drying. SIGNIFICANCE AND IMPACT OF THE STUDY: Inactivation mechanisms of L. helveticus during vacuum drying were partly elucidated. This information is useful for the improvement of the viability of vacuum-dried starter cultures.

Effect of carbohydrates on the survival of Lactobacillus helveticus during vacuum drying.

AIMS: To assess four carbohydrates for the protective effect against Lactobacillus helveticus cells inactivation during vacuum drying, and to study the effect of selected carbohydrate on changes of inactivation kinetics. METHODS AND RESULTS: Early stationary phase L. helveticus cells grown in MRS media were recovered from fermentation broth, washed with PBS buffer (pH 7.0), and then mixed with different concentrations of four carbohydrates, namely lactose, sorbitol, inulin, and xanthan gum. Cells were dried in a vacuum drier at 100 mbar, 43 degrees C for 12 h. Only cells with 1% sorbitol addition showed higher survival (18%) over cells without added carbohydrate (8%). Using in situ microbalance technique whereby cell weight during vacuum drying was continuously monitored via precision balances integrated into the vacuum chamber, drying and inactivation kinetics of cells and cells mixed with sorbitol were established. CONCLUSION: Survival of L. helveticus during the vacuum drying could be improved by the addition of optimal concentration of 1% sorbitol. Addition of sorbitol did not cause drastic changes in drying rate, water content and water activity of samples. The protection mechanisms of sorbitol seemed not to be due to a direct physical effect, which could be related to drying rate. SIGNIFICANCE AND IMPACT OF THE STUDY: The increase in survival of cells after vacuum drying by the addition of a protective carbohydrate may provide an alternative mean to preserve starter cultures at a higher level of activity.

Pressure-induced dissociation of casein micelles: size distribution and effect of temperature.

Pressure-induced dissociation of a turbid solution of casein micelles was studied in situ in static and dynamic light scattering experiments. We show that at high pressure casein micelles decompose into small fragments comparable in size to casein monomers. At intermediate pressure we observe particles measuring 15 to 20 nm in diameter. The stability against pressure dissociation increased with temperature, suggesting enhanced hydrophobic contacts. The pressure transition curves are biphasic, compatible with a temperature (but not pressure)-dependent conformational equilibrium of two micelle species. Our thermodynamic model predicts an increase in structural entropy with temperature.

Pressure-induced dissociation of casein micelles: size distribution and effect of temperature

Pressure-induced dissociation of a turbid solution of casein micelles was studied in situ in static and dynamic light scattering experiments. We show that at high pressure casein micelles decompose into small fragments comparable in size to casein monomers. At intermediate pressure we observe particles measuring 15 to 20 nm in diameter. The stability against pressure dissociation increased with temperature, suggesting enhanced hydrophobic contacts. The pressure transition curves are biphasic, compatible with a temperature (but not pressure)-dependent conformational equilibrium of two micelle species. Our thermodynamic model predicts an increase in structural entropy with temperature.

[Intubation with the Combitube-TM in massive hemorrhage from the locus Kieselbachii]. / Intubation mit dem Combitube-TM bei massiver Blutung aus dem Locus Kieselbachii.

Massive nasal haemorrhage occurred during an attempted nasal endotracheal intubation in a 52-year old patient, scheduled for bone grafting to the mandibula. Ventilation of the patient by face mask and conventional endotracheal intubation by laryngoscopy were not possible due to massive bleeding. This situation was successfully managed by the use of a Combitube. The role of the Combitube in difficult airway management as well as its extensive contraindications are discussed. Rare complications when using the Combitube, their diagnosis and treatment are mentioned.