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Concept: Freeze drying


Time of Flight secondary ion mass spectrometry (TOF-SIMS) has been used to explore the distribution of phospholipids in the plasma membrane of Tetrahymena pyriformis during cell division. The dividing cells were freeze dried prior to analysis followed by line scan and region of interest analysis at various stages of cell division. The results showed no signs of phospholipid domain formation at the junction between the dividing cells. Instead the results showed that the sample preparation technique had a great impact on one of the examined phospholipids, namely phosphatidylcholine (PC). Phosphatidylcholine and 2-aminoethylphosphonolipid (2-AEP) have therefore been evaluated in Tetrahymena cells that have been subjected to different sample preparation techniques: freeze drying ex situ, freeze fracture, and freeze fracture with partial or total freeze drying in situ. The result suggests that freeze-drying ex situ causes the celia to collapse and cover the plasma membrane.

Concepts: Cell membrane, Food preservation, Freeze drying, Vacuum, Sublimation


In spray freeze drying (SFD) solutions are frozen by spraying into a very cold environment and subsequently dried by sublimation. In contrast to conventional freeze drying, spray freeze drying has the possibility to produce flowable lyophilizates which offers a variety of new pharmaceutical applications. Here, a drop jet nozzle is proposed as liquid dispenser that is able to produce droplets with a very narrow size distribution compared to standard methods. The drop jet nozzle is mounted in a spray tower designed to prevent direct contact of the product with the freezing medium. Various formulations have been tested containing lysozyme as model protein and stabilizers such as bovine serum albumin, polyvinylpyrrolidone or dextran in various concentrations and mannitol. Excellent free flowing and nearly monodispersed, porous particles are produced where particle properties can be controlled by formulation and process conditions. The particle diameter varied between 231±3μm and 310±10μm depending on the formulation composition. The lysozyme activity was >94±5% for all formulations exhibiting a full preservation of enzyme activity. This new method is very promising for the production of nearly monodisperse particulate lyophilizates in various therapeutic applications.

Concepts: Enzyme, Serum albumin, Liquid, Bovine serum albumin, Lysozyme, Food preservation, Freeze drying, Sublimation


Polymeric micelles were studied as delivery carriers of diazepam, a practically insoluble drug in water, for rectal administration. The diazepam-loaded polymeric micelles were developed by using poloxamer 407 (P407), poloxamer 188, and D-α-tocopheryl poly(ethylene glycol) 1000 succinate (TPGS). Among the used polymers, TPGS resulted in polymeric micelles with good characteristics for encapsulation of diazepam which had the small particle size of 8-12 nm and narrow size distribution (PI 0.053-0.275). Additionally, 7.5% w/v of TPGS could entirely entrap the desired concentration of diazepam (5 mg/mL). To improve the physical stability upon lyophilization, an addition of P407 of 1% w/v prevented aggregation, increased physical stability, and maintained chemical stability of the lyophilized powders of diazepam-loaded polymeric micelles for 3 months storage at 4°C. The rate and amount of diazepam release from TPGS polymeric micelles mainly depended on the concentration of TPGS. The release data were fitted to Higuchi’s model suggesting that the drug release mechanism was controlled by Fickian diffusion. In conclusion, 10% w/v TPGS and 1% w/v P407 were the optimum formulation of lyophilized diazepam-loaded polymeric micelles.

Concepts: Polymer, Particle size distribution, Solution, Freeze drying, Sublimation, Polymers, Poloxamer, Poloxamer 407


Polyethylene glycol (PEG)-based low generation dendrimers are analyzed as single excipient or combined with trehalose in relation to their structure and efficiency as enzyme stabilizers during freeze-thawing, freeze-drying, and thermal treatment. A novel functional dendrimer (DGo -CD) based on the known PEG’s ability as cryo-protector and β-CD as supramolecular stabilizing agent is presented. During freeze-thawing, PEG and β-CD failed to prevent catalase denaturation, while dendrimers, and especially DGo -CD, offered the better protection to the enzyme. During freeze-drying, trehalose was the best protective additive but DGo -CD provided also an adequate catalase stability showing a synergistic behavior in comparison to the activities recovered employing PEG or β-CD as unique additives. Although all the studied dendrimers improved the enzyme remaining activity during thermal treatment of freeze-dried formulations, the presence of amorphous trehalose was critical to enhance enzyme stability. The crystallinity of the protective matrix, either of PEG derivatives or of trehalose, negatively affected catalase stability in the freeze-dried systems. When humidified at 52% of relative humidity, the dendrimers delayed trehalose crystallization in the combined matrices, allowing extending the protection at those conditions in which normally trehalose fails. The results show how a relatively simple covalent combination of a polymer such as PEG with β-CD could significantly affect the properties of the individual components. Also, the results provide further insights about the role played by polymer-enzyme supramolecular interactions (host-guest crosslink, hydrogen bonding, and hydrophobic interactions) on enzyme stability in dehydrated models, being the effect on the stabilization also influenced by the physical state of the matrix. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 2013.

Concepts: Stabilizer, Chemistry, Hydrogen bond, Relative humidity, Supramolecular chemistry, Freeze drying, Trehalose, Dendrimer


As liquid liposomal formulations are prone to chemical degradation and aggregation, these formulations often require freeze drying (e.g. lyophilization) to achieve sufficient shelf-life. However, liposomal formulations may undergo oxidation during lyophilization and/or during prolonged storage. The goal of the current study was to characterize the degradation of 1, 2-dilinolenoyl-sn-glycero-3-phosphocholine (DLPC) during lyophilization, and to also probe the influence of metal contaminants in promoting the observed degradation. Aqueous sugar formulations containing DLPC (0.01 mg/ml) were lyophilized, and DLPC degradation was monitored using HPLC/UV and GC/MS methods. The effect of ferrous ion and sucrose concentration, as well as lyophilization stage promoting lipid degradation, was investigated. DLPC degradation increased with higher levels of ferrous ion. After lyophilization, 103.1% ± 1.1%, 66.9% ± 0.8%, and 28.7% ± 0.7% DLPC remained in the sucrose samples spiked with 0.0 ppm, 0.2 ppm and 1.0 ppm ferrous ion, respectively. Lipid degradation predominantly occurs during the freezing stage of lyophilization. Sugar concentration and buffer ionic strength also influence the extent of lipid degradation, and DLPC loss correlated with degradation product formation. We conclude that DLPC oxidation during the freezing stage of lyophilization dramatically compromises the stability of lipid-based formulations. In addition, we demonstrate that metal contaminants in sugars can become highly active when lyophilized in the presence of a reducing agent.

Concepts: Iron, Hydrogen, Electrochemistry, Chemistry, Food preservation, Freeze drying, Vacuum, Sublimation


The aims of this study were to determine the stability of Podoviridae coliphage CA933P during lyophilization and storage in different media, and to establish similarities between the results obtained and those expected through mechanisms described for proteins stabilization during freeze-drying. PBS and SM buffer were assayed as lyophilization media. The effect of inorganic salts concentration as well as the addition of disaccharides on phage stability during freeze-drying and storage was also studied. The addition of low sucrose concentration (0.1 mol l(-1)) to SM buffer stabilized phage during freezing and drying steps of the lyophilization process, but higher sugar concentrations were detrimental to phage stability during freeze-drying. Sucrose stabilized phage during storage for at least 120 days. The lyoprotective effect of low concentrations of disaccharides during the drying step of the lyophilization of proteins as well as the stabilization of the freeze-dried product in time correlated with the results obtained for phage CA933P.

Concepts: Food preservation, Freeze drying, Trehalose, Vacuum, Sublimation


Micellar electrokinetic capillary chromatography with electrochemical detection has been used to quantify biogenic amines in freeze-dried Drosophila melanogaster brains. Freeze drying samples offers a way to preserve the biological sample while making dissection of these tiny samples easier and faster. Fly samples were extracted in cold acetone and dried in a rotary evaporator. Extraction and drying times were optimized in order to avoid contamination by red-pigment from the fly eyes and still have intact brain structures. Single freeze-dried fly-brain samples were found to produce representative electropherograms as a single hand-dissected brain sample. Utilizing the faster dissection time that freeze drying affords, the number of brains in a fixed homogenate volume can be increased to concentrate the sample. Thus, concentrated brain samples containing five or fifteen preserved brains were analyzed for their neurotransmitter content, and five analytes; dopamine N-acetyloctopamine, N-acetylserotonin, N-acetyltyramine, N-acetyldopamine were found to correspond well with previously reported values.

Concepts: Micellar electrokinetic chromatography, Food preservation, Freeze drying, Vacuum, Sublimation


Abstract Purpose: This paper presents a soft sensor that can be effectively used for in-line monitoring of the primary drying step of a pharmaceuticals freeze-drying process in vials. Methods: Process modeling and product temperature measurements are used to estimate the residual amount of ice in the vial and the heat transfer coefficient from the shelf to the product in the vial. The resistance of the dried cake to vapor flow is determined through the heat balance equation at the interface of sublimation. Mathematical simulation and experimental tests have been carried out to validate the estimations provided by the soft sensor. Results: Accurate estimations of the dynamics of the product until the end of primary drying are obtained, as well as of the heat and mass transfer coefficients, even in the case of a highly non-uniform batch. The reduction in the number of variables directly estimated by the soft sensor allows increasing the robustness of the tool with respect to other sensors presented in the literature. Conclusions: The proposed soft sensor is thus effective for process monitoring and it allows using model-based tools for cycle development in lab-scale units, where thermocouples are usually available, and for process monitoring in industrial-scale freeze-dryers, in case wireless sensors are used.

Concepts: Mathematics, Thermodynamics, Heat, Estimation, Pharmacy, Heat transfer, Sensors, Freeze drying


Red beet plants are rich in betalains that can be used as food natural colorants. Betalains were extracted from red beet and encapsulated with different carrier agents and freeze or spray dried. Effect of different encapsulating agents as maltodextrin, guar gum, gum Arabic, pectin and xanthan gum with different concentration (as encapsulating agents) were studied on the betalain stability. Encapsulated betalains with xanthan gum with maltodextrin showed about 65 % more recovery than the control. Encapsulation showed a higher recovery of betalains during freeze drying by 1.3 times than during spray drying. Spray dried samples has L* (lightness) higher than the freeze dried samples. The variations of maltodextrin with xanthan and guar gum freeze dried have highest chroma value of 21. The stabilization of pure betalain pigments may boost the use of these colouring molecules in the food industry and promote their application.

Concepts: E number, Food preservation, Freeze drying, Beet, Gums, Betalain, Guar gum, Instant coffee


The production of long shelf-life highly concentrated dried probiotic/starter cultures is of paramount importance for the food industry. The aim of the present study was to evaluate the protective effect of glucose, lactose, trehalose, and skim milk applied alone or combined upon the survival of potentially probiotic Lactobacillus rhamnosus CTC1679, Lactobacillus casei/paracasei CTC1677 and L. casei/paracasei CTC1678 during freeze-drying and after 39 weeks of storage at 4 and 22 °C. Immediately after freeze-drying, the percentage of survivors was very high (≥94%) and only slight differences were observed among strains and cryoprotectants. In contrast, during storage, survival in the dried state depended on the cryoprotectant, temperature and strain. For all the protectants assayed, the stability of the cultures was remarkably higher when stored under refrigeration (4 °C). Under these conditions, skim milk alone or supplemented with trehalose or lactose showed the best performance (reductions ≤0.9 log units after 39 weeks of storage). The lowest survival was observed during non-refrigerated storage and with glucose and glucose plus milk; no viable cells left at the end of the storage period. Thus, freeze-drying in the presence of appropriate cryoprotectants allows the production of long shelf-life highly concentrated dried cultures ready for incorporation in high numbers into food products as starter/potential probiotic cultures.

Concepts: Milk, Food, Probiotic, Food preservation, Freeze drying, Trehalose, Lactobacillus rhamnosus, Cryoprotectant