Some observations in freeze-drying of recombinant bioluminescent Escherichia coli for toxicity monitoring

A recombinant bioluminescent bacteria, containing a fabA::luxCDABE fusion gene, has been used to characterize freeze-drying methods, which may be conveniently used as a tool for the development of a portable biosensor. Through residual water, viability, biosensing activity and scanning electron microscopy analyses, the characteristics that four cryoprotectants, trehalose, sucrose, sorbitol, and mannitol, conferred on freeze-dried samples were elucidated, including the morphology, water content and activity of the cells. It was found that trehalose showed the best freeze-drying efficiency among the tested cryoprotectants and it might have a specific capacity limitation in protection of the cells during the freeze step. Humidity might result in damage to the cells, according to the viability, when exposed to air during storage, while the water remaining post freeze-drying showed good correlation with damage to the freeze-dried cells when under air-tight storage conditions. The results with other recombinant bioluminescent bacteria indicated that these findings might be general features of the freeze-drying processes.     

Optimisation of initial cell concentration enhances freeze-drying tolerance of Pseudomonas chlororaphis

The freeze-drying tolerance of Pseudomonas chlororaphis, an antifungal bacterium used as biocontrol agent was investigated. P. chlororaphis is freeze-drying sensitive and the viability drops more than 3 log units in the absence of protective freeze-drying medium. Of the freeze-drying media tested, lactose, sucrose, trehalose, glutamate, sucrose with glutamate, skimmed milk, and skimmed milk with trehalose, skimmed milk gave the lowest survival (0.6+/-0.2%) and sucrose the highest (6.4+/-1.2%). Cellular accumulation of sucrose from the freeze-drying medium and the protective effect of sucrose were dependent on sucrose concentration. The effect of initial cell concentration, from 1 x 10(7) to 5 x 10(10) CFU/ml, on survival after freeze-drying was studied for carbon starved cells with sucrose as freeze-drying medium. The highest freeze-drying survival values, 15-25%, were obtained for initial cell concentrations between 1 x 10(9) and 1 x 10(10) CFU/ml. For cell concentrations outside this window more than 10 times lower survival values were observed. P. chlororaphis was cultivated to induce stress response that could confer protection against freeze-drying inactivation. Carbon starvation and, to a lesser extent, heat treatment enhanced freeze-drying tolerance. By combining optimal cell concentration, optimal sucrose concentration and carbon starvation the survival after freeze-drying was 26+/-6%.     

The effects of freeze-drying on bacteriophage T4

The damage by freeze-drying to bacteriophage T4 was analysed in order to locate the site and the mechanism of damage. As a result of freeze-drying of bacteriophage T4, its head coat was damaged so as to lead to the loss of the DNA and emptying of the head. The tail assembly was generally undamaged and the freeze-dried phage preserved the biological activities concerned with absorption and injection (inhibition of host colony formation, inhibition of induction of beta-galactosidase, induction of changes in the potassium content in the host bacteria).The 2 mechanisms by which the freeze-drying damages the phage arc: osmotic shock, which occurs mainly during the resuspension of the FD phage, and the drying per se, i.e., the removal of water from the head protein.     

不同初始含水率下粘质土壤的入渗过程

土壤入渗是降雨渗入土体形成土壤水的基本水文过程,土壤渗透能力影响着地表径流和土壤侵蚀强度。土壤初始含水量决定了入渗初期的土水势,是影响土壤入渗过程的重要因素。利用环刀法,观测了三峡库区林地和草地的土壤入渗过程,对比分析了不同初始含水率下土壤入渗率和常用入渗模型的适宜性。结果表明,随土壤初始含水率的增大,林地和草地下土壤初始入渗率减小,入渗趋于稳定所需时间缩短,累积入渗量和稳定入渗率增大。土壤含水率为12%的林地初始入渗率为8.95 mm/min,是含水率40%林地初始入渗率的4倍,但1h累积入渗量仅是含水率40%林地的2/3。有机质含量丰富的草地土壤入渗过程对初始含水率的敏感性较弱,干湿草地相比较入渗参数的差异不如林地明显。随时间的延长,土壤入渗率逐渐降低,入渗曲线渐趋平缓,最小二乘法拟合结果显示Horton模型对林地和草地下土壤入渗过程的拟合效果较好,且模型参数具有物理意义,是分析和预测三峡库区林草覆盖下土壤入渗过程的适宜模型。     

Effect of Drying Medium on Residual Moisture Content and Viability of Freeze-Dried Lactic Acid Bacteria

The effect of various substances on the relationship between residual moisture content and the viability of freeze-dried lactic acid bacteria has been studied. Compounds such as polymers, which display considerable ability in displacing water, showed no protective action during freeze-drying. Adonitol, on the other hand, produced the smallest change in water content at various times during drying and allowed the highest rate of survival.     

Effect of culture age, protectants, and initial cell concentration on viability of freeze-dried cells of Metschnikowia pulcherrima

The effect of freeze-drying using different lyoprotectants at different concentrations on the viability and biocontrol efficacy of Metschnikowia pulcherrima was evaluated. The effects of initial yeast cell concentration and culture age on viability were also considered. Yeast cells grown for 36 h were more resistant to freeze-drying than were 48 h cells. An initial concentration of 10? cells·mL?1 favoured the highest survival after freeze-drying. When maltose (25%, m/v) was used as protectant, a high cell viability was obtained (64.2%). Cells maintained a high viability after 6 months of storage at 4 °C. The biocontrol efficacy of freeze-dried cells was similar to the activity of fresh cells on 'Gala' apples and was slightly lower on 'Golden Delicious' apples. After optimizing freeze-drying conditions, the viability of M. pulcherrima cells was similar to that obtained in other studies. The results constitute a first step towards the commercial development of M. pulcherrima as a biocontrol agent.     

Freeze-drying of live attenuated Vibrio anguillarum mutant for vaccine preparation

Vibrio anguillarum MVAV6203 is a mutant strain as a candidate of live attenuated vaccine. In vaccine preparation, the freeze-drying conditions of the strain were investigated to improve the survival after freeze-drying, including the protectant, rehydration medium, freezing temperature, and initial cell concentration. Vibrio anguillarum MVAV6203 is sensitive to freeze-drying and the viability was only 0.03% in the absence of protectant. Of the tested protectants, 5% trehalose with 15% skimmed milk gave the highest viability of 34.2%. Higher cell survival was obtained by quick freezing at -80 degrees C than slow freezing at -20 degrees C. Initial cell concentration was another important factor, preferable for 1-3 x 10(10)CFU/ml. The supplementation of 10% skimmed milk in rehydration medium improved obviously freeze-drying viability. The combination of the optimal conditions achieved 51.4% cell viability after freeze-drying.     

Effect of protective agents, rehydration media and initial cell concentration on viability of Pantoea agglomerans strain CPA-2 subjected to freeze-drying

The effect of initial cell density, protective agents and rehydration media on the viability of biocontrol agent Pantoea agglomerans CPA-2 when subjected to freeze-drying was studied. Several additives were tested as protective agents against freeze-drying injury. Maximum viability of the bacterial cells was obtained with disaccharides (survival levels > 60%). Freeze-dried samples were rehydrated with several media; the highest percentage viability was obtained with 10% non-fat skim milk (100%+). The effect of initial bacterial load on the final recovery was dependent on protectant but not on rehydration media. Sucrose was an effective protectant when a high initial concentration (10(10) cfu ml(-1) was used; the opposite occurred with non-fat skim milk. The use of 10(10) cfu ml(-1) as an initial concentration, sucrose as a protectant and non-fat skim milk as a rehydration medium enabled 100% of P. agglomerans viability to be conserved after freeze-drying. Results suggest the possibility of achieving a good formulation system for the studied biocontrol agent with a high number of viable cells to be used toward pathogens, which is desirable for the industrial development of the product.     

Kinetics of acrylamide formation/elimination reactions as affected by water activity

The influence of water activity on the kinetics of acrylamide formation and elimination reaction was investigated using low-moisture equimolar asparagine-glucose model systems, which were heated at temperatures between 120 and 200 degrees C for variable heating times. To determine the water content corresponding to the water activities tested, a sorption moisture isotherm was constructed experimentally. The acrylamide concentrations measured at different water activities could be modeled on the basis of a reaction scheme including not only acrylamide formation and elimination reactions but also an alternative Maillard reaction between both reactants. The corresponding rate constants and activation energies were estimated using nonlinear regression analysis. Whereas the rate constant for acrylamide formation varied only slightly with the initial water activity of the model system, the elimination rate constant showed a clear minimum around a water activity of 0.82. The opposite trend, namely, a maximum at a water activity of 0.82, was found for the Maillard reaction rate constant as a function of water activity, which confirms data from literature. The activation energies for the different reactions changed in a comparable way as the corresponding rate constant with water activity.     

Relations between water content, potential and permeability in stems of conifers

Abstract. The influence of sapwood water content on the conductivity of sapwood to water was measured on stem sections of Pinus contorta. A reduction in relative water content from 100 to 90% caused permeability to fall to about 10% of the saturated value.
Pressure–volume curves of branchwood and stem sapwood of Pinus contorta and Picea sitchensis have been analysed to definè the tissue capacitance and the time constant and resistance for water movement between stored water and the functional xylem as functions of tissue water potential. Three phases in water loss were discernible. In the initial phase at high water potentials (> –0.5 MPa), the capacitance was large, the time constant long and the resistance to flow large in comparison with intermediate water potentials (−0.5 to −1.5 MPa). At still lower water potentials (−1.5 to −3.0 MPa), the time constant and resistance declined still further but the capacitance had a tendency to increase again, especially in the stemwood of Sitka spruce. Typical values in the second phase were for the time constant 5 s, for the resistance 4 × 10⁻¹³ N s m⁻⁵ and for the capacitance (change in relative water content per unit change in potential) 1×10⁻¹¹ m³ Pa⁻¹. These parameters define the availability of stored water and are being used in a dynamic model of water transport in trees.     

Key composition optimization of meat processed protein source by vacuum freeze-drying technology

Vacuum freeze-drying technology is a high technology content, a wide range of knowledge of technology in the field of drying technology is involved, it is also a method of the most complex drying equipment, the largest energy consumption, the highest cost of drying method, but due to the particularity of its dry goods: the freeze-drying food has the advantages of complex water performance is good, cooler and luster of freezing and drying food to maintain good products, less nutrient loss, light weight, easy to carry transportation, easy to long-term preservation, and on the quality is far superior to the obvious advantages of other dried food, making it become the forefront of drying technology research and development. The freeze-drying process of Chinese style ham and western Germany fruit tree tenderloin is studied in this paper, their eutectic point, melting point and collapse temperature, freeze-drying curve and its heat and mass transfer characteristics are got, then the precool temperature and the highest limiting temperature of sublimation interface are determined. The effect of system pressure on freeze-dried rate in freeze-drying process is discussed, and the method of regulating pressure circularly is determined.     

高效液相法与硫酸-蒽酮法测定猪苓多糖含量比较

本研究目的是比较高效液相法与硫酸-蒽酮法在测定猪苓多糖含量上的差异。为此,研究采用水提醇沉、Sevag法除蛋白、透析和冷冻干燥制备相对纯化的猪苓多糖,通过凝胶渗透色谱及高效液相法对猪苓多糖的相对分子量分布、组成及含量进行研究,用硫酸-蒽酮分光光度法测定猪苓颗粒中猪苓多糖的含量。结果显示,猪苓多糖数均相对分子质量为48,232,重均相对分子质量为117,506,分子范围2.44,可能由海藻糖和葡萄糖组成,其含量分别为6.05%和62.28%。硫酸-蒽酮法侧的猪苓多糖含量为87.9%。对于猪苓多糖含量测定,高效液相法优于硫酸-蒽酮法。     

Freeze-drying of Lactobacillus coryniformis Si3--effects of sucrose concentration, cell density, and freezing rate on cell survival and thermophysical properties

Freeze-drying is commonly used to stabilize lactic acid bacteria. Many factors have been reported to influence freeze-drying survival, including bacterial species, cell density, lyoprotectant, freezing rate, and other process parameters. Lactobacillus coryniformis Si3 has broad antifungal activity and a potential use as a food and feed biopreservative. This strain is considered more stress sensitive, with a low freeze-drying survival, compared to other commercialized antifungal lactic acid bacterial strains. We used a response surface methodology to evaluate the effects of varying sucrose concentration, cell density and freezing rate on Lb. coryniformis Si3 freeze-drying survival. The water activity of the dry product, as well as selected thermophysical properties of importance for freeze-drying; degree of water crystallization and the glass transition temperature of the maximally freeze concentrated amorphous phase (Tg') were determined. The survival of Lb. coryniformis Si3 varied from less than 6% to over 70% between the different conditions. All the factors studied influenced freeze-drying survival and the most important factor for survival is the freezing rate, with an optimum at 2.8 degrees C/min. We found a co-dependency between freezing rate and formulation ingredients, indicating a complex system and the need to use statistical tools to detect important interactions. The degree of water crystallization decreased and the final water activity increased as a function of sucrose concentration. The degree of water crystallization and Tg' was not affected by the addition of 10(8)-10(10) CFU/ml. At 10(11) CFU/ml, these thermophysical values decreased possibly due to increased amounts of cell-associated unfrozen water.     

Effect of Water Diffusion Limitations on the Thermostability of Enzymes in Non-Aqueous Environments

The thermostability of anhydrous α-chymotrypsin has been analysed both in air and in organic solvents, with regard to the effect of the protein water-content on the course of deactivation. A higher initial water content increases the rate of inactivation.

Deactivation tests carried out under a constant thermodynamic activity of water indicate that reductions in dehydration rate lead to lower stability.

The effect of water diffusion phenomena has also been studied. Protein aggregates of larger size are less thermostable, thus indicating that diffusional limitations to water transfer can play a significant role in thermoinactivation.

The effect of water content on enzyme thermostability was also measured in the presence of two organic solvents of different hydrophobicity. In both cases, the resulting increased thermolability can be explained in terms of a limitation in water transfer towards the non-aqueous environment.     

Synthesis of alkylgalactosides using whole cells of Bacillus pseudofirmus species as catalysts

Whole cells of alkaliphilic Bacillus pseudofirmus AR-199, induced for beta-galactosidase activity, were used for the synthesis of 1-hexyl-beta-d-galactoside and 1-octyl-beta-d-galactoside, respectively, by transglycosylation reaction between lactose and the corresponding alcohol acceptor. The product yield was strongly influenced by the initial water content in the reaction mixture. Water content of 10% (v/v) was optimal providing 3.6-36 mM hexyl galactoside from 10 to 150 mM lactose, and no secondary product hydrolysis. Product yield could be enhanced by supplementing the reaction mixture with more cells or partly replacing the product with fresh substrate, but was decreased with time to the initial equilibrium level. Cell permeabilisation or disruption resulted in increased reaction rate and higher product yield but was followed by product hydrolysis. Octyl galactoside synthesis using whole cells was optimal at water content of 2% (v/v) with a yield of 26%. The cells were immobilised in cryogels of polyvinyl alcohol for use in continuous process, where hexyl galactoside was produced with a constant yield of 50% from 50mM lactose for at least a week.     

Effect of drying on conidial viability of Penicillium frequentans, a biological control agent against peach brown rot disease caused by Moniliniaspp

The effects of drying methods (freeze-, spray-, and fluid bed-drying) on viability of Penicillium frequentans conidia were compared. Viability, estimated by germination of fluid bed- and freeze-dried conidia, was similar to that of fresh conidia. Skimmed milk alone, or in combination with other protectants, was added to conidia before freeze-drying. After the freeze-drying process, all protectants used, except glycerol improved conidial viability. Freeze-dried P. frequentans conidia did not maintain viability after 30 days of storage at room temperature, while conidia dried by fluid bed-drying showed 28% viability following 180 days after drying. This work also demonstrated a relationship between conidial viability after 1 year of storage at room temperature, moisture content after fluid bed-drying and initial weight of sample. Conidial moisture contents must be reduced to 5-15% for optimal storage at room temperature. P. frequentans conidia dried by fluid bed-drying were as effective as fresh conidia in controlling brown rot of peaches.     

A study of water binding in lyophilized viral vaccine systems

Differential scanning calorimetry (DSC) has been used to estimate the amount of bound water in freeze-dried live measles vaccine in the presence of two cryoprotective mixtures, with and without sucrose. The purpose of the work was to see if a relationship exists between the amount of bound water in viral systems, the nature of the freeze-drying process, and the final stability of the virus preparations.It was shown that although the presence of sucrose in protective agents improves the retention of activity, the limiting amount of bound water (that for which the heat of fusion is zero) in the rehydrated samples was not related to the presence of sucrose, but depended on the method of freeze-drying.     

Aerosol Survival of Pasteurella tularensis Disseminated from the Wet and Dry States

The aerosol survival in air and in nitrogen was measured for Pasteurella tularensis live vaccine strain, disseminated from the wet and dry states. The results showed that most of the loss of viability occurred in less than 2 min of aerosol age, i.e., a rapid initial decay followed by a much slower secondary decay. In nitrogen and air, minimum survival occurred at 50 to 55% relative humidity (RH) for wet dissemination and at 75% RH for dry dissemination. This shift indicated that aerosols produced by wet and dry dissemination were not equivalent and suggested that survival might not be related to bacterial water activity or content. The results showed that rehydration is the key process with regard to survival, but that lysis on rehydration is not a primary death mechanism. The effects of oxygen were complex because it could be either protective or toxic, depending upon other conditions. The protective action of oxygen was through an effect on the spent culture suspending fluid. The latter contained a toxic component, the activity of which is suppressed by oxygen; possibly the component is pumped away during freeze-drying. A toxic effect of oxygen was not found in the presence of spent culture media because the toxicity of the latter masks such an effect. With other bacterial suspending fluids, oxygen was shown to be toxic at low RH. Similar effects with regard to oxygen toxicity were also found with a laboratory strain of P. tularensis. Differences in oxygen toxicity for aerosols generated from the wet and dry states also suggest that bacterial water content and activity do not control aerosol survival.     

Synthesis of alkylgalactosides using whole cells of Bacillus pseudofirmus species as catalysts

Whole cells of alkaliphilic Bacillus pseudofirmus AR-199, induced for β-galactosidase activity, were used for the synthesis of 1-hexyl-β- -galactoside and 1-octyl-β- -galactoside, respectively, by transglycosylation reaction between lactose and the corresponding alcohol acceptor. The product yield was strongly influenced by the initial water content in the reaction mixture. Water content of 10% (v/v) was optimal providing 3.6–36 mM hexyl galactoside from 10 to 150 mM lactose, and no secondary product hydrolysis. Product yield could be enhanced by supplementing the reaction mixture with more cells or partly replacing the product with fresh substrate, but was decreased with time to the initial equilibrium level. Cell permeabilisation or disruption resulted in increased reaction rate and higher product yield but was followed by product hydrolysis. Octyl galactoside synthesis using whole cells was optimal at water content of 2% (v/v) with a yield of 26%. The cells were immobilised in cryogels of polyvinyl alcohol for use in continuous process, where hexyl galactoside was produced with a constant yield of 50% from 50 mM lactose for at least a week.     

Effects of di- and polysaccharide formulations and storage conditions on survival of freeze-dried Sphingobium sp.

In this study we have compared the ability of the organic polymers Ficoll and hydroxyethylcellulose (HEC) and the disaccharides sucrose and trehalose to support cell survival during freeze-drying and subsequent storage of a gram-negative Sphingobium sp. In addition to determination of viability rates, cell integrity was evaluated using lipid peroxidation and RNA quality assays for the different storage conditions and formulation compositions. All formulations resulted in high initial cell survival rates after freeze-drying. However, the disaccharide formulations were superior to the polymer-based formulations in supporting cell survival during storage with the exception of Ficoll that upon storage under vacuum yielded bacterial survival rates equal to that of sucrose. Storage in the presence of both oxygen and moisture was detrimental for bacterial survival in all formulations tested, however, lipid peroxidation or RNA damages were not the controlling mechanisms for cell death in this system. The ability of Ficoll and HEC to support cell survival during freeze-drying show that organic polymers, expected to lack the water replacing capability of e.g. disaccharides, can successfully be used as lyoprotectants. For storage under vacuum conditions we suggest that the intracellular amount of sugars (i.e. trehalose), or other protective native cell components, is sufficient for a basic protection inside the bacteria cell and that the amorphous state is the most important aspect of the formulation excipient. However, when exposed to oxygen and moisture during storage this protection is not sufficient to prevent cell degeneration.