Novel Method for Enumeration of Viable Lactobacillus plantarum WCFS1 Cells after Single-Droplet Drying

Survival of probiotic bacteria during drying is not trivial. Survival percentages are very specific for each probiotic strain and can be improved by careful selection of drying conditions and proper drying carrier formulation. An experimental approach is presented, comprising a single-droplet drying method and a subsequent novel screening methodology, to assess the microbial viability within single particles. The drying method involves the drying of a single droplet deposited on a flat, hydrophobic surface under well-defined drying conditions and carrier formulations. Semidried or dried particles were subjected to rehydration, fluorescence staining, and live/dead enumeration using fluorescence microscopy. The novel screening methodology provided accurate survival percentages in line with conventional plating enumeration and was evaluated in single-droplet drying experiments with Lactobacillus plantarum WCFS1 as a model probiotic strain. Parameters such as bulk air temperatures and the carrier matrices (glucose, trehalose, and maltodextrin DE 6) were varied. Following the experimental approach, the influence on the viability as a function of the drying history could be monitored. Finally, the applicability of the novel viability assessment was demonstrated for samples obtained from drying experiments at a larger scale.     

Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying.

The microorganisms Escherichia coli DH5 alpha and Bacillus thuringiensis HD-1 show an increased tolerance to freeze-drying when dried in the presence of the disaccharides trehalose and sucrose. When the bacteria were dried with 100 mM trehalose, 70% of the E. coli and 57% of the B. thuringiensis organisms survived, compared with 56 and 44%, respectively, when they were dried with sucrose. Only 8% of the E. coli and 14% of the B. thuringiensis organisms survived drying without the sugars. Fourier transform infrared spectroscopy was used to investigate the role of membrane phase transitions in the survival of the organisms during drying and rehydration. Both E. coli and B. thuringiensis showed an increase of 30 to 40 degrees C in the temperature of their phospholipid phase transition when dried without the sugars, while phase transition temperatures of those dried with the sugars remained near those of the hydrated cells. A Fourier transform infrared spectroscopy microscope made it possible to investigate the effects of drying on the protein structure in the intact cells. The amide II peak shifts from 1,543 cm-1 in the hydrated cells to about 1,533 cm-1 in the cells dried without sugar. There is no shift in the amide II peak when the cells are dried with trehalose or sucrose. We attribute the increased survival to the sugars' ability to lower the membrane phase transition temperature and to protect protein structure in the dry state.(ABSTRACT TRUNCATED AT 250 WORDS)     

A small stress protein acts synergistically with trehalose to confer desiccation tolerance on mammalian cells

The ability to desiccate mammalian cells while maintaining a high degree of viability would be very important in many areas of biological science, including tissue engineering, cell transplantation, and biosensor technologies. Certain proteins and sugars found in animals capable of surviving desiccation might aid this process. We report here that human embryonic kidney (293H) cells transfected with the gene for the stress protein p26 from Artemia and loaded with trehalose showed a sharp increase in survival during air-drying. Further, we find vacuum-drying greatly improved the ability of the cells to survive, and that the physical shape and structure of the cellular sample had a large influence on recovery following rehydration. Cells suspended in a rounded droplet survived desiccation markedly better than those spread as a thin film. Finally, we used alamarBlue to monitor cellular metabolism and Hema 3 to assess colony formation after vacuum-drying. AlamarBlue fluorescence indicated that the transfected 293H cells expressing p26 (E11'L) grew much better than the control 293H cells. In fact, immediate survival and colony formation in E11'L cells increased as much as 34-fold compared with control cells when the samples were dried to a water content of 0.2 g H2O/g dry weight, as measured by gravimetric analysis. These results indicate that p26 improves cell survival following drying and rehydration, and suggest that dry storage of mammalian cells is a likely possibility in the future.     

海藻糖载入红细胞及其冷冻干燥的实验研究

冷冻干燥保存是长期保存人体红细胞的理想方案之一。冻干保护剂海藻糖渗入细胞内后,对细胞膜和细胞内物质有保护作用,其中的一个作用是增加细胞质的浓度,使冻干过程容易形成稳定的玻璃态。应用高渗法处理红细胞,通过考察胞内海藻糖含量、红细胞冻干后的存活率、腺苷三磷酸酶(ATPase)、超氧化物歧化酶(SOD)活力以及细胞形态变化,研究胞内海藻糖含量对红细胞冻干后活性的影响。结果显示:海藻糖对红细胞冻干具有明显的保护作用,随胞内海藻糖浓度升高,其保护性能逐渐增强;43.8mmol/L的胞内海藻糖浓度对红细胞保护最好,细胞存活率达到53.6%,形态保持良好,ATP和SOD活力均在正常的范围内。     

Desiccation and supra‐zero temperature storage of cat germinal vesicles lead to less structural damage and similar epigenetic alterations compared to cryopreservation

Understanding cellular and molecular damages in oocytes during exposure to extreme conditions is essential to optimize long‐term fertility preservation approaches. Using the domestic cat (Felis catus) model, we are developing drying techniques for oocytes’ germinal vesicles (GVs) as a more economical alternative to cryopreservation. The objective of the study was to characterize the influence of desiccation on nuclear envelope conformation, chromatin configuration, and the relative fluorescent intensities of histone H3 trimethylation at lysine 4 (H3K4me3) and at lysine 9 (H3K9me3) compared to vitrification. Results showed that higher proportions of dried/rehydrated GVs maintained normal nuclear envelope conformation and chromatin configuration than vitrified/warmed counterparts. Both preservation methods had a similar influence on epigenetic patterns, lowering H3K4me3 intensity to under 40% while maintaining H3K9me3 levels. Further analysis revealed that the decrease of H3K4me3 intensity mainly occurred during microwave dehydration and subsequent rehydration, whereas sample processing (permeabilization and trehalose exposure) or storage did not significantly affect the epigenetic marker. Moreover, rehydration either directly or stepwise with trehalose solutions did not influence the outcome. This is the first report demonstrating that the incidence of GV damages is lower after desiccation/rehydration than vitrification/warming.     

Trehalose as cryoprotectant for preservation of yeast strains

Preservation of genetic banks of yeast strains as well as of any kind of eukaryotic cells during dehydration and subsequent rehydration depends upon the maintenance of the integrity of the cell membrane. Trehalose has been successfully used as a non-toxic cryoprotectant for plant cells (Bhandal et al., 1985), as well as for lobster sarcoplasmic vesicles (Rudolph and Crowe, 1985). The hypothesis underlying these observations is that the disaccharide avoids fusion of membranes by replacing water molecules in the bilayer (Crowe et al., 1984). The viability of yeast strains submitted to different drying techniques is reported in this paper. Mutant strains with defects in the regulation of the trehalose-6-phosphate synthase complex were compared. Yeast strains dried in layers at 37°C for 6 h did not lose their viability, however, they died thereafter at 5°C, unless trehalose was used for resuspending the cells before drying. It should be noted that no trehalose accumulation was seen during drying at 37°C under our experimental conditions. In experiments in which cells were frozen at −120°C, addition of 10% trehalose to the suspending buffer had a significant protective effect. On the other hand, a mutant strain with an extremely high trehalose-6-phosphate synthase activity showed an intrinsic capacity for survival which did not depend upon addition of exogenous trehalose. This raises the question of the location of the internal trehalose pool and whether it could replace the externally added cryoprotectant.     

Compatible solutes of sclerotia of Mycoleptodiscus terrestris under different culture and drying conditions

Mycoleptodiscus terrestris is a plant pathogen which has been shown to be effective in controlling invasive aquatic weeds in inundative biocontrol applications. The preferred propagule for production and application is the sclerotium. In the current study, we evaluated the accumulation of carbohydrates and polyols in the sclerotia of M. terrestris under different culture and drying regimes. The carbohydrates and polyols screened for represent a class of compatible solutes found in fungi. The results show mannitol and trehalose are the major analytes identified in the sclerotia of M. terrestris. We identified higher levels of mannitol and trehalose in liquid-culture produced samples relative to solid-state produced samples. The impact of fermentation time was examined and shown to impact solute levels. In addition, the drying regime was varied to produce samples dried to different moisture contents. The experiments show greater drying led to higher mannitol levels, while trehalose levels remained constant. Rapid drying of the sclerotia in a fluidized-bed dryer also show rapid accumulation of mannitol and trehalose, which suggest the enzyme activities needed for production are readily available. The findings confirm mannitol and trehalose are important metabolites in M. terrestris and their concentrations are responsive to osmotic stress conditions.     

Augmented Dried versus Cryopreserved Amniotic Membrane as an Ocular Surface Dressing

Purpose

Dried amniotic membrane (AM) can be a useful therapeutic adjunct in ophthalmic surgery and possesses logistical advantages over cryopreserved AM. Differences in preservation techniques can significantly influence the biochemical composition and physical properties of AM, potentially affecting clinical efficacy. This study was established to investigate the biochemical and structural effects of drying AM in the absence and presence of saccharide lyoprotectants and its biocompatibility compared to cryopreserved material.

Methods

AM was cryopreserved or dried with and without pre-treatment with trehalose or raffinose and the antioxidant epigallocatechin (EGCG). Structural and visual comparisons were assessed using electron microscopy. Localisation, expression and release of AM biological factors were determined using immunoassays and immunofluorescence. The biocompatibility of the AM preparations co-cultured with corneal epithelial cell (CEC) or keratocyte monolayers were assessed using cell proliferation, cytotoxicity, apoptosis and migration assays.

Results

Drying devitalised AM epithelium, but less than cryopreservation and cellular damage was reduced in dried AM pre-treated with trehalose or raffinose. Dried AM alone, and with trehalose or raffinose showed greater factor retention efficiencies and bioavailability compared to cryopreserved AM and demonstrated a more sustained biochemical factor time release in vitro. Cellular health assays showed that dried AM with trehalose or raffinose are compatible and superior substrates compared to cryopreserved AM for primary CEC expansion, with increased proliferation and reduced LDH and caspase-3 levels. This concept was supported by improved wound healing in an immortalised human CEC line (hiCEC) co-cultured with dried and trehalose or raffinose membranes, compared to cryopreserved and fresh AM.

Conclusions

Our modified preservation process and our resultant optimised dried AM has enhanced structural properties and biochemical stability and is a superior substrate to conventional cryopreserved AM. In addition this product is stable and easily transportable allowing it to be globally wide reaching for use in clinical and military sectors.     

Action of trehalose on the preservation of Lactobacillus delbrueckii ssp. bulgaricus by heat and osmotic dehydration

AIM: This work determines the efficiency of trehalose on the preservation by heat or osmotic drying of a strain of Lactobacillus delbrueckii ssp. bulgaricus. Cell recovery at different trehalose concentrations during drying correlated with the surface properties and osmotic response of cells after rehydration. METHODS AND RESULTS: Bacteria were dried in the presence of glycerol, trehalose, sucrose at 70 degrees C and at 20 degrees C. Trehalose attenuates the loss of viability at 0.25 m. At this concentration, the osmotic response and zeta potential of the bacteria were comparable with the nondried ones. CONCLUSIONS: Trehalose diminishes significantly the damage produced by dehydration both when the bacteria are dried by heating or subjected to osmotic dehydration. This effect appears related to the preservation of the permeability to water and the surface potential of the bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Dehydration occurring during heating or during osmosis appears to have similar effects. As dehydration-induced damage is in correlation with osmotic response recovery and is hindered or buffered by the presence of trehalose, it may be related to water eliminated from biological structures involved in water permeation.     

Cryopreservation of spin-dried mammalian cells

This study reports an alternative approach to achieve vitrification where cells are pre-desiccated prior to cooling to cryogenic temperatures for storage. Chinese Hamster Ovary (CHO) cells suspended in a trehalose solution were rapidly and uniformly desiccated to a low moisture content (<0.12 g of water per g of dry weight) using a spin-drying technique. Trehalose was also introduced into the cells using a high-capacity trehalose transporter (TRET1). Fourier Transform Infrared Spectroscopy (FTIR) was used to examine the uniformity of water concentration distribution in the spin-dried samples. 62% of the cells were shown to survive spin-drying in the presence of trehalose following immediate rehydration. The spin-dried samples were stored in liquid nitrogen (LN(2)) at a vitrified state. It was shown that following re-warming to room temperature and re-hydration with a fully complemented cell culture medium, 51% of the spin-dried and vitrified cells survived and demonstrated normal growth characteristics. Spin-drying is a novel strategy that can be used to improve cryopreservation outcome by promoting rapid vitrification.     

The Effects of Rehydration and Oxygen on the Heat Resistance of High Vacuum Treated Spores

S ummary . Damage induced in bacterial spores by exposure to reduced pressures of the order of 10^-8 torr, has been assessed in terms of the differences in the heat resistance of the dried spores. The response of the spores has been assessed as a function of (a), the drying temperature from 0-65°; (b) the duration and level of rehydration; (c) the presence or absence of oxygen during heating. Comparison has also been made between spores dried to a given water level and spores rehydrated to the same water level after prolonged drying. Log survivors/heating time curves for treated spore samples have been constructed and have been shown to exhibit a shoulder at high survival levels and a linear portion below a surviving fraction of 0·1. These curves have been explained on the basis of the shoulder representing the time during which necessary structural changes occur in the spore, before the lethal mechanism responsible for the linear portion of the curve becomes operative. The heat response has been shown to be a function of the temperature of drying, and of the presence of oxygen during heating, the structural change itself being reversible by water.     

Spray drying as a method for preparing concentrated cultures of Lactobacillus bulgaricus

P. TEIXEIRA, H. CASTRO AND R. KIRBY. 1995. Spray drying and freeze drying as methods for concentration of Lactobacillus bulgaricus starter cultures were compared in terms of viability, lag phase until onset of pH decrease and total acid production. For the experimental conditions used, no significant differences were detected between the methods.
The effect of spray drying on the cell membrane of Lactobacillus bulgaricus was studied. Five separate methods were used to study the theory that spray drying causes cell membrane damage; three relating to leakage of intracellular components from the cell into the surrounding environment (260 and 280 nm absorbing materials, potassium ions and proteins); and two relating to increased cell permeability (increased sensitivity to NaCl and increased permeability to o -nitrophenyl-β-D-galactopyranoside (ONPG). Partial loss of some cytoplasmic material from the damaged cells was observed. The dried cells also became sensitive to NaCl and permeable to ONPG. Heat shock increased the survival of exponential cells as compared to controls but did not result in normal levels found with unshocked stationary phase cells. Heat shock had no effect on stationary phase cells. Different rehydration methods and media were investigated: slow rehydration increased survival.     

Preservation of Bacteria by Circulating-Gas Freeze Drying

Water-washed Serratia marcescens and Escherichia coli were freeze dried in a circulating-gas system at atmospheric pressure. This convective procedure resulted in a substantially higher survival of organisms than could be obtained by the vacuum method of freeze drying. There was little or no decrease in cell viability during convective drying when the residual moisture content was 15% or higher. Below this level, survival declined with decreasing moisture content. A detailed comparison of the convective and vacuum methods indicated that the advantage gained by freeze drying bacteria in air accrues in the early period of sublimation, at which time cells were found to be sensitive to vacuum drying but insensitive to air drying. An explanation for this difference is proposed, based upon the kinetics of water removal in the two processes. In brief, it is suggested that the convective method permits samples to be dried more uniformly; and regional over-drying, which may be deleterious even if transient, is thus avoided in achieving the optimal level of moisture.     

A study of the effect of sorbitol on osmotic tolerance during partial desiccation of bovine sperm

The goal of the study was to improve the partial desiccation survival of bovine sperm by decreasing the dehydration induced osmotic injury. The protective role of sorbitol, a polyol, was investigated by (i) studying the osmotic behavior of sperm in hypertonic Tyrode’s buffer in the presence of sorbitol and trehalose, (ii) studying the effect of sorbitol and trehalose on sperm motility following partial dehydration. The osmotic behavior studies included the assessment of motility and volumetric responses in the presence of the additives. For the drying experiments, motility was assayed after drying the samples to different end water content followed by immediate rehydration. Compared to the effect of “intracellular + extracellular” trehalose alone, results showed a much improved motility in the presence of sorbitol and trehalose. While the drying results suggest an enhanced osmotolerance in the presence of sorbitol, the study of motility under hypertonic conditions combined with the sperm volume excursion experiments suggest that sorbitol imparts the enhancement by permeating into the cell cytoplasm.     

Anhydrobiosis in yeast: is it possible to reach anhydrobiosis for yeast grown in conditions with severe oxygen limitation?

The yeast Saccharomyces cerevisiae was shown to be extremely sensitive to dehydration–rehydration treatments when stationary phase cells were subjected to conditions of severe oxygen limitation, unlike the same cells grown in aerobic conditions. The viability of dehydrated anaerobically grown yeast cells never exceeded 2 %. It was not possible to increase this viability using gradual rehydration of dry cells in water vapour, which usually strongly reduces damage to intracellular membranes. Specific pre-dehydration treatments significantly increased the resistance of anaerobic yeast to drying. Thus, incubation of cells with trehalose (100 mM), increased the viability of dehydrated cells after slow rehydration in water vapour to 30 %. Similarly, pre-incubation of cells in 1 M xylitol or glycerol enabled up to 50–60 % of cells to successfully enter a viable state of anhydrobiosis after subsequent rehydration. We presume that trehalose and sugar alcohols function mainly according to a water replacement hypothesis, as well as initiating various protective intracellular reactions.     

Beneficial effect of intracellular trehalose on the membrane integrity of dried mammalian cells

Recently, there has been much interest in using trehalose and other small carbohydrates to preserve mammalian cells in the dried state as an alternative to cryopreservation. Here, we report on the successful preservation of plasma membrane integrity after drying, as a first step toward full preservation of mammalian cells. Trehalose was introduced into cells using a genetically engineered version of alpha-hemolysin, a pore-forming protein; the cells were then dried and stored for weeks at different temperatures with approximately 90% recovery of the intact plasma membrane. We show that protection of the plasma membrane by internal trehalose is dose dependent and estimate the amount of internal trehalose required for adequate protection to be approximately 10(10) molecules/cell. In addition, a minimal amount of water (approximately 15 wt%) appears to be necessary. These results show that a key component of mammalian cells can be preserved in a dried state for weeks under mild conditions (-20 degrees C and 5% relative humidity) and thereby suggest new approaches to preserving mammalian cells.     

Physiological consequences of desiccation in the aquatic bryophyte <Emphasis Type="Italic">Fontinalis antipyretica</Emphasis>

The moss Fontinalis antipyretica, an aquatic bryophyte previously described as desiccation-intolerant, is known to survive intermittent desiccation events in Mediterranean rivers. To better understand the mechanisms of desiccation tolerance in this species and to reconcile the apparently conflicting evidence between desiccation tolerance classifications and field observations, gross photosynthesis and chlorophyll a fluorescence were measured in field-desiccated bryophyte tips and in bryophyte tips subjected in the laboratory to slow, fast, and very fast drying followed by either a short (30 min) or prolonged (5 days) recovery. Our results show, for the first time, that the metabolic response of F. antipyretica to desiccation, both under field and laboratory conditions, is consistent with a desiccation-tolerance pattern; however, drying must proceed slowly for the bryophyte to regain its pre-desiccation state following rehydration. In addition, the extent of dehydration was found to influence metabolism whereas the drying rate determined the degree of recovery. Photosystem II (PSII) regulation and structural maintenance may be part of the induced desiccation tolerance mechanism allowing this moss to recover from slow drying. The decrease in the photochemical quenching coefficient (qP) immediately following rehydration may serve to alleviate the effects of excess energy on photosystem I (PSI), while low-level non-photochemical quenching (NPQ) would allow an energy shift enabling recovery subsequent to extended periods of desiccation. The findings were confirmed in field-desiccated samples, whose behavior was similar to that of samples slowly dried in the laboratory.