Effect of culture medium and cryoprotectants on the growth and survival of probiotic lactobacilli during freeze drying

To investigate the effects of the medium and cryoprotective agents used on the growth and survival of Lactobacillus plantarum and Lactobacillus rhamnosus GG during freeze drying. A complex medium was developed consisting primarily of glucose, yeast extract and vegetable-derived peptone. Trehalose, sucrose and sorbitol were examined for their ability to protect the cells during freeze drying. Using standardized amount of cells and the optimized freeze drying media, the effect of the growth medium on cell survival during freeze drying was investigated. The results showed that glucose and yeast extract were the most important growth factors, while sucrose offered better protection than trehalose and sorbitol during freeze drying. When the cells were grown under carbon limiting conditions, their survival during freeze drying was significantly decreased. A clear relationship was observed between cell growth and the ability of the cells to survive during the freeze drying process. The survival of probiotic strains during freeze drying was shown to be dependent on the cryoprotectant used and the growth medium.

Freeze-frame: a new infant inhibition task and its relation to frontal cortex tasks during infancy and early childhood

The current study investigated a new, easily administered, visual inhibition task for infants termed the Freeze-Frame task. In the new task, 9-month-olds were encouraged to inhibit looks to peripheral distractors. This was done by briefly freezing a central animated stimulus when infants looked to the distractors. Half of the trials presented an engaging central stimulus, and the other half presented a repetitive central stimulus. Three measures of inhibitory function were derived from the task and compared with performance on a set of frontal cortex tasks administered at 9 and 24 months of age. As expected, infants' ability to learn to selectively inhibit looks to the distractors at 9 months predicted performance at 24 months. However, performance differences in the two Freeze-Frame trial types early in the experiment also turned out to be an important predictor. The results are discussed in terms of the validity of the Freeze-Frame task as an early measure of different components of inhibitory function. (C) 2007 Elsevier Inc. All rights reserved.

Survival of freeze dried Lactobacillus plantarum in instant fruit powders and reconstituted fruit juices

The aim of this study was to investigate the survival of freeze dried Lactobacillus plantarum cells mixed with several freeze dried instant fruit powders (strawberry, pomegranate, blackcurrant and cranberry) during storage for 12 months as well as after reconstitution with water each month. Inulin and gum arabic were also added to the instant fruit powders at two levels (10% and 20% w/w of dry weight) to improve the cell survival and functional properties of the product. The best cell survival over the 12 months of storage was observed for the blackcurrant powder (almost no decrease) followed by strawberry (~ 0.3 log decrease), pomegranate (~ 0.9 log decrease), whereas the worst survival was obtained in cranberry powder (~ 4.5 logs). To explain these results multiple regression analysis was conducted with the log decrease [log10N0 month − log10N12 months] as the dependent variable and water activity, pH, citric acid, dietary fibre and total phenol as the independent variables. The results indicated that among all the examined factors, the [log10N0 month − log10N12 months] depended only on the water activity (P < 0.05). Inulin and gum arabic demonstrated a substantial protective effect on cell survival (1–1.5 log) in the case of cranberry, which was likely due to a physical interaction between the cells and the carbohydrates. After reconstituting the dried fruit powders at room temperature and measuring cell viability for up to 4 h, it was shown that in the case of strawberry juice there was no decrease, and very little in the case of pomegranate and blackcurrant juices (< 0.5 log). On the other hand, a significant decrease was observed for cranberry juice (P < 0.05), which increased as the storage time of the dried cranberry powder increased, indicating that the cells became more susceptible with prolonged storage. Multiple regression analysis indicated that the main factors influencing cell survival were water activity and pH, while citric acid, dietary fibre and total phenol did not have an effect. Furthermore, inulin and gum arabic addition did not have a significant (P > 0.05) effect upon reconstitution of the dried fruit powder. This study showed that instant juice powders are very good carriers of probiotic cells and constitute good alternatives to highly acidic fruit juices.

The variation in transparency of amniotic membrane used in ocular surface regeneration

Background/aims: Scant consideration has been given to the variation in structure of the human amniotic membrane (AM) at source or to the significance such differences might have on its clinical transparency. Therefore, we applied our experience of quantifying corneal transparency to AM. Methods: Following elective caesarean, AM from areas of the fetal sac distal and proximal (ie, adjacent) to the placenta was compared with freeze-dried AM. The transmission of light through the AM samples was quantified spectrophotometrically; also, tissue thickness was measured by light microscopy and refractive index by refractometry. Results: Freeze-dried and freeze-thawed AM samples distal and proximal to the placenta differed significantly in thickness, percentage transmission of visible light and refractive index. The thinnest tissue (freeze-dried AM) had the highest transmission spectra. The thickest tissue (freeze-thawed AM proximal to the placenta) had the highest refractive index. Using the direct summation of fields method to predict transparency from an equivalent thickness of corneal tissue, AM was found to be up to 85% as transparent as human cornea. Conclusion: When preparing AM for ocular surface reconstruction within the visual field, consideration should be given to its original location from within the fetal sac and its method of preservation, as either can influence corneal transparency.

A simple model for predicting soil temperature in snow-covered and seasonally frozen soil: model description and testing

Microbial processes in soil are moisture, nutrient and temperature dependent and, consequently, accurate calculation of soil temperature is important for modelling nitrogen processes. Microbial activity in soil occurs even at sub-zero temperatures so that, in northern latitudes, a method to calculate soil temperature under snow cover and in frozen soils is required. This paper describes a new and simple model to calculate daily values for soil temperature at various depths in both frozen and unfrozen soils. The model requires four parameters average soil thermal conductivity, specific beat capacity of soil, specific heat capacity due to freezing and thawing and an empirical snow parameter. Precipitation, air temperature and snow depth (measured or calculated) are needed as input variables. The proposed model was applied to five sites in different parts of Finland representing different climates and soil types. Observed soil temperatures at depths of 20 and 50 cm (September 1981-August 1990) were used for model calibration. The calibrated model was then tested using observed soil temperatures from September 1990 to August 2001. R-2-values of the calibration period varied between 0.87 and 0.96 at a depth of 20 cm and between 0.78 and 0.97 at 50 cm. R-2 -values of the testing period were between 0.87 and 0.94 at a depth of 20cm. and between 0.80 and 0.98 at 50cm. Thus, despite the simplifications made, the model was able to simulate soil temperature at these study sites. This simple model simulates soil temperature well in the uppermost soil layers where most of the nitrogen processes occur. The small number of parameters required means, that the model is suitable for addition to catchment scale models.

Geomorphological Map of Ribbed Moraines on the Dubawnt Lake Palaeo-Ice Stream Bed: A Signature of Ice Stream Shut-down?

The beds of active ice streams in Greenland and Antarctica are largely inaccessible, hindering a full understanding of the processes that initiate, sustain and inhibit fast ice flow in ice sheets. Detailed mapping of the glacial geomorphology of palaeo-ice stream tracks is, therefore, a valuable tool for exploring the basal processes that control their behaviour. In this paper we present a map that shows detailed glacial geomorphology from a part of the Dubawnt Lake Palaeo-Ice Stream bed on the north-western Canadian Shield (Northwest Territories), which operated at the end of the last glacial cycle. The map (centred on 63 degrees 55 '' 42'N, 102 degrees 29 '' 11'W, approximate scale 1:90,000) was compiled from digital Landsat Enhanced Thematic Mapper Plus satellite imagery and digital and hard-copy stereo-aerial photographs. The ice stream bed is dominated by parallel mega-scale glacial lineations (MGSL), whose lengths exceed several kilometres but the map also reveals that they have, in places, been superimposed with transverse ridges known as ribbed moraines. The ribbed moraines lie on top of the MSGL and appear to have segmented the individual lineaments. This indicates that formation of the ribbed moraines post-date the formation of the MSGL. The presence of ribbed moraine in the onset zone of another palaeo-ice stream has been linked to oscillations between cold and warm-based ice and/or a patchwork of cold-based areas which led to acceleration and deceleration of ice velocity. Our hypothesis is that the ribbed moraines on the Dubawnt Lake Ice Stream bed are a manifestation of the process that led to ice stream shut-down and may be associated with the process of basal freeze-on. The precise formation of ribbed moraines, however, remains open to debate and field observation of their structure will provide valuable data for formal testing of models of their formation.

The radiative effect of a fir canopy on a snowpack

Models of snow processes in areas of possible large-scale change need to be site independent and physically based. Here, the accumulation and ablation of the seasonal snow cover beneath a fir canopy has been simulated with a new physically based snow-soil vegetation-atmosphere transfer scheme (Snow-SVAT) called SNOWCAN. The model was formulated by coupling a canopy optical and thermal radiation model to a physically based multilayer snow model. Simple representations of other forest effects were included. These include the reduction of wind speed and hence turbulent transfer beneath the canopy, sublimation of intercepted snow, and deposition of debris on the surface. This paper tests this new modeling approach fully at a fir site within Reynolds Creek Experimental Watershed, Idaho. Model parameters were determined at an open site and subsequently applied to the fir site. SNOWCAN was evaluated using measurements of snow depth, subcanopy solar and thermal radiation, and snowpack profiles of temperature, density, and grain size. Simulations showed good agreement with observations (e.g., fir site snow depth was estimated over the season with r(2) = 0.96), generally to within measurement error. However, the simulated temperature profiles were less accurate after a melt-freeze event, when the temperature discrepancy resulted from underestimation of the rate of liquid water flow and/or the rate of refreeze. This indicates both that the general modeling approach is applicable and that a still more complete representation of liquid water in the snowpack will be important.

Doppler lidar measurements of oriented planar ice crystals falling from supercooled and glaciated layer clouds

The properties of planar ice crystals settling horizontally have been investigated using a vertically pointing Doppler lidar. Strong specular reflections were observed from their oriented basal facets, identified by comparison with a second lidar pointing 4° from zenith. Analysis of 17 months of continuous high-resolution observations reveals that these pristine crystals are frequently observed in ice falling from mid-level mixed-phase layer clouds (85% of the time for layers at −15 °C). Detailed analysis of a case study indicates that the crystals are nucleated and grow rapidly within the supercooled layer, then fall out, forming well-defined layers of specular reflection. From the lidar alone the fraction of oriented crystals cannot be quantified, but polarimetric radar measurements confirmed that a substantial fraction of the crystal population was well oriented. As the crystals fall into subsaturated air, specular reflection is observed to switch off as the crystal faces become rounded and lose their faceted structure. Specular reflection in ice falling from supercooled layers colder than −22 °C was also observed, but this was much less pronounced than at warmer temperatures: we suggest that in cold clouds it is the small droplets in the distribution that freeze into plates and produce specular reflection, whilst larger droplets freeze into complex polycrystals. The lidar Doppler measurements show that typical fall speeds for the oriented crystals are ≈ 0.3 m s−1, with a weak temperature correlation; the corresponding Reynolds number is Re ∼ 10, in agreement with light-pillar measurements. Coincident Doppler radar observations show no correlation between the specular enhancement and the eddy dissipation rate, indicating that turbulence does not control crystal orientation in these clouds. Copyright © 2010 Royal Meteorological Society

Effect of growth time on the surface and adhesion properties of Lactobacillus rhamnosus GG

Aims: To investigate the changes in the surface properties of Lactobacillus rhamnosus GG during growth, and relate them with the ability of the Lactobacillus cells to adhere to Caco-2 cells. Methods and Results: Lactobacillus rhamnosus GG was grown in complex medium, and cell samples taken at four time points and freeze dried. Untreated and trypsin treated freeze dried samples were analysed for their composition using SDS-PAGE analysis and Fourier transform infrared spectroscopy (FTIR), hydrophobicity and zeta potential, and for their ability to adhere to Caco-2 cells. The results suggested that in the case of early exponential phase samples (4 and 8 h), the net surface properties, i.e. hydrophobicity and charge, were determined to a large extent by anionic hydrophilic components, whereas in the case of stationary phase samples (13 and 26 h), hydrophobic proteins seemed to play the biggest role. Considerable differences were also observed between the ability of the different samples to adhere to Caco-2 cells; maximum adhesion was observed for the early stationary phase sample (13 h). The results suggested that the adhesion to Caco-2 cells was influenced by both proteins and non-proteinaceous compounds present on the surface of the Lactobacillus cells. Conclusion: The surface properties of Lact. rhamnosus GG changed during growth, which in return affected the ability of the Lactobacillus cells to adhere to Caco-2 cells. Significance and Impact of the Study: The levels of adhesion of Lactobacillus cells to Caco-2 cells were influenced by the growth time and reflected changes on the bacterial surface. This study provides critical information on the physicochemical factors that influence bacterial adhesion to intestinal cells.

In vitro cumulative gas production techniques: History, methodological considerations and challenges

Methodology used to measure in vitro gas production is reviewed to determine impacts of sources of variation on resultant gas production profiles (GPP). Current methods include measurement of gas production at constant pressure (e.g., use of gas tight syringes), a system that is inexpensive, but may be less sensitive than others thereby affecting its suitability in some situations. Automated systems that measure gas production at constant volume allow pressure to accumulate in the bottle, which is recorded at different times to produce a GPP, and may result in sufficiently high pressure that solubility of evolved gases in the medium is affected, thereby resulting in a recorded volume of gas that is lower than that predicted from stoichiometric calculations. Several other methods measure gas production at constant pressure and volume with either pressure transducers or sensors, and these may be manual, semi-automated or fully automated in operation. In these systems, gas is released as pressure increases, and vented gas is recorded. Agitating the medium does not consistently produce more gas with automated systems, and little or no effect of agitation was observed with manual systems. The apparatus affects GPP, but mathematical manipulation may enable effects of apparatus to be removed. The amount of substrate affects the volume of gas produced, but not rate of gas production, provided there is sufficient buffering capacity in the medium. Systems that use a very small amount of substrate are prone to experimental error in sample weighing. Effect of sample preparation on GPP has been found to be important, but further research is required to determine the optimum preparation that mimics animal chewing. Inoculum is the single largest source of variation in measuring GPP, as rumen fluid is variable and sampling schedules, diets fed to donor animals and ratios of rumen fluid/medium must be selected such that microbial activity is sufficiently high that it does not affect rate and extent of fermentation. Species of donor animal may also cause differences in GPP. End point measures can be mathematically manipulated to account for species differences, but rates of fermentation are not related. Other sources of inocula that have been used include caecal fluid (primarily for investigating hindgut fermentation in monogastrics), effluent from simulated rumen fermentation (e.g., 'Rusitec', which was as variable as rumen fluid), faeces, and frozen or freeze-dried rumen fluid (which were both less active than fresh rumen fluid). Use of mixtures of cell-free enzymes, or pure cultures of bacteria, may be a way of increasing GPP reproducibility, while reducing reliance on surgically modified animals. However, more research is required to develop these inocula. A number of media have been developed which buffer the incubation and provide relevant micro-nutrients to the microorganisms. To date, little research has been completed on relationships between the composition of the medium and measured GPP. However, comparing GPP from media either rich in N or N-free, allows assessment of contributions of N containing compounds in the sample. (c) 2005 Published by Elsevier B.V.

Facultative scavenging as a survival strategy of entomopathogenic nematodes

Entomopathogenic nematodes cannot be considered only as parasitic organisms. With dead Galleria mellonella larvae, we demonstrated that these nematodes use scavenging as an alternative survival strategy. We consider scavenging as the ability of entomopathogenic nematodes to penetrate, develop and produce offspring in insects which have been killed by causes other than the nematode-bacteria complex. Six Steinernema and two Heterorhabditis species scavenged but there were differences among them in terms of frequency of colonisation and in the time after death of G. mellonella larvae that cadavers were penetrated. The extremes of this behaviour were represented by Steinernema glaseri which was able to colonise cadavers which had been freeze-killed 240 h earlier and Heterorhabditis indica which only colonised cadavers which had been killed up to 72 h earlier. Also, using an olfactometer, we demonstrated that entomopathogenic nematodes were attracted to G. mellonella cadavers. (c) 2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Suitability of cryopreservation for the long-term storage of rare and endangered plant species: a case history for Cosmos atrosanguineus

The suitability of cryopreservation for the secure, long-term storage of the rare and endangered species Cosmos atrosanguineus was investigated. Using encapsulation/dehydration of shoot tips in alginate strips, survival rates of up to 100 % and shoot regeneration of up to 35 % were achieved. Light and electron microscopy studies indicated that cellular damage to some regions of the shoot tip during the freeze/thaw procedure was high, although cell survival in and around the meristematic region allowed shoot tip regeneration. The genetic fingerprinting technique, amplified fragment length polymorphisms (AFLPs), showed that no detectable genetic variation was present between material of C. atrosanguineus at the time of initiation into tissue culture and that which had been cryopreserved, stored in liquid nitrogen for 12 months and regenerated. Wearied plantlets that were grown under glasshouse conditions exhibited no morphological variation from non-frozen controls. (C) 2003 Annals of Botany Company.

Suitability of cryopreservation for the long-term storage of rare and endangered plant species: a case history for cosmos atrosanguineus

The suitability of cryopreservation for the secure, long-term storage of the rare and endangered species Cosmos atrosanguineus was investigated. Using encapsulation/dehydration of shoot tips in alginate strips, survival rates of up to 100 % and shoot regeneration of up to 35 % were achieved. Light and electron microscopy studies indicated that cellular damage to some regions of the shoot tip during the freeze/thaw procedure was high, although cell survival in and around the meristematic region allowed shoot tip regeneration. The genetic fingerprinting technique, amplified fragment length polymorphisms (AFLPs), showed that no detectable genetic variation was present between material of C. atrosanguineus at the time of initiation into tissue culture and that which had been cryopreserved, stored in liquid nitrogen for 12 months and regenerated. Weaned plantlets that were grown under glasshouse conditions exhibited no morphological variation from non-frozen controls.

Effect of heat on rheology, surface hydrophobicity and molecular weight distribution of glutens extracted from flours with different bread-making quality

Gluten was extracted from flours of several different wheat varieties of varying baking quality. Creep compliance was measured at room temperature and tan 6 was measured over a range of temperatures from 25 to 95 degrees C. The extracted glutens were heat-treated for 20 min at 25, 40, 50, 60, 70 and 90 degrees C in a water bath, freeze-dried and ground to a fine powder. Tests were carried out for extractability in sodium dodecyl sulphate, free sulphydryl (SH) groups using Ellman's method, surface hydrophobicity and molecular weight (MW) distribution (MWD) using field-flow fractionation and multi-angle laser light scattering. With increasing temperature, the glutens showed a decrease in extractability, with the most rapid decreases occurring between 70 and 90 degrees C, a major transition in tan 6 at around 60 degrees C and a minor transition at 40 degrees C for most varieties, a decrease in free SH groups and surface hydrophobicity and a shift in the MWD towards higher MW. The poor bread-making variety Riband showed the highest values of tan delta and Newtonian compliance, the lowest content of free SH groups and the largest increase of HMW/LMW with increasing temperature. No significant correlations with baking volume were found between any of the measured parameters. (c) 2007 Elsevier Ltd. All rights reserved.