Effect of storage temperature on gentamicin release from antibiotic-coated bone chips

Freezing is the most common method for storing bones until use in skeletal reconstruction. However, the effect of freezing on antibiotic delivery from antibiotic-coated bone has not been evaluated. In this study, we compared antibiotic delivery in vitro from gentamicin-coated human bone stored at different temperatures. Bone chips obtained from human femur heads were chemically cleaned and mixed with gentamicin sulfate. Samples were stored for 4 months at ?20 °C, 4 months at ?80 °C, or evaluated immediately without freezing. Antibiotic release from the bone chips was measured using Bacillus subtilis as an indicator strain. Zones of inhibition and rates of gentamicin release were similar in all three groups. Storage at ?20 and ?80 °C for bone allografts has no effect on gentamicin release from chemically cleaned bone chips.     

Impact of some environmental factors on growth and production of ochratoxin A of/by <Emphasis Type="Italic">Aspergillus tubingensis,A. niger</Emphasis>, and <Emphasis Type="Italic">A. carbonarius</Emphasis> isolated from moroccan grapes

The effects of temperature, water activity (a_w), incubation time, and their combinations on radial growth and ochratoxin A (OTA) production of/by eight Aspergillus niger aggregate strains (six A. tubingensis and two A. niger) and four A. carbonarius isolated from Moroccan grapes were studied. Optimal conditions for the growth of most studied strains were shown to be at 25°C and 0.95 a_w. No growth was observed at 10°C regardless of the water activity and isolates. The optimal temperature for OTA production was in the range of 25°C∼30°C for A. carbonarius and 30°C∼37°C for A. niger aggregate. The optimal a_w for toxin production was 0.95∼0.99 for A. carbonarius and 0.90∼0.95 for A. niger aggregate. Mean OTA concentration produced by all the isolates of A. niger aggregate tested at all sampling times shows that maximum amount of OTA (0.24 μg/g) was produced at 37°C and 0.90 a_w. However, for A. carbonarius, mean maximum amounts of OTA (0.22 μg/g) were observed at 25°C and 0.99 a_w. Analysis of variance showed that the effects of all single factors (a_w, isolate, temperature and incubation time) and their interactions on growth and OTA production were highly significant.     

Whey Protein/Arabic Gum Gels Formed by Chemical or Physical Gelation Process

Whey proteins (WP) gelation process with addition of Arabic gum (AG) was studied. Two different driving processes were employed to induce gelation: (1) heating of 12% whey protein isolate (WPI) solutions (w/w) or (2) acidification of previous thermal denatured WPI solutions (5% w/w) with glucono-δ-lactone (GDL). Protein concentrations were different because they were minimal to form gel in these two processes, but denaturation conditions were the same (90 °C/30 min). Water-holding capacity and mechanical properties of the gels were evaluated. The BST equation was used to evaluate the nonlinear part of the stress–strain data. Cold-set gels were weaker than heat-set gels at the pH range near the isoelectric point (pI) of the main whey proteins, but heated gels were more deformable (did not exhibit rupture point) and showed greater elasticity modulus. However, gels formed by heating far from the pI (pH 6.7 or 3.5) showed more fragile structure, indicating that, in these mixed gels, there are prevailing biopolymers interactions. Cold-set and heat-set gels at pH near or below the WP pI showed strain-weakening behavior, but heated gels at neutral pH showed strong strain-hardening behavior. Such results suggest that differences in stress–strain curve at the nonlinear part of the data could be correlated to structure particularities obtained from different gelation processes.     

The Influence of Water Activity and Temperature on Germination,Growth and Sporulation of <Emphasis Type="Italic">Stachybotrys chartarum</Emphasis> Strains

The objectives were to determine the influence of water activity (a_w, 0.997–0.92) and temperature (10–37°C) and their interactions on conidial germination, mycelial growth and sporulation of two strains of Stachybotrys chartarum in vitro on a potato dextrose medium. Studies were carried out by modifying the medium with glycerol and either spread plating with conidia to evaluate germination and germ tube extension or centrally inoculating treatment media for measuring mycelial growth rates and harvesting whole colonies for determining sporulation. Overall, germination of conidia was significantly influenced by a_w and temperature and was fastest at 0.997–0.98 a_w between 15 and 30°C with complete germination within 24 h. Germ tube extension was found to be most rapid at similar a_w levels and 25–30°C. Mycelial growth rates of both strains were optimal at 0.997 a_w between 25 and 30°C, with very little growth at 37°C. Sporulation was optimum at 30°C at 0.997 a_w. However, under drier conditions, this was optimum at 25°C. This shows that there are differences in the ranges of a_w x temperature for germination and growth and for sporulation. This may help in understanding the role of this fungal species in damp buildings and conditions under which immune-compromised patients may be at risk when exposed to such contaminants in the indoor air environment.     

Effect of Soy Soluble Polysaccharide on the Stability of Soy-Stabilised Emulsions During In Vitro Protein Digestion

This study investigated physicochemical properties of soy soluble polysaccharide (SSP) and pectinase-hydrolysed soy soluble polysaccharide (PH-SSP) from okara, the residue from soy milk production, and their influences when used as a fibre source in oil-in-water (o/w) emulsions. Although pectinase hydrolysed only the carbohydrate fraction in SSP, it resulted in the self-association of PH-SSP to the large-size aggregates. When PH-SSP was added to liquid emulsion containing 3.33% (w/v) rice bran oil and 3.75% (w/v) heated soy protein, it regulated the contents of protein in serum phase, sediment phase and at oil–water interface. The types and contents of soy proteins in the serum phase and sediment phase could be manipulated by pre-heating of soy proteins at 80 °C for 30 min and the addition of PH-SSP. The presence of PH-SSP (0–6% w/v) induced different distribution of proteins to the sediment phase and subsequent in vitro protein digestion in the emulsion. Overall, this study proposed the means to design the distributions of proteins in different phases of o/w emulsion for different degrees of oil release, emulsion stability and protein-polysaccharide coacervation during the course of in vitro peptic and tryptic digestion.     

Stability Assessment of Injectable Castor Oil-Based Nano-sized Emulsion Containing Cationic Droplets Stabilized by Poloxamer–Chitosan Emulsifier Films

The objectives of the present work were to prepare castor oil-based nano-sized emulsion containing cationic droplets stabilized by poloxamer–chitosan emulgator film and to assess the kinetic stability of the prepared cationic emulsion after subjecting it to thermal processing and freeze–thaw cycling. Presence of cryoprotectants (5%, w/w, sucrose +5%, w/w, sorbitol) improved the stability of emulsions to droplet aggregation during freeze–thaw cycling. After storing the emulsion at 4°C, 25°C, and 37°C over a period of up to 6 months, no significant change was noted in mean diameter of the dispersed oil droplets. However, the emulsion stored at the highest temperature did show a progressive decrease in the pH and zeta potential values, whereas the emulsion kept at the lowest temperatures did not. This indicates that at 37°C, free fatty acids were formed from the castor oil, and consequently, the liberated free fatty acids were responsible for the reduction in the emulsion pH and zeta potential values. Thus, the injectable castor oil-based nano-sized emulsion could be useful for incorporating various active pharmaceutical ingredients that are in size from small molecular drugs to large macromolecules such as oligonucleotides.     

Biological fitness of Trichoderma atroviride during long-term storage,after production in different culture conditions

Identification of the production and storage factors that affect conidium germination and bioactivity (fitness) will assist the success of biological control agents. Effects of culturing conditions on conidium fitness of Trichoderma atroviride LU132 were examined in different storage conditions over time. Abiotic factors (temperature, nutrients, water activity and pH) during production were studied. Conidia from the culturing regimes which resulted in greatest and least bioactivity against Rhizoctonia solani in dual culture were selected to assess effects of storage conditions on conidium fitness. Fitness of the test conidia was examined after storage at 30°C and at 0% or 50% relative humidity (RH) over 6 months. Fitness declined over time, and the decline was greater for 50% RH than 0% RH, probably through reduced metabolic activity of conidia during long-term storage. Stored conidia were probably affected by dehydration, temperature and other factors such as oxidation, before and during storage, and also by rehydration after storage. The greatest number of conidia and germination percentage resulted from production at 25°C, but greatest bioactivity resulted from those produced at 30°C. No significant effects on bioactivity were detected between the conidium production treatments C?:?N 5?:?1 and C?:?N 160?:?1, indicating that C?:?N ratio in culture medium is not important for conidium survival of T. atroviride.     

Body heat responsive gelation of methylcellulose formulation containing betaine

We examined a methylcellulose (MC) formulation that gels at body temperature for enteral alimentation. Betaine was found to have a lowering effect on the gelation temperature of the MC solution. The thermal gelation temperature of a body heat-responsive (BHR) gelling MC formulation, consisting of 2% MC, 15% glucose, 1.2% sodium citrate, and 3.5% betaine mixture, was approximately 32 °C, indicating that it could gel in response to body heat. Glucose release from the BHR gels was delayed at 37 °C in an in vitro study. In rats, oral administration of BHR gelling MC formulation delayed an increase in blood glucose and appearance of ¹³CO₂ in expired air in a ¹³C-acetate breath test in comparison with the control. These results suggested that the BHR gelling MC formulation was gelled in the stomach and delayed gastric emptying after oral administration and glucose in the gels was absorbed slowly.     

Growth and sporulation of entomopathogenic Beauveria bassiana,Metarhizium anisopliae,Isaria farinosa and Isaria fumosorosea strains in relation to water activity and temperature interactions

This study examined six strains of Beauveria bassiana s.l. and Isaria farinosa, one strain of Isaria fumosorosea and five strains of Metarhizium anisopliae s.l. to identify the ability for (1) growth and (2) sporulation under interacting environmental factors of water activity (a_w) and temperature stress. Growth on Sabouraud Dextrose Agar (SDA; water activity, a_w = 0.995) or SDA modified with glycerol to 0.98, 0.96 and 0.94 a_w was measured at four different temperatures (25, 30, 35 and 37°C). All M. anisopliae strains grew at 25–35°C and 0.995 a_w while only two strains tolerated extreme water stress at 0.94 a_w.Three strains of B. bassiana were able to grow at 25–37°C and 0.995 a_w. Only one strain of I. farinosa was able to grow at 25–37°C and 0.995 a_w. A_w and temperature interactions resulted in different strain-dependent responses, in terms of growth and sporulation. Only one strain of I. farinosa and three of M. anisopliae grew at 0.94 a_w and none of the B. bassiana strains tolerated such water stress. At 0.96 and 0.94 a_w and 35–37°C, sporulation by all the strains of the three species were significantly affected. Under elevated temperatures and drought stress, very few of these strains of entomopathogenic fungi are able to grow and sporulate. Indeed, the B. bassiana strains were unable to tolerate the extreme conditions examined. Resilience to such abiotic interactions is critical for selecting strains for formulations. Tolerance to water and temperature stress could be good criteria for selection of strains with secondary spread potential for use as part of an integrated pest management system where secondary cycling may be important, especially in sub-tropical and tropical environments.     

Effect of temperature and water activity on growth and ochratoxin A production boundaries of two Aspergillus carbonarius isolates on a simulated grape juice medium

Aims: To develop and validate a logistic regression model to predict the growth and ochratoxin A (OTA) production boundaries of two Aspergillus carbonarius isolates on a synthetic grape juice medium as a function of temperature and water activity (a_w). Methods and Results: A full factorial design was followed between the factors considered. The a_w levels assayed were 0·850, 0·880, 0·900, 0·920, 0·940, 0·960, 0·980 and the incubation temperatures were 10, 15, 20, 25, 30, 35 and 40°C. Growth and OTA production responses were evaluated for a period of 25 days. Regarding growth boundaries, the degree of agreement between predictions and observations was >99% concordant for both isolates. The erroneously predicted growth cases were 3·4–4·1% false‐positives and 0·7–1·4% false‐negatives. No growth was observed at 10°C and 40°C for all a_w levels assayed, with the exception of 0·980 a_w/40°C, where weak growth was observed. Similarly, OTA production was correctly predicted with a concordance rate >98% for the two isolates with 0·7–1·4% accounting for false‐positives and 2·0–2·7% false‐negatives. No OTA production was detected at 10°C or 40°C regardless of a_w, and at 0·850 a_w at all incubation temperatures. With respect to time, the OTA production boundary shifted to lower temperatures (15–20°C) as opposed to the growth boundary that shifted to higher temperature levels (25–30°C). Using two literature datasets for growth and OTA production of A. carbonarius on the same growth medium, the logistic model gave one false‐positive and three false‐negative predictions out of 68 growth cases and 13 false‐positive predictions out of 45 OTA production cases. Conclusions: The results of this study suggest that the logistic regression model can be successfully used to predict growth and OTA production interfaces for A. carbonarius in relation to temperature and a_w. Significance and Impact of the Study: The proposed modelling approach helps the understanding of fungal‐food ecosystem relations and it could be employed in risk analysis implementation plans to predict the risk of contamination of grapes and grape products by A. carbonarius.     

Temperature and moisture requirements for conidial germination of an isolate of Beauveria bassiana,pathogenic to Rhodnius prolixus

The effects of temperature, relative humidity and water activity on germination of conidia of an isolate of Beauveria bassiana (Bals.) Vuill. pathogenic to the triatomine vector of Chagas' disease, Rhodnius prolixus Stål., were investigated in vitro. Germination occurred at temperatures between 15 °C and 35 °C under saturated atmosphere and the optima ranged from 25 °C to 3O °C. At the extreme temperatures tested (15 °C and 35 °C) the germination process was delayed, but germination rates reached more than 95%. Germination of B. bassiana conidia was strongly affected by moisture conditions. The availability of water, in both atmospheric and liquid conditions, caused changes in germination times as well as in germination rates. For example, at 25 °C + O.5 °C, germination took place within 20 h at 95.5% RH, whereas it needed 72 h of incubation at 90% RH. Germination times increased as the water activity declined from 0.96 a_w to 0.92 a_w. Below 0.92 a_w, o germination was observed after a 72 h incubation time.     

Observations on the spore release of Paxillus panuoides

Spore release of Paxillus panuoides was studied in a forest environment at temperatures from ?4 to 24°C and 30 to 100% relative humidity (RH) near Stoneville, Mississippi, from December 1983 through February 1985. Spores were released when temperatures were above 0°C, and daily peaks were usually associated with increased temperatures and decreased RH. In a controlled environment, spore release increased from a temperature of 2°C, to a maximum at 37°C, then ceased at 45°C. Light and RH treatments did not significantly affect spore release. Temperature was determined to be the stimulus for the natural spore release pattern.     

Gamma Irradiation on Fermentation Mashes Consisting Mainly of Cane Molasses

The application of the radiopasteurization method to fermentation media consisting mainly of molasses was investigated. γ-Irradiation was found to have an excellent pasteurization effect on the fermentation media and at the same time to bring about an increase in the fermentation rate and yield of ethanol. Percent survivals in molasses decreased to ca. 70% by heating at 80°C for 30 min, to ca. 10% by irradiation with 3.0 × 10⁵ rad and to ca. 1% by 6.0 × 10⁵ rad. Irradiated mash was suitable for the medium of the “starter”, since the rate and the degree of the growth of Saccharomyces cerevisiae in irradiated mash did not differ from those of the growth in heat-pasteurized mash.

In the case of the molasses mash supplemented with nitrogen sources, the fermentation rate and yield of ethanol in irradiated mash were larger than those in heated mash. Besides, in the absence of nitrogen sources a 14% difference in fermentation yield was seen between the mash irradiated with 3.0 × 10⁵ rad and the mash heated at 80°C. With the doses ranging from 1.0 × 10⁵ to 9.5 × 10⁵ rad, concentrations of total sugar and direct reducing sugar, pH, and optical density of molasses were little affected by irradiation.     

Naked Plasmid DNA Formulation: Effect of Different Disaccharides on Stability after Lyophilisation

Since plasmid DNA (pDNA) is unstable in solution, lyophilisation can be used to increase product shelf life. To prevent stress on pDNA molecules during lyophilisation, cryo- and lyoprotectants have to be added to the formulation. This study assessed the effect of disaccharides on naked pDNA stability after lyophilisation using accelerated stability studies. Naked pDNA was lyophilised with sucrose, trehalose, maltose or lactose in an excipient/DNA w/w ratio of 20. To one part of the vials extra residual moisture was introduced by placing the vials half opened in a 25°C/60% RH climate chamber, before placing all vials in climate chambers (25°C/60% RH and 40°C/75% RH) for stability studies. An ex vivo human skin model was used to assess the effect of disaccharides on transfection efficiency. Lyophilisation resulted in amorphous cakes for all disaccharides with a residual water content of 0.8% w/w. Storage at 40°C/75% RH resulted in decreasing supercoiled (SC) purity levels (sucrose and trehalose maintained approximately 80% SC purity), but not in physical collapse. The addition of residual moisture (values between 7.5% and 10% w/w) resulted in rapid collapse except for trehalose and decreasing SC purity for all formulations. In a separate experiment disaccharide formulation solutions show a slight but significant reduction (<3% with sucrose and maltose) in transfection efficiency when compared to pDNA dissolved in water. We demonstrate that disaccharides, like sucrose and trehalose, are effective lyoprotectants for naked pDNA.     

Processing and characterization of laser sintered hydroxyapatite scaffold for tissue engineering

The sintering processing of hydroxyapatite (HAP) powder was studied using selective laser sintering for bone tissue engineering. The effect of laser energy density on the microstructure, phase composition and mechanical properties of the sintered samples was investigated. The results indicate that the average grain size increases from 0.211 ± 0.039 to 0.979 ± 0.133 μm with increasing the laser energy density from 2.0 to 5.0 J/mm². The maximum value of Vickers hardness and fracture toughness were 4.0 ± 0.13 Gpa and 1.28 ± 0.033 MPam^1/2, respectively, when the laser energy density was 4.0 J/mm². The XRD results indicated that the nano-HAP was decomposed into TCP with the laser energy density of above 4.0 J/mm². In vitro bioactivity after soaking in simulated body fluid (SBF) for 3 ～ 12 days showed that a bone-like apatite layer on the surface of the sintered samples. It indicated that the HAP scaffold possesses favorable mechanical properties and bioactivity, and may be used for bone tissue engineering.     

Factors determining the microflora of stored barley grain

Colonisation of barley grain has been studied during storage at different water contents and with and without restriction of the air supply to simulate conditions in unsealed silos. Grain stored with a water activity >0·9 a_w (20% water content) heated spontaneously when aeration was unrestricted, the maximum temperature attained increasing with a_w to 58 °C at 1·0 a_w (39% water content). The presence of many unripe grains increased the tendency to heat at a given mean water content. Although heating was prevented by sheeting to restrict the air supply, it could occur subsequently when the sheeting was removed. Both heating and restriction of the air supply were associated with increased carbon dioxide (to >25%) and decreased oxygen concentrations (to <5%). Germination of grain after 6·9 months storage was correlated with a_w; germination levels approaching 100% were retained only at about 0·7 a_w (13·5% water content). Colonisation by Aspergillus species was correlated with a_w and temperature and similar correlations with Penicillium species were also found, with P. verrucosum var. cyclopium abundant at 0·85-0·90 a_w (17·20% water content) and P. piceum, P. funiculosum and P. capsulatum at 0·90-0·95 a_w (20–25% water content). In sealed containers P. roquefortii occurred at 1·00 a_w (39% water content) and P. hordei at 0·90-0·92 a_w (20–22% water content). Spores of fungi and actinomycetes formed during spontaneous heating of grain survived 6 months sealed storage although Absidia corymbifera and Micropolyspora faeni may have declined in numbers. Fungicides applied to the ripening grain had only limited effect on the colonisation of the grain during storage.     

Effect of high temperature treatment on disinfestation and quality characteristics of sorghum

Sorghum grain (two varieties) was modified to different water contents (12% to 16% wet weight basis) and heated to 60°C, 70°C and 80°C for periods of 4, 8 and 12 min. Germination, seedling vigour, seedling dry matter, free fatty acid (FFA) content, fungal contamination and infestation with the insect pest Rhyzopertha dominica (F.) were all markedly affected by heat treatment. The effectiveness of the heat treatment was also influenced by the size of the sample used. Heat treatment at 60°C and 70°C resulted in germination being unaffected or stimulated while at 80°C and the higher water contents significantly reduced, when compared to untreated controls. The dry matter of seedlings, and seedling vigour was positively correlated with germination and heat treatment. Heat treatment had no effect on FFA. All stages of the insect pest, R. dominica, were destroyed at 70°C and an 8 min exposure time. However, the water content of the sorghum was critical in determining the efficacy against this pest. The percentage fungal contamination of grain was reduced from 90% to about 25% by heat treatment. However, some grain fungi, particularly Eurotium spp., Aspergillus niger and Penicillium spp. could still be isolated from sorghum grain treated at 80°C for up to 12 minutes.     

The effect of gradual changes in temperature on the release of hormones from nerve endings isolated from bovine neural lobes.

Abstract— Homogenates of bovine neural lobe tissue were fractionated by differential centrifugation at 20°C or at 4°C and the distribution of activities of vasopressin and oxytocin among the fractions was compared. The ratio of total hormone to protein (mg) in the homogenate was similar at the two temperatures. At 20°C a much smaller proportion of the total hormone was recovered in the soluble fraction (100,000 g_av supernatant), than at 4°C with a corresponding increase in recovery in the nerve-ending fraction (800–3000 g sediment). Nerve endings isolated at 4°C did not, when incubated, release hormone in response to changes in temperature. Nerve endings isolated at 20°C released hormone when the temperature was reduced below 15°C. Gradual reduction in temperature led to hormone release unaccompanied by lactate dehydrogenase release. Incubation of nerve endings for 10 min at 10°C increased the release of vasopressin and of neurophysin without any increase in lactate dehydrogenase. These results demonstrate that release of vasopressin by cold stimulation occurs by way of exocytosis.     

Effects of temperature and water activity on <Emphasis Type="Italic">Lecanicillium</Emphasis> spp. conidia germination and growth,and mycosis of <Emphasis Type="Italic">Pissodes strobi</Emphasis>

Selecting entomopathogenic fungal isolates for use as biocontrol agents requires an assessment of their growth and virulence characteristics as affected by environmental conditions. Here we demonstrate a wide temperature and moisture range for colony growth, effective conidial germination and virulence against Pissodes strobi Peck (white pine weevil) of several isolates of Lecanicillium Gams and Zare, an entomopathogenic fungus distributed worldwide and indigenous to forests on Vancouver Island, British Columbia, Canada. In order to examine the potential Lecanicillium as a biological control agent, the pathogenicity of isolates collected from different geographical locations on P. strobi cadavers was assessed, and colony growth at different temperatures was evaluated. Colony growth was evident between 5 and 30°C, with optimal growth occurring at 25°C. Various combinations of water activity (0.55, 0.76, 0.85 and 0.99 a _w) and temperature (10, 15, 20, and 25°C) were also used to evaluate environmental impacts on conidial germination and cumulative mycosis of adult P. strobi. Certain Lecanicillium isolates displayed xerophilic (0.85 a _w) or psychrophilic (10°C) growth optima. Ultimately, identifying the abiotic limits of this entomopathogenic fungus will be used to determine which isolates have potential for future in situ biocontrol trials.