Optimal protection of stabilised dry live bacteria from bile toxicity in oral dosage forms by bile acid adsorbent resins

We previously found that dried live bacteria of a vaccine strain can be temporarily sensitive to bile acids and suggested that Bile Adsorbing Resins (BAR) can be used in oral vaccine tablets to protect dried bacteria from intestinal bile. Here, we report a quantitative analysis of the ability of BAR to exclude the dye bromophenol blue from penetrating into matrix tablets and also sections of hard capsule shells. Based on this quantitative analysis, we made a fully optimised formulation, comprising 25% w/w of cholestyramine in Vcaps™ HPMC capsules. This gave effectively 100% protection of viability from 4% bile, with 4200-fold more live bacteria recovered from this formulation compared to unprotected dry bacteria. From the image analysis, we found that the filler material or compaction force used had no measurable effect on dye exclusion but did affect the rate of tablet hydration. Increasing the mass fraction of BAR gave more exclusion of dye up to 25% w/w, after which a plateau was reached and no further dye exclusion was seen. More effective dye exclusion was seen with smaller particle sizes (i.e. cholestyramine) and when the BAR was thoroughly dried and disaggregated. Similar results were found when imaging dye penetration into capsule sections or tablets. The predictions of the dye penetration study were tested using capsules filled with dried attenuated Salmonella vaccine plus different BAR types, and the expected protection from bile was found, validating the imaging study. Surprisingly, depending on the capsule shell material, some protection was given by the capsule alone without adding BAR, with Vcaps™ HPMC capsules providing up to 174-fold protection against 1% bile; faster releasing Vcaps Plus™ HPMC capsules and Coni Snap™ gelatin capsules gave less protection.

Saturated salt solutions for humidity control and the survival of dry powder and oil formulations of Beauveria bassiana conidia

Oil-based formulated conidia sprayed on steel plates and conidia powder (control) of Beauveria bassiana isolate IMI 386243 were stored at temperatures from 10 to 40 degrees C in desiccators over saturated salt solutions providing relative humidities from 32 to 88%, or in hermetic storage at 40 degrees C, and moisture contents in equilibrium with 33 or 77% relative humidity. The negative semi-logarithmic relation (P < 0.005) between conidia longevity (at 40 degrees C) and equilibrium relative humidity did not differ (P > 0.25) between formulated conidia and conidia powder. Despite this, certain saturated salts provided consistently greater longevity (NaCl) and others consistently shorter longevity (KCl) for formulated conidia compared to conidia powder. These results, analysis of previous data, and comparison with hermetic storage, indicate that storage of conidia over saturated salt solutions provides inconsistent responses to environment and so may be problematic for bio-pesticide research. In hermetic storage, oil formulation was not deleterious to longevity and in the more moist environment enhanced survival periods. (c) 2005 Elsevier Inc. All rights reserved.

Survival and vigour of ultra-dry seeds after ten years of hermetic storage

Seeds of carrot, groundnut, lettuce, oilseed rape and onion were stored hermetically in laminated aluminium foil packets in four environments (dry or ultra-dry moisture contents combined factorially with temperatures of 20 degrees C or -20 degrees C), replicated at several sites. After ten years' hermetic storage, seed moisture content, equilibrium relative humidity, viability (assessed by ability to germinate normally in standard germination tests) and vigour were determined. After a decade, the change in seed moisture content of samples stored at -20 degrees C was small or nil. Except for groundnut and lettuce (where loss in viability was about 8 and 3%, respectively), no loss in viability was detected after 10 years' hermetic storage at -20 degrees C. In all cases, there was no difference in seed survival between moisture contents at this temperature (P > 0.25). Comparison of seed vigour (root length and rate of germination) also confirmed that drying to moisture contents in equilibrium with 10-12% r.h. had no detrimental effect to longevity when stored at -20 degrees C: the only significant (P < 0.05) differences detected were slightly greater root lengths for ultra-dry storage of four of the six seed lots. Seed moisture content had increased after a decade at 20 degrees C (generally to the level in equilibrium with ambient relative humidity). Hence, sub-zero temperature storage helped maintain the long-term integrity of the laminated aluminium foil packets, as well as that of the seeds within.

In situ degradability of dry matter and neutral-detergent fibre of thorn scrubland forage consumed by goats in the semi-arid region of north Mexico

Three goats provided with oesophageal and ruminal cannulae were used to determine variations in dry matter (DM) and neutral-detergent fibre (NDF) degradability of the forage consumed when grazing thorn scrubland in the semi-arid region of north Mexico, during two consecutive dry and wet periods. Ingesta samples were incubated intraruminally, the data were fitted to the exponential equation P = a + b (1-e(-ct)) and statistically analysed using a randomized-block design. Organic matter and crude protein (CP) contents were higher (P < 0.05) in the wet seasons. Values of NDF were similar in dry and wet season of both years whereas higher numerical values of acid-detergent fibre (ADF), lignin and cellulose were registered in the dry seasons. DM and NDF degradabilities after 24 and 48 h of ruminal incubation were higher (P < 0.05) in the wet seasons. Higher values (P < 0.05) in DM and NDF bag losses at zero time (A fraction) were registered in the two wet seasons. The insoluble but fermentable DM and NDF (B fractions) were higher (P < 0.05) in the 1999 wet season and variable in the rest of the studied period. Numerically higher values of DM and NDF c fraction were found in wet periods, whereas DM and NDF potential degradabilities were higher (P < 0.05) in the wet season in 1999 and similar across seasons in 2000. Lowest (P < 0.05) contents of CP in grazed forage, DM and NDF degradabilities after 48 h of ruminal incubation, and A, and B, and c fractions were observed in the dry seasons. Thus, these results may be related to both the lower feeding value of forage consumed by the animals and lower performance of livestock during this period. Then, the DM and NDF degradability after 48 h, together with the insoluble but fermentable matter and the c fraction permit the nutritive value of the forage consumed by grazing goats to be accurately described.

Dry matter intake is decreased more by abomasal infusion of unsaturated free fatty acids than by unsaturated Triglycerides

Previous experiments from our group have demonstrated that abomasal infusion of unsaturated free fatty acids (FFA) markedly decreases dry matter intake (DMI) in dairy cows. In contrast, experiments from other groups have noted smaller decreases in DMI when unsaturated triglycerides (TG) were infused postruminally. Our hypothesis was that unsaturated FFA would be more potent inhibitors of DMI than an equivalent amount of unsaturated TG. Four Holstein cows in late lactation were used in a single reversal design. Cows were fed a total mixed ration containing (DM basis) 23% alfalfa silage, 23% corn silage, 40.3% ground shelled corn, and 10.5% soybean meal. Two cows received soy FFA (UFA; 0, 200, 400, 600 g/d) and 2 received soy oil (TG) in the same amounts; cows then were switched to the other lipid source. Cows were abomasally infused with each amount for 5-d periods. The daily amount of lipid was pulse-dosed in 4 equal portions at 0600, 1000, 1700, and 2200 h; no emulsifiers were used and there was no sign of digestive disturbance. Both lipid sources linearly decreased DMI, with a significant interaction between lipid source and amount. Slope-ratio analysis indicated that UFA were about 2 times more potent in decreasing DMI than were TG. Decreased DMI led to decreased milk production. Milk fat content was increased linearly by lipid infusion. Milk fat yield decreased markedly for UFA infusion but was relatively unaffected by infusion of TG. Contents of short- and medium-chain fatty acids in milk fat decreased as the amount of either infusate increased. Contents of C-18:2 and C18: 3 in milk fat were increased linearly by abomasal infusion of either fat source; cis-9 C-18:1 was unaffected. Transfer of infused C18: 2 to milk fat was 35.6, 42.5, and 27.8% for 200, 400, and 600 g/d of UFA, and 34.3, 39.6, and 34.0% for respective amounts of TG. Glucagon-like peptide-1 (7-36) amide (GLP-1) concentration in plasma significantly increased as DMI decreased with increasing infusion amount of UFA or TG. Plasma concentration of cholecystokinin-octapeptide (CCK-8) was unaffected by lipid infusion. These results indicate that unsaturated FFA reaching the duodenum are more potent inhibitors of DMI than are unsaturated TG; the effect may be at least partially mediated by GLP-1.

Long-term response of the nematode community to elevated atmospheric CO2 in a temperate dry grassland soil

Long-term effects of the elevated atmospheric CO2 on biosphere have been in focus of research since the last few decades. In this experiment undisturbed soil monoliths of loess grassland were exposed to an elevated CO2 environment (two-times the ambient CO2 level) for a period of six years with the aid of the open top chamber method. Control without a chamber and CO2 elevation was applied as well. Elevated CO2 level had very little impact oil soil food web. It did not influence either root and microbial biomass or microbial and nematode community structure. The only significant response was that density of the bacterial feeder genus Heterocephalobus increased in the chamber with elevated CO2 concentration. Application of the open top chambers initiated more changes on nematodes than the elevated CO2 level. Open top chamber (OTC) method decreased nematode density (total and plant feeder as well) to less than half of the original level. Negative effect was found on the genus level in the case of fungal feeder Aphelenchoides, plant feeder Helicotylenchus and Paratylenchus. It is very likely that the significantly lower belowground root biomass and partly its decreased quality reflected by the increased C/N ratio are the main responsible factors for the lower density of the plant feeder nematodes in the plots of chambers. According to diversity profiles, MI and MI(2-15) parameters, nematode communities in the open top chambers (both on ambient and elevated CO2 level) seem to be more structured than those under normal circumstances six years after start of the experiment.

The nutritive value of forage sorghum genotypes developed for the dry tropical highlands of Kenya as feed source for ruminants

in vitro studies were conducted on five sorghum genotypes developed for the dry tropical highland climate of Kenya and which can be fed to ruminants fresh or as silage. The five sorghum genotypes consisted of two normal white mid-rib (WMR) genotypes, coded E1291 and E65181, and three brown-midrib (BMR) genotypes, coded Lan-5, Lan-6 and Lan-12. Whole mature plants (herbage plus grain) and silage made from E 1291 were used in the study. An in vitro manual gas production technique was used to compare the nutritive characteristics of these genotypes for ruminants. These sorghums differed significantly in true organic matter degraded (OMDeg), which ranged from 520 to 678 g/kg after 24 h incubation and 706 to 805 g/kg after 72 h incubation. All the BMR sorghums had a higher degradability than the WMR genotype, E6518, and the silage, with Lan-5 having the highest degradability. Methane produced per g OMDeg ranged from 40.6 to 46.4 mL/g after 24 h incubation and from 53.1 to 62.6 mL/g after 72 h incubation. It was similar for all genotypes after 24 h incubation but Lan-12 had the highest methane production after 72 h incubation. After 24 h and 72 h incubation all the genotypes produced a similar total amount of gas per OMDeg (293 to 309 and 357 to 385 mL/g, respectively) with similar total short chain fatty acid concentrations in the liquid digesta (7.8 to 10.4 and 9.5 to 10.3 mmol, respectively) and acetate to propionate ratios of 2.16 to 2.49 and 2.35 to 2.87, respectively. The sorghums showed great potential as ruminant feed sources in the region.

Modelling simultaneously water content and dry matter dynamics of wheat grains

A model was devised to describe simultaneously the grain masses of water and dry matter against thermal time during grain filling and maturation of winter wheat. The model accounted for a linear increase in water mass of duration anthesis-m(1) (end of rapid water assimilation phase) and rate a, followed by a more stable water mass until in,, after which water mass declined rapidly at rate e. Grain dry matter was described as a linear increase of rate bgf until a maximum size (maxgf) was attained at m(2).The model was fitted to plot data from weekly samples of grains taken from replicated field experiments investigating effects of grain position (apical or medial), fungicide (five contrasting treatments), sowing date (early or late), cultivar (Malacca or Shamrock) and season (2001/2002 and 2002/2003) on grain filling. The model accounted for between 83 and 99% of the variation ( 2) when fitted to data from individual plots, and between 97 and 99% when fitted to treatment means. Endosperm cell number of grains from early-sown plots in the first season were also counted. Differences in maxgf between grain positions and also between cultivars were mostly the result of effects on bgf and were empirically associated with water mass at nil. Fungicide application controlled S. tritici and powdery mildew infection, delayed flag leaf senescence, increased water mass at m(1) (wm(1)), and also increased m(2), bgf and maxgf. Fungicide effects on water mass were detected before fungicide effects on dry matter, but comparison of the effects of individual fungicide treatments showed no evidence that effects on wm(1), nor on endosperm cell numbers at about m(1), were required for fungicide effects on maxgf, (c) 2005 Elsevier B.V. All rights reserved.

Successful long-term ultra dry storage of seed of 15 species of Brassicaceae in a genebank: variation in ability to germinate over 40 years and dormancy

Seed of 15 species of Brassicaceae were stored hermetically in a genebank (at -5 degrees C to -10 degrees C with c. 3% moisture content) for 40 years. Samples were withdrawn at intervals for germination tests. Many accessions showed an increase in ability to germinate over this period. due to loss in dormancy. Nevertheless, some dormancy remained after 40 years' storage and was broken by pre-applied gibberellic acid. The poorest seed survival occurred in Hormatophylla spinosa. Even in this accession the ability to germinate declined by only 7% between 1966 and 2006. Comparison of seeds from 1966 stored for 40 years with those collected anew in 2006 from the original sampling sites, where possible, showed few differences, other than a tendency (7 of 9 accessions) for the latter to show greater dormancy. These results for hermetic storage at sub-zero temperatures and low moisture contents confirm that long-term seed storage can provide a successful technology for ex situ plant biodiversity conservation.

Examination of the persistency of milk fatty acid composition responses to fish oil and sunflower oil in the diet of dairy cows

Based on the potential benefits of cis-9, trans-11 conjugated linoleic acid (CLA) for human health, there is a need to develop effective strategies for enhancing milk fat CLA concentrations. Levels of cis-9, trans-11 CLA in milk can be increased by supplements of fish oil (FO) and sunflower oil (SO), but there is considerable variation in the response. Part of this variance may reflect time-dependent ruminal adaptations to high levels of lipid in the diet, which lead to alterations in the formation of specific biohydrogenation intermediates. To test this hypothesis, 16 late lactation Holstein-British Friesian cows were used in a repeated measures randomized block design to examine milk fatty acid composition responses to FO and SO in the diet over a 28-d period. Cows were allocated at random to corn silage-based rations (8 per treatment) containing 0 (control) or 45 g of oil supplement/ kg of dry matter consisting (1:2; wt/wt) of FO and SO (FSO), and milk composition was determined on alternate days from d 1. Compared with the control, the FSO diet decreased mean dry matter intake (21.1 vs. 17.9 kg/d), milk fat (47.7 vs. 32.6 g/kg), and protein content (36.1 vs. 33.3 g/kg), but had no effect on milk yield (27.1 vs. 26.4 kg/d). Reductions in milk fat content relative to the FSO diet were associated with increases in milk trans-10 18: 1, trans-10, cis-12 CLA, and trans-9, cis-11 CLA concentrations (r(2) = 0.74, 0.57, and 0.80, respectively). Compared with the control, the FSO diet reduced milk 4: 0 to 18: 0 and cis 18:1 content and increased trans 18:1, trans 18:2, cis-9, trans-11 CLA, 20: 5 n-3, and 22: 6 n-3 concentrations. The FSO diet caused a rapid elevation in milk cis-9, trans-11 CLA content, reaching a maximum of 5.37 g/100 g of fatty acids on d 5, but these increases were transient, declining to 2.35 g/100 g of fatty acids by d 15. They remained relatively constant thereafter. Even though concentrations of trans-11 18: 1 followed the same pattern of temporal changes as cis-9, trans-11 CLA, the total trans 18:1 content of FSO milk was unchanged because of the concomitant increases in the concentration of other isomers (Delta(4-10) and Delta(12-15)), predominantely trans-10 18:1. In conclusion, supplementing diets with FSO enhances milk fat cis-9, trans-11 CLA content, but the high level of enrichment declines because of changes in ruminal biohydrogenation that result in trans-10 replacing trans-11 as the major 18:1 biohydrogenation intermediate formed in the rumen.