Biodegradation of polyethylene glycol (PEG) in three tropical soils using radio labelled PEG

Polyethylene glycol (PEG) may be added to forage based diets rich in tannins for ruminant feeding because it binds to tannins and thus prevent the formation of potentially indigestible tannin-protein complexes. The objective of this work was to determine the in vitro biodegradation (mineralization, i.e., complete breakdown of PEG to CO2) rate of PEG. C-14-Polyethylene glycol (C-14-PEG) was added to three different tropical soils (a sandy clay loam soil, SaCL; a sandy clay soil, SaC; and a sandy loam soil, SaL) and was incubated in Bartha flasks. Free PEG and PEG bound to tannins from a tannin rich local shrub were incubated under aerobic conditions for up to 70 days. The biodegradation assay monitored the (CO2)-C-14 evolved after degradation of the labelled PEG in the soils. After incubation, the amount of (CO2)-C-14 evolved from the C-14-PEG application was low. Higher PEG mineralization values were found for the soils with higher organic matter contents (20.1 and 18.6 g organic matter/kg for SaCL and SaC, respectively) than for the SaL soil (11.9 g organic matter/kg) (P < 0.05). The extent of mineralization of PEG after 70 days of incubation in the soil was significantly lower (P < 0.05) when it was added as bound to the browse tannin than in the free form (0.040 and 0.079, respectively). (c) 2005 Elsevier B.V. All rights reserved.

Self assembly of a model amphiphilic phenylalanine peptide/polyethylene glycol block copolymer in aqueous solution

There has been great interest recently in peptide amphiphiles and block copolymers containing biomimetic peptide sequences due to applications in bionanotechnology. We investigate the self-assembly of the peptide-PEG amphiphile FFFF-PEG5000 containing the hydrophobic sequence of four phenylalanine residues conjugated to PEG of molar mass 5000. This serves as a simple model peptide amphiphile. At very low concentration, association of hydrophobic aromatic phenylalanine residues occurs, as revealed by circular dichroism and UV/vis fluorescence experiments. A critical aggregation concentration associated with the formation of hydrophobic domains is determined through pyrene fluorescence assays. At higher concentration, defined beta-sheets develop as revealed by FTIR spectroscopy and X-ray diffraction. Transmission electron microscopy reveals self-assembled straight fibril structures. These are much shorter than those observed for amyloid peptides, the finite length may be set by the end cap energy due to the hydrophobicity of phenylalanine. The combination of these techniques points to different aggregation processes depending on concentration. Hydrophobic association into irregular aggregates occurs at low concentration, well-developed beta-sheets only developing at higher concentration. Drying of FFFF-PEG5000 solutions leads to crystallization of PEG, as confirmed by polarized optical microscopy (POM), FTIR and X-ray diffraction (XRD). PEG crystallization does not disrupt local beta-sheet structure (as indicated by FTIR and XRD). However on longer lengthscales the beta-sheet fibrillar structure is perturbed because spheruilites from PEG crystallization are observed by POM. (C) 2009 Elsevier B.V. All rights reserved.

Orientational ordering in the nematic phase of a polyethylene glycol-peptide conjugate in aqueous solution

The orientational ordering of the nematic phase of a polyethylene glycol (PEG)-peptide block copolymer in aqueous solution is probed by small-angle neutron scattering (SANS), with the sample subjected to steady shear in a Couette cell. The PEG-peptide conjugate forms fibrils that behave as semiflexible rodlike chains. The orientational order parameters (P) over bar (2) and (P) over bar (4) are obtained by modeling the data using a series expansion approach to the form factor of uniform cylinders. The method used is independent of assumptions on the form of the singlet orientational distribution function. Good agreement with the anisotropic two-dimensional SANS patterns is obtained. The results show shear alignment starting at very low shear rates, and the orientational order parameters reach a plateau at higher shear rates with a pseudologarithmic dependence on shear rate. The most probable distribution functions correspond to fibrils parallel to the flow direction under shear, but a sample at rest shows a bimodal distribution with some of the rodlike peptide fibrils oriented perpendicular to the flow direction.

In vitro gas production and nitrogen degradability of tannin-containing tree fruits in response to incremental levels of polyethylene glycol

This study was designed to determine the response of in vitro fermentation parameters to incremental levels of polyethylene glycol (PEG) when tanniniferous tree fruits (Dichrostachys cinerea, Acacia erioloba, A. erubiscens, A. nilotica and Piliostigma thonningii) were fermented using the Reading Pressure Technique. The trivalent ytterbium precipitable phenolics content of fruit substrates ranged from 175 g/kg DM in A. erubiscens to 607 g/kg DM in A. nilotica, while the soluble condensed tannin content ranged from 0.09 AU550nm/40mg in A. erioloba to 0.52 AU550nm/40 mg in D. cinerea. The ADF was highest in P. thonningii fruits (402 g/kg DM) and lowest in A. nilotica fruits (165 g/kg DM). Increasing the level of PEG caused an exponential rise to a maximum (asymptotic) for cumulative gas production, rate of gas production and nitrogen degradability in all substrates except P. thonningii fruits. Dry matter degradability for fruits containing higher levels of soluble condensed tannins (D. cinerea and P. thonningii), showed little response to incremental levels of PEG after incubation for 24 h. The minimum levels of PEG required to maximize in vitro fermentation of tree fruits was found to be 200 mg PEG/g DM of sample for all tree species except A. erubiscens fruits, which required 100 mg PEG/g DM sample. The study provides evidence that PEG levels lower than 1 g/g DM sample can be used for in vitro tannin bioassays to reduce the cost of evaluating non-conventional tanniniferous feedstuffs used in developing countries in the tropics and subtopics. The use of in vitro nitrogen degradability in place of the favoured dry matter degradability improved the accuracy of PEG as a diagnostic tool for tannins in in vitro fermentation systems.

Effect of water-soluble polymers, polyethylene glycol and poly(vinylpyrrolidone),on the gelation of aqueous micellar solutions of Pluronic copolymer F127

The micellization of F127 (E98P67E98) in dilute aqueous solutions of polyethylene glycol (PEG6000 and PEG35000) and poly(vinylpyrrolidone) (PVP K30 and PVP K90) is studied. The average hydrodynamic radius (rh,app) obtained from the dynamic light scattering technique increased with increase in PEG concentration but decreased on addition of PVP, results which are consistent with interaction of the micelles with PEG and the formation of micelles clusters, but no such interaction occurs with PVP. Tube inversion was used to determine the onset of gelation. The critical concentration of F127 for gelation increased on addition of PEG and of PVP K30 but decreased on addition of PVP K90. Small-angle X-ray scattering (SAXS) was used to show that the 30 wt% F127 gel structure (fcc) was independent of polymer type and concentration, as was the d-spacing and so the micelle hard-sphere radius. The maximum elastic modulus (G0 max) of 30 wt% F127 decreased from its value for water alone as PEG was added, but was little changed by adding PVP. These results are consistent with the packed-micelles in the 30 wt% F127 gel being effectively isolated from the polymer solution on the microscale while, especially for the PEG, being mixed on the macroscale.

Assessment of wheat cultivars for drought tolerance via osmotic stress imposed at early seedling growth stages

A study was conducted in the Department of Plant Breeding and Genetics,Sindh Agriculture University, Tandojam, Pakistan during the year 2009. Sixteen spring wheat cultivars (Triticum aestivum L.) were screened under osmotic stress with three treatments i.e. control-no PEG (polyethylene glycol), 15 percent and 25 percent PEG-6000 solution. The analysis of variance indicated significant differences among treatments for all seedling traits except seed germination percentage. Varieties also differed significantly in germination percentage, coleoptile length, shoot root length, shoot weight, root/shoot ratio and seed vigour index. However, shoot and root weights were non-significant. Significant interactions revealed that cultivars responded variably to osmotic stress treatments; hence provided better opportunity to select drought tolerant cultivars at seedling growth stages. The relative decrease over averages due to osmotic stress was 0.8 percent in seed germination, 53 percent in coleoptile length 62.9 percent in shoot length, 74.4 percent in root length, 50.6 percent in shoot weight, 45.1 percent in root weight, 30.2 percent in root/shoot ratio and 68.5 percent in seed vigour index. However, relative decrease of individual variety for various seedling traits could be more meaningful which indicated that cultivar TD-1 showed no reduction in coleoptile length, while minimum decline was noted in Anmol. For shoot length, cultivar Sarsabz expressed minimum reduction followed by Anmol. However, cultivars Anmol, Moomal, Inqalab-91, and Pavan gave almost equally lower reductions for root length suggesting their higher stress tolerance. In other words, cultivars Anmol, Moomal, Inqalab-91, Sarsabz, TD-1, ZA-77 and Pavan had relatively longer coleoptiles, shoots and roots, and were regarded as drought tolerant. Correlation coefficients among seedlings traits were significant and positive for all traits except germination percentage which had no significant correlation with any of other trait. The results indicated that increase in one trait may cause simultaneous increase in other traits; hence selection for any of these seedling attributes will lead to develop drought tolerant wheat cultivars.

Structural features of condensed tannins affect in vitro ruminal methane production and fermentation characteristics

An in vitro study was conducted to investigate the effects of condensed tannins (CT) structural properties, i.e. average polymer size (or mean degree of polymerization); percentage of cis flavan-3-ols and percentage of prodelphinidins in CT extracts on methane production (CH4) and fermentation characteristics. CT were extracted from eight plants in order to obtain different CT types: black currant leaves, goat willow leaves, goat willow twigs, pine bark, red currant leaves, sainfoin plants, weeping willow catkins and white clover flowers. They were analysed for CT content and CT composition by thiolytic degradation, followed by HPLC analysis. Grass silage was used as a control substrate. Condensed tannins were added to the substrate at a concentration of 40 g/kg, with or without polyethylene glycol (+ or −PEG 6000 treatment) to inactivate tannins, and then incubated for 72 h in mixed buffered rumen fluid from three different lactating dairy cows per run. Total cumulative gas production (GP) was measured by an automated gas production system. During the incubation, 12 gas samples (10 μl) were collected from each bottle headspace at 0, 2, 4, 6, 8, 12, 24, 30, 36, 48, 56 and 72 h of incubation and analyzed for CH4. A modified Michaelis–Menten model was fitted to the CH4 concentration patterns and model estimates were used to calculate total cumulative CH4 production (GPCH4). Total cumulative gas production and GPCH4 curves were fitted using biphasic and monophasic modified Michaelis-Menten models, respectively. Addition of PEG increased GP, GPCH4, and CH4 concentration compared to the −PEG treatment. All CT types reduced GPCH4 and CH4 concentration. All CT increased the half time of GP and GPCH4. Moreover, all CT decreased the maximum rate of fermentation for GPCH4 and rate of substrate degradation. The correlation between CT structure and GPCH4 and fermentation characteristics showed that the proportion of prodelphinidins within CT had the largest effect on fermentation characteristics, followed by average 27 polymer size and percentage of cis-flavan-3-ols.

A high efficiency technique for the generation of transgenic sugar beets from stomatal guard cells

An optimized protocol has been developed for the efficient and rapid genetic modification of sugar beet (Beta vulgaris L.). A polyethylene glycol-mediated DNA transformation technique could be applied to protoplast populations enriched specifically for a single totipotent cell type derived from stomatal guard cells, to achieve high transformation frequencies. Bialaphos resistance, conferred by the pat gene, produced a highly efficient selection system. The majority of plants were obtained within 8 to 9 weeks and were appropriate for plant breeding purposes. All were resistant to glufosinate-ammonium-based herbicides. Detailed genomic characterization has verified transgene integration, and progeny analysis showed Mendelian inheritance.

Effects of volatile fatty acid supply on their absorption and on water kinetics in the rumen of sheep sustained by intragastric infusions

Three sheep fitted with a ruminal cannula and an abomasal catheter were used to study water kinetics and absorption of VFA infused continuously into the rumen. The effects of changing VFA concentrations in the rumen by shifting VFA infusion rates were investigated in an experiment with a 3 x 3 Latin square design. On experimental days, the animals received the basal infusion rate of VFA (271 mmol/h) during the first 2 h. Each animal then received VFA at a different rate (135, 394, or 511 mmol/h) for the next 7.5 h. Using soluble markers (polyethylene glycol and Cr-EDTA), ruminal volume, liquid outflow, apparent water absorption, and VFA absorption rates were estimated. There were no significant effects of VFA infusion rate on ruminal volume and water kinetics. As the VFA infusion rate was increased, VFA concentration and osmolality in the rumen were increased and pH was decreased. There was a biphasic response of liquid outflow to changes in the total VFA concentration in the rumen, as both variables increased together up to a total VFA concentration of 80.1 mM, whereas, beyond that concentration, liquid outflow remained stable at an average rate of 407 mL/h. There were significant linear (P = 0.003) and quadratic (P = 0.001) effects of VFA infusion rate on the VFA absorption rate, confirming that VFA absorption in the rumen is mainly a concentration-dependent process. The proportion of total VFA supplied that was absorbed in the rumen was 0.845 (0.822, 0.877, and 0.910 for acetate, propionate, and butyrate, respectively). The molar proportions of acetate, propionate, and butyrate absorbed were affected by the level of VFA infusion in the rumen, indicating that this level affected to a different extent the absorption of the different acids.

In vitro biological activity of tannins from Acacia and other tree fruits: Correlations with colorimetric and gravimetric phenolic assays

This Study was designed to investigate impact of tannins on in vitro ruminal fermentation parameters as well as relationships between concentration and in vitro biological activity of tannins present in tree fruits. Dry and mature fruits of known phenolic content harvested from Acacia nilotica, A. erubescens, A. erioloba, A. sieberiana, Piliostigima thonningii and Dichrostachys cinerea tree species were fermented with rumen fluid in vitro with or without polyethylene glycol (PEG). Correlation between in vitro biological activity and phenolic concentration was determined. Polyethylene glycol inclusion increased Cumulative gas production from all fruit substrates. The largest Increase (225%) after 48 h incubation was observed in D. cinerea fruits while the least (12.7%) increase was observed in A. erubescens fruits. Organic matter degradability (48 h) was increased by PEG inclusion for all tree species except A. erubescens and P. thonningii. For D. cinerea fruits, colorimetric assays were poorly correlated to Increases In gas production due to PEG treatment. Ytterbium precipitable phenolics (YbPh) were also poorly correlated with response to PEG for A. erioloba and P. thonningii fruits. However, YbPh were strongly and positively correlated to the increase In Cumulative gas production due to PEG for A. erubescens and A. nilotica. Folin-Ciocalteau assayed phenolics (SPh) were not correlated to response to PEG in P. thonningii and A. sieberiana. It was Concluded that the PEG effect oil in vitro fermentation was closely related to some measures of phenolic concentration but the relationships varied with tree species.

The effectiveness of adapted rumen fluid versus PEG to ferment tannin-containing substrates in vitro

This study investigated the potential of the goat's ruminal adaptation to reduce the negative effect of tannins on in vitro fermentation. Rumen fluid was obtained from goats fed a mixture of tannin-containing tree fruits (adapted rumen fluid) or tannin-free commercial protein supplements (unadapted rumen fluid) for 85 days. Dry, mature fruits of Acacia nilotica, Acacia erubescens, Acacia erioloba, Dichrostachys cinerea and Piliostigma thonningii were used as substrates for the in vitro fermentation. The effectiveness of adapted rumen fluid to ferment tannin-containing substrates was compared to the extent of fermentation when tannins were inactivated with polyethylene glycol (PEG), a known tannin-binding agent. Adapted rumen fluid (P < 0.05) increased gas production from all five substrates between 15.8% and 73.7%. In A. nilotica, D. cinerea and P thonningii, this increase was less than that obtained through PEG treatment. When PEG was added to adapted rumen fluid a further improvement in extent of fermentation was observed in four out of the five fruit samples. The largest PEG effect when incubated with adapted rumen fluid was observed in A. nilotica (43.1%) and D. cinerea (42.9%) fruits. It is concluded that some tannin-rich feedstuffs may still benefit from treatment even when these are offered to adapted animals. (c) 2006 Elsevier B.V. All rights reserved.

Tanniniferous Dichrostachys cinerea fruits do not require detoxification for goat nutrition: in sacco and in vivo evaluations

This study investigated the potential of Dichrostachys cinerea fruits as a protein supplement in semi-arid areas of Zimbabwe. The tanniniferous fruits were treated with aqueous solutions of polyethylene glycol (PEG) or sodium hydroxide (NaOH). Both treatments increased the soluble fraction, rate of degradation and effective degradability (ED) of nitrogen (N) in sacco. The PEG effects were higher than the NaOH effects (e.g. a 25% vs. 6% increase in effective N degradabilities, respectively). Five treatments were evaluated in a N-balance trial using Matebele goats: ground, PEG- or NaOH-treated D. cinerea fruits, a commercial protein supplement (CPS) and no supplement. Animals offered ground fruits or CPS retained most N (3.7 or 4.1 g N/day, respectively), while those offered NaOH- or PEG-treated fruits retained significantly less N (2.7 or 1.0 g/day, respectively). Unsupplemented animals were in negative N balance (-2.4 g/day). PEG treatment deactivated the tannins more than the NaOH treatment. PEG treatment resulted in excessive protein degradation in the rumen leading to high urine N loss. It is concluded that the D. cinerea fruits were beneficial for goat N-nutrition and that the tannins did not require inactivation. D. cinerea fruits can, therefore, replace the expensive commercial protein supplement. It is also suggested that the collection and grinding of fruits could be used as a management tool to control bush encroachment. (C) 2004 Elsevier B.V. All rights reserved.

Rates of production of acetate, propionate, and butyrate in the rumen of lactating dairy cows given normal and low-roughage diets

Five lactating dairy cows with a permanent cannula in the rumen were given ( kg DM/d) a normal diet (7.8 concentrates, 5.1 hay) or a low-roughage (LR) diet (11.5 concentrates, 1.2 hay) in two meals daily in a two-period crossover design. Milk fat (g/kg) was severely reduced on diet LR. To measure rates of production of individual volatile fatty acids (VFA) in the rumen, 0.5 mCi 1-C-14-acetic acid, 2-C-14-propionic acid, or 1-C-14-n-butyric acid were infused into the rumen for 22 h at intervals of 2 to 6 d; rumen samples were taken over the last 12 h. To measure rumen volume, we infused Cr-EDTA into the rumen continuously, and polyethylene glycol was injected 2 h before the morning feed. Results were very variable, so volumes measured by rumen emptying were used instead. Net production of propionic acid more than doubled on LR, but acetate and butyrate production was only numerically lower. Net production rates pooled across both diets were significantly related to concentrations for each VFA. Molar proportions of net production were only slightly higher than molar proportions of concentrations for acetate and propionate but were lower for butyrate. The net energy value (MJ/d) of production of the three VFA increased from 89.5 on normal to 109.1 on LR, equivalent to 55 and 64% of digestible energy, respectively. Fully interchanging, three-pool models of VFA C fluxes are presented. It is concluded that net production rates of VFA can be measured in non-steady states without the need to measure rumen volumes.

Do all tannins have similar nutritional effects? A comparison of three Brazilian fodder legumes

Three tropical legumes, namely Leucaena leucocephala, Sesbania sesban and Cajanus cajan, were subjected to chemical analysis plus in vitro, in situ and in vivo evaluations. Three different assays were used to determine total tannins: adsorption to polyvinyl pyrrolidine (PVPP-tannins), radial diffusion (RD-tannins) and protein precipitation capacity (BSA-tannins). Total phenols, total tannins and condensed tannins were highest for Sesbania. RD-tannins were correlated with total phenols (r(2) = 0.93), PVPP-tannins (r(2) = 0.92) and condensed tannins (r(2) = 0.99). The protein precipitation capacity of Sesbania, Leucaena and Cajanus were 25.9, 6.13 and 4.05 mu g BSA/g DM, respectively. Gas production at 24h was negatively correlated with total phenols (r(2) = 0.99), PVPP-tannins (r(2) = 0.99) and condensed tannins (r(2) = 0.91). The RD-, PVPP-tannins and the response to polyethylene glycol (PEG) in the gas production assay appeared to be useful as a first screen for tannins. In situ degradability did not reflect any adverse effects of tannins. However, in vivo experiments showed that the apparent DM digestibility of Sesbania and Leucaena was lower than the basal diet. The apparent protein digestibility was lower for all legumes compared to the basal diet. Most treatments caused a negative nitrogen balance. The problems associated with browse feeding were not only related to tannin contents, other factors such as inherently poor digestibility and low energy intake may also have lead to the poor animal performance on these diets. We propose, given the limitations of current tannin assays, that it is not possible to predict beneficial or harmful nutritional effects from total tannin concentrations per se. (C) 2004 Elsevier B.V. All rights reserved.

The effect of drying and urea treatment on nutritional and anti-nutritional components of browses collected during wet and dry seasons

Fibre, crude protein and tannin concentrations were measured in browse species from the semi-arid region of Northeast Brazil during the dry and wet seasons. The effects of oven-, sun- and shade-drying and of urea treatment were also determined. Crude protein (CP) content varied from 103 to 161 g/kg dry matter (DM) and the browses had similar CP content in the two seasons (during 2002) (102-161 and 107-153 g/kg DM in the wet and dry seasons, respectively). Total tannin concentrations ranged from 13 to 201 g/kg DM amongst the browses and were higher in the dry season. A 30-d treatment with urea reduced extractable tannins significantly (P < 0.05). The urea treatment was also most effective at reducing the in vitro effects of tannins compared to the other drying treatments. This was demonstrated by measuring the effect of polyethylene glycol (PEG) on gas production. Addition of PEG increased gas production of oven- (81.4%), sun- (78.5%) and shade-dried (76.7%) samples much more compared to urea treated samples (10.9%). (c) 2005 Elsevier B.V. All rights reserved.