Changes in Chemical Composition and Digestion Kinetics of Stockpiled Kura Clover in Late Fall

Stockpiled kura clover samples harvested on three different winter dates were used to determine changes in chemical composition and N digestion kinetics. Kura clover was harvested from four different plots at 14 d intervals and analyzed for neutral detergent fiber (NDF), acid detergent fiber (ADF), crude protein (CP), acid detergent insoluble nitrogen (ADIN), and in vitro digestible dry matter (IVDMD), and in situ digestion kinetics of N. Crude protein concentrations decreased, but ADIN concentrations increased with later date of harvest. Digestible N pool-size and the rate of digestion was the lowest in third-harvest kura clover. Although the proportion of protein that is soluble or nondigestible increased, proportion of protein that is potentially digestible decreased with maturity.

Development of food ingredients for modulation of glycemia

Starches are a source of digestible carbohydrate and are frequently used in formulated food products in the presence of other carbohydrates, proteins and fat. This thesis explored the effect of addition of neutral (Konjac glucomannan) or charged (milk proteins) polymers on the physical characteristics and digestion kinetics of waxy maize starch. The aim was to identify mechanisms to modulate the pasting properties and subsequent susceptibility to amylolytic digestion. Addition of αs- or β-caseinate protein fractions to waxy maize starch restricted granular swelling during gelatinisation, increasing granule integrity. It was shown that, while β-caseinate can adsorb to starch granules during pasting, αscaseinate can be absorbed into maize starch granules. The resultant effect was a reduction in granule size after heating, more intact granules and a subsequent decrease in starch digestion in vitro as determined by analysis of reducing sugars. The ability of αs-caseinate to reduce the level of amylolytic digestion was confirmed through in vivo pig (Teagasc, Moorepark) and human (University of Surrey, UK) trials. The scope of the thesis extended to the development of a new automated cell for attachment to a rheometer to measure digestion kinetics of starch-protein mixtures. In conclusion, the thesis offers new approaches to modulation of the physical characteristics of unmodified starch during gelatinisation and suggests that the type of protein and/or polysaccharide used in starch-based food systems may influence the ability of the food to modulate glycemia. This is an important consideration in the design of foods with positive health benefits.

Evaluation of in vitro gas production of roughages and their mixtures using the curves subtraction method

The in vitro gas production of four single roughages and their paired combinations (1:1 on dry matter basis) were evaluated. Two roughage samples (100 mg) per treatment were fermented with ruminal fluid during a 48 h incubation period. Total 48 h gas volumes of fermentation dry matter (DM), neutral detergent fiber (NDF) and soluble compounds in neutral detergent (NDS) were for sugarcane = 16.8, 11.2, 6.9 mL; sugarcane + corn silage = 20.1, 12.6, 9.1 mL; sugarcane + 60-day elephantgrass = 16.5, 17.6 mL; sugarcane + 180-day elephantgrass = 13.8, 8.2, 5.9 mL; corn silage = 18.8, 16.8, 4.7 mL; corn silage + 60-day elephantgrass = 16.3, 15.4, 2.4 mL; corn silage + 180-day elephantgrass = 16.1, 11.8, 4.2 mL; 60-day elephantgrass = 16.9, 19.0 mL and 180-day elephantgrass = fermented 10.7, 12.2 mL, respectively. The NDS gas production was not possible to estimate for sugarcane + 60-day elephantgrass, 60-day elephantgrass and 180-day elephantgrass. The present data shows that the curves subtraction method can be an option to evaluate the contribution of the soluble fractions in roughages to digestion kinetics. However, this method underestimates the NDS gas contribution when roughages are low in crude protein and soluble carbohydrates. It is advisable to directly apply the two-compartmental mathematical model to the digestion curves for roughage DM, when determining the NDS gas volume and the digestion rate. This method is more straightforward and accurate when compared to the curve subtraction method. Non-structural carbohydrates combined with fiber and protein promoted a positive associative effect in sugarcane + corn silage (50:50) mixture. Therefore, it can be concluded that the soluble fraction of roughages greatly contributes to gas production. (C) 2004 Elsevier B.V. All rights reserved.

Chromatin structure and DNase I hypersensitivity in the transcriptionally active and inactive porcine tumor necrosis factor gene locus

We have analyzed the chromatin structure of the porcine tumor necrosis factor gene locus (TNF-alpha and TNF-beta). Nuclei from porcine peripheral blood mononuclear cells were digested with different nucleases. As assessed with micrococcal nuclease, the two TNF genes displayed slightly faster digestion kinetics than bulk DNA. Studies with DNaseI revealed distinct DNaseI hypersensitive sites (DH-sites) within the porcine TNF locus. Four DH-sites could be observed in the promoter and mRNA leader regions of the TNF-beta gene. Two DH-sites could be observed for the TNF-alpha gene, one located in the promoter region close to the TATA-box and the other site in intron 3. This pattern of DH-sites was present independently of the activation state of the cells. Interestingly in a porcine macrophage-like cell line, we found that the TNF-alpha promoter DH-site disappeared and another DH-site appeared in the region of intron 1. Additionally, the DH-site of intron 3 could be enhanced by PMA-stimulation in these cells. TNF-beta sites were not detected in this cell line. However, DH-sites were totally absent in fibroblasts (freshly isolated from testicles) and in porcine kidney cells (PK15 cell line) both of which do not transcribe the TNF genes. Therefore, the pattern of DH-sites corresponds to the transcriptional activity of analyzed cells.

Monensin supplementation of lactating cows fed tropical grasses and cane molasses or grain

Effects of monensin (Mon) on performance of Holstein-Friesian cows fed tropical grasses and cane molasses (M) or cereal grain were examined in three experiments. In experiment I (incomplete 4 x 4 Latin square), three rumen-fistulated cows [188 I I days in milk (DIM)] were fed mixed diets based on rhodes grass (Chloris gayana cv. Callide) bay where M was substituted for wheat grain (W) at rates of 0 (MO), 125 (M 125) or 250 (M250) g/kg dry matter (DM). A fourth diet contained M250 plus 0.02 g Mon/kg DM (M250 + Mon). Substituting M for W tended (P < 0.10) to decrease the ratio of rumen molar proportions of acetate+butyrate (Bu):propionate (Pr) (4.3 versus 3.8 and 4.0 for M0, M125 and M250, respectively). There were no treatment effects (P> 0.10) on intake, organic matter digestibility, milk production or liveweight (LW) change. In experiment 2, 48 cows (173 &PLUSMN; 28.3 DIM) grazing kikuyu (Pennisetum clandestinum cv. common) pastures and supplemented with maize silage and a grain-based concentrate were offered either M (2.6 kg DM/(cow day)) or barley grain (B) (2.7 kg DM/(cow day)). Within each supplement type, half were fed 0 or 320 mg of Mon/(cow day). There were Mon x supplement interactions (Mon x S; P < 0.05) on the rumen molar proportion of Pr and Bu at 15:00 h, with B + Mon having the highest value for Pr (0.259 mmol/mmol) and lowest value for Bu (0.121 mmol/mmol). A Mon x S effect (P < 0.05) on milk fat content was noted with Mon causing a lower value regardless of energy source (31 and 36 g/l versus 40 and 38 g/l for B + Mon, M + Mon, B - Mon and M - Mon, respectively). As a main effect, M as opposed to B, reduced yields of milk (P < 0.05; 16.21/(cow day) versus 18.01/(cow day)) and protein (P < 0.05; 479 g/(cow day) versus 538 g/(cow day)). Monensin reduced milk fat yield (P < 0.05; 669 g/(cow day) versus 562 g/(cow day)), raised milk protein concentration (P < 0.05; 31 g/l versus 29 g/l) and caused LW gain rather than loss (P < 0.05; +0.06 kg/(cow day) versus -0.30 kg/(cow day)). No treatment effects on pasture intake were noted. In experiment 3, 48 cows (91 &PLUSMN; 16.1 DIM) grazing kikuyu pasture and supplemented with grain-based concentrate, sugar cane silage and 2.7 kg DM(cow day) of M were supplemented with either 0 or 320 mg Mon/(cow day). Monensin reduced (P < 0.05) milk fat content (33 g/l versus 30 g/l) and tended (P < 0.10) to reduce milk protein content (29 g/l versus 28 g/l). No effects of Mon on other milk production parameters, LW change or pasture intake were noted. Feeding monensin to mid-lactation Holstein-Friesian cows offered diets based on tropical grasses, and cane molasses or grain, improves rumen fermentation efficiency, thereby improving energy efficiency resulting in higher LW gain. Monensin had no effect on milk yield, but reduced milk fat concentration.

Magnitude differences in bioactive compounds, chemical functional groups, fatty acid profiles, nutrient degradation and digestion, molecular structure and metabolic characteristics of protein in newly developed yellow-seeded and black-seeded canola lines.

Recently, new lines of yellow-seeded (CS-Y) and black-seeded canola (CS-B) have been developed with chemical and structural alteration through modern breeding technology. However, no systematic study was found on the bioactive compounds, chemical functional groups, fatty acid profiles, inherent structure, nutrient degradation and absorption, or metabolic characteristics between the newly developed yellow- and black-seeded canola lines. This study aimed to systematically characterize chemical, structural, and nutritional features in these canola lines. The parameters accessed include bioactive compounds and antinutrition factors, chemical functional groups, detailed chemical and nutrient profiles, energy value, nutrient fractions, protein structure, degradation kinetics, intestinal digestion, true intestinal protein supply, and feed milk value. The results showed that the CS-Y line was lower (P ≤ 0.05) in neutral detergent fiber (122 vs 154 g/kg DM), acid detergent fiber (61 vs 99 g/kg DM), lignin (58 vs 77 g/kg DM), nonprotein nitrogen (56 vs 68 g/kg DM), and acid detergent insoluble protein (11 vs 35 g/kg DM) than the CS-B line. There was no difference in fatty acid profiles except C20:1 eicosenoic acid content (omega-9) which was in lower in the CS-Y line (P < 0.05) compared to the CS-B line. The glucosinolate compounds differed (P < 0.05) in terms of 4-pentenyl, phenylethyl, 3-CH3-indolyl, and 3-butenyl glucosinolates (2.9 vs 1.0 μmol/g) between the CS-Y and CS-B lines. For bioactive compounds, total polyphenols tended to be different (6.3 vs 7.2 g/kg DM), but there were no differences in erucic acid and condensed tannins with averages of 0.3 and 3.1 g/kg DM, respectively. When protein was portioned into five subfractions, significant differences were found in PA, PB1 (65 vs 79 g/kg CP), PB2, and PC fractions (10 vs 33 g/kg CP), indicating protein degradation and supply to small intestine differed between two new lines. In terms of protein structure spectral profile, there were no significant differences in functional groups of amides I and II, α helix, and β-sheet structure as well as their ratio between the two new lines, indicating no difference in protein structure makeup and conformation between the two lines. In terms of energy values, there were significant differences in total digestible nutrient (TDN; 149 vs 133 g/kg DM), metabolizable energy (ME; 58 vs 52 MJ/kg DM), and net energy for lactation (NEL; 42 vs 37 MJ/kg DM) between CS-Y and CS-B lines. For in situ rumen degradation kinetics, the two lines differed in soluble fraction (S; 284 vs 341 g/kg CP), potential degradation fraction (D; 672 vs 590 g/kg CP), and effective degraded organic matter (EDOM; 710 vs 684 g/kg OM), but no difference in degradation rate. CS-Y had higher digestibility of rumen bypass protein in the intestine than CS-B (566 vs 446 g/kg of RUP, P < 0.05). Modeling nutrient supply results showed that microbial protein synthesis (MCP; 148 vs 171 g/kg DM) and rumen protein degraded balance (DPB; 108 vs 127 g/kg DM) were lower in the CS-Y line, but there were no differences in total truly digested protein in small intestine (DVE) and feed milk value (FMV) between the two lines. In conclusion, the new yellow line had different nutritional, chemical, and structural features compared to the black line. CS-Y provided better nutrient utilization and availability.

Biological phosphorus removal during high-rate, low-temperature, anaerobic digestion of wastewater.

We report, for the first time, extensive biologically-mediated phosphate removal from wastewater during high-rate anaerobic digestion (AD). A hybrid sludge bed/fixed-film (packed pumice stone) reactor was employed for low-temperature (12°C) anaerobic treatment of synthetic sewage wastewater. Successful phosphate removal from the wastewater (up to 78% of influent phosphate) was observed, mediated by biofilms in the reactor. Scanning electron microscopy and energy dispersive X-ray analysis revealed the accumulation of elemental phosphorus (~2%) within the sludge bed and fixed-film biofilms. 4’, 6-diamidino-2-phenylindole (DAPI) staining indicated phosphorus accumulation was biological in nature and mediated through the formation of intracellular inorganic polyphosphate (polyP) granules within these biofilms. DAPI staining further indicated that polyP accumulation was rarely associated with free cells. Efficient and consistent chemical oxygen demand (COD) removal was recorded, throughout the 732-day trial, at applied organic loading rates between 0.4-1.5 kg COD m-3 d-1 and hydraulic retention times of 8-24 hours, while phosphate removal efficiency ranged from 28-78% on average per phase. Analysis of protein hydrolysis kinetics and the methanogenic activity profiles of the biomass revealed the development, at 12˚C, of active hydrolytic and methanogenic populations. Temporal microbial changes were monitored using Illumina Miseq analysis of bacterial and archaeal 16S rRNA gene sequences. The dominant bacterial phyla present in the biomass at the conclusion of the trial were the Proteobacteria and Firmicutes and the dominant archaeal genus was Methanosaeta. Trichococcus and Flavobacterium populations, previously associated with low temperature protein degradation, developed in the reactor biomass. The presence of previously characterised polyphosphate accumulating organisms (PAOs) such as Rhodocyclus, Chromatiales, Actinobacter and Acinetobacter was recorded at low numbers. However, it is unknown as yet if these were responsible for the luxury polyP uptake observed in this system. The possibility of efficient phosphate removal and recovery from wastewater during AD would represent a major advance in the scope for widespread application of anaerobic wastewater treatment technologies.

Animal Guts as Nonideal Chemical Reactors: Partial Mixing and Axial Variation in Absorption Kinetics

Animal guts have been idealized as axially uniform plug-flow reactors (PFRs) without significant axial mixing or as combinations in series of such PFRs with other reactor types. To relax these often unrealistic assumptions and to provide a means for relaxing others, I approximated an animal gut as a series of n continuously stirred tank reactors (CSTRs) and examined its performance as a Function of n. For the digestion problem of hydrolysis and absorption in series, I suggest as a first approximation that a tubular gut of length L and diameter D comprises n=L/D tanks in series. For n greater than or equal to 10, there is little difference between performance of the nCSTR model and an ideal PFR in the coupled tasks of hydrolysis and absorption. Relatively thinner and longer guts, characteristic of animals feeding on poorer forage, prove more efficient in both conversion and absorption by restricting axial mixing, in the same total volume, they also give a higher rate of absorption. I then asked how a fixed number of absorptive sites should be distributed among the n compartments. Absorption rate generally is maximized when absorbers are concentrated in the hindmost few compartments, but high food quality or suboptimal ingestion rates decrease the advantage of highly concentrated absorbers. This modeling approach connects gut function and structure at multiple scales and can be extended to include other nonideal reactor behaviors observed in real animals.

A dynamic mathematical model for sequential leach bed anaerobic digestion of organic fraction of municipal solid waste

A mathematical model that describes the operation of a sequential leach bed process for anaerobic digestion of organic fraction of municipal solid waste (MSW) is developed and validated. This model assumes that ultimate mineralisation of the organic component of the waste occurs in three steps, namely solubilisation of particulate matter, fermentation to volatile organic acids (modelled as acetic acid) along with liberation of carbon dioxide and hydrogen, and methanogenesis from acetate and hydrogen. The model incorporates the ionic equilibrium equations arising due to dissolution of carbon dioxide, generation of alkalinity from breakdown of solids and dissociation of acetic acid. Rather than a charge balance, a mass balance on the hydronium and hydroxide ions is used to calculate pH. The flow of liquid through the bed is modelled as occurring through two zones-a permeable zone with high flushing rates and the other more stagnant. Some of the kinetic parameters for the biological processes were obtained from batch MSW digestion experiments. The parameters for flow model were obtained from residence time distribution studies conducted using tritium as a tracer. The model was validated using data from leach bed digestion experiments in which a leachate volume equal to 10% of the fresh waste bed volume was sequenced. The model was then tested, without altering any kinetic or flow parameters, by varying volume of leachate that is sequenced between the beds. Simulations for sequencing/recirculating 5 and 30% of the bed volume are presented and compared with experimental results. (C) 2002 Elsevier Science B.V. All rights reserved.

Concurrent microscopic observations and activity measurements of cellulose hydrolyzing and methanogenic populations during the batch anaerobic digestion of crystalline cellulose

This study compares process data with microscopic observations from an anaerobic digestion of organic particles. As the first part of the study, this article presents detailed observations of microbial biofilm architecture and structure in a 1.25-L batch digester where all particles are of an equal age. Microcrystalline cellulose was used as the sole carbon and energy source. The digestions were inoculated with either leachate from a 220-Lanaerobic municipal solid waste digester or strained rumen contents from a fistulated cow. The hydrolysis rate, when normalized by the amount of cellulose remaining in the reactor, was found to reach a constant value 1 day after inoculation with rumen fluid, and 3 days after inoculating with digester leachate. A constant value of a mass specific hydrolysis rate is argued to represent full colonization of the cellulose surface and first-order kinetics only apply after this point. Additionally, the first-order hydrolysis rate constant, once surfaces were saturated with biofilm, was found to be two times higher with a rumen inoculum, compared to a digester leachate inoculum. Images generated by fluorescence in situ hybridization (FISH) probing and confocal laser scanning microscopy show that the microbial communities involved in the anaerobic biodegradation process exist entirely within the biofilm. For the reactor conditions used in these experiments, the predominant methanogens exist in ball-shaped colonies within the biofilm. (C) 2005 Wiley Periodicals, Inc.

Structure of a cellulose degrading bacterial community during anaerobic digestion

It is widely accepted that cellulose is the rate-limiting substrate in the anaerobic digestion of organic solid wastes and that cellulose solubilisation is largely mediated by surface attached bacteria. However, little is known about the identity or the ecophysiology of cellulolytic microorganisms from landfills and anaerobic digesters. The aim of this study was to investigate an enriched cellulolytic microbial community from an anaerobic batch reactor. Chemical oxygen demand balancing was used to calculate the cellulose solubilisation rate and the degree of cellulose solubilisation. Fluorescence in situ hybridisation (FISH) was used to assess the relative abundance and physical location of three groups of bacteria belonging to the Clostridium lineage of the Firmicutes that have been implicated as the dominant cellulose degraders in this system. Quantitation of the relative abundance using FISH showed that there were changes in the microbial community structure throughout the digestion. However, comparison of these results to the process data reveals that these changes had no impact on the cellulose solubilisation in the reactor. The rate of cellulose solubilisation was approximately stable for much of the digestion despite changes in the cellulolytic population. The solubilisation rate appears to be most strongly affected by the rate of surface area colonisation and the biofilm architecture with the accepted model of first order kinetics due to surface area limitation applying only when the cellulose particles are fully covered with a thin layer of cells. (c) 2005 Wiley Periodicals, Inc.