Particle size of insoluble fibres and gelation of soluble fibres influence digesta passage rate throughout the gastrointestinal tract of finishing pigs.

Fibres, as abundant in agricultural by-products, exhibit a large range of physicochemical properties that can influence digestive processes such as digesta mean retention time (MRT), thereby affecting nutrient digestion kinetics. In this study, we investigated the effects of particle size of insoluble fibres, and gelation of soluble fibres on MRT of liquids, fine solids, and fibrous particles in the different segments of the gastrointestinal tract (GIT) of pigs. Twenty-four boars (51.6 ± 4.90 kg) were allocated to four diets; two diets contained 15% wheat straw, either coarsely chopped or finely ground (1-mm screen), two diets contained 27% wheat bran without or with the addition of 10% low-methylated pectin. After 14 days of adaptation to the diet, a total collection of faeces was performed to determine the total tract digestibility of nutrients. Thereafter, pigs were fed diets supplemented with tracers for at least 5 days and dissected following a frequent feeding procedure to approach steady-state passage of digesta. The MRT of liquids (Co-EDTA), fine solids (TiO2), and fibrous particles (Chromium-mordanted fibres) in the different segments of the GIT were quantified. In the stomach, particle size reduction of straw decreased the MRT of fine solids by 02:39 h, and fibrous particles by 07:21 h (P < 0.10). Pectin addition to the wheat bran diet reduced the MRT of fine solids by 03:09 h, and fibrous particles by 07:10 h (P < 0.10), but not of liquids, resulting in less separation between digesta phases in the stomach compared with the bran diet (P < 0.05). In the mid-small intestine (SI), pectin addition reduced the MRT of fibrous particles and the separation between fibrous particles and fine solids. No further effects of particle size reduction of straw nor pectin addition on MRT and digestibility of starch, nitrogen, or fat were observed in the SI. In the large intestine (LI), particle size reduction of straw reduced separation between fibrous particles and fine solids (P < 0.10), while pectin addition had no effects. Total tract, non-starch polysaccharide degradation of straw was poor (∼31%), and unaffected by particle size reduction (P > 0.10). The complete fermentation of pectin did not influence the degradation of wheat bran fibres (∼51%). In conclusion, the effects of particle size of insoluble fibres and gelling properties of soluble fibres on the passage of digesta phases were most pronounced in the stomach, but less prominent in distal segments of the GIT.

Dynamics of the Phanerochaete carnosa transcriptome during growth on aspen and spruce.

BACKGROUND: The basidiomycete Phanerochaete carnosa is a white-rot species that has been mainly isolated from coniferous softwood. Given the particular recalcitrance of softwoods to bioconversion, we conducted a comparative transcriptomic analysis of P. carnosa following growth on wood powder from one softwood (spruce; Picea glauca) and one hardwood (aspen; Populus tremuloides). P. carnosa was grown on each substrate for over one month, and mycelia were harvested at five time points for total RNA sequencing. Residual wood powder was also analyzed for total sugar and lignin composition. RESULTS: Following a slightly longer lag phase of growth on spruce, radial expansion of the P. carnosa colony was similar on spruce and aspen. Consistent with this observation, the pattern of gene expression by P. carnosa on each substrate converged following the initial adaptation. On both substrates, highest transcript abundances were attributed to genes predicted to encode manganese peroxidases (MnP), along with auxiliary activities from carbohydrate-active enzyme (CAZy) families AA3 and AA5. In addition, a lytic polysaccharide monooxygenase from family AA9 was steadily expressed throughout growth on both substrates. P450 sequences from clans CPY52 and CYP64 accounted for 50% or more of the most highly expressed P450s, which were also the P450 clans that were expanded in the P. carnosa genome relative to other white-rot fungi. CONCLUSIONS: The inclusion of five growth points and two wood substrates was important to revealing differences in the expression profiles of specific sequences within large glycoside hydrolase families (e.g., GH5 and GH16), and permitted co-expression analyses that identified new targets for study, including non-catalytic proteins and proteins with unknown function.

Characterisation of non-degraded oligosaccharides in enzymatically hydrolysed and fermented, dilute ammonia-pretreated corn stover for ethanol production.

BACKGROUND: Corn stover is lignocellulosic biomass that has potential to be used as raw material for bioethanol production. In the current research, dilute ammonia pretreatment was used to improve the accessibility of corn stover carbohydrates to subsequently added hydrolytic enzymes. Some carbohydrates, however, were still present after enzymatic hydrolysis and fermentation. Hence, this research was aimed to characterise the recalcitrant carbohydrates, especially the oligosaccharides that remained after hydrolysis and fermentation of dilute ammonia-pretreated corn stover (DACS). RESULTS: About 35% (w/w) of DACS carbohydrates remained after enzymatic hydrolysis and fermentation of the released monosaccharides. One-third of these recalcitrant carbohydrates were water soluble and composed of diverse oligosaccharides. By using UHPLC-MS n , more than 50 oligosaccharides were detected. Glucurono-xylooligosaccharides (UAXOS) with a degree of polymerisation (DP) less than 5 were the most abundant oligosaccharides. The (4-O-methyl) glucuronosyl substituent was mostly attached onto the terminal xylosyl residue. It was shown that the glucuronosyl substituent in some UAXOS was modified into a hexenuronosyl, a glucuronamide or a hexenuronamide residue due to the dilute ammonia pretreatment. Another group of abundant oligosaccharides comprised various xyloglucan oligosaccharides (XGOS), with a DP 5 annotated as XXG as the most pronounced. In addition, disaccharides annotated as xylosyl-glucose with different ß linkages as well as larger carbohydrates were present in the fermentation slurry. CONCLUSIONS: Around one-third of the 35% (w/w) recalcitrant DACS carbohydrates remained as water-soluble saccharides. In this study, more than 50 recalcitrant oligosaccharides were detected, which mostly composed of xylosyl and/or glucosyl residues. The most pronounced oligosaccharides were UAXOS and XGOS. Hence, α-glucuronidase and α-xylosidase were suggested to be added to the enzyme mixture to degrade these oligosaccharides further, and hence the fermentation yield is potentially increased.

Different action patterns of glucoamylases on branched gluco-oligosaccharides from amylopectin.

A bottleneck in enzymatic starch hydrolysis, like in biofuel industry, is relatively slow degradation of branched structures compared to linear ones. This research aimed to evaluate glucoamylases for their activity towards branched gluco-oligosaccharides. The activity of seven modified glucoamylases and two homologs was compared to that of a reference glucoamylase obtained from a commercial enzyme cocktail 'Distillase® SSF'. All enzymes were evaluated for their activity towards panose (glc(α1-6)glc(α1-4)glc), pullulan and a purified branched gluco-oligosaccharide with a degree of polymerisation of 5 (bDP5) identified as glc(α1-4)[glc(α1-4)glc(α1-6)]glc(α1-4)glc. The enzymes degraded bDP5 differently, which was mainly due to variation in their capability to cleave α-(1→6)-linked or the α-(1→4)-linked glucosyl residue at the non-reducing end of the branched glucosyl residue. By comparing the enzyme activity towards bDP5 with those towards panose and pullulan, it was suggested that the activity towards bDP5 could be estimated only when the activity towards both commercial substrates was evaluated.

Characterisation of branched gluco-oligosaccharides to study the mode-of-action of a glucoamylase from Hypocrea jecorina.

In the conversion of starch to fermentable glucose for bioethanol production, hydrolysis of amylopectin by α-amylases and glucoamylases is the slowest step. In this process, α-1,6-branched gluco-oligosaccharides accumulate and are slowly degraded. Glucoamylases that are able to degrade such branched oligosaccharides faster are economically beneficial. This research aimed at the isolation and characterisation of branched gluco-oligosaccharides produced from amylopectin digestion by α-amylase, to be used as substrates for comparing their degradation by glucoamylases. Branched gluco-oligosaccharides with a DP between five and twelve were purified using size exclusion chromatography. These structures were characterised after labelling with 2-aminobenzamide using UHPLC-MS(n) analysis. Further, the purified oligosaccharides were used to evaluate the mode-of-action of a glucoamylase from Hypocrea jecorina. The enzyme cleaves the α-1,4-linkage adjacent to the α-1,6-linkage at a lower rate than that of α-1,4-linkages in linear oligosaccharides. Hence, the branched gluco-oligosaccharides are a suitable substrate to evaluate glucoamylase activity on branched structures.

Effects of acid extrusion on the degradability of maize distillers dried grain with solubles in pigs.

Commonly used feed processing technologies are not sufficient to affect recalcitrant nonstarch polysaccharides (NSP) such as arabinoxylans present in maize distillers dried grain with solubles (DDGS). Instead, hydrothermal treatments combined with acid catalysts might be more effective to modify these NSP. The objective of this experiment was to investigate the effects of hydrothermal maleic acid treatment (acid extrusion) on the degradability of maize DDGS in growing pigs. It was hypothesized that acid extrusion modifies DDGS cell wall architecture and thereby increases fermentability of NSP. Two diets, containing either 40% (wt/wt) unprocessed or acid-extruded DDGS, were restrictedly fed to groups of gilts (n=11, with 4 pigs per group; initial mean BW: 20.8±0.2 kg) for 18 d and performance and digestibility were analyzed. Acid extrusion tended to decrease apparent ileal digestibility (AID) of CP (approximately 3 percentage units [% units]); P=0.063) and starch (approximately 1% unit; P=0.096). Apparent digestibility of CP and starch measured at the mid colon (2% units, P=0.030, for CP and 0.3% units, P<0.01, for starch) and apparent total tract digestibility (ATTD; 3% units, P<0.01, for CP and 0.2% units, P=0.024, for starch) were lower for the acid-extruded diet compared with the control diet. Hindgut disappearance was, however, not different between diets, indicating that reduced CP and starch digestibility were mainly due to decreased AID. Acid extrusion tended to increase AID of NSP (6% units; P=0.092) and increased digestibility of NSP measured at the mid colon (6% units; P<0.01), whereas hindgut disappearance and ATTD of NSP did not differ between diets. Greater NSP digestibility was mainly due to greater digestibility of arabinosyl, xylosyl, and glucosyl residues, indicating that both arabinoxylan and cellulose degradability were affected by acid extrusion. In conclusion, these results show that acid extrusion did not improve degradation of DDGS for growing pigs. Although acid extrusion seemed to facilitate more rapid degradation of NSP and shifted fermentation to more proximal gastrointestinal segments, total extent of NSP degradation was not affected. More than 35% of the NSP from DDGS remained undegraded, independent of technological processing. Enzyme technologies that specifically target ester-linked acetyl, feroloyl, or coumaroyl groups were identified to be of interest for future research.

Separation of digesta fractions complicates estimation of ileal digestibility using marker methods with Cr2O3 and cobalt-ethylenediamine tetraacetic acid in broiler chickens.

Marker methodologies to measure ileal and total tract digestibilities of diets varying in content and degradability of dietary fiber in broiler chickens were evaluated. Chromium sesquioxide (Cr2O3) and cobalt-ethylenediamine tetraacetic acid (Co-EDTA) were used as markers of solid and soluble fractions, respectively, and compared with digestibility values obtained with the total collection method. Groups of broilers (n = 17, 11 broilers/group) were assigned to a low-fiber diet or 1 of 2 high-fiber diets, the latter 2 containing 35% rapeseed meal (RSM). Pectolytic enzymes were added to one RSM diet to improve degradability of the fiber fraction. Excreta were quantitatively collected for 96 h, and contents from ileum and ceca were collected at slaughter at 29, 30, or 31 d of age. Chromium recovery in excreta ranged between 86 and 95%, whereas cobalt recovery was considerably lower (66 to 70%). Chromium:cobalt ratio was higher in ileal digesta than in feed. Hardly any chromium was found in the ceca, indicating that separation of the marker and specific digesta fractions occurs. Estimates of apparent total tract digestibility (ATTD) were lower when calculated using the marker method compared with the total collection method, particularly in high-fiber diets. Using Cr2O3 as a marker, differences were relatively small and effects due to enzyme addition were generally similar. Using Co-EDTA as a marker, ATTD of all components were lower compared with values obtained using the collection method (3 to 45 percentage units, P < 0.001), likely related to the low Co recovery. When estimating apparent ileal digestibility (AID), separation of marker and digesta resulted in unrealistically high estimates for the digestibility of nonglucose polysaccharides (54 to 66%), exceeding ATTD values by 16 to 42 percentage units. Moreover, the effect of pectolytic enzyme addition on the AID of nonglucose polysaccharides was in the opposite direction when compared with total collection. The data illustrate that fractionation of digesta, particularly in high-fiber diets, complicates accurate AID measurements in broilers, regardless of the choice of markers used.

Effects of processing technologies and pectolytic enzymes on degradability of nonstarch polysaccharides from rapeseed meal in broilers.

Rapeseed meal (RSM) contains a high level of nonstarch polysaccharides (NSP) that are not well degraded in poultry and interfere with digestion of other nutrients as protein, starch, and fat. By altering physicochemical properties of NSP from RSM, processing and enzyme technologies might improve digestive utilization of RSM, enhancing its potential as a source of nutrients in poultry diets. The effects of wet milling and extrusion in combination with pectolytic enzymes on the degradability of RSM in broilers were investigated in a 3 × 2 factorial arrangement. Wet milling and extrusion did not affect total tract apparent digestibility of DM, CP, crude fat, and nonglucose polysaccharides (NGP). Addition of pectolytic enzymes did not affect total tract apparent digestibility of CP and crude fat, but improved degradability of NGP by 9 to 20% units (P < 0.001), independent of prior technological processing of RSM. This coincided with an increased NGP concentration in the ceca (4 to 7 g/g of cobalt, P < 0.001), indicating that more NGP were solubilized such that they could enter the ceca and become available for fermentation. Particle size reduction facilitated solubilization of polysaccharides from RSM, increasing the concentration of NGP found in the ceca (4 g/g of cobalt, P = 0.008). Without help of additional pectolytic enzymes, those solubilized structures could, however, still not be degraded by the cecal microbiota. Feed intake, BW gain, and feed conversion ratio were not affected. No interaction between processing technologies and enzyme addition was found. Apparently, the processing technologies studied were not facilitating accessibility of NSP to pectolytic enzymes added to the feed in vivo.

Characterization and mode of action of two acetyl xylan esterases from Chrysosporium lucknowense C1 active towards acetylated xylans.

Two novel acetyl xylan esterases, Axe2 and Axe3, from Chrysosporium lucknowense (C1), belonging to the carbohydrate esterase families 5 and 1, respectively, were purified and biochemically characterized. Axe2 and Axe3 are able to hydrolyze acetyl groups both from simple acetylated xylo-oligosaccharides and complex non-soluble acetylglucuronoxylan. Both enzymes performed optimally at pH 7.0 and 40 °C. Axe2 has a clear preference for acetylated xylo-oligosaccharides (AcXOS) with a high degree of substitution and Axe3 does not show such preference. Axe3 has a preference for large AcXOS (DP 9-12) when compared to smaller AcXOS (especially DP 4-7) while for Axe2 the size of the oligomer is irrelevant. Even though there is difference in substrate affinity towards acetylated xylooligosaccharides from Eucalyptus wood, the final hydrolysis products are the same for Axe2 and Axe3: xylo-oligosaccharides containing one acetyl group located at the non-reducing xylose residue remain as examined using MALDI-TOF MS, CE-LIF and the application of an endo-xylanase (GH 10).

Endoglucanase V and a phosphatase from Trichoderma viride are able to act on modified exopolysaccharide from Lactococcus lactis subsp. cremoris B40.

EPS B40 from Lactococcus lactis subsp. cremoris consists of a repeating unit of-->4)-beta-D-Glcp-(1-->4)-[alpha-L-Rhap-(1 -->2)][alpha-D-Galp-1-PO4-3]-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->. A phosphatase from Trichoderma viride was able to release phosphate, but only after removal of rhamnosyl and galactosyl residues by mild CF3CO2H treatment. Purified endoV from T. viride was able to act on the backbone of the polymer, but only if rhamnosyl substituents and phosphate had been removed. After complete removal of phosphate and partial removal of rhamnosyl residues by HF treatment, incubation with endoV resulted in a homologous series of oligomers. Purification of these oligomers and subsequent characterisation by NMR demonstrated that endoV was able to cleave the beta-(1-->4) linkage between two glucopyranosyl residues when the galactopyranosyl residue towards the nonreducing end is unsubstituted. The mode of action of endoV on HF-treated EPS B40 is discussed on the basis of the subsite model described for endoV [J.-P. Vincken, G. Beldman, A.G.J. Voragen, Carbohydr. Res., 298 (1997) 299-310].