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Replacing dairy components from milk replacer (MR) with vegetable products has been previously associated with decreased protein and fat digestibility in milk-fed calves resulting in lower live weight gain. In this experiment, the major carbohydrate source in MR, lactose, was partly replaced with gelatinized corn starch (GCS) to determine the effect on protein and fat digestibility in milk-fed calves. In total, 16 male Holstein-Friesian calves received either MR with lactose as the carbohydrate source (control) or 18% GCS at the expense of lactose. In the adaptation period, calves were exposed to an increasing dose of GCS for 14 weeks. The indigestible marker cobalt ethylenediaminetetraacetic acid was incorporated into the MR for calculating apparent nutrient digestibility, whereas a pulse dose of chromium (Cr) chloride was fed with the last MR meal 4 h before slaughter as an indicator of passage rates. The calves were anesthetized and exsanguinated at 30 weeks of age. The small intestine was divided in three; small intestine 1 and 2 (SI1 and SI2, respectively) and the terminal ileum (last ~100 cm of small intestine) and samples of digesta were collected. Small intestinal digesta was analysed for α-amylase, lipase and trypsin activity. Digestibility of protein was determined for SI1, SI2, ileum and total tract, whereas digestibility of fat was determined for SI1, SI2 and total tract. Apparent protein digestibility in the small intestine did not differ between treatments but was higher in control calves at total tract level. Apparent crude fat digestibility tended to be increased in SI1 and SI2 for GCS calves, but no difference was found at total tract level. Activity of α-amylase in SI2 and lipase in both SI1 and SI2 was higher in GCS calves. Activity of trypsin tended to be higher in control calves and was higher in SI1 compared with SI2. A lower recovery of Cr in SI2 and a higher recovery of Cr in the large intestine suggest an increased rate of passage for GCS calves. Including 18% of GCS in a milk replacer at the expense of lactose increased passage rate and decreased apparent total tract protein digestibility. In the small intestine, protein digestion did not decrease when feeding GCS and fat digestion even tended to increase. Overall, effects on digestion might be levelled when partially replacing lactose with GCS, because starch digestion is lower than that of lactose but fat digestion may be slightly increased when feeding GCS.

Genes encoding pectic enzymes were introduced to wild-type potato Karnico. Cell wall materials were extracted from Karnico and transgenic lines expressing β-galactosidase (β-Gal-14 mutant) or rhamnogalacturonan lyase (RGL-18 mutant). After sequential extraction, β-Gal-14 hot buffer-soluble solids (HBSS) of pectin contained 54% less galactose than Karnico HBSS, representing shorter galactan side chains. The individual pectin populations of β-Gal-14 HBSS showed different modifications extended to the two sub-populations as obtained by ion-exchange chromatography. Compared to wild-type, RGL-18 HBSS contained 27% more galacturonic acid and 55% less Gal on fresh potato weight basis, which was due to the removal of galactan-rich rhamnogalacturonan I (RG-I) segments. All pectin populations of RGL-18 showed consistently low levels of RG-I segments. Transgenic modification showed side effects on the methyl-esterification and acetyl substitution of RGL-18 HBSS (DM = 53, DA = 21), but not of the β-Gal-14 HBSS in comparison to wild-type (DM = 29, DA = 54).

ScopeArabinoxylan is one of the most commonly consumed dietary fiber. Immunomodulation by arabinoxylan is documented but the mechanisms by which these immune-effects are accomplished are unknown.Methods and resultsBy applying reporter cell lines for Toll-like receptors (TLRs) and Dectin-1, we demonstrated that arabinoxylan interacts with Dectin-1 receptors and not with TLRs. Arabinoxylan activates Dectin-1 to a similar magnitude as soluble -glucans. Soluble beta-glucans are known to inhibit the particulate beta-glucan-induced activation of Dectin-1. As arabinoxylan is also soluble, the inhibiting capacity of arabinoxylan on particulate -glucan-activated Dectin-1 cell lines was studied. It was found that this inhibition was similar to that of soluble -glucan and was caused predominantly by inhibition of the Dectin-1A transcript variant. The Dectin-1 inhibitory function of arabinoxylan was further confirmed in human dendritic cells that demonstrated reduced production of IL-10 and TNF-alpha The production of the antifungal cytokines IL-4 and IL-23 were increased in dendritic cells stimulated with arabinoxylan and particulate -glucan. In contrast to soluble -glucan, arabinoxylan did not enhance production of IL-10, TNF-alpha, and IL-23.ConclusionArabinoxylan activates Dectin-1 and supports antifungal immune responses in human dendritic cells. The mode of action of arabinoxylan is similar but not identical to that of soluble beta-glucans.

Background: The proportion of starch disappearing from the small intestinal lumen is generally lower in ruminants than in monogastric animals, and there are indications that the starch digestion capacity in ruminants is limited.Objectives: Milk-fed calves were used to study the rate-limiting enzyme in starch hydrolysis and to quantify starch fermentation in ruminants.Methods: Forty male Holstein-Friesian calves were fed milk replacer containing either lactose (control) or 1 of 4 corn starch products. The following starch products differed in the enzyme ratios required for their complete hydrolysis to glucose: gelatinized starch [alpha-amylase and (iso)maltase], maltodextrin [(iso)maltase and alpha-amylase], maltodextrin with alpha-1,6-branching (isomaltase, maltase, and alpha-amylase), and maltose (maltase). In the adaptation period, calves were stepwise exposed to an increasing dose of the starch product for 14 wk to allow maximal adaptation of all enzyme systems involved. In the experimental period, apparent total tract and ileal starch product disappearance, total tract starch product fermentation, and alpha-amylase, maltase, and isomaltase activities were determined at 18% inclusion of the starch product.Results: Maltase and isomaltase activities in the brush border did not increase for any of the starch product treatments. Luminal alpha-amylase activity was lower in the proximal (3 9 +/- 3.2 and 2.7 +/- 1.7 U/mg Co for control and starch product calves, respectively) but greater in the distal small intestine of starch-fed calves than in control calves (0.0 +/- 0.0 and 6.4 +/- 1.5 U/mg Co for control and starch product calves, respectively; means +/- SEs for control and means +/- pooled SEMs for starch product treatments). Apparent deal (61.6% +/- 6.3%) and total tract (99.1% +/- 0.4%) starch product disappearance did not differ between starch product treatments, suggesting that maltase activity limits starch digestion in ruminants. Total tract starch product fermentation averaged 414 +/- 43 g/d, corresponding to 89% of intake, of which Half was fermented before the terminal ileum, regardless of starch product treatment.Conclusion: Fermentation, rather than enzymatic digestion, is the main reason for small intestinal starch disappearance in milk-fed calves.

Scope: This study characterize intestinal fermentation of isomalto/malto-polysaccharides (IMMPs), by monitoring degradation of IMMPs, production of short chain fatty acids (SCFAs), lactic acid, and succinic acid as well as enzyme activity and microbiota composition. Methods and results: IMMP-94 (94% α-(1→6) glycosidic linkages), IMMP-96, IMMP-27, and IMMP-dig27 (IMMP-27 after removal of digestible starch segments) are fermented batchwise in vitro using human fecal inoculum. Fermentation digesta samples are taken for analysis in time up till 48 h. The fermentation of α-(1→6) glycosidic linkages in IMMP-94, IMMP-96, and IMMP-dig27 starts after 12 h and finishes within 48 h. IMMP-27 fermentation starts directly after inoculation utilizing α-(1→4) linked glucosyl residues; however, the utilization of α-(1→6) linked glucoses is delayed and start only after the depletion of α-(1→4) linked glucose moieties. SCFAs are produced in high amounts with acetic acid and succinic acid being the major products next to propionic acid and butyric acid. The polysaccharide fraction is degraded into isomalto-oligosaccharides (IMOs) mainly by extracellular enzymes. The smaller IMOs are further degraded by cell-associated enzymes. Overall microbial diversity and the relative abundance of Bifidobacterium and Lactobacillus, significantly increase during the fermentation of IMMPs. Conclusion: IMMP containing segments of α-(1→6) linked glucose units are slowly fermentable fibers with prebiotic potential.

Isomalto/malto-polysaccharides (IMMPs) are soluble dietary fibres produced by the enzymatic modification of starch with 4,6-α-glucanotransferase (GTFB). The structure, size, and linkage distribution of these IMMPs has remained largely unknown, since most structural information has been based on indirect measurements such as total α-(1→6) content, iodine staining and GTFB hydrolytic activity. This study provides a deeper understanding of IMMP structure in relation to its respective starch substrate, by combining preparative fractionation with linkage compositition analysis. IMMPs were produced from a variety of amylose-rich and amylose-free starches. The extent of modification was investigated per IMMP molecular weight (Mw)-fraction, distinguishing between linear α-(1→6) linkages introduced by GTFB and starch's native α-(1→4,6) branching points. It emerged that the amount of α-(1→6) linkages was consistently higher in IMMP low Mw-fractions and that GTFB activity was limited by native α-(1→4,6) linkages. The presence of amylose turned out to be a prerequisite for the incorporation of linear α-(1→6) linkages in amylopectin.

To study the variability in human milk oligosaccharide (HMO) composition of Chinese human milk over a 20-wk lactation period, HMO profiles of 30 mothers were analyzed using CE-LIF. This study showed that total HMO concentrations in Chinese human milk decreased significantly over a 20-wk lactation period, independent of the mother's SeLe status, although with individual variations. In addition, total acidic and neutral HMO concentrations in Chinese human milk decreased over lactation, and levels are driven by their mother's SeLe status. Analysis showed that total neutral fucosylated HMO concentrations in Chinese human milk were higher in the two secretor groups as compared to the nonsecretor group. On the basis of the total neutral fucosylated HMO concentrations in Chinese human milk, HMO profiles within the Se+Le+ group can be divided into two subgroups. HMOs that differed in level between Se+Le+ subgroups were 2′FL, DF-L, LNFP I, and F-LNO. HMO profiles in Dutch human milk also showed Se+Le+ subgroup division, with 2′FL, LNT, and F-LNO as the driving force.

Separation and characterization of complex mixtures of pectic oligosaccharides still remains challenging and often requires the use of multiple analytical techniques, especially when isomeric structures are present. In this work, it is demonstrated that the coupling of hydrophilic interaction chromatography (HILIC) to traveling-wave ion mobility mass spectrometry (TWIMMS) enabled the simultaneous separation and characterization of complex mixtures of various isomeric pectic oligosaccharides. Labeling of oligosaccharides with 3-aminoquinoline (3-AQ) improved MS-ionization efficiency of the oligosaccharides and reduced the complexity of the product ion mass spectra, without losing resolution of the HILIC separation. In addition, labeling enabled quantification of oligosaccharides on molar basis using in-line fluorescence detection. Isomeric structures were distinguished using TWIMMS. The 3-AQ-HILIC–TWIMMS method was used to characterize a series of isomeric sugar beet rhamnogalacturonan I derived oligosaccharides carrying a glucuronic acid substituent. Thereby, some novel structural features were identified for the first time: glucuronic acid was attached to O-3 or to O-2 of galacturonic acid residues and a single galacturonic acid residue within an oligomer could contain both an acetyl group and a glucuronic acid substituent.

A commercially available resistant maltodextrin (RMD), which is made out of corn starch by chemically and enzymatically modification, consists of atypical starch linkages and a low average molecular mass of 2 kDa. These characteristics of RMD make it rather complicated to identify with any chromatographic or masspectrometric method at this moment. A batch fermentation with human inocula over 48 h under anaerobic conditions showed that the degradation of the indigestible RMD is slow in comparison to dietary fibres like FOS. The RMD is only fermentable for ca. 60% and no specific oligosaccharide within the RMD population seems to be preferentially utilised by the microbiota present. Short chain fatty acid (SCFA) production, with acetic acid as the main SCFA, started after ca. 5 h of fermentation, while the increase of SCFA at 11 h was concurrent with the major degradation of the oligosaccharides of RMD. The microbiota composition as analysed by HITchip, revealed that RMD slightly stimulated the growth of bifidobacteria in comparison to the blank. The activity of carbohydrate degrading enzymes, produced by the microbiota during the in vitro fermentation, revealed that potential capacity to degrade typical starch linkages was high, but the RMD was only slowly and partly degradable during the incubation of 20 h.

A gene encoding a putative pectinesterase from Bacillus licheniformis DSM13 was cloned and expressed in Escherichia coli. The resulting recombinant enzyme ( Bli PME) was purified and characterized as a pectin methylesterase. The enzyme showed maximum activity at pH 8.0 and 50 °C. Bli PME is able to release up to 100% of the methylesters from lime pectin (DM 34–76 → DM 0) and up to 73% of all methylesters from SBPs (DM 30–73 → DM 14). Bli PME efficiently de-methylesterifies lemon pectins and SBPs in a blockwise manner and is quite tolerant towards the acetyl groups present within the SBPs. Detailed analysis of the Bli PME-modified pectins using HILIC–MSn and the classical calcium reactivity measurement showed that the enzyme generates pectins with low methylesterification (lime and SBP) and high acetyl content (SBP) while creating blocks of nonmethylesterified galacturonic acid residues. The high activity of Bli PME towards highly methylesterified and acetylated pectins makes this novel esterase more efficient in removing methylesters from highly esterified beet pectin compared to other PMEs, e.g. Aspergillus niger PME.

An acid-extracted polysaccharide from alchohol-insoluble solids of leek was obtained. The sugar composition indicated that galactose and galacturonic acid were the major sugars, followed by small amounts of rhamnose and arabinose. The fraction contained a relatively high methyl-esterified homogalacturonan next to rhamnogalacturonan type I decorated with galactose-rich side chains. The fraction consisted of three high Mw populations, covering the range of 10–100 kDa. Enzymatic fingerprinting was performed with HG/RG-I degrading enzymes to elucidate the structure. The oligomers were analysed using LC-HILIC-MS, HPAEC, and MALDI-TOF MS. The data revealed the presence of GalA sequences, having different patterns of methyl-esterification, RG-I composed of unbranched segments and segments heavily substituted with β-(1→4)-linked galactan chains of varying length. The rheological study showed the shear-thinning, weak thixotropic, anti-thixotropic, and non-Newtonian behavior of the polysaccharide. The pectin exhibited higher water holding capacity than oil-holding capacity and the fraction did form stable foams at high concentration.

Research was conducted to understand cell wall polysaccharide (CWPs) degradation in chicory root pulp (CRP) during an in vitro fermentation by human faecal microbiota. Enzyme extracts (EEs) from CRP fermentation liquids (2, 12 and 24 h) were tested for enzyme activities towards p-nitrophenyl-glycosides and polysaccharides. The EEs were also incubated for 24 h on polysaccharides and cell wall residues (CWRs) derived from CRP, to determine the mechanism and efficiency of enzyme action. The study showed that the presence of arabinofuranosidase, β-galactosidase, endo-arabinanase, endo-galactanase and exo-polygalacturonase increased for the 24 h EE, whereas the activity of enzymes degrading (hemi)cellulose was highest in the 12 h EE. It was hypothesised that increased levels of arabinofuranosidase, β-galactosidase, endo-arabinanase, endo-galactanase, exo-polygalacturonase, pectin de-esterifying enzymes and endo-polygalacturonase contributed to a synergy in degrading pectin in CRP from 12 to 24 h of fermentation. The higher degradability of arabinan compared to galactan in the network is due to the architecture in CRP involving more accessible arabinan than galactan.

Screening of eight carbohydrate acetyl esterases for their activity towards xanthan resulted in the recognition of one active esterase. AXE3, a CAZy family CE1 acetyl xylan esterase originating from Myceliophthora thermophila C1, removed 31% of all acetyl groups present in xanthan after a 48 h incubation. AXE3 activity towards xanthan was only observed when xanthan molecules were in the disordered conformation. Optimal performance towards xanthan was observed at 53 °C in the complete absence of salt, a condition favouring the disordered conformation. AXE3-deacetylated xanthan was hydrolyzed using cellulases and analyzed for its repeating units using UPLC–HILIC–ELSD/ESI–MS. This showed that AXE3 specifically removes the acetyl groups positioned on the inner mannose and that acetyl groups positioned on the outer mannose are not removed at all. After a prolonged incubation at optimal conditions, 57% of all acetyl groups, representing 70% of all acetyl groups on the inner mannose units, were hydrolyzed.

The aim of this research was to study the in vitro fermentation of chicory root pulp (CRP) and ensiled CRP (ECRP) using human fecal inoculum. Analysis of carbohydrate levels in fermentation digests showed that 51% of all CRP carbohydrates were utilized after 24 h of fermentation. For ECRP, having the same cell wall polysaccharide composition as CRP, but with solubilization of 4 times more of CRP pectin due to ensiling, the fermentation was quicker than with CRP as 11% more carbohydrates were utilized within the first 12 h. The level of fiber utilization for ECRP after 24 h was increased by 8% compared to CRP. This effect on fiber utilization from ECRP seemed to arise from (i) increased levels of soluble pectin fibers (arabinan, homogalacturonan, and galactan) and (ii) ahypothesized more open structure of the remaining cell walls in ECRP, which was more accessible to degradation than the CRP cell wall network. KEYWORDS: chicory root pulp, ensiled chicory root pulp, insoluble

Unprocessed and acid-extruded rapeseed meal (RSM) was fed to broiler chickens, with and without addition of commercial pectolytic enzymes. Nonstarch polysaccharide (NSP) fermentability and unfermented NSP structures from RSM were studied in the excreta in detail. From unprocessed RSM, 24% of the nonglucose polysaccharides could be fermented. Acid treatment did not have a significant effect, but enzyme addition did improve fermentability to 38%. Most likely, the significant increase in NSP fermentability can be ascribed to the addition of pectolytic enzymes, which decreased branchiness of the water-soluble arabinan. Mainly xyloglucan, (glucurono-)xylan, (branched) arabinan, and cellulose remained in the excreta. The proportion of unextractable carbohydrates increased in excreta from broilers fed acid-extruded RSM. Probably, acid extrusion resulted in a less accessible NSP matrix, also decreasing the accessibility for pectolytic enzymes added in the diet. During alkaline extraction of the excreta, 39 to 52% (wt/wt) of the insoluble carbohydrates was released as glucosyl- and uronyl-rich carbohydrates, probably originally present via ester linkages or hydrogen bonding within the cellulose-lignin network. These linkages are expected to hinder complete NSP fermentation and indicate that digestibility of RSM may benefit substantially from an alkaline treatment or addition of esterases.

Replacing part of glucose with galactose in the post-weaning diet beneficially affects later life metabolic health in female mice. The liver is the main site of galactose metabolism, but the direct effects of this dietary intervention on the liver in the post-weaning period are not known. The aim of this study was to elucidate this. Weanling female mice (C57BL/6JRccHsd) were fed a starch containing diet with glucose (32 en%) monosaccharide (GLU), or a diet with glucose and galactose (1:1 both 16 en%) (GLU+GAL). Body weight, body composition, and food intake were determined weekly. After 3 weeks, mice were sacrificed, and serum and liver tissues were collected. Global hepatic mRNA expression was analyzed and hepatic triglyceride (TG) and glycogen contents were determined by enzymatic assays. Body weight and body composition were similar in both groups, despite higher food intake in mice on GLU+GAL diet. Hepatic TG content was lower in GLU+GAL-fed than GLU-fed females, while glycogen levels were unaffected. Analysis of global expression patterns of hepatic mRNA showed that mainly inflammation-related pathways were affected by the diet, which were predominantly downregulated in GLU+GAL-fed females compared to GLU-fed females. This reduction in inflammation in GLU+GAL-fed females was also reflected by decreased serum concentrations of acute phase protein Serum amyloid A 3. In conclusion, replacing part of glucose with galactose in the post-weaning diet reduces hepatic TG content and hepatic inflammation.

Enzymatic fingerprinting was applied to sugar beet pectins (SBPs) modified by either plant or fungal pectin methyl esterases and alkali catalyzed de-esterification to reveal the ester distributions over the pectin backbone. A simultaneous pectin lyase (PL) treatment to the commonly used endo-polygalacturonase (endo-PG) degradation showed to be effective in degrading both high and low methylesterified and/or acetylated homogalaturonan regions of SBP simultaneously. Using LC-HILIC–MS/ELSD, we studied in detail all the diagnostic oligomers present, enabling us to discriminate between differently prepared sugar beet pectins having various levels of methylesterification and acetylation. Furthermore, distinction between commercially extracted and de-esterified sugar beet pectin having different patterns of substitution was achieved by using novel descriptive pectin parameters. In addition to DBabs approach for nonmethylesterified sequences degradable by endo-PG, the “degree of hydrolysis” (DHPG) representing all partially saturated methylesterified and/or acetylated galacturonic acid (GalA) moieties was introduced as a new parameter. Consequently, the description DHPL has been introduced to quantify all esterified unsaturated GalA oligomers.

Supplementation of a Trichoderma longibrachiatum preparation to an industrial Aspergillus niger/Talaromyces emersonii enzyme mixture demonstrated synergy for the saccharification of corn silage water-unextractable solids (WUS). Sub-fractions of the crude T. longibrachiatum preparation obtained after chromatography were analyzed regarding their hydrolytic activity. An acetyl xylan esterase 1 [Axe1, carbohydrate esterase (CE) family 5]-enriched sub-fraction closely mimicked the hydrolytic gain as obtained by supplementation of the complete, crude enzyme mixture (increase of 50%, 62% and 29% for Xyl, Ara and Glc, respectively). The acetic acid released from model polysaccharides (WUS) and oligosaccharides [neutral (AcXOS) and acidic (AcUXOS) xylo-oligosaccharides] by Axe1 was two and up to six times higher compared to the acetic acid released by acetyl xylan esterase A (AxeA, CE 1). Characterization of Axe1 treated AcXOS and AcUXOS revealed deacetylation of oligosaccharides that were not deacetylated by AxeA or the A. niger/T. emersonii preparation.

Dietary fiber intake is associated with lower incidence and mortality from disease, but the underlying mechanisms of these protective effects are unclear.We hypothesized that β2→1-fructan dietary fibers confer protection on intestinal epithelial cell barrier function via Toll-like receptor 2 (TLR2), and we studied whether β2→1-fructan chain-length differences affect this process. T84 human intestinal epithelial cell monolayers were incubated with 4 β2→1-fructan formulations of different chain-length compositions and were stimulated with the proinflammatory phorbol 12-myristate 13-acetate (PMA). Transepithelial electrical resistance (TEER) was analyzed by electric cell substrate impedance sensing (ECIS) as a measure for tight junction-mediated barrier function. To confirm TLR2 involvement in barrier modulation by β2→1-fructans, ECIS experiments were repeated using TLR2 blocking antibody. After preincubation of T84 cells with short-chain β2→1-fructans, the decrease in TEER as induced by PMA (62.3 ± 5.2%, P < 0.001) was strongly attenuated (15.2 ± 8.8%, P < 0.01). However, when PMA was applied first, no effect on recovery was observed during addition of the fructans. By blocking TLR2 on the T84 cells, the protective effect of short-chain β2→1-fructans was substantially inhibited. Stimulation of human embryonic kidney human TLR2 reporter cells with β2→1-fructans induced activation of nuclear factor kappa-light-chain-enhancer of activated B cells, confirming that β2→1-fructans are specific ligands for TLR2. To conclude, β2→1-fructans exert time-dependent and chain length-dependent protective effects on the T84 intestinal epithelial cell barrier mediated via TLR2. These results suggest that TLR2 located on intestinal epithelial cells could be a target of β2→1-fructan-mediated health effects. © 2014 American Society for Nutrition.Chemicals/CAS: inulinase, 9025-67-6; phorbol 13 acetate 12 myristate, 16561-29-8; toll like receptor 2, 203811-81-8

Prebiotics are considered to have potential to reduce disturbances in the gut microbiota induced by antibiotics. Results in literature are, however, not consistent. The current in vitro study conducted in a fermentation screening platform allowed to unambiguously compare the impact of galacto-oligosaccharides (GOS) on adult gut microbiota composition and activity upon treatment with four antibiotics at two doses. The changes in relative abundance of bacteria upon antibiotic treatment and the growth of Bifidobacterium and Lactobacillus upon GOS addition were antibiotic and dose dependant. This conclusion explains discrepancies in literature and indicates that particular combinations of GOS antibiotic should be studied. The combination GOS–Amoxicillin was especially of interest as, after decrease in Bifidobacterium levels, a recovery of mainly Bifidobacterium longum was observed and could be correlated with specific degradation patterns of GOS. Next to different degradation profiles of individual GOS, an accumulation of monosaccharides and intermediate organic acids was observed in antibiotic-treated microbiota as compared to nontreated microbiota. This showed that although GOS were utilized and beneficial bacteria could grow in 3 of 4 antibiotics tested, the metabolic activity of an antibiotic-treated microbiota was still disturbed as compared to the nontreated microbiota.

Two major types of food-grade surfactants used to stabilize foams are proteins and low molecular weight (LMW) surfactants. Proteins lower the surface tension of interfaces and tend to unfold and stabilize the interface by the formation of a visco-elastic network, which leads to high surface moduli. In contrast, LMW surfactants lower the surface tension more than proteins, but do not form interfaces with a high modulus. Instead, they stabilize the interface through the Gibbs-Marangoni mechanism that relies on rapid diffusion of surfactants, when surface tension gradients develop as a result of deformations of the interface. A molecule than can lower the surface tension considerably, like a LMW surfactant, but also provide the interface with a high modulus, like a protein, would be an excellent foam stabilizer. In this article we will discuss molecules with those properties: oligofructose fatty acid esters, both in pure and mixed systems. First, we will address the synthesis and structural characterization of the esters. Next, we will address self-assembly and rheological properties of air/water interfaces stabilized by the esters. Subsequently, this paper will deal with mixed systems of mono-esters with either di-esters and lauric acid, or proteins. Then, the foaming functionality of the esters is discussed.

Endo-xylogalacturonan hydrolase is a member of glycoside hydrolase family28 (GH28) that hydrolyzes the glycosidic bond between two -xylose-substituted galacturonic acid residues in pectin. Presented here is the X-ray crystal structure of the endo-xylogalacturonan hydrolase from Aspergillustubingensis (XghA) at 1.75 angstrom resolution. The high degree of structural conservation in the active site and catalytic apparatus compared with polygalacturonases indicates that cleavage of the substrate proceeds in essentially the same way as found for the other GH28 enzymes. Molecular modeling of a xylosylated tri-galacturonate in the active site identified the amino acid residues involved in substrate binding. They border a substrate-binding cleft that is much wider than in other polygalacturonases, and can accommodate xylosylated substrates. The most extensive interactions appear to occur at subsite +2, in agreement with the enzyme kinetics results, which showed enhanced activity on substrates with a xylose attached to the galacturonic acid bound at subsite +2.DatabaseStructural data are available in the Protein Data Bank database under accession number 4C2L.

Five xanthans were hydrolyzed to their repeating units using cellulases. Hydrophilic interaction chromatography with online electrospray ionization ion trap mass spectrometry and evaporative light scattering detection was used to analyze the oligomers released. It was concluded that six different pentamer repeating units (RUs) exists within a xanthan sample. The most abundant RU shows acetylation on the inner mannose and pyruvylation on the outer mannose. The second most abundant RU shows acetylation on both the inner and the outer mannose. It becomes clear that more variations in the xanthan structure exist than generally recognized. Comparison of five different xanthan samples revealed that, although the molecular composition of xanthan samples can be exactly the same, the ratio in which the RUs occur can differ significantly. It is, therefore, concluded that xanthan samples should be characterized for both, their molecular composition and the relative abundance of the RUs present.

RATIONALE Mass spectrometry has become a useful technique for elucidating the chemical structures of oligosaccharides. The combined use of chromatography and mass spectrometry for the separation and identification of oligosaccharides has shown much progress in recent years. However, no powerful method has yet been developed to quickly identify isomeric oligosaccharides in complex mixtures. METHODS A rapid travelling-wave ion mobility mass spectrometry (TWIMS-MS) method was developed for the identification of various isomeric oligogalacturonic acids in mixtures and determined their structures, using 3-aminoquinoline (3-AQ) as a labelling agent. RESULTS TWIMS successfully distinguished isomeric oligogalacturonic acids of various degrees of polymerisation (DPs) and levels of methyl-esterification. After derivatisation by 3-AQ, isomeric oligosaccharides of galacturonic acid, with the DP ranging from 2 to 9 and the number of methyl esters ranging from 1 to 5, were identified by 3-AQ-TWIMS-MS. The isomeric oligosaccharides with varying sites of methyl ester substitution were identified by the post-fragmentation mode of TWIMS using 3-AQ labelling to obtain simplified mass spectra. CONCLUSIONS Using the 3-AQ-TWIMS-MS method, the precise distribution of methyl esters within the pectin molecule and isomeric oligogalacturonic acids after enzyme degradation was determined. Simplified product ion mass spectra and precise analysis of the isomers were achieved by labelling 3-AQ at the reducing end of the oligosaccharides. Series of methyl-esterified galacturonic acid oligomers have predictable drift times, depending on the precise position of the methyl ester.

This invention relates generally to compositions and methods for combating inflammatory disease and particularly to the use of food and/or feed compositions for preventing, reducing and/or treating inflammatory disorders, diseases, or discomforts, wherein the compositions comprising at least one specific pectin.

Scope: We aimed to investigate the effects of three different soluble pectins on the digestion of other consumed carbohydrates, and the consequent alterations of microbiota composition and SCFA levels in the intestine of pigs.Methods and results: Piglets were fed a low-methyl esterified pectin enriched diet (LMP), a high-methyl esterified pectin enriched diet (HMP), a hydrothermal treated soybean meal enriched diet (aSBM) or a control diet (CONT). LMP significantly decreased the ileal digestibility of starch resulting in more starch fermentation in the proximal colon. In the ileum, low-methyl esterified pectin present was more efficiently fermented by the microbiota than high-methyl esterified pectin present which was mainly fermented by the microbiota in the proximal colon. Treated soybean meal was mainly fermented in the proximal colon and shifted the fermentation of cereal dietary fiber to more distal parts, resulting in high SCFA levels in the mid colon. LMP, HMP, and aSBM decreased the relative abundance of the genus Lactobacillus and increased that of Prevotella in the colon.Conclusion: The LMP, HMP, and aSBM, differently affected the digestion processes compared to the control diet and shaped the colonic microbiota from a Lactobacillus-dominating flora to a Prevotella-dominating community, with potential health-promoting effects.

Contains fulltext : 170614.pdf (Publisher’s version ) (Open Access) It has been shown in vitro that only specific dietary fibers contribute to immunity, but studies in vivo are not conclusive. Here, we investigated degree of polymerization (DP) dependent effects of beta2-->1-fructans on immunity via microbiota-dependent and -independent effects. To this end, conventional or germ-free mice received short- or long-chain beta2-->1-fructan for 5 days. Immune cell populations in the spleen, mesenteric lymph nodes (MLNs), and Peyer's patches (PPs) were analyzed with flow cytometry, genome-wide gene expression in the ileum was measured with microarray, and gut microbiota composition was analyzed with 16S rRNA sequencing of fecal samples. We found that beta2-->1-fructans modulated immunity by both microbiota and microbiota-independent effects. Moreover, effects were dependent on the chain-length of the beta2-->1-fructans type polymer. Both short- and long-chain beta2-->1-fructans enhanced T-helper 1 cells in PPs, whereas only short-chain beta2-->1-fructans increased regulatory T cells and CD11b-CD103- dendritic cells (DCs) in the MLN. A common feature after short- and long-chain beta2-->1-fructan treatment was enhanced 2-alpha-l-fucosyltransferase 2 expression and other IL-22-dependent genes in the ileum of conventional mice. These effects were not associated with shifts in gut microbiota composition, or altered production of short-chain fatty acids. Both short- and long-chain beta2-->1-fructans also induced immune effects in germ-free animals, demonstrating direct effect independent from the gut microbiota. Also, these effects were dependent on the chain-length of the beta2-->1-fructans. Short-chain beta2-->1-fructan induced lower CD80 expression by CD11b-CD103- DCs in PPs, whereas long-chain beta2-->1-fructan specifically modulated B cell responses in germ-free mice. In conclusion, support of immunity is determined by the chemical structure of beta2-->1-fructans and is partially microbiota independent.

This study simulates the fermentation process of barley ?-glucan and sugar beet pectin in the human colon and monitors the degradation products formed. Additionally, immune effects of the degradation products were investigated.Immunostimulatory activity of fermentation digesta was investigated using bone marrow derived dendritic cells (BMDCs) from toll-like receptor 2/4 (TLR2/4) knockout mice, which were unresponsive to microbe-associated molecular patterns. Cytokine responses were elicited to dietary fibers and not to the SCFA and microbiota. The fermentation digesta were analyzed for their SCFA profiles and glycan metabolites over time. During fermentation the amount of insoluble precipitating fibers increased and induced as well as soluble molecules of lower molecular mass greater amounts of cytokines in BMDCs than the parental fiber. Additionally, high amounts of cytokines can be attributed to soluble galactose-rich beet pectin molecules.The fermentation of the two fibers led to fiber-specific amounts of SCFA, glycosidic metabolites, and different immunomodulatory properties. BMDC from TLR2/4 knockout mice did not respond to the digest microbiota and SCFA, making it a useful approach to study temporal effects of fermentation on the immunomodulatory effects of fibers.? 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Antibiotic treatments can lead to a disruption of the human microbiota. In this in-vitro study, the impact of antibiotics on adult intestinal microbiota was monitored in a new high-throughput approach: a fermentation screening-platform was coupled with a phylogenetic microarray analysis (Intestinal-chip). Fecal inoculum from healthy adults was exposed in a fermentation screening-platform to seven widely-used antibiotics during 24. h in-vitro fermentation and the microbiota composition was subsequently determined with the Intestinal-chip.Phylogenetic microarray analysis was first verified to be reliable with respect to variations in the total number of bacteria and presence of dead (or inactive) cells. Intestinal-chip analysis was then used to identify and compare shifts in the intestinal microbial composition after exposure to low and high dose (1μgml-1 and 10μgml-1) antibiotics. Observed shifts on family, genus and species level were both antibiotic and dose dependent. Stronger changes in microbiota composition were observed with higher doses. Shifts mainly concerned the bacterial groups Bacteroides, Bifidobacterium, Clostridium, Enterobacteriaceae, and Lactobacillus. Within bacterial groups, specific antibiotics were shown to differentially impact related species.The combination of the in-vitro fermentation screening platform with the phylogenetic microarray read-outs has shown to be reliable to simultaneously analyze the effects of several antibiotics on intestinal microbiota. © 2012 Elsevier B.V.

This research aimed to develop a method for analyzing specific alginate oligosaccharides (AOS) in a complex matrix such as pig feces. The data obtained were used to study alginate degradation by the microbiota in the large intestine during adaptation, including the individual variation between pigs. A method using an UHPLC system with an ethylene bridged hybrid (BEH) amide column coupled with MS(n) detection was able to distinguish saturated and unsaturated AOS with DP 2-10. Isomers of unsaturated trimer and tetramer could be separated and annotated. In the feces, saturated and unsaturated AOS were present. The presence of unsaturated AOS indicates that the microbiota produced alginate lyase. The microbiota utilized unsaturated AOS more than saturated AOS. The results also suggested that guluronic acid at the reducing end of AOS inhibits the utilization by microbiota during the first weeks of adaptation. After adaptation, the microbiota was able to utilize a broader range of AOS.

In order to use corn fiber as a source for bioethanol production the enzymatic hydrolysis of the complex glucuronoarabinoxylans present has to be improved. Several oligosaccharides present in the supernatant of mild acid pretreated and enzymatically saccharified corn fiber that resist the current available enzymes were (semi)purified for structural analysis by NMR or ESI-MSn. The structural features of 21 recalcitrant oligosaccharides are presented. A common feature of almost all these oligosaccharides is that they contain (part of) an α- l -galactopyranosyl-(1→2)-β- d -xylopyranosyl-(1→2)-5-O-trans-feruloyl- l -arabinofuranose side chain attached to the O-3 position of the β-1–4 linked xylose backbone. Several of the identified oligosaccharides contained an ethyl group at the reducing end hypothesized to be formed during SSF. The ethyl glycosides found are far more complex than previously described structures. A new feature present in more than half of the oligosaccharides is an acetyl group attached to the O-2 position of the same xylose to which the oligomeric side chain was attached to the O-3 position. Finding enzymes attacking these large side chains and the dense substituted xylan backbone will boost the hydrolysis of corn fiber glucuronoxylan.

To study the applicability of oligofructose palmitic acid esters (OF-C16) as novel surfactants in food systems, the functional properties of OF-C16 were studied in the presence of whey protein isolate (WPI). Surface tension measurements, surface dilatational rheology, foam stability tests and Brewster angle microscopy were used to study the competitive adsorption of WPI and OF-C16 and the displacement of WPI by OF-C16. Pure WPI stabilized interfaces had a moderate surface tension (48 mN/m) and a dilatational modulus of 90 mN/m, while pure OF-C16 stabilized interfaces had a low surface tension (30 mN/m) and a dilatational modulus of 50 mN/m. The stabilization mechanisms of WPI (elastic network formation) and of OF-C16 (surface solidification) are very different, and the combined adsorption of these two components led to a structure with a much lower dilatational modulus. At the lowest WPI concentrations (0.5% and 1%), the equilibrium surface tension was similar to a pure OF-C16 stabilized interface, pointing to a low WPI surface concentration. However, apparently still sufficient WPI had adsorbed either at or just below the interface, to prevent the OF-C16 from solidifying. Despite the low moduli, the foam stability for the mixed systems was high. The interfaces were probably stabilized by the Gibbs–Marangoni mechanism. In contrast, at the highest WPI concentration (2%), the equilibrium surface concentration of WPI was sufficiently high to decrease the interfacial mobility of OF-C16, which decreased the Gibbs–Marangoni effect and resulted in decreased foam stability. Finally, OF-C16 could also displace a fully developed WPI network from the interface.

Mass spectrometric analysis was used to compare the roles of two acetyl esterases (AE, carbohydrate esterase family CE16) and three acetyl xylan esterases (AXE, families CE1 and CE5) in deacetylation of natural substrates, neutral (linear) and 4-O-methyl glucuronic acid (MeGlcA) substituted xylooligosaccharides (XOS). AEs were similarly restricted in their action and apparently removed in most cases only one acetyl group from the non-reducing end of XOS, acting as exo-deacetylases. In contrast, AXEs completely deacetylated longer neutral XOS but had difficulties with the shorter ones. Complete deacetylation of neutral XOS was obtained after the combined action of AEs and AXEs. MeGlcA substituents partially restricted the action of both types of esterases and the remaining acidic XOS were mainly substituted with one MeGlcA and one acetyl group, supposedly on the same xylopyranosyl residue. These resisting structures were degraded to great extent only after inclusion of α-glucuronidase, which acted with the esterases in a synergistic manner. When used together with xylan backbone degrading endoxylanase and β-xylosidase, both AE and AXE enhanced the hydrolysis of complex XOS equally.

Xylan is an important part of plant biomass and represents a renewable raw material for biorefineries. Contrary to cellulose, the structure of hemicellulose is quite complex. Therefore, the biodegradation of xylan needs the cooperation of many enzymes. For industrial production of xylanase multienzyme complexes (cocktails) and selected monocomponent xylanases, different Trichoderma reesei mutants and recombinants are used. T. reesei QM 6a (wild-type parent of best existing mutants) was selected as a starting material in the 1960s when the modern in-depth analytical methods were not yet in use. Therefore, screening of fungi genetically close to T. reesei in biodegradation of xylan may have a scientific value. Fifteen different strains from Trichoderma section Longibrachiatum have been tested for extracellular xylan-degrading enzyme production on three carbon sources (wheat straw, corn fiber, and eucalyptus wood) in shake flask cultivation. The enzyme activities were evaluated by traditional colorimetric enzyme assays and by HPLC and matrix assisted laser desorption/ionization time-of-flight mass spectrometry. Degradation of xylan was studied on four different xylan-rich model substrates. T. reesei CPK 155, Trichoderma parareesei TUB F-2535, and Trichoderma gracile TUB F-2543 isolates were equally good or better in degradation of the wheat arabinoxylan (WAX) and corn fiber alcohol insoluble solids as hydolysis substrates than the well-known T. reesei QM 6a and RUT C30 strains. Though Trichoderma saturnisporum ATCC 18903 gave relatively low volumetric enzyme activities by traditional colorimetric assays, it could release quite large amount of hydrolysis products (mono- and oligosaccharides) from WAX. Therefore, these fungi may be potential candidates for further experiments. Enzyme production on wheat straw and corn fiber carbon sources was more effective than on eucalyptus wood.

The acetyl (AcE), feruloyl (FE), and p-coumaroyl (pCE) ester contents of different cereal and grass polysaccharides were determined by a quantitative 1H NMR-based method. The repeatability and the robustness of the method were demonstrated by analyzing different plant polysaccharide preparations. Good sensitivity and selectivity for AcE, FE, and pCE were observed. Moreover, an optimized and easy sample preparation allowed for simultaneous quantification of AcE, FE, and pCE. The method is suitable for high-throughput analysis, and it is a good alternative for currently used analytical procedures. A comparison of the method presented to a conventional HPLC-based method showed that the results obtained are in good agreement, whereas the combination of the optimized sample preparation and analysis by the 1H NMR-based methodology results in significantly reduced analysis time.

Pressed potato fibre (PPF) has a high water holding capacity (WHC) affecting its processing as an animal feed. The aim of this study was to characterize cell wall polysaccharides (CWPs) in PPF and investigate their WHC. This was done via sequential extractions. Half of all CWPs were recovered in the hot buffer soluble solids extract as pectins (uronic acid and rhamnose) and galactans wherein most pectins (76%) from PPF were water soluble. Most likely, the network of CWPs is loosened during processing of potatoes. PPF showed a WHC of 7.4 expressed as the amount of water held per g of dry matter (mL/g). Reconstituting hot buffer soluble solids with buffer insoluble solids in water gave a WHC comparable to that of PPF. Removal of alkali soluble solids, which mainly comprised xyloglucans, lowered the WHC of the final residue. The results indicated that interactions between CWPs could affect the WHC of PPF.

In this study, we analyze 31 transgenic lines and their respective untransformed background lines to determine the transgene effects on targeted structures including the pectin components rhamnogalacturonan I (RG-I) and homogalacturonan (HG), neutral side chains (galactan/arabinangalactan), acetylation of pectin, and cellulose level. Modification arising from the pectin backbone- or pectin side chain transgenic lines either increased or decreased the HG:RG-I ratio, side chain length, and methyl esterification of pectin in the tuber cell wall. The pectin esterification transgenic line exhibited only limited side effects. The cellulose level-targeting transgenic lines yielded an unexpectedly high HG:RG-I ratio and longer pectic side chains. These results clearly demonstrate that in effects of a transgene are not restricted to the direct activity of the targeted enzyme but have consequences for the structure of the cell wall matrix. Analysis of whole cell wall structure is therefore necessary to assess the complete effect, direct and indirect, of a transgene.

Genes encoding pectic enzymes were introduced into wild-type potato Karnico. Cell wall materials were extracted from Karnico and transgenic lines expressing β-galactosidase (β-Gal-14) or rhamnogalacturonan lyase (RGL-18). Pectic polysaccharides from the β-Gal-14 transgenic line exhibited rhamnogalacturonan-I structural elements with shorter galactan side chains, whereas the RGL-18 transgenic line had less rhamnogalacturonan-I structures than Karnico. Xyloglucan in primary cell walls interacts with pectin and other cell wall polysaccharides and controls cell growth.Xyloglucan extracts from transgenic lines had different levels of monosaccharides compared to wild-type. Most XXGG-type xyloglucans from Karnico and RGL-18 alkali-extractable extracts predominantly consisted of XXGG and XSGG building blocks. Karnico and RGL-18 4 mol LAfter transgenic modification on pectin, the xyloglucan building blocks may have been changed. The β-Gal-14 lines mostly present XXXG-type repeating units instead of the XXGG-type in 4 mol L

Sufficient dietary fibre intake reduces the risk of several diseases, but the mechanisms linking fibre structure and health effects remain unclear. To analyse the influence of the amount of methyl groups esterified to the backbone, lemon pectins of different degrees of methyl esterification (30, 56, and 74DM) were studied for immune receptor activating potential and epithelial barrier protection. In reporter assays, the pectins demonstrated TLR/MyD88 dependent activation of NF-kappa B/AP-1, which increased with increasing pectin DM. To analyse the importance of backbone structure, the pectins were enzymatically digested into oligomers, which abrogated TLR activating potential. The 30 and 74DM pectins induced strong protection of the epithelial barrier measured by T84 transepithelial electrical resistance (TEER). These results indicate that activation of immune cells by lemon pectins is TLR dependent, and the intact polymer backbone is indispensable for activation. In addition, DM is a determining factor in activating potential and epithelial barrier protective effects. (C) 2016 Elsevier Ltd. All rights reserved.

Research on prebiotics and other novel health-promoting food components has been active for over a decade. Arabinoxylan (AX) derived arabinoxylooligosaccharides (AXOS), which may have various chemical structures, depending on the xylan source and the degradation method used, stand increasingly in the spotlight as potential prebiotics. During the past decade, the studies of the possibilities to produce the AXOS by using biocatalytic conversion have received more attention. In addition, there is an interest in the use of novel cereal biomass for the production of AXOS. The aim of this study was to investigate the influence of various commercial enzyme preparations on the degradability of insoluble arabinoxylans in wheat and rye wholemeal treated by extrusion, identify and quantify xylooligosaccharides (XOS) and arabinoxylooligosaccharides (AXOS) in treated media. The enzymatic degradation of rye and wheat cell wall materials was monitored by HPSEC, HPAEC and MALDI-TOF-MS techniques. It was noticed that there is no significant difference between extruded and natural cereals, and type of cereals had not significant influence on XOS and AXOS production. The most effective biocatalysts were hemicellulases expressed in the enzyme preparations from Trichoderma and Aspergillus spp. (Depol 692), Humicola and Bacillus spp. (Ceremix Plus). Degradability of rye and wheat cell wall materials by these enzyme preparations obtained break down percentages of 70–87% and 67–77%, respectively. After enzymatic treatment, only small amounts of xylose, xylobiose, and xylotriose was eluted compare to the amount of more complex oligosaccharides with higher degree of polymerization (DP). The mass spectra of oligosaccharides indicated the presence of a homologous series of pentoses ranging from DP 4 to 15. This indicates that chosen enzyme preparations acted well on wheat and rye biomass, and released quite high amounts of XOS and AXOS.

The objective was to determine the effects of dietary fibre with bulking, viscous and gel-forming properties on satiation, and to identify the underlying mechanisms. We conducted a randomised crossover study with 121 men and women. Subjects were healthy, non-restrained eaters, aged 18–50 years and with normal BMI (18·5–25 kg/m2). Test products were cookies containing either: no added fibre (control), cellulose (bulking, 5 g/100 g), guar gum (viscous, 1·25 g/100 g and 2·5 g/100 g) or alginate (gel forming, 2·5 g/100 g and 5 g/100 g). Physico-chemical properties of the test products were confirmed in simulated upper gastrointestinal conditions. In a cinema setting, ad libitum intake of the test products was measured concurrently with oral exposure time per cookie by video recording. In a separate study with ten subjects, 4 h gastric emptying rate of a fixed amount of test products was assessed by 13C breath tests. Ad libitum energy intake was 22 % lower for the product with 5 g/100 g alginate (3·1 (sd 1·6) MJ) compared to control (4·0 (sd 2·2) MJ, Psd 1·9) min) was 48 % longer than for control (1·6 (sd 0·9) min, P= 0·01). Gastric emptying of the 5 g/100 g alginate product was faster compared to control (P

Filamentous fungi are a good source of hemicellulolytic enzymes for biomass degradation. Enzyme preparations were obtained as culture supernatants from 78 fungal isolates grown on wheat straw as carbon source. These enzyme preparations were utilized in the hydrolysis of insoluble wheat straw and corn fiber xylan rich fractions. Up to 14% of the carbohydrates in wheat straw and 34% of those in corn fiber were hydrolyzed. The degree of hydrolysis by the enzymes depended on the origin of the fungal isolate and on the complexity of the substrate to be degraded. Penicillium, Trichoderma or Aspergillus species, and some non-identified fungi proved to be the best producers of hemicellulolytic enzymes for degradation of xylan rich materials. This study proves that the choice for an enzyme preparation to efficiently degrade a natural xylan rich substrate, is dependent on the xylan characteristics and could not be estimated by using model substrates.

Xylanases are mostly classified as belonging to glycoside hydrolase (GH) family 10 and 11, which differ in catalytic properties and structures. However, within one family, differences may also be present. The influence of solubility and molecular structure of substrates towards the efficiency of two GH10 xylanases from Myceliophthora thermophila C1 was investigated. The xylanases differed in degradation of high and low substituted substrate and the substitution pattern was an important factor influencing their efficiency. Alkali-labile interactions, as well as the presence of cellulose within the complex cell wall structure hindered efficient hydrolysis for both xylanases. The presence of a carbohydrate binding module did not enhance the degradation of the substrates. The differences in degradation could be related to the protein structure of the two xylanases. The study shows that the classification of enzymes does not predict their performance towards various substrates.

In vitro fermentation of 12 dietary fibres by faecal inocula from pigs and humans were performed. The fibres included homoglucans, mannans, fructans, polyuronides, and complex heteroglycans. Gas production, short chain fatty acid production and fibre degradation products were monitored during fermentation. Human inoculum has more ability to ferment resistant starch and fibres containing uronic acids. In contrast, pig inoculum is able to ferment cellulose, which is hardly fermented by human inoculum. The sugar and linkage composition of the fibres has an important influence on fibre fermentation patterns. Fibres containing uronic acids induced the production of acetate, whereas fibres containing neutral sugars induced the production of propionate or butyrate. Fermentation of the fructans showed that molecular size could be an influential factor, and fermentation of complex heteroglycans showed that the arrangement of sugars in the molecules may also affect the fermentation patterns. This experiment also shows that monitoring of fibre degradation products is important for understanding how fibres are degraded during fermentation.

a-Lactalbumin was glycated via the Maillard reaction in the dry state using various mono- and oligosaccharides. The reaction resulted not only in coupling of the saccharides to a-lactalbumin but also in cross-linked proteins. The glycation rate and the extent of cross-link formation were highly dependent on the saccharide used. Glycation by arabinose and xylose led to a very fast protein cross-link formation, whereas glucose showed a relatively low protein cross-linking ability. The stability of foams, created using the various glycated protein samples, depended on the type of saccharide used, the extent of glycation, and possibly the amount of cross-linked protein. Compared to nonmodified a-lactalbumin, glycation with rhamnose and fucose improved foam stability, whereas application of glucose, galacturonic acid, and their oligosaccharides did not exert a clear effect. Mass spectrometric analysis revealed that dehydration of the Amadori products is an indicator of the formation of protein cross-links.

The characterization of oligosaccharides in the feces of breast-fed babies is a valuable tool for monitoring the gastrointestinal fate of human milk oligosaccharides (HMOs). In the present study we monitored fecal oligosaccharide profiles together with the HMO-profiles of the respective breast milks up to six months postpartum, by means of capillary electrophoresis-laser induced fluorescence detection and mass spectrometry. Eleven mother/child pairs were included. Mother's secretor- and Lewis-type included all combinations [Le(a-b+), Le(a+b-), Le(a-b-)]. The fecal HMO-profiles in the first few months of life are either predominantly composed of neutral or acidic HMOs and are possibly effected by the HMO-fingerprint in the respective breast milk. Independent of the initial presence of acidic or neutral fecal HMOs, a gradual change to blood-group specific oligosaccharides was observed. Their presence pointed to a gastrointestinal degradation of the feeding-related HMOs, followed by conjugation with blood group specific antigenic determinants present in the gastrointestinal mucus layer. Eleven of these 'hybrid'-oligosaccharides were annotated in this study. When solid food was introduced, no HMOs and their degradation- and metabolization products were recovered in the fecal samples.

Pectins, complex plant-derived polysaccharides, are novel candidates for biomaterial nanocoatings. Pectic rhamnogalacturonan-I regions (RG-I) can be enzymatically treated to so-called modified hairy regions (MHR). We surveyed the growth and differentiation of murine preosteoblastic MC3T3-E1 cells on Petri dishes coated with RG-Is from native or genetically engineered potato tubers. Uncoated tissue culture polystyrene (TCPS) and aminated (AMI) dishes served as controls. MHRPTR_GAL sample was depleted of galactose (9 mol % galactose; 23 mol % arabinose) and MHRPTR_ARA of arabinose (61 mol % galactose; 6 mol % arabinose). Wild-type (modified hairy region from potato pectin (MHRP)_WT) fragment contained default amounts (58 mol % galactose; 13 mol % arabinose) of both sugars. Focal adhesions (FAs) indicating cellular attachment were quantified. Reverse transcriptase polymerase chain reaction (RT-PCR) of alkaline phosphatase and osteocalcin genes indicating osteoblastic differentiation was performed along with staining the produced calcium with tetracycline as an indicator of osteoblastic differentiation. Osteoblasts proliferated on all the samples to some extent. The control surfaces performed better than any of the pectin samples, of which the MHRP_WT seemed to function best. FA length was greater on MHRPTR_GAL than on other pectin samples, otherwise the mutants did not significantly deviate. RT-PCR results indicate that differences between the samples at the gene expression level might be even subtler. However, tetracycline-stained calcium-containing mineral was detected merely only on uncoated TCPS. These results indicate the possibility to affect bone cell growth with in vivo-modified pectin fragments, consecutively providing information on the significance of certain monosaccharides on the biocompatibility of these polysaccharides. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.

Brewer’s spent grain (BSG) is an important coproduct of the brewing industry and is generally used in animal feed. Recently, there has been considerable research into the use of enzymes to convert BSG into more value-added products. In this study, the efficiency of enzymatic fractionation of freshly produced BSG was studied in pilot scale. Carbohydrateand protein-degrading enzymes were applied sequentially to produce three fractions: carbohydrate hydrolysate, protein hydrolysate, and a solid lignin- carbohydrate residue. To minimize microbial contamination, processing was performed at 60°C for 4 hr. In all, 14 and 36% of the original dry matter was liberated by carbohydrase and protease, respectively. The carbohydrate- degrading enzyme cocktail produced cellulose- and hemicellulose- derived mono- and oligosaccharides. The protease treatment released peptide-enriched hydrolysate. Approximately half of the original BSG solids were solubilized during the two-stage enzymatic process. Although the protease efficiently released the majority of BSG proteins, the carbohydrate matrix was much less accessible to enzyme attack. The impact of scale-up to pilot scale was compared with previous laboratory-scale studies, and the results were found to be in good agreement.

Two β-xylosidases of glycoside hydrolase family 3 (GH 3) from Aspergillus nidulans FGSC A4, BxlA and BxlB were produced recombinantly in Pichia pastoris and secreted to the culture supernatants in yields of 16 and 118 mg/L, respectively. BxlA showed about sixfold higher catalytic efficiency (k(cat)/K(m)) than BxlB towards para-nitrophenyl β-D-xylopyranoside (pNPX) and β-1,4-xylo-oligosaccharides (degree of polymerisation 2-6). For both enzymes k(cat)/K(m) decreased with increasing β-1,4-xylo-oligosaccharide chain length. Using pNPX as donor with 9 monosaccharides, 7 disaccharides and two sugar alcohols as acceptors 18 different β-xylosyl-oligosaccharides were synthesised in 2-36% (BxlA) and 6-66% (BxlB) yields by transxylosylation. BxlA utilised the monosaccharides D-mannose, D-lyxose, D-talose, D-xylose, D-arabinose, L-fucose, D-glucose, D-galactose and D-fructose as acceptors, whereas BxlB used the same except for D-lyxose, D-arabinose and L-fucose. BxlB transxylosylated the disaccharides xylobiose, lactulose, sucrose, lactose and turanose in upto 35% yield, while BxlA gave inferior yields on these acceptors. The regioselectivity was acceptor dependent and primarily involved β-1,4 or 1,6 product linkage formation although minor products with different linkages were also obtained. Five of the 18 transxylosylation products obtained from D-lyxose, D-galactose, turanose and sucrose (two products) as acceptors were novel xylosyl-oligosaccharides, β-D-Xylp-(1→4)-D-Lyxp, β-D-Xylp-(1→6)-D-Galp, β-D-Xylp-(1→4)-α-D-Glcp-(1→3)-β-D-Fruf, β-D-Xylp-(1→4)-α-D-Glcp-(1→2)-β-D-Fruf, and β-D-Xylp-(1→6)-β-D-Fruf-(2→1)-α-D-Glcp, as structure-determined by 2D NMR, indicating that GH3 β-xylosidases are able to transxylosylate a larger variety of carbohydrate acceptors than earlier reported. Furthermore, transxylosylation of certain acceptors resulted in mixtures. Some of these products are also novel, but the structures of the individual products could not be determined.

Eucalyptus globulus wood samples were subjected to double hydrothermal processing to remove extractives in the first stage, and to cause the selective solubilisation of 4-O-methylglucuronoxylan in the second stage. The hemicellulose-derived products present in the liquors from the second hydrothermal stage (substituted xylooligosaccharides, denoted XOS) were refined by treatments with membranes and ion exchange. The purified XOS product was assayed for composition and characterised by HPLC-RI, HPAEC-PAD, HPSEC, MALDI-TOF-MS and NMR techniques. The results suggested the presence of neutral and acidic XOS with a degree of acetylation of about 0.6. The fermentability of the refined XOS product by faecal inocula was assessed by measuring both substrate consumption and formation of short-chain fatty acids.