Engineering of xylanases for the development of biotechnologically important characteristics.

Xylanases are the main biocatalysts used for the reduction of the xylan backbone from hemicellulose, randomly splitting off ß-1,4-glycosidic linkages between xylopyranosyl residues. Xylanase market has been annually estimated at 500 million US Dollars and they are potentially used in broad industrial process ranges such as paper pulp biobleaching, xylo-oligosaccharide production, and biofuel manufacture from lignocellulose. The highly stable xylanases are preferred in the downstream procedure of industrial processes because they can tolerate severe conditions. Almost all native xylanases can not endure adverse conditions thus they are industrially not proper to be utilized. Protein engineering is a powerful technology for developing xylanases, which can effectively work in adverse conditions and can meet requirements for industrial processes. This study considered state-of-the-art strategies of protein engineering for creating the xylanase gene diversity, high-throughput screening systems toward upgraded traits of the xylanases, and the prediction and comprehensive analysis of the target mutations in xylanases by in silico methods. Also, key molecular factors have been elucidated for industrial characteristics (alkaliphilic enhancement, thermal stability, and catalytic performance) of GH11 family xylanases. The present review explores industrial characteristics improved by directed evolution, rational design, and semi-rational design as protein engineering approaches for pulp bleaching process, xylooligosaccharides production, and biorefinery & bioenergy production.

How Do Prebiotics Affect Human Intestinal Bacteria?-Assessment of Bacterial Growth with Inulin and XOS In Vitro.

Prebiotics are believed to exhibit high specificity in stimulating the growth or activity of a limited number of commensal microorganisms, thereby conferring health benefits to the host. However, the mechanism of action of prebiotics depends on multiple factors, including the composition of an individual's gut microbiota, and is therefore difficult to predict. It is known that different bacteria can utilize inulin and xylooligosaccharides (XOS), but an overview of which bacteria in the human gut may be affected is lacking. Detailed knowledge of how bacterial growth is affected by prebiotics is furthermore useful for the development of new synbiotics, which combine a living microorganism with a selective substrate to confer a health benefit to the host. Hence, we developed a statistical model to compare growth in vitro among typical human gut bacteria from different phylogenetic lineages. Based on continuous observation of the optical density (OD600), we compare maximal growth rates (rmax), maximal attained OD600 (ODmax), and area under the growth curve (AUC) of bacteria grown on inulin or XOS. The consideration of these three parameters suggests strain-specific preferences for inulin or XOS and reveals previously unknown preferences such as Streptococcus salivarius growth on XOS.

Hazelnut (Corylus avellana L.) shells as a potential source of dietary fibre: impact of hydrothermal treatment temperature on fibre structure and degradation compounds.

BACKGROUND: Hemicellulose extraction from lignocellulosic biomasses has gained interest over the years, and hydrothermal treatment is one of the most common methods employed for this purpose. This work aimed to deeply study hazelnut (Corylus avellana L.) shells as a new source of dietary fibre, evaluating the effect of hydrothermal treatment temperatures on the type and structure of fibre extracted, but also on the formation of side-products derived from lignocellulose degradation. RESULTS: Different process temperatures led to diverse polysaccharides in the hydrothermal extract. Pectin was identified for the first time in hazelnut shells when experimenting with extraction at 125 °C, whereas at 150 °C a heterogeneous mixture of pectin, xylan, and xylo-oligosaccharides was present. The highest yield in terms of total fibre was gained at 150 and 175 °C, and then decreased again at 200 °C. Finally, more than 500 compounds from different chemical classes were putatively identified and they appeared to be present in the extracted fibre with a different distribution and relative amount, depending on the heat treatment severity. A generally high content of phenols, phenyls, oligosaccharides, dehydro-sugars, and furans was observed. CONCLUSIONS: Modulation of the hydrothermal treatment temperature allows fibre extracts with very different compositions, and therefore different potential end uses, to be obtained from hazelnut shells. A sequential temperature-based fractionation approach, as a function of the severity of the extraction parameters, can also be considered. Nevertheless, the study of the side-compounds formed from lignocellulosic matrix degradation, as a function of the applied temperature, needs to be fully addressed for a safe introduction of the fibre extract within the food chain. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Statistical investigation of the bioprocess conditions of alkali combined twin-screw extrusion pretreatment to enhance fractionation and enzymatic hydrolysis of bulgur bran.

BACKGROUND: Bulgur bran (BB) is a potential source for the production of value-added products such as fermentable sugars and xylooligosaccharides (XOs). In this study, alkali combined twin-screw extrusion pretreatment was performed and statistically optimized to enhance fractionation and enzymatic hydrolysis of BB. The pretreatment conditions (barrel temperature, screw speed and alkali impregnation) were optimized by Box-Behnken design (BBD) to obtain the highest hemicellulose separation from BB. The obtained fractions were analyzed for the production of fermentable sugars and XOs. RESULTS: The results revealed that twin-screw extrusion of BB performed at 67 °C barrel temperature and 250 rpm screw speed after alkali impregnation at 0.02 g alkali g-1 biomass concentration provided 40.4% higher hemicellulose separation yield compared to the untreated BB. Alkali combined twin-screw extrusion pretreatment increased the enzymatic hydrolysis yield of BB fourfold, whereas a 13-fold increase was achieved after the separation of hemicellulose from pretreated BB. Xylose (X1)-free xylobiose (X2) was the main product after xylanase hydrolysis of hemicellulose fraction. SEM images confirmed the morphological modifications in BB, which were in agreement with the enhanced fractionation performance and the higher enzymatic hydrolysis yield. CONCLUSION: The results of this study suggested that pretreatment by alkali combined twin-screw extrusion followed by alkali extraction could be a reliable and effective process for fractionation of BB and production of fermentable sugars and XOs. © 2022 Society of Chemical Industry.

Expression of coagulin A with low cytotoxic activity by Pediococcus pentosaceus ST65ACC isolated from raw milk cheese.

AIMS: We aimed to evaluate some specific conditions for growth of Pediococcus pentosaceus ST65ACC and its bacteriocin expression through ABC transporters; to purify the bacteriocin and determine its sequence; and to evaluate the cytotoxicity potential of the purified bacteriocin(s). METHODS AND RESULTS: The results presented for growth behaviour of P. pentosaceus ST65ACC showed that the bacterial growth was slightly influenced when cultured in MRS broth with different amounts of inoculum: 1, 2, 5 and 10%. The bacteriocin activity increased when 5 and 10% inocula were used. The carbon source (glucose) used in different amounts (1, 2, 3 or 4%) had no significant effect on growth and bacteriocin production. The studied strain P. pentosaceus ST65ACC was able to metabolize xylooligosaccharide (XOS) as the sole carbon source, resulting in the production of an antimicrobial peptide. The genes involved in the ABC transport system and sugar metabolism of P. pentosaceus ST65ACC were expressed at different levels. The bacteriocin produced by P. pentosaceus ST65ACC was partially purified by precipitation with ammonium sulphate (40% saturation), followed by reversed-phase liquid chromatography, resulting in the identification of an active bacteriocin. Tandem mass spectrometry was used to identify the partial sequence KYYGNGVTCGKHSCSVDWGK sharing high similarity to coagulin A. The semi-purified bacteriocin had low cytotoxicity based on estimated values for maximal nontoxic concentration (MNC) and cytotoxicity concentration (CC50 ). CONCLUSIONS: The bacteriocin produced by P. pentosaceus ST65ACC is similar to coagulin, with low cytotoxicity, strong antimicrobial activity and possible additional metabolite routes in the producer cell. In addition to MRS broth, bacteriocin was produced also in medium containing XOS (as the single carbon source). SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this is the first report of evaluation of the role of ABC transporters in the expression of bacteriocin by P. pentosaceus, cultured in MRS and XOS.

Dose-Dependent Effects of Dietary Xylooligosaccharides Supplementation on Microbiota, Fermentation and Metabolism in Healthy Adult Cats.

In order to investigate the effect and appropriate dose of prebiotics, this study evaluated the effect of two levels of xylooligosaccharides (XOS) in cats. Twenty-four healthy adult cats were divided into three groups: no-XOS control diet with 1% cellulose; low XOS supplementation (LXOS) with 0.04% XOS and 0.96% cellulose; and high XOS supplementation (HXOS) with 0.40% XOS and 0.60% cellulose. Both XOS groups increased blood 3-hydroxybutyryl carnitine levels and decreased hexadecanedioyl carnitine levels. Both XOS treatments displayed an increased bacterial abundance of Blautia, Clostridium XI, and Collinsella and a decreased abundance of Megasphaera and Bifidobacterium. LXOS groups increased fecal pH and bacterial abundance of Streptococcus and Lactobacillus, decreased blood glutaryl carnitine concentration, and Catenibacterium abundance. HXOS group showed a more distinct microbiome profile and higher species richness, and an increased bacterial abundance of Subdoligranulum, Ruminococcaceae genus (unassigned genus), Erysipelotrichaceae genus, and Lachnospiraceae. Correlations between bacterial abundances and blood and fecal parameters were also observed. In conclusion, XOS could benefit feline gut health by altering microbiota; its effects dependant on the dose. The higher-dose XOS increased bacterial populations that possibly promoted intestinal fermentation, while the lower dose altered populations of carbohydrate-metabolic microbiota and possibly modulated host metabolism. Low-dose prebiotics may become a trend in future studies.

The effects of exogenous xylanase supplementation on the in vivo generation of xylooligosaccharides and monosaccharides in broilers fed a wheat-based diet.

1. This study quantified xylanase-induced changes in soluble monosaccharides, xylooligosaccharides (XOS) and volatile fatty acid (VFA) contents of the different sections of the gastrointestinal tract (GIT) and whether these were related to altered bird performance. 2. An in vitro digestion of the wheat-based diet was carried out with the xylanase (Econase XT at 16,000BXU/kg diet) to compare the in vitro and in vivo generation of these XOS and monosaccharides. For the in vivo study, 80 male Ross 508 b roiler chicks were split into two groups fed a wheat-based diet with or without Econase XT (16,000BXU/kg diet) for 21 days. 3. There were no effects of Econase XT inclusion on growth performance characteristics, likely a result of the high-quality wheat diet, the corresponding high performance of the control group (FCR average of 1.45 in controls) and the relatively young age of the birds (from four to 26 days of age). 4. Econase XT supplementation increased the xylotetraose (X4) content in the colon (P = 0.046, enzyme x GIT section interaction) and the xylose contents in the colon and caeca (P < 0.001, enzyme x GIT section interaction). 5. The trend for increased acetate production in the caeca of Econase XT treated birds (P = 0.062) suggested that the XOS generated were subsequently fermented in the caeca, potentially impacting upon the types of microbiota present. 6. The present study suggested that wheat arabinoxylan degradation was enhanced by xylanase supplementation, which may have increased the production of beneficial volatile fatty acids (VFA) in the caeca, and thereby potentially modulated the caecal microbiome, but without affecting bird performance at this early age.

Xylanase and xylo- oligosaccharide prebiotic improve the growth performance and concentration of potentially prebiotic oligosaccharides in the ileum of broiler chickens.

1. The objective of this study was to investigate the effect of supplementing broiler diets with xylanase or xylo- oligosaccharide (XOS) on growth performance, the concentration of non-starch polysaccharide (NSP) hydrolysis products in the ileum and concentration of short chain fatty acids (SCFA) in the caeca of broiler chickens.2. In total, 500 male Ross 308 broilers were used in this 29-day (d) study. The treatments were organised into a 2 × 2 plus 1 factorial arrangement consisting of two additives (xylanase or XOS) at two levels (low or high) plus a control treatment with no additives. This gave five treatments with 100 birds in each treatment group. The diets were slightly deficient in protein by 20 g/kg and energy by 1 MJ/kg.3. On d 14 and 28, two birds per pen were euthanised, the caeca content collected and analysed for short chain fatty acid (SCFA) concentration. On d 29, six birds per pen were euthanised and ileal digesta were collected and analysed for the concentration of NSP fractions.4. On d 14, caecal acetic acid, iso-butyric acid, iso-valeric acid, n-valeric acid and total SCFA concentrations were significantly greater (P ≤ 0.05) when diets were supplemented with XOS compared with xylanase.5. Ileal concentration of arabinose, galactose and glucuronic acid (GlucA2) were significantly greater (P ≤ 0.05) in the insoluble NSP fraction when diets were supplemented with a high level of xylanase, compared with the control treatment. Ileal concentration of fructose was significantly greater (P ≤ 0.05) in the water soluble NSP when a high level of xylanase or low level of XOS were included in the diet compared with the control.6. It was concluded that xylanase and XOS had similar effects on NSP concentration and SCFA in the caeca, although there was little effect on performance. This observation demonstrated further benefits of xylanase supplementation in wheat-based broiler diets beyond digesta viscosity reduction and the release of extra nutrients.

The prebiotics (Fructo-oligosaccharides and Xylo-oligosaccharides) modulate the probiotic properties of Lactiplantibacillus and Levilactobacillus strains isolated from traditional fermented olive.

This study aimed to examine the influence of two prebiotics, fructo-oligosaccharides (FOS) and xylo-oligosaccharides (XOS), on probiotic properties (resistance to low pH and bile salt, hydrophobicity and auto-aggregation), metabolites production, and antimicrobial activity of probiotic Lactiplantibacillus (L. pentosus S42 and L. plantarum S61) and Levilactobacillus (L. brevis S27) strains isolated from fermented olive. The results demonstrated the ability of strains to ferment XOS more than FOS as a sole carbon source, resulting in pH reduction. The prebiotics (FOS and XOS) significantly increased (p < 0.05) their survival in gastro-intestinal conditions (low pH and 0.3% of bile salts), as well as their hydrophobicity, auto-aggregation and production of proteins, compared to glucose (control). The major organic acids produced by Lactiplantibacillus and Levilactobacillus strains, were oxalic, malic and lactic acids from FOS, XOS and glucose, respectively. No antimicrobial activity was observed from cell-free supernatant (CFS) of Lactiplantibacillus and Levilactobacillus strains obtained from FOS. In the presence of XOS the organic acids, produced by Lactiplantibacillus and Levilactobacillus strains, were more diverse with high contents, and exhibited higher antifungal and antibacterial activities, more than that of FOS and glucose. The combination of L. plantarum S61 and XOS demonstrated the highest inhibition zones ranges of 20.7-22.2 mm against pathogenic bacteria and 29.2-30 mm against yeasts. This combination can be used in production of antifungal preservatives and pharmaceuticals, against pathogenic and spoilage yeasts.

Targeting gut microbiota and barrier function with prebiotics to alleviate autoimmune manifestations in NOD mice.

AIMS/HYPOTHESIS: Adopting a diet containing indigestible fibre compounds such as prebiotics to fuel advantageous bacteria has proven beneficial for alleviating inflammation. The effect of the microbial changes on autoimmunity, however, remains unknown. We studied the effects of prebiotic xylooligosaccharides (XOS) on pancreatic islet and salivary gland inflammation in NOD mice and tested whether these were mediated by the gut microbiota. METHODS: Mother and offspring mice were fed an XOS-supplemented diet until diabetes onset or weaning and were compared with a control-fed group. Diabetes incidence was monitored, insulitis and sialadenitis were scored in histological sections from adult mice, and several metabolic and immune variables were analysed in mice before the development of diabetes. Gut barrier function was assessed using an in vivo FITC-dextran permeability test. The importance of XOS-mediated gut microbial changes were evaluated in antibiotic-treated mice fed either XOS or control diet or given a faecal microbiota transplant from test animals. RESULTS: Diabetes onset was delayed in the XOS-fed mice, which also had fewer cellular infiltrations in their pancreatic islets and salivary glands. Interestingly, insulitis was most reduced in the XOS-fed groups when the mice were also treated with an antibiotic cocktail. There was no difference in sialadenitis between the dietary groups treated with antibiotics; the mice were protected by microbiota depletion regardless of diet. Faecal microbiota transplantation was not able to transfer protection. No major differences in glucose-insulin regulation, glucagon-like peptide-1, or short-chain fatty acid production were related to the XOS diet. The XOS diet did, however, reduce gut permeability markers in the small and large intestine. This was accompanied by a more anti-inflammatory environment locally and systemically, dominated by a shift from M1 to M2 macrophages, a higher abundance of activated regulatory T cells, and lower levels of induction of natural killer T cells and cytotoxic T cells. CONCLUSIONS/INTERPRETATION: Prebiotic XOS have microbiota-dependent effects on salivary gland inflammation and microbiota-independent effects on pancreatic islet pathology that are accompanied by an improved gut barrier that seems able to heighten control of intestinal diabetogenic antigens that have the potential to penetrate the mucosa to activate autoreactive immune responses.

Delineating thermophilic xylanase from Bacillus licheniformis DM5 towards its potential application in xylooligosaccharides production.

In this present study novel endoxylanase producing Bacillus licheniformis DM5 isolated, identified based on 16S rDNA from Garampani hotspring, Assam, India and enzyme was purified. RNA secondary structure predicted the similarity of B. licheniformis DM5 with B. licheniformis ATCC14580. Highest production of xylanase from B. licheniformis DM5 was achieved in the TY medium with cell densities 12 g/l and extracellular protein concentration containing xylanase 400 mg/l. Partially purified extracellular xylanase displayed optimum pH 6.5 and temperature 50 °C. Thermostability of the xylanase at the elevated temperature showed stability between 50 and 60 °C retaining its 99% activity. Kinetic parameters of thermophilic xylanase revealed Km 1.5 ± 0.2 mg/ml, Vmax 2.7 ± 0.2 U/ml and and Kcat 1.8 ± 0.2 s-1 against beechwood xylan but ruled out any exo-acting activity against synthetic pNP-xylopyranoside substrate. Time dependent enzymatic hydrolysis of beechwood xylan and preprocessed agrowaste corncob exhibited the release of xylotriose and xylobiose oligosaccharide (XOS) significantly high. Xylobiose and xylotriose exhibited higher binding affinities with BIAXP transporter protein of probiotic bacteria explaining their easy uptake by the cells. Mixed oligosaccharides also exhibited better prebiotic activity by promoting growth of Bifidobacterium infantis and Lactobacillus delbrueckii. Mixed XOS when tested for their cytotoxicity on Hela cell lines in in vitro MTT assay displayed significant lowering of cell viability after 48 h and 24 h at 100 µg/ml to 60% and 50%, respectively. In contrast, cytotoxicity wasn't observed against normal cervical cell line (VK2/E6E7-ATCC-CRL-2616). Therefore, thermophilic endoxylanase from B. licheniformis DM5 could be attributed for the production of prebiotic and anti-inflammatory XOS from agrowaste.

Structural insights of RmXyn10A - A prebiotic-producing GH10 xylanase with a non-conserved aglycone binding region.

Hydrolysis of arabinoxylan (AX) by glycoside hydrolase family 10 (GH10) xylanases produces xylo- and arabinoxylo-oligosaccharides ((A)XOS) which have shown prebiotic effects. The thermostable GH10 xylanase RmXyn10A has shown great potential to produce (A)XOS. In this study, the structure of RmXyn10A was investigated, the catalytic module by homology modelling and site-directed mutagenesis and the arrangement of its five domains by small-angle X-ray scattering (SAXS). Substrate specificity was explored in silico by manual docking and molecular dynamic simulations. It has been shown in the literature that the glycone subsites of GH10 xylanases are well conserved and our results suggest that RmXyn10A is no exception. The aglycone subsites are less investigated, and the modelled structure of RmXyn10A suggests that loop ß6α6 in the aglycone part of the active site contains a non-conserved α-helix, which blocks the otherwise conserved space of subsite +2. This structural feature has only been observed for one other GH10 xylanase. In RmXyn10A, docking revealed two alternative binding regions, one on either side of the α-helix. However, only one was able to accommodate arabinose-substitutions and the mutation study suggests that the same region is responsible for binding XOS. Several non-conserved structural features are most likely to be responsible for providing affinity for arabinose-substitutions in subsites +1 and +2. The SAXS rigid model of the modular arrangement of RmXyn10A displays the catalytic module close to the cell-anchoring domain while the carbohydrate binding modules are further away, likely explaining the observed lack of contribution of the CBMs to activity.

Engineering improved thermostability of the GH11 xylanase from Neocallimastix patriciarum via computational library design.

Xylanases, which cleave the ß-1,4-glycosidic bond between xylose residues to release xylooligosaccharides (XOS), are widely used as food additives, animal feeds, and pulp bleaching agents. However, the thermally unstable nature of xylanases would hamper their industrial application. In this study, we used in silico design in a glycoside hydrolase family (GH) 11 xylanase to stabilize the enzyme. A combination of the best mutations increased the apparent melting temperature by 14 °C and significantly enhanced thermostability and thermoactivation. The variant also showed an upward-shifted optimal temperature for catalysis without compromising its activity at low temperatures. Moreover, a 10-fold higher XOS production yield was obtained at 70 °C, which compensated the low yield obtained with the wild-type enzyme. Collectively, the variant constructed by the computational strategy can be used as an efficient biocatalyst for XOS production at industrially viable conditions.

EcXyl43 ß-xylosidase: molecular modeling, activity on natural and artificial substrates, and synergism with endoxylanases for lignocellulose deconstruction.

Biomass hydrolysis constitutes a bottleneck for the biotransformation of lignocellulosic residues into bioethanol and high-value products. The efficient deconstruction of polysaccharides to fermentable sugars requires multiple enzymes acting concertedly. GH43 ß-xylosidases are among the most interesting enzymes involved in hemicellulose deconstruction into xylose. In this work, the structural and functional properties of ß-xylosidase EcXyl43 from Enterobacter sp. were thoroughly characterized. Molecular modeling suggested a 3D structure formed by a conserved N-terminal catalytic domain linked to an ancillary C-terminal domain. Both domains resulted essential for enzymatic activity, and the role of critical residues, from the catalytic and the ancillary modules, was confirmed by mutagenesis. EcXyl43 presented ß-xylosidase activity towards natural and artificial substrates while arabinofuranosidase activity was only detected on nitrophenyl α-L-arabinofuranoside (pNPA). It hydrolyzed xylobiose and purified xylooligosaccharides (XOS), up to degree of polymerization 6, with higher activity towards longer XOS. Low levels of activity on commercial xylan were also observed, mainly on the soluble fraction. The addition of EcXyl43 to GH10 and GH11 endoxylanases increased the release of xylose from xylan and pre-treated wheat straw. Additionally, EcXyl43 exhibited high efficiency and thermal stability under its optimal conditions (40 °C, pH 6.5), with a half-life of 58 h. Therefore, this enzyme could be a suitable additive for hemicellulases in long-term hydrolysis reactions. Because of its moderate inhibition by monomeric sugars but its high inhibition by ethanol, EcXyl43 could be particularly more useful in separate hydrolysis and fermentation (SHF) than in simultaneous saccharification and co-fermentation (SSCF) or consolidated bioprocessing (CBP).

In Vitro Fermentation of Selected Prebiotics and Their Effects on the Composition and Activity of the Adult Gut Microbiota.

Recently, the concept of prebiotics has been revisited to expand beyond non-digestible oligosaccharides, and the requirements for selective stimulation were extended to include microbial groups other than, and additional to, bifidobacteria and lactobacilli. Here, the gut microbiota-modulating effects of well-known and novel prebiotics were studied. An in vitro fermentation screening platform (i-screen) was inoculated with adult fecal microbiota, exposed to different dietary fibers that had a range of concentrations (inulin, alpha-linked galacto-oligosaccharides (alpha-GOS), beta-linked GOS, xylo-oligosaccharides (XOS) from corn cobs and high-fiber sugar cane, and beta-glucan from oats), and compared to a positive fructo-oligosaccharide (FOS) control and a negative control (no fiber addition). All dietary fibers displayed prebiotic activity, with beta-glucan showing more distinct effects on the microbial composition and metabolism compared to the other fibers. Beta-glucan induced the growth of Prevotella and Roseburia with a concomitant increase in propionate production. Inulin and both forms of GOS and XOS had a strong bifidogenic effect on the microbial composition. A dose-response effect was observed for butyrate when exposed to beta-glucan and inulin. The findings of this study support the potential for alpha-GOS, XOS, and oat beta-glucan to serve as novel prebiotics, due to their association with the positive shifts in microbiome composition and short-chain fatty acid production that point to potential health benefits.

Effect of probiotics and xylo-oligosaccharide supplementation on nutrient digestibility, intestinal health and noxious gas emission in weanling pigs.

OBJECTIVE: This study was conducted to evaluate the effect of probiotics (Bacillus subtilis and Enterococcus faecium) and xylo-oligosaccharide (XOS) supplementation on growth performance, nutrient digestibility, serum profiles, intestinal health, fecal microbiota and noxious gas emission in weanling pigs. METHODS: A total of 240 weanling pigs ([Yorkshire×Landrace]×Duroc) with an average body weight (BW) of 6.3±0.15 kg were used in this 28-day trial. Pigs were randomly allocated in 1 of the following 4 dietary treatments in a 2×2 factorial arrangement with 2 levels of probiotics (0 and 500 mg/kg probiotics) and XOS (0 and 200 mg/kg XOS) based on the BW and sex. RESULTS: Administration of probiotics or XOS improved average daily gain (p<0.05) during 0 to 14 d and the overall period, while pigs that were treated with XOS had a greater average daily gain and feed efficiency (p<0.05) compared with unsupplemented treatments throughout 15 to 28 d and the whole experiment. Either probiotics or XOS treatments increased the apparent total tract digestibility of nutrients (p<0.05) during 0 to 14 d. No effects on serum profiles were observed among treatments. The XOS increased villus height: crypt depth ratio in jejunum (p<0.05). The supplementation of probiotics (500 mg/kg) or XOS (200 mg/kg) alone improved the apparent total tract digestibility of dry matter, nitrogen and gross energy on d 14, the activity of trypsin and decreased fecal NH3 concentration (p<0.05). Administration of XOS decreased fecal Escherichia coli counts (p<0.05), while increased lactobacilli (p<0.05) on d 14. There was no interaction between dietary supplementation of probiotics and XOS. CONCLUSION: Inclusion of XOS at 200 mg/kg or probiotics (Bacillus subtilis and Enterococcus faecium) at 500 mg/kg in diets containing no antibiotics significantly improved the growth performance of weanling pigs. Once XOS is supplemented, further providing of probiotics is not needed since it exerts little additional effects.

HPAEC-PAD for oligosaccharide analysis-novel insights into analyte sensitivity and response stability.

The rising importance of accurately detecting oligosaccharides in biomass hydrolyzates or as ingredients in food, such as in beverages and infant milk products, demands for the availability of tools to sensitively analyze the broad range of available oligosaccharides. Over the last decades, HPAEC-PAD has been developed into one of the major technologies for this task and represents a popular alternative to state-of-the-art LC-MS oligosaccharide analysis. This work presents the first comprehensive study which gives an overview of the separation of 38 analytes as well as enzymatic hydrolyzates of six different polysaccharides focusing on oligosaccharides. The high sensitivity of the PAD comes at cost of its stability due to recession of the gold electrode. By an in-depth analysis of the sensitivity drop over time for 35 analytes, including xylo- (XOS), arabinoxylo- (AXOS), laminari- (LOS), manno- (MOS), glucomanno- (GMOS), and cellooligosaccharides (COS), we developed an analyte-specific one-phase decay model for this effect over time. Using this model resulted in significantly improved data normalization when using an internal standard. Our results thereby allow a quantification approach which takes the inevitable and analyte-specific PAD response drop into account. Graphical abstract HPAEC-PAD analysis of oligosaccharides and determination of PAD response drop leading to an improved data normalization.

Gut morphology and hepatic oxidative status of European sea bass (Dicentrarchus labrax) juveniles fed plant feedstuffs or fishmeal-based diets supplemented with short-chain fructo-oligosaccharides and xylo-oligosaccharides.

The effects of short-chain fructo-oligosaccharides (scFOS) and xylo-oligosaccharides (XOS) on gut morphology and hepatic oxidative status were studied in European sea bass juveniles weighing 60 g. Fish were fed diets including fishmeal (FM diets) or plant feedstuffs (PF diets; 30 FM:70 PF) as main protein sources (control diets). Four other diets were formulated similar to the control diets but including 1 % scFOS or 1 % XOS. At the end of the trial, fish fed PF-based diets presented histomorphological alterations in the distal intestine, whereas only transient alterations were observed in the pyloric caeca. Comparatively to fish fed FM-based diets, fish fed PF diets had higher liver lipid peroxidation (LPO), superoxide dismutase (SOD) and catalase (CAT) activities, and lower glutathione peroxidase, glutathione reductase and glucose 6-phosphate dehydrogenase activities. In fish fed the PF diets, prebiotic supplementation decreased SOD activity and XOS supplementation further decreased CAT activity. In fish fed the FM diets, XOS supplementation promoted a reduction of all antioxidant enzyme activities. Overall, dietary XOS and scFOS supplementation had only minor effects on gut morphology or LPO levels. However, dietary XOS reduced antioxidant enzymatic activity in both PF and FM diets, which indicate a positive effect on reduction of hepatic reactive oxygen species production.

The effects of dietary xylooligosaccharide on mucosal parameters, intestinal microbiota and morphology and growth performance of Caspian white fish (Rutilus frisii kutum) fry.

The present study investigates the effects of different levels of dietary xylooligosaccharide (XOS) on skin mucus bactericidal activity and protein content, intestinal microbiota and morphology as well as growth performance of Caspian white fish (Rutilus frisii kutum) fry. The specimens (1.54 ± 0.03 g) were fed experimental diets supplemented with different levels of XOS [0% (control), 1%, 2% and 3%] for 8 weeks. The results revealed that feeding on XOS significantly increased skin mucus antibacterial activity and protein levels (P < 0.05) and the highest levels were observed in 3% XOS treatment. The total autochthonous intestinal heterotrophic bacteria significantly increased following XOS administration in diet (P < 0.05). Administration of 2 or 3% XOS in fry diet significantly elevated the autochthonous lactic acid bacteria (P < 0.05). However, dietary XOS had no significant effect on intestinal morphology, growth performance and diet utilisation of Caspian white fish fry (P > 0.05). These results demonstrate beneficial effects of dietary XOS on different parameters of mucosal immunity (both skin mucus and intestinal microbiota) and encourage further studies regards various aspect of XOS administration in early life stages of fish.

Gene expression of enzymes involved in utilization of xylooligosaccharides by Lactobacillus strains.

Prebiotics are defined as food components that confer health benefits on the host through modulation of the microbiota. Xylooligosaccharides (XOS) are non-digestible oligosaccharides that have recently received increasing attention as potential prebiotic candidates. XOS are sugar oligomers composed of 1,4-linked xylopyranosyl backbone and are obtained by either chemical or, more commonly, enzymatic hydrolysis of xylan polysaccharides, extracted from the plant cell wall. The bifidogenic effect of XOS was demonstrated by both in vitro studies and small-scale in vivo human studies. Some intestinal bacterial strains are able to grow on XOS, yet numerous studies have demonstrated that the ability to utilize these oligosaccharides varies considerably among these bacteria. The aim of this study is to investigate the ability of several strains Lactobacillus to use XOS. Fifteen Lactobacillus strains, allifiated to L. plantarum, L. brevis and L. sakei, were studied. Screening procedure was performed for the ability of the strains to utilize XOS as an alternative carbon source. Only some of them utilize XOS. The growth kinetics show the presence of two lag phases, indicating that these bacteria utilize probably some monosaccharides present in the used XOS. XOS were fermented with high specificity by Bifidobacteria strains, but Lactobacilli did not metabolize XOS efficiently.