Arabinoxylooligosaccharides Research Articles

Arabinoxylo-oligosaccharides production from unexploited agro-industrial sesame (Sesamum indicum L.) hulls waste

This work demonstrates that sesame (Sesamum indicum L.) hull, an unexploited food industrial waste, can be used as an efficient source for the extraction of hemicellulose and/or pectin polysaccharides to further obtain functional oligosaccharides. Different polysaccharides extraction methods were surveyed including alkaline and several enzymatic treatments. Based on the enzymatic release of xylose, arabinose, glucose, and galacturonic acid from sesame hull by using different enzymes, Celluclast®1.5 L, Pectinex®Ultra SP-L, and a combination of them were selected for the enzymatic extraction of polysaccharides at 50 °C, pH 5 up to 24 h. Once the polysaccharides were extracted, Ultraflo®L was selected to produce arabinoxylo-oligosaccharides (AXOS) at 40 °C up to 24 h. Apart from oligosaccharides production from extracted polysaccharides, alternative approaches for obtaining oligosaccharides were also explored. These were based on the analysis of the supernatants resulting from the polysaccharide extraction, alongside a sequential hydrolysis performed with Celluclast®1.5 L and Ultraflo®L of the starting raw sesame hull. The different fractions obtained were comprehensively characterized by determining low molecular weight carbohydrates and monomeric compositions, average Mw and dispersity, and oligosaccharide structure by MALDI-TOF-MS. The results indicated that sesame hull can be a useful source for polysaccharides extraction (pectin and hemicellulose) and derived oligosaccharides, especially AXOS.

Structure and function characterization of the α-L-arabinofuranosidase from the white-rot fungus Trametes hirsuta

α-L-Arabinofuranosidases (Abfs) play a crucial role in the degradation of hemicelluloses, especially arabinoxylans (AX). Most of the available characterized Abfs are from bacteria, while fungi, as natural decomposers, contain Abfs with little attention given. An arabinofuranosidase (ThAbf1), belonging to the glycoside hydrolase 51 (GH51) family, from the genome of the white-rot fungus Trametes hirsuta, was recombinantly expressed, characterized, and functionally determined. The general biochemical properties showed that the optimal conditions for ThAbf1 were pH 6.0 and 50°C. In substrate kinetics assays, ThAbf1 preferred small fragment arabinoxylo-oligosaccharides (AXOS) and could surprisingly hydrolyze di-substituted 23,33-di-L-arabinofuranosyl-xylotriose (A2,3XX). It also synergized with commercial xylanase (XYL) and increased the saccharification efficiency of arabinoxylan. The crystal structure of ThAbf1 indicated the presence of an adjacent cavity next to the catalytic pocket which led to the ability of ThAbf1 to degrade di-substituted AXOS. The narrow binding pocket prevents ThAbf1 from binding larger substrates. These findings have strengthened our understanding of the catalytic mechanism of GH51 family Abfs and provided a theoretical foundation for the development of more efficient and versatile Abfs to accelerate the degradation and biotransformation of hemicellulose in biomass. KEY POINTS: • ThAbf1 from Trametes hirsuta degraded di-substituted arabinoxylo-oligosaccharide. • ThAbf1 performed detailed biochemical characterization and kinetics. • ThAbf1 structure has been obtained to illustrate the substrate specificity.

Arabinoxylan-based substrate preferences and predicted metabolic properties of Bifidobacterium longum subspecies as a basis to design differential media

Arabinoxylan (AX) and arabinoxylo-oligosaccharides (AXOS) derived therefrom are emergent prebiotics with promising health promoting properties, likely linked to its capacity to foster beneficial species in the human gut. Bifidobacteria appear to be one taxa that is frequently promoted following AX or AXOS consumption, and that is known to establish metabolic cross-feeding networks with other beneficial commensal species. Therefore, probiotic bifidobacteria with the capability to metabolize AX-derived prebiotics represent interesting candidates to develop novel probiotic and synbiotic combinations with AX-based prebiotics. In this work we have deepen into the metabolic capabilities of bifidobacteria related to AX and AXOS metabolization through a combination of in silico an in vitro tools. Both approaches revealed that Bifidobacterium longum and, particularly, B. longum subsp. longum, appears as the better equipped to metabolize complex AX substrates, although other related subspecies such as B. longum subsp. infantis, also hold some machinery related to AXOS metabolization. This correlates to the growth profiles exhibited by representative strains of both subspecies in AX or AXOS enriched media. Based on these results, we formulated a differential carbohydrate free medium (CFM) supplemented with a combination of AX and AXOS that enabled to recover a wide diversity of Bifidobacterium species from complex fecal samples, while allowing easy discrimination of AX metabolising strains by the appearance of a precipitation halo. This new media represent an appealing alternative to isolate novel probiotic bifidobacteria, rapidly discriminating their capacity to metabolize structurally complex AX-derived prebiotics. This can be convenient to assist formulation of novel functional foods and supplements, including bifidobacterial species with capacity to metabolize AX-derived prebiotic ingredients.

Insights into the xylan degradation system of Cellulomonas sp. B6: biochemical characterization of rCsXyn10A and rCsAbf62A.

Valorization of the hemicellulose fraction of plant biomass is crucial for the sustainability of lignocellulosic biorefineries. The Cellulomonas genus comprises Gram-positive Actinobacteria that degrade cellulose and other polysaccharides by secreting a complex array of enzymes. In this work, we studied the specificity and synergy of two enzymes, CsXyn10A and CsAbf62A, which were identified as highly abundant in the extracellular proteome of Cellulomonas sp. B6 when grown on wheat bran. To explore their potential for bioprocessing, the recombinant enzymes were expressed and their activities were thoroughly characterized. rCsXyn10A is a GH10 endo-xylanase (EC 3.2.1.8), active across a broad pH range (5 to 9), at temperatures up to 55°C. rCsAbf62A is an α-L-arabinofuranosidase (ABF) (EC 3.2.1.55) that specifically removes α-1,2 and α-1,3-L-arabinosyl substituents from arabino-xylo-oligosaccharides (AXOS), xylan, and arabinan backbones, but it cannot act on double-substituted residues. It also has activity on pNPA. No differences were observed regarding activity when CsAbf62A was expressed with its appended CBM13 module or only the catalytic domain. The amount of xylobiose released from either wheat arabinoxylan or arabino-xylo-oligosaccharides increased significantly when rCsXyn10A was supplemented with rCsAbf62A, indicating that the removal of arabinosyl residues by rCsAbf62A improved rCsXyn10A accessibility to β-1,4-xylose linkages, but no synergism was observed in the deconstruction of wheat bran. These results contribute to designing tailor-made, substrate-specific, enzymatic cocktails for xylan valorization. KEY POINTS: • rCsAbf62A removes α-1,2 and α-1,3-L-arabinosyl substituents from arabino-xylo-oligosaccharides, xylan, and arabinan backbones. • The appended CBM13 of rCsAbf62A did not affect the specific activity of the enzyme. • Supplementation of rCsXyn10A with rCsAbf62A improves the degradation of AXOS and xylan.

In vivo formation of arabinoxylo-oligosaccharides by dietary endo-xylanase alters arabinoxylan utilization in broilers

Previously, arabinoxylan (AX) depolymerization by dietary endo-xylanase was observed in the broiler ileum, but released arabinoxylo-oligosaccharides (AXOS) were not characterized in detail. This study aimed at extracting and identifying AXOS released in vivo in broilers, in order to delineate the influence of endo-xylanase on AX utilization. Hereto, digesta from the gizzard, ileum, ceca and excreta of broilers fed a wheat-soybean diet without (Con) or with endo-xylanase supplementation (Enz) were assessed. Soluble AX content in the ileum was higher for Enz diet (26.9%) than for Con diet (18.8%), indicating a different type and amount of AX entering the ceca. Removal of maltodextrins and fructans enabled monitoring of AX depolymerization to AXOS (Enz diet) using HPSEC-RI and HPAEC-PAD. A recently developed HILIC-MSn methodology allowed AXOS (DP 4–10) identification in ileal digesta and excreta. Xylanase-induced AXOS formation coincided with decreased total tract AX recovery, which indicated improved AX hindgut utilization.

Strategy to identify reduced arabinoxylo-oligosaccharides by HILIC-MSn

Identification of arabinoxylo-oligosaccharides (AXOS) within complex mixtures is an ongoing analytical challenge. Here, we established a strategy based on hydrophilic interaction chromatography coupled to collision induced dissociation-mass spectrometry (HILIC-MSn) to identify a variety of enzyme-derived AXOS structures. Oligosaccharide reduction with sodium borohydride remarkably improved chromatographic separation of isomers, and improved the recognition of oligosaccharide ends in MS-fragmentation patterns. Localization of arabinosyl substituents was facilitated by decreased intensity of Z ions relative to corresponding Y ions, when fragmentation occurred in the vicinity of substituents. Interestingly, the same B fragment ions (MS2) from HILIC-separated AXOS isomers showed distinct MS3 spectral fingerprints, being diagnostic for the linkage type of arabinosyl substituents. HILIC-MSn identification of AXOS was strengthened by using specific and well-characterized arabinofuranosidases. The detailed characterization of AXOS isomers currently achieved can be applied for studying AXOS functionality in complex (biological) matrices. Overall, the present strategy contributes to the comprehensive carbohydrate sequencing.

Two α-L-arabinofuranosidases from Bifidobacterium longum subsp. longum are involved in arabinoxylan utilization.

Arabinoxylan (AX) and arabinoxylooligosaccharides (AXOs) are carbohydrate sources utilized by Bifidobacterium longum subsp. longum. However, their degradation pathways are poorly understood. In this study, we characterized two genes, BLLJ_1850 and BLLJ_1851, in the hemicellulose-degrading gene cluster (BLLJ_1836-BLLJ_1859) of B. longum subsp. longum JCM 1217. Both recombinant enzymes expressed in Escherichia coli exhibited exo-α-L-arabinofuranosidase activity toward p-nitrophenyl-α-L-arabinofuranoside. BlArafE (encoded by BLLJ_1850) contains the glycoside hydrolase family 43 (GH43), subfamily 22 (GH43_22), and GH43_34 domains. The BlArafE GH43_22 domain was demonstrated to release α1,3-linked Araf from AX, but the function of BlArafE GH43_34 could not be clearly identified in this study. BlArafD (encoded by BLLJ_1851) contains GH43 unclassified subfamily (GH43_UC) and GH43_26 domains. The BlArafD GH43_UC domain showed specificity for α1,2-linked Araf in α1,2- and α1,3-Araf double-substituted structures in AXOs, while BlArafD GH43_26 was shown to hydrolyze α1,5-linked Araf in the arabinan backbone. Co-incubation of BlArafD and BlArafE revealed that these two enzymes sequentially removed α1,2-Araf and α1,3-Araf from double-substituted AXOs in this order. B. longum strain lacking BLLJ_1850-BLLJ_1853 did not grow in the medium containing α1,2/3-Araf double-substituted AXOs, suggesting that BlArafE and BlArafD are important for the assimilation of AX. KEY POINTS: • BlArafD GH43 unclassified subfamily domain is a novel α1,2-L-arabinofuranosidase. • BlArafE GH43 subfamily 22 domain is an α1,3-L-arabinofuranosidase. • BlArafD and BlArafE cooperatively degrade α1,2/3-Araf double-substituted arabinoxylan.

Hydrolysis of Arabinoxylo-oligosaccharides by α-L-Arabinofuranosidases and β-D-Xylosidase from Bifidobacterium dentium.

Two α-L-arabinofuranosidases (BfdABF1 and BfdABF3) and a β-D-xylosidase (BfdXYL2) genes were cloned from Bifidobacterium dentium ATCC 27679, and functionally expressed in E. coli BL21(DE3). BfdABF1 showed the highest activity in 50 mM sodium acetate buffer at pH 5.0 and 25°C. This exo-enzyme could hydrolyze p-nitrophenyl arabinofuranoside, arabino-oligosaccharides (AOS), arabinoxylo-oligosaccharides (AXOS) such as 32-α-L-arabinofuranosyl-xylobiose (A3X), and 23-α-Larabinofuranosyl-xylotriose (A2XX), whereas hardly hydrolyzed polymeric substrates such as debranched arabinan and arabinoxylans. BfdABF1 is a typical exo-ABF with the higher specific activity on the oligomeric substrates than the polymers. It prefers to α-(1,2)-L-arabinofuranosidic linkages compared to α-(1,3)-linkages. Especially, BfdABF1 could slowly hydrolyze 23,33-di-α-L-arabinofuranosyl-xylotriose (A2+3XX). Meanwhile, BfdABF3 showed the highest activity in sodium acetate at pH 6.0 and 50°C, and it has the exclusively high activities on AXOS such as A3X and A2XX. BfdABF3 mainly catalyzes the removal of L-arabinose side chains from various AXOS. BfdXYL2 exhibited the highest activity in sodium citrate at pH 5.0 and 55°C, and it specifically hydrolyzed p-nitrophenyl xylopyranoside and xylo-oligosaccharides (XOS). Also, BfdXYL2 could slowly hydrolyze AOS and AXOS such as A3X. Based on the detailed hydrolytic modes of action of three exo-hydrolases (BfdABF1, BfdABF3, and BfdXYL2) from Bf. dentium, their probable roles in the hemiceulloseutilization system of Bf. dentium are proposed in the present study. These intracellular exo-hydrolases can synergistically produce L-arabinose and D-xylose from various AOS, XOS, and AXOS.

Microwave-assisted enzymatic hydrolysis to produce xylooligosaccharides from rice husk alkali-soluble arabinoxylan

The prebiotic properties of xylooligosaccharides (XOS) and arabino-xylooligosaccharides (AXOS) produced from rice husk (RH) using microwave treatment combined with enzymatic hydrolysis were evaluated. The RH was subjected to microwave pretreatment at 140, 160 and 180 °C for 5, 10 and 15 min to obtain crude arabinoxylan (AX). Increasing microwave pretreatment time increased sugar content. Crude AX was extracted with 2% (w/v) sodium hydroxide at 25 °C for 24 h and used as a substrate for XOS production by commercial xylanases. Results showed that oligosaccharides produced by Pentopan Mono BG and Ultraflo Max provided xylobiose and xylotriose as the main products. AXOS was also present in the oligosaccharides that promoted growth of Lactobacillus spp. and resisted degradation by over 70% after exposure to simulated human digestion.

Enzymatic Production of Xylo-oligosaccharides from Destarched Wheat Bran and the Impact of Their Degree of Polymerization and Substituents on Their Utilization as a Carbon Source by Probiotic Bacteria.

The enzymatic production of xylo-oligosaccharides (XOs) from destarched wheat bran with a GH11 xylanase was studied. Xylo-oligosaccharides (XOs) produced were separated into different fractions according to their degree of polymerization (DP) and the nature of their substituents: arabinoxylo-oligosaccharides (AXOs) with a DP from 2 to 3 and DP from 2 to 6 and feruloylated arabinoxylo-oligosaccharides (FAXOs) esterified by ferulic and p-coumaric acids with a DP from 3 to 6. Both AXOs (short and long DP) and FAXOs stimulated the growth of Bifidobacterium adolescentis, Faecalibacterium prausnitzii, and Prevotella copri similarly but not Lactobacillus rhamnosus. The utilization of AXOs and FAXOs as a carbon source resulted in the increase in turbidity, decrease in pH, and production of short-chain fatty acids (SCFAs) in the culture broth. The highest amount of SCFAs was produced by F. prausnitzii using FAXOs. Results suggest that FAXOs and AXOs have the potential to be considered as prebiotics.

Interaction between xylanase and a proton pump inhibitor on broiler chicken performance and gut function.

Three hundred thirty-six Ross 308 male broiler chicks were used in a 21-d study to explore performance and gut function when treated with a proton pump inhibitor (PPI; 0 or 89 mg/kg) in a 2 × 2 factorial arrangement with a xylanase (Xyl; 0 or 0.1 g/kg) to determine if the beneficial activity of arabinoxylan (AX) depolymerisation, through arabinoxylo-oligosaccharides (AXOS) production, starts in the upper gastrointestinal tract. Treatment with the PPI started from d 14, and by d 21 animal performance had deteriorated (P < 0.001). An interaction was observed between PPI and Xyl for feed conversion ratio (FCR) (P < 0.05), whereby the combination reduced the negative effect of PPI on FCR. Application of PPI raised digesta pH in the gizzard and caecum (P < 0.05), increased protein concentrations in the lower gut (P < 0.05) and reduced intake of digestible nutrients (P < 0.05). Caecal concentrations of indole, p-cresol, ammonia and the ratio of total volatile fatty acid (VFA) to butyric acid were increased with PPI (P < 0.05), indicating enhanced protein fermentation. Xylanase activity in the digesta were greatest in the caeca, especially when Xyl was supplemented (P < 0.001). The concentration of total soluble AX was greater in the gizzard and ileal digesta with Xyl supplementation (P < 0.05), supporting the depolymerisation action of xylanase even under acidic conditions. These data suggest xylanase may function in the gizzard even though pH is not optimal for activity and emphasises the importance of chlorohydric acid secretions in ensuring overall optimum gut function. AX depolymerisation benefits animal performance although it is still unknown how the AXOS produced with xylanase supplementation in the upper gastrointestinal tract influence the microbial populations and overall gut functionality.

Endo-xylanases from Cohnella sp. AR92 aimed at xylan and arabinoxylan conversion into value-added products.

The genus Cohnella belongs to a group of Gram-positive endospore-forming bacteria within the Paenibacillaceae family. Although most species were described as xylanolytic bacteria, the literature still lacks some key information regarding their repertoire of xylan-degrading enzymes. The whole genome sequence of an isolated xylan-degrading bacterium Cohnella sp. strain AR92 was found to contain five genes encoding putative endo-1,4-β-xylanases, of which four were cloned, expressed, and characterized to better understand the contribution of the individual endo-xylanases to the overall xylanolytic properties of strain AR92. Three of the enzymes, CoXyn10A, CoXyn10C, and CoXyn11A, were shown to be effective at hydrolyzing xylans-derived from agro-industrial, producing oligosaccharides with substrate conversion values of 32.5%, 24.7%, and 10.6%, respectively, using sugarcane bagasse glucuronoarabinoxylan and of 29.9%, 19.1%, and 8.0%, respectively, using wheat bran-derived arabinoxylan. The main reaction products from GH10 enzymes were xylobiose and xylotriose, whereas CoXyn11A produced mostly xylooligosaccharides (XOS) with 2 to 5 units of xylose, often substituted, resulting in potentially prebiotic arabinoxylooligosaccharides (AXOS). The endo-xylanases assay displayed operational features (temperature optima from 49.9 to 50.4°C and pH optima from 6.01 to 6.31) fitting simultaneous xylan utilization. Homology modeling confirmed the typical folds of the GH10 and GH11 enzymes, substrate docking studies allowed the prediction of subsites (- 2 to + 1 in GH10 and - 3 to + 1 in GH11) and identification of residues involved in ligand interactions, supporting the experimental data. Overall, the Cohnella sp. AR92 endo-xylanases presented significant potential for enzymatic conversion of agro-industrial by-products into high-value products.Key points• Cohnella sp. AR92 genome encoded five potential endo-xylanases.• Cohnella sp. AR92 enzymes produced xylooligosaccharides from xylan, with high yields.• GH10 enzymes from Cohnella sp. AR92 are responsible for the production of X2 and X3 oligosaccharides.• GH11 from Cohnella sp. AR92 contributes to the overall xylan degradation by producing substituted oligosaccharides.

Hemicellulose-Derived Oligosaccharides: Emerging Prebiotics in Disease Alleviation.

The gut microbiota in the human body is an important component that plays a pivotal role in the ability of the host to prevent diseases and recover from these diseases. If the human microbiome changes for any reason, it affects the overall functioning of the host. Healthy and vigorous gut microbiota require dietary fiber supplementation. Recently, oligosaccharides have been found to play a significant role in the modulation of microbiota. Several such oligosaccharides, i.e., xylooligosaccharides (XOS), mannooligosaccharides (MOS), and arabino-xylooligosaccharides (AXOS), are derived from hemicellulosic macromolecules such as xylan, mannan, and arabino-xylan, respectively. These oligosaccharides serve as substrates for the probiotic production of health-promoting substances (short-chain fatty acids, branched chain amino acids etc.), which confer a variety of health benefits, including the prevention of some dreaded diseases. Among hemicellulose-derived oligosaccharides (HDOs), XOS have been largely explored, whereas, studies on MOS and AXOS are currently underway. HDOs, upon ingestion, help reduce morbidities by lowering populations of harmful or pathogenic bacteria. The ATP-binding cassette (ABC) transporters are mainly utilized for the uptake of oligosaccharides in probiotics. Butyrate generated by the selective fermentation of oligosaccharides, along with other short-chain fatty acids, reduces gut inflammation. Overall, oligosaccharides derived from hemicelluloses show a similar potential as conventional prebiotics and can be supplemented as functional foods. This review summarizes the role of HDOs in the alleviation of autoimmune diseases (inflammatory bowel disease, Crohn's disease), diabetes, urinary tract infection, cardiovascular diseases, and antimicrobial resistance (AMR) through the modulation of the gut microbiota. The mechanism of oligosaccharide utilization and disease mitigation is also explained.

Arabinoxylo- and Arabino-Oligosaccharides-Specific α-L-Arabinofuranosidase GH51 Isozymes from the Amylolytic Yeast Saccharomycopsis fibuligera.

Two genes encoding probable α-L-arabinofuranosidase (E.C. 3.2.1.55) isozymes (ABFs) with 92.3% amino acid sequence identity, ABF51A and ABF51B, were found from chromosomes 3 and 5 of Saccharomycopsis fibuligera KJJ81, an amylolytic yeast isolated from Korean wheat-based nuruk, respectively. Each open reading frame consists of 1,551 nucleotides and encodes a protein of 517 amino acids with the molecular mass of approximately 59 kDa. These isozymes share approximately 49% amino acid sequence identity with eukaryotic ABFs from filamentous fungi. The corresponding genes were cloned, functionally expressed, and purified from Escherichia coli. SfABF51A and SfABF51B showed the highest activities on p-nitrophenyl arabinofuranoside at 40~45°C and pH 7.0 in sodium phosphate buffer and at 50°C and pH 6.0 in sodium acetate buffer, respectively. These exo-acting enzymes belonging to the glycoside hydrolase (GH) family 51 could hydrolyze arabinoxylo-oligosaccharides (AXOS) and arabino-oligosaccharides (AOS) to produce only L-arabinose, whereas they could hardly degrade any polymeric substrates including arabinans and arabinoxylans. The detailed product analyses revealed that both SfABF51 isozymes can catalyze the versatile hydrolysis of α-(1,2)-and α-(1,3)-L-arabinofuranosidic linkages of AXOS, and α-(1,2)-, α-(1,3)-, and α-(1,5)-linkages of linear and branched AOS. On the contrary, they have much lower activity against the α-(1,2)-and α-(1,3)-double-substituted substrates than the single-substituted ones. These hydrolases could potentially play important roles in the degradation and utilization of hemicellulosic biomass by S. fibuligera.

Enzyme synergy for the production of arabinoxylo-oligosaccharides from highly substituted arabinoxylan and evaluation of their prebiotic potential

Wheat bran arabinoxylan can be converted by enzymatic hydrolysis into short arabinoxylo-oligosaccharides (AXOS) with prebiotic potential. Alkali extraction of arabinoxylan from wheat-bran offers advantages in terms of yield and results in arabinoxylan with highly-substituted regions which has been a challenge to hydrolyse using endoxylanases. We show that this hurdle can be overcome by selecting an arabinoxylanase that attacks these regions. The yield of AXOS can be increased by enzyme synergy, involving the hydrolysis of some arabinoxylan side groups. Thus, arabinoxylanase (CtXyl5At) from Clostridium thermocellum, belonging to subfamily 34 of glycoside hydrolase (GH) family 5 was investigated pertaining to its specificity for highly-substituted regions in the arabinoxylan-backbone. CtXyl5At preferentially hydrolysed the water-soluble fraction of alkali-extracted arabinoxylan. AXOS with DP 2–4 were determined as major products from CtXyl5At catalyzed hydrolysis. Increase in AXOS yield was observed with enzyme synergy, involving an initial treatment of soluble arabinoxylan with a GH43 α-l-arabinofuranosidase from Bifidobacterium adolescentis termed BaAXHd3 (30 °C, 6h), followed by hydrolysis with CtXyl5At (50 °C, 24h). The prebiotic potential of AXOS was shown by growth analysis using the human gut bacteria Bifidobacterium adolescentis ATCC 15703 and Roseburia hominis DSM 6839. Importantly, AXOS were utilized by the bacteria and short-chain fatty acids were produced.

Structural features, interaction with the gut microbiota and anti-tumor activity of oligosaccharides.

Some oligosaccharides are regarded as biological constituents with benefits to human health in an indirect way. They enter the intestinal tract to be fermented by the gut microbiota, causing changes in the abundance and composition of the gut microbiota and producing fermentation products such as short-chain fatty acids (SCFAs). In this review, the structural features and biological activities of eight common natural oligosaccharides were summarized, including human milk oligosaccharides (HMOS), xylo-oligosaccharides (XOS), arabinoxylo-oligosaccharides (AXOS), isomaltooligosaccharides (IMOS), chitin oligosaccharides (NACOS), mannan-oligosaccharides (MOS), galacto-oligosaccharides (GOS) and fructo-oligosaccharides (FOS). Furthermore, XOS were selected to explain the anti-tumor mechanism mediated by gut microbiota. The review aims to reveal primary structural features of natural functional oligosaccharides related to the biological activities and also provide an explanation of the anti-tumor activity of functional oligosaccharides mediated by the gut microbiota.

Differential antioxidant activity of glucuronoxylooligosaccharides (UXOS) and arabinoxylooligosaccharides (AXOS) produced by two novel xylanases

XOS are particularly interesting bioactive molecules. Bacillus safensis CBLMA18, a xylanolytic bacterium has been isolated and two of its xylanases have been identified and fully characterized. Xyn11A is an extracellular 22.5-kDa GH11 xylanase while a second xylanase, Xyn10B, corresponds to an intracellular 48-kDa GH10 enzyme. Both unimodular xylanases showed activity only on xylan substrates with important differences in their catalytic pattern. Xyn11A displays higher activity on glucuronoxylans, with an optimum at pH 8 and 50 °C, and a Vmax of 5281 U/mg on beechwood xylan, meanwhile Xyn10B prefers arabinoxylans, with an optimum of pH 7 and 60 °C, and a Vmax of 50.29 U/mg on rye arabinoxylan. The antioxidant activity of xylanase-generated XOS obtained from glucuronoxylans (UXOS) and arabinoxylans (AXOS) was tested with the ABTS (2, 2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) method. UXOS showed higher antioxidant activity than AXOS (>80% of antioxidant capacity). Thin layer chromatography and MALDI-TOF MS analysis showed that UXOS comprise neutral and acidic XOS with methylglucuronic acid (MeGlcA) ramifications, while AXOS contain only neutral molecules with arabinose decorations. The MeGlcA ramifications seem to have an important role in the antioxidant capacity of oligosaccharides. Besides, the increase of UXOS size correlates with an increase in their activity.

Oligosaccharides Preparation from Rice Bran Arabinoxylan by Two Different Commercial Endoxylanase Enzymes.

Xylooligosaccharides (XOS) and arabinoxylooligosaccharides (AXOS), potential prebiotics, can be produced from rice bran arabinoxylan (RBAX) using commercial endoxylanase enzymes. However, differences in rice bran cultivars and endoxylanase enzyme types may affect extracted oligosaccharides (OS) yields. This study investigated extracted OS structures derived from three different RBAX using two commercial endoxylanase enzymes. RBAX extracted from commercially defatted rice bran (CDRB) yielded the greatest OS amount (456.69 mg/g RBAX), followed by that of the San-Pah-Tawng1 (SPT1) cultivar (231.7 mg/g RBAX), and lastly, the Chai-Nat1 (CN1) cultivar (172.57 mg/g RBAX), as evidenced via Ultraflo L enzyme hydrolysis. Ultraflo Max enzyme hydrolysis produced a similar trend, however, OS amounts from all RBAX sources were remarkably lower (83.39 mg/g RBAX extracted from CDRB, 27.05 mg/g RBAX from SPT1 cultivar, and 21.53 mg/g RBAX from CN1 cultivar). Interestingly, 32-α-L-Araf-(1-4)-β-D-xylobiose (A3X) was the primary AXOS product in all RBAX hydrolysates prepared by Ultraflo Max. RBAX extracted from CDRB solubilized by Ultraflo L (45.67% weight of RBAX) had higher OS yields than that obtained via Ultraflo Max (8.3% weight of RBAX). Ultraflo L was therefore a suitable commercial enzyme for short-chain OS conversion from RBAX.

Structure driven immunomodulatory properties of xylo-/arabinoxylo-oligosaccharides and arabinoxylans in RAW264.7 macrophages

Cereal derived arabinoxylans (AX) have been shown to possess immunomodulatory properties. However, AX are complex molecules that can exist with different degree of polymerization (DP) and degree of arabinose substitution (DS). We investigated the effect of these structural differences on the immunomodulatory property of xylo-oligosaccharides (XOS), arabinoxylo-oligosaccharides (AXOS) and AX polysaccharides. We investigated the effect of these substrates on RAW264.7 cells with respect to nitric oxide (NO) and TNF-α production. XOS did not display an immunomodulatory activity with respect to NO production except for X3, which decreased NO production compared to control. AXOS which contained arabinose substitution were better capable of decreasing NO production in macrophages. AX polysaccharides on the other hand were inducers of NO production acting as inmmunostimulators. High molecular weight AX polysaccharide (AX H) was immunostimulators with respect to TNF-α production as well. AX H also displayed a dose dependent relationship with TNF-α production in RAW264.7 cells. These results suggest that exploring high molecular weight AX polysaccharides as immunomodulators could be beneficial compared to smaller molecular weight unsubstituted oligosaccharides.

Single-step production of arabino-xylooligosaccharides by recombinant Bacillus subtilis 3610 cultivated in brewers’ spent grain

Brewers’ spent grain (BSG) is an inexpensive and abundant brewery by-product that can be used to produce prebiotic arabino-xylooligosaccharides (AXOS). In this study, Bacillus subtilis 3610 was used, for the first time, to produce AXOS through direct fermentation of BSG. Additionally, the microorganism was genetically modified to improve the AXOS production. The xylanase gene xyn2 from Trichoderma reesei coupled with a secretion tag endogenous to B. subtilis was cloned in pDR111 and integrated into its chromosome. After optimization by experimental design, AXOS with a degree of polymerization ranging from 2 to 6 were obtained. The maximum production yield expressed in xylose equivalents per amount of BSG (54.2 ± 1.1 mg/g) represents an increase of 33% comparing to the wild type. When compared with the enzymatic hydrolysis process, single-step fermentation with B. subtilis proved to be a very promising low-cost strategy for the simultaneous production of AXOS and valorization of BSG.