β-Glucan (Barley; Low Viscosity)

Fermentation of β-glucan fractions from barley [average molecular mass (MM), of 243, 172, and 137 kDa] and oats (average MM of 230 and 150 kDa) by the human faecal microbiota was investigated. Fractions were supplemented to pH-controlled anaerobic batch culture fermenters inoculated with human faecal samples from three donors, in triplicate, for each substrate. Microbiota changes were monitored by fluorescent in situ hybridization; groups enumerated were: Bifidobacterium genus, Bacteroides and Prevotella group, Clostridium histolyticum subgroup, Ruminococcus-Eubacterium-Clostridium (REC) cluster, Lactobacillus-Enterococcus group, Atopobium cluster, and clostridial cluster IX. Short-chain fatty acids and lactic acid were measured by HPLC. The C. histolyticum subgroup increased significantly in all vessels and clostridial cluster IX maintained high populations with all fractions. The Bacteroides-Prevotella group increased with all but the 243-kDa barley and 230-kDa oat substrates. In general β-glucans displayed no apparent prebiotic potential. The SCFA profile (51 : 32 : 17; acetate : propionate : butyrate) was considered propionate-rich. In a further study a β-glucan oligosaccharide fraction was produced with a degree of polymerization of 3-4. This fraction was supplemented to small-scale faecal batch cultures and gave significant increases in the Lactobacillus-Enterococcus group; however, the prebiotic potential of this fraction was marginal compared with that of inulin.

A commercially available enzymic method for the quantitative measurement of (1→3),(1→4)-β-glucan has been simplified to allow analysis of up to 10 grain samples in 70 min or of 100–200 samples by a single operator in a day. These improvements have been achieved with no loss in accuracy or precision and with an increase in reliability. The glucose oxidase/peroxidase reagent has been significantly improved to ensure colour stability for periods of up to 1 h after development. Some problems experienced with the original method have been addressed and resolved, and further experiments to demonstrate the quantitative nature of the assay have been designed and performed.

A method developed for the quantification of (1→3)(1→4)-β-D-glucan in barley flour has been modified to allow its use in the measurement of this component in malt, wort, beer and spent grain. For malt samples, free D-glucose was first removed with aqueous ethanol. Quantification of the polymer in wort and beer samples involved precipitation of the β-glucan with ammonium sulphate followed by washing with aqueous ethanol to remove free D-glucose. Spent grain was lyophilised and milled and then analysed by the method developed for malt. In all cases, the β-glucan was depolymerised with lichenase and the resultant β-gluco-oligosaccharides hydrolysed to D-glucose with β-D-glucosidase. The released D-glucose was then specifically determined using glucose oxidase-peroxidase reagent.

Mixed linkage β-glucane and pentosanes (mainly arabinoxylanes) are the major endosperm cell-wall polysaccharides of barley and wheat respectively. These polysaccharides, although minor components of the whole grain, significantly affect the industrial utilization of these cereals. The modification of barley corns during malting requires the dissolution of the β-glucan in the cell-wall of the starch endosperm. High β-glucane concentration in wort and beer effect the rate of filtration and can also lead to precipitate or gel formation in the final product. In a similar manner, pentosane is thought to cause filtration problems with wheat starch hydrolysates by increasing viscosity and by producing gelatinous precipitate which blocks filters. Ironically, it is this same viscosity building and water binding capacity which is considered to render pentosanes of considerable value in dough development and bread storage (anti-staling functions). In the current paper, some aspects of the beneficial and detrimental effects of pentosans and β-glucan in the industrial utilization of wheat and barley are discussed. More specifically, enzymic methods for the preparation, analysis and identification of these polysaccharides and for the removal of their functional properties, are described in detail.

A simple and quantitative method for the determination of (1→3) (1→4)-β-D-glucan in barley flour and malt is described. The method allows direct analysis of β-glucan in flour and malt slurries. Mixed-linkage β-glucan is specifically depolymerized with a highly purified (1→3) (1→4)-β-D-glucanase (lichenase), from Bacillus subtilis, to tri-, tetra- and higher degree of polymerization (d.p.) oligosaccharides. These oligosaccharides are then specifically and quantitatively hydrolysed to glucose using purified β-D-glucosidase. The glucose is then specifically determined using glucose oxidase/peroxidase reagent. Since barley flours contain only low levels of glucose, and maltosaccharides do not interfere with the assay, removal of low d.p. sugars is not necessary. Blank values are determined for each sample allowing the direct measurement of β-glucan in values are determined for each sample allowing the direct measurement of β-glucan in malt samples. α-Amylase does not interfere with the assay. The method is suitable for the routine analysis of β-glucan in barley samples derived from breeding programs; 50 samples can be analysed by a single operator in a day. Evaluation of the technique on different days has indicated a mean standard error of 0-1 for barley flour samples containing 3-8 and 4-6% (w/w) β-glucan content.

Endo-β-1,4-glucanase is a crucial glycoside hydrolase (GH) involved in the decomposition of cellulosic materials. In this study, to discover a novel cold-adapted β-1,4-d-glucan-degrading enzyme, the gene coding for an extracellular endo-β-1,4-glucanase (GluL) from Lichenicola cladoniae PAMC 26568, an Antarctic lichen (Cladonia borealis)-associated bacterium, was identified and recombinantly expressed in Escherichia coli BL21. The GluL gene (1044-bp) encoded a non-modular polypeptide consisting of a single catalytic GH8 domain, which shared the highest sequence identity of 55% with that of an uncharacterized protein from Gluconacetobacter takamatsuzukensis (WP_182950054). The recombinant endo-β-1,4-glucanase (rGluL: 38.0 kDa) most efficiently degraded sodium carboxymethylcellulose (CMC) at pH 4.0 and 45°C, and showed approximately 23% of its maximum degradation activity even at 3°C. The biocatalytic activity of rGluL was noticeably enhanced by >1.3-fold in the presence of 1 mM Mn²⁺ or NaCl at concentrations between 0.1 and 0.5 M, whereas the enzyme was considerably downregulated by 1 mM Hg²⁺ and Fe²⁺ together with 5 mM N-bromosuccinimide and 0.5% sodium dodecyl sulfate. rGluL is a true endo-β-1,4-glucanase, which could preferentially decompose d-cellooligosaccharides consisting of 3 to 6 d-glucose, CMC, and barley β-glucan, without other additional glycoside hydrolase activities. The specific activity (15.1 U mg^–1) and k_cat/K_m value (6.35 mg^–1 s^–1mL) of rGluL toward barley β-glucan were approximately 1.8- and 2.2-fold higher, respectively, compared to its specific activity (8.3 U mg^–1) and k_cat/K_m value (2.83 mg^–1 s^–1mL) toward CMC. The enzymatic hydrolysis of CMC, d-cellotetraose, and d-cellohexaose yielded primarily d-cellobiose, accompanied by d-glucose, d-cellotriose, and d-cellotetraose. However, the cleavage of d-cellopentaose by rGluL resulted in the production of only d-cellobiose and d-cellotriose. The findings of the present study imply that rGluL is a novel, acidic, and cold-adapted GH8 endo-β-1,4-glucanase with high specific activity, which can be exploited as a promising candidate in low-temperature processes including textile and food processes.

A novel membraneless β-glucan/O₂ enzymatic fuel cell was developed by combining a bioanode based on buckypaper modified with co-immobilized Agaricus meleagris pyranose dehydrogenase (AmPDH) and Rhodothermus marinus β-glucosidase (RmBgl3B) (RmBgl3B-AmPDH/buckypaper) with a biocathode based on solid graphite modified with Myrothecium verrucaria bilirubin oxidase (MvBOx/graphite). AmPDH was connected electrochemically with the buckypaper using an osmium redox polymer in a mediated reaction, whereas MvBOx was connected with graphite in a direct electron transfer reaction. The fuel for the bioanode was produced by enzymatic hydrolysis of β-glucan by the exoglucanase RmBgl3B into d-glucose, which in turn was enzymatically oxidised by AmPDH to generate a current response. This design allows to obtain an efficient enzymatic fuel cell, where the chemical energy converted into electrical energy is higher than the chemical energy stored in complex carbohydrate based fuel. The maximum power density of the assembled β-glucan/O₂ biofuel cell reached 26.3 ± 4.6 μWcm⁻² at 0.36 V in phosphate buffer containing 0.5 % (w/v) β-glucan at 40°C with excellent stability retaining 68.6 % of its initial performance after 5 days. The result confirms that β-glucan can be employed as fuel in an enzymatic biofuel cell.

A multigene polysaccharide utilization locus (PUL) encoding enzymes and surface carbohydrate (glycan)-binding proteins (SGBPs) was recently identified in prominent members of Bacteroidetes in the human gut and characterized in Bacteroides ovatus. This PUL-encoded system specifically targets mixed-linkage β1,3-1,4-glucans, a group of diet-derived carbohydrates that promote a healthy microbiota and have potential as prebiotics. The BoSGBP_MLG-A protein encoded by the BACOVA_2743 gene is a SusD-like protein that plays a key role in the PUL's specificity and functionality. Here, we perform a detailed analysis of the molecular determinants underlying carbohydrate binding by BoSGBP_MLG-A, combining carbohydrate microarray technology with quantitative affinity studies and a high-resolution X-ray crystallography structure of the complex of BoSGBP_MLG-A with a β1,3-1,4-nonasaccharide. We demonstrate its unique binding specificity toward β1,3-1,4-gluco-oligosaccharides, with increasing binding affinities up to the octasaccharide and dependency on the number and position of β1,3 linkages. The interaction is defined by a 41-Å-long extended binding site that accommodates the oligosaccharide in a mode distinct from that of previously described bacterial β1,3-1,4-glucan-binding proteins. In addition to the shape complementarity mediated by CH-π interactions, a complex hydrogen bonding network complemented by a high number of key ordered water molecules establishes additional specific interactions with the oligosaccharide. These support the twisted conformation of the β-glucan backbone imposed by the β1,3 linkages and explain the dependency on the oligosaccharide chain length. We propose that the specificity of the PUL conferred by BoSGBP_MLG-A to import long β1,3-1,4-glucan oligosaccharides to the bacterial periplasm allows Bacteroidetes to outcompete bacteria that lack this PUL for utilization of β1,3-1,4-glucans. 

We proposed a framework with parametric modeling to obtain biological relevant parameters from the total probiotic growth pattern and metabolite production curves. The lag phase, maximum increase rate, and maximum capacity were obtained via a 205-h exploratory In vitro fermentation of a library of 13 structural-characterized prebiotic candidates against an exclusively breastfed infant fecal inoculum. We also conducted 16S rRNA amplicon sequencing of the infant fecal inoculum. Moreover, we introduce a robust composite metabolite-based indicator that reflects the eubiosis/dysbiosis of microbiota to complement the conventional microbial markers. In terms of short-chain fatty acid, we discovered that polymeric beta-glucans from barley demonstrated potential as prebiotic candidates, while alpha-glucans as glycogen showed the least dissolved ammonia production. In terms of total probiotic, beta-glucans from oat and mushroom sclerotia of Pleurotus tuber-regium showed comparable sustainability when compared to alpha-glucans after 48 h. Being classical prebiotic, galacto-oligosaccharides gave the second-highest metabolite-based indicator, followed by lactose. While limited improvement could be made to lactose and oligosaccharides, polymeric beta-glucans from barley avails more capacity for novel prebiotic development, such as structural modification. We anticipate that more similar parallel screening with the element of time and parametric modeling will provide more novel insights

Microorganisms residing within a biofilm become more tolerant to antibiotics and other types of adverse impact, and biofilm formation by pathogenic bacteria is an important problem of current medicine. Polysaccharides that prevent biofilm formation are among the promising candidates to help tackle this problem. Earlier we demonstrated the ability of a potato polysaccharide galactan to inhibit biofilm formation by a Pseudomonas aeruginosa clinical isolate. Here we investigate the effect of potato galactan on P. aeruginosa biofilms in more detail. Microscopic analysis indicated that the galactan did not interfere with the adhesion of bacterial cells to the substrate but prevented the build-up of bacterial biomass. Moreover, the galactan not only inhibited biofilm formation, but partially destroyed pre-formed biofilms. Presumably, this activity of the galactan was due to the excessive aggregation of bacterial cells, which prohibited the formation and maintenance of proper biofilm architecture, or due to some other mechanisms of biofilm structure remodeling. This led to an unexpected effect, i.e., P. aeruginosa biofilms treated with an antibiotic and the galactan retained more viable bacterial cells compared to biofilms treated with the antibiotic alone. Galactan is the first polysaccharide demonstrated to exert such effect on bacterial biofilms.

The sco6546 gene of Streptomyces coelicolor A3(2) was annotated as a putative glycosyl hydrolase belonging to family 48. It is predicted to encode a 973-amino acid polypeptide (103.4 kDa) with a 39-amino acid secretion signal. Here, the SCO6546 protein was overexpressed in Streptomyces lividans TK24, and the purified protein showed the expected molecular weight of the mature secreted form (934 aa, 99.4 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. SCO6546 showed high activity toward Avicel and carboxymethyl cellulose, but low activity toward filter paper and β-glucan. SCO6546 showed maximum cellulase activity toward Avicel at pH 5.0 and 50°C, which is similar to the conditions for maximum activity toward cellotetraose and cellopentaose substrates. The kinetic parameters k_cat and K_M, for cellotetraose at pH 5.0 and 50°C were 13.3 s^-1 and 2.7 mM, respectively. Thin layer chromatography (TLC) of the Avicel hydrolyzed products generated by SCO6546 showed cellobiose only, which was confirmed by mass spectral analysis. TLC analysis of the cello-oligosaccharide and chromogenic substrate hydrolysates generated by SCO6546 revealed that it can hydrolyze cellodextrins mainly from the non-reducing end into cellobiose. These data clearly demonstrated that SCO6546 is an exo-β-1,4-cellobiohydrolase (EC 3.2.1.91), acting on nonreducing end of cellulose.

The aim of this study was to evaluate the effects of dietary levels of xylanase on production performance, egg quality, nutrient digestibility, and excreta microbiota shedding of laying hens in a 12-week trial. Two-hundred-forty Hy-Line brown laying hens (44 wk old) were distributed according to a randomized block experimental design into one of 4 dietary treatments with 10 replicates of 6 birds each. The 4 dietary treatments were corn-soybean-meal-wheat-based diets supplemented with 0, 225, 450, or 900 U/kg xylanase. Daily feed intake, egg production, egg weight, egg mass, feed conversion ratio, and damaged egg rate showed no significant response to increasing xylanase supplementation during any phase (P > 0.05). No significant responses were observed for apparent total tract digestibility of dry matter, nitrogen, or gross energy (P > 0.05). A significant linear increase to increasing xylanase supplementation was seen for lactic acid bacteria numbers, although coliforms and Salmonella counts were not affected. Increasing the dietary xylanase resulted in a significant linear increase in eggshell thickness in wk 3, 6, 9, and 12 (P < 0.05). In addition, a significant linear increase occurred for Haugh unit and albumen height in wk 12 (P < 0.05). In summary, the inclusion of xylanase in corn-soybean-meal-wheat-based diets increased eggshell thickness, Haugh unit, albumen height, and excreta lactic acid bacteria count but had no effect on production performance or nutrient digestibility.

Biological hydrolysis of microcrystalline cellulose is an uncommon feature in the microbial world, especially among bacteria and archaea growing optimally above 70°C (the so‐called extreme thermophiles). In fact, among this group only certain species in the genus Caldicellulosiruptor are capable of rapid and extensive cellulose degradation. Four novel multi‐domain glycoside hydrolases (GHs) from Caldicellulosiruptor morganii and Caldicellulosiruptor danielii were produced recombinantly in Caldicellulosiruptor bescii and characterized. These GHs are structurally organized with two or three catalytic domains flanking carbohydrate binding modules from Family 3 (CBM3). Collectively, these enzymes represent GH families 5, 9, 10, 12, 44, 48, and 74, and hydrolyze crystalline cellulose, glucan, xylan, and mannan, the primary carbohydrates in plant biomass. Degradation of microcrystalline cellulose by cocktails of GHs from three Caldicellulosiruptor species demonstrated that synergistic interactions enable mixtures of multiple enzymes to outperform single enzymes, suggesting a community mode of action for lignocellulose utilization in thermal environments.

Scope: This study simulates the fermentation process of barley β-glucan and sugar beet pectin in the human colon and monitors the degradation products formed. Additionally, immune effects of the degradation products were investigated. Methods and results: 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. Conclusions: 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.

Prebiotics are being added to infant formula in order to mimic the effects of human milk oligosaccharides (HMOs) for the growth of probiotic bacteria especially bifidobacteria and lactobacilli in the infant gut. This preliminary study compares the in vitro fermentation of 13 different carbohydrates including monosaccharides, disaccharides, oligosaccharides and polysaccharides by infant fecal samples collected from 3-month old breast-milk fed babies. The growth of the total anaerobic bacteria and two probiotic bacteria (bifidobacteria and lactobacilli) during the fermentation period was measured by total plate count (TPC) and was expressed as colony forming units (CFUs). Among other things, beta-glucans seem to selectively enhance the growth of lactobacilli for a longer period of fermentation time than most of the carbohydrates tested. The selective enrichment of the probiotic bacteria by these carbohydrates and their potential use as prebiotics in the infant formula are discussed.

Beer contains low-molecular-weight β-linked oligosaccharides that originate from the degradation of β-glucan in the barley cell wall during malting and mashing. Over 90% of these oligosaccharides contain three or four glucosyl units. They remain intact through a static oral, gastric and small intestinal in vitro human digestive system model, indicating that they should be available to beneficial organisms known to be present in the human large intestine. Several intestine-associated Lactobacillus strains were shown to be capable of growth on these β-linked oligosaccharides, thereby leading us to tentatively propose that these compounds may represent prebiotics.

Deconstruction of cellulose, the most abundant plant cell wall polysaccharide, requires the cooperative activity of a large repertoire of microbial enzymes. Modular cellulases contain non-catalytic type A carbohydrate-binding modules (CBMs) that specifically bind to the crystalline regions of cellulose, thus promoting enzyme efficacy through proximity and targeting effects. Although type A CBMs play a critical role in cellulose recycling, their mechanism of action remains poorly understood. Here we produced a library of recombinant CBMs representative of the known diversity of type A modules. The binding properties of 40 CBMs, in fusion with an N-terminal GFP domain, revealed that type A CBMs possess the ability to recognize different crystalline forms of cellulose and chitin over a wide range of temperatures, pH levels, and ionic strengths. A Spirochaeta thermophila CBM64, in particular, displayed plasticity in its capacity to bind both crystalline and soluble carbohydrates under a wide range of extreme conditions. The structure of S. thermophila StCBM64C revealed an untwisted, flat, carbohydrate-binding interface comprising the side chains of four tryptophan residues in a co-planar linear arrangement. Significantly, two highly conserved asparagine side chains, each one located between two tryptophan residues, are critical to insoluble and soluble glucan recognition but not to bind xyloglucan. Thus, CBM64 compact structure and its extended and versatile ligand interacting platform illustrate how type A CBMs target their appended plant cell wall-degrading enzymes to a diversity of recalcitrant carbohydrates under a wide range of environmental conditions.

A set of fungal polysaccharide samples was characterised by elemental analysis and FTIR spectroscopy and compared with reference chitins, chitosans and β-D-glucans. The nitrogen to carbon (N/C) values and FTIR spectra were used to compare the samples based on their composition. It was found that the N/C ratio correlates well with deacetylation degree (DD) of chitosans and chitin/glucan ratio R_chit of fungal chitin – β-D-glucan complexes with the exception of some samples having significant nitrogen and/or carbon admixtures. FTIR spectroscopy was indicative for the N-acetylation of chitins (chitosans) as well as for the chitin (chitosan) contribution to fungal polysaccharide preparations. Multivariate analyses of the FTIR data (HCA, PCA) discriminated samples and reference materials into several clusters depending on their similarity. Chitosan lactates, chitosan – β-D-glucans and chitin – β-D-glucans of high and low amounts of chitin were successfully discriminated from the reference polysaccharides and from each other. The proposed procedures based on the N/C ratio and multivariate analyses of FTIR spectra may be used in screening fungal polysaccharide preparations.

The recently isolated bacterial strain 80/3 represents one of the most abundant 16S rRNA phylotypes detected in the healthy human large intestine and belongs to the Ruminococcaceae family of Firmicutes. The completed genome sequence reported here is the first for a member of this important family of bacteria from the human colon. The genome comprises two large chromosomes of 2.24 and 0.73 Mbp, leading us to propose the name Ruminococcus bicirculans for this new species. Analysis of the carbohydrate active enzyme complement suggests an ability to utilize certain hemicelluloses, especially β-glucans and xyloglucan, for growth that was confirmed experimentally. The enzymatic machinery enabling the degradation of cellulose and xylan by related cellulolytic ruminococci is however lacking in this species. While the genome indicated the capacity to synthesize purines, pyrimidines and all 20 amino acids, only genes for the synthesis of nicotinate, NAD⁺, NADP⁺ and coenzyme A were detected among the essential vitamins and co-factors, resulting in multiple growth requirements. In vivo, these growth factors must be supplied from the diet, host or other gut microorganisms. Other features of ecological interest include two type IV pilins, multiple extracytoplasmic function-sigma factors, a urease and a bile salt hydrolase.

Components of modular cellulases, type-A cellulose-binding modules (CBMs) bind to crystalline cellulose and enhance enzyme effectiveness, but structural details of the interaction are uncertain. We analyzed cellulose binding by EXLX1, a bacterial expansin with ability to loosen plant cell walls and whose domain D2 has type-A CBM characteristics. EXLX1 strongly binds to crystalline cellulose via D2, whereas its affinity for soluble cellooligosaccharides is weak. Calorimetry indicated cellulose binding was largely entropically driven. We solved the crystal structures of EXLX1 complexed with cellulose-like oligosaccharides to find that EXLX1 binds the ligands through hydrophobic interactions of three linearly arranged aromatic residues in D2. The crystal structures revealed a unique form of ligand-mediated dimerization, with the oligosaccharide sandwiched between two D2 domains in opposite polarity. This report clarifies the molecular target of expansin and the specific molecular interactions of a type-A CBM with cellulose.