Enzymic quantification of (1→3) (1→4)-β-D-glucan in barley and malt.
McCleary, B. V. & Glennie-Holmes, M. (1985). Journal of the Institute of Brewing, 91(5), 285-295.
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 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.
Measurement of (1→3)(1→4)-β-D-glucan in malt, wort and beer.
McCleary, B. V. & Nurthen, E. (1986). Journal of the Institute of Brewing, 92(2), 168-173.
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.
Enzymic hydrolysis and industrial importance of barley β-glucans and wheat flour pentosans.
McCleary, B. V., Gibson, T. S., Allen, H. & Gams, T. C. (1986). Starch-Starke, 38(12), 433-437.
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-glucane 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 pentosanes and β-glucane 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.
Measurement of (1→3),(1→4)-β-D-glucan in barley and oats: A streamlined enzymic procedure.
McCleary, B. V. & Codd, R. (1991). Journal of the Science of Food and Agriculture, 55(2), 303-312.
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.
In Vitro fermentation of oat and barley derived β-glucans by human faecal microbiota.
Hughes, S. A., Shewry, P. R., Gibson, G. R., McCleary, B. V. & Rastall, R. A. (2008). FEMS Microbiology Ecology, 64(3), 482–493.
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.
Preparation of arabinoxylobiose from rye xylan using family 10 Aspergillus aculeatus endo-1,4-β-D-xylanase.
Rantanen, H., Virkki, L., Tuomainen, P., Kabel, M., Schols, H. & Tenkanen, M. (2007). Carbohydrate Polymers, 68(2), 350-359.
Commercial xylanase preparation Shearzyme®, which contains the glycoside hydrolase family 10 endo-1,4-β-D-xylanase from Aspergillus aculeatus, was used to prepare short-chain arabinoxylo-oligosaccharides (AXOS) from rye arabinoxylan (AX). A major AXOS was formed as a hydrolysis product. Longer AXOS were also produced as minor products. The pure GH10 xylanase from A. aculeatus was used as a comparison to ensure that the formed AXOS were consequence of the endoxylanase‘s function instead of some side enzymes present in Shearzyme. The major AXOS was purified and the structure confirmed with various analysis methods (TLC, HPAEC-PAD, MALDI-TOF-MS, and one- and two-dimensional NMR spectroscopy with nano-probe) as α-L-Araf-(1→3)-β-D-Xylp-(1→4)-D-Xylp (arabinoxylobiose). This is the first report on 13C NMR data of pure arabinoxylobiose. The yield of arabinoxylobiose was 12% from the quantified hydrolysis products. In conclusion, GH10 endoxylanase from A. aculeatus is thus able to cut efficiently the xylosidic linkage next to the arabinofuranosyl-substituted xylose unit which is not typical for all the GH10 endoxylanases. Interestingly, pure A. aculeatus xylanase showed notably activity towards p-nitrophenyl-β-D xylopyranose. In previously studies longer AXOS have been produced with Shearzyme but the formation of short-chain AXOS by A. aculeatus GH10 xylanase has not been studied before.
New enzyme-based method for analysis of water-soluble wheat arabinoxylans.
Virkki, L., Maina, H. N., Johansson, L. & Tenkanen, M. (2008). Carbohydrate Research, 343(3), 521-529.
Arabinoxylans (AX) are the predominant cell-wall polysaccharides in wheat flour. Water-extractable AX are essential for dough and bread properties and performance. However, there is no specific and accurate way of determining the content and structure of AX. An enzyme-assisted method employing an efficient enzyme mixture for the total hydrolysis of AX was developed in the present work. Enzymatic hydrolysis (EH) is a gentle method during which no unwanted sugar destruction occurs. Following EH, liberated monosaccharides were analysed by gas chromatography (GC) and liquid chromatography using HPAEC–PAD. The results were compared with acid methanolysis (AM) and acid hydrolysis (AH). EH performed better on commercially isolated AX samples than the reference method AM. Its action in the water extract from wheat flour was also more efficient than that of AM and comparable to the efficiency of AH. HPAEC–PAD revealed a significant amount of fructose in the water extract following EH, originating from fructans in wheat flour not detected in the GC analysis. The wheat flour examined contained 0.29% water-extractable AX. The arabinose/xylose ratio was 0.32. The enzyme-based method developed is applicable for comparison of different wheat flours and can be used in evaluating the effect of processing on the content and structure of water-extractable AX.
Water extract of Triticum aestivum L. and its components demonstrate protective effect in a model of vascular dementia.
Han, H. S., Jang, J. H., Jang, J. H., Choi, J. S., Kim, Y. J., Lee, C., Lim, S. H., Lee, H. K. & Lee, J. (2010). Journal of Medicinal Food, 13(3), 572-578.
Although vascular dementia is the second leading cause of dementia and often underdiagnosed, there are no drugs yet approved for the treatment of vascular dementia. In this study, it is demonstrated that water extract of Triticum aestivum L. (TALE) and some of its components have protective effects against vascular dementia-induced damage by preserving the myelin sheath and inhibiting astrocytic activation. The memory test used a vascular dementia model utilizing bilateral ligation of the carotid arteries of rats. TALE, some of its components, such as starch, total dietary fiber (TDF), arabinoxylan, β-glucan, and degraded products of arabinoxylan, such as arabinose and xylose, were administered to the animals from day 8 to day 14, following the surgery. Twenty-one days after the surgery, the water maze test was performed for 5 days, and the time taken to find the platform during training trials (mean escape latency) was measured. The mean escape latency was decreased consistently in the TALE-, starch-, TDF-, arabinoxylan-, and arabinose-treated groups, compared with that in the vascular dementia group. To measure brain damage, Luxol fast blue staining and immunohistochemistry of myelin basic protein (MBP) were performed to observe myelin sheath in the white matter, and immunohistochemistry of glial fibrillary acidic protein (GFAP) was performed to observe the astrocytic reaction. Vascular dementia reduced the MBP level and increased the GFAP level. Arabinose effectively inhibited the MBP and GFAP change, whereas arabinoxylan inhibited the GFAP change only. These results suggest that TALE and some of its components can be used as a medicinal material for the development of neuroprotective agents against vascular dementia.
Yeasts in an industrial malting ecosystem.
Laitila, A., Wilhelmson, A., Kotaviita, E., Olkku, J., Home, S. & Juvonen, R. (2006). Journal of Industrial Microbiology and Biotechnology, 33(11), 953-966.
The malting ecosystem consists of two components: the germinating cereal grains and the complex microbial community. Yeasts and yeast-like fungi are an important part of this ecosystem, but the composition and the effects of this microbial group have been largely unknown. In this study we surveyed the development of yeasts and yeast-like fungi in four industrial scale malting processes. A total of 136 malting process samples were collected and examined for the presence of yeasts growing at 15, 25 and 37°C. More than 700 colonies were isolated and characterized. The isolates were discriminated by PCR-fingerprinting with microsatellite primer (M13). Yeasts representing different fingerprint types were identified by sequence analysis of the D1/D2 domain of the 26S rRNA gene. Furthermore, identified yeasts were screened for the production of α-amylase, β-glucanase, cellulase and xylanase. A numerous and diverse yeast community consisting of both ascomycetous (25) and basidiomycetous (18) species was detected in the various stages of the malting process. The most frequently isolated ascomycetous yeasts belonged to the genera Candida, Clavispora, Galactomyces, Hanseniaspora, Issatchenkia, Pichia, Saccharomyces and Williopsis and the basidiomycetous yeasts to Bulleromyces, Filobasidium, Cryptococcus, Rhodotorula, Sporobolomyces and Trichosporon. In addition, two ascomycetous yeast-like fungi (black yeasts) belonging to the genera Aureobasidium and Exophiala were commonly detected. Yeasts and yeast-like fungi produced extracellular hydrolytic enzymes with a potentially positive contribution to the malt enzyme spectrum. Knowledge of the microbial diversity provides a basis for microflora management and understanding of the role of microbes in the cereal germination process.
Differential adjuvant effects of soluble beta glucans from barley and Saccharomyches cerevisia in primary and secondary humoral immune responses.
Uslu, K. & Bagriacik, E. U. (2011). International Journal of Hematology & Oncology/UHOD: Uluslararasi Hematoloji Onkoloji Dergisi, 21(3).
The purpose of this study was to investigate and compare adjuvant effects of soluble β-glucans from barley and Saccharomyches cerevisia in induction of antigen specific humoral immune responses. Mice were immunized with conalbumin at a relatively low concentration in the presence of beta glucans. Anti-conalbumin antibodies in the sera of immunized and control mice were quantified by ELISA. At high doses, both of glucans increased effectively levels of circulating IgM and IgG antibodies which were specific for conalbumin. However, coadministration of glucan from barley at 1 μg dose resulted in lower yield in IgG1, IgG2a, IgG2b, and IgA levels in comparison to that of yeast-derived glucan at the same dose. We also found that antigen (conalbumin) specific antibody levels enhanced by β-glucan from Saccharomyches cerevisia were always higher than those of the glucan from barley. Based on these findings, we concluded that (1,3),(1,6)-β-D-glucan from yeast cell wall might have superior immunostimulant activity in induction of antigen specific humoral immune responses over (1,3),(1,4)-β-D-glucan from barley.
In vitro fermentation kinetics and end-products of cereal arabinoxylans and (1,3;1,4)-β-glucans by porcine faeces.
Williams, B. A., Mikkelsen, D., Le Paih, L. & Gidley, M. J. (2011). Journal of Cereal Science, 53(1), 53-58.
Purified and semi-purified polysaccharides characteristic of cereals were fermented in vitro with a pig faecal inoculum, using the cumulative gas production technique, to examine the kinetics and end-products of fermentation after 48 h. It was shown that arabinoxylan and mixed linkage (1,3;1,4) β-glucan were rapidly fermented if soluble, while less soluble substrates (insoluble arabinoxylan, maize and wheat starch granules, and bacterial cellulose) were more slowly fermented. Relevant monosaccharides were fermented at very similar rates to soluble polymeric arabinoxylan and β-glucan, showing that depolymerisation was not a limiting step, in contrast to some previous studies. Bacterial cellulose is shown to be a useful model substrate for fermentation of plant cellulose which is difficult to obtain without harsh chemical treatments. Fermentation end-products were related to kinetics, with slow carbohydrate fermentation resulting in increased protein fermentation. Ratios of short-chain fatty acid products were similar for all arabinoxylan and β-glucan substrates.
Role of (1,3)(1,4) β-glucan in cell walls: Interaction with cellulose.
Kiemle, S. N., Zhang, X., Esker, A. R., Toriz, G., Gatenholm, P. & Cosgrove, D. J. (2014). Biomacromolecules, 15 (5), 1727-1736.
(1,3)(1,4)-β-D-Glucan (mixed-linkage glucan or MLG), a characteristic hemicellulose in primary cell walls of grasses, was investigated to determine both its role in cell walls and its interaction with cellulose and other cell wall polysaccharides in vitro. Binding isotherms showed that MLG adsorption onto microcrystalline cellulose is slow, irreversible, and temperature-dependent. Measurements using quartz crystal microbalance with dissipation monitoring showed that MLG adsorbed irreversibly onto amorphous regenerated cellulose, forming a thick hydrogel. Oligosaccharide profiling using endo-(1,3)(1,4)-β-glucanase indicated that there was no difference in the frequency and distribution of (1,3) and (1,4) links in bound and unbound MLG. The binding of MLG to cellulose was reduced if the cellulose samples were first treated with certain cell wall polysaccharides, such as xyloglucan and glucuronoarabinoxylan. The tethering function of MLG in cell walls was tested by applying endo-(1,3)(1,4)-β-glucanase to wall samples in a constant force extensometer. Cell wall extension was not induced, which indicates that enzyme-accessible MLG does not tether cellulose fibrils into a load-bearing network.
A fibrolytic potential in the human ileum mucosal microbiota revealed by functional metagenomics.
Patrascu, O., Béguet-Crespel, F., Marinelli, L., Le Chatelier, E., Abraham, A., Leclerc, M., Klopp, C., Terrapon, N., Henrissat, B., Blottière, H. M., Doré, J. & Christel Béra-Maillet. (2017). Scientific Reports, 7, 40248.
The digestion of dietary fibers is a major function of the human intestinal microbiota. So far this function has been attributed to the microorganisms inhabiting the colon, and many studies have focused on this distal part of the gastrointestinal tract using easily accessible fecal material. However, microbial fermentations, supported by the presence of short-chain fatty acids, are suspected to occur in the upper small intestine, particularly in the ileum. Using a fosmid library from the human ileal mucosa, we screened 20,000 clones for their activities against carboxymethylcellulose and xylans chosen as models of the major plant cell wall (PCW) polysaccharides from dietary fibres. Eleven positive clones revealed a broad range of CAZyme encoding genes from Bacteroides and Clostridiales species, as well as Polysaccharide Utilization Loci (PULs). The functional glycoside hydrolase genes were identified, and oligosaccharide break-down products examined from different polysaccharides including mixed-linkage β-glucans. CAZymes and PULs were also examined for their prevalence in human gut microbiome. Several clusters of genes of low prevalence in fecal microbiome suggested they belong to unidentified strains rather specifically established upstream the colon, in the ileum. Thus, the ileal mucosa-associated microbiota encompasses the enzymatic potential for PCW polysaccharide degradation in the small intestine.
Bonds broken and formed during the mixed-linkage glucan: xyloglucan endotransglucosylase reaction catalysed by Equisetum hetero-trans-β-glucanase.
Simmons, T. J. & Fry, S. C. (2017). Biochemical Journal, 474(7), 1055-1070.
Mixed-linkage glucan : xyloglucan endotransglucosylase (MXE) is one of the three activities of the recently characterised hetero-trans-β-glucanase (HTG), which among land-plants is known only from Equisetum species. The biochemical details of the MXE reaction were incompletely understood - details that would promote understanding of MXE's role in vivo and enable its full technological exploitation. We investigated HTG's site of attack on one of its donor substrates, mixed-linkage (1→3),(1→4)-β-D-glucan (MLG), with radioactive oligosaccharides of xyloglucan as acceptor substrate. Comparing three different MLG preparations, we showed that the enzyme favours those with a high content of cellotetraose blocks. The reaction products were analysed by enzymic digestion, thin-layer chromatography, HPLC and gel-permeation chromatography. Equisetum HTG consistently cleaved the MLG at the third consecutive β-( 1→4)-bond following (towards the reducing terminus) a β-( 1→3)-bond. It then formed a β-( 1→4)-bond between the MLG and the non-reducing terminal glucose residue of the xyloglucan oligosaccharide, consistent with its XTH subfamily membership. Using size-homogeneous barley MLG as donor substrate, we showed that HTG does not favour any particular region of the MLG chain relative the polysaccharide's reducing and non-reducing termini; rather, it selects its target cellotetraosyl unit stochastically along the MLG molecule. This work improves our understanding of how enzymes can exhibit promiscuous substrate specificities and provides the foundations to explore strategies for engineering novel substrate specificities into transglycanases.
Multi-scale characterisation of deuterated cellulose composite hydrogels reveals evidence for different interaction mechanisms with arabinoxylan, mixed-linkage glucan and xyloglucan.
Martínez-Sanz, M., Mikkelsen, D., Flanagan, B. M., Gidley, M. J. & Gilbert, E. P. (2017). Polymer, 124, 1-11.
The interactions of cellulose with other major plant cell wall polysaccharides - arabinoxylan (AX), xyloglucan (XG) and mixed linkage glucans (MLG) - have been investigated by characterising the architecture of composite deuterated cellulose hydrogels by means of SAXS and SANS, combined with XRD, NMR and microscopy. The results indicate that cellulose-AX interactions, limited to the ribbons' surface, take place via a non-specific adsorption mechanism. In contrast, XG and MLG interact specifically with cellulose, forming two different fractions: (i) interfibrillar domains interacting with the cellulose microfibrils and (ii) surface domains, responsible for the cross-linking of ribbons. XG co-crystallises with cellulose, promoting the formation of Iβ-richer microfibrils and forming intercalated amorphous regions. On the other hand, MLG interacts with cellulose forming a paracrystalline coating layer. This structural role of XG and MLG in preventing microfibril aggregation may help explain their key function in the cell expansion process of growing plant tissues.