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 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.
Induction, screening and identification of Coniothyrium minitans mutants with enhanced β-glucanase activity.
Zantinge, J. L., Huang, H. C. & Cheng, K. J. (2003). Enzyme and Microbial Technology, 32(2), 224-230.
The production of β-glucanase in a mycoparasitic fungus, Coniothyrium minitans, was investigated using a wild type strain 2134. Through ultraviolet (UV) irradiation of strain 2134, four mutants, M11-3B2, A7-3D, A8-1 and A10-4, exhibiting enhanced β-glucanase activities were isolated. Strains A8-1 and A10-4 were constitutive mutants that expressed barley β-glucan hydrolysing activity in the absence of a supplemental inducer (β-glucan substrate). Supernatant from A8-1 and A10-4 cultures grown in potato dextrose broth (PDB), the medium without β-glucan, had maximum levels of β-glucanase activity on average 10 times greater than the wild type strain 2134. M11-3B2 had low levels of constitutive β-glucanase expression and enhanced laminarin hydrolysing activity when grown in presence of β-glucan-rich substrate.
Synergism between cucumber α-expansin, fungal endoglucanase and pectin lyase.
Wei, W., Yang, C., Luo, J., Lu, C., Wu, Y. & Yuan, S. (2010). Journal of Plant Physiology, 167(14), 1204-1210.
Several recombinant fungal enzymes (endoglucanase and pectinase) were studied for their interactions with α-expansin in cell wall extension and polysaccharide degradation. Both Cel12A and Cel5A were able to hydrolyze cellulose CMC-Na and mixed-linkage β-glucan. In contrast to Cel5A, Cel12A could also hydrolyze xyloglucan and induce wall extension of cucumber hypocotyls in an in vitro assay. Combining α-expansin, even at high concentrations, with Cel12A did not enhance the maximum/final wall extension rate induced by Cel12A alone. These results strongly suggest that modification/degradation of the xyloglucan molecule/network is the key for cell wall extension, and α-expansin and Cel12A may share the same acting site in the substrate. Pectinase (Pel1, a pectin lyase) enhanced α-expansin-induced wall extension in a concentration-dependent manner, suggesting that the pectin network may normally regulate accessibility of expansin to the xyloglucan–cellulose complex. α-Expansin enhanced Cel12A's hydrolytic activity on cellulose CMC-Na but not on xyloglucan and β-glucan. Expansin did not affect Cel5A's hydrolytic activity. Interestingly, expansin also enhanced Pel1's activity on degrading high esterified pectin. A potential explanation for why expansin could synergistically interact with only certain enzymes on specific polysaccharides is discussed. Additional results also suggested that cell wall swelling may not be a significant event during the action of expansin and hydrolases.
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.