ENZYMIC QUANTIFICATION OF (1→3) (1→4)-β-D-GLUCAN IN BARLEY AND MALT

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 maltsamples.α-Amylasedoes not interfere with the assay. The method issuitable for the routineanalysis 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.     

Molecular weight and structure of water soluble (1→3), (1→4)-β-glucans affect pasting properties of oat flours

Seven experimental oat lines with high (5.9% to 7.2%), medium (5.3% to 5.5%), and low (4.4%) β-glucan concentrations were evaluated for the effects of β-glucan molecular weight (MW) and structure on viscosities of oat-flour slurries. The MW of β-glucans was determined by size-exclusion high-performance liquid chromatography. The structural features of β-glucans were measured by using fluorophore-assisted capillary-electrophoresis after complete hydrolysis with lichenase. The oat-slurry viscosities were measured on a Rapid Visco Analyser under 4 conditions: (1) without starch (amylolysis, removal of starch by α-amylase); (2) without β-glucan (removal of β-glucan by lichenase); (3) natural action of enzymes (autolysis, in sodium buffer); and (4) inhibition of enzymes (in silver nitrate solution). Excluding one line (regression outlier), significant correlations (P < 0.05) between peak MW of β-glucan and viscosities of oat slurries were obtained under inhibition. The ratio of degree of polymerization (DP) 3/DP4 negatively correlated with viscosity under amylolysis, autolysis, and inhibition (P < 0.05). The amount of DP ≥ 5 negatively correlated with pasting final viscosity after β-glucan removal by lichenase (P < 0.05). Positive correlations (P < 0.05) between the ratio of β-(1→4)/β-(1→3) linkages and viscosities under autolysis and inhibition were found. Overall, these findings demonstrated that the peak MW, ratio of DP3/DP4, amount of DP ≥ 5, and ratio of β-(1→4)/β-(1→3) linkages of β-glucans impacted pasting properties of oat-flour slurries.     

Barley β-glucan is effective as a hypocholesterolaemic ingredient in foods

Barley contains high levels of soluble dietary fibre, including mixed linked 1→3, 1→4β-D -glucans (β-glucan). An extract of β-glucan from waxy, hulless barley containing 56% total dietary fibre (TDF) was incorporated into flour tortillas, cornstarch pudding and apple granola bars to provide 2 g soluble fibre as β-glucan per serving. The foods were tested for objective functional properties. Flour tortillas with β-glucan were incorporated into rat diets and compared to diets containing an equivalent amount of cellulose, to test the fibre effect on growth and lipid metabolism parameters. Rats fed β-glucan tortillas had lower feed consumption and body weight (P<0·05) compared to those fed the cellulose tortillas, although feed/gain ratios were not different (P>0·05). Plasma LDL-cholesterol of rats fed β-glucan was lower (P<0·05) than cellulose-fed controls, although total cholesterol and triglycerides did not differ (P>0·05). Rats fed β-glucan tortillas had higher (P<0·05) faecal fat excretion, suggesting impairment of intestinal fat absorption. Liver composition data showed lower (P<0·05) levels of total lipid and cholesterol in β-glucan-fed rats. The results suggest that the barley β-glucan concentrate has potential as a food ingredient to provide supplemental soluble fibre which may be beneficial in reducing plasma LDL-cholesterol in humans. © 1998 SCI.     

COMPARATIVE STUDIES OF THE β-D-GLUCAN RELEASED INTO SORGHUM AND BARLEY WORTS

Worts prepared from two cultivars of Nigerian grown sorghum six day melts — LI87 end SK5912 had β-D-glucan levels off five to seven times more than that of proctor barley. In contrast to barley, malting of the sorghums results in the release off more β-D-glucan into wort. Apparently, this is due to increasing levels of β-glucan solubilase and (1→3)-β-glucanase during malting with no significant (1→3, 1→4)-β-glucanase activity.     

Measurement of (1 → 3),(1 → 4)-β-D-glucan in barley and oats: A streamlined enzymic procedure

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.     

CHANGES IN BARLEY ENDOSPERMS DURING EARLY STAGES OF GERMINATION*

Kernels of 4 barley cultivars were germinated at 18°C and samples were removed for analysis at short time intervals for the first 30 h and at longer intervals during the ensuing 90 h. α-Amylase, (1 → 3) (1 → 4)-β-glucanase and (1 → 3) β-glucanase activities were measured in each sample. Analysis of kernel sections stained with Calcofluor showed that hydrolysis of β-glucan in the crushed cell layer commenced 6–9 h after the initiation of germination. Hydrolysis proceeded from the ventral edge to the dorsal edge of the kernel. Starch granule hydrolysis followed a similar pattern in the endosperm region adjacent to the crushed cell layer, but starch hydrolysis was always preceded by β-glucan hydrolysis.     

A comparison of the non-starch carbohydrates in cereal grains

The carbohydrates soluble in 80% ethanol and in water at 100°C were extracted from two varieties of wheat, barley, oats, rye, triticale, rice and malt. The highest concentrations of sugars soluble in 80% ethanol were found in malt and rye with the lowest in rice. Barley contained the most raffinose and rye contained the most fructan. Rye had the highest levels of water-soluble and total pentosans and barley the highest levels of water-soluble and total (1→3), (1→4)-β-glucans. The total arabinose:xylose ratio varied from 1.2 in wheat and triticale to 0.3 in oats. All were very low in uronic acids.     

ENZYMATIC DETERMINATION OF 1,3:1,4-β-GLUCANS IN BARLEY GRAIN AND OTHER CEREALS†

A direct and specific enzymatic method is described for the determination of 1,3:1,4-β-glucans in barley grain and other cereals. In the procedure, purified, amylase-free, bacterial 1,3:1,4-β-glucan hydrolase is used to depolymerize the 1,3:1,4-β-glucan in autoclaved and ethanol-extracted flour prepared from whole grain. The liberated oligoglucosides are extracted with 80% (vol/vol) ethanol and, following acid hydrolysis, measured by the glucose oxidase method. The method can be used to measure total β-glucan and β-glucan in 65°C water-soluble and water-insoluble fractions of cereal grains. The procedure has been applied to barley grains allowed to develop under controlled environmental conditions and to a series of barleys of different geographical origins. The results for Canadian cultivars are compared with estimates based on viscometric data for the same samples. The 1,3:1,4-β-glucan content of a number of other cereals has also been measured.     

SUBSTRATE SPECIFICITY AND NATURE OF ACTION OF BARLEY β-GLUCAN SOLUBILASE*

Barley β/glucan solubilase was shown to be active, to differing extends, towards hot water (65°C) soluble β-glucan, CM-cellulose and cellodextrins (DP 2–8). However, the enzyme did not affect the viscosity of CM-pachyman or appear to solubilise cotton cellulose. When β/glucan was treated with lichenase a mixture of small molecular weight products was obtained including a DP 9 dextrin. This dextrin was not obtained when the β-glucan was treated with β-glucan solubilase prior to hydrolysis by lichenase. It has been concluded, therefore, that this β-glucan solubilase is an endo-type glucanase, which appears to attack the small proportion of long blocks of (1→4)-β-linked glucosyl residues reputed to be present in barley β-glucan.     

Preparation and characterization of molecular weight standards of low polydispersity from oat and barley (1→3)(1→4)-β- -glucan

Purified (1→3)(1→4)-β- -glucans (β-glucans) from oat and barley with broad molecular weight (MW) distribution were separated into seven fractions using gradient precipitation with ammonium sulfate (NH₄)₂SO₄. The MW of each fraction decreased consecutively with the concentration of (NH₄)₂SO₄ at which it was precipitated. The MW distribution of each fraction was much narrower compared to the parent sample and is comparable to commercially available pullulan MW standards. To determine whether the fractionation process was separating sub-fractions of different structure, the original β-glucan sample and each fraction were hydrolyzed by a (1→3)(1→4)-D-β-glucan-4-glucanohydrolase (lichenase, E.C.3.2.1.73) and the liberated oligosaccharides were analyzed by high performance anion exchange chromatography. The analysis revealed no differences in oligosaccharide pattern (DP 2–9) derived from each fraction and the parent sample. In particular, the tri/tetra oligosaccharide ratio remained constant for all fractions, indicating no fractionation based on structural features had taken place. The effect of starting β-glucan concentration on the fractionation process was studied. The results showed that it was possible to achieve good separation at overlapping parameter c[η] lower than 3.5. Further increase in starting β-glucan concentration hindered clear separation of the fractions. Temperature also affected the fractionation efficiency. The higher the temperature, the lower the amount of (NH₄)₂SO₄ that was necessary to precipitate the samples of same MW. A Mark Houwink relationship was derived from the measured MW and intrinsic viscosity for fractions from oat and barley, respectively.     

MEASUREMENT OF (1 → 3)(1 → 4)-β-d-GLUCAN IN MALT,WORT AND BEER

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.     

Cryogelation of cereal β-glucans: structure and molecular size effects

The effects of molecular size and fine structure of mixed-linkage cereal (1→3), (1→4) β- -glucans (β-glucans) on their cryogelation behavior were investigated. Values of apparent molecular weight (M_w) for oat β-glucans ranged between 65 and 200×10³, whereas the respective values for both barley and wheat β-glucan preparations were about 200×10³. The fine structure of cereal β-glucans, as assessed by high-performance anion-exchange chromatography of the cellulosic oligomers released by the action of lichenase, revealed differences in the relative amounts of 3-O-β-cellobiosyl- -glucose (DP3) and 3-O-β-cellotriosyl- -glucose (DP4) units only among the different genera of cereals; the weight percent of DP3 units estimated as 67.1, 63.3, and 55.3–55.8% for wheat, barley, and oat β-glucans, respectively. Aqueous β-glucan solutions (1–3% w/v) were subjected to 12 freezing (−18 °C for 24 h) and thawing (5 °C for 24 h) cycles. The phenomenological appearance of the gelled materials obtained after this process as well as the yield of cryostructurates were influenced by the initial solution concentration, the number of freeze–thaw cycles, as well as by the molecular features of the β-glucans. Such effects were unraveled by studying the cryogelation process with differential scanning calorimetry (DSC), small strain dynamic rheometry, and large deformation mechanical measurements. For the cereal β-glucan cryogels the storage modulus, G′, increased and the tan δ decreased with decreasing polysaccharide molecular size and with increasing initial solution concentration, number of freeze–thaw cycles, and trisaccharide segments in the polymeric chains. The apparent melting enthalpy values (ΔH) of β-glucan cryostructurates, as determined from the DSC endothermic peaks, increased with decreasing molecular size and with increasing amount of cellotriose units, but they were independent of the number of freeze–thaw cycles. The DSC melting temperature of the gel network was found to increase with the molecular size and amount of DP3 units of β-glucans. Moreover, large deformation mechanical tests (compression mode) revealed an increase in strength of cereal β-glucan cryogels with increasing molecular size and decreasing trisaccharide units in the polysaccharide preparation.     

AN APPROACH TO THE IDENTIFICATION OF A ß-GLUCAN SOLUBILASE FROM BARLEY1

A ß-glucan solubilase activity was demonstrated in barley extract. This enzyme catalyzed the dissolution of barley ß-glucan by releasing a product having a narrow molecular weight distribution and a molecular weight of about 20,000. The enzyme was partially purified by ion exchange chromatography on DEAE-cellulose and gel permeation chromatography on Bio-Gel P-100. Although carboxypeptidase activity was present in the crude extract of barley flour the partially purified ß-glucan solubilase did not hydrolyse N-CBZ-Phe-ala. Examination of extracts from different barley tissues indicated that the ß-glucan solubilase activity was associated with the husks only; a large portion of the activity was extractable from whole barley kernels. About 85% of the enzyme activity in crude extracts from barley flour was retained after 40 min at 62°C. However, the enzyme was much more heat-labile in extracts of whole barley kernels. The pH of maximal activity was found to be about pH 5.7 and results from column chromatography suggested that the enzyme had a low pl value and a MW between 5 × 10⁴ and 6 × 10⁴.     

Changes in β-glucan and other carbohydrate components of barley during malting

Changes in total (1→3), (1→4)-β-glucan content were followed during the micro-malting of nine varieties of barley with a wide range of malting qualities. These changes were related to estimates of endosperm modification based upon staining with Calcofluor. β-Glucan content declined from an average of 3.54% in the barley to 0.75% in the malt. Pentosan and total starch (including starch-derived oligosaccharides) levels showed comparatively little change during malting. β-Glucan composition of the barley was a poor indicator of malting performance. However, the β-glucan, starch and xylose contents of the malt all showed significant correlations with malt extract. Estimation of malt β-glucan content gave the best indication of malt quality. Direct determination of β-glucan may be of more value in assessing malt quality than indirect techniques based upon assessing modification of stained grains.     

Structural Characterization,Technological Functionality,and Physiological Aspects of Fungal β-D-glucans: A Review

β-D-glucans are a (1→3)-linked glucose polymer with (1→6)-linked side chains and a major component of fungal cell walls. They exhibit structural integrity to the fungal cell wall. In addition, β-glucans are widely used as food adjuvant in food and pharmaceutical industries because of their physico-chemical properties. Several studies have focused on different isolation processes of (1→3) (1→6)-β-glucan that could affect the physico-chemical and functional properties of β-glucan such as chemical composition, solubility, viscosity, hydration properties, and oil binding capacity. Immunological activity is one of the most important properties of β-glucans. Thus, they are effective in inhibiting growth of cancer cells and metastasis and preventing bacterial infection. In humans, β-glucans reduce blood cholesterol, improve glucose absorption by body cells, and so help wound healing. This review described the prebiotic potentiality of fungal β-D-glucans with the objective to detail the methodologies applied for their extraction, their structure and techno-functional properties, and finally their biological effects.     

Comparison of the solution properties of (1→3),(1→4)-β-d-glucans extracted from oats and barley

The solution properties of β-glucan isolated from whole grains of oats and barley were studied by viscosity and oscillatory measurements both in water and in aqueous cuoxam, an ammoniacal Cu(OH)₂/CuCl-solution. The viscosity measurements of water solutions show stronger shear thinning for oat than for barley β-glucans. The crossover points of the moduli G′ and G″ in cuoxam solutions were different for the two β-glucans, i.e. 10 Hz for oat β-glucan and 1.5 Hz for barley β-glucan. The molecular weights of the β-glucans were similar. Therefore, the difference in solution properties seems to be related to the difference in structure. This difference is revealed by the ratio DP3:DP4 of the main building blocks of β-glucans, which was higher for barley than for oat β-glucan.     

THE EFFECT OF β-GLUCAN MOLECULAR WEIGHT ON THE SENSITIVITY OF DYE BINDING ASSAY PROCEDURES FOR β-GLUCAN ESTIMATION

An assay procedure has been developed for quantifying β-glucan, based upon its reaction with the dye Congo Red. The sensitivity of this method to changes in β-glucan molecular weight has been determined using β-glucans prepared from standard material by acid hydrolysis and comparison has been made with a Calcofluor-based method for quantifying β-glucans. The Congo Red assay was found to be optimally sensitive to β-glucans with molecular weights of approximately 2.5 × 10⁵ Daltons, whereas the Calcofluor assay was most sensitive to β-glucans with molecular weights in excess of 5 × 10⁴ Daltons. The purity of the β-glucan used during this investigation was determined using an enzyme-based procedure in which the polysaccharide was degraded to glucose by the addition of β-glucanase. The β-glucanase employed was partially-purified from Trichoderma viride cellulase using a novel batch ion-exchange method which is also described.     

β-GLUCANASE: THE SUCCESSFUL APPLICATION OF GENETIC ENGINEERING

The now well established principles of genetic engineering are described in relation to the solution of problems associated with β-glucans in beer. The application of these techniques has enabled the isolation of a Bacillus subtilis endo-1, 3–1, 4-β-D-glucanase gene which expresses a biologically active enzyme in yeast.^15,16 Although this enzyme is capable of hydrolysing beer β-glucans during fermentation, thereby enhancing beer filtration, insufficient β-glucanase is produced in yeast to enable successful commercial implementation. The requirements for the efficient production of β-glucanase in genetically manipulated brewing yeast are described.     

VARIATION IN THE BIOCHEMICAL COMPOSITION OF ACID EXTRACTS FROM BARLEYS OF CONTRASTING MALTING QUALITY

Acid extracts from barleys of poor malting quality were characterized by high viscosity caused by enhanced concentrations of high molecular weight carbohydrates of which more than 80 per cent was β-glucan. Up to 67 per cent of the β-glucan in these barleys was soluble in the acid extracting solution. In contrast, only about 25 per cent of the β-glucan in good malting barleys was soluble, resulting in extracts having low viscosity. No evidence was found for autolysis in any of the extracts, and extracts from good malting varieties were characterized by high concentrations of fructose and sucrose. Protein concentration in the extracts varied little, although the protein content of the barleys ranged from 9·1 per cent to 15·4 per cent. Soluble protein has no effect on extract viscosity or the gel filtration properties of the carbohydrates. Some quantitative variation in acid-soluble and salt-soluble proteins was seen after electrophoresis on polyacrylamide.     

STRUCTURE OF TOTAL BARLEY BETA-GLUCAN1

The fine structure of total barley β-glucan, as extracted by hot perchloric acid, was investigated by partial enzymatic hydrolysis. Molecular weight profiles of the resulting oligomeric products were similar to those from hydrolysed 40°C water-soluble β-glucan. Concentrations of individual oligosaccharides from total β-glucan were found to vary between oats and barley and among barley varieties, suggesting variability in β-glucan structure. Methylation studies, using HPLC to separate methylated sugars, showed no evidence for the presence of contiguous β-1,3 links in total barley β-glucan, although not all fractions of total β-glucan were analysed.