AN ENZYMIC METHOD FOR THE MEASUREMENT OF TOTAL AND WATER-SOLUBLE β-GLUCAN IN BARLEY

Crude cellulase preparations from Trichoderma reesei, freed of amyloglucosidase by heating, will completely hydrolyse barley β-glucan to glucose. Apparent non-quantitative recovery of glucose is due to its adsorption by the high levels of protein present. Methods are given for the measurement of total and water-soluble β-glucan. Dehusking does not lower the amount of β-glucan which is measured.     

PENICILLIUM FUNICULOSUM AS A SOURCE OF β-GLUCANASE FOR THE ESTIMATION OF BARLEY β-GLUCAN

The β-glucanase in commercial |cellulases prepared from Penicillium funiculosum is more stable to heating than is the equivalent enzyme system from Trichoderma reesei. Consequently the heat-labile amyloglucosidase can be destroyed more reliably in Penicillium cellulase, and it follows that this enzyme is to be preferred for use in the enzymic method for measuring β-glucan.     

THE RELATIONSHIP BETWEEN β-GLUCAN SOLUBILASE,BARLEY AUTOLYSIS AND MALTING POTENTIAL

There is a correlation between the autolysis of barleys and their β-glucan solubilase activities. There is no correlation between autolysis and nitrogen content, β-glucan level, Milling Energy or Zeleny sedimentation value of the barley. Activities of endo-β-glucanase are inversely related to coarse-grind Hot Water Extract obtained from malts grown for 4 days. Whilst β-glucanase is not involved in the early stages of autolysis, β-glucan solubilase, present in large amounts in untreated barley, has a role both in extracting and degrading β-glucan. Barleys with low β-glucan content or high β-glucan solubilase modify more rapidly.     

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.     

THE RELATIONSHIP BETWEEN TOTAL β-GLUCAN OF MALT AND MALT QUALITY

The total β-glucan content of barley and malts has been determined using a direct enzyme degradation method incorporating measures to ensure inactivation of any contaminating amyloglucosidase. Results for barleys range between 2·7 and 4·4% w/w and indicate genetic variation in the β-glucan content. Malts produced by both a laboratory micromalting procedure and commercially have been analysed for total β-glucan, extract and 70°C mash viscosity at different stages of germination and in the end product. A very good correlation has been found between total β-glucan and fine-concentrated extract difference values showing that in a fully modified malt having a negligible extract difference value, all the β-glucan material is degraded. The extract difference value had been demonstrated earlier to be closely linked with brewhouse extract yield. The total β-glucan of malt, therefore, is directly associated with achievable extract in the brewhouse and is the most important biochemical factor determining the extractability of a malt.     

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.     

β-GLUCAN AND β-GLUCAN SOLUBILASE IN MALTING AND MASHING

During malting the water-insoluble β-glucan of barley is diminished whilst water-soluble gum is little decreased. The amount of β-glucan surviving into malt depends on variety but barleys rich in glucan give malts with high β-glucan levels. The β-glucan content of barley depends on variety and growth site. β-Glucan solubilase survives mashing and catalyses the release of hemicellulose into solution. There is no correlation between the β-glucan content of malt and the amount released into wort. However, barley adjuncts containing high levels of β-glucan give worts rich in β-glucan. β-Glucan dissolution in mashing is dependent on time, temperature, grist particle size and liquor: grist ratio. Use of adjuncts derived from barley contribute relatively more β-glucan in wort, coinciding with reduced rates of wort separation, but these can be increased by using a β-glucanase produced by growing the fungus Trichoderma viride on spent grains.     

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.     

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.     

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.     

THE DEVELOPMENT OF β-GLUCAN SOLUBILASE DURING BARLEY GERMINATION

β-Glucan solubilase in either germinating barley or in endosperm slices treated with gibberellic acid is synthesized before endo-β-glucanase, α-amylase and protease. In common with these enzymes, β-glucan solubilase is synthesized much sooner in endosperm slices than in whole grain. Gibberellic acid stimulates β-glucan solubilase synthesis in endosperm slices and most of the activity is rapidly released into the surounding medium, irrespective of whether the hormone is present. Inhibitors of RNA and protein synthesis block the formation of β-glucan solubilase. Unlike β-glucanase, α-amylase and protease, β-glucan solubilase is present in significant quantity in untreated barley where it is concentrated in the embryo-containing half of the grain. The only β-glucan solubilase activity in barley is due to an acidic carboxypeptidase. Malt contains a small amount of a second solubilizing enzyme which appears to be an endo-β1, 3-glucanase.     

ASSAY OF MALT β-GLUCANASE USING AZO-BARLEY GLUCAN: AN IMPROVED PRECIPITANT

A procedure recently described for the assay of malt β-glucanase, which employs a dye-labelled and chemically-modified barley β-glucan substrate, has been improved by changing the precipitant solution used to terminate the reaction. The new precipitant solution contains 0·4% (w/v) zinc acetate and 4% (w/v) sodium acetate dissolved in 80% (v/v) aqueous methyl cellosolve. With this precipitant the procedure can be directly applied to the assay of cellulase activity, and with minor modification, to the assay of lichenase activity.     

FIELD FUNGI AND β-GLUCAN SOLUBILASE IN BARLEY KERNELS*

Significant amounts of β-glucan solubilase activity have been found in barleys harvested from a number of test sites. Enzyme activity appeared to be related to the climatic conditions at crop maturity, indicating that β-glucan solubilase was generated, possibly, by microflora on the barley grain. Species of the most common field fungi genera, Alternaria, Cladosporium, Epicoccum and Helminthosporium and two bacterial cultures were isolated from barley kernels and incubated on autoclaved barley for solubilase examinations. All the fungal isolates studied showed abilities to reduce the viscosity of carboxymethyl cellulose and to solubilise barley β-glucan. The molecular size distribution of the solubilised β-glucan products resembled that obtained for products formed by a partially purified preparation of solubilase from barley. It has been concluded, therefore, that the common field fungi associated with the hull and seed cot of barley may be the source of β-glucan solubilase.     

Effects of Tocols and β-Glucan on Serum Lipid Parameters in Chickens*

Cereal grain diets affect serum lipids by their soluble fibre and tocotrienols. Chickens were fed diets containing an oat bran fraction or waxy hulless barley that were enriched or depleted in β-glucan and/or tocotrienols. Serum cholesterol and triacylglycerides and enzymes of cholesterol metabolism were measured. Weight gains appeared to be lower in birds on oat bran fraction-containing diets and higher in those on barley-containing diets supplemented with β-glucanase. All diets containing oat bran fraction or barley lowered serum total cholesterol and low-density-lipoprotein (LDL) cholesterol relative to the corn control diet. LDL cholesterol was reduced more by oat bran fraction supplemented with tocotrienols than by either oat bran fraction or tocotrienols alone. LDL cholesterol levels were the same for all barley-based diets. Activities of β-hydroxy-β-methylglutaryl coenzyme A (HMG CoA) reductase and cholesterol 7α-hydroxylase were inversely affected by the diets. Oat bran fraction plus tocotrienols, barley and solvent-extracted barley decreased HMG CoA reductase by 50% and increased cholesterol 7α-hydroxylase by 100%; other diets caused lesser effects. It was concluded that both β-glucan and tocotrienols affected cholesterol levels and metabolism, and the effects were additive or less. Removal of β-glucan from barley diets abolished or diminished effects on enzyme activities but did not alter effects on cholesterol levels, indicating the possibility of another component in barley that affected cholesterol levels. © 1997 SCI.     

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.     

SOME BARLEY GRAIN AND GREEN MALT PROPERTIES AND THEIR INFLUENCE ON MALT HOT-WATER EXTRACT: I. β-GLUCAN, β-GLUCAN SOLUBILASE AND ENDO-β-GLUCANASE

A study has been made of the variation between varieties in some properties of barley and malt and how this variation relates to malt hot water extract (HWE). The development of enzyme activity along the grain during germination was investigated. In this first paper we have examined β-glucan-related characters and found significant varietal variation in maximum enzyme activities and in the activities in different sections of grain during germination. Varietal variation was greater than environmental variation for each character. The fraction of β-glucan soluble in acid was the character most highly correlated with HWE.     

THE MAJOR BIOCHEMICAL CONSTITUENTS OF AN ACID EXTRACT OF BARLEY FLOUR AS USED IN VISCOSITY DETERMINATIONS BY THE FALLING BALL METHOD

The viscosity of an acid extract of barley flour, measured as its ‘falling time’, has been used as a guide to malting potential.²² One such extract, made from a sample of Maris Mink, was examined in detail to identify the principle biochemical constituents and to investigate their contribution to viscosity. The extract contained a high concentration of carbohydrate (3·30 mg/ml) divided into two discrete fractions of high and low molecular weight (in the weight ratio of 39:61) by gel filtration. The high molecular weight component was polydisperse and consisted principally of β-glucan with some starch and pentosan also present. The low molecular weight fraction contained mostly fructose, glucose and some pentose. Protein present in the extract (1·07 mg/ml) was derived almost exclusively from the albumin and globulin (salt soluble) fraction of barley protein. Studies on the viscosities of reconstituted preparations of these components indicated that β-glucan made the major contribution to the viscosity of the extract. This conclusion is supported by observed changes in viscosity after the extract was treated with β-glucanase, α-amylase and protease.     

EFFECTS OF CULTIVAR AND ENVIRONMENT ON β-GLUCAN CONTENT AND MALTING QUALITY OF TURKISH BARLEYS

Eight barley cultivars grown under the same agronomic conditions and samples of Tokak cultivar grown at six different sites of Turkey were used in this study. There were significant differences among the barley cultivars and growing locations in terms of β-glucan content (p<0.05). Among malt quality criteria tested for the 8 barley cultivars; friability, viscosity, Kolbach index and extract difference showed significant correlations (p<0.05) with the total β-glucan content. Similar correlations were also observed between the malt quality criteria (Kolbach index and extract difference) and β-glucan contents for the Tokak samples grown at different sites.     

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.     

VARIATION IN A BARLEY COLLECTION FOR ENDOSPERM ATTRIBUTES THAT RELATE TO MALTING QUALITY

Éstimates of grain hardness from measurements of milling energy were made on four hundred and eighty barley cultivars. The same cultivars were also subjected to an infra-red analysis used to predict soluble β-glucan and nitrogen contents. The results demonstrated variation for these endosperm characters, with eighteen cultivars having a lower milling energy, and more than a hundred cultivars with a lower soluble β-glucan content than Gerkra, a barley with good malting quality. Of these, nine cultivars were lower for both characteristics. Information about this variation is, therefore, potentially useful when breeding for malting quality.