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.     

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.     

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.     

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.     

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.     

TOTAL β-GLUCAN CONTENT OF SOME BARLEY CULTIVARS

The use of cellulase preparations from Trichoderma reesii for measuring the total β-glucan content of barley was examined. The activities of amyloglucosidase and β-glucanase in the cellulase varied considerably between batches, and different heat treatments were necessary to ensure that amyloglucosidase was reduced to an insignificant level while adequate β-glucanase activity was retained. After suitable treatment the cellulase was used to study variation of total β-glucan concentration in some barley cultivars. Significant varietal variation was found in the fifteen genotypes examined. These had β-glucan concentrations in the range 2.7% to 5.2% dry weight.     

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.     

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.     

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.     

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.     

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.     

EVALUATION OF MALTING QUALITY IN A BARLEY BREEDING PROGRAMME USE OF α-AMYLASE AND β-GLUCAN LEVLES IN MALT AS PRESELECTION TOOLS

Analysis according to the EBC protocol, immunological determination of a α-amylase and estimation of malt β-glucan using the Calcofluor-FIA method, were used to screen 327 barley breeding lines for malting quality. The results obtained with the α-amylase and β-glucan methods are highly correlated to the important malt quality paramters: extract yield and β-glucan content in the wort. It is recommended that either of the two methods, which are simple to perform are used as prescreening tools in breeding programmes for malting barley.     

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.     

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.     

THE DEGRADATION OF β-GLUCAN DURING MALTING AND MASHING: THE ROLE OF β-GLUCANASE

Significant β-glucanolysis takes place during mashing and is catalysed by a β-glucanase which is specific to mixed-linkage β-glucans. The enzyme develops during the germination of barley, but is rapidly and extensively destroyed in kilning. Partially-purified preparations of β-glucanase are protected from denaturation by heat if their solutions are adjusted to pH 4 or if bovine serum albumin is added. However the most effective stabiliser of the enzyme is reduced glutathione. Oligosaccharides containing three and four glucosyl units are produced by the action of β-glucanase and they are further converted during malting and mashing by a different enzyme(s) to disaccharides and glucose.     

KINETICS OF β-GLUCAN DEGRADATION IN WORT BY EXOGENOUS β-GLUCANASES TREATMENT

Three commercial β-glucanases, one bacterial (Cereflo 200L from Novo) and two fungal (Biobeta from Gist-Brocades and Filtrase from Biocon) have been studied with regard to the hydrolysis of β-glucan in sweet and hopped wort. At temperatures below 70°C these processes follow first order kinetics with rate constants being directly proportional to the enzyme concentrations. The rate constant for bacterial β-glucanase Cereflo 200L shows a negative dependence on temperature but positive with wort pH, whereas the reverse is the case for the two fungal β-glucanases. Within the ranges of pH and temperature tested the bacterial β-glucanase has 2–5 times the activity of the fungal ones. No evidence for synergic or competitive effects between bacterial and fungal β-glucanases have been found.     

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.     

A ROLE FOR CARBOXYPEPTIDASE IN THE SOLUBILIZATION OF BARLEY β-GLUCAN

An enzyme is described which catalyzes the release of soluble β-glucan from insoluble barley endosperm cell walls. This enzyme increases in activity throughout malting. It has been partially purified and found to behave in the same way as an acidic carboxypeptidase on isoelectric focusing and in its sensitivity to inhibitors and activators and to heating. The importance of the β-glucan solubilizing enzyme in malting and mashing is discussed. An improved method for β-glucan determination is described.     

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.     

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.