LEVELS OF ALKALI-SOLUBLE β-D-GLUCANS IN CEREAL GRAINS

Extraction of β-D-glucans (primarily from barley grains) using sodium hydroxide solution, gave similar results to those obtained using hydrazine extraction, suggesting that the alkali is as effective as hydrazine for the extraction of β-D-glucans. Commercial growth conditions in different European countries caused limited shifts in the β-D-glucan levels of malting barley samples. Sorghum and wheat grains contained significantly less β-D-glucans than barley. Potassium bromate solution inhibited initial breakdown of β-D-glucans during the malting of barley grains. The breakdown of β-D-glucan in Sonja barley was much slower than in Golden Promise barley, during malting.     

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.     

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.     

Comparative studies of the degradation of non-starchy polysaccharides by sorghums and barleys during malting

The β-D-glucan of whole grains of four sorghum (Sorghum bicolor (L) Moench) varieties grown in Nigeria, unlike those of barley, is not significantly reduced during malting. Pentosan level, while enhanced in barley (Hordeum rulgare L) during the same period, dropped in sorghum. Apparently sorghums have very low β-D-glucan degrading endo β 1–3,1-4 glucanase activity as observed from their failure to hydrolyse their isolated endosperm cell walls and barley β-D-glucan extracted in water at 40°C.     

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 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.     

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.     

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.     

Effect of Mashing Temperatures and Endo-β-Glucanase on β-Glucan Content of Malt Worts

Similar basal levels of β-D-glucans were released into worts produced at 45°C from enzymically active or inactivated flours of milled malts. In contrast, significantly higher levels of β-D-glucans were found in worts derived from either enzymically active or inactivated malt flours mashed at 65°C. In general, mashing temperature may play a more important role in releasing β-D-glucans during mashing than enzymes described as β-glucan-releasing. In this context, the physical release of β-D-glucan during mashing should be separated from the enzymic release and degradation of β-D-glucan which occur during malting.     

Influence of Heat Pretreatments of Oat Grain on the Viscosity of Flour Slurries

Heat treatment of oat grain had significant effects on the viscosity of flour slurries. Steamed oats produced highly viscous flour slurries, whose viscosity increased with time, whereas the viscosity of flour slurries produced from raw or roasted (104°C for 120 min) oats was much lower and degraded rapidly. Slurry viscosity was correlated with (1→3), (1→4)-β-D -glucan concentration in the flours and treatment of slurries with lichinase lowered viscosity significantly. Effects of steaming were partially reversed by roasting treatment and vice versa. Mixtures of equal amounts of raw and steamed flour resulted in slurries more viscous than either flour alone, but that viscosity degraded after 3 h to less than the mean viscosity of the steamed and raw controls. Water-soluble extracts from steamed flour had about twice the viscosity of raw or roasted flour extracts, but contained only 80% of the (1→3), (1→4)-β-D -glucan present in those extracts. Molecular weight analysis of soluble carbohydrates from raw, roasted and steamed soluble extracts indicated the molecular weight of β-glucans in these extracts was similar. However, if extracts were made from slurries that had incubated for 3 h, extensive degradation of β-glucans was evident in raw and roasted samples. It is likely that enzymic degradation of (1→3),(1→4)-β-D -glucans is responsible for much of the decreased raw and roasted flour slurry viscosity over extended time periods, but different heat treatments appear to also affect (1→3), (1→4)-β-D -glucan polymer interaction. © 1997 SCI.     

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.     

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.     

THE DEVELOPMENT OF β-D-GLUCANASES DURING THE GERMINATION OF BARLEY AND THE EFFECT OF KILNING ON INDIVIDUAL ISOENZYMES†

Two endo-1,3;1,4-β-D-glucanase isoenzymes developed in response to gibberellic acid, during the germination of barley. Two endo-1, 3-β-D-glucanases, one present in ungerminated, steeped grain, also developed but did not appear to be markedly stimulated by the hormone. A comparison of crude and partially purified malt extracts highlighted the errors that are involved in the specific determination of endo-1, 3;1, 4-β-glucanase activity in crude extracts. The development and effect of kilning on individual malt isoenzymes was demonstrated by carboxymethylcellulose (CM-cellulose) chromatography profiles. Kilning and dry-milling of germinated barley caused losses of 80–90% in the specific endo-1,3;1,4-glucanase activity. The effect was less pronounced if wet-milling was substituted for dry-milling. Extraction studies and CM-cellulose chromatography profiles indicated that both endo-1,3;1, 4-β-glucanase isoenzymes were heat labile and were particularly susceptible to oxidation. In contrast, endo-1,3-β-glucanase activity and cellobiase activity in malt extracts were less affected by the kilning process or extraction procedures. Preliminary results suggested that one of the endo-1,3-β-glucanase isoenzymes was more sensitive to kilning.     

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.     

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.     

IMPROVED EXTRACTION AND ASSAY OF β-AMYLASE BROM BARLEY AND MALT

β-Amylase was extracted from barley or malt using four physical techniques to break up grists which had been prepared using a Moulinex coffee grinder. Grinding with a Polytron homogeniser apparently completely disrupted all cells, as determined by transmission electron microscopy, and increased the efficiency of extraction of β-amylase from barley by more than 30%. The other treatments tested were without value . The β-amylase activity in extracts of barley or malt was assayed by measuring the production of reducing sugars from reduced soluble starch, using a PAHBAH reagent. α-Amylase, which interferes with the quantitation of β-amylase in extracts of malt, was not totally inactivated by the chelating buffer used for enzyme extraction or by several other chelating agents. α-Amylase activity was quantified specifically using Phadebas. Using purified α-amylase a calibration was developed which related activity, as determined using Phadebas, to reducing power units. Thus the α-amylase activity present in an extract containing β-amylase could be determined using Phadebas and the reducing power equivalent activity subtracted from the total “apparent” activity to give the actual β-amylase activity. α-Glucosidase and limit dextrinase activities are believed to be too low to have a significant effect on the apparent β-amylase . The soluble and bound β-amylase activities were measured in samples taken from micromalting barley (Alexis). Dry weight losses increased to over 10% after 8 days germination. Antibiotics, applied during steeping, were used to control microbes in one experiment. However, their use checked germination and reduced malting losses to 8.4% in 8 days germination. The soluble enzyme present in extracts from steeped barley and early stages of germination was activated (20–40%) by additions of the reducing agent DTT .     

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.     

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.