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.     

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.     

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.     

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.     

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.     

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.     

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 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.     

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.     

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.     

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.     

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.     

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.     

Structural Characterization of Oat Bran (1→3), (1→4)-β-D-Glucans by Lichenase Hydrolysis Through High-Performance Anion Exchange Chromatography with Pulsed Amperometric Detection

Cereal mixed linkages (1 → 3) (1 → 4)-β-D-glucan is a linear polysaccharide composed of glucose units. Oat β-glucan is a natural polymer. The main products of β-glucanase are oligosaccharides with DP3 and DP4, i.e., 3-Ob-cellobiosyl-D-glucose and 3-Ob-cellotriosyl-D-glucose, which represent over 90% of the molecule. Keeping in mind all the benefits of oat bran, the present study was planned to investigate the structural properties of oat bran, high-performance anion exchange chromatography with pulsed amperometric detection was used to examine these oligosaccharides. The structural analysis of oat bran of two oat varieties revealed that the ratio of soluble and insoluble triose to tetraose in β-glucan fraction was 1.44 and 1.78, respectively, for Avon variety; while the ratio of soluble and insoluble triose to tetraose in β-glucan fraction for Sargodha-81 was 1.49 and 1.77. The major units determined were cellotriose and cellotetraose. Other units cellopentaose and hexaoses were also existed but in minor fractions. Lichenase hydrolysis high-performance anion exchange chromatography with pulsed amperometric detection appeared to be the best choice for structural analysis of purified samples of mixed-linkage β-glucan.     

STARCH DEGRADATION IN ENDOSPERMS OF DEVELOPING BARLEY KERNELS*

Shortly after anthesis, empty endosperm cells adjacent to the embryo were crushed between the developing embryo and endosperm tissues to form the crushed cell layer. Starch granules in cells adjacent to this layer were hydrolysed and the empty cells were added to the crushed layer. In this way, the crushed layer increased in thickness throughout kernel development. α-Amylase 2 was detected in the crushed cell layer region of barley endosperms during the period that starch granule hydrolysis was occurring.     

THE EFFECTS OF INCREASED STEEPING TEMPERATURES ON ENZYME DEVELOPMENT IN MALT

Malts produced by steeping at 30 °C for a total of 24 h including two air rests, frequently give the same results in routine analyses as do malts produced by steeping at 16°C for longer periods. However, the corresponding worts often filter more slowly under controlled laboratory conditions. The increased steeping temperatures can delay or reduce development of endopeptidase and β-glucan solubitase, but generally have little effect on the activity of α-amylase, carboxypeptidase, or endo-β-glucanase. The problems with wort separation are most probably due to high molecular weight β-glucan and disulphide-linked hordein persisting at the end of malting.     

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.     

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.     

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.