THE ACTION OF ENDO-β-1,3-GLUCANASES ON BARLEY AND MALT β-GLUCANS

The β-glucan extracted from ungerminated barley with water at 40 °C has a much lower specific viscosity than the corresponding material isolated from a wort prepared at 65 °C from a two-day germinated barley malt. Both glucans are similar in that they are polymers of β-D-glucose, with approximately 74% of the linkages in the β-1,4 configuration and 26% in the β-1,3 configuration. However, the two glucans are not hydrolysed to the same extent either by a partially purified bacterial endo-β-1,3-glucanase or by a homogeneous endo-β-1,3-glucanase from malted barley. The malt glucan is readily hydrolysed, causing a rapid decrease in specific viscosity but with no measurable increase in reducing power, whereas barley glucan undergoes only limited hydrolysis under similar conditions. Thus, different β-glucan preparations from barley or malt may be identical in the proportion of β-1,3 to β-1,4-linkages but the overall arrangement of linkages, and hence susceptibility to enzyme attack, differs according to the source and the method of extraction of the glucan. The molecular weights of both β-glucan preparations and the products of their enzyme hydrolysis have been determined by agarose gel permeation chromatography. A simple model which illustrates the underlying structural relationships of the β-glucans from barley and malt is suggested.     

STUDIES ON CARBOHYDRATE METABOLIZING ENZYMES PART XXII. THE β-GLUCANASE SYSTEM OF MALTED BARLEY

Extracts of malted barley have been shown by molecular sieve chromatography to contain at least five enzymes which hydrolyse β-glucosidic linkages. In order of decreasing molecular weight, these are two β-glucosidases, an endo-β-1,4-glucanase, an endo-barley-β-glucanase and an endo-β-1,3-glucanase. This last-named enzyme was purified about 60-fold and shown to be specific for substrates containing only β-1,3-glucosidic linkages. This enzyme was activated by calcium ions, and deactivated by EDTA, but was not affected by chloride ions or a sulphydryl reagent. The endo-β-1,4-glucanase, which was purified about 50-fold, hydrolysed glucans containing only β-1,4- or a mixture of β-1,3- and β-1,4-linkages. The two β-glucosidases were shown to be true glycosidases and not exo-β-glucanases. The changes which occur in the levels of the various enzymic activities during the malting process have been measured.     

Effect of barley β-glucan on murine RAW264.7 macrophages against virulent Salmonella enterica serovar Typhimurium

Heavily evidenced in antimicrobial effects of microbial β-(1,3)(1,6)-glucan, the botanical β-(1,3)(1,4)-glucan is mostly shown to effectively control blood cholesterol level. S. Typhimurium belongs to food-borne zoonoses often causing worldwide epidemic outbreaks in animals and human with severe diarrhea and gastrointestinalitis. Cereal soups are commonly prescribed as neutraceutical for rehydration purpose and a sustaining therapy. Botanical β-(1,3)(1,4)-glucan is easily released in boiled cereal soup. β-(1,3)(1,4)-Glucans on modulating host defense to enteric infectious agents is seldom reported. Our results demonstrated that barley β-(1,3)(1,4)-glucans effectively increased the murine macrophage cell line RAW264.7 against S. Typhimurium infection through antibacterial lysozyme activity (P < 0.001), not the production of intracellular reactive oxygen species. Furthermore, barley β-(1,3)(1,4)-glucans upregulated the gene expressions of its receptor dectin-1. In conclusion, barley β-(1,3)(1,4)-glucan induces a mild immune response with increasing antibacterial lysozymes through up-regulating its receptors dectin-1 and lysozyme M gene expressions.     

Direct comparison of β-glucan content in wild and cultivated barley

β-Glucan is an important chemical found in cereals, tremendously beneficial to human health. In this study, β-glucan contents in wild and cultivated barley from representative regions worldwide were investigated. Results exhibited that coe?cient of variation of β-glucan content of wild and cultivated barley was 24.18% and 13.99%, respectively. The β-glucan contents of studied wild barley accessions were ranged from 3.26% to 7.67% while cultivated barley varieties were ranged from 2.68% to 4.74%. A significant difference of β-glucan content (p ≤ 0.001) was found between wild and cultivated barely. Wild barley showed a higher β-glucan content and variation than cultivated barley. This study gives an idea about elite germplasms for genetic improvement and shed light to trace barley domestication in relation to grain metabolite view.     

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.     

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.     

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.     

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 structure of large-scale extracted β-glucan from barley and oat: Identification of a significantly changed block structure in a high β-glucan barley mutant

Health effects of β-glucan are typically related to dose, size and viscosity without taking the specific molecular structure into account. High β-glucan mutant barley, mother barley and oat β-glucans were large-scale extracted by comparable protocols using hot water, enzyme assisted hydrolysis and ethanol precipitation leading to similar molecular masses (200–300 kDa). Multivariate data analysis on all compositional, structural and functional features demonstrated that the main variance among the samples was primarily explained by block structural differences as determined by HPSEC–PAD. In particular the barley high β-glucan mutant proved to exhibit a unique block structure with DP3 and DP4 contributions of: 78.9% and 16.7% as compared to the barley mother (72.1% and 21.4%) and oat (66.1% and 29.1%). This unique block structure was further confirmed by the ¹H NMR determination of the β-1,4 to β-1,3 linkage ratio. Low solubility of the barley samples was potentially an effect of substructures consisting of longer repetitive cellotriosyl sequences. FT-Raman and NMR spectroscopy were useful in measuring sample impurities of α-glucans and prediction of β-linkage characteristics.     

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.     

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.     

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.     

DETERMINATION OF TOTAL β-GLUCAN IN MALT

An enzymic method for the estimation of total β-glucan in barley has been modified to make it suitable for determination of the small amounts of β-glucan present in malt. Interference from the high levels of reducing sugars in malt has been eliminated by reducing the free sugars in the sample with sodium borohydride rather than extracting them using 80% (v/v) ethanol. The reduction procedure also inactivates endogenous carbohydrate hydrolases in the sample. Because it is no longer necessary to extract the samples with ethanol and centrifuge repeatedly, the modified method is also advantageous in the analysis of barley β-glucan. Errors associated with extraction are eliminated and the speed of analysis of large batches is greatly increased.     

THE USE OF INFRA RED REFLECTANCE FOR THE RAPID ESTIMATION OF THE SOLUBLE β-GLUCAN CONTENT OF BARLEY

It has previously been reported that the malting quality of barley is related to the amount of β-glucan dissolved at low pH under defined conditions. An infra red reflectance instrument, the ‘InfraAlyzer’, can be used to estimate this ‘acid-soluble’ β-glucan content of barley. This is a rapid test (approximately one min/sample) and is therefore suitable when screening for low soluble β-glucan in the early generations of malting quality breeding programmes. The relationship between predicted and measured values for soluble β-glucan differs from comparisons made for other quality components in forages, in that it is curvilinear rather than linear. A logarithmic transformation of the soluble β-glucan values, however, allows the current ‘InfraAlyzer’, which produces linear regressions only, to predict values for soluble β-glucan that correlate well (r = 0.87) with the measured results.     

THE VISCOSITY OF WORTS IN RELATION TO THEIR CONTENT OF β-GLUCAN

A procedure is given for assessing that proportion of wort viscosity which is attributable to β-glucan. Worts obtained from unkilned samples of malt which have been processed for 54 or 72 h show enhanced viscosity. This is principally due to β-glucan although the contribution of other constituents, absent from the wort of fully modified malt, is of significance. Barley variety is shown to have a pronounced effect on wort viscosity. Insoluble β-glucan is brought into solution in mashes at 65° C. The β-glucan isolated from malt which has been inactivated using aqueous ethanol prior to extraction at 65° C, is of higher specific viscosity than that isolated from control worts prepared at the same temperature. The introduction of a rest by mashing initially at 40° C results in the production of wort of lower viscosity, a decrease in the β-glucan content of the wort and a reduction in the specific viscosity of the β-glucan. There is no apparent relationship between the endo-β-glucanase content of the malts and either the viscosity of derived worts or the degree of breakdown of β-glucan which occurred during malting and mashing. Abrasion of barley, which is a factor assisting the distribution of enzymes during malting, acts to reduce wort viscosity.     

A new colorimetric method to quantify β-1,3-1,6-glucans in comparison with total β-1,3-glucans in edible mushrooms

Mushroom β-glucans are known for their activity as biological response modifiers and anticarcinogenic agents. β-1,3-1,6 Branched glucans with a triple helix tertiary structure are recognised as the most potent ones. In the present work, a colorimetric method for β-1,3-1,6-glucan quantification based on the dye Congo red is introduced. This method is specific for β-glucans with a triple helix. The β-1,3-1,6-glucan content of mycelia and fruiting bodies from various mushrooms was determined and compared with the total β-1,3-glucan content, measured by a fluorimetric method. The results show equal amounts of β-1,3-1,6- and total β-1,3-glucans in the analysed species but obvious differences between mycelia and fruiting bodies. On the average, 3% of mycelia and 8% of fruiting body dry mass consist of β-1,3-1,6-glucans. The average percentage of β-1,3-1,6-glucans in the total β-1,3-glucan content differs between mycelia (46%) and fruiting bodies (87%).     

Analysis of mixed-linked (1→3), (1→4)-β-d-glucans in cereal grains from Turkey

In this study, (1→3), (1→4)-β-d-glucan content of some culture type cereal grains grown in Turkey, such as barley, wheat, oats, rye, corn, beans, peas and chick peas; and barley, wheat (white, shain, lanser), rye, orabyehis sativa (adi yonca in Turkish) and medicaga satien (adi korunga in Turkish) grown in the plant research institute of the faculty of Atatürk University in Erzurum (Turkey) were determined quantitatively by enzymatic methods. By using pure β-d-glucanase and β-d-glucosidase in the experiments, (1→3), (1→4)-glycosidic bonds of linear polysaccharides found in cell-wall endosperm of plant seeds were hydrolyzed and the resulting β-d-glucans were determined by glucose oxidase/peroxidase solution and measuring the absorbances at 510 nm in a UV-spectrophotometer. The β-d-glucan levels of cultured cereal grains were compared to those obtained from other countries. Mean whole grain β-glucan levels (dry weight basis) for each grain type analyzed in this study were barley, 3.6%, oats, 4.1%, rye, 1.4% and wheat, 0.8%, corns (I, II), 1.4%, 0.5%, beans (I, II) 2.9%, 2.8%, peas (I, II), 0.9% and 1.4% and chickpeas, 0.9%. β-d-Glucan levels of each grain type grown in the Plant Research Institute of Ataturk University analyzed in this study were: barley, 2.8%, rye, 0.9%, wheats (shain, white, lanser), 5.4%, 0.5%, 0.6%, Orabyehis sativa, 1.4%, and Medicaga satien, 1.1%.     

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.