A RAPID,SIMPLE VISCOMETRIC TECHNIQUE FOR INDIRECT ESTIMATION OF SOLUBLE β-GLUCAN CONTENT OF RAW BARLEY

A Simple, rapid method of estimating the viscosity of raw barley extract is presented which involves measurement of the time of passage of a ball falling between two points in a precision bore tube filled with the extract. This value for falling time is directly related with β-glucan. The technique requires only 0·6 g of ground grain which means that it is now practicable to test material from a single plant. In view of the considerable number of samples involved in a breeding programme the high testing rate of up to 144 samples a day is of considerable practical importance.     

THE ESTIMATION OF β-GLUCAN IN BARLEY

The method described involves a differential assay in which the glucose content of a total acid hydrolysate of an extract of barley is estimated by glucose oxidase, and the α-glucan content similarly determined after treatment of the extract with amyloglucosidase. The difference between these estimations represents the glucose derived from β-glucan. The method provides reproducible results which are relatively insensitive to minor variations in the assay conditions, apart from temperature of extract preparation.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

THE EBC METHODS FOR DETERMINATION OF HIGH MOLECULAR WEIGHT β-GLUCAN IN BARLEY,MALT, WORT AND BEER

Two new methods for the determination of β-glucan in barley, malt, wort and beer are accepted as EBC Standard Methods for β-glucan by the Analysis Committee of the European Brewery Convention. One method (The fluorimetric method) is based upon the specific interaction of the fluorochrome Calcofluor towards high molecular weight β-glucan. The method is suitable where a high number of determinations have to be done routinely. The other method (The enzymatic method) utilizes a specific degradation of the β-glucan by purified enzymes. This method is more laborious, but it can be carried out using apparatus available in an analytical laboratory. The methods, which are described in detail, have been tested in a EBC Collaborative Test with 14 participating laboratories. Barley, malt and beer samples at 6 levels of concentration were tested for β-glucan. The collaborative test resulted in acceptable statistical values for repeatability and reproductibility for both methods. Comparison of the results from the two methods, showed that they are highly correlated and that the actual values of β-glucan content are comparable.     

SEVERAL NEW FACTORS INFLUENCING THE MEASUREMENT OF β-GLUCAN CONTENT USING CALCOFLUOR FLOW-INJECTION ANALYSIS METHOD

Factors such as temperature, calcofluor concentration, sample injection volume, void volume of the mixing tube and other factors have been reported to influence the calcofiuor flow-injection analysis (FIA) of β-glucan. In addition to these factors, we revealed the following factors and elucidated their effects on the FIA of β-glucans: the inside diameter and arrangement of the mixing tube, calibration method using peak area or peak height of the edition patterns, and the quality of the calcofluor reagent. An apparently lower value was obtained when (1) the mixing tube had a smaller inside diameter, (2) the mixing tube was looped in a ring of smaller diameter, or (3) the mixing tube was wound as double coils in a figure eight instead of a loop. Furthermore, it was indicated that the peak area calibration produced higher measures values than the peak height calibration. The reagent also affected the results; a calcofluor reagent from Sigma Chemical Co. gave a higher value than one from Polyscience Inc. It was concluded that these troublesome phenomena were derived from the difference between the test sample and the standard solution of β-glucan, that is, the molecular weight distribution of β-glucans and/or the sugar and ethanol in the sample solutions. Based on these findings, it is suggested that international standardization of the FIA method for β-glucan be made.     

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.     

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.     

SOLUBILIZATION OF β-GLUCAN DURING MASHING

β-Glucan can be solubilized during mashing by an enzyme or enzymes differing from, and more heat-stable than, the endo-barley-β-glucanase which reduces the viscosity of solutions of barley β-glucan.