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.     

RELEASE AND ACTIVATION OF BARLEY β-AMYLASE

The aim of this study was to determine the role of low molecular weight thiols both in the release and activation of β-amylase during grain germination. In quiescent barley grains (Hordeum vulgare L. cv. Torrent) about 55% of the β-amylase was extracted with buffer, the remaining 45% was in the bound form. During micromalting the bound form was progressively solubilised between germination days 1 and 4. When free β-amylase, extracted from ungerminated grains, was incubated with dithiothreitol the enzymic activity increased by 15%-20%. This activation did not occur when free β-amylase, from grain germinated for 3 days or more, was incubated with DTT. The release of bound β-amylase with thiols was pH dependant, occurring most rapidly at and above pH 8.0. At the onset of germination the embryo released soluble thiol (approximately 5 nmol per embryo) into the endosperm. Degermed grains were dosed with reduced glutathione and incubated for 72 h. The addition of 60 nmol glutathione caused the release of about 80% of the bound β-amylase. When less glutathione was used, 5 nmol (an amount similar to that released by the embryo in vivo) no significant release of the bound enzyme was detected. When degermed grains were dosed with oxidised glutathione (60 nmol), no bound β-amylase was released. However, addition of the disulphide bis-hydroxyethyldisulphide (60 nmol) did cause the release of about 90% of the bound enzyme. The aleurone layer reduced the bis-hydroxyethyldisulphide to a thiol, presumably 2-mercaptoethanol. Oxidised glutathione and cystine were not significantly reduced to thiols by isolated aleurone layers. The aleurone layer did cause the disappearance of cysteine from solution. When preparations of bound β-amylase were incubated with extracts from the endosperms of grains germinated for three days, the bound enzyme was released. This release was due to the high molecu lar weight material (>5 kDa) in the extract and not to low molecular weight thiols. It seems unlikely that simple thiols, such as glutathione, are solely responsible for the release of bound β-amylase.     

THE CHROMATOGRAPHIC PROPERTIES OF BARLEY AND MALT β-AMYLASES

Barley β-amlyase occurs as a heterogeneous, polydisperse enzyme in thiol-free extracts of Conquest barley. During malting, the polydisperse enzyme is altered, resulting in the formation of four distinct enzyme components which increase in activity as germination progresses. Addition of thioglycerol to a thiol-free extract of barley, or initial extraction with thioglycerol, produces extracts containing two discrete β-amylase enzymes. β-amylase I is the major component of the extract; β-amylase II occurs as a minor component. Similarly, malt extracts containing thioglycerol have two β-amylase enzymes, β-amylase III and IV. Barley β-amylase II and malt β-amylase III have similar chromatographic properties on CM-cellulose but it is not known whether these enzymes are identical. During the early stages of germination, barley β-amylase I disappears and cannot be detected in extracts of 1-day malt; β-amylase III is the major β-amylase enzyme in this extract. Malt β-amylase IV cannot be detected in barley extracts. It develops during germination until it becomes the major β-amylase in malt extracts.     

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 .     

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 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.     

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.     

α-GLUCOSIDASE ACTIVITY OF SORGHUM AND BARLEY MALTS

Sorghum malt α-glucosidase activity was highest at pH 3.75 while that of barley malt was highest at pH 4.6. At pH 5.4 employed in mashing sorghum malt α-glucosidase was more active than the corresponding enzyme of barley malt. α-Glucosidase was partly extracted in water but was readily extracted when L-cysteine was included in the extraction buffer, pH 8. Sorghum malt made at 30°C had higher α-glucosidase activities than the corresponding malts made at 20°C and 25°C. Nevertheless, the sorghum malts made at 20°C and 25°C produced worts which contained more glucose than worts of malt made at 30°C. Although barley malts contained more α-glucosidase activity than sorghum malts, the worts of barley had the lowest levels of glucose. The limitation to maltose production in sorghum worts, produced at 65°C, is due to inadequate gelatinization of starch and not to limitation to β-amylase and α-amylase activities. Gelatinization of the starch granules of sorghum malt in the decantation mashing procedure resulted in the production of sorghum worts which contained high levels of maltose, especially when sorghum malt was produced at 30°C. Although the β-amylase and α-amylase levels of barley malt was significantly higher than those of sorghum malted optimally at 30°C, sorghum worts contained higher levels of glucose and equivalent levels of maltose to those of barley malt. It would appear that the individual activities of α-glucosidase, α-amylase and β-amylase of sorghum malts or barley malts do not correlate with the sugar profile of the corresponding worts. In consequence, specifications for enzymes such as α-amylase and β-amylase in malt is best set at a range of values rather than as single values.     

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.     

SOLUBILITY OF HOP α- AND β-ACIDS IN LIQUID CARBON DIOXIDE*: ADDENDUM: SOLUBILITY OF PESTICIDES IN LIQUID CARBON DIOXIDE

A procedure is described for determining the solubility of hop α- and β-acids in liquid carbon dioxide. Results have shown that the optimum temperature range for the extraction of hops with liquid carbon dioxide is +5 to +10°C. A number of pesticides used by hop growers are appreciably soluble in liquid carbon dioxide.     

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.     

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.     

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.     

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.     

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 MODE OF BINDING OF β-GLUCANS AND PENTOSANS IN BARLEY ENDOSPERM CELL WALLS

β-Glucans in barley endosperm cell walls exist as polymers of very high molecular weight (about 4 × 10⁷ daltons) containing firmly linked peptide sequences. This peptidic material is an essential part of the structure of the β-glucan complex as it exists in the cell wall. Rupture of peptide bonds by hydrazinolysis or with the proteolytic enzyme thermolysin gives β-glucans similar in size to those from short-grown green malts (about 10⁶ daltons). This suggests that proteolysis is the first step in β-glucan degradation. Large β-glucans are not all precipitated in 30% (w/v) ammonium sulphate; only 34% of the β-glucan in a hot aqueous extract of cell walls is precipitated. The amount is increased to 63% if the cell walls have been previously dehydrated. Prolonged incubation of cell wall β-glucan at 40°C, mechanical stress, chromatography lasting 8–10 h at or above 65°C, or chromatography in M sodium chloride causes some disassociation of high molecular weight β-glucan to a size of about 10⁷ daltons. Heating a solution for 1 h at 100°C does not disassociate the β-glucan. Pentosans isolated from cell walls are not covalently linked to the β-glucans and can be separated from them by molecular sieve chromatography. They have a higher xylose/arabinose ratio than previously reported for barley pentosans. The pentosan molecules extracted by water are smaller (10⁶ daltons) than those extracted by alkali (5 × 10⁶ daltons). Little difference was observed in the chemical or physical properties of cell wall materials of barley cultivars of different malting qualities.     

DETERMINATION OF HIGH MOLECULAR WEIGHT β-GLUCAN IN WORT AND BEER USING A POST-COLUMN CALCOFLUOR FLOW-INJECTION-ANALYSIS (FIA) METHOD

To eliminate the influence of maltose, ethanol, low molecular weight β-glucan and an inhibitor of the calcofluor fluorescence reaction in wort and beer on the measured values of a calcofluor-FIA, a post-column calcofluor-FIA method has been developed using a short-size gel permeation chromatography column (6.0 × 50 mm). A column packed with polyhydroxymethacrylate gel (molecular weight exclusion limit, 100,000) was found to be the most appropriate for this system. This short column allowed rapid and specific measurement of high molecular weight β-glucan in a few minutes without the influence of the fluorescence inhibitor, maltose and ethanol which have molecular weights of less than 1000 daltons. Because the low molecular weight β-glucan responsible for the scatter caused by a slight difference in measuring conditions such as temperature, calcofluor concentrations, sample volumes, etc., was separated through the use of the column, the measured values by the post-column calcofluor-FIA method hardly fluctuated under different conditions. Though it has been recognized that dilution of a sample could affect a calcofluor FIA, the new system was not influenced. This also made it possible to measure the β-glucan content in dark-coloured samples (even over 100 EBC colour units). The measured values by the post-column FIA showed a high correlation (r² = 0.993) with those obtained by the enzymatic method (the McCleary method).     

TISSUE DISTRIBUTION OF α-AMYLASE AND PHOSPHORYLASE IN DEVELOPING BARLEY GRAIN

Approximately half the total α-amylase and phosphorylase activity detectable in crude homogenates of the tissues of developing barley grain was localized in the pericarp. This tissue is particularly active in the early stages of grain development. It is suggested that this activity may influence the starch type and content of the mature barley grain.