THE MALTING QUALITY OF SOME SPRING BARLEY VARIETIES GROWN IN ENGLAND AND WALES BETWEEN 1880 AND 1980

The malting behaviour of fifteen barley varieties widely grown between 1880 and 1980 has been compared using material grown at two sites in England in 1980. All the varieties examined were considered to be of malting quality at their time of widespread commercial use. Varieties introduced after 1950 gave higher Hot Water Extracts (mean 74·7%) than varieties introduced before 1950 (mean 71·0%), and when the yield data were included to calculate the yield of extract/ha the post-1950 varieties were found to exceed the older varieties by 48·7%. The Kolbach Index of the modern varieties was higher, although there was no evidence of an increase in cold water extract.     

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.     

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 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.     

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 RELATIONSHIP BETWEEN TOTAL β-GLUCAN OF MALT AND MALT QUALITY

The total β-glucan content of barley and malts has been determined using a direct enzyme degradation method incorporating measures to ensure inactivation of any contaminating amyloglucosidase. Results for barleys range between 2·7 and 4·4% w/w and indicate genetic variation in the β-glucan content. Malts produced by both a laboratory micromalting procedure and commercially have been analysed for total β-glucan, extract and 70°C mash viscosity at different stages of germination and in the end product. A very good correlation has been found between total β-glucan and fine-concentrated extract difference values showing that in a fully modified malt having a negligible extract difference value, all the β-glucan material is degraded. The extract difference value had been demonstrated earlier to be closely linked with brewhouse extract yield. The total β-glucan of malt, therefore, is directly associated with achievable extract in the brewhouse and is the most important biochemical factor determining the extractability of a malt.     

RELATIONSHIPS BETWEEN β-AMYLASE POLYMORPHISMS IN DEVELOPING,MATURE AND GERMINATING GRAINS OF BARLEY

A β-amylase polymorphism (electrophoretic forms Sd1 and Sd2) in barley malt is shown to be closely associated with the proportion of free to total (free plus latent) β-amylase, but not with the level of total β-amylase in the mature grains of 46 cultivars. All of the cultivars with Sd1 malts have a proportion of free β-amylase less than 50% (usually from 30% to 40%) of total whereas Sd2 types have free to total β-amylase (F/T) ratios greater than 50% (usually between 62% and 78%). These polymorphisms are also correlated with forms of β-amylase in the developing grain, although, in the latter, Sd1 cultivars can be divided into two types, Sd^d and Sd^e which cannot be distinguished either in malts or on the basis of F/T ratio. Unusual F/T ratios of an intermediate type (approximately 50%) and a very low type (under 30%) also occurred in these experiments. These may result from environmental effects or may be new genetic types.     

‘FREE’ AND ‘BOUND’ β-AMYLASES DURING MALTING OF BARLEY. CHARACTERIZATION BY TWO-DIMENSIONAL IMMUNOELECTROPHORESIS

Major qualitative and quantitative changes in the β-amylases and in other salt soluble barley proteins occurred during the first four days of germination. Two soluble forms of barley β-amylase, ‘free’ β-amylase and β-amylase aggregated with a non-active protein Z, were found in extracts from all stages. A third enzyme form appeared during malting. Immunoelectrophoretic characterization seemed to support the possibility that this enzyme form could be a product of ‘bound’ β-amylase solubilization. All soluble forms of β-amylase and of protein Z in malt were electrophoretically heterogeneous. Two different, immunochemically related forms of protein Z present after malting retained their immunoelectrophoretic properties during brewing and were found to be dominant antigens in beer.     

EFFECTS OF CULTIVAR AND ENVIRONMENT ON β-GLUCAN CONTENT AND MALTING QUALITY OF TURKISH BARLEYS

Eight barley cultivars grown under the same agronomic conditions and samples of Tokak cultivar grown at six different sites of Turkey were used in this study. There were significant differences among the barley cultivars and growing locations in terms of β-glucan content (p<0.05). Among malt quality criteria tested for the 8 barley cultivars; friability, viscosity, Kolbach index and extract difference showed significant correlations (p<0.05) with the total β-glucan content. Similar correlations were also observed between the malt quality criteria (Kolbach index and extract difference) and β-glucan contents for the Tokak samples grown at different sites.     

CHANGES IN THE CATIONIC ISOENZYMES OF PEROXIDASE DURING THE MALTING OF BARLEY II: THE EFFECT OF GIBBERELLIC AND ABSCISIC ACIDS

During germination, alpna-amylase activity in barley (variety: Chariot) increased 29-fold, while peroxidase activity increased 4.2-fold. Increasing concentrations of GA₃ increased the rate of development of alpha-amylase activity while the rate of increase of peroxidase activity remained unchanged, but there was an overall increase in the amount of peroxidase synthesized. ABA caused a concentration-dependent suppression of activity and a decrease in the rate of expression of both enzymes. Three different types of response of individual peroxidase isoenzymes occurred during germination. Six isoenzymes increased in activity during malting, four decreased in activity and nine showed a rise at the beginning of germination followed by a significant fall. The majority of the isoenzymes were positively GA₃-sensitive, ie increased in activity or were synthesized earlier in the presence of GA₃, and negatively ABA-sensitive. Only four isoenzymes were negatively GA₃-sensitive, and only three were positively ABA-sensitive. Detailed data for the synthesis during germination of the six major peroxidase isoenzymes that survive into the finished malt is presented .     

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.     

ASSAY OF MALT β-GLUCANASE USING AZO-BARLEY GLUCAN: AN IMPROVED PRECIPITANT

A procedure recently described for the assay of malt β-glucanase, which employs a dye-labelled and chemically-modified barley β-glucan substrate, has been improved by changing the precipitant solution used to terminate the reaction. The new precipitant solution contains 0·4% (w/v) zinc acetate and 4% (w/v) sodium acetate dissolved in 80% (v/v) aqueous methyl cellosolve. With this precipitant the procedure can be directly applied to the assay of cellulase activity, and with minor modification, to the assay of lichenase activity.     

A COMPARISON OF TWO METHODS FOR THE ESTIMATION OF β-GLUCANASE ACTIVITY IN MALT

A new method for estimating malt β-glucanase activity, based on a chemically modified, azo dye-labelled barley β-glucan substrate, has been compared with a viscometric procedure. The azo-barleyglucan method was found to be simple to perform, to give excellent precision with good reproducibility between laboratories and to have a high correlation (r=0·903***) with the slower viscometric procedure currently used by the industry.     

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.     

ENZYMATIC DETERMINATION OF 1,3:1,4-β-GLUCANS IN BARLEY GRAIN AND OTHER CEREALS†

A direct and specific enzymatic method is described for the determination of 1,3:1,4-β-glucans in barley grain and other cereals. In the procedure, purified, amylase-free, bacterial 1,3:1,4-β-glucan hydrolase is used to depolymerize the 1,3:1,4-β-glucan in autoclaved and ethanol-extracted flour prepared from whole grain. The liberated oligoglucosides are extracted with 80% (vol/vol) ethanol and, following acid hydrolysis, measured by the glucose oxidase method. The method can be used to measure total β-glucan and β-glucan in 65°C water-soluble and water-insoluble fractions of cereal grains. The procedure has been applied to barley grains allowed to develop under controlled environmental conditions and to a series of barleys of different geographical origins. The results for Canadian cultivars are compared with estimates based on viscometric data for the same samples. The 1,3:1,4-β-glucan content of a number of other cereals has also been measured.     

OPTIMIZED McCLEARY METHOD FOR MEASUREMENT OF TOTAL β-AMYLASE IN BARLEY AND ITS APPLICABILITY

The McCleary method for determination of β-amylase in malt has been modified in order to allow determination of total β-amylase in barley as well as malt. A ruggedness test, performed on the modified method, demonstrated that the method is quite robust and highly reproducible. When the variables α-amylase, β-amylase and diastatic power were measured in 90 malt samples, only β-amylase was significantly correlated to diastatic power (r² = 0.85 and p < 0.0001). The same high correlation was found between total β-amylase in 20 barley samples and diastatic power in the corresponding malts. The validity of this relationship was tested by predicting diastatic power in malt from total β-amylase in barley. Predicted values correlated highly to measured values (r² = 0.95). In breeding material a positive relationship was found between total β-amylase in barley and protein content. This relationship must be considered when evaluating new barley lines.     

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.     

THE MEASUREMENT OF DIMETHYL SULPHOXIDE IN BARLEY AND MALT AND ITS REDUCTION TO DIMETHYL SULPHIDE BY YEAST

Dimethyl sulphoxide (DMSO) is a normal component of malt and barley. A method is described for its extraction and estimation. DMSO is produced by the oxidation of dimethyl sulphide (DMS), particularly during kilning of malt, and higher levels are found in malts subjected to ale kilning schedules. DMS may also be oxidized during wort preparation. DMSO can be reduced to DMS by yeast in glucose/salts medium, by yeast cell suspensions and by a cell-free extract. Reduction of DMSO is inhibited by methionine sulphoxide. The results suggest that reduction of DMSO may account for the DMS produced during fermentation of ale and lager worts.     

ISOLATION,PURIFICATION AND ELECTROPHORETIC PROPERTIES OF AN α-AMYLASE FROM MALTED BARLEY

An α-amylase component from malted barley was isolated and purified using aqueous extraction at pH 8·0, heat treatment of the extract at 70°C, specific precipitation with glycogen and ion exchange chromatography on carboxymethyl (CM) and diethylaminoethyl (DEAE) cellulose. The enzyme preparation was shown to be pure by disc electrophoresis at pH 8·9 and iso-electricfocusing on polyacrylamide gel in a pH 4–8 gradient.     

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.