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.     

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.     

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.     

VARIATION IN A BARLEY COLLECTION FOR ENDOSPERM ATTRIBUTES THAT RELATE TO MALTING QUALITY

Éstimates of grain hardness from measurements of milling energy were made on four hundred and eighty barley cultivars. The same cultivars were also subjected to an infra-red analysis used to predict soluble β-glucan and nitrogen contents. The results demonstrated variation for these endosperm characters, with eighteen cultivars having a lower milling energy, and more than a hundred cultivars with a lower soluble β-glucan content than Gerkra, a barley with good malting quality. Of these, nine cultivars were lower for both characteristics. Information about this variation is, therefore, potentially useful when breeding for malting quality.     

VARIATION IN THE BIOCHEMICAL COMPOSITION OF ACID EXTRACTS FROM BARLEYS OF CONTRASTING MALTING QUALITY

Acid extracts from barleys of poor malting quality were characterized by high viscosity caused by enhanced concentrations of high molecular weight carbohydrates of which more than 80 per cent was β-glucan. Up to 67 per cent of the β-glucan in these barleys was soluble in the acid extracting solution. In contrast, only about 25 per cent of the β-glucan in good malting barleys was soluble, resulting in extracts having low viscosity. No evidence was found for autolysis in any of the extracts, and extracts from good malting varieties were characterized by high concentrations of fructose and sucrose. Protein concentration in the extracts varied little, although the protein content of the barleys ranged from 9·1 per cent to 15·4 per cent. Soluble protein has no effect on extract viscosity or the gel filtration properties of the carbohydrates. Some quantitative variation in acid-soluble and salt-soluble proteins was seen after electrophoresis on polyacrylamide.     

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.     

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 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.     

NEW ANALYSES IN MALTING AND BREWING*

Recent advances in our knowledge of the plant physiological and biochemical processes surrounding malting and brewing have prompted the development of a new generation of analytical techniques. These techniques are based upon the measurement of biochemical and physical parameters which have lately been shown to directly influence the quality of both malt and beer. Recognition of these parameters, coupled with a deep insight of the physical and chemical changes that can occur during the entire malting and brewing production chain, allow strategic emplacement of new analyses in order to provide a superior specific monitoring of malting and brewing. In the present context we submit several examples of these novel analyses, spanning from barley control to analysis of pasteurising efficiency. Characteristic for all the analyses discussed, is the ease and rapidity with which they can be performed. The following methods are described: (1) Measurement of both pre-harvest sprouting and germination efficiency in barley. (2) Malt modification analysis to measure malting efficiency, time of kilning and malt homogeneity. (3) Rapid β-glucan analysis in malt, wort and beer. (4) Immunological analysis to determine the amount and type of additives in finished beer as well as addition of adjunct. (5) Enzymic and immunological control of beer pasteurisation.     

LEVELS OF ALKALI-SOLUBLE β-D-GLUCANS IN CEREAL GRAINS

Extraction of β-D-glucans (primarily from barley grains) using sodium hydroxide solution, gave similar results to those obtained using hydrazine extraction, suggesting that the alkali is as effective as hydrazine for the extraction of β-D-glucans. Commercial growth conditions in different European countries caused limited shifts in the β-D-glucan levels of malting barley samples. Sorghum and wheat grains contained significantly less β-D-glucans than barley. Potassium bromate solution inhibited initial breakdown of β-D-glucans during the malting of barley grains. The breakdown of β-D-glucan in Sonja barley was much slower than in Golden Promise barley, during malting.     

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.     

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.     

QUANTITATIVE CHANGES IN THE CONCENTRATIONS OF THE MAJOR COMPONENTS OF FOUR GENOTYPES OF BARLEY GRAINS DURING MALTING AND MASHING

Four genotypes of barley, including good and poor malting varieties, were sampled as grain, green malt, kilned malt and spent grains. Each of these samples were analysed for total protein, aggregated protein, total and soluble β-glucan, starch and husk contents. Protein sub-units were separated using sodium dodecyl sulphate polyacrylamide electrophoresis. Activities of β-glucanase, endopeptidase and α-amylase were measured and starch from each sample was purified and separated into large and small granules, and analysed for total protein and sub-unit protein content. Results calculated as % of dry weight and as a proportion of the weight of dry grain showed quantitatively the changes which occurred in the components of the grain during malting and mashing. Comparisons of the composition of the genotypes at the various stages showed that the best malting variety studied, Ark Royal, was better because of moderate superiority in several characters rather than a fundamental difference in a single attribute and supports the thesis that to further improve malting quality plant breeders should select for several characters which are independently inherited.     

‘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.     

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.     

SOME BARLEY GRAIN AND GREEN MALT PROPERTIES AND THEIR INFLUENCE ON MALT HOT-WATER EXTRACT: I. β-GLUCAN, β-GLUCAN SOLUBILASE AND ENDO-β-GLUCANASE

A study has been made of the variation between varieties in some properties of barley and malt and how this variation relates to malt hot water extract (HWE). The development of enzyme activity along the grain during germination was investigated. In this first paper we have examined β-glucan-related characters and found significant varietal variation in maximum enzyme activities and in the activities in different sections of grain during germination. Varietal variation was greater than environmental variation for each character. The fraction of β-glucan soluble in acid was the character most highly correlated with HWE.     

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.     

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.     

THE MAJOR BIOCHEMICAL CONSTITUENTS OF AN ACID EXTRACT OF BARLEY FLOUR AS USED IN VISCOSITY DETERMINATIONS BY THE FALLING BALL METHOD

The viscosity of an acid extract of barley flour, measured as its ‘falling time’, has been used as a guide to malting potential.²² One such extract, made from a sample of Maris Mink, was examined in detail to identify the principle biochemical constituents and to investigate their contribution to viscosity. The extract contained a high concentration of carbohydrate (3·30 mg/ml) divided into two discrete fractions of high and low molecular weight (in the weight ratio of 39:61) by gel filtration. The high molecular weight component was polydisperse and consisted principally of β-glucan with some starch and pentosan also present. The low molecular weight fraction contained mostly fructose, glucose and some pentose. Protein present in the extract (1·07 mg/ml) was derived almost exclusively from the albumin and globulin (salt soluble) fraction of barley protein. Studies on the viscosities of reconstituted preparations of these components indicated that β-glucan made the major contribution to the viscosity of the extract. This conclusion is supported by observed changes in viscosity after the extract was treated with β-glucanase, α-amylase and protease.     

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.