MALTING AND BREWING WITH BARLEYS HAVING BLUE ALEURONES

Blue aleurones in barley are associated with elevated levels of polyphenolic materials such as anthocyanins and anthocyanidins. A rapid method has been developed for assessing the anthocyanin content of barleys, malts and worts. Malts were prepared from a range of barleys, some of which had intensely blue aleurones, while others were only slightly blue or showed no visible pigmentation. The malting quality of barley was not affected by aleurone colour and ales and lagers of sound flavour as well as acceptable analytical parameters were brewed from malts having pronounced blue aleurones. In some cases sweet worts prepared from blue aleurone malts had a slight pinkish tinge, but this disappeared during wort boiling, and beer colours were normal. Levels of anthocyanins in barley correlated with those in malt and in wort. However, the concentration of anthocyanins was unrelated to the amount of anthocyanogens or total polyphenols. High anthocyanin levels in either barley or beer had no deleterious effects on beer flavour or the rate of haze development.     

INFLUENCE OF SOIL NUTRIENT LEVELS ON HARVEST YIELD AND MALTING QUALITY OF BREWING BARLEY

Four levels of N, P and K nutrition (poor, moderate, satisfactory and high) and all their possible combinations with 64 treatments in two replications (128 plots) were studied in a long term field trial on barley yield and malting quality. A standard East-European spring barley “Opal” (bred in Czechoslovakia) was grown in 1986, 13th year of the agricultural experiment, involving various crops in previous years, on a calcareous loamy chernozem soil. The optimum fertility levels for yield enhancement resulted in the poorest malting quality: low modification and extract but long saccharification time and high protein. To solve this problem the brewing industry will have to apply the well-known technological methods available since growers are not likely to give up their fertilizers. Applying soil and plant analysis data, having knowledge about both soil and plant optimum values, the danger of the excessive use of fertilizers can be realized and decreased.     

PROANTHOCYANIDIN-FREE BARLEY—MALTING AND BREWING

Colloidal haze in beer is due to the precipitation of proteins by proanthocyanidins. Carlsberg Research Center reported in 1977 that the use of barley mutants which block the biosynthesis of catechins and proanthocyanidins in the grain prevents the formation of haze in beer. The results from ten years of malting and brewing research with proanthocyanidin-free barley and malt are reviewed.     

CAMBRIDGE PRIZE LECTURE INDUSTRIAL SCALE RESEARCH INTO MALTING AND BREWING PROBLEMS*

A series of investigations into production processes is described. The interaction of grain and its environment during commercial barley steeping and germination has been examined. Deficiencies which may be induced by anaerobic conditions in steeping appear to be eliminated during germination. Conversely, malt quality may be affected by excessive steep aeration. In a study of commercial brewery fermentations the importance of wort production procedures in allowing an adequate supply of unsaturated fatty acids, essential for satisfactory attenuation, has been demonstrated. Differences in fermentation behaviour cannot be fully explained by the observed yeast sterol contents. Yeast glycogen does not appear to limit fermentation performance. The application of cross-flow membrane filtration (ultra- and microporous filtration) to the recovery of beer from conditioning tank residues has been examined. The pilot-scale production of high quality permeates, and the successful blending of permeate into finished beer, have been demonstrated. The economic advantages of the ultrafiltration process are assessed briefly.     

TECHNICAL OPPORTUNITIES FOR MALTING AND BREWING IN THE ′90's*

That the malting and brewing and associated industries will change technically in the 90's is not in question. The areas described represent some realistic suggestions about where such change may occur. However, because of scientific and technical knowledge available within the community at large no-one should be in any doubt that the present ‘mature’ phase of the industry's technical activities is coming to an end. Consequently the highlighted topics should be viewed as the beginnings of change and the opening up of new opportunities.     

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.     

QUANTITATIVE IMMUNOELECTROPHORESIS OF BARLEY AND MALT PROTEINS

Quantitative immunoelectrophoresis techniques were applied to the study of barley and malt proteins. By crossed immunoelectrophoresis of malt more than 54 immunochemically distinct proteins were distinguished, whereas only 24 proteins have been included in the E.B.C. system of reference based on electro-immunodiffusion. * 1 Electrophoretic separation followed by immunodiffusion (immunoelectrophoresis ad modum Grabar¹¹) is termed “electro-immunodiffusion” in this paper. The use of this term is substantiated in the initial section of “Results and Discussion”.
Crossed immunoelectrophoresis was also used to estimate four aminopeptidases in organs of germinating barley, and to demonstrate non-identity, identity and partial identity between barley and malt proteins. Tandem crossed immunoelectrophoresis was used to compare the proteins in extracts of barley and malt and rocket immunoelectrophoresis to determine an α-amylase in germinating barley. Fused rocket immunoelectrophoresis was used to detect elution patterns of individual barley proteins after ion exchange chromatography, and line immunoelectrophoresis to compare three barley antisera. Advantages of quantitative immunoelectrophoresis over electro-immunodiffusion are demonstrated and discussed. A new system of reference for barley and malt proteins based on crossed immunoelectrophoresis is suggested.     

The Advantages of Using Natural Substrate‐Based Methods in Assessing the Roles and Synergistic and Competitive Interactions of Barley Malt Starch‐Degrading Enzymes

Existing methods of assay of malt starch‐degrading enzymes were critically appraised. New methods based on natural substrates, namely starch and its natural intermediate‐derivative, were developed for all the enzymes, except limit dextrinase for which pullulan was used. Thermostability, optimal temperatures and pHs were established. α‐Amylase and limit dextrinase were the most thermostable and β‐amylase, α‐glucosidase and maltase were the least stable while diastase occupied an intermediate position. The optimal temperatures were congruent with thermostability, β‐ amylase having the lowest (50°C) and α‐amylase the highest (65°C) with the remaining enzymes, including diastase, falling in between. In contrast, α‐amylase has the lowest optimal pH (pH 4.5) and β amylase the highest (pH 5.5) while the others have pHs in between the two values. The roles of the enzymes were evaluated taking into account the level of activity, thermostability, optimum pH, the nature of the product(s), and the relevance to brewing. β‐Amylase production of maltose was synergistically enhanced, mostly by α‐amylase but also limit dextrinase. α‐Glucosidase and maltase are unimportant for brewing, because of their low activity and the negative impact on β‐amylase activity and the negative effect of glucose on maltose uptake by yeast. The starch‐degrading enzymes (diastase) in a gram of malt were able to degrade more than 8 g boiled starch into reducing sugars in 10 min at 65°C. The latter, suggests that it will be possible to gelatinise most of the malt starch at a higher temperature and ensure its hydrolysis to fermentable sugars by mixing with smaller portions of malt and mashing at lower temperatures e.g. 50–60°C.     

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.     

NON-STARCH POLYSACCHARIDES FROM BARLEY: STRUCTURAL FEATURES AND BREAKDOWN DURING MALTING

An extraction procedure is described for the complete solubilisation and fractionation of non-starch polysaccharides (NSP) from barley and malt, using ethanol, water and 4-methylmorpholine-N-oxide. Important structural features of the NSP are preserved with this non-destructive procedure. The fractionation method enables the investigation of structural features of NSP fractions and of NSP breakdown during malting.     

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.     

TOTAL AND INDIVIDUAL BARLEY (1–3), (1–4)-β-D-GLUCANASE ACTIVITIES IN SOME GREEN AND KILNED MALTS

Total β-glucanase activity was measured in extracts of malts prepared from three winter and three spring cultivars of barley. Samples were taken at intervals during modification and, after 116 hr, from malt kilned to 65°C. Good malting varieties showed highest levels of total β-glucanase activity, with a high correlation (r = 0.926) with HWE. Angora had the highest activity in the intermediate stages of malting, least loss of activity after heating extracts to 48°C for 10 mins and least loss of activity on kilning. Separation of isozymes by HPLC⁹ confirmed the greater heat stability of isozyme II, but, unlike previous studies on Australian cultivars, we found considerable activity of isozyme I after kilning, even up to 85°C. However, total β-glucanase activity was destroyed by heating extracts of all varieties to 60°C. Angora showed the highest proportion of total activity as isozyme II after kilining. Cultivar differences suggested some scope for breeding varieties with increased total activity by combining high activities of each isozyme. The high correlation of total activity with HWE suggests β-glucanase activity as a rapid test of malting quality.     

STUDIES ONα-GLUCOSIDASE IN BARLEY AND MALT*

A specific and sensitive method was used to determine α-glucosidase activity in barley and malt. Reliable results were obtained only after extracts of barley and malt had been dialyzed extensively to remove low molecular weight carbohydrates that interfered with the enzyme assay, α-Glucosidase was present in immature kernels of Bonanza and Ellice barley shortly after anthesis but enzyme levels fell rapidly as the kernels matured. A high proportion of the activity was present in pericarp tissue. Enzyme activity increased rapidly in Bonanza and Klages barley during initial stages of germination and fell only slightly during kilning. A high proportion of enzyme activity was present in the scutellum of 4-day germinated barley with lesser amounts in the aleurone and endosperm.     

EXTRACTION OF α-GLUCOSIDASE FROM GERMINATED BARLEY KERNELS

α-Glucosidase activities were extracted from flour of lyophilized, germinated barley seeds. To establish conditions resulting in maximal extraction of α-glucosidase activity from flour of germinated barley, we tested different buffer pH's, extraction times and temperatures, and additions of sulfhydryl reducing reagents, salts, and detergents to the extraction buffer. More enzyme activity was extracted as the extraction buffer pH increased from 3 to 9. The sulfhydryl reagents studied did not significantly increase extractable activity. The extractable activity rose with increased NaCl concentrations up to 2 M. Of the six detergents tested, the zwitterionic detergent CHAPS was most effective in solubilizing α-glucosidase activity, and when combined with NaCl resulted in a doubling in extractable activity as compared with phosphate buffer at pH 8.0. Extraction time and temperature were not critical for the actual solubilization of activity but temperature was quite critical in retaining solubilized α-glucosidase activity in crude extracts.     

‘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.     

THE CAMBRIDGE PRIZE LECTURE 1995: INDIRECT AND DIRECT CONTRIBUTIONS OF BARLEY MALT TO BREWING

The suitability of barley malt as a raw material for brewing is determined by an amalgamation of “indirect” and “direct” contributions to the beer produced. Indirect contributions are considered as those which affect the quality of the brewing process performance whereas direct contributions are considered as those which affect the quality of the product. As a potential indirect contribution of malt to brewing quality evidence is presented that barley malt contains a flocculent which influences mash filterability. As a potential direct contribution of barley malt to beer quality evidence is presented that the mineral silicate found in beer may have a role in moderating dietary aluminium.     

ENVIRONMENTAL AND VARIETAL DIFFERENCES IN TOTAL β GLUCAN CONTENTS OF BARLEY AND THE EFFECTIVENESS OF ITS BREAKDOWN UNDER DIFFERENT MALTING CONDITIONS

The total β glucan contents of a number of barley varieties grown at different sites in England have been determined using a direct enzyme degradation method. Variations in values for a single variety between sites or between harvests indicates that environmental factors are involved in regulating β glucan formation while the involvement of genetic factors is inferred from the finding that the ranking order for different varieties at each site and at each harvest is similar. A single variety of barley has been micromalted under varying conditions of moisture, temperature, gibberellic acid concentration and germination time and a very close correlation between the total β glucan levels and the fine-concentrated difference values has been obtained. This work confirms the relationship obtained earlier with different varieties of malts under a specific malting schedule, indicating that the relationship is one which is widely applicable. The measurement of total β glucan can therefore be used in the estimation of modification and the prediction of brewhouse yield.     

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.     

STUDIES ON BARLEY AND MALT WITH THE RAPID VISCOANALYSER: [III] THE PREDICTION OF MALTING POTENTIAL FROM VISCOGRAMS

Examination of the relationships between extract and viscogram data of malts suggested the measurement of Time to Peak as a cultivar-independent measure of quality potential. This was tested against a large number of samples from the routine assessment programme. Both barleys and malts were examined. Some comparisons with the Falling Number Apparatus are reported. Prediction of malting potential from the differences between viscograms of barleys with and without added enzyme was also assessed. No measurement from the viscogram of barley was sufficiently accurate for predicting malting potential of a population of crossbreds from a breeding programme.