DEGRADATION OF HORDEIN BY THE PROTEOLYTIC ENZYMES OF BARLEY AND MALT

Development of the endo- and exo-peptidases that degrade the major barley storage protein, hordein, during malting is affected by treatment with gibberellic acid and potassium bromate, and also by the moisture levels attained during malting. For a given set of malting conditions, cultivars had different peptidase activities, but there were no consistent comparable differences between cultivars in amylase or endo-β-1,3 glucanase activities.     

VARIETAL IDENTIFICATION OF BARLEY AND MALT*

A vertical polyacrylamide — SDS electrophoretic technique, including whole protein extraction and staining steps, was improved with a view to developing it for routine laboratory use with single barley kernels. The pattern consisted of 4 zones: A (albumins-globulins). B and C (hordeins) and D (possibly glutelins) displaying unequal varietal polymorphisms (1, 13, 13 and 4 types respectively). 28% of the barley samples (77 varieties), including most cultivars grown in France, could be unambiguously identified from qualitative differences only, in the B, C and D zones. Adding three other characteristics (hairs and furrow hairiness, peroxidase, zymogram, esterase zymogram), as many as 78% of the varieties could be identified, the other 22% consisting of very closely related barleys. After slight modification of protein extraction conditions, the same methods could be used with malt, based on the same electrophoretic types. A graphic tablet connected to a microcomputer was used for automatic acquisitions of records and comparisons of electrophoretic data.     

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.     

BARLEY AND MALT TANNINOGENS AND BEER QUALITY

Anthocyanogen and catechin contents (tanninogen values) were determined for ten two-row and thirteen six-row barleys and for their corresponding malts. Four barley-malts were then selected for brewing, one with high, one with low, and two with intermediate tanninogen contents. The brews were made using bottom-fermenting (lager) as well as top-fermenting (ale) yeasts, both at 50–55° F. and at 68° F. The quality of the beers, as expressed by standard analyses and flavour evaluation, is discussed in the light of the tanninogen contents of the barleys and the different brewing parameters (yeast type and fermentation temperature).     

BARLEY AND MALT ANALYSIS—A REVIEW

This review describes the developments in barley and malt analysis since 1960, the suitability of analyses commonly used at present, and changes which are likely to occur in future. The review is not restricted to analyses suitable for brewers and distillers but also discusses methods used in the control of malting and in the selection of barley for malting.     

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.     

RAPID SMALL-SCALE DETERMINATION OF MALT EXTRACT IN BARLEY BREEDING

A simple procedure for the determination of malt extract requiring only 0·5 g of malt is described. A constant volume of hot water is added and the mash is centrifuged rather than filtered. The extract is then estimated directly from a refractometer reading. The relationship between refractometer reading and wort solids was linear and was not found to vary with barley variety or grain nitrogen. The effect of varying the grist/mash ratio was investigated. The method had similar precision to other methods, but was more rapid, making it useful in barley breeding.     

A RAPID AND SIMPLE METHOD FOR THE DETERMINATION OF STARCH AND β-GLUCAN IN BARLEY AND MALT

A simple and precise method suitable for the routine determination of starch and β-glucan in barley and malt is described. Perchloric acid (50 mM) was used to effect rapid (3 min) and exhaustive extraction of both glucans which were then measured directly from this single extract by specific enzymic hydrolysis of the individual glucans to glucose. The glucose was also measured enzymically. Little or no acid hydrolysis of starch or β-glucan was observed under the extraction conditions used; most or all of the free glucose could be attributed to hydrolysis of sucrose. Complete solubilisation of the gum and hemicellulosic components of β-glucan was achieved. Preincubation of the acid extracts with protease prior to amyloglucosidase digestion resulted in higher measurements (approximately 4% w/w) of starch. The method was used to measure the levels of starch and β-glucan in five varieties of barley with contrasting malting quality, in micro-malts prepared from these samples and in commercial lager and ale malts.     

THE RELATIONSHIP BETWEEN SOME BARLEY AND GREEN MALT PROPERTIES AND MALT HOT WATER EXTRACT

Further studies have been made on the relationships between malt hot-water extract (HWE) and some barley and green malt characters previously identified as influencing HWE.^17,18 Relationships were found to be relatively consistent between spring and winter barleys and over years, suggesting that these characters play an important fundamental role in determining the HWE attainable under standard malting conditions.     

DETERMINATION OF THE MOISTURE AND NITROGEN CONTENTS OF BARLEY AND MALT BY NEAR INFRARED SPECTROSCOPY (NIRS)

The determination of the moisture and nitrogen contents of barley and malt by near infrared spectroscopy (NIRS) has been tested by the Analysis Committee of the European Brewery Convention. In the collaborative trial four samples of barley and malt were analysed by 17 laboratories. Repeatability (r₉₅) and reproducibility (R₉₅) values of 0.3 and 1.5% m/m respectively were obtained for barley moisture over the range 12.7 to 15.8% m/m. For malt moisture these values were 0.2 and 1.3% m/m over the range 4.0 to 4.3% m/m, for barley nitrogen 0.1 and 0.3% m/m on dry matter over the range 1.57 to 2.14% m/m, and for malt nitrogen 0.1 and 0.2% m/m on dry matter over the range 1.58 to 1.82% m/m, respectively.     

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.     

QUANTIFICATION OF MOULD CONTAMINATION IN SULPHURED AND UNSULPHURED MALT

The use of different techniques for assesment of mould contamination in barley and malt is illustrated by reference to an examination of the effect of sulphuring of malt. Reductions in levels of mould contamination were detected in sulphured malts, compared with corresponding unsulphured controls, using direct and dilution plating techniques. Viable counts of wild yeasts, bacteria and thermophilic actinomycetes were also lower in sulphured malts.     

EVALUATION OF BARLEY AND MALT QUALITY USING NEAR-INFRARED REFLECTANCE TECHNIQUES

A scanning near-infrared reflectance spectrophotometer was calibrated for the prediction of barley aleurone colour and malt moisture. The malt moisture was predicted on malt ground for the determination of malt extract (coarse grind) making the method suitable for moisture correction in malt extract estimation. Calibrations for the prediction of malt extract and endosperm modification from barley and malt were also attempted. A correlation (r= 0.851 n = 135) was found between malt hot water extract and the percentage of the endosperm estimated as being modified by microscopy following staining with Calcofluor. Probably because of this influence of modification on malt extract, the use of near-infrared reflectance to predict malt extract was most successful at predicting the malt extract values obtained following micro-malting in the absence of the additives, gibberellic acid and potassium bromate.     

EFFECTS OF GAMMA IRRADIATION OF BARLEY AND MALT ON MALTING QUALITY

Two two-rowed barley cultivars, Tokak and Clerine, were irradiated at two different dose ranges (0.05–0.75 kGy and 0.5–5.0 kGy) using a ⁶⁰Co source. Irradiation of barley at the medium levels before malting had detrimental effects on most of the malt quality criteria. The detrimental effects of irradiation was lower at doses up to 0.25 kGy. Irradiation of malt samples caused either slight or no deterioration of quality characteristics .     

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.     

ACTIVITIES OF VARIOUS PEPTIDASES IN BARLEY,GREEN MALT AND MALT

The three groups of malt peptidases (carboxypeptidases, “naphthylamidases” and (amino) peptidases acting on Leu-Tyr and Ala-Gly) are present in unmalted barley; the activities are low and of a similar order of magnitude. On germination the activities of the different carboxypeptidases increase from 10- to 20-fold; the “naphthylamidases” increase only 2-fold, and the peptidase activities increase from 3- to 6-fold. None of these enzymes is inactivated during kilning to any significant extent. There are considerable differences between the carboxypeptidase activities of malts derived from different varieties of barley and the activities are correlated with α-amylase activity.     

RAPID PREDICTION OF MALT HOT WATER EXTRACT BY NEAR INFRARED REFLECTANCE SPECTROSCOPY STUDIES ON BARLEY

In recent years there has been an increasing awareness of the need for rapid screening tests for malting quality in barley. An infrared reflectance instrument has been calibrated against malt hot water extract (HWE). Results suggest that this type of instrument can be used to estimate HWE on barley and that this might provide a suitable screening method for use in a breeding programme. Greater accuracy is achieved if separate calibrations are made for winter and spring barleys and the correlation coefficients with HWE were 0.70 (n = 168) and 0.82 (n = 134) respectively.     

LIPIDS OF BARLEY,MALT AND ADJUNCTS

Barley contains up to 4·4% of its dry weight as lipid when measured as total fatty acid. Commercial malts can contain up to 3·4% lipid and the proportions of the constituent fatty acids are similar to those of barley. About 70% of the fatty acids of barley and malt are present as triglycerides whilst up to 8% of the lipids in malt are found as free fatty acids. Germination results in a fall of up to 30% in the lipid content, mainly due to the hydrolysis of triglycerides and metabolism of the released fatty acids. The immediate products of triglyceride breakdown, namely fatty acids and mono- and diglycerides, do not accumulate in malt to any significant extent. Common brewing adjuncts contain up to 4% lipid.     

EVALUATION OF THE INFRAALYZER 400 FOR THE ANALYSIS OF BARLEY AND MALT

A collaborative study is described of the use of infra-red reflectance for the evaluation of the analytical characteristics of barley and malt. Transfer of centrally-prepared calibrations to other reflectance machines was found to be possible for moisture content and protein content in barley and malt but not for malt extract or malt modification. The examination of infra-red reflectance results, obtained at different wavelengths, by step-wise ascending regression rather than step-wise descending regression showed that the former was more satisfactory, especially in being more rapid in execution.     

A MICROBIOLOGICAL EVALUATION OF BARLEY MALT PRODUCTION

The development of the microflora of barley malt was examined by direct and dilution plating. At all stages of the malting process mesophilic bacteria predominated. Viable counts of bacteria on green malt were 85–600 times greater than on the original barley, but fell to less than one-half of the original level with kilning. Corresponding increases in yeast and, especially, mould counts during malting were smaller. The yeast-like mould Geotrichum candidum was prominent in green malt. Although counts of yeasts and most moulds were considerably reduced by kilning, Mucor spp. proliferated during kilning.