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.     

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.     

RECOGNITION OF TWO LIPASES FROM BARLEY AND GREEN MALT

Lipase was extracted from ground samples of germinating and ungerminated barley, using the detergent Triton X-100. Enzyme activity, measured by the release of oleic acid from radioactively labelled triolein, was approximately half that in suspensions of barley flour. Two distinct lipases, both active at neutral pHs, and having similar molecular weights (around 400,000) but differing in their ionic properties, were isolated from barley. The more abundant lipase was found mainly in the embryo, while the other was located in the endosperm. Both total and extractable lipase activity increased during germination.     

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).     

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.     

COMPARATIVE MALTING PERFORMANCE OF OLD AND NEW BARLEY VARIETIES

A comparison was made of the malting behaviour of eight barley varieties representing a range from ancient to modern types. Plants were grown under controlled conditions in a glasshouse with two different nitrogen treatments and the grain obtained was subjected to a factorial micro-malting analysis. Under the conditions of this experiment there was little evidence of an improvement in malting ability between Plumage Archer and Ark Royal and there is limited scope for further increases in potential hot water extract. It would therefore appear that emphasis should now be given to the production of varieties with good agronomic characteristics which maintain this potential and modify rapidly without the need for abrasion and exogenous GA₃.     

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.     

HORDEIN IN BARLEY AND MALT—A REVIEW

The chemical nature of hordein and current methods of extraction and fractionation are reviewed. The importance of hordein in identifying barley varieties and in predicting and assessing malting performance is discussed. Proteolytic breakdown of hordein during malting and mashing is summarized and the composition of hordein from barley, malt and spent grains is compared.     

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.     

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.     

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.     

ANALYSIS OF WHOLE BARLEY KERNELS USING NEAR INFRARED REFLECTANCE SPECTROSCOPY

Near infrared (NIR) reflectance spectroscopy has been evaluated for the rapid analysis of whole barley from the 1984 and 1985 crops. Excellent agreement was obtained between the NIR and the reference method for moisture with a correlation coefficient (r) of 0-980 and a standard error of prediction (SEP) of 0-15% for the 1984 crop. Similar results were obtained when the data for both crops were combined. The total nitrogen analysis was less accurate (r 0-844, SEP 0-096%) using a calibration comprising five varieties. Better accuracy was achieved using a single variety calibration (r 0-948, SEP 0-052%). It was also possible to estimate Hot Water Extract values (r 0-920, SEP 3-2 1°/kg) from scans on whole barley without the need for malting or mashing.     

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.     

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.     

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 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.     

INSTITUTE OF BREWING: ANALYSIS COMMITTEE RECOMMENDED MILL FOR MALT ANALYSIS

The Miag Disc Mill is now the standard mill for malt analysis. A method of setting the gap between the grinding discs by mean of a feeler gauge has been adopted. Using the recommended setting 5.0 for the measurement of Hot Water Extract the replication error is 0.8 litre °/kg. The error due to the mills is negligible compared with the errors arising from the remaining parts of the measurement.     

CHANGES IN THE CATIONIC ISOENZYMES OF PEROXIDASE DURING THE MALTING OF BARLEY. I: TISSUE LOCATION STUDIES

Sixteen cationic isoenzymes of peroxidase were detected in barley grain (variety: Chariot) and twenty in green malt, falling to fifteen after kilning. The distribution of these among the different seed tissues was examined with a view to determining which isoenzymes were likely to survive mashing and be active during brewing. In isolated barley tissues, the aleurone layer contained one major band (Rm 0.77), and the endosperm only the four fastest running bands (Rm values 0.84–0.97). In green malt the growing embryo tip contained fifteen isoenzyme bands (Rm values 0.04–0.70), with an absence of only the faster running bands. In kiln-dried malt, isoenzymes with Rm values 0.04–0.10 increased in activity, two extra light bands appeared (Rm 0.26 and 0.61) and three other bands present in barley were lost (Rm values 0.63, 0.70 and 1.0). Much of the activity in the aleurone layer was lost. Seven of the fifteen isoenzymes in finished malt accounted for over 75% of the cationic peroxidase activity. Variation in aleurone and endosperm isoforms between different cultivars was observed, whereas the embryo showed little variation. The endosperm isoforms seem to be the ones most likely to cause oxidation problems during brewing.     

DETERMINATION OF TOTAL NITROGEN IN BARLEY AND MALT BY COMBUSTION METHOD—COLLABORATIVE TRIAL

A combustion method, relying on the Dumas principle, for the determination of total nitrogen in barley and malt, has been collaboratively tested by the Analysis Committee of the European Brewery Convention. Repeatability, r₉₅, and reproducibility, R₉₅, values were 0.063 and 0.116% of dry matter, respectively, for samples with nitrogen contents in the range 1.23 to 1.86% N of dry matter. There was no significant difference between these values for barley and malt. The Analysis Committee approved the adoption of the combustion method for inclusion in Analytica EBC as an alternative method.