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.     

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY OF BARLEY PROTEINS: RELATIVE QUANTITIES OF HORDEIN FRACTIONS CORRELATE WITH MALT EXTRACT

Several high performance liquid chromatography (HPLC) approaches have been used to study the relationships between amounts of particular groups of grain storage proteins (hordeins) and malt extract, using two sets of barley samples. The areas of several chromatogram peaks varied between samples in a manner that correlated with malt extract. Size-exclusion HPLC of protein aggregates extracted by sonication gave negative correlations of the relative areas of the three largest chromatogram peaks with malt extract in a set of 39 samples of the one variety, but not in a set of samples of 8 varieties grown at 4–5 sites. Under reducing conditions, disulphide-bonded hordeins were quantitatively extracted and the proportion of hordein in the largest peak, comprised of D- and B-hordeins, correlated well with malt extract in both sets of samples. The hordeins sequentially extracted into 50% (v/v) 1-propanol and 50% (v/v) 1-propanol-50 mM dithiothreitol (DTT) were also analysed by reversed-phase HPLC, and could be resolved into several peaks of B-, C- and D- hordeins. Relationships between the amount of certain C- hordeins in the propanol extract, and certain B- hordeins in the propanol-DTT extract and malt extract were seen. As well as providing a better understanding of the relationship between barley grain protein composition and malting quality, these HPLC methods are useful additional methods to enable partial prediction of malt extract using un-malted barley.     

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.     

PURIFICATION AND PROPERTIES OF MALT CARBOXYPEPTIDASES ATTACKING HORDEIN

Carboxypeptidases capable of releasing amino acids from barley hordein have been extracted and purified from malted barley. Three enzymes can be distinguished, with similar molecular weights but slightly different ionic characteristics and specificities. The malt carboxypeptidases preferentially attack peptides containing an aromatic amino acid, although other peptides, including those containing proline, are also attacked. The purified carboxypeptidase fractions reduce the viscosity of β-glucan extracted from endosperm cell walls. It is suggested that these enzymes may be instrumental in the initial solubilization of endosperm cell walls, possibly by the hydrolysis of the peptide component of these walls. The pH optima and heat stability data indicate that carboxypeptidases are crucial for the release of amino acids both from hordein, and from peptides derived from hordein during both malting and mashing.     

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.     

AN APPROACH TO THE ESTIMATION OF BREWING QUALITY IN BARLEY AND MALT

Methods of assessing the brewing qualities of new varieties are reviewed. A new method for making this assessment is proposed. This is ideally a three-step process but it would be possible to eliminate the third, costly, step without severe loss of effectiveness. The use of the new method is illustrated by reference to trials using 251 samples from recent trials in France.     

AREAS OF ABSORPTION RELATING TO MALT EXTRACT VALUE IN MODIFIED NEAR INFRA-RED SPECTRA OF BARLEY FLOUR

Determinations of milling energy, nitrogen, acid-soluble beta glucan and malt extract after micromalting, were made on grain samples of twenty nine barley cultivars. In addition the flour from unmalted grains was scanned over the near infra-red spectrum, and the scan data were subjected to a principal components analysis (PC). Predicted malt extract values resulting from an NIR multiple regression equation with PC terms, correlated well (r = 0.934) with the manual extract values. This confirms previous evidence that malt extract value depends largely on constituents in the resting grain, rather than on malt enzymes. An attempt to locate NIR absorptions relating to malting quality was made by restructuring the NIR spectrum of the samples according to the weightings modified by the regression coefficients in the PC regression equation for hot water extract value. The spectrum reconstructed in this way showed a number of absorption peaks and troughs. From comparison with previous NIR scans it was concluded that a strong peak at the wavelength 2100 nm was due to a starch absorption and this together with minor starch overtone peaks correlated positively with malt extract. Troughs at 2180 nm, 1980 nm and 1700 nm indicated a negative association between malt extract and some proteins. There were also wide troughs at 1830 and 2330 nm indicating a negative relationship between malt extract and beta glucan. Other peaks and troughs in the reconstructed spectrum could not readily be assigned to a constituent. A reconstructed protein spectrum consisted of peaks and troughs that agreed with previous assignations for protein absorbancies. A milling energy reconstruction was approximately the inverse of the malt extract spectrum, and an acid-soluble beta glucan reconstruction was relatively featureless and appeared to be due to the effects of particle size variations.     

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 .     

RELATIONSHIP BETWEEN FUSARIUM INFESTATION OF BARLEY AND THE GUSHING POTENTIAL OF MALT

Fifty barley samples, displaying a range of 0 to 100% kernels infested with Fusarium, were collected in North Dakota, South Dakota, and Minnesota during the harvest of 1994. Samples were micromalted, and the levels of the fungal metabolites, deoxynivalenol and ergosterol, were determined. Fusarium infestation and the levels of fungal metabolites were evaluated as predictors of gushing in laboratory trials. Malt samples which were infested with Fusarium or contaminated with the fungal metabolites exhibited a propensity to gush. However, only the levels of deoxynivalenol and ergosterol were found to be strongly correlated with the actual amount of gushing observed. This suggests that their production may parallel that of the component which actual causes gushing, and that screening barley and malt for these metabolites, may offer a means of reducing gushing problems in the brewery. Determination of deoxynivalenol is rapid, when it is present, and necessary because of food safety and malt quality concerns.     

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.     

THE USE OF THE FRIABILIMETER TO DETERMINE MALT MODIFICATION AND HOMOGENEITY

The Analysis Committee has tested collaboratively the Friabilimeter of Kretschmer and Chapon for the determination of malt modification. The version of the method in which friability is determined by measuring the residue remaining in the Friabilimeter drum has been approved for inclusion in the Recommended Methods of the Institute of Brewing. The development of the method, advanced by Baxter and O'Farrell for the measurement of homogeneity, has also been recommended.     

THE EFFECT OF KILNING ON THE CAPABILITY OF MALT TO OXIDISE LIPIDS

The influence of kilning on the ability of malt to oxidise lipids was studied. Barley was malted with a standard programme and subsequently dried with varying kilning procedures. The capability of samples taken during kilning to oxidise lipids was determined by the lipoxygenase (LOX) reaction, i.e. by measuring O₂ consumption in an aqueous suspension of samples upon addition of excess amounts of exogenous linoleic acid. Various kilning programmes from isothermal low-temperature kilnings to kilnings with varying temperature profiles induced a two- to threefold increase in the rate of the LOX reaction in the samples taken during the first two to six hours of kilning. However, when lipid changes in the intact kernels were measured, oxidation of the endogenous malt lipids was very limited during the first half of kilning. Furthermore, the rate of the LOX reaction decreased in samples taken during the latter part of kilning, the rate of decrease depending on the kilning programme and especially the final curing temperatures. These results indicate that a possible risk of a marked ability to oxidise lipids remains at moderate kilning temperatures. In conclusion, kilning induces a significant capability to oxidise lipids but does not affect the fatty acid composition of whole kernels. However, in aqueous suspensions of ground malt the oxidative instability created might lead to oxidation of lipids .     

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.     

EXTRACTION AND ASSAY OF PROTEOLYTIC ACTIVITIES IN SORGHUM MALT

A method has been developed to assay the proteinase and carboxypeptidase activities in sorghum malt. The method specifically addresses two problems encountered with some other assays for malt proteolytic activity; that of enzyme inextractability and the use of alien substrates. It was found that as with barley malt proteolytic enzymes, a high proportion of sorghum malt proteolytic activity was inextractable with sodium chloride solution. Systematic investigation into the extraction of proteolytic enzymes from sorghum malt led to the development of a novel extractant viz. 0.6 M Lithium iodide LiI plus 3.33 mM dithiothreitol. This extractant appears to solubilize a significantly higher proportion of the malt proteolytic activities with a single extraction than previously used buffers. Assay is carried out Using purified Kafirin, the sorghum prolamin storage protein, as substrate. Proteinase is measured in terms of total nitrogen solubilized and carboxypeptidase as free α-amino nitrogen solubilized.     

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.     

USE OF THE FRIABILIMETER TO ASSESS HOMOGENEITY OF MALT

A method has been developed for assessing the homogeneity of a sample of malt using the Friabilimeter. Material retained by the Friabilimeter sieve is further fractionated using a sieve with slits 2·2mm wide by 23mm long, as recommended by the EBC for assessing corn size. Material composed of unmodified grains and large pieces of endosperm is collected from the sieve and weighed. This gives a rapid and reproducible estimate of the proportion of unmodified material in the sample. The technique readily detects small quantities of unmodified grain added to a sample of malt. Staining half corns with Calcofluor gives similar scores for homogeneity but with poorer reproducibility and the technique is much slower. Staining with methylene blue gives much lower figures for homogeneity and is also slower and less reproducible than the Friabilimeter method.     

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.     

THE FATE OF THE SULPHUR CONTENT OF MALATHION WHEN APPLIED TO BARLEY USED TO PRODUCE MALT WHISKY

The use of malathion to protect malting barley from infestation by insects led to fears that malt whisky could be tainted by odorous sulphur compounds such as mercaptans derived from the malathion. Laboratory scale experiments in which barley was treated with sulphur-35 radio-labelled malathion, showed that the majority of the sulphur from the malathion was lost in the early stages of processing and that none penetrated to the final distilled spirit. Treatment of malt with the radio-labelled malathion showed that although the majority of the sulphur from the malathion was lost after the infusion and fermentation stages, a small percentage was found in the distilled spirit. However, re-use of the final infusion liquor as the first liquor on the next batch of malt as is common practice, could lead to a slight risk of an accumulation of sulphur compounds until equilibrium is reached.     

APPLICATION OF A COMMERCIAL ENZYME PREPARATION IN THE BARLEY MALTING PROCESS

This paper presents data on barley micromalting with addition of the CELLUCLAST enzyme complex. This is a commercial, multicarbohydrase with distinct β-glucanase and proteinase activities. The enzyme was added to steeping and germination phases, in different quantities (0.05%; 0.75% and 0.1% of initial barley). The enzyme was added to different malting phases: to the 2nd and 3rd steep water, at the beginning of germination on the 1st day by spraying, on the 2nd day of germination and in combination of addition to 3rd steeping water and in germination start (50% of total quantity of each). CELLUCLAST enzyme had a significant effect on reduction of wort viscosity, extract difference, wort filterability and protein breakdown, depending on the quantity of added enzyme and the malting phase to which it was added. There was no negative effect on other malt quality parameters. The best values of cytolytic breakdown parameters (viscosity, extract difference, filtration rate) were obtained with addition of 0.075% of CELLUCLAST, on the first day of germination.     

THE USE OF CHEMICAL AGENTS TO OVERCOME DORMANCY IN MALTING BARLEY*

Treatments were applied to dormant grains pre-drying, during dry storage and in the immediate pre-steep period. The effects on rates of recovery from dormancy were assessed. Only applications of sulphuric acid and gibberellic acid in the pro-drying period subsequently enhanced germination. No treatments applied during dry storage were beneficial. It appeared that oxygen was not needed during storage for grain maturation. Atmospheres of carbon dioxide applied during warm storage reduced subsequent germination but this gas was without effect on grain stored at ambient temperatures. Steeping grains in dilute solutions of mineral acids and some sulphur containing compounds improved their germinability.