ORIGIN OF A DOMINANT BEER PROTEIN IMMUNOCHEMICAL IDENTITY WITH A β-AMYLASE-ASSOCIATED PROTEIN FROM BARLEY

Two-dimensional immunoelectrophoretic techniques were used to elucidate the origin of a dominant beer antigen 1. Immunochemical identity was found between a protein Z associated with β-amylase in extracts of barley and one form of the beer antigen 1a. Immunochemical identity was also found between a papain-modified form of protein Z and the antigen form 1b from beer. The results indicate that protein Z is very resistant to degradation and denaturation during malting and brewing.     

THE CHROMATOGRAPHIC PROPERTIES OF BARLEY AND MALT β-AMYLASES

Barley β-amlyase occurs as a heterogeneous, polydisperse enzyme in thiol-free extracts of Conquest barley. During malting, the polydisperse enzyme is altered, resulting in the formation of four distinct enzyme components which increase in activity as germination progresses. Addition of thioglycerol to a thiol-free extract of barley, or initial extraction with thioglycerol, produces extracts containing two discrete β-amylase enzymes. β-amylase I is the major component of the extract; β-amylase II occurs as a minor component. Similarly, malt extracts containing thioglycerol have two β-amylase enzymes, β-amylase III and IV. Barley β-amylase II and malt β-amylase III have similar chromatographic properties on CM-cellulose but it is not known whether these enzymes are identical. During the early stages of germination, barley β-amylase I disappears and cannot be detected in extracts of 1-day malt; β-amylase III is the major β-amylase enzyme in this extract. Malt β-amylase IV cannot be detected in barley extracts. It develops during germination until it becomes the major β-amylase in malt extracts.     

IMPROVED EXTRACTION AND ASSAY OF β-AMYLASE BROM BARLEY AND MALT

β-Amylase was extracted from barley or malt using four physical techniques to break up grists which had been prepared using a Moulinex coffee grinder. Grinding with a Polytron homogeniser apparently completely disrupted all cells, as determined by transmission electron microscopy, and increased the efficiency of extraction of β-amylase from barley by more than 30%. The other treatments tested were without value . The β-amylase activity in extracts of barley or malt was assayed by measuring the production of reducing sugars from reduced soluble starch, using a PAHBAH reagent. α-Amylase, which interferes with the quantitation of β-amylase in extracts of malt, was not totally inactivated by the chelating buffer used for enzyme extraction or by several other chelating agents. α-Amylase activity was quantified specifically using Phadebas. Using purified α-amylase a calibration was developed which related activity, as determined using Phadebas, to reducing power units. Thus the α-amylase activity present in an extract containing β-amylase could be determined using Phadebas and the reducing power equivalent activity subtracted from the total “apparent” activity to give the actual β-amylase activity. α-Glucosidase and limit dextrinase activities are believed to be too low to have a significant effect on the apparent β-amylase . The soluble and bound β-amylase activities were measured in samples taken from micromalting barley (Alexis). Dry weight losses increased to over 10% after 8 days germination. Antibiotics, applied during steeping, were used to control microbes in one experiment. However, their use checked germination and reduced malting losses to 8.4% in 8 days germination. The soluble enzyme present in extracts from steeped barley and early stages of germination was activated (20–40%) by additions of the reducing agent DTT .     

THE USE OF INFRA RED REFLECTANCE FOR THE RAPID ESTIMATION OF THE SOLUBLE β-GLUCAN CONTENT OF BARLEY

It has previously been reported that the malting quality of barley is related to the amount of β-glucan dissolved at low pH under defined conditions. An infra red reflectance instrument, the ‘InfraAlyzer’, can be used to estimate this ‘acid-soluble’ β-glucan content of barley. This is a rapid test (approximately one min/sample) and is therefore suitable when screening for low soluble β-glucan in the early generations of malting quality breeding programmes. The relationship between predicted and measured values for soluble β-glucan differs from comparisons made for other quality components in forages, in that it is curvilinear rather than linear. A logarithmic transformation of the soluble β-glucan values, however, allows the current ‘InfraAlyzer’, which produces linear regressions only, to predict values for soluble β-glucan that correlate well (r = 0.87) with the measured results.     

Breeding of lipoxygenase‐1‐less malting barley variety ‘Satuiku 2 go’

The lipoxygenase‐1‐less (LOX‐less) trait has positive effects on beer quality, in particular, improvement of flavour stability related to the reduction of beer‐deteriorating substances such as trans‐2‐nonenal. ‘Ryohfu’ is the only spring‐sown malting barley variety grown in Hokkaido, located in the northern part of Japan, and has been used in the Japanese brewing industry for over 20 years. ‘Satuiku 2 go’ was developed as the first LOX‐less malting barley variety in Japan by successive back‐crossing with molecular marker‐assisted selection to introduce the LOX‐less trait into the recurrent parent ‘Ryohfu’. The agronomic performance and general malt quality of ‘Satuiku 2 go’ were almost equivalent to those of ‘Ryohfu’. Wort and beer analyses at the pilot‐scale brewing trial indicated that the LOX‐less trait had little effect on the general characteristics. In contrast, the beers made from ‘Satuiku 2 go’ malt exhibited reduced levels of trans‐2‐nonenal and trihydroxyoctadecenoic acid. The sensory evaluation demonstrated the superiority of ‘Satuiku 2 go’ beers stored under differing conditions in terms of staleness. It can be concluded that the LOX‐less trait was effective in different genetic backgrounds of the recurrent parents used for the development of LOX‐less malting barley varieties. Copyright © 2018 The Institute of Brewing & Distilling     

Relationship Between β-amylase Activity,Steeliness, Mealiness,Nitrogen Content and the Nitrogen Fractions of the Barley Grain

Steely grains tend to have a higher total nitrogen than corresponding mealy grains. β-Amylase activities of different barley varieties appeared to be associated, to different degrees, with steeliness, high total nitrogen content, and high levels of total salt soluble proteins and hordein proteins. The consistent relationship between β-amylase activity and these parameters suggests that β-amylase contents of barley not only reflect genetic differencies in varieties, but also differences in protein type and grain structure. Notwithstanding, the β-amylase content of the grain may not be indicative of the potential of the grain to develop β-amylase activity during malting.     

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.     

Use of acid gel electrophoresis in the characterisation of ‘B’ hordein protein in relation to malting quality and mildew resistance of barley

The ‘B’ polypeptides of the storage protein (hordein) of barley were studied in 84 varieties. On the basis of acid gel electrophoresis the varieties were classified into 13 distinct groups. Contrary to the suggestion that varieties preferred for malting tend to show less intensity of staining of the faster moving bands of the ‘B’ fraction of hordein, no consistent association was found in this study. Thus the malting potential of some varieties may be less affected by protein composition than by other factors. The genetic linkage between the Hor-2 locus, specifying the ‘B’ group of polypeptides, and the Mlalocus, determining resistance to powdery mildew (Erysiphe graminis), was illustrated by the similar groupings of varieties carrying the same resistance alleles, but exceptions were observed. Examples of the inheritance of hordein patterns are illustrated and discussed.     

THE RELEASE OF BOUND β-AMYLASE BY MACROMOLECULES

The aim of this study was to clarify the roles of endogenous proteases and higher molecular weight thiols in the release of bound β-amylase, which occurs during barley germination. In resting barley grains (Hordeum vulgare L. cv. Torrent) five major β-amylase monomers were found in the thiol reduced, salt soluble extract. Only the two smallest monomers were present in large amounts in the bound form of the enzyme that had been released with 2-mercaptoethanol. In vitro, bound β-amylase was solubilised by “releasing factors” extracted from the endosperm of decorticated grains germinated for three days. The releasing factors were in the higher molecular weight fraction (>5 kDa) and their formation was induced in degermed grains by gibberellic acid. When an endosperm extract, containing only higher molecular material (>5 kDa) prepared from three day germinated grain, was incubated with a preparation of bound β-amylase, about 75% of the release of the enzyme could be prevented by a mixture of proteolytic inhibitors. The dominant class of proteases in malt were the sulphydryl proteases. These were not fully active when extracted, but could be activated approximately six fold by the addition of 2-mercaptoethanol. Heating the extract to a limited extent destroyed all the proteolytic activity, but 10% of the bound β-amylase could still be released by the extract.     

SOME RELATIONSHIPS BETWEEN THE PROTEIN NITROGEN OF BARLEY AND THE PRODUCTION OF AMYLOLYTIC ENZYMES DURING MALTING

Barleys studied (Chariot and Delibes) contained different levels of extractable β-amylase enzymes. The potential levels of β-amylase enzymes of the two varieties studied were similar at 1.4 to 1.7% total nitrogen. Higher values of potential β-amylase enzyme were observed in the Delibes barley of higher total nitrogen of 1.9%. The higher level of β-amylase found in the barleys with the highest total nitrogen was not reflected in the protein banding patterns as revealed by SDS-PAGE protein fractionation. Extraction of barley proteins was largely influenced by the different extractants used. The alcohol soluble proteins, M_r 97 kDa (D-hordeins), were only extracted when mercaptoethanol was included in the extracting solution. Although barleys with the highest nitrogen (1.9%) had the highest apparent potential to develop β-amylase enzymes, the better modified low nitrogen barleys produced higher levels of β-amylase and α-amylase when malted. Dehusking revealed that the high nitrogen barleys contained more steely grains. In contrast, the low nitrogen barleys contained more mealy grains. Steely grains contained more nitrogen than mealy grains and had the greater potential to develop β-amylase. Notwithstanding, the results of this study suggested that the proteins of the lower nitrogen barleys (1.4–1.7%) were capable of producing higher levels of β-amylase and α-amylase than the higher nitrogen barleys (1.9%) over comparable periods of malting. The high apparent β-amylase potential of the barley was linked to high nitrogen levels and associated high levels of steeliness, whilst the corresponding high β-amylase levels of malt were linked to the efficiency of endosperm modification of the malted grain.     

A ROLE FOR CARBOXYPEPTIDASE IN THE SOLUBILIZATION OF BARLEY β-GLUCAN

An enzyme is described which catalyzes the release of soluble β-glucan from insoluble barley endosperm cell walls. This enzyme increases in activity throughout malting. It has been partially purified and found to behave in the same way as an acidic carboxypeptidase on isoelectric focusing and in its sensitivity to inhibitors and activators and to heating. The importance of the β-glucan solubilizing enzyme in malting and mashing is discussed. An improved method for β-glucan determination is described.     

ASSESSMENT OF BACILLUS LICHENIFORMIS α-AMYLASE AS A CANDIDATE ENZYME FOR GENETIC ENGINEERING OF MALTING BARLEY1

Bacillus licheniformis α-amylase, a thermostable starch-degrading enzyme, has been assessed as a candidate enzyme for the genetic transformation of malting barley. The temperature optimum, pH optimum and thermostability of B. licheniformis α-amylase were compared with those of barley α-amylase. The bacterial enzyme has a higher pH optimum (?9), a higher temperature optimum (?90°C) and much higher thermostability at elevated temperatures than the barley enzyme. The specific activity of the bacterial enzyme under conditions of pH and temperature relevant to the brewing process (pH 5.5, 65°C) is ?1.5-fold higher than that of the barley enzyme. Measurements of α-amylase activity during a micro-mash showed that the bacterial enzyme is at least as stable as the barley enzyme under these conditions, and that a level of expression for the bacterial enzyme corresponding to ?0.5% of total malt protein would approximately double the α-amylase activity in the mash. B. licheniformis α-amylase activity was rapidly eliminated by boiling following mashing as would occur during brewing. The combined results suggest that barley expressing the bacterial enzyme may be useful in the brewing process.     

Kaffircorn malting and brewing studies. XVI. The distribution and activity of alpha- and beta- amylases in germinating kaffircorn

Unlike barley which possesses β-amylase activity before germination, sorghum grain is devoid of this enzyme. Both α- and β-amylase are produced during the germination of sorghum and, in any particular malting trial, the ratio of the enzymes to one another remains approximately constant throughout the trial. The actual value at which the ratio remains constant depends on the temperature of the malting and the variety but not on the watering treatment given during malting. The amylase concentration in the embryo is usually higher than in the endosperm but the total amount of amylase in the embryo is much less than in the endosperm. Although the embryo of sorghum is a minor contributor to the total amylase, it contributes more thsn does the embryo of barley to barley malt. The ratio of α- to β-amylase in the embryo differs from that found in the endosperm. In Short Red kaffircorn, a common malting variety, the embryo contained ii significantly lower percentage of αamylase than the endosperm. The opposite was found with the Birdproof and Sugar Drip varieties, the embryo being the richer in αamylase.     

THE MILLING ENERGY OF MALTED BARLEY AND ITS RELATIONSHIP WITH HOT WATER EXTRACT AND α-AMYLASE ACTIVITY

Hot water extract is dependant on endosperm structure and its breakdown during malting. These endosperm characteristics can be rapidly assessed on 5 g samples by measuring the milling energy of malted barley. α-amylase determinations on the resultant flour indicate that good malting barleys have either moderate or high levels of the enzyme. High levels of α-amylase will not confer good malting quality where endosperm modification has proceeded slowly.     

Utilization of the Linear Mode of MALDI‐TOF Mass Spectrometry in the Study of Glycation During the Malting Process

The process of glycation during the malting process was monitored by the linear mode of matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐TOF MS). Water‐soluble proteins were investigated and two hulled barley varieties, Jersey and Tolar, were compared to the hulless line KM 1910. The crude extracts of the proteins obtained from the grain, the malt, and aliquots collected every 24 h during the malting process, were mixed with the matrix (2,6‐dihydroxyacetophenone) and analyzed by mass spectrometry. The protein composition of the barley changed during the malting process. The protein patterns did not differ significantly between the three varieties of the barley grains. However, significant differences between the malts were evident. Results showed the influence of the malting process on the glycation of certain water‐soluble barley proteins, nonspecific lipid transfer protein 1 (LTP1) and protein Z, of which the glycated forms survived the brewing process. These major barley proteins are very important for the formation and stability of beer foam and glycation may prevent their precipitation. Analysis results indicated that slight glycation of the proteins had occurred on the second day of malting. The linear mode of MALDI‐TOF mass spectrometry was used as a fast and simple method for monitoring the patterns of low‐molecular weight barley proteins with regard to barley variety discrimination. This procedure also enables the selection of barley varieties suitable for the malting industry.     

DIFFERENCES IN MALTING PERFORMANCE BETWEEN BARLEYS GROWN IN SPAIN AND SCOTLAND

Two barley genotypes were grown, in 2 seasons, at sites in both Scotland and Spain. The development of enzyme levels and endosperm modification were assayed, over the final 3 days of malting. Spanish grown samples demonstrated faster and more extensive synthesis of both α-amylase and β-glucanase, more rapid cell wall modification and a greater reduction in milling energy during malting than Scottish grown samples. Malt milling energy was strongly associated with cell wall breakdown, which was a limiting step in modification of Scottish, but not Spanish, grown samples. Extract levels were not related to α-amylase activity, but Kolbach index exhibited an association with extract at both sites.     

MULTIPLE FORMS OF α-AMYLASE IN MALTING BARLEY VAR. EMMA.

Multiple forms of α-amylase arise from translation of separate messages and post-translational proteolytic modification during malting. α-amylase activity is tolerant of proteolysis. The enzyme appears to have two domains one of which is associated with starch cleavage. The second site which binds cyclodextrin is destroyed by proteolysis. Cleavage yields a low molecular weight form which retains α-amylase activity.     

THE BREWING POTENTIAL OF “ACHA” (DIGITARIA EXILIS) MALT COMPARED WITH PEARL MILLET (PENNISETUM TYPHOIDES) MALTS AND SORGHUM (SORGHUM BICOLOR) MALTS

The malting and brewing characteristics of millets (Pennisetum typhoides and Digitaria exilis) and sorghum (Sorghum bicolor) were compared. Diastase, α-amylase, amyloglucosidase and proteases increased with malting time and the increase was associated with the modification. Development of hydrolytic enzymes was significantly higher in pearl millet and Digitaria exilis (“acha”) than in sorghum at P ≥ 0.01. The major starch degrading enzyme in the three varieties of pearl millet (SE composite, SE.13 and SE 2124) was α-amylase. On the other hand, β-amylase was the major starch degrading enzyme in “acha” (Digitaria exilis) which is similar to the pattern in barley. Gibberellic acid had a stimulating effect on the diastatic activity of pearl millets, Digitaria exilis (“acha”) and sorghum (KSV-4), but inhibited the diastatic activities of sorghum (Farafara). Gibbereltic acid inhibited the proteolytic activities in all the pearl millet varieties, Digitaria exilis and sorghum varieties. Potassium bromate had little or no effect in the reduction of malting losses. Although “acha” (Digitaria exilis) had a high β-amylase content, a high malting loss makes it uneconomical to brew with “acha” mart. A blend of “acha” malt with pearl millet malt or sorghum malt (composite malt) will produce a malt of the same profile as barley malt and this will enhance the quality of sorghum and pearl millet malt during the mashing process. Wort quality of all the samples was suitable for brewing conventional beer.     

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.     

RELEASE AND ACTIVATION OF BARLEY β-AMYLASE

The aim of this study was to determine the role of low molecular weight thiols both in the release and activation of β-amylase during grain germination. In quiescent barley grains (Hordeum vulgare L. cv. Torrent) about 55% of the β-amylase was extracted with buffer, the remaining 45% was in the bound form. During micromalting the bound form was progressively solubilised between germination days 1 and 4. When free β-amylase, extracted from ungerminated grains, was incubated with dithiothreitol the enzymic activity increased by 15%-20%. This activation did not occur when free β-amylase, from grain germinated for 3 days or more, was incubated with DTT. The release of bound β-amylase with thiols was pH dependant, occurring most rapidly at and above pH 8.0. At the onset of germination the embryo released soluble thiol (approximately 5 nmol per embryo) into the endosperm. Degermed grains were dosed with reduced glutathione and incubated for 72 h. The addition of 60 nmol glutathione caused the release of about 80% of the bound β-amylase. When less glutathione was used, 5 nmol (an amount similar to that released by the embryo in vivo) no significant release of the bound enzyme was detected. When degermed grains were dosed with oxidised glutathione (60 nmol), no bound β-amylase was released. However, addition of the disulphide bis-hydroxyethyldisulphide (60 nmol) did cause the release of about 90% of the bound enzyme. The aleurone layer reduced the bis-hydroxyethyldisulphide to a thiol, presumably 2-mercaptoethanol. Oxidised glutathione and cystine were not significantly reduced to thiols by isolated aleurone layers. The aleurone layer did cause the disappearance of cysteine from solution. When preparations of bound β-amylase were incubated with extracts from the endosperms of grains germinated for three days, the bound enzyme was released. This release was due to the high molecu lar weight material (>5 kDa) in the extract and not to low molecular weight thiols. It seems unlikely that simple thiols, such as glutathione, are solely responsible for the release of bound β-amylase.