DIFFERENT RATES OF DEVELOPMENT OF α-AMYLASE IN DISTAL ENDOSPERM ENDS OF GERMINATED (MALTED) CHARIOT AND TIPPER BARLEY VARIETIES

Chariot Barley had a faster malting rate than Tipper. Even when the total levels of the aleurone-produced enzyme α-amylase were similar in both Chariot and Tipper, the distal ends of the grains of Chariot developed α-amylase at a faster rate than the corresponding endosperm ends of Tipper. The excised aleurone layers of Chariot and Tipper had similar potentials to produce α-amylase. Therefore the faster rate of development of α-amylase in the distal ends of the grains of Chariot may reflect faster transport of active gibberellic acid through the aleurone layer. Differences in the rates of transport of gibberellic acid through the plasmodesmata of the aleurone layer may determine the efficiency of production and distribution of endosperm-degrading enzymes during malting. The transport of gibberellic acid in the aleurone layer was facilitated by high moisture levels.     

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.     

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.     

RELATIONSHIP BETWEEN LEVELS OF GIBBERELLIC ACID AND THE PRODUCTION AND ACTION OF CARBOHYDRASES OF BARLEY

Different hydrolytic enzymes require different levels of gibberellic acid to induce their maximal production and release into the endosperm of barley. Barley-endo-β-glucanase requires a higher level of gibberellic acid to induce maximal production than does α-amylase. Although gibberellic acid also increases the level of barley endo-β-1,3 glucanase, this enzyme, unlike the barley-endo-β-glucanase, develops to significant levels when gibberellic acid is absent. In gibberellic acid-treated aleurone layers β-glucanases degrade the cell wall mainly to glucose. Xylose and cellobiose appear when the aleurone wall has undergone extensive enzymic hydrolysis. Laminaribiose and arabinose are found whether or not gibberellic acid is present in the medium. In addition to the degradation of the endosperm cell walls, β-glucanases may also play an important role in the release of enzymes from the aleurone into the endosperm during malting.     

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.     

The Development of Enzymes During Germination and Seedling Growth in Nigerian Sorghum

The levels of enzymes responsible for the enzymic modification of sorghum endosperm have been followed during germination and seedling growth. Sorghum β-glucanase was shown to be inactive towards barley β-glucan. Gibberellic acid does not appear to control the levels of α-amylase. In contrast to barley, the synthesis of this enzyme occurs in the embryo but it subsequently acts on the starch granules in the endosperm. Limit dextrinase, on the other hand appears to be present in the endosperm as a zymogen. Proteases were also examined during germination and seedling growth. Amino acid-releasing enzymes develop in the embryo and are absent from the endosperm, whereas endoproteases can be detected in the embryo and to a greater extent in the endosperm. Amylolytic attack on endosperm starch in sorghum is very extensive during the early stages of grain growth. The significance of these results to the malting properties of sorghum is discussed.     

MORPHOLOGY OF STARCH GRANULES IN CEREAL GRAINS AND MALTS

During malting, amylases have limited action on large starch granules of barley endosperm but rapidly degrade the small granules. In contrast, the small starch granules of wheat endosperm are resistant to enzymic attack. High levels of exogenous gibberellic acid increase the production of α-amylase and encourage the appearance of radial channels in the partially-degraded large starch granules. These endo-corroded granules are mainly found in the proximal (embryo) half of the endosperm where levels of α-amylase are much higher than at the distal end. Degradation of malt starch can therefore result from enzymic attack both outside and inside the granules. Malting of barley reduces the population of small starch granules which are slower to gelatinize than large granules at the infusion mashing temperatures of 65° C. During germination of barley multiple starch granules are rapidly synthesized in single amyloplasts in the scutellum. The endosperm of high amylose barley is devoid of small starch granules and the average size of the large granules is reduced. Steeliness in sorghum is related to the close packing of the starch-protein matrix rather than to unequal distribution of protein. The significance of these results is discussed, particularly in relation to the morphology of starch granules, the nature of their outer covering, the distribution of amylopectin and amylose within the granule, and the site of enzymic attack.     

The Effect of Nitrogen Level on the Performance of Malting Barley Varieties during Germination

The malting characteristics of high and low nitrogen barleys were compared. Results confirmed that low nitrogen facilitates malting rate and extract development. Modification rate was not related to β-glucan content. In general, these low nitrogen barleys and high nitrogen barleys had similar levels of α-amylase but high nitrogen barleys tend to have higher levels of β-amylase. Since barleys of similar nitrogens had different levels of α-amylase and β-amylase, nitrogen may not be a reliable indicator of the potential of the grain to develop amylolytic enzymes.     

IMPROVED MALTING PERFORMANCE OF FRESHLY HARVESTED BARLEY AFTER ABRASION

Abrasion improved the malting performance of dried, freshly-harvested barley and provided an alternative to storage. The maltability of undried, unstored barley was improved by abrasion, but the undried grain was more difficult to abrade than was dried grain. Unabraded barley required storage periods of at least 3 weeks before adequate extracts could be obtained. Although acidulation reduced the malting losses of dried grain, it only improved the yield of extract with barleys stored for at least 9 weeks before malting. The potential of dried freshly-harvested barley to produce α-amylase and endo-β-glucanase, as indicated by the response of endosperm slices to gibberellic acid, was initially low and increased gradually as the barley was stored. It is considered that the dominant factor in the accelerated malting of freshly-harvested barley is the improved distribution of enzymes in the endosperm which results from abrasion.     

THE RELATION BETWEEN α-AMYLASE ACTIVITY AND NUCLEIC ACID CONTENT OF BARLEY GRAIN*

Wolfe and Olli varieties of barley, which are poor and good malting varieties respectively, were analysed during germination for the activity of α-amylase, the amount of RNA and DNA, and the nucleotide composition of RNA. Olli, the variety that was higher in α-amylase, was also high in RNA content, but there were no significant differences in the DNA content or the nucleotide composition of RNA between the two varieties. The proportional increase in α-amylase activity during germination was much greater than the increase in RNA, which suggests that specific components or states of RNA are responsible for directing enzyme synthesis. Rate of α-amylase development appears to be a function of RNA and the value of the function differed for the two varieties. The development of α-amylase activity may reflect, by association, the synthesis of other enzymes, and thus may also reflect extent of malt modification. Varietal characteristics are also involved.     

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.     

α-1,4-GLUCANS. PART XX. THE MOLECULAR STRUCTURE OF THE STARCHES FROM OATS AND MALTED OATS*

Starch was isolated from samples of oats and malted oats, and fractionated into amylose and amylopectin components. Analysis showed that malting caused a very limited decrease in the molecular size of the amylose, and a small but significant reduction in the exterior chain length of the amylopectin. These changes are very similar to those which occur when oat starch granules are subjected to limited α-amylolysis. It is suggested that α-amylase is the predominant enzyme involved in the changes which occur in cereal starches during malting.     

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.     

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 .     

EFFECT OF BORON ON BARLEY GERMINATION AND MALTING

Following reports of increasing concentrations of boron, essentially as borate, in water supplies, the effect of boron on barley germination, enzyme development in malting and the analysis of the malts has been examined. At concentrations up to 100 p.p.m. boron has no effect on the germination of either mature or water-sensitive barley, but root growth and α-amylase development are reduced.     

EFFECT OF MALTING TEMPERATURE AND TIME ON ENZYME DEVELOPMENT AND SORGHUM WORT PROPERTIES

The effect of malting temperature and time on enzyme development and wort properties of an improved Nigerian sorghum cultivar (Ex-Kwara) were investigated. Malting was carried out at two temperature regimes, 20°C and 25°C for eight days. Parameters evaluated included α- and β;-amylase development, hot water extract (HWE), soluble extract, fermentability, fermentable extract, viscosity, filtration rate, reducing sugars, α;-amino nitrogen and total soluble nitrogen (TSN). For virtually all the parameters studied, germination at 25°C produced higher values on the 4th day after which temperature appeared to have little influence. α;-Amylase development continued throughout the germination period while β;-amylase peaked on the 6th day. Optimal values of total soluble nitrogen (TSN) were recorded at both 25°C and 20°C at the 6th and 8th day of germination respectively .     

EFFECTS OF FINAL WARM WATER STEEP AND AIR REST CYCLES ON MALT PROPERTIES OF THREE IMPROVED NIGERIAN SORGHUM CULTIVARS

The effects of final warm steeping at 40°C and air-rest cycles on malting loss and other malt quality parameters of three improved Nigerian sorghum cultivars were investigated. Grains were steeped for 48 h under four different steep regimes then germinated for 4 days at 30°C. The effect of final warm steeping on kernel growth, malting loss, diastatic power, α and β-amylase and extracts of the malts was significantly affected by air-resting, cultivar and the pair-wise interactions of cultivar and steep regime. Generally, malting loss and kernel growth reduced significantly (p< 0.001) when the steep cycle was a combination of air-resting and final warm water steep at 40°C. Diastatic power, α-amylase, β-amylase and extract also improved significantly in both ICSV 400 and KSV 8 when these grains were malted by a steep regime which combined both air-rest cycles and final warm water steep. However steeping these grains under a final warm water steep without air-resting led to a decrease in extract recovery and enzyme activity. Conversely steeping SK 5912 by a combination of air-resting and final warm water steep generally, inhibited rather than enhanced extract and enzyme development.     

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.     

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.     

A REASSESSMENT OF THE PATTERN OF ENDOSPERM HYDROLYSIS (MODIFICATION) IN GERMINATED BARLEY

Microscopic and enzymic studies of germinated barley have confirmed that excised barley embryos can produce α-amylase because the peripheral areas of the scutellar tissue contain aleurone cells. In contrast, the aleurone-free tissue of the scutellum is incapable of producing significant quantities of α-amylase. The potential of excised embryos to develop α-amylase is not correlated with the in vivo elongation of the scutellar epithelial cells in the grain because these cells do not elongate in excised embryos. Detailed anatomical studies revealed that the highly insoluble Intermediate layer of cell wall material, which is located between the embryo and the starchy endosperm, is broken-down asymmetrically, thus confirming that enzymic modification of the endosperm is under aleurone rather than scutellar control, in germinated barley. Other studies which have sited the scutellum of barley as inducing symmetric break-down of the endosperm have not linked structural changes, in vivo, with fluorescent or non-fluorescent staining patterns. Some of these studies have failed to recognise the possibility that grains such as sorghum and rice may have a different pattern of endosperm break-down from that of barley.