INCREASED ENDOSPERM MODIFICATION OF ABRADED BARLEY GRAIN AFTER GIBBERELLIC ACID TREATMENT*

Physiological and structural observations on barley grains revealed that the pericarp is impermeable to exogenous gibberellic acid. The use of a mechanical hand-mill abraded the pericarp at the non-embryo (distal) end of the grain without damaging the embryo or the overlying husk. When grown in gibberellic acid, the α-amylase content of these abraded grains was significantly higher than that of the controls. Malts prepared from abraded grains were visually identical with control malts prepared with gibberellic acid, but they gave higher hot water extracts. Grain abrasion permitted exogenous gibberellic acid to reach a larger number of aleurone cells, thus increasing the extent of endosperm modification during growth.     

INHIBITION BY HORDEIN OF STARCH DEGRADATION

Large and small starch granules prepared from Proctor barley contain high levels of firmly bound protein. Experiments with α-amylase under simulated mashing conditions suggest that this protein limits the rate of starch breakdown during mashing. Gel electrophoresis shows hordein to be a principal component. Treatment with cysteine or malt endopeptidase changes the nature of the associated protein.     

FACTORS AFFECTING THE PRODUCTION OF α-AMYLASE IN BARLEY GRAINS

The production of α-amylase by the endosperm of barley varieties, both commercial and non-commercial, in normal germination and after incubation with gibberellic acid, has been measured. The level of activities produced differed considerably between varieties. All varieties tested produced considerably more enzyme when supplied with a source of exogenous gibberellic acid than under normal germination. In general the differences between varieties were less in the de-embryonated treatment.     

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.     

Structural and Enzymic Changes in Germinated Barley and Rye

Germination of barley and rye grains at 15°C and 56% moisture content resulted in the production of α-amylase, β-glucanase, endo-β-xylanase and α-arabinosidase. During germination of rye, enzyme activity increased significantly after the first day, whereas in the case of barley a rapid increase was not observed until after the third or fourth day. Rye varieties produced more xylanase, whereas barley produced more α-amylase and β-glucanase. Of the barley samples, germinated Pokko had the highest activities of β-glucanase, xylanase and α-amylase, and at 46% moisture content highest degradation of cell walls on the basis of image analysis. Arve had the highest α-arabinofuranosidase activity. Of the rye samples, Amando produced more xylanase and α-arabinosidase than Anna. The M_w of barley and rye β-glucan decreased significantly during germination. In rye samples, residual high molecular weight arabinoxylans were still present after 4 days of germination. The mass average molecular weight (M_w) of this fraction was 3 × 10⁶.     

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 .     

α-GLUCOSIDASE ACTIVITY OF SORGHUM AND BARLEY MALTS

Sorghum malt α-glucosidase activity was highest at pH 3.75 while that of barley malt was highest at pH 4.6. At pH 5.4 employed in mashing sorghum malt α-glucosidase was more active than the corresponding enzyme of barley malt. α-Glucosidase was partly extracted in water but was readily extracted when L-cysteine was included in the extraction buffer, pH 8. Sorghum malt made at 30°C had higher α-glucosidase activities than the corresponding malts made at 20°C and 25°C. Nevertheless, the sorghum malts made at 20°C and 25°C produced worts which contained more glucose than worts of malt made at 30°C. Although barley malts contained more α-glucosidase activity than sorghum malts, the worts of barley had the lowest levels of glucose. The limitation to maltose production in sorghum worts, produced at 65°C, is due to inadequate gelatinization of starch and not to limitation to β-amylase and α-amylase activities. Gelatinization of the starch granules of sorghum malt in the decantation mashing procedure resulted in the production of sorghum worts which contained high levels of maltose, especially when sorghum malt was produced at 30°C. Although the β-amylase and α-amylase levels of barley malt was significantly higher than those of sorghum malted optimally at 30°C, sorghum worts contained higher levels of glucose and equivalent levels of maltose to those of barley malt. It would appear that the individual activities of α-glucosidase, α-amylase and β-amylase of sorghum malts or barley malts do not correlate with the sugar profile of the corresponding worts. In consequence, specifications for enzymes such as α-amylase and β-amylase in malt is best set at a range of values rather than as single values.     

Control of wheat α-amylase using inhibitors from cereals

A survey of 46 varieties of cereals and related species (including 27 different species from the Poaceae) indicated the presence of a strong inhibitor of wheat α-amylase in all seven Hordeum species tested. Rye contained a lower level of inhibitor activity, but the other species contained insignificant amounts of wheat α-amylase inhibitor activity. The partially purified barley inhibitor was most effective in inhibiting wheat α-amylase activity at high pH. The addition of chromosome 2 of barley to wheat (Chinese Spring addition line 2H) resulted in an apparent increase in the molecular weight of the α-amylase produced during germination. This was probably due to the formation of a complex between the inhibitor encoded by the asi gene on chromosome 2 of barley and wheat α-amylase 2. Breeding of wheat with the barley inhibitor gene may reduce the impact of the high α-amylase levels that result from pre-harvest sprouting in wheat.     

PRODUCTION OF α-AMYLASES IN GERMINATING WHOLE AND INCUBATING DE-EMBRYONATED BARLEY KERNELS*

α-Amylases produced in germinated barley and incubated de-embryonated barley kernels (cv. Bonanza), in the absence and presence of gibberellic acid (GA₃), were analyzed qualitatively by polyacrylamide gel isoelectric focusing (PAG-IEF) and quantitatively by chromatofocusing. Identical patterns of α-amylase components were obtained for both germinated barley and incubated de-embryonated barley kernels at each germination/incubation stage, in the absence or presence of GA₃. Total α-amylase increased rapidly in the germinating whole seed whereas in the incubating de-embryonated grain the α-amylase activity increase was much slower. Addition of exogenous GA₃ did not induce production of higher levels of α-amylase in either the germinating whole or incubating de-embryonated barley kernel. Quantitative chromatofocusing analysis revealed that the proportion of α-amylase III to α-amylase II activity decreased linearly with germination time in the whole grain but remained constant in the incubating de-embryonated grain in the absence or presence of GA₃. The major proportion of α-amylase activity in the germinating whole grain and incubating de-embryonated grain was synthesized in the form of α-amylase II components. However, α-amylase I represented a larger proportion of the total α-amylase activity produced in the incubating de-embryonated grain, as compared to the germinating whole seed in the absence or presence of GA₃. These results suggest that embryo excision differentially affects production of α-amylase II as compared to α-amylase I.     

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.     

Genotypic variation in oil and protein in barley grain

Oil content and composition, protein content and grain weight have been estimated in 86 barley genotypes from diverse sources grown in a replicated field experiment. Overall range between genotypes in composition was: oil content, 1.9-4.1 %; palmitic, 21.4-28.7%; stearic, 0.6-1.8%; oleic, 10.4-16.9%; linoleic, 52.4-58.3%; linolenic, 4.5-7.3%. Modern European varieties varied from 2.2-2.8% oil, and oil content was higher in two-rowed than six-rowed types and higher in naked than husked types. Protein content was higher in naked than husked types, but there was no significant difference between the protein content of two- and six-rowed types. Mean grain weight was higher in both two-rowed and in husked types. Oil was positively correlated with grain protein and the proportion of oleic acid, and negatively correlated with the proportions of palmitic and stearic acid. The highest oil contents were found in Rise 1508 and Hiproly which are high-lysine genotypes.     

Performance of Husked,Acid Dehusked and Hull‐less Barley and Malt in Relation to Alcohol Production

Studies carried out on normal husked barley, normal hull‐less (naked) barley, acid dehusked barley and acid dehusked hull‐less barley, as well as the malts derived from them, showed that when acid dehusked barley samples (obtained from either husked or hull‐less barley), were processed using commercial enzyme preparations, they produced more alcohol when compared with the alcohol yield obtained from the barley samples from which the acid dehusked samples were derived. When the husked (Optic) control, acid dehusked and hull‐less barley samples were malted, Optic control barley produced malt that gave higher dextrinising units (DU) and diastatic power (DP), whilst acid dehusked Optic and hull‐less barley produced malts that gave similar DU results on day 5 of the germination time. When mashed, acid dehusked (Optic) barley malt produced wort that filtered faster than the wort obtained from the malt made from hull‐less barley. This observation is very important because it shows that the husk of the barley is not the only factor that determines the filtration performance of the malted barley, since both the malt samples made from husked and acid dehusked barley had similar filtration rates on day 5 of the germination time. The slow filtration rate observed for the wort made from hull‐less barley suggests that other factors play some role during the filtration of the mash made from hull‐less barley malt. Although hull‐less malt appeared to develop lower DU and DP enzyme activities, when compared with the values obtained for the Optic control, hull‐less barley malted faster and produced optimum predicted spirit yield (PSY) at day 4 of the germination time. In contrast, the control husked Optic barley malt that had higher DU and DP produced equivalent (optimum) predicted spirit yield one day later at 5 days germination time. This is an advantage for hull‐less barley, both in terms of time and energy saving during the malting of barley. Although the acid dehusked Optic barley produced more alcohol than the husked control when commercial enzyme preparation was used to process barley, it was surprising that when the derived malt was assessed, it gave a lower predicted spirit yield than the husked control, even though it produced a higher amount of hot water extract (HWE). The higher extract yield and lower predicted spirit yield obtained from the malt made from acid dehusked malt confirmed that high extract yield is not necessarily associated with high fermentable extract.     

Genotypic and environmental variation in the total phenol and flavanol contents of barley grain

Flavanol and total phenol contents have been determined in samples of barley grown in Britain under a wide range of environmental conditions. An assessment has also been made of the genetic variation in flavanol and total phenol contents among exotic genotypes and current UK varieties. Total phenol content decreased in the first 3 months immediately after harvest but no indication was observed that prolonged storage significantly reduced the levels of flavanols and, after the initial decline, no systematic decreases were found in total phenol content. Significant differences were found between trial sites for both constituents but these could not be related to geographical locations. An examination of inter-varietal variation revealed significant differences among both the current UK varieties (flavanols 0.11–0.17%, total phenols 0.43–0.53%) and 85 diverse genotypes (flavanols 0.08–0.15%, total phenols 0.37–0.54%). The levels of both constituents appeared to be independent of grain size, malting quality, oil content and protein content and no significant differences were found between either two and six-rowed varieties or husked and naked grain types.     

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.     

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.     

Prediction of Final Grain Protein Content with Plant Protein Content of Malting Barley Under Mediterranean Conditions

The prediction of protein content in mature barley grain using the protein content of the plant at heading time is assessed in two experiments. In the first, 32 barley varieties of wide genetic origin were grown in two contrasting environments in Spain, with two sowing dates in one of them. In the second, a collection of 102 doubled haploid (DH) lines from a cross between two high quality two-row spring malting barleys Beka (European, high protein) and Logan (North-American, low protein), was grown in three contrasting environments in Spain, the former two plus a third, with a high incidence of drought and heat stresses during grain filling. The regression equations obtained from the data of both experiments showed a very good fit, as did the equation calculated over all data. The range of protein values used for the overall calculation was from 9 to 21.5%, thus covering the complete spectrum of variation. We conclude that it is possible to predict grain protein in this way, but we suggest that prediction for each combination of variety and growing region should be calculated.     

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.     

大豆β-淀粉酶在生产麦芽糖浆上的优势

介绍了β-淀粉酶和α-淀粉酶的酶种来源及其在生产麦芽糖浆中的作用机理,并对大豆β-淀粉酶、大麦β-淀粉酶、小麦β-淀粉酶、真菌α-淀粉酶、普鲁兰酶的适用条件、失活条件进行了比较,得出大豆β-淀粉酶在生产麦芽糖浆上的优势。     

OPTIMIZED McCLEARY METHOD FOR MEASUREMENT OF TOTAL β-AMYLASE IN BARLEY AND ITS APPLICABILITY

The McCleary method for determination of β-amylase in malt has been modified in order to allow determination of total β-amylase in barley as well as malt. A ruggedness test, performed on the modified method, demonstrated that the method is quite robust and highly reproducible. When the variables α-amylase, β-amylase and diastatic power were measured in 90 malt samples, only β-amylase was significantly correlated to diastatic power (r² = 0.85 and p < 0.0001). The same high correlation was found between total β-amylase in 20 barley samples and diastatic power in the corresponding malts. The validity of this relationship was tested by predicting diastatic power in malt from total β-amylase in barley. Predicted values correlated highly to measured values (r² = 0.95). In breeding material a positive relationship was found between total β-amylase in barley and protein content. This relationship must be considered when evaluating new barley lines.     

ISOELECTRICFOCUSING AND IMMUNOCHEMICAL ANALYSIS OF GERMINATED BARLEY α-AMYLASES AFTER FREEZE-DRYING AND KILNING

Isoelectricfocusing analysis showed that germinated barley contained three groups of α-amylase—α-amylase I a minor component and major components α-amylases II and III. During kilning there was a significant conversion of α-amylase III to α-amylase II. These two enzymes were shown to have immunochemical identity.