Genetic and environmental variation in the diastatic power of australian barley

Variation in the diastatic power of Australian barley, and the relationships between diastatic power and the starch-degrading enzymes contributing to diastatic power, were investigated in 11 cultivars of barley grown at six diverse locations in Australia. Diastatic power varied with genotype and location, with the levels ranging from 3·1 to 16·5 U/kg. For alpha-amylase activity, levels across cultivar and location ranged from 52 to 214 U/g, for beta-amylase activity they ranged from 201 to 1550 U/g; and, for limit dextrinase activity, they ranged from 56 to 636 U/kg. Alpha-amylase (r = 0·64) and beta-amylase (r=0·77) activities were correlated more strongly with diastatic power than was limit dextrinase (r=0·37). Grain nitrogen content was correlated positively with diastatic power (r=0·71), largely because of the relationship between nitrogen content and beta-amylase activity (r=0·82). High grain nitrogen contents were also associated with small grain sizes (r=−0·76) and low hot-water extracts (r=−0·75). The levels of alpha-amylase activity were correlated more closely with limit dextrinase activity (r=0·65) than with beta-amylase activity (r=0·28). The results indicate the need to select barley cultivars separately for alpha-amylase and beta-amylase activities to achieve high levels of diastatic power.     

Measurement ofBeta-amylase in Malting Barley (Hordeum vulgareL.). I. Development of a Quantitative ELISA forBeta-amylase

A double antibody, sandwich enzyme-linked immunosorbent assay (ELISA) was developed using polyclonal antibodies specific tobeta-amylase to estimate the amount of ‘free’ (soluble in aqueous saline solution) or ‘combined’ (extracted with saline solution including reducing agent)beta-amylase protein in barley grain and malt. This ELISA was used to quantify the amount ofbeta-amylase in barley grain and malt from four varieties grown at nine sites in South Australia in 1993. The antibody used to develop the ELISA reacted differently withbeta-amylase depending on whether the source was barley grain or malt, and on thebeta-amylase band pattern in isoelectric focussing (IEF) of the barley variety. On the basis of their IEF band patterns barley varieties were divided into two types, designatedBmy1-Sd1 andBmy1-Sd2. Malting resulted in proteolytic cleavage of thebeta-amylase peptide with a reduction in the apparent molecular weight of up toM_r4000 and the appearance of new maltbeta-amylase IEF bands that were more basic. The new maltbeta-amylase IEF band patterns still allowed the identification of theBmy1-Sd1 andBmy1-Sd2 IEF types despite the change in molecular weight and pI. The data obtained using thebeta-amylase ELISA were highly correlated withbeta-amylase activity for both the free and combined fractions when the IEF band pattern and its source, barley grain or malt, were taken into account.     

Variation of Beta -Amylase Activity in Barley as Affected by Cultivar and Environment and its Relation to Protein Content and Grain Weight

The cultivar and environmental variation of beta -amylase activity was studied using two barley cultivars with contrasting growth properties. There was a significant difference in beta -amylase activity between the two cultivars used, 92-11 being significantly higher than Xiumai 3. A significant variation in beta -amylase activity was detected between grains at different positions within a spike. The two cultivars showed the same pattern, with top grains showing the highest and bottom ones the lowest activities. The relative difference within a spike varied between the cultivars, with 92-11 being larger than Xiumai 3. Both seeding rate and timing of N application dramatically affected the beta -amylase activity. With N application at the booting stage, beta -amylase activity increased, mainly due to the significantly increased beta-amylase activity in the topmost grains. The bottom grains showed a lower response to timing of N application. The variation in protein content and grain weight between cultivars and among the various treatments was also examined. The possible influence of these factors on beta -amylase activity are discussed.     

Beta -Amylase Activity and Thermostability in Two Mutants Derived from the Malting Barley cv. Triumph

The malting barley cultivar, Triumph, and two mutants derived from it with higher (TL9) and lower (TL43) dormancy, respectively, were grown in replicated trials at Lleida, Spain and Dundee, Scotland, in 1999. Measurement ofbeta -amylase in the mature grain showed both mutants to have higher enzyme activity than the parental type with Spanish-grown samples higher in beta -amylase than Scottish-grown. There were no genotypic differences in beta -amylase thermostability or in the portion of the enzyme that was water-soluble. However, for all three genotypes, Scottish-grown samples had a higher proportion of water-soluble beta -amylase and total beta -amylase thermostability was also higher in the Scottish-grown samples. Data from the Spanish-grown samples suggested that water-soluble beta -amylase was slightly more thermostable than the portion of the enzyme released by papain.     

Effect of hydrogen peroxide and ozone treatment on improving the malting quality

The effect of hydrogen peroxide (HP) and ozone (O₃) treatment during barley steeping on the quality of malt produced from two barley varieties (GrangeR and AC Metcalfe) by micro-malting was investigated. The two steeping oxidation treatments that was observed to promote barley acrospire growth. Ozone treatment improved the malt enzyme activity of endo-protease, α-amylase, free beta-amylase and total limit dextrinase to differing extents, with GrangeR improving to a greater degree. HP treatment contributed to the increase of α-amylase, β-glucanase and endo-protease. Surprisingly, HP or ozone oxidation during malting resulted in different and novel outcomes for total beta-amylase in GrangeR and AC Metcalfe. In GrangeR, total beta-amylase activity reduced with respect to the control in both treatments. In comparison with AC Metcalfe there was a substantial increase of 78% with HP and 90% O₃ in total beta-amylase activity. Malt quality including wort free amino nitrogen, β-glucan, turbidity and diastatic power was differentially increased by the oxidation induction treatment during steeping in malting. Gene expression analysis indicated that the effects of the steep oxidation treatments on enzyme and malt quality were putatively linked with the up-regulation of certain genes involved in GA synthesis (GA20ox1) and ABA catabolism (ABA8′OH). Barley grain germination assay results also showed that moderate HP induction could improve barley germination tolerance to the ABA effect. Malting including steep oxidation induction was shown to be beneficial to malt quality by improving the resultant wort quality and the efficiency of the beer brewing process. These observations point the way towards improving malt quality and the efficiency of the malting process.     

The Relationship Between D Hordein and Malting Quality in Barley

The contribution of grain protein to the malting quality of barley (Hordeum vulgareL.) was investigated by comparing the hordein composition and the malting quality in barley produced under a range of field conditions. Two malting cultivars, Schooner and Arapiles, and one feed cultivar, Galleon, were grown under five nitrogen regimes in each of two years. Hordein composition of the grain was determined at maturity using a combination of sodium dodecyl sulphate-polacrylamide gel electrophoresis and laser densitometry. Malt extract was determined on all samples after micromalting. Variation in growth conditions resulted in a wide range of grain protein contents and malt extract values, as well as variation in the proportions of the individual B, C and D hordeins in the grain. D hordein in particular varied over a 10-fold range. High levels of all protein fractions were associated with low malt extract. Total protein, as expected, displayed a strong, negative correlation with malt extract. The relationship was cultivar specific and separate regression lines were generated for each cultivar. Of the individual hordein fractions, D hordein displayed the strongest negative correlation with malt extract and its relationship to malt extract was independent of cultivar. A consistent relationship between D hordein and malt extract was observed across seasons, treatments and cultivars that was indicative of a causal relationship between D hordein and malting quality. D hordein therefore offers an alternative measurement to total protein for the prediction of malting quality over a wide range of environmental conditions and cultivars.     

氮肥施用时期对啤酒大麦籽粒醇溶蛋白组分含量和β-淀粉酶活性的影响

氮肥施用时期显著影响籽粒蛋白质含量、B醇溶蛋白和C醇溶蛋白组分含量,而对D醇溶蛋白组分含量的影响不显著;与前期施氮相比,后期施用氮肥显著增加籽粒蛋白质和三种醇溶蛋白组分的含量。品种对B、C醇溶蛋白含量的影响要比氮肥施用时期大,而籽粒蛋白质和D醇溶蛋白含量的差异主要是由氮肥施用时期的差异引起的。生育后期施用氮肥增加β-淀粉酶活性。     

氮肥运筹对啤酒大麦籽粒品质及产量的影响

为了揭示氮肥运筹对啤酒大麦籽粒品质和产量的调节效应,以啤酒大麦花30为材料,在施纯氮总量225kg.hm-2条件下,研究了不同氮肥运筹对千粒重、β-淀粉酶活性、蛋白质组分含量动态变化和产量及其构成因子的影响。结果表明:(1)随花后天数的增加,大麦籽粒千粒重、β-淀粉酶活性、醇溶蛋白和谷蛋白含量呈现由低到高的变化,而清蛋白和球蛋白含量先急剧上升,后略微下降;(2)增加拔节肥比例能显著增加β-淀粉酶活性、醇溶蛋白和谷蛋白含量,而对清蛋白和球蛋白含量影响较小;(3)千粒重、β-淀粉酶活性及蛋白质组分含量的相关性均达极显著水平;(4)随着氮肥用量后移,有效穗数、产量呈上升趋势,千粒重呈下降趋势。综合考虑各项指标,建议在类似本试验条件下的啤酒大麦生产区,基肥、苗肥、拔节肥比例以6∶3∶1为宜。     

Comparative Enzyme Kinetics of Two Allelic Forms of Barley (Hordeum vulgare L.) Beta -amylase

The barley (Hordeum vulgare L.) varieties, Franklin and Schooner, contain two different allelic forms of beta -amylase (EC 3.2.1.2) encoded on chromosome 4H by the Bmy 1-Sd1 and Bmy 1-Sd2L alleles, respectively. The corresponding enzymes, referred to as Sd1 and Sd2L, were purified from both mature barley grain and germinated barley (green malt), and their physical and kinetic properties studied. Approximately 4 kDa were cleaved from both Sd1 and Sd2Lbeta -amylases after germination. The K_mvalue for green malt beta -amylase was less than that of mature grain beta -amylase for both varieties when potato starch was used as a substrate, although V_maxwas similar. This indicated that proteolysis after germination increased the affinity of beta -amylase for potato starch. No significant kinetic differences were observed between beta -amylase from mature grain and green malt of the two barley varieties when amylose (degree of polymerisation 100 and 18) and maltopentaose were used as substrates. Kinetic differences were also observed between the two allelic forms of beta -amylase. Sd1 beta -amylase from green malt exhibited a lower K_mvalue for potato starch than Sd2L beta -amylase, demonstrating that at non-saturating starch concentrations Sd1 beta -amylase is better able to hydrolyse starch than Sd2L beta -amylase. As the degree of polymerisation of the substrates decreased from approximately 740 (potato starch) to 5 (maltopentaose), the K_mvalues for beta -amylase increased, whereas V_maxvalues decreased. Maltose, the hydrolytic product of beta -amylase, was found to be a weak competitive inhibitor of both Sd1 and Sd2L green malt beta -amylases with respect to potato starch and amylose. Taken together the kinetic observations for bet a-amylase suggest that the allelic differences and C-terminal proteolysis might be exploited to improve the efficiency of starch hydrolysis during the mashing stage of the brewing process.     

Developmental Expression of Amylases During Barley Malting

Amylase activity and qualitative changes in amylase isoenzymes as a function of barley seedling age were investigated in 10 Brazilian barley cultivars. All cultivars showed few isoenzymes in early germination. An increase in general activity ensued in the following days when new isoenzymes were detected and those already observed since early germination had their activity increased. All cultivars disclosed increase in amylase activity until the third or fourth day of germination. Some cultivars maintained this high activity until the last day analysed. Other cultivars presented a decrease in activity in the fifth or sixth day. No electrophoretic pattern or allelomorph responsible for a higher amylase activity were detected. Beta -amylase activity was always superior to alpha -amylase activity. High beta -amylase activity was already observed on the second day of germination while alpha -amylase activity began to increase only from the third day on. The results obtained suggest that, at least for the cultivars analysed, there is a high general amylase activity around the fourth day of germination, indicating that germination could stop at this moment, ensuring that hydrolitic enzyme activity required in the brewing process is met. Beta -amylase was lightly correlated with diastatic power (r=0·565) but no correlation was observed between alpha -amylase and diastatic power (r=-0·128), or neither betweenalpha - and beta -amylase with malting quality (r=0·153 andr =−0·348, respectively). These results indicate that beta -amylase activity in barley grains, more than alpha -amylase, can be a good predictor of diastatic power.     

Relationships Between Barley Hordeins and Malting Quality in a Mutant of cv. Triumph I. Genotype by Environment Interaction of Hordein Content

The genetic (G), environmental (E) and G×E effects on total grain protein and hordein fractions, have been studied in a mutant (TL43) and its parent cultivar, the malting barley Triumph, in various seasons at Dundee (E Scotland) and Lleida (NE Spain). The grain protein content of TL43 was consistently about 2% higher than that of Triumph across environments. Differences in hordein composition were shown by SDS-PAGE electrophoresis. TL-43 had three B-hordein bands not present in Triumph which had a further three bands of the same hordein group not present in TL43. TL43 showed higher B-hordein content than Triumph in Scotland but lower in Spain, whereas the mutant showed consistently higher C- and D-hordein content than Triumph across environments, i.e. there was crossover G×E interaction for B-hordein and non-crossover for C- and D-hordein content. There were also differences in grain ultra-structure between the two lines, as TL43 showed a more dense protein matrix than Triumph, together with thinner pericarp, testa and aleurone layers. It was concluded that the mutation in TL43 had a significant effect on storage protein composition.     

Mapping of Barley (Hordeum vulgare L.) Beta -amylase Alleles in which an Amino Acid Substitution Determines Beta -amylase Isoenzyme Type and the Level of Free Beta -amylase

The three beta -amylase genes (Bmy1, 2 and 3) in cultivated barley were mapped to chromosomes 4HL, 2HL And 4HL respectively using RFLP analysis. No recombinants between Bmy1 andBmy3 were detected among 264 DH lines. Polymorphism of the Sd1 and Sd2 isoenzymes of beta -amylase co-segregated with the Bmy loci on chromosome 4HL in a doubled-haploid population of the cross Chebec (Sd2)×Harrington (Sd1). This locus also explained 90·5% of the variation in the level of free enzyme between the two parents. Two cDNAs ofbeta -amylase were isolated by RT-PCR from the developing grains of Harrington (Sd1) and Galleon (Sd2). Alignment of the deduced amino acid sequences identified three amino-acid substitutions between the Sd2 and Sd1 forms of beta -amylase (Arg115 – Cys, Asp165 – Glu, and Val430 – Ala). Three allele-specific PCR primer pairs based on the three amino acid substitutions were used to amplify the beta -amylase genes in genomic DNA of sixteen barley cultivars/lines. Only the Arg115(Sd2)/Cys(Sd1) substitution was consistent with the isoenzyme form. This amino acid replacement reduced the pI of the Sd1 beta -amylase consistent with the fact that the Sd2 form is more basic than the Sd1 form when separated by IEF. The mutation from Arg115 to Cys in the Sd1 form also provides one more -SH group to form S-S-bridges. As bound beta -amylase is linked to the insoluble proteins of the endosperm and its inhibitor via disulphide bridges this could explain the higher level of binding exhibited by Sd1 vs Sd2. Thus a single amino acid substitution determines both the isoenzyme type and beta -amylase binding.     

Barley grain β-glucan content as affected by nitrogen and irrigation

Barley (Hordeum vulgare L.) with high grain β-glucan content in the soluble dietary fiber fraction may be useful as a specialty crop for human food. In contrast, low β-glucan content is desirable for brewing or animal feed. The objective of this study was to evaluate the effect of nitrogen and irrigation on barley grain β-glucan content. Relationships between barley grain β-glucan content and some agronomic traits were examined. Generally, high nitrogen levels increased barley grain β-glucan content in both years. The N₂ treatment gave the best grain β-glucan contents. A negative effect of irrigation was observed for barley grain β-glucan content. Increased levels of irrigation tended to decrease grain β-glucan content in barley. Non-irrigated treatments commonly had the greatest values for grain β-glucan content in all treatments. Higher positive correlations between β-glucan and protein content were observed in both years. A negative correlation between β-glucan content and 1000 grain weight was significant in 1999 while a negative correlation between β-glucan content and sieve analysis was significant in 2000. It may be concluded that increased nitrogen is desirable for high grain β-glucan content in barley but not irrigation.     

Thermostability variation in alleles of barley beta-amylase

Thermostability assays in conjunction with IEF and molecular mapping were used to identify three beta-amylase alleles (Bmyl-Sd1, -Sd2L, -Sd2H) in cultivated barley and an additional allele (Bmy1-Sd3) in an accession of wild barley Hordeum vulgare ssp. spontaneum. The four forms of beta-amylase exhibit different rates of thermal inactivation in barley extracts. This variation was shown to persist after the proteolytic processing of the enzyme that occurs during germination. Three forms of beta-amylase representing the range of thermostabilities were purified and shown to have T₅₀ temperatures of 56·8°C for the Sd2L enzyme, 58·5°C for the Sd1 enzyme, and 60·8°C for the Sd3 beta-amylase from wild barley. Analysis of the relationship between beta-amylase thermostability and fermentability, i.e. the yield of fermentable sugars obtained from starch hydrolysis during brewing in 42 commercial malt samples suggests that increased thermostability results in more efficient starch degradation. Screening for specific beta-amylase alleles is proposed as a method for increasing fermentability in malting barley.     

Hormonal changes in grains of cv. Triumph and its mutant TL43 as affected by nitrogen fertilizer at heading time

A pot experiment was conducted to study the hormonal changes during the grain filling stage in barley lines of Triumph and its mutant TL43 with or without nitrogen fertilizer at heading time. The ABA, ZR and IAA contents in grains were higher under nitrogen treatment (HN) for both genotypes at 30 days post anthesis (DPA). No genotypic differences were observed in GA₃, ABA and IAA content over the grain filling stage and ZR content on 10 DPA exhibited the major difference, which might be associated with the decrease of A-type starch granules in endosperm of TL43. Triumph also showed significantly higher grain weight, lower hordein and glutelin contents than TL43. The changes of these characters might be correlated with the ZR deficiency of TL43 at early grain filling stage.     

Beta-amylase degradation by serine endoproteinases from green barley malt

Proteolytic degradation of barley proteins is examined in green (unkilned) malt and germinating seeds from Hordeum vulgare L. cv. Harrington. Zymographic analysis of the Harrington green malt extracts using commercial preparations of barley beta-amylase incorporated as a proteolytic substrate in 2-D SDS gels shows multiple proteolytic activities. A developmental study shows that the several green malt beta-amylase-degrading activities appear at around day 2 of germination. The several activities appear to increase and decrease through 7 days of germination in a coordinated fashion. Gels treated with class-specific proteinase inhibitors show that serine-class proteinase activities are responsible for barley beta-amylase degradation seen on the zymograms. Western blot analysis also shows that proteolytic enzymes recovered from 1-D electrophoretic gels degrade barley beta-amylase, and that the degradation is inhibited by PMSF. This is the first demonstration that malt proteinases are capable of degrading important metabolic enzymes in germinating barley, and the first postulated physiological role for the serine class proteinases in barley malt.     

Modelling the Contribution ofAlpha-Amylase,Beta-Amylase and Limit Dextrinase to Starch Degradation During Mashing

Response surface methodology was used to determine the levels ofalpha-amylase,beta-amylase and limit dextrinase enzymes required for efficient conversion of starch to fermentable sugars during mashing. Micro-scale mashes with purified barley starch and malt enzymes were performed in a Brewing Research Foundation mash bath, and mash liquors were analysed for solubilised starch, reducing sugars (neocuproine assay) and fementable sugars (anion exchange HPLC). Fermentable sugars in the mash liquor were positively correlated with reducing sugars (R²=0·94) and the percentage of starch solubilised during mashing (R²=0·68). A multiple regression equation relating the levels of the three starch degrading enzymes to the percentage of starch hydrolysed to fermentable sugars gave a good fit to the second order response surface (R²=1·00, RMSE=1·37%). Addition of limit dextrinase to the mashes resulted in a substantial increase in levels of fermentable sugars, and limit dextrinase showed a synergistic effect in increasing levels of maltose in the mash liquor when combined with high levels ofbeta-amylase. The efficiency of any one starch degrading enzyme in a mash is influenced by the presence of other starch degrading enzymes. Commercial malts contain excess levels ofbeta-amylase and below optimal levels of limit dextrinase. Malt extract may not be a good indicator of the level of fermentable carbohydrates produced during mashing.     

Use of falling number and rapid visco analyser instruments to estimate sorghum malt diastatic power

Diastatic power (DP), a measure of joint alpha- and beta-amylase activities, is the most important quality criterion of sorghum malt. There is a need for a rapid method to estimate sorghum malt DP. Such methods have been developed using both the Falling Number (FN) and Rapid Visco Analyser (RVA) instruments, which measure alpha-amylase activity. Maize starch is used as substrate at a ratio of malt to maize starch of 1:29. Good estimates of DP can be obtained with malts prepared from grain of a single cultivar (FN, r = −0·872; RVA, r = −0·993). The estimate is less good with malts prepared from different cultivars (FN, r = −0·759; RVA, r = −0·759), probably a result of the different cultivars having varying proportions of alpha-amylase relative to DP. The methods are well suited, therefore, to quality control in maltings and breweries, but less suitable for evaluating the malting quality of different cultivars.     

Use of two fungal strains as test organisms for the evaluation of relative nutritive value in cereals and legumes

Two fungal strains were used to evaluate the relative nutritive value of various cereals and legumes.Aspergillus flavus Link ex Fr., isolated from spoiling bread crumbs, was tested on two sets of samples at equal nitrogen level. One comprised of 4 cereals and 19 legumes and the second included 25 barley strains, ranging in protein content from 7.7 to 17.8 percent. Total dry weight of the mycelium produced was used was used as the index of biomass.A. flavus was able to differentiate between different species of grain and also between different varieties within a species. A lysine dependent strain ofNeurospora crassa Shear and Dodge, was used to test 25 barley strain. The milled samples were administered, at equal nitrogen level with excess of starch and vitamins. In both the fungi biomass was positively correlated with protein quality.     

Novel food and non-food uses for sorghum and millets

Sorghum and millets have considerable potential in foods and beverages. As they are gluten-free they are suitable for coeliacs. Sorghum is also a potentially important source of nutraceuticals such antioxidant phenolics and cholesterol-lowering waxes. Cakes, cookies, pasta, a parboiled rice-like product and snack foods have been successfully produced from sorghum and, in some cases, millets. Wheat-free sorghum or millet bread remains the main challenge. Additives such as native and pre-gelatinised starches, hydrocolloids, fat, egg and rye pentosans improve bread quality. However, specific volumes are lower than those for wheat bread or gluten-free breads based on pure starches, and in many cases, breads tend to stale faster. Lager and stout beers with sorghum are brewed commercially. Sorghum's high-starch gelatinisation temperature and low beta-amylase activity remain problems with regard to complete substitution of barley malt with sorghum malt . The role of the sorghum endosperm matrix protein and cell wall components in limiting extract is a research focus. Brewing with millets is still at an experimental stage. Sorghum could be important for bioethanol and other bio-industrial products. Bioethanol research has focused on improving the economics of the process through cultivar selection, method development for low-quality grain and pre-processing to recover valuable by-products. Potential by-products such as the kafirin prolamin proteins and the pericarp wax have potential as bioplastic films and coatings for foods, primarily due to their hydrophobicity.