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.     

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.     

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.     

CerealBeta-Amylases

Cerealbeta-amylases are perhaps best known in terms of the vital role they play in releasing easily fermentable sugars from cereal grain starch to fuel the production of alcohol by yeast in brewing. The extent to which they have been investigated is indeed largely due to their significance in this economically important industry. However, cerealbeta-amylases are also, or could be, employed in many other aspects of the food industry and the analysis of starch, and they constitute valuable markers in cereal assessment and breeding studies. Quite apart from their practical significance, they are rewarding objects of biochemical and physiological research. They are interesting models for the study of enzyme polymorphism, post-translational modification and the differential expression of isoenzymes. In spite of their often high activitiesin situand all that is known about their generation, they are an enigma in that their physiological function, or even necessity, remains unclear. It has been recently recognised that there are two different categories of cerealbeta-amylases which exhibit different tissue and taxonomic specificities and physiological developmental patterns. The «classical»beta-amylases present at high activities in cereal seeds appear to be limited to the endosperm of the species of the Triticeae tribe of the Festucoideae subfamily of the Gramineae (wheat, barley and rye), whereas all cereals exhibit a different, tissue-«ubiquitous» form of the enzyme which is present at much lower activity levels. The physiological phenomenology and the usage of cerealbeta-amylases are discussed in relation to these two categories of enzyme.     

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.     

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.     

In VitroDigestion of Wheat Microstructure with Xylanase and Cellulase fromTrichoderma reesei

Resistant starch (RS), producedin vitroby hydrolysis of retrograded pea starch gels and amylose gels by porcine pancreaticalpha-amylase, was characterised by X-ray diffraction, size exclusion chromatography and methylation analysis. These techniques showed that RSin vitroconsisted of semi-crystalline, mostly linear material that was present in two main molecular size subfractions (DP_n>100 andDP_n20–30) with a third, minor subfraction (DP_n≤5). The extent of retrogradation of amylose was found to be of primary importance in determining the RS content of starch. Analysis ofin vivoRS, recovered during an ileostomy study, produced results that were similar to those obtained from RSin vitro. Anin vitromodel for the structure of resistant starch is proposed.     

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.     

Physicochemical Studies on Resistant StarchIn VitroandIn Vivo

Resistant starch (RS), producedin vitroby hydrolysis of retrograded pea starch gels and amylose gels by porcine pancreaticalpha-amylase, was characterised by X-ray diffraction, size exclusion chromatography and methylation analysis. These techniques showed that RSin vitroconsisted of semi-crystalline, mostly linear material that was present in two main molecular size subfractions (DP_n>100 andDP_n20–30) with a third, minor subfraction (DP_n≤5). The extent of retrogradation of amylose was found to be of primary importance in determining the RS content of starch. Analysis ofin vivoRS, recovered during an ileostomy study, produced results that were similar to those obtained from RSin vitro. Anin vitromodel for the structure of resistant starch is proposed.     

Potential longevity (Ki) of malting barley (Hordeum vulgare L.) grain lots relates to their degree of pre-germination assessed through different industrial quality parameters

Barley (Hordeum vulgare L.) grain germination is required to perform the malting process. Maintenance of barley seed viability during storage is crucial for the malt industry; and modern cultivars are bred for rapid grain dormancy release after physiological maturity. Low dormancy level combined with rain close to harvest induces pre-germination/pre-harvest sprouting damage. Pre-germination might not affect viability in the short term after harvest, but it could reduce potential longevity (K_i) of a barley seed lot. K_i value is inherent for each barley lot; however, its determination is time-consuming which precludes its assessment at an industrial scale. In this study we sought quantitative relationships between K_i and the pre-germination degree of barley grain lots, assessed through quality tests routinely performed by malthouses [Falling Number (FN), α-Amylase Activity and Carlsberg]. Field pre-germinated lots from one old barley cultivar (Quilmes Palomar) and artificially pre-germinated lots from major varieties currently grown in Argentina were used. Associations between K_i and values obtained from all quality tests analysed were found for Q. Palomar. However, FN was the parameter that yielded the best and simplest explanation of K_i variability. A significant positive linear K_i -FN relationship was also obtained for each modern barley cultivar.     

Protein Inhibitors ofAlpha-amylase in Mature and Germinating Grain of Rye (Secale cereale)

The activities of endogenous (R-type) and exogenous acting (D-type) protein inhibitors ofalpha-amylase and the activities ofalpha- and total amylase were determined in milling fractions of rye. High D-type amylase inhibitor activities were detected in the embryo (255 IU/g) and in the endosperm fraction (64·9 IU/g), low inhibitor activities were found in the aleurone layer fraction (25·9 IU/g). The highest R-typealpha-amylase inhibitor activity was found in the aleurone layer fraction (32·6 IU/g), and the lowest value in the epidermis containing fraction (5·0 IU/g). The D- and R-typealpha-amylase inhibitor activities varied with growing conditions. D-type amylase inhibitor activities were found to be high in those samples which grew under drought conditions and low in samples cultivated under wet and cool weather. Higher R-typealpha-amylase inhibitor activities were found in rye genotypes cultivated under wet conditions and lower values under dry weather. There were small variations inalpha-amylase inhibitor activities between sprout-stable and sprout-sensitive rye genotypes. The D- and R-typealpha-amylase inhibitor activities of all varieties were stable during 72 h of germination. Similar soil conditions will therefore lead to differentialalpha-amylase inhibitor activities depending on weather conditions during growth.     

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.     

The endogenous endoprotease inhibitors of barley and malt and their roles in malting and brewing

Endoproteases play an important role in barley germination by controlling the hydrolysis of the grain's storage proteins into peptides and amino acids that are needed by the young plant. During malting, the commercial version of this process, many high M_r barley biopolymers are converted into malt nutrients that can be utilized by yeasts during brewing. However, barley and malt both contain endogenous proteins that inhibit the enzymatic activities of these proteases. High levels of these inhibitors can cause brewing problems by preventing the proteases from producing optimal levels of soluble proteins and amino acids. Both high and low M_r inhibitors of cysteine proteases occur in barley and malt. Two of the high M_r inhibitors, lipid transfer protein 1 (LTP1) and LTP2, have been purified and studied. Recently, members of the trypsin/alpha-amylase inhibitor protein family (CM proteins) have been shown to inhibit the activity of SEP-1, a purified serine class barley protease. No inhibitors of aspartic proteases or metalloproteases have yet been purified, but it has been reported that endogenous metalloprotease inhibitors do exist. The inhibitors of the cysteine proteases and metalloproteases are probably the ones most important for brewing, because members of these two protease classes apparently catalyse most of the protein hydrolysis that occurs during malt mashing and, presumably, also during malting. More biochemical studies are needed to clarify how these proteins interact with the proteases to control protein hydrolysis during germination.     

Production of Proteases by Fusarium Species Grown on Barley Grains and in Media Containing Cereal Proteins

The production of proteases by the cereal plant pathogens Fusarium culmorum, F. graminearum and F. poae was followed through seven days of cultivation. The fungi were grown in mineral and in gluten culture media, and on autoclaved barley grains. The proteolytic activities of each sample were analysed at pH 2·2, 5·0 and 8·0 and the pH optima of the most active proteases were determined. All of the fungi grown in the gluten medium produced proteases that were active at pH levels between 6 and 10 and were most active at about pH 9·0. Fusarium poae also produced acid protease(s) with pH optima between 3.0 and 3.5 when grown in the gluten medium. No protease activity was detected in the cultures that were grown in the mineral medium, except that a small amount was formed after the glucose substrate was depleted. When grown on the barley grain medium the Fusarium species produced protease activities that were similar to the neutral and alkaline ones present in the gluten cultures, but no pH 2·2 protease activity was detected. The alkaline proteases had some characteristics that were similar to those of chymotrypsin.     

啤酒大麦与饲用大麦籽粒结构和淀粉粒的比较研究

为了探索大麦籽粒结构和淀粉粒与其用途的关系，利用扫描电子显微镜（SEM）和差示扫描量热分析仪（DSC）对啤酒大麦（港啤1号和扬农啤2号）与饲用大麦（扬饲麦1号）的籽粒结构和淀粉粒进行了比较研究。结果表明，3个品种表现相似的胚乳结构，但啤酒大麦的淀粉粒与蛋白质结合不紧密，饲用大麦的淀粉粒和蛋白质结合较紧密，且饲用大麦的蛋白质基质含量高于啤酒大麦。采用碱处理和差速沉淀法分离了籽粒胚乳大、中、小淀粉粒，虽然品种间淀粉粒的形态无明显差别，但大、中、小淀粉粒的大小和含量差异很大。DSC分析所得参数表明，大淀粉粒糊化时的起始温度（To）、峰值温度（Tp）和终结温度（n）高于相同品种的中、小淀粉粒，且糊化过程所需的热焓（△H）也较高。     

Protein and hordein content in barley seeds as affected by nitrogen level and their relationship to beta-amylase activity

Two barley cultivars differing in grain size and protein content were used to investigate the effects of nitrogen nutrition, cultivar and their interaction on grain protein content, hordein content and beta-amylase activity and the relationship between hordein content and beta-amylase activity during in vitro spike culture. The content of protein and hordein fraction, and beta-amylase activity in barley grains increased as the nitrogen level in culture solution increased. Grain protein content was significantly affected by nitrogen treatment and cultivar, and there was no significant interaction between nitrogen treatment and cultivar. Hordein content and beta-amylase activity were significantly affected by nitrogen treatment and cultivar as well as their interaction. Beta-amylase activity was positively correlated with grain protein and hordein contents, and the ratio of hordein B:C was negatively correlated with total protein content and beta-amylase activity.     

Hydrothermal Processing of Barley (cv. Blenheim): Optimisation of Phytate Degradation and Increase of FreeMyo-inositol

Optimal conditions were developed for hydrothermal processing of whole barley kernels (cv. Blenheim) to degrade phytate (myo-inositol hexaphosphate) and to increase the content of freemyo-inositol. The hydrothermal treatment comprised of two wet steeps, where lactic acid solution of different concentrations was used, and two dry steeps followed by successive drying. Experiments were performed as a central composite design and evaluated by multiple linear regression. The variables in the experiments were temperature in the first wet and dry steep (T₁), temperature in the second wet and dry steep (T₂) and lactic acid solution concentration in both wet steeps (C) and mathematical models were developed in these variables. Optimal conditions for maximal phytate degradation and for maximal increase of freemyo-inositol wereT₁=48 °C,T₂=48–50 °C andC=0·8%, at these conditions the amount of phytate was reduced by 95–96% and the freemyo-inositol concentration was increased from 0·56 to 2·45 μmol/g d.m. We conclude that this hydrothermal process can be used to produce a barley product (cv. Blenheim) with a low phytate content and a high level of freemyo-inositol.     

Effect of particle size on kinetics of starch digestion in milled barley and sorghum grains by porcine alpha-amylase

The influence of milled grain particle size on the kinetics of enzymatic starch digestion was examined. Two types of cereals (barley and sorghum) were ground, and the resulting grounds separated by size using sieving, with sizes ranging from 0.1 to 3 mm. In vitro enzymatic digestion was performed, using pancreatic alpha-amylase, amyloglucosidase and protease, to determine fractional-digestion rates over 24 h. The resulting glucose production rate data were well fitted by simple first-order kinetics. For each sieve screen size, the digestion rate of barley was always higher than that of sorghum. The rate coefficients for digestion showed a decrease with increasing size, and could be well fitted by an inverse square relationship. This is consistent with the supposition that starch digestion in these systems is controlled by diffusion of enzyme through the grain fragment. Apparent diffusion coefficients of alpha-amylase obtained by fitting the size dependence were 0.76 (sorghum) and 1.7 (barley) × 10⁻⁷ cm² s⁻¹, 9 (sorghum) and 4 (barley) times slower than predicted for a molecule of the size of alpha-amylase in water.     

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.     

The endogenous endoprotease inhibitors of barley and malt and their roles in malting and brewing

Endoproteases play an important role in barley germination by controlling the hydrolysis of the grain's storage proteins into peptides and amino acids that are needed by the young plant. During malting, the commercial version of this process, many high M_r barley biopolymers are converted into malt nutrients that can be utilized by yeasts during brewing. However, barley and malt both contain endogenous proteins that inhibit the enzymatic activities of these proteases. High levels of these inhibitors can cause brewing problems by preventing the proteases from producing optimal levels of soluble proteins and amino acids. Both high and low M_r inhibitors of cysteine proteases occur in barley and malt. Two of the high M_r inhibitors, lipid transfer protein 1 (LTP1) and LTP2, have been purified and studied. Recently, members of the trypsin/alpha-amylase inhibitor protein family (CM proteins) have been shown to inhibit the activity of SEP-1, a purified serine class barley protease. No inhibitors of aspartic proteases or metalloproteases have yet been purified, but it has been reported that endogenous metalloprotease inhibitors do exist. The inhibitors of the cysteine proteases and metalloproteases are probably the ones most important for brewing, because members of these two protease classes apparently catalyse most of the protein hydrolysis that occurs during malt mashing and, presumably, also during malting. More biochemical studies are needed to clarify how these proteins interact with the proteases to control protein hydrolysis during germination.