Limit Dextrinase Activity Research Articles

The effect of L-cysteine on starch and protein degradation during barley germination.

In order to investigate the impact of L-cysteine (L-Cys) on starch and protein degradation during barley germination. The amylase activities, degradation of macromolecules during germination were determined in this study. Barley was germinated in petri dish for 0 to 5days with different levels of L-Cys (0mM, 2.5mM, 5mM, 10mM). L-Cys addition increased the total limit dextrinase (LD) activities and decreased the LD inhibitor activities during whole germination stage. The activities of α-amylase, β-amylase and free LD were increased with the addition of 2.5, 5mM L-Cys at germination days 1 to 4. Due to higher amylase in malt with the addition of L-Cys, the non-fermentable sugars were reduced and the glucose, maltotriose were improved. Furthermore, the protein degradation analysis showed that low molecular weight protein increased and middle molecular weight protein decreased obviously in wort from the malt germinated with L-Cys, demonstrating that the L-Cys promote the protein degradation. Lastly, the filtration performance of malt with the addition of L-Cys during malting was better than the control. In conclusion, L-Cys can promote the degradation of storage material (starch, protein) during barley germination, leading to a better green malt quality.

Investigating the Malting Suitability and Brewing Quality of Different Rice Cultivars

Nineteen globally diverse rice cultivars were analyzed for various chemical parameters important to malting, including germination energy, protein, apparent amylose content, and gelatinization temperatures (GT). The rice cultivars were then malted, and congress mashes were produced. Several parameters important to brewing were then assessed in the malts and worts (i.e., extract, soluble protein, free amino nitrogen (FAN), GT, etc.). The rice malts produced were saccharified to varying degrees, had high limit dextrinase activities, and contained sufficient FAN/protein concentrations. This suggests their potential to yield robust fermentations in beer styles with high adjunct inclusions without requiring additional nitrogen supplementation. Rice cultivars with purple-pigmented bran were found to yield unique wort colors and could serve as novel natural gluten-free colorants for future recipes. Overall, these findings suggest that malted rice could offer a more local and gluten-free source of starch for brewers and beverage/food producers.

Effects of post-heading high temperature on some quality traits of malt barley

Global change is bringing barley with more frequency of suffering from high temperature. However, little has been known about the influence of high temperature on malt quality traits. In this study, we investigated the impact of 1-wk heat stress (32°C/26°C, day/night, 12 h/12 h) initiating from the 7th (HT7) and 14th (HT14) days after heading on some grain and malt quality traits of two barley cultivars. In comparison with normal temperature (24°C/18°C, day/night, 12 h/12 h), heat stress significantly reduced kernel weight, seed setting rate and grains per spike: HT7 having a larger effect than HT14. Meanwhile, total protein and β-glucan contents, and β-amylase and limit dextrinase activities were significantly increased under high temperature, with HT7-treated plants showing larger changes. Moreover, the different changes of four protein fractions under heat stress were found in the two barley cultivars, indicating the possibility of breaking positive association between protein content and enzyme activity.

The influence of drought stress on malt quality traits of the wild and cultivated barleys

As a major abiotic stress, drought causes instability and deterioration of malt barley quality. There is distinct difference among barley cultivars in the responses of the main malt quality traits to drought stress. In the previous study, we identified some Tibetan wild barley accessions with relatively less change of malt quality traits under drought. In this study, we examined the impact of drought stress during grain filling stage on grain weight and several important malt quality traits, including total protein content, β-glucan content, limit dextrinase activity, β-amylase activity, and protein fractions in four barley genotypes (two Tibetan wild accessions and two cultivars). Drought treatment reduced grain weight, β-glucan content, and increased total protein content, β-amylase activity. These changes differed among barley genotypes and treatments, and are closely associated with grain filling process and kernel weight. All the results indicated Tibetan wild barley had great potential for developing drought tolerant barley cultivars. Relatively stable kernel weight or filling process under water stress should be highlighted in malt barley breeding in order to reduce the effect of water stress on malt barley quality.

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.

Comparisons of Modern United States and Canadian Malting Barley Cultivars with Those from Pre-Prohibition: IV. Malting Quality Assessments Using Standard and Nonstandard Measures

This study was conducted to identify which traits or combination of traits associated with malting quality and mashing performance could best define the differences between and within a population of six pre-Prohibition malting barley varieties and a population of five modern elite malting barley cultivars. To accomplish this, standard and nonstandard metrics of malt quality and of performance during Congress mashing were analyzed by simple linear correlations and multivariate statistics. Analyses of the two populations combined revealed that activities of α-amylase at each time sampled during mashing were positively and significantly correlated with the concentrations of glucose at that same time, were not significantly correlated with maltose concentrations at any time, and were only significantly correlated with maltotriose concentrations for the first 30 min of mashing. Activities of β-amylase were not significantly correlated with concentrations of glucose, maltose, or maltotriose at any time during mashing. Analysis of α-glucosidase activities showed no consistent trends in correlations with either glucose or maltotriose and no significant correlation with maltose at any time. Analysis of limit dextrinase activities showed significant and positive correlations with glucose and maltotriose concentrations prior to the mash temperature reaching the starch conversion stage. Although no individual amylolytic enzyme activity was significantly correlated with maltose concentration, diastatic power was positively and significantly correlated with maltose at five of six times sampled during mashing. Diastatic power was not significantly correlated with glucose or maltotriose concentrations at any time. Malt extract values and osmolyte concentrations were positively and significantly correlated with mash glucose concentrations at all times sampled, with maltose concentrations at the first four times sampled, and with maltotriose for the first five times. Principal component analysis (PCA) of the traits analyzed in the two populations combined showed that the modern malting barleys were generally well separated from the pre-Prohibition barleys by three principal components that accounted for 75% of the variation among the 11 cultivars. The traits that collectively contributed the most to separation of the modern from the pre-Prohibition barleys were osmolyte concentrations, malt extract values and glucose concentration, which were higher in the modern cultivars, and α-glucosidase thermostability and concentrations of maltopentaose, maltohexaose and maltoheptaose, which were higher in the pre-Prohibition cultivars. PCA of the individual populations also separated Tradition from the other modern cultivars due to its lower levels of glucose, maltotriose, and α-amylase activity and by its higher levels of maltose and % plump. PCA of the pre-Prohibition population separated Hanna from the other pre-Prohibition cultivars primarily by its higher levels of maltopentaose through maltoheptaose and its low levels of osmolytes. Silver King was somewhat displaced from other pre-Prohibition cultivars due to its low levels of osmolytes and to its very high levels of α-glucosidase thermostability.

Comparisons of Modern U.S. and Canadian Malting Barley Cultivars with Those from Pre-Prohibition: II. Amylolytic Enzyme Activities and Thermostabilities

ABSTRACTAmylolytic enzyme activities and thermostabilities in North American pre-Prohibition and modern malting barley cultivars were compared throughout Congress mashing. The mean α-amylase activity was significantly higher in modern cultivars throughout mashing than in pre-Prohibition varieties, although the reverse was observed for mean thermostabilities, expressed as percent of initial activity. The mean limit dextrinase activity in modern cultivars was higher early in mashing than in pre-Prohibition varieties, but modern cultivar mean thermostability was less than in pre-Prohibition varieties at the end of mashing. There were no significant differences in population means for β-amylase or α-glucosidase activities during mashing although mean thermostabilities for both enzymes were greater in pre-Prohibition cultivars than in modern cultivars. A comparison of individual genotypes showed the four highest α-amylase activities were in modern cultivars, with Charles having the most thermostable (116%) and Tradition the least thermostable (21%). Among pre-Prohibition cultivars, Oderbrucker (132%) and Manchuria (127%) had the most thermostable α-amylases. Hanna and Manchuria had the highest β-amylase activities at 30 and 45 min of mashing. No cultivar had β-amylase activity beyond 45 min of mashing. At the end of mashing, Silver King, Hannchen and Oderbrucker had the highest α-glucosidase activities and thermostabilities (88, 52, and 53%, respectively). Among modern cultivars, highest α-glucosidase activities and thermostabilities were in Metcalfe (39%), Tradition (47%) and Charles (42%). The highest limit dextrinase activities, followed by their thermostabilities, at the end of mashing were in Manchuria (57%) and Hannchen (60%) followed by Tradition (44%), Metcalfe (26%) and Charles (37%).

The relationship of limit dextrinase, limit dextrinase inhibitor and malt quality parameters in barley and their genetic analysis

Limit dextrinase (LD) and limit dextrinase inhibitor (LDI) are the two important traits affecting malt quality. The genetic variation and controlling of LD activity and LDI content in barley grains and malt are not well understood. In this study, we measured LD activity and LDI content in both grains and malt of 68 cultivated barley genotypes. The results show that there is a wide difference among barley genotypes in both LD activity and LDI content. LD in malt is not correlated with LD in grains, but negatively correlated with LDI in malt. LD in malt is positively correlated with diastatic power (DP), Kolbach index (KI) and soluble nitrogen content (SN), and negatively correlated with viscosity (VC). LDI in malt is positively correlated with DP and total nitrogen (TN), and negatively correlated with KI. Association analysis identifies 5 QTLs associated with LD and 3 QTLs associated with LDI in malt. Three major QTLs controlling LD in malt account for 35.7%, 35.7% and 28.4% of phenotypic variation, respectively. A total of 17 QTLs associated with malt quality are identified. The current results address the importance of both LD and LDI in affecting malt quality and the identified QTLs could be useful in barley breeding.

A Chromogenic Assay Suitable for High-Throughput Determination of Limit Dextrinase Activity in Barley Malt Extracts

Twenty-four malt samples were assayed for limit dextrinase activity using a chromogenic assay developed recently in our group. The assay utilizes a small soluble chromogenic substrate which is hydrolyzed selectively by limit dextrinase in a coupled assay to release the chromophore 2-chloro-4-nitrophenol. The release of the chromophore, corresponding to the activity of limit dextrinase, can be followed by measuring the UV absorption at 405 nm. The 24 malt samples represented a wide variation of limit dextrinase activities, and these activities could be clearly differentiated by the assay. The results obtained were comparable with the results obtained from a commercially available assay, Limit-Dextrizyme from Megazyme International Ireland. Furthermore, the improved assay uses a soluble substrate. That makes it well suited for high-throughput screening as it can be handled in a 96-well plate format.

The genotypic difference in the effect of water stress after anthesis on the malt quality parameters in barley

It is well known that water stress is a major abiotic factor causing barley yield loss and unstable malt quality. However it is not quite clear about the altered pattern of the main malt quality traits for the barley genotypes differing in water stress tolerance. In this study, the effect of water stress during grain filling stage on grain weight, β-glucan content, β-amylase and limit dextrinase (LD) activities of three Tibetan wild and two cultivated barley genotypes were investigated. The results showed that water stress reduced biomass accumulation and grain weight of all genotypes, and increased protein content, but the reduced or increased extent differed significantly among genotypes, with XZ147 being less affected. In general, water stress increased β-amylase activity, reduced β-glucan content, had little effect on LD activity, which are attributed to increased protein and hordein contents, reduced grain weight, and its ineffective form in grains, respectively. Tibetan wild barley XZ147 is characterized by little change in grain weight and β-glucan content, dramatically increase of β-amylase activity under water stress, indicating its great potential for use in developing barley cultivars with high tolerance to water stress and high malt quality.

Crystal Structure of Barley Limit Dextrinase-Limit Dextrinase Inhibitor (LD-LDI) Complex Reveals Insights into Mechanism and Diversity of Cereal Type Inhibitors

Molecular details underlying regulation of starch mobilization in cereal seed endosperm remain unknown despite the paramount role of this process in plant growth. The structure of the complex between the starch debranching enzyme barley limit dextrinase (LD), hydrolyzing α-1,6-glucosidic linkages, and its endogenous inhibitor (LDI) was solved at 2.7 Å. The structure reveals an entirely new and unexpected binding mode of LDI as compared with previously solved complex structures of related cereal type family inhibitors (CTIs) bound to glycoside hydrolases but is structurally analogous to binding of dual specificity CTIs to proteases. Site-directed mutagenesis establishes that a hydrophobic cluster flanked by ionic interactions in the protein-protein interface is vital for the picomolar affinity of LDI to LD as assessed by analysis of binding by using surface plasmon resonance and also supported by LDI inhibition of the enzyme activity. A phylogenetic analysis identified four LDI-like proteins in cereals among the 45 sequences from monocot databases that could be classified as unique CTI sequences. The unprecedented binding mechanism shown here for LDI has likely evolved in cereals from a need for effective inhibition of debranching enzymes having characteristic open active site architecture. The findings give a mechanistic rationale for the potency of LD activity regulation and provide a molecular understanding of the debranching events associated with optimal starch mobilization and utilization during germination. This study unveils a hitherto not recognized structural basis for the features endowing diversity to CTIs.

Genetic diversity and QTL mapping of thermostability of limit dextrinase in barley.

Limit dextrinase (LD) is an essential amylolytic enzyme for the complete degradation of starch, and it is closely associated with malt quality. A survey of 51 cultivated barley and 40 Tibetan wild barley genotypes showed a wide genetic diversity of LD activity and LD thermostability. Compared with cultivated barley, Tibetan wild barley showed lower LD activity and higher LD thermostability. A doubled haploid population composed of 496 DArT and 28 microsatellite markers was used for mapping Quantitative Trait Loci (QTLs). Parental line Yerong showed low LD activity and high LD thermostability, but Franklin exhibited high LD activity and low LD thermostability. Three QTLs associated with thermostable LD were identified. The major QTL is close to the LD gene on chromosome 7H. The two minor QTLs colocalized with previously reported QTLs determining malt-extract and diastatic power on chromosomes 1H and 2H, respectively. These QTLs may be useful for a better understanding of the genetic control of LD activity and LD thermostability in barley.

Colourimetric and fluorimetric substrates for the assay of limit dextrinase

The measurement of limit-dextrinase (LD) (EC 3.2.1.142) in grain samples such as barley, wheat or rice can be problematic for a number of reasons. The intrinsic LD activity in these samples is extremely low and they often contain a limit-dextrinase inhibitor and/or high levels of reducing sugars. LD also exhibits transglycosylation activity that can complicate the measurement of its hydrolytic activity. A minor modification to the industrial standard Limit-Dextrizyme tablet test is suggested here to overcome this transglycosylation issue.In addition, two new substrates are described that can be adopted for use in an auto-analyser format. 4,6-O-benzylidene-2-chloro-4-nitrophenyl-β-63-α-d-maltotriosyl-maltotrioside (BzCNPG3G3, Hexachrom) is not susceptible to transglycosylation and serves amiably as a routine quantitative assay tool with the potential to run kinetic assays due to the low pKa (∼5.5) of the chromogenic moiety while 4,6-O-benzylidene-4-methylumbelliferyl-β-63-α-d-maltotriosyl-maltotrioside (BzMUG3G3, Hexafluor) was found to be susceptible to transglycosylation with LD. It is anticipated that Hexafluor may find extensive use in applications where high sensitivity is required such as high throughput screening studies.

Oligosaccharide and Substrate Binding in the Starch Debranching Enzyme Barley Limit Dextrinase

Complete hydrolytic degradation of starch requires hydrolysis of both the α-1,4- and α-1,6-glucosidic bonds in amylopectin. Limit dextrinase (LD) is the only endogenous barley enzyme capable of hydrolyzing the α-1,6-glucosidic bond during seed germination, and impaired LD activity inevitably reduces the maltose and glucose yields from starch degradation. Crystal structures of barley LD and active-site mutants with natural substrates, products and substrate analogues were sought to better understand the facets of LD–substrate interactions that confine high activity of LD to branched maltooligosaccharides. For the first time, an intact α-1,6-glucosidically linked substrate spanning the active site of a LD or pullulanase has been trapped and characterized by crystallography. The crystal structure reveals both the branch and main-chain binding sites and is used to suggest a mechanism for nucleophilicity enhancement in the active site. The substrate, product and analogue complexes were further used to outline substrate binding subsites and substrate binding restraints and to suggest a mechanism for avoidance of dual α-1,6- and α-1,4-hydrolytic activity likely to be a biological necessity during starch synthesis.

Colourimetric and fluorometric substrates for measurement of pullulanase activity

Specific and highly sensitive colourimetric and fluorometric substrate mixtures have been prepared for the measurement of pullulanase and limit-dextrinase activity and assays employing these substrates have been developed. These mixtures comprise thermostable α- and β-glucosidases and either 4,6-O-benzylidene-2-chloro-4-nitrophenyl-β-maltotriosyl (1-6) α-maltotrioside (BzCNPG3G3, 1) as a colourimetric substrate or 4,6-O-benzylidene-4-methylumbelliferyl-β-maltotriosyl (1-6) α-maltotrioside (BzMUG3G3, 2) as a fluorometric substrate. Hydrolysis of substrates 1 and 2 by exo-acting enzymes such as amyloglucosidase, β-amylase and α-glucosidase is prevented by the presence of the 4,6-O-benzylidene group on the non-reducing end d-glucosyl residue. The substrates are not hydrolysed by any α-amylases studied, (including those from Aspergillus niger and porcine pancreas) and are resistant to hydrolysis by Pseudomonas sp. isoamylase. On hydrolysis by pullulanase, the 2-chloro-4-nitrophenyl-β-maltotrioside (3) or 4-methylumbelliferyl-β-maltotrioside (4) liberated is immediately hydrolysed to d-glucose and 2-chloro-4-nitrophenol or 4-methylumbelliferone. The reaction is terminated by the addition of a weak alkaline solution leading to the formation of phenolate ions in solution whose concentration can be determined using either spectrophotometric or fluorometric analysis. The assay procedure is simple to use, specific, accurate, robust and readily adapted to automation.

Comparisons of Amylolytic Enzyme Activities and β-Amylases with Differing Bmy1 Intron III Alleles to Osmolyte Concentration and Malt Extract during Congress Mashing with North American Barley Cultivars

This study was conducted to determine the relationships between patterns of activity of malt amylolytic enzymes (α-amylase, β-amylase, and limit dextrinase) and the patterns of osmolyte concentration (OC) and malt extract (ME) production in two- and six-row North American barley cultivars over the course of Congress mashing and to test three hypotheses: 1) that maximal activity of and rates of increase in β-amylase activity during the initial phases of mashing would correlate better than the other amylolytic enzymes with OC and ME production, 2) that positive rates of change of OC would continue for a longer period than ME during mashing regardless of cultivar β-amylase intron III allelic variation, and 3) that β-amylase intron III allelic variation would have no significant association with the magnitude or OC and ME production during mashing. Malts of 12 barley cultivars were mashed in a micromasher and assayed for amylolytic enzyme activities and OC and ME levels at six time points during the 115-min Congress mashing regime. Maximal β-amylase activity correlated positively and significantly with OC (r = 0.701, P = 0.011) and better than with ME (r = 0.419, P = 0.175). Maximal α-amylase activity correlated positively and significantly with OC (r = 0.582, P = 0.047) but not as well as did β-amylase, nor did it significantly correlate with ME. Maximal limit dextrinase activity did not correlate significantly with either OC or ME. Rates of increase of β-amylase activity correlated well with rates of increase of OC (r = 0.656, P = 0.0205) but not with ME (r = 0.269. P = 0.398) during the initial phase of mashing (5 to 30 min). Rates of increase of α-amylase and limit dextrinase activities did not correlate significantly with OC or ME over this period. Rates of increase for wort OC and ME for each cultivar were greatest over the period from 5 to 30 min of mashing. Between 55 and 75 min, rates of increase of wort ME plummeted to near nil and remained significantly the same (P < 0.0001) for the remainder of mashing. In contrast, rates of increase in wort OC for each cultivar were still positive between 55 and 75 min, albeit at a lower level than earlier in mashing. For most cultivars, rates of increase in OC, although decreasing, remained positive until the end of mashing. β-Amylase intron III allelic variation had no apparent association with this pattern of OC change. The OC data indicate continued degradation of storage compounds for a significantly longer duration than increases in ME after the mass of material in wort had stabilized. Throughout mashing, cultivars with either the β-amylase Bmy1.a or Bmy1.b intron III alleles were not significantly different for the highest OC or ME. OC measurements resulted in more significant differences between cultivars than ME measurements. Also, OC did not consistently rank cultivars in the same pattern as ME during mashing, indicating that the intrinsic differences in the two methods yield slightly differing results. These data support all three hypotheses.

Effect of teff (Eragrostis tef) variety and storage on malt quality attributes

Varieties of a cereal may have a considerable influence on malting qualities owing to variations in the physicochemical properties of the grains. This research was aimed at assessing the influence of five teff varieties on malt quality attributes. The teff samples were malted using previously optimized malting conditions and mashed with the congress mashing procedure. In this research, the Kuncho teff variety was malted in a one year period after harvesting, whereas the other four varieties were malted after three years of storage. Alpha- and β-amylase, and limit dextrinase activity, were in the ranges 14–68, 10–440 and 375–1072 U/kg, respectively. Extracts ranged from 54% for Dessie to 74% for Ivory teff. Free amino nitrogen, protein content, soluble nitrogen, Kolbach index, viscosity and wort colour were in the ranges 160–364 mg/L, 8.6–13.6%, 532–1048 mg/100 g, 24–50%, 1.441–1.629 mPa s and 5.9–9.0 EBC units, respectively. High-performance liquid chromatographic analysis for individual fermentable sugars revealed that the highest value in all varieties was recorded for glucose followed by maltose. The concentration of glucose ranged from 9.49 g/L in Brown teff, to 19.42 g/L in Ivory teff, whereas maltose ranged from 2.95 g/L in Dessie teff to 16.1 g/L in Kuncho teff. All of the malt quality attributes considered in this study were markedly influenced (p < 0.05) by the type of teff cultivar. It was concluded that the use of different teff varieties yielded malts with significantly different malt quality attributes. Copyright © 2013 The Institute of Brewing & Distilling

Association of HvLDI with limit dextrinase activity and malt quality in barley

Limit dextrinase (LD) is a unique de-branching enzyme involved in starch mobilization of barley grains during malting, and closely related to malt quality. Genotypic variation of LD activity is controlled by genetic factors and also affected by environmental conditions. Correlation analysis between LD activity and four malt quality parameters showed that LD activity was positively correlated with diastatic power, Kolbach index and the quality of malt extract, while negatively correlated with viscosity. The structure-based association analysis demonstrated that HvLDI, a gene encoding limit dextrinase inhibitor, was a major determinant of LD activity and malt quality. The single nucleotide polymorphisms associated with LD activity could be used in early generation selection for barley breeding.

Effect of Drying Temperature and Time on Alpha-Amylase, Beta-Amylase, Limit Dextrinase Activities and Dimethyl Sulphide Level of Teff (Eragrostis tef) Malt

Teff is a gluten-free cereal with attractive nutritional profile. This research was aimed to study the influence of kilning on the enzyme activities and dimethyl sulphide (DMS) level of DZ-Cr-387 teff variety and suggest a kilning condition that yields teff malt with low DMS with no or little damage on its enzyme activities. The malts were dried using isothermal conditions at 30, 40, 50, 60 and 70 °C for 40 h with sampling in certain time interval. To set up kilning program, two temperature regimens 18 h at 30 °C + 1h at 60 °C + 3 or 5 h at 65 °C (R1) and 18 h at 30 °C + 1 h at 60 °C + 3 or 5 h at 80 °C (R2) were selected. Results from isothermal kilning indicated that enzyme activities, DMS and moisture contents were affected (P < 0.05) by time and temperature. The values of α-amylase, β-amylase, limit dextrinase activities and DMS content while using the first regimen (R1) with 3 h curing at 65 °C were 68 U/g, 440 U/g, 1,072 U/kg and 3.3 mg/kg, respectively. Whereas in the second regimen with 3 h curing at 80 °C, the values were 42 U/g, 406 U/g, 736 U/kg and 2.15 mg/kg, respectively. Prolonged curing in both kilning regimens caused an adverse effect on the amylolytic enzyme activities. R1 with shorter curing time is considered to be the best condition in preserving enzymes. The enzyme activities and DMS level show that teff can be an alternative raw material for production of gluten-free malt.

Tracking Amylolytic Enzyme Activities during Congress Mashing with North American Barley Cultivars: Comparisons of Patterns of Activity and β-Amylases with Differing Bmy1 Intron III Alleles and Correlations of Amylolytic Enzyme Activities

This study was conducted to test three hypotheses: 1) that α-amylase will have less consistent patterns of activity during mashing than will β-amylase and limit dextrinase; 2) that differing β-amylase 1 intron III alleles (Bmy1.a and Bmy1.b) would not be useful in predicting high or low activities or thermostabilities among cultivars; and 3) that correlations of α-amylase versus β-amylase activities would be more significant than for either amylase versus limit dextrinase during the initial 55 min of mashing, when the bulk of wort sugars are produced. Malts of six two-row and six six-row barley cultivars were mashed, and amylolytic enzymes were assayed at six time points during mashing. α-Amylase activities of four cultivars peaked at 30 min (45°C), six at 55 min (70°C), and two at 75 min (70°C). β-Amylase activities of all cultivars peaked sharply at 30 min (45°C) into mashing and declined precipitously thereafter, rapidly approaching near nil by 75 min. Limit dextrinase activities of 11 of 12 cultivars peaked at 55 min (70°C), and one peaked at 30 min (45°C). These data indicate no uniform pattern for the development of α-amylase activity compared with patterns for β-amylase and limit dextrinase during mashing. Steptoe, a feed barley cultivar, had much lower peak activities for all three amylolytic enzymes than did malting cultivars. The highest β-amylase activities during mashing of cultivars with either the Bmy1.a (Legacy, Lacey, and Morex) or Bmy1.b (Merit and Harrington) intron III alleles were not significantly different as determined by least significant difference (LSD) analysis (P < 0.0001). As temperatures increased from 45 to 70°C (55 min), percentages of the highest β-amylase activities decreased from 11.9% (Rasmusson) to 41.5% (Steptoe). LSD analysis revealed that Steptoe (Bmy1.a allele) had significantly higher β-amylase thermostability than did malting cultivars. The highest malting barley thermostabilities for β-amylases (Lacey [Bmy1.a allele] and Harrington [Bmy1.b allele]) were not significantly different (P < 0.0001). Correlations of α-amylase versus β-amylase activities were positive and significant in seven of nine possible correlations from 5 to 55 min of mashing, whereas α-amylase and β-amylase versus limit dextrinase activity correlations over the same period were significant in two of nine and one of nine correlations, respectively. This study supports all three of the proposed hypotheses.