J. Eglinton

Thermostability variation in alleles of barley beta-amylase

Abstract 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.

Assessing the impact of the level of diastatic power enzymes and their thermostability on the hydrolysis of starch during wort production to predict malt fermentability.

In this study, commercially produced malts were used for small-scale simulated mashing trials to investigate the impact of differences in the level and thermostability of malt diastatic power (DP) enzymes on the resultant wort fermentability. A modified European Brewery Convention/American Society Brewing Chemists mashing protocol was used with mash-in temperatures ranging between 45 and 76°C for full-malt and 30% rice adjunct mashes. Malt extract yield varied little with mashing temperature for most varieties in this temperature range. However, the fermentability, maltose content, and free amino nitrogen of that extract was considerably affected by mashing temperature with 65°C achieving the highest fermentability for all malt varieties. Multilinear regression analysis of full-malt and rice adjunct mashing trials at 65°C using 43 commercial malts showed that the level of α-amylase and total limit dextrinase activity, Kolbach Index, and the total β-amylase activity level and thermostability were the most important malt quality predictors of wort fermentability. These conclusions suggest that the conventional DP assessment could be replaced with the measurement of its component enzymes outlined above so that maltsters could better satisfy brewers malt quality expectations by blending and defining their malt quality in terms of these fermentability predicting factors. This information would be particularly useful to brewers who brew with multiple varieties and blends from different suppliers. The focus on individual enzyme characteristics by barley breeders is likely to provide selection targets that are more accurate and achievable.

QTL mapping of chromosomal regions conferring reproductive frost tolerance in barley (Hordeum vulgare L.)

Spring radiation frost is a major abiotic stress in southern Australia, reducing yield potential and grain quality of barley by damaging sensitive reproductive organs in the latter stages of development. Field-based screening methods were developed, and genetic variation for reproductive frost tolerance was identified. Mapping populations that were segregating for reproductive frost tolerance were screened and significant QTL identified. QTL on chromosome 2HL were identified for frost-induced floret sterility in two different populations at the same genomic location. This QTL was not associated with previously reported developmental or stress-response loci. QTL on chromosome 5HL were identified for frost-induced floret sterility and frost-induced grain damage in all three of the populations studied. The locations of QTL were coincident with previously reported vegetative frost tolerance loci close to the vrn-H1 locus. This locus on chromosome 5HL has now been associated with response to cold stress at both vegetative and reproductive developmental stages in barley. This study will allow reproductive frost tolerance to be seriously pursued as a breeding objective by facilitating a change from difficult phenotypic selection to high-throughput genotypic selection.

Genotyping single nucleotide polymorphisms for selection of barley β-amylase alleles

A high-throughput single nucleotide polymorphism (SNP) genotyping system was developed and used to select barley seedlings carrying superior alleles of β-amylase. In the malting process, β-amylase is a key enzyme involved in the degradation of starch. Four allelic forms of the enzyme are found in barley, each exhibiting a different rate of thermal inactivation, or thermostability. The level of thermostability influences starch degradation, which determines the yield of fermentable sugars for alcohol production during brewing. Control of the fermentability level is important for barley breeding programs to allow targeting quality profiles of new varieties to suit end-user requirements. Alignment of the cDNA sequences encoding the 4 enzyme forms revealed 6 SNPs (cSNPs). The 4 alleles could be identified unambiguously by codominantly genotyping 2 of the cSNPs using a duplex single nucleotide primer extension (SNuPE) assay. Two genotyping primers with their 3′ ends directly flanking the selected SNPs were annealed to the amplified target sequences and extended by single dideoxynucleotides complementary to the polymorphic nucleotides. Extended primers were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS). Making use of the inherent molecular weight difference between DNA bases, incorporated nucleotides were identified by the increase in mass of the extended primers. A cleaved amplified polymorphic sequence (CAPS) assay enabling broader classification of the alleles was also developed to facilitate the transfer of this marker to other laboratories. Plants carrying alternative β-amylase alleles were selected at the seedling stage for barley breeding.

The determinants and genome locations influencing grain weight and size in barley (Hordeum vulgare L.)

Grain weight and size are traits important to malting and feed barley. Understanding the determinants of grain weight and size, especially under stressful growing environments, will aid breeding efforts to improve these traits. The determinants of grain weight and size are discussed in relation to the pre- and post-anthesis periods of barley development. Genetic mapping of the loci influencing grain weight and size has provided a fundamental understanding of these traits, and a summary of mapped quantitative trait loci (QTLs) from Australian and international mapping populations is presented. The influence of developmental loci on grain weight and size QTLs, approaches to discovering non-developmentally related loci, and prospects for a marker assisted selection approach to improving grain weight and size are discussed.

The properties and genetics of barley malt starch degrading enzymes

The properties and quality of barley malt starch degrading enzymes are of primary importance to the efficiency and profitability of brewing (beer and whiskey), and the bio-fuel (bio-ethanol) industries. The barley starch degrading enzymes hydrolyse starch into fermentable sugars that yeast converts into alcohol. This process is key for the alcohol producing industries as the starch substrate makes up approximately 60% of grain weight (Holtekjolen et al., 2006). Malted barley is the main source of the diastase or diastatic power (DP) enzymes that hydrolyse starch. The DP enzymes comprise the combined activity of α-amylase, β-amylase, α-glucosidase and limit dextrinase whose concerted action hydrolyse the α-(1,4) and a-(1,6) glucosyl linkages in starch (Fig. 6.1) into fermentable sugars (i.e., glucose, maltose, etc.), dextrins and limit dextrins. The actions of the DP enzymes are summarised as follows: (1) α-amylase cleaves α-(1,4)-linkages internally (endo-acting) to primarily produce oligosaccharides, limit dextrins and some fermentable sugars; (2) β-amylase cleaves α-(1,4)-linkages from the non-reducing ends (exo-acting) to produce maltose; (3) Limit dextrinase hydrolyses internal a-(1,6)-linkages (endo), to remove branch-points in amylopectin or α-limit dextrins; (4) α-Glucosidase primarily cleaves α-(1,4)-linkages from the non-reducing ends to produce glucose. Open image in new window Fig. 6.1 Schematic representation of starch hydrolysis

Prediction of starch pasting properties in barley flour using ATR-MIR spectroscopy

The objective of this study was to evaluate the feasibility of using attenuated total reflectance (ATR) mid infrared (MIR) spectroscopy to predict starch pasting properties in barley flour samples. A total of 180 barley flour samples sourced from the University of Adelaide germplasm collection, harvested over three seasons (2009, 2010 and 2011) were analysed using both ATR-MIR and the rapid visco analyser (RVA) techniques. Calibrations (n=100) were developed using partial least squares (PLS1) regression and full cross validation. The coefficient of determination (R(2)) and the standard error in cross validation (SECV) were 0.74 (SECV=875 RVU) for peak viscosity (PV), 0.63 (SECV=561 RVU) for trough (THR), 0.80 (SECV=173 RVU) for breakdown (BRK), 0.74 (SECV=126 RVU) for setback (STB), 0.77 (SECV=679 RVU) for final viscosity (FV), and 0.73 (SECV=0.57 s) for time to peak (TTP). The RPD values (SD/SEP) from the validation indicated that only BRK can be accurately predicted (RPD=4). We have demonstrated that ATR-MIR spectroscopy has the potential to significantly reduce analytical time and cost during the analysis of barley flour for starch pasting properties.

A Novel Approach to Monitor the Hydrolysis of Barley (Hordeum vulgare L) Malt: A Chemometrics Approach

Malting barley is a process that has been profusely studied and is known to be influenced by several physical and biochemical properties of the grain. In particular, the amount of material that can be extracted from the malt (malt extract) is an important measure of brewing performance and end quality. The objectives of this study were (a) to compare the time course of hydrolysis of different malting barley (Hordeum vulgare L) varieties and (b) to evaluate the usefulness of mid-infrared (MIR) spectroscopy as high-throughput method to monitor malt hydrolysis. Differences in the pattern of hydrolysis were observed between the malt samples analyzed where samples from the same variety that have similar hot water extract (HWE) values tend to have the same pattern of hydrolysis. Principal component score plots based on the MIR spectra showed similar results. Partial least-squares discriminate analysis (PLS-DA) was used to classify malt samples according to their corresponding variety and time course of hydrolysis. The coefficient of determination (R(2)) and the standard error of cross validation (SECV) obtained for the prediction of variety and time course of hydrolysis were 0.67 (1.01) and 0.38 (19.90), respectively. These differences might be the result of the different composition in sugars between the barley varieties analyzed after malting, measured as wort density and not observed when only the HWE value at the end point is reported. This method offers the possibility to measure several parameters in malt simultaneously, reducing the time of analysis as well as requiring minimal sample preparation.

An informative set of SNP markers for molecular characterisation of Australian barley germplasm

The identification of genetic variation using molecular markers is fundamental to modern plant breeding and research. The present study was undertaken to develop a resource of informative single nucleotide polymorphism (SNP) markers for molecular characterisation of Australian barley germplasm. In total, 190 SNP markers were developed and characterised using 88 elite barley lines and varieties, sampling genetic diversity relevant to Australian breeding programs, and a core set of 48 SNPs for distinguishing among the barley lines was identified. The utility of the core 48-SNP set for distinguishing barley lines and varieties using DNA extracted from grain samples was also assessed. Finally, the 48 SNPs in the core set were converted into simple PCR markers to enable co-dominant SNP genotyping on agarose gel. The SNP markers developed, and in particular the core 48-SNP set, provide a useful marker resource for assessing genetic relationships between individuals and populations of current Australian barley germplasm. They are also useful for identity and purity testing of inbred lines in research, breeding, and commercial applications.

Feasibility study on the use of attenuated total reflectance MIR spectroscopy to measure the fructan content in barley

The aim of this study was to evaluate the feasibility of using attenuated total reflectance mid-infrared (ATR-MIR) spectroscopy to predict the fructan content in both barley and malt flour samples. Samples (n = 60) were sourced from commercial and experimental barley grain varieties and their corresponding malts. The fructan content in grain and malt flour was determined using the enzymatic kit from Megazyme (K-FRUC, Megazyme International Ireland). Samples were scanned in a MIR instrument using an ATR single bounce cell (Bruker Optics, Germany). The coefficients of determination in cross-validation (R2) and the standard error of cross-validation (SECV) obtained for the prediction of the fructan content in the calibration set were 0.76 and 0.20%, respectively. The residual predictive deviation (RPD = SD/SECV) value obtained was 2.3, indicating that these calibrations can be used for qualitative determination of the fructan content (e.g. low, medium and high) in the set of samples analysed. This study showed that ATR-MIR spectroscopy might be used as approximate estimates of the true fructan concentration in barley and malt in order to rank samples (low, medium, high) in the context of a breeding program.