Germinating Barley Grain Research Articles

Production and application of barley malt extract with high peptidase activity for the degradation of gluten in wort

Special malt with high gluten-specific peptidase activity was prepared by germinating barley grain for 8 days at 18 °C and 48 % humidity. Endogenous malt peptidases were characterized and used to degrade gluten in barley wort. Peptidases of aqueous malt extracts degraded celiac-active peptides by cleaving peptide bonds involving proline residues. The peptidase activity was not affected by cultivar, area of cultivation, and high temperatures up to 80 °C during kiln drying. Temperatures up to 50 °C were tolerated without loss of full activity in aqueous solution. The presence of ethanol decreased the activity significantly. Cross-flow filtration, freeze-drying, and evaporation under reduced pressure at 50 °C were used to produce a concentrated enzyme-active malt extract. The latter method yielded the best results, and the obtained concentrate was used to study gluten degradation in barley wort as affected by concentration of the extract, incubation time, and temperature. The gluten content was determined by a competitive R5-ELISA. The addition of 10 % concentrated extract (grist-to-water ratio 1:2.5; 40.4 °Brix) to wort and incubation for 24 h at 50 °C resulted in a gluten-free wort.

The effects of GA and ABA treatments on metabolite profile of germinating barley

Sugar degradation during grain germination is important for malt quality. In malting industry, gibberellin (GA) is frequently used for improvement of malting quality. In this study, the changes of metabolite profiles and starch-degrading enzymes during grain germination, and as affected by GA and abscisic acid (ABA) were investigated using two wild barley accessions XZ72 and XZ95. Totally fifty-two metabolites with known structures were detected and the change of metabolite during germination was time- and genotype dependent. Sugars and amino acids were the most dramatically changed compounds. Addition of GA enhanced the activities of starch-degrading enzymes, and increased most metabolites, especially sugars and amino acids, whereas ABA had the opposite effect. The effect varied with the barley accessions. The current study is the first attempt in investigating the effect of hormones on metabolite profiles in germinating barley grain, being helpful for identifying the factors affecting barley germination or malt quality.

Structural Changes of Starch Molecules in Barley Grains During Germination

ABSTRACTThe germination of barley grains is an important step in malt production for beer brewing. This study examines the changes in starch molecular structure in barley grains under optimal conditions for sprouting. Two barley varieties were tested: Schooner and Grimmett, which are low and moderate dormancy grains, with >90 and <5% of grains germinating within two days in sprouting conditions, respectively. The barley grains were germinated in humid conditions after being soaked in excess water for 24 h. The grain weight and starch content of Schooner decreased significantly (P < 0.05) with germination time, whereas those of Grimmett showed no significant changes (P > 0.05). The starch chain‐length distributions, analyzed from enzymatically debranched starch molecules with size‐exclusion chromatography, indicated that the shorter amylopectin branches of Schooner were degraded at a faster rate than its longer amylopectin branches, which was also confirmed by the decreasing degree of branching with germination time. The changes in starch molecular structure of Grimmett grains were less apparent. The results of this study showed that barley grains containing shorter starch chains can produce sugars more quickly, which indicates that starch structure can serve as one of the specifications when selecting barley grains for brewing.

Application of Proteomics to Hordein Screening in the Malting Process

This study was undertaken to investigate the effect of the malting process on hordein composition. For this purpose, combination of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and the method of isotopic peptides labeling iTRAQ was used. Barley proteins are essential components determining the quality of both malt and beer. Since hordeins represent the most abundant proteins accounting for about 40-50% of total protein fraction of mature barley grain, our research was focused on them. In this respect, the proteins of interest were extracted from milled samples of barley grain, germinated barley grain (samples collected at different time intervals), green malt and malt, respectively. Particular hordein extracts were firstly fractionated via SDS- PAGE, which was used as a relatively rapid and reliable technique providing information about hordein profile of analyzed samples. Then, separated proteins were in-gel digested and resulting peptides were measured by mass spectrometry. In addition, the chosen proteins, after in-gel digestion, were subjected to the iTRAQ method and the screening of proteins during malting process was evaluated. Our results have revealed that most of the hordein components present in the barley grain can be found in all stages of the malting process as well as in the final malt. The amount of hordeins decreases during the malting process; in the case of C hordein, the protein decrease is approximately 65%. On the other hand, significant degradation of D hordein was detected. The suggested procedure can be used to follow the development of the hordein profile during germination, which is of great technological importance in beer production.

Assessment of Chemical Properties of Raw, Germi-nated Barley Grains, Talbina, and Biscuits En-riched with Talbina.

Assessment of Chemical Properties of Raw, Germi-nated Barley Grains, Talbina, and Biscuits En-riched with Talbina.

Chemical genetics and cereal starch metabolism: structural basis of the non-covalent and covalent inhibition of barley β-amylase

There are major issues regarding the proposed pathway for starch degradation in germinating cereal grain. Given the commercial importance but genetic intractability of the problem, we have embarked on a program of chemical genetics studies to identify and dissect the pathway and regulation of starch degradation in germinating barley grains. As a precursor to in vivo studies, here we report systematic analysis of the reversible and irreversible inhibition of the major β-amylase of the grain endosperm (BMY1). The molecular basis of inhibitor action was defined through high resolution X-ray crystallography studies of unliganded barley β-amylase, as well as its complexes with glycone site binder disaccharide iminosugar G1M, irreversible inhibitors α-epoxypropyl and α-epoxybutyl glucosides, which target the enzyme's catalytic residues, and the aglycone site binders acarbose and α-cyclodextrin.

The Role of α-Glucosidase in Germinating Barley Grains

The importance of α-glucosidase in the endosperm starch metabolism of barley (Hordeum vulgare) seedlings is poorly understood. The enzyme converts maltose to glucose (Glc), but in vitro studies indicate that it can also attack starch granules. To discover its role in vivo, we took complementary chemical-genetic and reverse-genetic approaches. We identified iminosugar inhibitors of a recombinant form of an α-glucosidase previously discovered in barley endosperm (ALPHA-GLUCOSIDASE97 [HvAGL97]), and applied four of them to germinating grains. All four decreased the Glc-to-maltose ratio in the endosperm 10 d after imbibition, implying inhibition of maltase activity. Three of the four inhibitors also reduced starch degradation and seedling growth, but the fourth did not affect these parameters. Inhibition of starch degradation was apparently not due to inhibition of amylases. Inhibition of seedling growth was primarily a direct effect of the inhibitors on roots and coleoptiles rather than an indirect effect of the inhibition of endosperm metabolism. It may reflect inhibition of glycoprotein-processing glucosidases in these organs. In transgenic seedlings carrying an RNA interference silencing cassette for HvAgl97, α-glucosidase activity was reduced by up to 50%. There was a large decrease in the Glc-to-maltose ratio in these lines but no effect on starch degradation or seedling growth. Our results suggest that the α-glucosidase HvAGL97 is the major endosperm enzyme catalyzing the conversion of maltose to Glc but is not required for starch degradation. However, the effects of three glucosidase inhibitors on starch degradation in the endosperm indicate the existence of unidentified glucosidase(s) required for this process.

The transcriptome analysis of barley (Hordeum vulgare L.) using the Affymetrix Barley1 GeneChip

An alternative to complete genome sequencing is development and analysis of ESTs-fragments of transcribed coding DNA sequences. The EST collections also enhanced the development of cDNA microarray technologies, which make possible assessing the transcription levels of several thousand genes in a studied tissue of an organism in the same experiment. This paper provides an overview of the results of experiments with a barley microarray, Affymetrix Barley1 GeneChip. The variation in transcription levels of over 22000 genes in germinating barley grain of 150 barley double haploid lines produced by crossing cultivars Steptoe and Morex. Variation in gene expression of each gene is a quantitative trait, which can be mapped in population of double haploids as the genetic loci determining its variation (expressed QTL or eQTL). A regulatory locus (eQTL) can colocalize with the corresponding gene on genetic map (cis-eQTL) or be distant from it, frequently on another chromosome (trans-eQTL). Thus, it is possible to detect and analyze cis- and trans-regulatory loci for genes on a genome-wide scale. The design of the Affymetrix oligonucleotide arrays makes it possible not only to concurrently test the transcription level of several thousand genes, but also to simultaneously detect the polymorphic regions in cDNA sequences, thereby finding a considerable fraction of all nucleotide substitutions between the compared genotypes. Two types of data (the expression levels of several thousand genes and the presence of polymorphic sites in their sequences) can be obtained concurrently when processing the results of the same experiment. The details of both procedures are illustrated with explanatory examples.

Effects of steeping and anaerobic treatment on GABA (γ-aminobutyric acid) content in germinated waxy hull-less barley

Effects of steeping conditions (time, temperature and soaking solution) and anaerobic storage on the gamma-aminobutyric acid (GABA) content in waxy hull-less barley grains during germination was examined. The barley kernel was steeped for 16 h at different temperatures (5, 15 or 35 °C) either in water or in a buffer solution (pH 6.0, 50 mmol/L sodium acetate) and then germinated at 15 °C for 72 h. To reach the optimum water content (36–44 g/100 g) for germination, a longer steeping period was required when steeping temperature was lower (16 h at 5 °C vs. 8 h at 15 °C). At 35 °C for steeping, however, the water content in the grains increased excessively, and thus germination percentage became much less than those at 5 and 15 °C. The GABA content increased with increasing germination time and was higher in the buffer solution than water. These findings indicate that the glutamate decarboxylase (GAD), which is the rate-limiting enzyme for GABA synthesis, is more activated by extending germination at controlled pH (6.0). An anaerobic storage with nitrogen in the dark for the germinated barley grains substantially raised the GABA content: 14.3 mg/100 g after the treatment for 12 h, which was four times higher than that of control sample (3.7 mg/100 g). Overall results suggest that the steeping prior to germination greatly affects the GABA production during the germination of barley, and the anoxia storage with nitrogen after the germination increases the GABA content.

Unprocessed barley aleurone endo-β-1,4-xylanase X-I is an active enzyme

Endo-β-1,4-xylanase X-I is a major hydrolase produced by the aleurone tissue of germinating barley grain. It was previously reported that this cytosolic enzyme is synthesized as an inactive precursor which is proteolytically processed to active forms upon its programmed cell death dependent release. We here demonstrate, however, that the precursor form of X-I is an active enzyme. Purified recombinant precursor X-I was characterised with respect to its molecular weight, iso-electric point and temperature and pH activity and stability. Analysis of the hydrolysis products showed that it is an endo-acting enzyme which has the striking ability to release xylose from both polymeric xylan as well as from small xylo-oligosaccharides. The implications of these findings in relation to the putative role of the N-terminal propeptide as a carbohydrate binding module and the possible consequences for the way X-I fulfils its role in the germination process, are discussed.

Accumulation and Degradation of Two Functional Constituents, GABA and .BETA.-Glucan, and Their Varietal Differences in Germinated Barley Grains

The changes in the contents of two functional constituents, γ-aminobutyric acid (GABA) and (1→3)(1→4)-β-D-glucans (β-glucan), during germination were investigated in two malting barley varieties in order to determine the optimal conditions for accumulation and degradation of these constituents in dried barley grains after germination treatment. A time-course analysis during germination treatment revealed that the content of GABA remarkably increased in the 48 hour imbibition process and decreased in the subsequent germination process. On the other hand, β-glucan, which is known to be decomposed during malting, did not undergo substantial degradation after the 48 hour imbibition process. These findings thus indicated that it is possible to produce germinated barley grains containing these functional constituents at high levels by optimal germination treatment. A total of 43 barley varieties were surveyed for the GABA and β-glucan contents in germinated grains. Our results also indicated that there were varietal differences in the content of each ingredient. A waxy starch variety in which the contents of both β-glucan and GABA were higher than those in the other barley varieties tested, could become a promising source of the two functional constituents.

Evaluation of Czech spring malting barleys with respect to the β‐amylase allele incidence

AbstractStrong enzymatic activities in the germinating barley grain, together with protein and starch content, are crucial for high extraction values in the resulting malt and, therefore, barley malting quality. The efficient characterization of registered barley cultivars and genetic resources with respect to one of the relevant thermostability enzymes (β‐amylase) is an essential requirement. The template‐directed dye‐terminator incorporation (TDI) assay: based on flourescence resonance energy transfer (TDI‐FRET) (Chen et al. 1995) was used to detect single nucleotide polymorphisms (SNPs) in the β‐amylase coding sequence resulting in low (Sd2L), intermediate (Sd1) and high (Sd2H and Sd3) thermostability enzyme across 84 Czech barley cultivars and genetic resources used over a period of time in the Czech Republic. The incidence of different alleles has changed during the last 100 years. Also the new resources with high thermostabile β‐amylase were identified. They can be used effectively to breed for malting quality improvement.

A role for phosphorylation in the regulation of the barley scutellar peptide transporter HvPTR1 by amino acids

Protein reserves in the cereal endosperm are sequentially degraded to small peptides and amino acids during germination and these are translocated across the scutellum to support growth of the embryo. Peptide transport in the germinating barley grain is mediated by specific carriers localized to the plasma membrane of the scutellar epithelium. In isolated barley embryos peptide transport is rapidly inhibited by amino acid concentrations comparable with those found in the post-germination barley grain. However, this inhibition of HvPTR1 activity is not effected at either the transcriptional or translational level. The protein phosphatase inhibitor okadaic acid repressed transport of Ala-[14C]Phe, but not [14C]Ala, into the barley scutellar epithelium. In vivo [32P]orthophosphate labelling studies of barley scutellar tissue in combination with immunoprecipitation studies using antiserum raised to HvPTR1 showed that HvPTR1 (66 kDa) is phosphorylated in the presence of amino acids. Immunopurified HvPTR1 was further demonstrated to be phosphorylated on serine residues. Digestion with the N-glycosidase enzyme PNGase F results in a shift in the molecular mass of the protein by 10 kDa, indicating that HvPTR1 is an N-linked glycoprotein. These results provide strong circumstantial evidence that HvPTR1 peptide transport activity in the germinating barley grain is regulated at the post-translational level by phosphorylation in response to rising levels of amino acids emanating from the endosperm as a result of storage protein breakdown and mobilization. This is potentially an important element in balancing the flux of organic nitrogen and carbon from the endosperm to embryo during germination and seedling establishment.

Developmental Patterns of Enzymes and Proteins During Mobilization of Endosperm Stores in Germinating Barley Grains

Mobilization of stored reserves in the endosperm of barley grains involves the interaction of enzymes and other proteins synthesized during grain development and those synthesized during germination. In order to obtain information on this interaction, localization of α-amylase, the bifunctional subtilisin/α-amylase inhibitor (BASI), the unspecific lipid transfer protein (LTP), and the antimicrobial proteins chitinase and ribosome inactivating protein (RIP) were determined by immunofluorescence microscopy in the different tissues of resting and germinating grains of the Hordeum vulgare L. genotypes Alexis, Klages, and Steptoe. Targeting of α-amylase from the proximal to the distal part of the endosperm progresses with different rates in the three genotypes. LTP is localized in the endosperms, the aleurone layers and in the embryo of the ungerminated grains. LTP is depleted during the first days of germination but then re-synthesized in the embryo and in the aleurone during the following phases of germination and released to the endosperm independent of cell wall hydrolysis. Chitinase is present in the aleurone layers and, depending on variety, in variable amounts in the endosperm of the mature and the germinating grains. Secretion of chitinase from the aleurone and scutellum is indicated by the increasing amount found in the endosperm during germination. Chitinase accumulates in the distal aleurone layers before a-amylase IS detected and thus must be able to penetrate the intact endosperm β-glucan walls. BASI and RIP are localized in all parts of the mature grain and at later stages of germination the amount in the scutellum and aleurone layer increases. The analysed enzymes and proteins fulfill different functions during germination and are of importance for the industrial use of the barley crop. The efficient immunohistochemical demonstration of the genotypic variability in these characteristics provides a possibility to identify important breeding targets.

Differential gene expression during seed germination in barley (Hordeum vulgare L.).

A barley cDNA macroarray comprising 1,440 unique genes was used to analyze the spatial and temporal patterns of gene expression in embryo, scutellum and endosperm tissue during different stages of germination. Among the set of expressed genes, 69 displayed the highest mRNA level in endosperm tissue, 58 were up-regulated in both embryo and scutellum, 11 were specifically expressed in the embryo and 16 in scutellum tissue. Based on Blast X analyses, 70% of the differentially expressed genes could be assigned a putative function. One set of genes, expressed in both embryo and scutellum tissue, included functions in cell division, protein translation, nucleotide metabolism, carbohydrate metabolism and some transporters. The other set of genes expressed in endosperm encodes several metabolic pathways including carbohydrate and amino acid metabolism as well as protease inhibitors and storage proteins. As shown for a storage protein and a trypsin inhibitor, the endosperm of the germinating barley grain contains a considerable amount of residual mRNA which was produced during seed development and which is degraded during early stages of germination. Based on similar expression patterns in the endosperm tissue, we identified 29 genes which may undergo the same degradation process.

The diffusive transport of gibberellins and abscisic acid through the aleurone layer of germinating barley grain: a mathematical model.

A mathematical model of the diffusive transport of abscisic acid (ABA) and gibberellins (GAs) through the aleurone layer of barley (Hordeum vulgare L.) grain is presented. The model consists of two partial differential equations describing the accumulation of phytohormone in the apoplastic and symplasmic compartments of the aleurone layer, both spatially and temporally. The mathematical model contains the morphology of the barley grain and the physicochemical properties of the two phytohormones. A mathematical derivation of the accumulation ratios for the two phytohormones between the symplast and apoplast under equilibrium conditions resulted in different distribution mechanisms for GAs and ABA. A sensitivity analysis of the accumulation ratio for GAs indicated high sensitivity to the apoplastic pH and the membrane potential, whereas the accumulation ratio for ABA proved to be most sensitive to the pH difference between the apoplast and symplast. The diffusive transport time for GAs to the basal site of the aleurone layer as calculated with the mathematical model is within a physiologically plausible timescale according to experimental data from the literature. Abscisic acid cannot be transported by diffusion to the end of the aleurone layer as quickly as GAs, according to model simulations. Therefore, the functional role of ABA in germination is likely to be in the vicinity of the embryo.

Binding interactions between barley thaumatin-like proteins and (1,3)-beta-D-glucans. Kinetics, specificity, structural analysis and biological implications.

The specificity and kinetics of the interaction between the pathogenesis-related group of thaumatin-like proteins (PR5) in higher plants and (1,3)-beta-D-glucans have been investigated. Two thaumatin-like proteins with 60% amino-acid sequence identity were purified from extracts of germinated barley grain, and were designated HvPR5b and HvPR5c. Purified HvPR5c interacted with insoluble (1,3)-beta-D-glucans, but not with cellulose, pustulan, xylan, chitin or a yeast mannoprotein. Tight binding was observed with unbranched and unsubstituted (1,3)-beta-D-glucans, and weaker binding was seen if (1,6)-beta-linked branch points or beta-glucosyl substituents were present in the substrate. The HvPR5b protein interacted weakly with insoluble (1,3)-beta-D-glucans and did not bind to any of the other polysaccharides tested. This indicated that only specific barley PR5 isoforms interact tightly with (1,3)-beta-D-glucans. The complete primary structures of HvPR5b and HvPR5c were determined and used to construct molecular models of HvPR5b and HvPR5c, based on known three-dimensional structures of related thaumatin-like proteins. The models were examined for features that may be associated with (1,3)-beta-D-glucan binding, and a potential (1,3)-beta-D-glucan-binding region was located on the surface of HvPR5c. No obvious structural features that would prevent binding of (1,3)-beta-D-glucan to HvPR5b were identified, but several of the amino acids in HvPR5c that are likely to interact with (1,3)-beta-D-glucans are not present in HvPR5b.

Synthesis, processing and export of cytoplasmic endo-beta-1,4-xylanase from barley aleurone during germination.

We have identified the major endo-beta-1,4-xylanase (XYN-1) in the aleurone of germinating barley grain, and show that it is expressed as a precursor of Mr 61 500 with both N- and C-terminal propeptides. XYN-1 is synthesized as an inactive enzyme in the cytoplasm, and only becomes active at a late stage of germination when the aleurone ceases to secrete hydrolases. A series of processing steps, mediated in part by aleurone cysteine endoproteases, yields a mature active enzyme of Mr 34 000. Processing and extracellular release of the mature enzyme coincide with the programmed cell death (PCD)-regulated disintegration of aleurone cells. We discuss the significance of delayed aleurone cell-wall degradation by endoxylanases in relation to the secretory capacity of the aleurone, and propose a novel role for aleurone PCD in facilitating the export of hydrolases.

Contrasting effects of ethylene perception and biosynthesis inhibitors on germination and seedling growth of barley (Hordeum vulgare L.)

The effects of the plant growth regulator ethylene, and of ethylene inhibitors, on barley (Hordeum vulgare L.) germination and seedling growth were investigated. Exogenous 1-aminocyclopropane-1-carboxylic acid (ACC) at 100 microM enhanced ethylene production by barley seedlings and stimulated shoot growth, whereas both germination and seedling growth were inhibited by antagonists of ethylene perception (75 microM silver ions, 100 microM 2,5-norbornadiene (NBD)). In contrast, germination was unaffected by, and root and shoot growth of seedlings was strongly stimulated by inhibitors of ethylene biosynthesis (10 microM cobalt chloride, 10 microM aminoethoxyvinylglycine (AVG)). Since the ethylene and polyamine biosynthetic pathways are linked through S:-adenosylmethionine, this prompted further explorations into the role of polyamines in germination and seedling growth. Exogenous polyamines (putrescine, spermidine and spermine) at 1 microM concentration stimulated barley seedling growth in a similar fashion to the ethylene biosynthetic inhibitors. Both polyamines and ethylene biosynthetic inhibitors reversed the inhibitory effects of ethylene perception inhibitors on germination and seedling growth. Blocking endogenous ethylene production with aminoethoxyvinylglycine enhanced the free putrescine and spermidine content of germinating barley grains. Thus endogenous polyamines may play a complementary, growth-promotive, role to ethylene in the normal course of barley germination. Further, experiments that have been carried out using inhibitors of ethylene biosynthesis may have to be re-evaluated to take the possible effect of polyamines into account.

The barley scutellar peptide transporter: biochemical characterization and localization to the plasma membrane

Thiol‐affinity labelling was used to identify and characterize components of the peptide transport system in the barley (Hordeum vulgare) scutellar epithelium. SDS‐PAGE and 2D‐PAGE in conjunction with fluorography were used to study derivatized proteins. Membrane proteins of 42 kDa and 66 kDa were identified using a strategy devized to label substrate protectable protein with the thiol specific reagent [14C]N‐ethylmaleimide (NEM). The scutellar plasma membrane is the anticipated site of transporters involved in the mobilization of endosperm storage reserves in the germinating barley grain. The subcellular localization of these proteins to the plasma membrane was demonstrated by thiol‐affinity labelling of high purity plasma membrane vesicles isolated from barley scutellar tissue. A peptide transporter, HvPTR1, specific to the barley scutellum has recently been cloned in this laboratory. A 66 kDa protein, comparable to the predicted molecular mass of HvPTR1, was identified by [14C]NEM labelling studies of Xenopus laevis oocytes expressing HvPTR1 cRNA, but not water injected controls. Peptide antiserum raised to HvPTR1 also cross‐reacted with a 66 kDa membrane protein in barley scutellar tissue. This confirms that the 66 kDa protein identified here by thiol‐affinity labelling studies is the barley scutellum peptide transporter HvPTR1, and demonstrates that this protein is localized to the plasma membrane of scutellar epithelial cells during germination.