Quantitative study of the formation of endoproteolytic activities during malting and their stabilities to kilning

The proteinases of germinating barley (Hordeum vulgare L.) hydrolyze storage proteins into amino acids and small peptides that can be used by the growing plant or, during brewing, by yeast. They are critical for the malting and brewing processes because several aspects of brewing are affected by the amounts of protein, peptide, and amino acids that are in the wort. This study was carried out to quantitatively measure when endoproteinases form in green malt and whether they are inactivated at the high temperatures that occur during malt kilning. Little endoproteolytic activity was present in ungerminated barley, but the activities began forming 1 day into the "germination" phase of malting, and they were nearly maximal by the third germination day. Quantitative studies with azogelatin "in solution" assays showed that the green malt endoproteolytic activities were not inactivated under commercial kilning conditions that use temperatures as high as 85 degrees C but that some actually increased during the final kilning step. Qualitative (2-D, IEF x PAGE) analyses, which allow the study of individual proteases, showed that some of the enzymes were affected by heating at 68 and 85 degrees C, during the final stages of kilning. These changes obviously did not, however, decrease the overall proteolytic activity.     

The effect of mashing on malt endoproteolytic activities

During malting and mashing, the proteinases of barley (Hordeum vulgare L.) and malt partially hydrolyze their storage proteins. These enzymes are critical because several aspects of the brewing process are affected by the soluble proteins, peptides and/or amino acids that they release. To develop improved malting barleys and/or malting and brewing methods, it is imperative to know whether and when the green malt endoproteinases are inactivated during malting and mashing. These enzyme activities are totally preserved during kilning and, in this study, we have determined when they were inactivated during mashing. Samples were removed from experimental mashes that mirrored those used in commercial breweries and their endoproteolytic activities were analyzed. The malt endoproteinases were stable through the 38 degrees C protein rest phase, but were quickly inactivated when the mash temperature was raised to 72 degrees C for the conversion step. All of the proteinase activities were inactivated at about the same rate. These findings indicate that the soluble protein levels of worts can be varied by adjusting the protein rest phase of mashing, but not by altering the conversion time. The rates of hydrolysis of individual malt proteins probably cannot be changed by altering the mash temperature schedule, since the main enzymes that solubilize these proteins are affected similarly by temperature.     

Improvement of malt modification by use of Rhizopus VII as starter culture

The development of a selected starter culture on malting barley and its effects on malt quality aspects were studied. Application of Rhizopus sporangiospores in a malting process resulted in increased beta-glucanase and xylanase contents of the malting barley and improved starchy endosperm cell-wall degradation. Activation of the sporangiospores and optimization of the inoculation procedure led to a further increase in enzyme levels and to larger and more consistent impacts on cell-wall modification. Whereas the main effect of the starter culture on beta-glucan degradation was observed during malting, a further decrease in beta-glucan during mashing suggests that the microbial enzymes that survived the kilning step were active during mashing. Other quality aspects that were influenced by the starter culture activity were protein modification, wort color, and wort pH. The level of microbial enzymes produced was related to the amount of barley kernels infected with the starter culture.     

Evolution of phenolic compounds and antioxidant activity during malting

Two barley varieties, Gan4 and Hamelin, were malted to investigate the evolution of phenolic compounds and antioxidant activity during malting. The antioxidant activity was evaluated with DPPH radical scavenging activity, ABTS radical cation scavenging activity, reducing power, and metal chelating activity. Results showed that malting had significant influences on individual and total phenolic contents as well as antioxidant activities of two barley varieties. The contents of some phenolic compounds and the antioxidant activities decreased significantly during steeping and the early stages of germination and then increased remarkably during the later stages of germination and subsequent kilning. The most phenolic compounds identified in barley were (+)-catechin and ferulic acid, which both changed significantly during malting. Moreover, results from the Pearson correlation analysis showed that there were good correlations among DPPH radical scavenging activity, ABTS radical cation scavenging activity, reducing power, total phenolic content and sum of individual phenolic contents during malting.     

Endosperm structure affects the malting quality of barley (Hordeum vulgare L.)

Twenty-seven barley (Hordeum vulgare L.) samples collected from growing sites in Scandinavia in 2001 and 2002 were examined to study the effect of endosperm structure on malting behavior. Samples were micromalted, and several malt characteristics were measured. Samples were classified as having a mealier or steelier endosperm on the basis of light transflectance (LTm). Because endosperm structure is greatly dependent on protein content, three barley sample pairs with similar protein contents were chosen for further analysis. During malting, the steelier barley samples produced less root mass, but showed higher respiration losses and higher activities of starch-hydrolyzing enzymes. Malts made from steelier barley had a less friable structure, with more urea-soluble D hordein and more free amino nitrogen and soluble protein. The reason for these differences may lie in the structure or localization of the hordeins as well as the possible effects of endosperm packing on water uptake and movement of enzymes.     

Retardation of Discoloration in Barley Flour Gel and Dough

Dark discoloration negatively influences the aesthetic properties of barley‐based food products. The effects of abrasion and heat treatment of grains, exclusion of oxygen, and the use of antibrowning agents on the retardation of darkening in barley flour gel or dough were determined in four types of barley, including hulled proanthocyanidin‐containing and hulled proanthocyanidin‐free, hulless regular, and hulless waxy barley. Abrasion by >30% in hulled barley and by >15% in hulless barley significantly increased the brightness (L*) of barley flour dough by 0.1–7.1. Steam heating of abraded grains also significantly increased the L* of barley flour gels by 1.8–3.4. Ascorbic acid at 1,500 ppm was most effective for retarding discoloration of barley flour dough, followed by 50 ppm of 4‐hexylresorcinol, which is an enzyme competitive inhibitor. The discoloration of barley flour dough was also effectively reduced by storing the dough sheets at 4°C under nitrogen gas to exclude oxygen or under anaerobic conditions at 20°C. Discoloration of barley‐based food products may be effectively controlled by selecting genotypes with low discoloration development such as proanthocyanidin‐free genotypes, by lowering total polyphenol content or polyphenol oxidase (PPO) activity through abrasion, by heat treatment, by exclusion of oxygen, and by the use of enzyme inhibitors.     

Improved Methods for High-Throughput Extraction and Assay of Green Barley Malt Proteinase Activity Facilitating Examination of Proteinase Activity Across Large-Scale Barley Populations

We report efficient sample extraction and assay methods allowing quantitative determinations of proteinase activities from barley malt. The improved methods are used to assay >2,200 developmental lines of malting barley for two subsets of proteinase activity. The distributions of the resulting activities suggest differences in population structures between the two types of proteinases. Comparison of the activities of the green malt proteinases with standard malting quality measurements show highly significant correlations that differ between the proteinase subsets. The pH 4.5 hydrolysis of the artificial substrate Z-Phe-Arg-AMC correlates well with the traditional malting quality measurements, supporting the role of cysteine-class proteinases in mobilization of grain reserves during malting and mashing. Results from assays of gelatin hydrolysis at pH 6.0 suggest that these proteolytic activities may be involved in other aspects of seed C and N dynamics also linked to malting quality measurements. The differences between the pH 4.5 and 6.0 activities assayed here and their association with malting quality measurements suggest different physiological roles for the two proteinase activities in several aspects of seed germination. Either assay could be useful for population surveys, depending on the particular facet of seed metabolism under study.     

Proteolysis and bioconversion of cereal proteins to glutamate and γ-Aminobutyrate (GABA) in Rye malt sourdoughs

This study aimed to achieve the conversion of cereal proteins to the alternative end products glutamate or γ-aminobutyrate (GABA). Rye malt, fungal proteases, and lactobacilli were employed to convert wheat gluten or barley proteins. Glutamate and GABA formations were strain-dependent. Lactobacillus reuteri TMW1.106 and Lactobacillus rossiae 34J accumulated glutamate; L. reuteri LTH5448 and LTH5795 accumulated GABA. Glutamate and GABA accumulation by L. reuteri TMW1.106 and LTH5448 increased throughout fermentation time over 96 h, respectively. Peptides rather than amino acids were the main products of proteolysis in all doughs, and barley proteins were more resistant to degradation by rye malt proteases than wheat gluten. However, addition of fungal protease resulted in comparable degradation of both substrates. Glutamate and GABA accumulated to concentrations up to 63 and 90 mmol kg(-1) DM, respectively. Glutamate levels obtained through bioconversion of cereal proteins enable the use of hydrolyzed cereal protein as condiment.     

Influence of Barley and Malt Storage on Lipoxygenase Reaction

The dioxygenation of linoleic acid (LA) by aqueous flour suspensions of barley and malting samples was studied. The rate of this lipoxygenase (LOX) reaction varied as the malting process proceeded, giving a characteristic LOX reaction profile for a malting. The differences in the profiles from one malting to another were dramatic. It also appeared that during storage of dry, intact kernel samples from a single malting, a reduction in the rate of LOX reaction always occurred, and the rates of reduction with time were dependent on the stage of malting at the time of sampling. The kinetics of this aging could roughly be divided into four categories representing different stages of malting. Consequently, greatly varying LOX reaction profiles can be obtained from a single malting depending on the time of storage of kernels before assays. The results indicate that steeping, germination and the subsequent drying render the state of kernels unstable with respect to the LOX reaction for at least two to three weeks. Homogeneity of malt quality is important in the further applications of malt, especially in the brewing industry. Therefore, the rate of LOX reaction should be considered as a quality factor of malt.     

Starch Lipids of Barley and Malt

Barley and malt starches were compared with respect to their lipid content and composition. The starch lipids were first fractionated into internal and surface lipid fractions followed by lipid class and fatty acid analyses of each fraction. Barley starch contained 13 mg/g lipids, of which 9.3 mg were internal lipids and 3.7 mg were surface lipids. The total lipid content of malt starches varied between 11 and 13 mg/g of starch. However, malt starch contained only 1 mg of surface lipids; therefore, the internal lipid contents were as high as or even higher than those in the corresponding fraction of barley starch. Lipid class analyses suggested that the ability for hydrolysis of starch surface lipids was increased in malt. The hydrolysis occurred during the malting or the isolation process, resulting in reduced surface lipid content in malt starch. However, no reduction in the portion of polyunsaturated fatty acids was seen; therefore, lipid oxidation could not have been responsible for the lower content of malt starch surface lipids. Also, not only was the content of starch internal lipids higher in malt, but the composition of these lipids was different when compared to barley starch. The increase in starch internal lipids during malting may be due to transportation and reacylation of free fatty acids that had been liberated by hydrolysis from surface lipids.     

Functionality of Barley Proteins Extracted and Fractionated by Alkaline and Alcohol Methods

This research optimized the extraction of different protein fractions from barley grains and assessed the physicochemical properties of the fractions obtained. Pearling was first used to remove the grain's outer layers (mainly bran and germ) so that the barley cytoplasmic proteins (albumin and globulin) would be enriched in the pearling flour (PF), while endosperm proteins (hordein and glutelin) would be enriched in the pearled grain flour (PGF). Salt, alcohol, and alkaline solutions were then used to extract different barley protein fractions from PF and PGF. The effects of extraction solvent type, pH, temperature, and extraction time on protein content and extraction efficiency were studied. Aqueous ethanol (55%, v/v) efficiently extracted barley hordein from PGF at 60°C, whereas pH 11.5 alkaline solution was the most efficient for extracting both cytoplasmic and endosperm proteins from barley PF and PGF at 23°C. Subunit molecular weight, amino acid composition, and the functional properties of each isolated barley protein fraction were investigated. Barley glutelin demonstrated superior oil‐binding property and emulsifying stability, whereas barley hordein exhibited good foaming capacity.     

Effects of Four Independent Low‐Phytate Mutations in Barley (Hordeum vulgare L.) on Seed Phosphorus Characteristics and Malting Quality

Conversion of the seed phosphorus storage compound phytic acid, which is poorly digested by nonruminants, to available forms of phosphorus will have nutritional and environmental benefits. Low‐phytate (LP) barley (Hordeum vulgare L.) cultivars are in development and their commercialization will be facilitated by understanding their phosphorus profiles and malting quality. To study these issues, LP and normal types derived from mutagenized populations of barley cultivar Harrington (sets of sib lines homozygous for the wild‐type [WT] allele, or for one of four low‐phytic acid mutations, lpa1‐1, lpa2‐1, lpa3‐1, or M955), were developed through backcrosses to Harrington. Grain was produced in irrigated and rain‐fed environments. WT phosphorus profiles were similar to those of Harrington, suggesting that the major variable was the presence or absence of mutant alleles. All mutations conferred increased inorganic phosphorus. Total P was reduced for lpa1‐1. Phosphorus profiles were relatively stable across environments, which will facilitate the inclusion of LP barley in animal rations. Utilization of LP cultivars for malting may be difficult, as the LP trait was associated with substantial reductions in diastatic power. All mutations, except for lpa2‐1, affected wort β‐glucan levels, which could not be attributed to altered grain β‐glucan levels.     

Esterases in Barley and Malt

Several esterases from barley and malt have been separated on polyacrylamide gels. The slowest moving bands appear to represent a single enzyme displaying a spread of migration owing to differences in surface charge. During malting, this enzyme, which is located in the starchy endosperm, shifts to a more migratory form. Two other main esterase groups are identified through gel electrophoresis, notably a highly anionic, highly labile enzyme, MW 62,000, located in the aleurone. The slowest and fastest moving bands have been partially purified using salt fractionation and ion‐exchange chromatography. The former, MW 47,000, has strong capability for hydrolyzing acetylxylan and it is speculated that its role in the starchy endosperm is as part of the enzyme system that hydrolyzes the cell walls.     

Comparative Study of Activity and Heat Stability of Limit Dextrinase in 16 Barley Cultivars

We conducted a comprehensive study of 15 cultivars of Chinese barley and one cultivar of European barley to examine and compare the activity and heat stability of limit dextrinase (LD) among them. Our results revealed that the enzymatic activity of LD drops dramatically at incubation temperatures >65°C, indicating that 65°C is the point of incubation temperature sensitivity for this enzyme. Compared with the European barley cultivar Triumph, the levels of enzymatic activity and heat stability of LD in most Chinese barley cultivars included in this study were low, except for Jipi 2 and 92001, which were nearly equal to Triumph. We also found that the activity and heat stability of LD were strongly influenced by the genotype and growth environment of the cultivar, which suggests that genetic manipulation and optimal cultivation conditions may be useful in producing high‐quality barley for the brewing industry.     

Hydrolysis of low-molecular-weight soil peptides by immobilized proteases in column and batch reaction systems

Summary Organic matter was extracted from three soils, a cultivated Berwick sandy loam, a cultivated Franklin loamy sand, and an uncultivated Cumberland silty loam. Gel-permeation chromatography was used to separate organic matter extracts into high- (HMW) and low-molecular-weight (LMW) fractions. Reversed-phase high performance liquid chromatography was used to separate and collect the LMW peptide fractions. Peptide samples were hydrolyzed with immobilized proteases attached to beaded agarose and carboxymethyl cellulose in column and batch reaction systems. The chromatograms suggested that peptides are bound to common soil components. The amino acids released in the greatest percentages were relatively non-polar. Large percentages of serine, glycine, alanine, threonine, and valine were observed in the LMW soil peptides. Little aspartic acid, asparagine, glutamic acid, glutamine, arginine, and no histidine was detected in the LMW soil peptides. The soil peptides released different amino acid percentages and quantities when hydrolyzed by immobilized proteases attached to different supports. The quanitities of amino acids released by batch hydrolysis differed from those obtained with column hydrolysis. Greater quantities of amino acids were released (by both types of immobilized protease) from the LMW peptide hydrolysates of the two cultivated soils than from the uncultivated soil.     

Barley Contains Two Cationic Acetylxylan Esterases and One Anionic Feruloyl Esterase

Various carbohydrate‐active esterases are detected in extracts of malted barley when analyzed by polyacrylamide gel electophoresis. The slowest migrating and most heat‐resistant of these are relatively cationic acetylxylan esterases. Two such activities, one with a high affinity for esterase substrates including acetylated xylan, and one with a low affinity, are indicated. These enzymes did not hydrolyze methyl ferulate. A relatively heat‐labile anionic feruloyl esterase has also been purified. It has some, albeit low, ability to act on acetylated xylan. The feruloyl esterase effects extensive release of ferulate from endosperm cell walls isolated from barley, whereas the acetylxylan esterases are only capable of very limited release of acetate.     

Inhibition by soil humic acids of native and acetylated proteolytic enzymes

The activities of acetyltrypsin and acetylcarboxypeptidase were unaffected by neutralized solutions of soil humic acids in concentrations which markedly inhibited the non-acetylated enzymes. Further, acetyltrypsin, unlike trypsin, did not coprecipitate with humic acids. The results support the conclusion that humic acids bind the proteases by a cationexchange mechanism whereby protease amino groups are linked to humic acid carboxyl groups.     

Nitrogen Uptake by Malting Barley Grown under Conditions Found in Buenos Aires Province,Argentina

Abstract

There is a lack of data associated with applications of nitrogen (N) fertilizer to increase yield while not increasing seed protein to levels exceeding those acceptable for malting barley (Hordeum vulgare L.) in the Buenos Aires province of Argentina. The effect of rates and timing of N application on yield and grain N concentration of malting barley was evaluated at eight sites in 1999 and 2000. Aboveground dry matter accumulation and N‐uptake pattern through the growing season were evaluated. Dry matter production and N-uptake were measured at four sampling times: tillering, head emergence, grain filling, and maturity. The N fertilizer increased grain yield, but its response varied between sites. Under appropriate conditions, the yield increased and maintained the grain N concentration within a desirable range for malting barley. Split applications were as effective in increasing grain yield as one addition at emergence, but they invariably increased grain N concentration. The season affected the yield response to N fertilizer and its levels in the grain, but the nitrogen harvest index was not affected by the rate of N application.     

Isolation and Characterization of Water‐Extractable Arabinoxylan from Hull‐less Barley Flours

A new procedure was developed for the isolation of highly purified water‐extractable arabinoxylan (WE‐AX) from hull‐less barley flour. It included inactivation of endogenous enzymes, removal of proteins with silica gel, and removing β‐glucans, arabinogalactan‐peptides, and starch fragments by enzyme or solvent precipitation steps. WE‐AX recovered by this isolation procedure represented, on average, 47% of all WE‐AX present in hull‐less barley flour. Purified WE‐AX from flour of different hull‐less European barley cultivars contained 84.9–91.8% AX and showed small structural differences. The apparent peak molecular weight of the purified WE‐AX was 730,000–250,000, and the arabinose‐to‐xylose ratio was 0.55–0.63. Proton nuclear magnetic resonance spectroscopy showed that the levels of un‐, O‐2 mono‐, O‐3 mono‐, and O‐2,O‐3 disubstituted xylose residues were 59.1–64.7%, 8.2–10.0%, 5.7–10.6%, and 17.6– 23.1%, respectively, and the ratio of di‐ to monosubstituted xylose was 0.90–1.54. Both O‐3 mono‐ and disubstituted xylose residues occurred isolated or next to disubstituted xylose residues in the WE‐AX chain.     

Image Analysis of Grain and Chemical Composition of the Barley Plant as Predictors of Malting Quality in Mediterranean Environments

We have explored the possibility of predicting the malting quality of barley grain, indicated by malt extract yield, by characteristics measured either on plants at anthesis or in mature dry grain by image analysis. To produce barley samples with varying levels of all the characteristics studied, we used grain from an experiment designed to study the influence of lowinput husbandry practices on malting quality of barley by growing five malting genotypes at each of four environments (site × season) and with two different agronomic treatments (N fertilization and herbicide-mechanical roguing of weeds). The results showed that nitrogen content in the plant at anthesis was a good predictor of grain protein content, this characteristic in turn being positively correlated with embryo size and grain volume, as estimated by image analysis, and negatively correlated with nonstructural carbohydrate content in the plant at anthesis. Extract yield was positively correlated with Kolbach index (ratio of soluble to total wort protein) and negatively correlated with wort viscosity and barley grain protein content. Thus, the only practical predictor of malt extract was grain protein content.