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

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

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

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

Effects of added enzymes on sorted,unsorted and sorted-out barley: A model study on realtime viscosity and process potentials using rapid visco analyser

Barley sorting is an important step for selecting grain of required quality for malting prior to brewing. However, brewing with unmalted barley with added enzymes has been thoroughly proven, raising the question of whether traditional sorting for high quality malting-barley is still necessary. To gain more insight on this, we examine realtime viscosity of sorted-out and unsorted barley during downscaled mashing with added enzymes in comparison with malting quality sorted barley. A rapid visco analyser was used to simulate brewery mashing process at lab scale together with two commercial enzymes (Ondea^®-Pro and Cellic^®-CTec2). During downscaled mashing, viscosity profile of sorted-out barley was markedly different from others, irrespective of enzyme type, whereas a small difference was observed between the sorted and un-sorted barley. Furthermore, whilst sorted-out barley generated lowest sugar-concentration, unsorted and sorted barley resulted in higher sugar-content, regardless of the enzyme used. In terms of filterability, the Ondea^®-Pro treatment resulted in significantly lower-turbidity and smaller particle-size compared to Cellic^®-CTec2; however, this effect was observed in sorted and unsorted barley but not in sorted-out barley. Consequently, we find that unsorted barley demonstrates great potential for brewing with added enzymes and its use may help to improve sustainability of the brewing process.     

How various malt endoproteinase classes affect wort soluble protein levels

During the germination of seeds, storage proteins are degraded and the resulting amino acids are utilized by the growing seedling. In barley, this process is commercially important because it forms the basis for the malting and brewing industries. In this study, barleys and malts were mashed in the presence of compounds that specifically inhibited the four common proteinase classes. The efficacies of the proteinases in solubilizing proteins were in the order cysteine≈metallo>aspartic>serine≈0, which roughly reflected how the inhibitors affected the mash endoproteolytic activities. It was previously believed that only the cysteine enzymes were involved. All four enzyme classes affected the free amino nitrogen concentration but none altered any of the other measured wort characteristics. With either single inhibitors or inhibitor mixtures, the effect of pH was as expected, based on earlier studies that indicated that cysteine and aspartic proteinases were most active at low pH values and the metalloproteinases were only active at high pH. At the North American commercial mashing pH of 6.0, about one third of the soluble protein of a typical wort came from ungerminated barley, half was solubilized during malting and the remaining 22% was released during mashing.     

Fusarium Species Synthesize Alkaline Proteinases in Infested Barley

Barley (Hordeum vulgare L.) that is infested with Fusarium head blight (FHB, ‘scab’) is unsuitable for malting and brewing because it may contain mycotoxins and has unacceptable malting quality. Fungal proteinases are apparently often involved in plant-microbe interactions, where they degrade storage proteins, but very little is known about the enzymes that the fungi produce in the infected grain. We have shown previously that one plant pathogenic fungus, Fusarium culmorum, produced subtilisin- and trypsin-like enzymes when grown in a cereal protein medium. To establish whether these proteinases were also synthesized in FHB-infested barley in vivo, field-grown barley was infested as the heads emerged. Extracts were prepared from the grain as it developed and matured and their proteolytic activities were measured with N-succinyl-Ala-Ala-Pro-Phe p -nitroanilide and N-benzoyl-Val-Gly-Arg p -nitroanilide. The heavily infested barleys contained both subtilisin- and trypsin-like activities. These enzymes reacted with antibodies prepared against each of the two F. culmorum proteinases, indicating that those produced in the laboratory cultures and in the field-infested barley were the same. The presence of these proteinases correlated with the degradation of specific buffer-soluble proteins in the infested grains. These enzymes readily hydrolyzed barley grain storage proteins (C- and D-hordeins) in vitro. The presence of these Fusarium proteinases in the barley indicates that they probably play an important role in the infestation, but exactly how and when they function is not clear.     

Phenotypic,physiological and malt quality analyses of US barley varieties subjected to short periods of heat and drought stress

Drought and heat are major abiotic stresses that significantly reduce crop yield and seed quality. In this study, we examined the impact of heat, drought and combined effect of heat and drought stress imposed during the grain filling stage in 18 US spring barley varieties. These impacts were assessed based on dry biomass, seed yield and six important malting quality traits, namely, beta-glucan, free amino nitrogen, soluble protein, refractive index, diastatic power and alpha-amylase activity. Singly applied heat or drought stress evoked a diverse set of responses among these varieties with respect to biomass, seed yield and malt quality traits suggesting these varieties can be exploited for enhancing barley production based on local conditions. Majority of the tested varieties performed poorly with reference to seed yield when the stresses were applied in combination, suggesting a lack of genetic diversity in the currently grown spring barley varieties to overcome co-occurring episodic drought and heat regimes, especially during heading stages. In the wake of global climate change, enhancing adaptive capacity of barley varieties by introducing novel germplasm into breeding programs or via new technologies is vital to sustain US barley production and meet the demands of the rapidly growing brewing industry.     

法国啤酒大麦的质量控制体系

为了促进我国啤麦生产种植的管理规范化和大规模工业化发展，本文分析了法国啤麦的质量控制体系，以供我国相关领域借鉴参考。法国是世界最大的酿造大麦生产国，也是向我国出口啤酒大麦的三大国家之一。法国啤酒大麦的生产建立了一整套严格的管理体系，从品种的选择、生产过程的监控到产后销售储运都有严格的管理办法。基于这套完整的对啤酒大麦的管理、考核体系，法国啤酒大麦的生产、销售质量能够得到充分的保证，其产品在国际市场上有很强的竞争力。借鉴法国啤酒大麦质量管理的方法扣经验，对于提高我国啤酒大麦的质量，实行啤麦自给降低市场风险和成本有着十分重要的意义。     

大麦种子贮藏期间的生理变化及其安全贮藏年限

为了探明常温条件下贮藏的大麦种子劣变生理表现及不同用途大麦安全贮藏期限,以1997～2000年收获、贮藏的8个大麦品种32份种子为材料,对种子过氧化物酶活性、种子生活力、种子浸出液电导率变化规律进行了分析研究。结果表明,随着大麦种子贮藏年限的延长,种子过氧化物酶活性与种子生活力均呈下降趋势,种子浸出液电导率呈上升趋势。种子过氧化物酶活性下降与种子浸出液电导率上升均是导致种子生活力下降的原因。在长江流域常温条件下,大麦种质可以安全贮藏两年半;生产用种的安全贮藏期不到一年半,但品种间存在一定的差异;啤麦原料均可安全贮藏到当年11月份。欲延长大麦种质、生产用种及啤麦原料的安全贮藏期,须改善贮藏条件。     

Development of proteolytic activities during barley malting and their localization in the green malt kernel

The water-insoluble storage proteins of barley seeds reside in the starchy endosperm tissue. This tissue, when expressed from germinating barley, has a pH of 4·8. The hydrolysis of storage proteins during germination (malting) occurs mainly in the endosperm, so proteinases that are located in endosperm and are active at pH 4·8 are probably important to the storage protein hydrolytic process. This study reports our continued investigations of the endoproteinases of germinating barley (Hordeum vulgare L., cv. Morex) with a two-dimensional gel separation method that uses isoelectric focusing (IEF) and non-denaturing polyacrylamide gel electrophoresis (PAGE) in gels containing incorporated substrate protein. We identified the endoproteinases that were active at pH 4·8 and determined when they appeared during germination and where they were located in 4-day germinated barley (green malt). A total of nine cysteine, four aspartic, and two serine class proteolytic activities that were active at pH 4·8 were extracted from the endosperm tissue of green malt. It seems probable that some or all of these endosperm endoproteinases, especially proteinases C7, C8, C11, D3, E3 and E4, are the ones most intimately involved in hydrolyzing the storage proteins during malting.     

Characterization of germinated barley endoproteolytic enzymes by two-dimensional gel electrophoresis

Germinating barley proteolytic enzymes hydrolyze insoluble seed storage proteins into soluble proteins, peptides and amino acids. This process is of vital importance to both seed germination and the commercial malting process. This study reports the development of a two-dimensional IEF × PAGE separation method utilizing protein substrates incorporated into the PAGE gel to analyze and partially characterize the endoproteinases of germinating barley grain. The method separated 42 different activities, which fell into five groups on the basis of their pI values, PAGE mobilities and biochemical characteristics. Multiple representatives of each of the four proteinase classes were present, but about 64% of the enzymes were cysteine proteinases. The majority of the endoproteinases had pH optima considerably below neutrality, but those of the serine proteinases were generally eight or higher. Most of the endoproteinases hydrolyzed gelatin faster than edestin, but the four aspartic class proteinases and one of an undetermined class only hydrolyzed edestin. These results show that the protein-hydrolyzing system of green malt (4-day-old germinated barley) is very complex.     

Current perspectives on the role of enzymes in brewing

The brewing of beer is a very traditional process, based upon a complex endogenous enzymology occurring during the malting of grain, mashing of grist and fermentation. The relevant cereal-derived enzymes that are involved are reviewed. It is likely that the production of alcoholic beverages in breweries in the projected future will assume different paradigms, procedures which are already realistic for the production of flavoured alcoholic beverages (malternatives) and “near-beer” drinks (happoshu and other beer-like beverages) in Japan. A range of exogenous enzymes—such as glucanases, acetolactate decarboxylase and prolyl endopeptidase—are available for enhancement of the existing brewing process.     

Current perspectives on the role of enzymes in brewing

The brewing of beer is a very traditional process, based upon a complex endogenous enzymology occurring during the malting of grain, mashing of grist and fermentation. The relevant cereal-derived enzymes that are involved are reviewed. It is likely that the production of alcoholic beverages in breweries in the projected future will assume different paradigms, procedures which are already realistic for the production of flavoured alcoholic beverages (malternatives) and “near-beer” drinks (happoshu and other beer-like beverages) in Japan. A range of exogenous enzymes—such as glucanases, acetolactate decarboxylase and prolyl endopeptidase—are available for enhancement of the existing brewing process.     

LTP is not a Cysteine Endoprotease Inhibitor in Barley Grains

Several different protease inhibitors have been identified in the mature barley grain, which are proposed to play a defensive role against potential barley pathogens. Cysteine protease inhibitors have been detected in mature grain and in the early stages of germination. The nature of these inhibitors has recently been investigated, and barley lipid transfer protein (LTP1) has been identified as an effective inhibitor of both cysteine and serine endoprotease activity expressed in germinating grain. We show that barley LTP1, in its native state, is not a cysteine protease inhibitor, but in a denatured state becomes a preferred substrate for the barley endoprotease EP-B and, as such, behaves as a competitive inhibitor for poorer substrates of EP-B. The presence of significant amounts of LTP1 in barley malt beer suggests that this very compact protein is highly resistant to proteolytic attack during malting and mashing and its denaturation during wort boiling coincides with inactivation of the malt endoproteases. Analysis of the cleavage products of denatured LTP1, generated by EP-B, provides further evidence for the cleavage site specificity of this barley cysteine endoprotease, where a hydrophobic residue in the P₂position is strongly preferred.     

Evaluation of malting barley quality using exploratory data analysis. I. Extraction of information from micro-malting data of spring and winter barley

This paper presents an exploratory multivariate approach for analysis of malting barley quality data. By using principal component analysis (PCA) and partial least squares regression (PLSR) complex malting quality data are combined into functional factors which are used for malting barley quality characterisation. Fifty barley samples were used in this investigation, representing 15 spring barley and 10 winter barley varieties grown at two locations in Denmark. The samples were micro-malted and mashed and analysed for 13 quality parameters according to official methods of the European Brewery Convention. These data were combined and reduced into a few latent (functional) factors using PCA by which it is demonstrated that the modification of β-glucan plays a major role in both spring and winter barleys. Additionally, the spring barley and winter barley samples display different covariate latent structures, mainly in the nitrogen and diastatic power patterns. It is furthermore shown that graphic display as facilitated by exploratory data analysis, can be utilized in order to evaluate genotype-environmental interactions by considering the position and movements of the individual objects (genotypes in this instance) in the score plots. Thus, in contrast to the classical analysis of variance, the samples can be individually evaluated and the corresponding loadings can be used to validate the genetic and environmental effect of a given sample in a quality perspective.Several of the investigated malting quality parameters are highly intercorrelated. This fact is utilized by applying PLSR to barley and malt data for the prediction of wort quality in order to exclude the mashing step. This approach was successful for the modification-dependent wort parameters, extract, wort β-glucan and viscosity.     

The effects of malting and mashing on barley protein extractability

Proteins in unmalted and malted barley and in brewers’ spent grain (BSG) obtained after mashing were fractionated on the basis of their differential extractability in different media and characterised by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance liquid chromatography (HPLC). Albumins and globulins were first extracted with 5.0% NaCl and hordeins (barley prolamins) were extracted with 55.0% 1-propanol in the presence, or absence, of 1.0% DTT. Glutelins were then extracted with 2.0% SDS/6.0 M urea/1.0% DTT or with 55.0% 1-propanol/6.0 M urea/1.0% DTT/0.036 M Tris-HCl (pH 8.4). Under non-reducing conditions, monomeric C hordeins and some B hordeins were extracted from unmalted barley, whereas most if not all B, C and D hordeins were extracted under reducing conditions. During malting, disulfide bonds are reduced and B and D hordeins are broken down by proteolysis. No D hordeins were extracted from malt and nearly the same levels of malt B hordeins were extracted both under non-reducing and reducing conditions. B hordeins present in BSG proteins were only extractable under reducing conditions. Whereas most of the C hordeins were extracted from BSG under non-reducing conditions, more C hordeins were extracted under reducing conditions. Mashing probably induced disulfide bond formation resulting in aggregation. Although earlier literature suggested the formation of an aggregate composed of B and D hordein (and glutelin) during mashing, the present work suggests the formation of an aggregate composed of B hordeins in which C hordeins are entrapped.     

啤酒大麦种子在成熟过程中品质形成的研究

以 4个啤酒大麦品种 ,研究啤酒大麦种子在种子发育过程中啤酒大麦品质的形成 ,结果表明 :啤酒大麦种子的千粒重随种子的成熟呈直线上升的趋势 ,而蛋白质的含量在种子成熟过程中变化较小 ,呈波状变化 ;抽穗后 36d内随种子的发育成熟 ,发芽率呈上升的趋势 ,抽穗后 36d是大麦种子最佳收获时间 ,此时收获的大麦种子具较高的发芽势与发芽率相对应的 ,此时收获的大麦种子活力、POD和a -淀粉酶活性也最高     

Evaluation of malting barley quality using exploratory data analysis. II. The use of kernel hardness and image analysis as screening methods

This paper presents an exploratory investigation of the use of image analysis and hardness analysis of barley kernels for characterisation and prediction of malting quality. A sample set of fifty barley samples representing 15 spring barley and 10 winter barley varieties grown at two locations in Denmark was used. The samples were micro-malted and mashed and analysed for 13 quality parameters according to the official methods of the European Brewery Convention. A sub-sample of the barley samples was analysed on two different single kernel instruments: (1) Foss Tecator GrainCheck was applied for non-destructive recording of single kernel size and shape (width, length, roundness, area, volume and total light reflectance) and (2) Perten Single Kernel Characterization System 4100 was applied for single kernel hardness and weight determinations. The eight variables from these single seed analyses have been used in two different ways, either as means and standard deviations, or as appended histogram spectra representing 250 kernels from each bulk sample. By the two methods, it has been possible to obtain reasonable Partial Least Squares Regression (PLSR) models for the structural and physical part of the malting quality complex associated to malt modification, but it was as expected impossible to predict the biochemical parameters associated with nitrogen chemistry and enzymatic power. The best model was achieved for (1→3, 1→4)-β-D-glucan in barley. The hardness of the barley kernels is by far the most important variable for describing malting performance. The additional use of the morphological data as acquired by fast non-destructive image analysis, however, also reveals some malting quality information by improving the calibration models based on hardness alone. The brightness of the kernels is by far the most important GrainCheck variable but also kernel size and shape is associated to malting performance. In general, the utilisation of the single kernel readings (used as histogram spectra), compared to sample mean and standard deviation, did not provide additional information for an improved prediction of the malting quality parameters.     

啤酒大麦籽粒蛋白质含量基因型与环境效应的研究进展

综述了目前国内外啤酒大麦籽粒蛋白质含量的基因型和环境效应研究的进展。内容包括蛋白质含量的遗传模型、一般配合力和特殊配合力 ,以温度、光照、水分、湿度为代表的气象因子和氮肥运筹方式作主导的栽培措施等环境条件对啤酒大麦籽粒蛋白质含量的效应 ,蛋白质含量与其它品质性状之间的相关性 ,生产中降低啤酒大麦籽粒蛋白质的调控措施。