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.     

ABTS and DPPH methods as a tool for studying antioxidant capacity of spring barley and malt

Many analytical methods for antioxidant determination in foodstuffs and raw materials based on various principles have been published so far. However, not all of them are applicable to barley and malt. The results of total antioxidant capacity (TEAC) of barley and malt obtained with methods based on ability to eliminate radicals of 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH) were compared and statistically evaluated. The average TEAC of barley determined using ABTS and DPPH ranged from 2.1 to 2.5 μmol g⁻¹ and from 1.2 to 1.7 μmol g⁻¹, respectively. The TEAC ranges in malt were 2.7–3.0 μmol g⁻¹ (ABTS) and 1.8–2.6 μmol g⁻¹ (DPPH). TEAC of barley and malt were affected by the weather conditions (year), variety and application of Zn²⁺ fertilizer. The ABTS and the DPPH methods represent an effective tool for the assessment of antioxidant capacity of spring barley and malt and the factors having an impact on it.     

Effects of sulphur on yield and malting quality of barley

Eight field experiments were conducted at four sites in the UK in 2003 and 2004 to investigate the effects of sulphur (S) application on yield and malting quality of barley. Significant yield responses to S additions were obtained in five out of the eight experiments, with yield increases ranging from 0.2 to 1.2 t/ha (4.7–22.5%). At the two most S-deficient sites, S application significantly increased malt diastatic power, alpha-amylase activity, friability and homogeneity, and decreased (1→3,1→4)-β-glucan concentration in the wort, indicating an improved endosperm modification during malting. Sulphur applications also significantly increased the concentration of S-methylmethionine (the precursor of dimethylsulphide) in kilned malt, which could impact on beer flavour. When the supply of N was limiting, S applications decreased grain N concentration due to a dilution effect as a result of increased grain yield. In some cases, S applications resulted in decreased grain size. At sites non-deficient or marginally deficient in S, applications of S had little effect on grain or malting quality parameters. The need to maintain an adequate S supply to barley for both yield and malting quality was demonstrated.     

啤用二棱大麦产量构成因素的表型相关分析与应用

应用简单相关、回归分析、偏相关、通径分析方法对沪麦4号等10个啤用二棱大麦品种（系）的产量构成因素进行统计分析和研究,结果表明,3个产量构成因素对单株产量均有正向效应,其中以每株穗数的贡献最大,每穗粒数次之,千粒重贡献最小。因此,提出了高产优质啤用二棱大麦的育种模式：以增加每株穗数“多穗型”为主要选择目标,兼顾每穗粒重,增加千粒重为配合选择指标,并适当协调好千粒重与穗粒数的关系。     

Influence of sulphur application on hordein composition and malting quality of barley (Hordeum vulgare L.) in northern European growing conditions

The influence of sulphur (S) application on yield formation, hordein composition and malting quality of 2-rowed spring barley (Hordeum vulgare L.) was studied in Nordic conditions for the first time. In a greenhouse experiment, S deficiency was indicated when 10 mg S/kg soil or less was available, by an increase in the malate:sulphate ratio in leaves. The contents of aspartic acid and cystine in grains increased and decreased, respectively. Also a substantial decrease of total hordein and the proportion of B hordein, and an increase in the proportion of C hordein were associated with S deficiency. The effect of S was further assessed in field experiments by applying N and S in four different combinations prior to sowing. No apparent S deficiency occurred in the field conditions based on the malate:sulphate and grain N:S ratio. However, in a site where the grain S content was slightly but significantly increased by S application, a change in hordein composition and malting quality was observed. Thus even in S-sufficient conditions, the end use quality of malting barley may be affected by S application.     

Individual polyphenolic profiles and antioxidant activity in sorghum grains are influenced by very low and high solar UV radiation and genotype

Sorghum is becoming more widely recognised around the world as a valuable crop for its polyphenol antioxidant health-promoting properties and adaptation to harsh environments. The antioxidant capacity of diverse polyphenols in sorghum grain can vary with climatic conditions and genotype. To explore this further, the potential role of UV radiation on the profile and concentration of polyphenols was investigated in six diverse sorghum genotypes in a controlled environment facility using natural UV and visible radiation under either UV-transmitting or UV-blocking treatments. The polyphenol content and antioxidant activity were significantly reduced under the UV-blocking treatment, with contents of individual polyphenols differing among the genotypes. This information is valuable for sorghum breeders enabling them to select genotypes and UV growth conditions to produce sorghum grain with target levels of polyphenols and antioxidant capacity for human foods to meet the nutritional and health needs of different consumers.     

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 enzymatic hydrolysis on molecular structure and antioxidant activity of barley hordein

Hordein, the major storage protein of barley (Hordeum vulgare L.), was hydrolysed by three selected proteases, including alcalase, flavourzyme and pepsin. The effects of protease type and hydrolysis time on hordein molecular weight, surface hydrophobicity, secondary structure and antioxidant activity were investigated. Flavourzyme hydrolysis of hordein was relatively more extensive and rapid, resulting in the formation of medium- and small-sized peptides with a broad distribution within 30 min. Alcalase and pepsin more gradually and less extensively hydrolysed hordein into medium- and larger-sized peptides, respectively. Protein surface hydrophobicity decreased with an increasing degree of hydrolysis. The flavourzyme and alcalase hydrolysates had superior DPPH (1,1-diphenyl-2-picryl hydrazyl) free radical scavenging activity (44-70 and 48-58%, respectively, at 0.5 mg/mL), Fe²⁺-chelating ability (21-64% and 39-73%, respectively, at 1 mg/mL), and superoxide radical scavenging capacity. It is proposed that the large- and medium-size hydrolysate fractions were most likely responsible for the antioxidant activities of hordein hydrolysates, and could be used as antioxidant peptides in food and nutraceutical applications.     

QTLs for malting flavour component associated with pre-harvest sprouting susceptibility in barley (Hordeum vulgare L.)

Lipoxygenase (LOX) is a key factor affecting quality of beer in terms of foam stability and flavour. Low LOX content is a desirable trait for malting quality. A doubled haploid (DH) population was made from a cross of Australian malting barley Stirling and Canadian malting barley Harrington and mapped with 513 molecular markers. The 120 DH lines with their parents were planted in field trials and the harvested grains were micro-malted for analysis of LOX content in two consecutive years. LOX content was controlled by both genetic effects and environment conditions. Three QTLs were consistently detected. One QTL flanked by the markers E6216 and SCssr03907 at the telomere region of chromosome 5HL contributed 39% of genetic variation in LOX content. The second QTL close to the centromere region of chromosome 5H accounted for 17% of genetic variation. A minor QTL on chromosome 2H explained 6% of genetic variation but was significant in both years. The Australian variety Stirling contributed to higher LOX content for the three QTLs. The two QTLs mapped at chromosome 5H for LOX content coincided with the QTLs for seed dormancy/pre-harvest sprouting from the same population. The pre-harvest sprouting susceptible alleles were associated with low LOX content, which indicated that the low LOX QTL from the Canadian malting barleys are only useful in the barley growing areas where the pre-harvest sprouting risk is low. New genetic sources for low LOX should be exploited in different germplasm with different mechanisms.     

Reduction of polyphenol and phytic acid content of pearl millet grains by malting and blanching

This work was undertaken to evaluate the changes in polyphenol and phytic acid content in malted and blanched pearl millet grains. For malting, grains were steeped for 16 hours, germinated for 48 or 72 hours and then kilned at 50 ^°C for 24 hours. Blanching was done for 30 seconds in boiling water at 98 ^°C. Results indicated that blanching resulted in significant reduction in polyphenol (28%) and phytic acids (38%). Destruction of polyphenols (38 to 48%) and phytic acid (46 to 50%) was significantly higher in grains subjected to malting than blanching. The overall results suggested that malting with 72 hours of germination was most effective in reducing the antinutrient levels of pearl millet grains.     

Black currant polyphenols: Their storage stability and microencapsulation

Interest in the consumption of fresh fruits is, to a large extent, due to its content of bioactive nutrients and their importance as dietary antioxidants. There is a growing demand for delivery of antioxidants through functional foods with the related challenge of protecting their bioactivity during food processing and subsequent passage through the gastrointestinal tract. This study focuses on the evaluation of concentration of bioactive compounds in black currant berries (Ribes nigrum L.) and retention of black currant polyphenol compounds and their antioxidant activity after microencapsulation by spray-drying. The polyphenol contents and antioxidant activities were assessed for 5 black currant cultivars grown in Canada in fresh and stored fruits at −20 °C for 9 months. The Ben Alder cultivar was the richest in anthocyanins and flavonols content with high antioxidant potential measured with ABTS and DPPH radicals (4.5 and 2.4 mM/100 g). Black currant polyphenols were stable during 9 month of storage at −20 °C. The results of this study indicated that in black currants storage did not affect negatively the antioxidant capacity. The good stability of bioactive compounds of black currants during freezer storage allows prolonging the storage time of these berries. Maltodextrins with different DE (dextrose equivalent): DE11, DE18, DE21 and inulin were selected as wall material. The stability of encapsulated polyphenols during 12 months storage at 8 °C and 25 °C was evaluated. Compared to maltodextrins DE18 and DE21, maltodextrin DE11 had not only higher drying yield but also offered better protection for phenolics during storage. Radical scavenging activity studies demonstrated significant antioxidant activity of microencapsulated powders before and after storage. The black currant polyphenol microcapsules described in this study represent a promising food additive for incorporation into functional foods due to antioxidant content.     

Functional study of raw and cooked blue maize flour: Starch digestibility,total phenolic content and antioxidant activity

The purpose of this study was to determine the chemical composition and antioxidant capacity of blue maize (BM) (Zea mays L.) flour and to investigate the effects of polyphenol-containing extracts and BM wholegrain flour on starch digestion under uncooked and cooked conditions; commercial white maize flour was used as control. Total phenolic content in BM flour (BMF) (164 ± 14 mg gallic acid/g of dry matter) was higher than white maize (127 ± 7 mg gallic acid/g of dry matter), and the presence of anthocyanins (2.0 ± 0.5 mg cyanidin 3-glucoside/100 g) was detected. Also, an important scavenging activity against ABTS (2,2’-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid) and DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals and ferric reducing power (FRAP) was determined. Extracts of BMF reduced amylase activity (>90% of inhibition). BMF showed higher slowly digestible and resistant starch contents, thus exhibiting lower predicted glycemic index than white maize. Total anthocyanins (r = −0.89 and r = −0.79, p<0.05), antioxidant capacity (r = −0.86 and r = −0.96, p<0.05), total starch (r = 0.99 and 0.92, p<0.05) and resistant starch content (r = −0.99 and r = −0.92, p<0.05) were correlated with pGI for uncooked and cooked flours, respectively. These results indicate the potential use of BMF and its phenolic-rich extract as functional ingredients to develop antioxidant and indigestible carbohydrate-rich foods with potential health benefits.     

Volatile metabolite profiling of malt contaminated by Fusarium poae during malting

A volatile metabolite-profiling approach based on solid-phase microextraction (SPME) combined with gas chromatography–mass spectrometry was used to investigate volatile metabolic changes over the course of a malting process contaminated by Fusarium poae to assess its influence on malt flavor. GC–MS analysis of time-serial samples revealed a broad spectrum of volatile compounds including aldehydes, ketones, alcohols, organic acids, aromatics and furans. Statistical assessment of the data via principal component analysis demonstrated that at the initial phase of germination, the influence of F. poae was relatively small. However, its influence increased over time, resulting in differences between contaminated and controlled samples at 120 h and 144 h. Analysis of the corresponding loadings showed that volatile metabolites from green malt were influenced by contamination with F. poae fungi during malting to an extent, especially for the volatile aldehyde fractions. These compounds can be formed during both primary and secondary metabolism, and the potential exists for metabolic perturbation resulting from F. poae.     

Fundamental study on protein changes taking place during malting of oats

During the malting process, storage proteins are degraded by proteolytic enzymes into small peptides and amino acids. The activity of these enzymes was measured during malting of oats and was found to be increased. To quantify proteolytic degradation, proteins of unmalted, germinating and malted grains were fractionated. After extracting the oat proteins (Osborne fractionation), protein fractions were analysed using a Lab-on-a-Chip technique, which separates the proteins – based on their molecular weight – by capillary electrophoresis. This new technique for the analysis of proteins was supported by using two-dimensional gel electrophoresis. In addition, amino acid analysis was carried out. In general a degradation of proteins to small peptides and amino acids could be observed in the globulin, prolamin and glutelin fractions. In the albumin fraction a protein increase was observed, which is due to the fact that this fraction contains the majority of the metabolically active proteins. Amino acid analysis supported the observation of increased protein amount in the albumin fraction and decreased protein amounts in the other fractions. Some proteins, which have not been described in the literature, were detected in the albumin and glutelin fraction, since Lab-on-a-Chip technique allows detection of proteins with low molecular weights of 4.5 kDa.     

Investigations into the thiamine and riboflavin content of malt and the effects of malting and roasting on their final content

A simple and effective method for the analysis of thiamine (B₁) and riboflavin (B₂) vitamers, in unmalted and malted grains, by high performance liquid chromatography (HPLC) has been developed. This method makes use of trichloroacetic acid (TCA) as an extraction medium to effectively clean up the sample and analyze the vitamer content with high accuracy (R² > 0.9992; %RSD < 5%). This method was employed in the routine analysis of a wide range of malted and unmalted grains, and it was found that lighter colored malts contain higher concentrations (2–5 times higher) of thiamine and riboflavin vitamers compared to darker colored malts. The malting process has no effect on the overall riboflavin content; however, both steeping and kilning processes cause increases in thiamine vitamer content. Roasting profiles show that thiamine and riboflavin vitamer concentrations are significantly effected when the temperature reaches 120 °C, which explains why roasted products have lower vitamer content than paler malts.     

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.     

Potential longevity (Ki) of malting barley (Hordeum vulgare L.) grain lots relates to their degree of pre-germination assessed through different industrial quality parameters

Barley (Hordeum vulgare L.) grain germination is required to perform the malting process. Maintenance of barley seed viability during storage is crucial for the malt industry; and modern cultivars are bred for rapid grain dormancy release after physiological maturity. Low dormancy level combined with rain close to harvest induces pre-germination/pre-harvest sprouting damage. Pre-germination might not affect viability in the short term after harvest, but it could reduce potential longevity (K_i) of a barley seed lot. K_i value is inherent for each barley lot; however, its determination is time-consuming which precludes its assessment at an industrial scale. In this study we sought quantitative relationships between K_i and the pre-germination degree of barley grain lots, assessed through quality tests routinely performed by malthouses [Falling Number (FN), α-Amylase Activity and Carlsberg]. Field pre-germinated lots from one old barley cultivar (Quilmes Palomar) and artificially pre-germinated lots from major varieties currently grown in Argentina were used. Associations between K_i and values obtained from all quality tests analysed were found for Q. Palomar. However, FN was the parameter that yielded the best and simplest explanation of K_i variability. A significant positive linear K_i -FN relationship was also obtained for each modern barley cultivar.     

In vitro antioxidant activity of ragweed (Ambrosia artemisiifolia L., Asteraceae) herb

The antioxidant activity in 70% aqueous acetone extracts of the herb of Ambrosia artemisiifolia L., Asteraceae was evaluated with regards to total polyphenol and flavonoid contents. Antioxidant activity has been assessed by two commonly used in vitro tests, based on determination of ferric-reducing antioxidant power (FRAP assay) and DPPH free radical scavenging ability (DPPH assay), against butylated hydroxytoluene (BHT), ascorbic acid and quercetin, which were used as positive control substances. The results of both antioxidant tests showed that the plant material expressed a considerable activity (DPPH IC₅₀ = 27.6 μg/ml; FRAP value = 2.37 mmol Fe²⁺/g), attributed to both flavonoid aglyca and resembling glycosides, as verified by dot-blot TLC analysis.     

磷酸一铵在啤酒大麦上的应用效果初报

在石灰性土壤条件下通过田间小区和大区试验初步研究了磷酸一铵在啤酒大麦上的应用效果。结果表明，与磷酸二铵和低浓度混合肥相比，磷酸一铵作基肥效果显著，且成本较低，平均增产8．41％，而肥料成本降低6．02％。啤酒大麦的最佳经济施肥量宜为磷酸一铵15kg／亩，配合纯氮10．5kg／亩。     

Physicochemical changes of starch during malting process of sorghum grain

The objective of this study was to evaluate the physicochemical changes that occur in sorghum starch during malting to determine its potential in the beer industry. The results showed that the physicochemical properties of amorphous amylose and amylopectin of starch over time are modified during the germination while its crystalline structure is not affected. During the different stages of malting the SEM images showed the progress of the enzymatic attack in the starch granule. Germination and the increase in reducing sugars supported amorphous starch degradation. The X-ray diffraction pattern confirmed that during malting, the crystalline structure in starch remained without changes. The pasting profile showed high values for peak and final viscosity, once the sorghum was soaked and decreasing over the malting process. These changes are associated with starch degradation and amylopectin debranching, increasing the reducing sugars.