Dual positional and stereospecificity of lipoxygenase isoenzymes from germinating barley (green malt): biotransformation of free and esterified linoleic acid

The lipoxygenase isoenzymes LOX1 and LOX2 from green malt were separated by isoelectric focusing, and their catalytic properties regarding complex lipids as substrates were characterized. The regio- and stereoisomers of hydroperoxy octadecadienoates (HPODE) resulting from LOX1 and LOX2 enzymatic transformations of linoleic acid, methyl linoleate, linoleic acid glycerol esters monolinolein, dilinolein, and trilinolein, and 1-palmitoyl-2-linoleoyl-glycero-3-phosphocholine (PamLinGroPCho) were determined. In addition, biotransformations of polar and nonpolar lipids extracted from malt were performed with LOX1 and LOX2. The results show that LOX2 catalyzes the oxidation of esterified fatty acids at a higher rate and is more regioselective than LOX1. The dual position specificity of LOX2 (9-HPODE:13-HPODE) with trilinolein as the substrate (6:94) was higher than the resultant ratio (13:87) when free linoleic acid was transformed. A high (S)-enantiomeric excess of 13-HPODE was analyzed with all esterified substrates confirming the formation of 13-HPODE through the LOX2 enzyme; however, 9-HPODE detected after LOX2 biotransformations showed (R)-enantiomeric excesses. PamLinGroPCho was oxygenated by LOX1 with the highest regio- and stereoselectivities among the applied substrates.     

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.     

Determination of ergosterol levels in barley and malt varieties in the Czech Republic via HPLC

Ergosterol is considered to be a suitable indicator of mold infestation in barley and malt. In this study ergosterol levels in different varieties of barley and malt produced in the Czech Republic were determined. A modified high-performance liquid chromatography (HPLC) method was statistically processed, validated (Effivalidation program), and applied to 124 samples of barley and malt. Ergosterol was isolated by extraction and saponification, and the quantification was performed using HPLC with diode array detection. The content of ergosterol ranged between the limit of detection (LOD) and 36.3 mg/kg in barley and between the LOD and 131.1 mg/kg in malt. Ergosterol is presumably connected with metabolites generated when barley grain is attacked by pathogens, and such barley often shows a high overfoaming (gushing) value. However, it was found that the content of ergosterol does not correlate with the degree of beer gushing.     

Plant,grain, and soil response of irrigated malt barley as affected by cultivar,phosphorus, and sulfur applications on an alkaline soil

Information on the effects of phosphorus (P) and sulfur (S) applications on crop response and soil-P status of two-row malt barley (Hordeum vulgare L.) under high-input conditions are limited in alkaline soils despite widespread fertilizer-P and -S use. A field study was conducted during the 2015 and 2016 growing seasons where the barley cultivars (ABI-Voyager and Moravian 69) were grown at five rates of P (0, 37, 73, 110, and 147?kg P ha^?1) and three rates of S (0, 112, 224?kg S ha^?1). ABI-Voyager had significantly greater biological yield (17,023?kg ha^?1) and grain yield (7433?kg ha^?1) but a lower (44%) harvest index (HI) than Moravian 69 (15,037?kg ha^?1, 7168?kg ha^?1 and 49%, respectively). Grain yield increased with rate of P-application until 37?kg P ha^?1 where the maximum calculated yield was obtained at 98?kg P ha^?1 by a quadratic model. Sulfur application had no significant effect on any of the measured crop or soil parameters. Olsen P increased linearly with greater fertilizer-P applications, indicating grain-P removal was not sufficient to reduce or retain STP concentrations at initial levels when P was applied. Crop-P uptake and soil-P response to fertilizer P applications are important, as remaining soil P is available for subsequent crop usage and may have potential negative environmental impacts. Thus, cultivar selection and appropriate fertilizer-P and S management will ensure optimal agronomic and economic returns while minimizing potentially negative environmental impacts for two-row malt barley produced in the western United States.     

Conjugated linoleic acid, cis-9,trans-11, is a substrate for pulmonary 15-lipoxygenase-1 in rat

The objective of this study was to determine whether two of the major conjugated linoleic acid (CLA) isomers, cis-9,trans-11 (c9,t11) and trans-10,cis-12 (t10,c12), are possible substrates for pulmonary 15-lipoxygenase-1 (15-LOX-1) and, therefore, they are also involved in the production of 13(S)-hydroxyoctadecadienoic acid [13(S)-HODE] in biological systems. 13(S)-HODE, a major bioactive metabolite of linoleic acid, is an important intracellular signal agent and is involved in cell proliferation and differentiation in various biological systems. Nordihydroguaiaretic acid (NDGA), a known LOX inhibitor, was used as a control for measuring 15-LOX-1 enzyme activity. It was found that c9,t11-CLA was 25% as active as linoleic acid as a substrate for 15-LOX-1; however, t10,c12-CLA was not a substrate for 15-LOX-1 as measured by 13(S)-HODE production. The authenticity of the production of 13(S)-HODE from c9,t11 as a substrate was established by isolation and cochromatography with pure standard on HPLC using non-radioactive and [14C]-c9,t11-CLA.     

Variability in the release of free and bound hydroxycinnamic acids from diverse malted barley (Hordeum vulgare L.) cultivars during wort production

Volatile phenols have long been recognized as important flavor contributors to the aroma of various alcoholic beverages. The two main flavor-active volatile phenols in beer are 4-vinylguaiacol and 4-vinylphenol. They are the decarboxylation products of the precursors ferulic acid and p-coumaric acid, respectively, which are released during the brewing process, mainly from malt. In this study, the variability in the release of free and ester-bound hydroxycinnamic acids from nine malted barley ( Hordeum vulgare L.) varieties during wort production was investigated. A large variability between different barley malts and their corresponding worts was observed. Differences were also found between free ferulic acid levels from identical malt varieties originating from different malt houses. During mashing, free hydroxycinnamic acids in wort are both water-extracted and enzymatically released by cinnamoyl esterase activity. Esterase activities clearly differ between different barley malt varieties. Multiple linear regression analysis showed that the release of ferulic acid during mashing did not depend only on the barley malt esterase activity but also on the amount of ester-bound ferulic acid initially present in the wort and on its endoxylanase activity. The study demonstrates the importance of selecting a suitable malt variety as the first means of controlling the final volatile phenol levels in beer.     

Antioxidant vitamins in barley green biomass

Two malting hulled varieties (Sebastian, Malz) and one nonmalting hull-less variety (AF Lucius) were used to assess vitamins C and E in the green biomass of young plants of spring barley ( Hordeum vulgare L.) in three stages of growth and development (BBCH 29, 31, 32-33). The samples from sampling I (BBCH 29) had statistically significantly higher vitamin C content and vitamin E activity compared to sampling I (BBCH 31). The highest average vitamin content was determined in the malting variety Sebastian (vitamin C, 520 mg 100 g(-1) DM; vitamin E, 73.06 mg kg(-1) DM) compared to the varieties Malz (501 mg 100 g(-1) DM; 61.84 mg kg(-1) DM) and AF Lucius (508 mg 100 g(-1) DM; 67.81 mg 100 g(-1) DM). The locality Krome?r?i?z? (Czech Republic, CR), with vitamin C and E contents of 524 mg 100 g(-1) DM and 68.74 mg kg(-1) DM, respectively, proved to be more suitable for growing green biomass compared to the locality Z?abc?ice (CR) (content of vitamins C and E, 477 mg 100 g(-1) DM and 66.39 mg kg(-1) DM, respectively). During the research period (2005-2007), it was determined that the green mass of young plants of spring barley was a significant source of vitamins C and E in the growth stage BBCH 29; in later samplings (BBCH 32-33) the vitamin levels dropped (by as much as 48%). These vitamins are important antioxidants for human health. Therefore, "green barley" can be recommended for the preparation of natural dietary supplements and is preferred to synthetic vitamin preparations.     

Polymorphism of Aspartic Proteinases in Resting and Germinating Barley Seeds

Both resting and germinated barley seeds (Hordeum vulgare L. ‘Morex’) contain aspartic endopeptidase activities, and the activities increase during germination. We have extracted and partially purified aspartic endopeptidases from both resting seeds and green malt (four-day germinated barley). Six aspartic proteinase activities were found in resting barley seeds while only four activities were detected in green malt. All of the aspartic proteinases had similar pH activity optima (pH 3.5–4.5) and pI values (≈4.5). The purified green malt aspartic proteinases selectively digested a group of barley seed proteins, postulated to serve as defensive proteins, that are coded by the amylase-trypsin inhibitor super gene family. The aspartic proteinases that bound to a pepstatin A affinity column at pH 4.5 cross-reacted with antiserum raised against aspartic proteinases purified from barley seed. However, those that did not bind the affinity column also did not cross-react with the antiserum, indicating that there are two distinct groups of aspartic proteases in germinating barley.     

The effect of fungicidal treatment on selected quality parameters of barley and malt

Protection of barley grain against contamination by fungi such as Fusarium spp., particularly by those producing mycotoxins, secondary metabolites with adverse health effects, is of principal importance. Fungicides applied immediately after full heading of spring barley is one method of direct protection. In this work, extensive two-year field experiments combined with a detailed chemical laboratory analysis (barley and malt) were performed with the aim to study the effect of previous crops, different fungicides, and other conditions on the selected barley and malt quality parameters (content of beta-glucans, pentosans, oxalic acid, deoxynivalenol, and gushing), while the main task was to follow the effect of the fungicide (used as a treatment to protect against pathogens, mostly Fusarium) on changes of the chemical composition in barley and malt, and gushing. It was found that the relationship between the studied factors and the parameters usually applied to the evaluation of barley and malt quality is quite complex and not straightforward. The responses show typical features of a multifactorial influence with both positive and negative correlations resulting in a decrease or increase in grain quality (concentrations of beta-glucans, pentosans, deoxynivalenol, and other studied parameters). The role of previous crops was also found to be important. The fungicides should be applied at the time of heading but not at the very beginning of this period.     

Purification and partial characterization of a second cysteine proteinase inhibitor from ungerminated barley (Hordeum vulgare L.)

It was previously shown that ungerminated barley contains inhibitors that suppress the activities of green malt cysteine proteinases. This paper reports the purification and partial characterization of a second barley cysteine endoproteinase inhibitor, a protein called lipid transfer protein 2 (LTP2). The chromatographically purified inhibitor had a molecular mass of 7112. The amino acid composition and sequence data of the purified inhibitor indicated that it was a protein whose gene, but not the protein itself, was isolated earlier from barley aleurone tissue. The purified protein inhibited the activities of electrophoretically separated green malt cysteine proteinases but not the activities of the serine- or metalloproteinases. The purified LTP2 inhibited the same proteases as the LTP1 that was characterized previously but was present in the mature seed in much smaller amounts. Neither LTP1 nor LTP2 has been proven to transport lipids in vivo, and it seems possible that both serve to keep cysteine endoproteinases that are synthesized during barley seed development inactive until the plant needs them. The small amount of LTP2 in the seed made it impossible to determine whether it, like LTP1, is involved in beer foam formation. Because of its proteinase-inhibiting ability and its resistance to heat inactivation, some of the LTP2 may persist in beer.     

灌水与肥密配置对甘啤7号大麦产量和蛋白质的影响

为筛选高产、优质、节水啤酒大麦新品种甘啤7号合理种植方式和适宜的肥密组合,2012~2013年,研究两种灌水条件下不同施肥水平、栽培密度对啤酒大麦甘啤7号产量和蛋白质含量的影响。结果表明,全生育期灌水一次(S1)和全生育期灌水两次(S2)处理下均为高肥水平(F4)大麦籽粒产量高于其它施肥水平,两种灌水条件下,F4较不施肥处理F1(CK)增产幅度均达到60%以上。S1与S2处理均显示:随着施肥量的增加大麦籽粒蛋白质含量增加,且各施肥水平之间籽粒蛋白质含量差异达显著水平。在F1、F2与F3施肥水平下其蛋白质含量均达到国家一级或者优级酿造标准。播种密度对啤酒大麦产量影响不显著,对啤酒大麦蛋白质含量无影响。通过肥料和密度互作效应结果分析得出:组合F3M2产量在所有组合中始终位于前四,并且其酿造品质一直良好。在相同肥密配置下,S1与S2处理的产量表现出相同的变化趋势,基本表现为大麦籽粒产量S1S2。综合生态、经济以及其酿造价值等诸多因素,建议啤酒大麦新品种甘啤7号全生育期灌水一次,肥料施用量为180 kg/hm2,种植密度为600万株/hm2。     

Evaluation of antioxidant activity and electronic taste and aroma properties of antho-beers from purple wheat grain

Moderate consumption of beer is known to be beneficial for health. Thus, antioxidant, likely taste, and aroma properties of antho-beers made from purple wheat grain (antho-grain) were evaluated. The 2,2-diphenyl-1-picryhydrazyl free radical (DPPH*) scavenging activity, total phenolic content (TPC), oxygen radical absorbance capacity (ORAC), and phenolic acid compositions of antho-bran were also investigated. DPPH* scavenging activity at 60 min was 50.6-59.9% for control and antho-beer extracts, 15.0-54.1% for antho-bran extracts and hydrolysates. The TPC ranged from 410 to 609 mg/L, from 84 to 95 mg/L, and from 2473 to 7634 mg/kg for control (from barley malt) and antho-beer original samples, control and antho-beer extracts, and antho-bran extracts and hydrolysates, respectively. The corresponding ORAC values were 3050-4181 mg/L, 2961-3184 mg/L, and 74-213 g/kg, respectively. The major known phenolic acids comprised four types in control beer, five types in antho-beers, and seven types in antho-bran hydrolysates. Total anthocyanin content of antho-bran was up to 1160 mg/kg. Differences in likely taste and aroma were found between control and antho-beers by using electronic tongue and nose methods. Brewing materials had an effect on the antioxidant, likely taste, and aroma properties of beers; however, antho-grain may have potential as a novel brewing material.     

Contents of phenolic acids, alkyl- and alkenylresorcinols, and avenanthramides in commercial grain products

The contents of free and total phenolic acids and alk(en)ylresorcinols were analyzed in commercial products of eight grains: oat (Avena sativa), wheat (Triticum spp.), rye (Secale cerale), barley (Hordeum vulgare), buckwheat (Fagopyrum esculentum), millet (Panicum miliaceum), rice (Oryza sativa), and corn (Zea mays). Avenanthramides were determined in three oat products. Free phenolic acids, alk(en)ylresorcinols, and avenanthramides were extracted with methanolic acetic acid, 100% methanol, and 80% methanol, respectively, and quantified by HPLC. The contents of total phenolic acids were quantified by HPLC analysis after alkaline and acid hydrolyses. The highest contents of total phenolic acids were in brans of wheat (4527 mg/kg) and rye (4190 mg/kg) and in whole-grain flours of these grains (1342 and 1366 mg/kg, respectively). In other products, the contents varied from 111 mg/kg (white wheat bread) to 765 mg/kg (whole-grain rye bread). Common phenolic acids found in the grain products were ferulic acid (most abundant), ferulic acid dehydrodimers, sinapic acid, and p-coumaric acid. The grain products were found to contain either none or only low amounts of free phenolic acids. The content of avenanthramides in oat flakes (26-27 mg/kg) was about double that found in oat bran (13 mg/kg). The highest contents of alk(en)ylresorcinols were observed in brans of rye (4108 mg/kg) and wheat (3225 mg/kg). In addition, whole-grain rye products (rye bread, rye flour, and whole-wheat flour) contained considerable levels of alk(en)ylresorcinols (524, 927, and 759 mg/kg, respectively).     

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.     

Synthesis and phytotoxicity of new ionic liquids incorporating chiral cations and/or chiral anions

The aim of this work was to synthesize chiral ionic liquids as chiral solvents for organic synthesis and to evaluate the phyto(eco)toxicity of the new products and starting N-alkylimidazoles and their potential environmental influence on soil and plants. Chiral ionic liquids containing anions such as Cl-, Br-, TsO-, PF6(-), NO3(-), CF3SO3(-), and (+)- and (-)-C6H5CH(OH)C(O)O- were synthesized using (-)-(1R)-6,6-dimethylbicyclo[3.1.1]hept-2-ene-2-ethyl [(-)-(1R)-nopyl] halides (X = Cl, Br) and tosylate in 62-100% yields. The chloride 7 and the nitrate 13 ionic liquids possessed a toxicity dependent on the applied concentration. The lowest concentration causing a distinct reduction in plant germination/growth was 100 mg/kg. Spring barley better tolerated the ionic liquids (200 mg/kg) than common radish (100 mg/kg). The nitrate liquid did not exhibit an inhibiting effect on the germination ability of seeds. The starting N-methylimidazole used in lower concentrations (1 and 10 mg/kg of soil dry weight) was not phytotoxic, in contrast to higher doses (>1000 mg/kg).     

Relationships Between Fatty Acid Contents of Barley Grain,Malt, and Wort with Malt Quality Measurements

Although lipids and fatty acids (FA) represent only 1–3% of the grain weight, they can play an important role in regulating, modulating, and determining several chemical and physical properties of the grain and corresponding malts. The aim of this study was to evaluate the relationships between the content of FA in grain, malt, and wort with malt quality characteristics such as hot water extract (HWE) and apparent attenuation limit (AAL) in different commercial malting barley varieties. High and positive correlations were found between myristic acid and HWE (r = 0.71) and between stearic acid and AAL (r = 0.76), with intermediate correlations between palmitic, oleic, linoleic, and linolenic acids and AAL (r = >0.50) in grain. High and negative correlations were found between stearic acid and HWE (r = –0.66), and high and negative correlations were found between palmitic (r = −0.74) and linoleic (r = −0.60) acids and AAL in the wort. Results from this study showed that lipids, as well as the combination of unsaturated and saturated FA, might play a role in determining differences in HWE and AAL between the barley varieties analyzed. No clear evidence on HWE was observed when grain and malt samples from the same variety were compared. These results indicated that lipids and FA should be considered together with starch properties to explain differences between HWE and AAL.     

Trypsin/alpha-amylase inhibitors inactivate the endogenous barley/malt serine endoproteinase SEP-1

Barley (Hordeum vulgare L.) malt contains endoproteinases belonging to all four of the commonly occurring classes, including serine proteinases. It also contains low molecular weight proteins that inhibit the activities of many of these endoproteinases, but it had never been shown that any barley or malt serine proteinases could be inhibited by any of these endogenous proteins. It is now reported that some proteins that were concentrated using an "affinity" method inhibited the activity of a malt serine endoproteinase. Two-dimensional electrophoretic and in vitro analyses showed that the inhibited enzyme was serine endoproteinase 1 (SEP-1) and that the inhibition could be quantified using a semipurified preparation of this enzyme. Amino acid sequencing and MALDI-TOF MS were used to identify the components of the partially purified inhibiting fractions. Only the "trypsin/alpha-amylase inhibitors" or chloroform/methanol (CM) proteins, most of which had truncated N and C termini, and one fragment of beta-amylase were present in the inhibitory fractions. When a CM protein fraction was prepared from barley according to traditional methods, some of its component proteins inhibited the activity of SEP-1 and some did not. This is the first report of the purification and identification of barley malt proteins that can inhibit an endogenous serine proteinase. It shows that some of the CM proteins probably play a role in controlling the activity of barley proteinases during germination, as well as possibly protecting the seed and young plant from microbes or pests.     

Effect of several germination conditions on total P, phytate P, phytase, and acid phosphatase activities and inositol phosphate esters in rye and barley.

Two assays were conducted to study the evolution of rye and barley phosphatases (phytase and acid phosphatase) and the degradation of its substrates (inositol phosphate esters) during seed germination. In this manner we could obtain a low-phytate, endogenous phosphatase rich ingredient to be used in animal nutrition. In the first assay, the seeds were soaked for 1 and 14 h and germinated for 3 and 5 days with and without the addition of gibberellic acid (GA3). In the second assay, the seeds were soaked for 1 h and germinated for 1, 3, and 5 days with GA3. Phytase (up to 5739 and 3151 U x kg(-1)) and acid phosphatase (up to 18288 and 3151 U x g(-1)) activities, and IP6 (6.09 and 6.01 mg x g(-1)), IP5 (0.48 and 0.48 mg x g(-1)), and IP4 (0.13 and 0.06 mg x g(-1)) were detected in ungerminated rye and barley, respectively. The germination process caused a significant increase of Phy and AcPh activities in rye (up to 112 and 213%) and barley (up to 212 and 634%) and a reduction in the phytate phosphorus content (up to 84 and 58%, respectively). Phytate phosphorus content was affected only by soaking time in the case of rye. Finally, during the course of germination, IP6 and IP5 were rapidly degraded in rye (88 and 79%) and barley (67 and 52%), and IP4 was only a short-living intermediate, which was increased during hydrolysis and degraded to IP3. In conclusion, a marked increase of Phy and AcPh activities in rye and barley with a concomitant decrease in phytate phosphorus content and an increase in the content of lower inositol phosphates were observed during the rye and barley germination.     

Degradation of starchy endosperm cell walls in nongerminating sterilized barley by fungi

Strains of fungi from different origins, including isolates of the natural microflora of barley, were screened for their ability to modify barley starchy endosperm cell walls in situ. In an initial step, fungi were selected that degrade the major component of the cell walls, that is, (1-->3),(1-->4)-beta-D-glucan, in vitro on artificial media. Nongerminating, sterilized barley, obtained by gamma-irradiation, was inoculated with such fungi and subjected to solid state fermentation under conditions resembling those of a traditional malting process. For some strains of fungi, a clear correlation between the production of endo-beta-glucanase and the friability of the treated kernels was found. Image analysis of Calcofluor stained longitudinal sections of barley kernels fermented with the endo-beta-glucanase producing strains showed that starchy endosperm cell walls were modified. As malt quality is inversely related to its (1-->3),(1-->4)-beta-D-glucan content, the selected strains have high potential to be used as starter cultures during malt production, contributing to the processing quality of the final product.     

Stability of barley and malt lipid transfer protein 1 (LTP1) toward heating and reducing agents: relationships with the brewing process

Barley lipid transfer protein (LTP1) is a heat-stable and protease-resistant albumin that concentrates in beer, where it participates in the formation and stability of beer foam. Whereas the barley LTP1 does not display any foaming properties, the corresponding beer protein is surface-active. Such an improvement is related to glycation by Maillard reactions on malting, acylation on mashing, and structural unfolding on brewing. The structural stability of purified barley and glycated malt LTP1 toward heating has been analyzed. Whatever the modification, lipid adduction or glycation, barley LTP1s are highly stable proteins that resisted temperatures up to 100 degrees C. Unfolding of LTP1 occurred only when heating was conducted in the presence of a reducing agent. In the presence of sodium sulfite, the lipid-adducted barley and malt LTP1 displayed higher heat stability than the nonadducted protein. Glycation had no or weak effect on heat-induced unfolding. Finally, it was shown that unfolding occurred on wort boiling before fermentation and that the reducing conditions are provided by malt extract.