Qualitative and quantitative changes in barley seed protein patterns during the malting process analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with respect to malting quality.

Seeds of two barley cultivars, similar in total protein content and malt extract yield but different in their final attenuation values, were malted. Samples taken at daily intervals during the malting process were extracted sequentially with Tris-HCl buffer, aqueous 2-propanol, aqueous 2-propanol containing 0.5% dithiothreitol, and 4 M urea, containing 0.5% dithiothreitol and 1% Nonidet P-40. The protein composition of these extracts was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and computer densitometry to determine whether differences observed in the rate or extent of protein modification are related to the malting quality character final attenuation. It was found that, common to both cultivars, the albumin and globulin proteins were relatively resistant to proteolysis, whereas the hordeins suffered a dramatic breakdown during malting, with the D hordein being degraded most rapidly, followed by the B and C hordeins. Besides these similarities, differences between both cultivars were observed in the relative rates of D hordein degradation, as this rate was considerably higher in the cultivar with high malting quality. Similar, but much less distinct kinetics were seen with certain B hordeins. Since a possible relationship might exist between the rate of proteolysis of the D hordeins and the character final attenuation, we analyzed a larger number of barley cultivars with different final attenuation values with a simplified technique. For the ten cultivars examined, differences during germination were again seen in the rates of modification of the D hordeins. However, significant correlations between the D hordein breakdown and final attenuation values were not obtained, so that we propose that there exists at best a loose correlation between the relative rate of proteolysis of these proteins and the malting quality character final attenuation.     

MALDI‐TOF mass spectrometry of hordeins: rapid approach for identification of malting barley varieties

A procedure for identification of malting barley varieties using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) of ethanol‐soluble barley proteins (hordeins) is described. The hordeins were first extracted from milled barley grains by several extraction protocols (using different extraction agents and conditions). Hordein extracts were then analyzed directly via MALDI‐TOF MS without any preliminary purification or separation step, and the protein profiles of analyzed hordein extracts were compared in order to find out the most suitable extraction procedure for mass spectrometric analysis. The optimized procedure was successfully applied to identification of 13 malting barley varieties. Our results revealed that the proposed mass spectrometry‐based approach provides characteristic mass patterns of extracted hordeins, which can be advantageously used for barley variety identification. Copyright © 2009 John Wiley & Sons, Ltd.     

Two-dimensional polyacrylamide gel electrophoresis, with immobilized pH gradients in the first dimension, of barley seed proteins: discrimination of cultivars with different malting grades.

The suitability of high-resolution two-dimensional gel electrophoresis for barley cultivar discrimination and for classification with respect to their malting properties was studied. Seed proteins of 14 barley cultivars with different malting qualities were extracted with urea/dithiothreitol/Nonidet P-40 buffer and subjected to two-dimensional gel electrophoresis with immobilized pH gradients in the first dimension (IPG-DALT). The results of IPG-DALT were compared to the protein patterns obtained by a standard technique, sodium dodecyl sulfate polyacrylamide gel electrophoresis of hordeins. Sodium dodecyl sulfate-gel electrophoresis yielded seven different "B" and four different "C" hordein patterns; "A" and "D" hordein patterns were uniform in all cultivars tested. Four cultivars could be distinguished unequivocally, the others were classified into three groups containing between two and five cultivars. In contrast to these findings. IPG-DALT yielded three different "A", eight different "B", four different "C" and two different "D" hordein patterns. When the "A", "B", "C" and "D" hordein patterns were combined, ten cultivars exhibited unique hordein patterns whereas the remaining ones were classified into two groups containing two cultivars each. Moreover, when albumin and globulin proteins were used for evaluation in addition to the hordeins, all cultivars could be discriminated by IPG-DALT. IPG-DALT, performed on small-scale and/or ready-made gels, proved to be an ideal complementary system to one-dimensional electrophoretic methods for routine seed testing purposes because of its speed, reliability, and simplicity. IPG-DALT was also applied to study the relationship between the different polypeptide patterns and the malting quality. Although cultivars with identical one-dimensional protein patterns but different malting quality could be successfully differentiated by IPG-DALT, a direct correlation between specific protein spots or protein patterns to the malting quality was not found within the cultivars tested.     

Proteomic identification of technologically modified proteins in malt by combination of protein fractionation using convective interaction media and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A method for the fast separation of proteins and identification of their modifications based on the use of monolithic chromatographic media and mass spectrometric techniques is described. This method has been developed and applied to the analysis of malt proteins and its posttranslational modifications (glycation). Glycation, one of the most common forms of posttranslational modifications (PTM), can be detected in both biological and industrial samples. Our attention was focused on the investigations of possible chemical modifications of water-soluble barley proteins during malting process by combination of anion-exchange chromatography with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The malt extract was directly fractioned by anion-exchange chromatography using short monolithic columns and a linear gradient from 0 to 700 mM NaCl. Sufficient fractionation was obtained for malt sample, which demonstrates the potential of anion-exchange chromatography on this type of column. Proteins in separated fractions were identified by MALDI-TOF/TOF MS. Our proteomic analysis provided the identification of the major proteins present in the malt that were found to be heterogeneously glycated after malting. One of these proteins: nonspecific lipid transfer protein 1 (LTP1) can be used as a marker for characterization of glycation during malting. This protein and its modifications can be easily determined by the developed method.     

Fluctuation in the ergosterol and deoxynivalenol content in barley and malt during malting process

This paper describes determination of the deoxynivalenol and ergosterol in samples from different varieties of barley and, consequently, malt produced from this barley. In total, 20 samples of barley and 20 samples of barley malt were analyzed. The alkaline hydrolysis with consequent extraction into hexane was applied to obtain the ergosterol from cereals. Extraction to acetonitrile/water and subsequent solid-phase extraction (SPE) were used for deoxynivalenol. The determination of the samples was performed on high-performance liquid chromatography using UV detection (ergosterol) and mass spectrometric detection (deoxynivalenol). The influence of the malting process on the production of two compounds of interest was assessed from obtained results. Ergosterol concentration ranged 0.88–15.87 mg/kg in barley and 2.63–34.96 mg/kg in malt, where its content increased to 95% compared to samples before malting. The malting process was observed as having a significant effect on ergosterol concentration (P = 0.07). The maximum concentration of deoxynivalenol was found to be 641 μg/kg in barley and 499 μg/kg in malt. Its concentration was lower than the legislative limit for unprocessed cereals (1,250 μg/kg). The statistic effect of the malting process on deoxynivalenol production was not found. Linear correlation between ergosterol and deoxynivalenol content was found to be very low (barley R = 0.02, malt R = 0.01). The results revealed that it is not possible to consider the ergosterol content as the indicator of deoxynivalenol contamination of naturally molded samples.     

Beer: An Ancient Yet Modern Biotechnology

The brewing of beer is a complex process that draws on a diversity of sciences and technology, of which chemistry is but one. This paper focuses on the chemistry of the brewing process and of the finished product. It examines each of the main classes of molecule found in beer, considers their contribution to quality and their origins in the brewing process. The study of beer and its production provides an excellent illustrative example for teaching how raw materials and the manner by which they are processed determine the acceptability of a product. Beer, whilst 90%+ water, contains a wide range of chemical species which establish its properties. Apart from ethanol (the common denominator amongst all alcoholic beverages), beer contains substances that determine its flavor, foam, and color. The flavorsome components of beer include the bitter iso-a-acids and aromatic essential oils from hops, along with esters, acids, sulfur-containing compounds and vicinal diketones from yeast. The foaminess of beer depends on the presence of carbon dioxide but also of surface-active materials like amphipathic polypeptides from malt and the bitter substances from hops. The color is due to Maillard reaction products generated largely during the kilning of malt. The malting and brewing processes (which are briefly described) are designed to maximize the extraction and digestion of barley starch and protein, yielding highly fermentable wort. The processes are also designed to eliminate materials that can have an adverse effect on beer quality, such as the haze-forming polyphenol from barley and hops and the lipids and oxygen that, together, can cause beer to stale.     

Construction of a comprehensive beer proteome map using sequential filter‐aided sample preparation coupled with liquid chromatography tandem mass spectrometry

The quality traits of beer, which include flavor, texture, foam stability, gushing, and haze formation, rely on contributions from beer proteins and peptides. Large‐scale proteomic analysis of beer is gaining importance, not only with respect to authenticity of raw material in beer but also to improve quality control during beer production. In this work, foam proteins were first isolated from beer by virtue of their high hydrophobicity. Then sequential filter‐aided sample preparation coupled with liquid chromatography and tandem mass spectrometry was used to analyze both beer protein and foam protein. Finally, 4692 proteins were identified as beer proteins, and 3906 proteins were identified as foam proteins. In total, 7113 proteins were identified in the beer sample. Several proteins contributing to beer quality traits, including lipid transfer protein, serpin, hordein, gliadin, and glutenin, were detected in our proteins list. This work constructed a comprehensive beer proteome map that may help to evaluate potential health risks related to beer consumption in celiac patients.     

Barley cultivar discrimination: I. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and glycoprotein blotting.

Two different methods of detecting electroblotted glycoproteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Tris-buffer soluble barley seed proteins were examined for their applicability for barley cultivar discrimination. These are the highly specific, lectin-based concanavalin A/peroxidase method and the more general periodate/danyslhydrazine method. The results of the periodate/dansylhydrazine method enabled us to divide the 20 examined cultivars into three groups, whereas the more sensitive concanavalin A/peroxidase method revealed six different glycoprotein patterns. In comparison, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of the alcohol-soluble barley seed proteins (hordeins) gave nine different banding patterns. A combination of hordein electrophoresis together with glycoprotein staining by the concanavalin A/peroxidase method made it possible to classify the cultivars into twelve groups, the largest of which contained four cultivars. The qualitative expression of the glycoprotein patterns seemed to be independent of growth conditions, whereas the band intensities obviously were not. As a whole, glycoprotein blotting is a valuable supplement to sodium dodecyl sulfate-polyacrylamide gel electrophoresis of hordeins in barley cultivar discrimination.     

Scotch Single Malt Whisky. Zur Chemie einer besonderen Spirituose

Malt whiskies bottled by one single distillery are called single malts. The ingredients for producing Scotch single malts are peat‐smoked barley malt and yeast. After fermentation, these yield an alcoholic liquid which is distilled in copper pot stills. Having been stored in oak casks for a legaly ordered time of at least three years, the new spirit may be sold as Scotch whisky. Its flavour is mainly due to chemical components forming during storage in a maturation process, but the peat used in malting and the pot stills also have some influence on its character.     

Phosphopeptide quantitation using amine-reactive isobaric tagging reagents and tandem mass spectrometry: application to proteins isolated by gel electrophoresis

Polyacrylamide gel electrophoresis is widely used for protein separation and it is frequently the final step in protein purification in biochemistry and proteomics. Using a commercially available amine-reactive isobaric tagging reagent (iTRAQ) and mass spectrometry we obtained reproducible, quantitative data from peptides derived by tryptic in-gel digestion of proteins and phosphoproteins. The protocol combines optimized reaction conditions, miniaturized peptide handling techniques and tandem mass spectrometry to quantify low- to sub-picomole amounts of (phospho)proteins that were isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Immobilized metal affinity chromatography (FeIII-IMAC) was efficient for removal of excess reagents and for enrichment of derivatized phosphopeptides prior to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis. Phosphopeptide abundance was determined by liquid chromatography/tandem mass (LC/MS/MS) using either MALDI time-of-flight/time-of-flight (TOF/TOF) MS/MS or electrospray ionization quadrupole time-of-flight (ESI-QTOF) MS/MS instruments. Chemically labeled isobaric phosphopeptides, differing only by the position of the phosphate group, were distinguished and characterized by LC/MS/MS based on their LC elution profile and distinct MS/MS spectra. We expect this quantitative mass spectrometry method to be suitable for systematic, comparative analysis of molecular variants of proteins isolated by gel electrophoresis.     

Characterization of a β-glucanase produced by Rhizopus microsporus var. microsporus, and its potential for application in the brewing industry

Background  

In the barley malting process, partial hydrolysis of β-glucans begins with seed germination. However, the endogenous 1,3-1,4-β-glucanases are heat inactivated, and the remaining high molecular weight β-glucans may cause severe problems such as increased brewer mash viscosity and turbidity. Increased viscosity impairs pumping and filtration, resulting in lower efficiency, reduced yields of extracts, and lower filtration rates, as well as the appearance of gelatinous precipitates in the finished beer. Therefore, the use of exogenous β-glucanases to reduce the β-glucans already present in the malt barley is highly desirable.     

Short monolithic columns for purification and fractionation of peptide samples for matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry analysis in proteomics

This study records a novel application of methacrylate-based monolithic columns for MALDI-TOF/TOF MS analyses in proteomics for pre-concentration and separation of peptides derived from protein digestion. Reversed-phase monolithic capillary columns (30 mm × 0.32 mm i.d.) were created inside the fused silica capillary via thermal-initiated free-radical polymerization of ethylene glycol dimethacrylate and lauryl methacrylate monomers in the presence of 1-propanol and 1,4-butandiol as a porogen system. The elution of peptides was achieved using a linear gradient of acetonitrile from 0 to 60% in water with 0.1% trifluoroacetic acid formed in a microsyringe. Individual fractions of separated peptides were collected on the MALDI target spots covered with alpha-cyano-4-hydroxycinnamic acid used as a matrix and then they were analyzed using MALDI-TOF/TOF mass spectrometry. The developed method was tested with a mixture of tryptic peptides from bovine serum albumin and its applicability was also tested for tryptic in-gel digests from barley grain extracts of water soluble proteins separated using SDS gel electrophoresis. The number of detected peptides was approximately three to four times higher compared to the analysis without previous separation. These results show an improved quality of sample information with the higher amount of identified peptides which increased protein sequence coverage and improved sensitivity of mass spectrometry measurements.     

Preparation and characterization of enzymatically hydrolyzed prolamins from wheat,rye, and barley as references for the immunochemical quantitation of partially hydrolyzed gluten

Celiac disease (CD) is a permanent gastrointestinal disorder characterized by the intolerance to a group of proteins called gluten present in wheat, rye, barley, and possibly oats. The only therapy is a strict lifelong gluten-free diet. The standard method for gluten determination in foods produced for CD patients is the R5-enzyme-linked immunosorbent assay (ELISA) as proposed by the recent Codex Alimentarius Draft Revised Standard. This test is based on the determination of prolamins, the alcohol-soluble proteins of gluten, and is available as a sandwich ELISA for intact proteins and as a competitive ELISA for gluten-derived peptides. While the suitability of the sandwich ELISA including a wheat prolamin (gliadin) reference for calibration has been shown by various studies and a ring test, the competitive ELISA still lacks a convenient reference for the quantitation of gluten peptides in fermented cereal foods (e.g., sourdough products, starch syrup, malt extracts, beer). Therefore, the aim of the present study was to prepare a suitable reference for the quantitation of partially hydrolyzed gluten in fermented wheat, rye, and barley products. The prolamin fractions from barley (hordein) and rye (secalin) were isolated from corresponding flours by means of a modified preparative Osborne fractionation. The prolamin fraction from wheat was obtained as reference gliadin from the Prolamin Working Group. The prolamin fractions were successively digested by pepsin and trypsin or pepsin and chymotrypsin procedures, which have been used for CD-specific toxicity tests on cereal storage proteins for many years. The protein/peptide content (N × 5.7) of the prolamin fractions and digests, which was the basis for the calculation of the gluten content by means of ELISA, varied between 67.1% and 96.0%. The prolamin fractions and enzymatic digests were then tested for their response in both sandwich and competitive assays. Intact prolamins responded similarly in both ELISA showing no important differences between the cereals. In the case of digested proteins, however, the sandwich ELISA was considerably less sensitive than the competitive ELISA. The former provided approximately 40% and the latter 70% of the signal intensity obtained with the intact prolamins. Thus, the combination of the competitive ELISA and the enzymatic digests of prolamin fractions as reference was considered to be an adequate system for the analysis of partially hydrolyzed gluten. The limit of detection using a peptic-tryptic hordein digest as reference was 2.3 μg prolamin equivalent per milliliter, and the limit of quantitation was 6.7 μg prolamin equivalent per milliliter. This system was applied for the determination of gluten equivalents in five commercial beverages based on fermented cereals.      

Low‐molecular‐weight color pI markers to monitor on‐line the peptide focusing process in OFFGEL fractionation

High‐throughput mass spectrometry‐based proteomic analysis requires peptide fractionation to simplify complex biological samples and increase proteome coverage. OFFGEL fractionation technology became a common method to separate peptides or proteins using isoelectric focusing in an immobilized pH gradient. However, the OFFGEL focusing process may be further optimized and controlled in terms of separation time and pI resolution. Here we evaluated OFFGEL technology to separate peptides from different samples in the presence of low‐molecular‐weight (LMW) color pI markers to visualize the focusing process. LMW color pI markers covering a large pH range were added to the peptide mixture before OFFGEL fractionation using a 24‐wells device encompassing the pH range 3–10. We also explored the impact of LMW color pI markers on peptide fractionation labeled previously for iTRAQ. Then, fractionated peptides were separated by RP_HPLC prior to MS analysis using MALDI‐TOF/TOF mass spectrometry in MS and MS/MS modes. Here we report the performance of the peptide focusing process in the presence of LMW color pI markers as on‐line trackers during the OFFGEL process and the possibility to use them as pI controls for peptide focusing. This method improves the workflow for peptide fractionation in a bottom‐up proteomic approach with or without iTRAQ labeling.     

不同富硒条件对麦芽硒含量的影响

探讨了不同浸麦度、Na2S3O3及制麦添加剂浓度对麦芽富硒的影响。结果表明,在浸麦度为42%时使用Na2SeO3处理最佳,麦芽硒含量比对照提高35倍;抗生素P和赤霉素浓度分别为100mg/L和0.05mg/L时表现出对麦芽富硒最大的促进作用,麦芽中硒含量分别比对照增加1.24和3.35倍。本实验的研究为工业化富硒麦芽的生产提供了科学的理论依据。     

Separation of hordein proteins from european barley by high-performance liquid chromatography: Its application to the identification of barley cultivars

Summary High-performance liquid chromatography using either reversed phase or anion-exchange techniques was used for the fractionation of hordein proteins extracted from European barley. A reversed phase method is presented which utilises an Ultrapore column packed with a wide pore (30 nm) C-3 alkylbonded silica support. Using this method up to 20 components may be separated in 54 min. Elution profiles were found to be reproducible. A further method using rapid anion-exchange chromatography indicated that up to 13 components may be separated, a number which is comparable to that found with electrophoresis. The separation of proteins extracted from different barley cultivars indicated that on the basis of elution profiles high-performance liquid chromatography using either reversed phase or anion-exchange offers considerable potential as a method for barley cultivar identification.     

Determination of cadmium,aluminium, and copper in beer and products used in its manufacture by electrothermal atomic absorption spectrometry

Procedures were developed for determining cadmium, aluminium, and copper in beer and the products used in its manufacture by electrothermal atomic absorption spectrometry. Beer samples were injected into the furnace and solid samples were introduced as suspensions after preparation in a medium containing hydrogen peroxide, nitric acid, and ammonium dihydrogen phosphate for cadmium atomization. Calibration was performed with aqueous standards, and characteristic masses and detection limits were, respectively, 1 and 0.3 pg for cadmium, 18 and 5.4 pg for aluminium, and 5.6 and 6.8 pg for copper. Different samples of beer, wort, brewer's yeast, malt, raw grain, and hops were analyzed by the proposed procedures. Cadmium was found in low concentrations (0.001-0.08 microg/g and 0-1.3 ng/mL); copper (3-13 microg/g and 25-137 ng/mL) and aluminium (0.6-9 microg/g and 0.1-2 microg/mL) were found at higher levels. The reliability of the procedure was confirmed by comparing the results obtained with others based on microwave oven sample digestion, and by analyzing several certified reference materials.     

Novel staining-free proteomic method for simultaneous identification of proteins and determination of their pI values by using low-molecular-mass pI markers

A new proteomic staining-free method for simultaneous identification of proteins and determination of their pI values by using low-molecular-mass pI markers is described. It is based on separation of proteins in gels by IEF in combination with mass spectrometric analysis of both peptides derived by in-gel digestion and low-molecular-mass pI markers extracted form the same piece excised from the gel. In this method, the pI markers are mixed with a protein mixture (a commercial malted barley protein extract) deposited on a gel and separated in a pH gradient. Color pI markers enable supervision of progress of focusing process. Several separated bands of the pI markers (including separated proteins) were excised and the pI markers were eluted from each gel piece by water/ethanol and identified by MALDI-TOF/TOF MS. The remaining carrier ampholytes were then washed out from gel pieces and proteins were in-gel digested with trypsin or chymotrypsin. Obtained peptides were measured by MALDI-TOF/TOF MS and proteins were identified via protein database search. This procedure allows omitting time-consuming protein staining and destaining procedures, which shortens the analysis time. For comparison, other IEF gels were stained with CBB R 250 and proteins in the gel bands were identified. Similarity of the results confirmed that our approach can give information about the correct pI values of particular proteins in complex samples at significantly shorter analysis times. This method can be very useful for identification of proteins and their post-translational modifications in prefractioned samples, where post-translational modifications (e.g., glycation) are frequent.     

Optimization of Modern Analytical SPME and SPDE Methods for Determination of <Emphasis Type="Italic">Trans</Emphasis>-2-nonenal in Barley,Malt and Beer

Trans-2-nonenal is an aldehyde contributing to an unpleasant off-flavor and odor of rancid butter in stored beer. The automated solid-phase microextraction technique (SPME) coupled with gas chromatography (GC) and solid-phase dynamic extraction (SPDE) coupled with gas chromatography were optimized and introduced to determine trans-2-nonenal in barley, malt and beer. Five types of SPME fibers coated with different stationary phases (100 μm PDMS, 65 μm PDMS/DVB, 85 μm CAR/PDMS, 50/30 μm DVB/CAR/PDMS, 85 μm PA) and two needles (PDMS, PDMS/AC) were compared and tested for their efficiencies in the headspace (HS) SPME and SPDE determination of trans-2-nonenal in barley, malt and beer. The highest extraction efficiency of HS-SPME was achieved with the PDMS/DVB fiber, and addition of 1.5 g of NaCl, extraction time was 20 min at 60 °C. The highest extraction efficiency of HS-SPDE was obtained with the PDMS needle, 15 extraction strokes at 60 °C and addition of 1.5 g of NaCl. Trans-2-nonenal was identified with the method of HS-SPME coupled gas chromatography-mass spectrometry (GC–MS); the samples were analyzed using the HS-SPME-GC-coupled gas chromatography-flame ionization detector (GC-FID) technique.