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Barley Proteomic Studies Related to Beer Production
Benkovská, Dagmar ; Márová, Ivana (oponent) ; Ehrenbergerová, Jaroslava (oponent) ; Zdráhal, Zbyněk (oponent) ; Bobáľová, Janette (vedoucí práce)
This work is focused on barley proteomic studies in relation to the beer production. Barley belongs between the most important crops in the world and its greatest use is for malting purposes, most commonly for the brewing industry. Studies of barley proteins during malting and brewing provide information about changes in protein composition or their post translational modifications. Since the protein composition and their modifications are essential for the quality of malt and beer, barley proteomic studies have a potential to improve the malting and brewing process. The main goal of this thesis is to investigate barley water-soluble proteins and their changes that occur during the malting and brewing process. The differences in protein composition were investigated using gel electrophoresis, reversed phase and size exclusion liquid chromatography, and MALDI-TOF mass spectrometry. The amount of some proteins is increasing and some new proteins are created in the germinated grain during the malting process. Contrary, many proteins are decomposed during the brewing process due to the high temperature and enzymatic activity of some proteases. Only some proteins belonging to the family of pathogenesis related proteins resist these harsh conditions and pass into the beer where they can influence several important quality properties. Furthermore, various barley varieties and their differences were investigated. Varieties allowed for the production of certified Czech beer were compared to one variety with well proven malting properties and one non-malting barley variety. In addition, alcohol soluble barley proteins and their changes during the malting process were investigated as well. A special attention was paid to selected post-translational modifications of proteins, namely glycosylations. Non-enzymatically glycosylated barley proteins (or glycated proteins) are formed during the malting process considering the large amount of glucose released from the starch degradation, and influence the protein stability as well as the beer quality, especially foaming properties. Enzymatic N-glycosylation represents the most frequently studied post translational modification in plants because glycoproteins play a key role in various biological functions. Since glycoproteins are often present in a small amount, their enrichment from a complex mixture is required for their analysis. Lectin concanavalin A affinity chromatography was used for barley glycoproteins investigation. Moreover, the analysis of the carbohydrate part of glycoproteins was optimized. This doctoral thesis brings important information about barley proteins, their modifications and analysis that are useful for further studies.
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Stanovení kyseliny propionové v pekařských výrobcích kapalinovou chromatografií
Benkovská, Dagmar ; Vítová, Eva (oponent) ; Kučera, Pavel (vedoucí práce)
Tato práce se zabývá vývojem metody stanovení kyseliny propionové v pekařských výrobcích pomocí vysokoúčinné kapalinové chromatografie (HPLC). Práce vycházela z potřeb a podmínek laboratoře Státní zemědělské a potravinářské inspekce v Brně. V teoretické části jsou popsány vlastnosti kyseliny propionové, její výroba, metabolismus, toxicita a konzervační účinek. Dále je pojednáno o kapalinové chromatografii, validaci analytické metody a způsobech stanovení kyseliny propionové. Praktická část je zaměřena na stanovení kyseliny propionové ve většině dostupných výrobcích na českém trhu, kde byl předpoklad použití této konzervační látky. Kyselina byla ze vzorku extrahována ve vodě, zfiltrována a filtrát přímo stanoven HPLC. Separace byla provedena na koloně XBridge C18 (3 x 150 mm, 3,5 µm) s použitím fosfátového pufru (pH upravené na 2,8) jako mobilní fáze a UV detekcí při 210 nm. Použitá metoda izolace kyseliny propionové z matrice byla porovnána s metodami opakované extrakce ve vodě, extrakce v roztoku mobilní fáze a destilace s vodní parou jak u komerčních vzorků, tak u laboratorně upečených chlebů se známým přídavkem kyseliny propionové. Technika HPLC s využitím extrakce ve vodě byla vyhodnocena jako vhodná metoda stanovení kyseliny propionové v pekařských výrobcích.
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Barley Proteomic Studies Related to Beer Production
Benkovská, Dagmar ; Márová, Ivana (oponent) ; Ehrenbergerová, Jaroslava (oponent) ; Zdráhal, Zbyněk (oponent) ; Bobáľová, Janette (vedoucí práce)
This work is focused on barley proteomic studies in relation to the beer production. Barley belongs between the most important crops in the world and its greatest use is for malting purposes, most commonly for the brewing industry. Studies of barley proteins during malting and brewing provide information about changes in protein composition or their post translational modifications. Since the protein composition and their modifications are essential for the quality of malt and beer, barley proteomic studies have a potential to improve the malting and brewing process. The main goal of this thesis is to investigate barley water-soluble proteins and their changes that occur during the malting and brewing process. The differences in protein composition were investigated using gel electrophoresis, reversed phase and size exclusion liquid chromatography, and MALDI-TOF mass spectrometry. The amount of some proteins is increasing and some new proteins are created in the germinated grain during the malting process. Contrary, many proteins are decomposed during the brewing process due to the high temperature and enzymatic activity of some proteases. Only some proteins belonging to the family of pathogenesis related proteins resist these harsh conditions and pass into the beer where they can influence several important quality properties. Furthermore, various barley varieties and their differences were investigated. Varieties allowed for the production of certified Czech beer were compared to one variety with well proven malting properties and one non-malting barley variety. In addition, alcohol soluble barley proteins and their changes during the malting process were investigated as well. A special attention was paid to selected post-translational modifications of proteins, namely glycosylations. Non-enzymatically glycosylated barley proteins (or glycated proteins) are formed during the malting process considering the large amount of glucose released from the starch degradation, and influence the protein stability as well as the beer quality, especially foaming properties. Enzymatic N-glycosylation represents the most frequently studied post translational modification in plants because glycoproteins play a key role in various biological functions. Since glycoproteins are often present in a small amount, their enrichment from a complex mixture is required for their analysis. Lectin concanavalin A affinity chromatography was used for barley glycoproteins investigation. Moreover, the analysis of the carbohydrate part of glycoproteins was optimized. This doctoral thesis brings important information about barley proteins, their modifications and analysis that are useful for further studies.
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Stanovení kyseliny propionové v pekařských výrobcích kapalinovou chromatografií
Benkovská, Dagmar ; Vítová, Eva (oponent) ; Kučera, Pavel (vedoucí práce)
Tato práce se zabývá vývojem metody stanovení kyseliny propionové v pekařských výrobcích pomocí vysokoúčinné kapalinové chromatografie (HPLC). Práce vycházela z potřeb a podmínek laboratoře Státní zemědělské a potravinářské inspekce v Brně. V teoretické části jsou popsány vlastnosti kyseliny propionové, její výroba, metabolismus, toxicita a konzervační účinek. Dále je pojednáno o kapalinové chromatografii, validaci analytické metody a způsobech stanovení kyseliny propionové. Praktická část je zaměřena na stanovení kyseliny propionové ve většině dostupných výrobcích na českém trhu, kde byl předpoklad použití této konzervační látky. Kyselina byla ze vzorku extrahována ve vodě, zfiltrována a filtrát přímo stanoven HPLC. Separace byla provedena na koloně XBridge C18 (3 x 150 mm, 3,5 µm) s použitím fosfátového pufru (pH upravené na 2,8) jako mobilní fáze a UV detekcí při 210 nm. Použitá metoda izolace kyseliny propionové z matrice byla porovnána s metodami opakované extrakce ve vodě, extrakce v roztoku mobilní fáze a destilace s vodní parou jak u komerčních vzorků, tak u laboratorně upečených chlebů se známým přídavkem kyseliny propionové. Technika HPLC s využitím extrakce ve vodě byla vyhodnocena jako vhodná metoda stanovení kyseliny propionové v pekařských výrobcích.
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Optimization of N-glycopeptides analysis methods and their preliminary application to barley proteins study
Benkovská, Dagmar ; Flodrová, Dana ; Bobálová, Janette ; Laštovičková, Markéta
N-glycosylation is the most frequently studied plant protein post-translational modification. The analysis of Nglycopeptides after protein proteolytic digestion offers information about the structure of both oligosaccharide and peptide moiety. However, this method has so far been less commonly used. Since glycopeptides hardly ionize during MS analysis in the presence of non-glycosylated peptides, they need to be separated from the complex peptide mixture. In this study, the glycopeptides enrichment, purification and analysis methods were successfully optimized on two standard N-glycoproteins. Concanavalin A (ConA) lectin tips were used for glycopeptide capturing, and obtained fractions were purified on carbon tips and analyzed using MALDI-TOF mass spectrometry. The differences in the CID fragmentation of certain types of glycopeptides were found. This technique was then applied to glycopeptide analysis of barley grain and malt proteins. Several barley glycopeptides were found, however, their identification was very difficult. More proteins separation techniques will be required before this enrichment procedure in further studies.
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