Studiengang Lebensmitteltechnologie und Bioprodukttechnologie WS 2015/2016
Masterthesis
Einfluss der Temperatur auf Proteinmodifikationen von Gerste
(Hordeum vulgare) beim Darren.
Influence of temperature on proteinmodifications of barley
(Hordeum vulgare) during kilning.
Author: Elena Sosna
First supervisor: Prof. Dr. Leif-Alexander Garbe Second supervisor: Prof. Dr.-Ing. Heralt Schöne
URN: urn:nbn:de:gbv:519-thesis2015-0648-4
Submission Date: 20.01.2016
Abstract
Barley proteins are crucial for the quality of malt and beer. However, these proteins are degraded and modified by high temperature treatments as during kilning. It is expected that higher kilning temperatures even enhance these processes. Therefore, degradation and modification of different kilned malts were studied by measuring protein concentrations colorimetrically and applying SDS-PAGE after cold extraction and congress mashing. Already after five hours at 120 °C most proteins were degraded. Subsequent congress mashing revealed that only one protein with a molecular mass of around 16 kDa survived these harsh conditions.
Zusammenfassung
Gerstenproteine sind entscheidend für die Qualität von Malz und Bier. Prozesse mit hohen Temperaturen z.B. Darren des Malzes führen jedoch dazu, dass Proteine modifiziert oder abgebaut werden. Um den Temperatureinfluss zu bestimmen, wurden verschieden gedarrte Malzproben angefertigt, deren Proteine mittels Kaltextraktion und Kongressmaischen isoliert wurden. Der Proteingehalt wurde kolorimetrisch ermittelt, das Proteinprofil mittels SDS-PAGE visualisiert. Bereits nach fünf Stunden darren bei 120 °C konnte ein Abbau der meisten Proteine im Kaltextrakt beobachtet werden. Das Kongressmaischen hat dazu geführt, dass lediglich ein Protein der Masse 16 kDa nachgewiesen werden konnte.
Introduction
Beer proteins originating from barley (Hordeum vulgare L.) influence the entire brewing process. Appearing as enzymes, they are responsible for the degradation of starch, β-glucans and proteins. Protein-protein linkages ensure foam and flavor stability, mouthfeel and combined with polyphenols they also lead to haze formation. As amino acids and peptides, they serve as nitrogen source for the brewer´s yeast (Steiner et al., 2011a; Steiner & Back, 2009). The most important proteins originating from barley that survive the brewing process are protein Z, non-specific lipid transfer protein (ns-LTPs) LTP1 and LTP2, hordein fractions and trypsin/α-amylase inhibitors (Benkovska et al., 2011).
During the whole beer production protein profiles undergo major changes due to degradation, coagulation and modification including glycation, acylation and partial digestion (Iimure & Sato, 2013; Iimure et al, 2015). Enzymatic modifications i.e. glycosylation occur during mashing and malting for instance by proteolytic enzymes. During kilning, malting and wort boiling several complex Maillard reaction products are generated, due to non-enzymatic glycation (Curioni et al., 1995; Lapolla et al., 1993; Leiper et al., 2003a ; Leiper et al., 2003b). Glycated proteins are more stable, resist proteolysis, survive the malting process and positively impact beer properties (Lastovickova et al., 2010; Bobalova et al., 2008). All these structural changes as well as post-translational modification can regulate beer´s flavor, texture and appearance. Gaining knowledge about these changes will help brewers and maltsters to produce better products with desired properties.
Besides brewing, malting is one of the determining processes concerning protein modifications. Malting consists of steeping, germination and kiln-drying (Wunderlich & Back, 2008; Wittmann & Eichner, 1989). During kilning, Maillard reaction products especially melanoidines are generated, due to chemical reactions. Malts brewed at higher kilning temperatures are known to bestow color, flavor and antioxidative activity of wort and beer. Increased levels of antioxidants distribute to flavor stability and an elongated shelf life. Furthermore, the rising melanoidine levels, due to excessive Maillard reaction, contribute to foam stability and mouthfeel. However, worts of darker malts contain fewer amounts of reducible sugars and amino acids, that can provide to yeast nutrition, leading to a lower fermentation rate (Coghe et al., 2005). Thus, the kilning temperature
directly affect the final composition of beer, and therefore highly contribute to properties and quality of the end-product.
In the first, theoretical part of this thesis, the most important proteins concerning beer quality that undergo modifications during beer production are described. Specific protein modifications during germination, kiln-drying, mashing, wort boiling and fermentation are elucidated for each single step of the beer production. Since quality and quantity of barley proteins influence beer quality, proteins are extensively studied by the use of proteomics, that allows monitoring changes in protein composition during the whole beer production (Coghe et al., 2005; Coghe et al., 2006). The use of proteomics is an established method for monitoring intact and modified barley and beer proteins. The state-of-the-art of proteomics in monitoring beer and barley protein profiles is presented in this part.
As kilning has a high impact on barley proteins, in the second, experimental part, studies on the influence of the kilning temperature on protein profiles were investigated. Therefore, samples kilned at different temperatures were prepared and the simple and fast one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to monitor changes in pattern. Moreover, the protein concentration was measured in dry matter by using Kjeldahl. The water-soluble protein concentration of cold extracted and congress mash extracted different kilned barley samples was calculated. For this purpose, Lowry and Bradford assay were applied. Furthermore, the color of the samples was measured by using the Commission internationale de l'éclairage (CIE) L*a*b* parameters. Colored Maillard reaction products are generated during kilning, due to the reaction of sugars with free amino residues of proteins. Therefore, the color may provide information about the degree of altered proteins.
Table of content
Theoretical Part: Monitoring protein modifications - Part I. Recent proteomic approaches from barley to beer. Abstract ... 7 1. Introduction ... 8 2. Proteomics ... 9 2.1. Gel-based Proteomics ... 10 2.2. Gel-free Proteomics ... 11 3. Barley Proteins ... 12
3.1. Non-specific lipid transfer proteins (ns-LTP) ... 14
3.2. Protease/α-amylase inhibitors ... 14
3.3. Other pathogen-related proteins ... 15
3.4. Hordeins ... 16
4. Protein modifications during beer production ... 17
4.1. Protein modifications during malting ... 22
4.1.1. Germination ... 23
4.1.2. Kiln-Drying ... 24
4.2. Protein modifications during brewing ... 25
4.2.1. Mashing ... 25
4.2.2. Wort boiling ... 26
4.2.3. Fermentation ... 27
5. Conclusion ... 29
Experimental Part: Monitoring protein modifications - Part II. Impact of kiln temperature on barley (H. vulgare) protein profile. Abstract ... 30
1. Introduction ... 31
2. Experimental ... 32
2.1. Raw materials ... 33
2.2. Kiln-drying and Milling ... 33
2.3. Measurement of color by tristimulus values... 33
2.5. Kjeldahl method ... 33
2.6. Colorimetric assays ... 34
2.6.1. Sample preparation ... 34
2.6.2. Lowry assay ... 34
2.6.3. Bradford assay ... 35
2.7. Sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) ... 35
3. Results and Discussion ... 36
3.1. Color measurement ... 36
3.2. Protein concentrations ... 37
3.3. SDS-PAGE ... 39
4. Conclusion ... 40
I. References ... 41
II. Summarized discussion ... 54 III. Enclosures
Declaration of Originality (Eigenständigkeitserklärung) List of figures
List of tables List of abbreviations
Theoretical Part: Monitoring protein modifications - Part I. Recent proteomic
approaches from barley to beer.
Authors: Prof. Dr. Leif-Alexander Garbe, Hochschule Neubrandenburg, University of Applied Sciences, Fachbereich Agrarwirtschaft und Lebensmittelwissenschaften, Brodaer Straße 2, 17033 Neubrandenburg, garbe@hs-nb.de
Prof. Dr.-Ing. Heralt Schöne, Hochschule Neubrandenburg, University of Applied Sciences, Fachbereich Agrarwirtschaft und Lebensmittelwissenschaften, Brodaer Straße 2, 17033 Neubrandenburg, schoene@hs-nb.de
Elena Sosna*, Hochschule Neubrandenburg, University of Applied Sciences, Fachbereich Agrarwirtschaft und Lebensmittelwissenschaften, Brodaer Straße 2, 17033 Neubrandenburg, al14009@hs-nb.de
* corresponding author
Descriptors
Proteomics, Barley (Hordeum vulgare), Malt, Wort, Beer Abstract
Proteins that originate from barley highly contribute to beer properties and quality. In almost all steps of the brewing process, these proteins are affected. They are either increasing or decreasing, disappearing or arising anew or they are somehow modified. All these changes have impact on the final product, namely beer. In order to monitor protein profiles and their changes, proteomic techniques are inevitable. Recent studies using proteomics have discovered a multitude of proteins and modified proteins. In this review, the findings of the latest studies are presented with focus on each single step from barley to beer. Protein profiles regarded separately will help to understand what changes occur at which step of the beer production. Thus, it may help maltsters and brewers optimizing processes.
1. Introduction
Barley (Hordeum vulgare L.) is after wheat, rice and corn the fourth most important cereal crop worldwide (Steiner et al., 2011a; Yalcin et al., 2008; Jadhav et al., 1992; Mohamed et al., 2007). It possesses economical relevance in malting and brewing industry (Gorjanovic, 2009). Beer proteins originating from barley seed influence the entire brewing process. Appearing as enzymes, they are responsible for the degradation of starch, β-glucans and proteins. Protein-protein linkages ensure foam and flavor stability, mouthfeel and combined with polyphenols they also lead to haze formation. As amino acids and peptides, they serve as nitrogen source for the brewer´s yeast (Steiner et al., 2011a; Steiner & Back, 2009). The most crucial proteins originating from barley that survive the brewing process are protein Z, non-specific lipid transfer protein (ns-LTPs) LTP1 and LTP2, hordein fractions and trypsin/α-amylase inhibitors (Benkovska et al., 2011).
During malting and brewing barley proteins undergo several modifications. The malting process leads to an activation of amylases and more than 40 different proteases and thus, to a partially
degradation of barley storage proteins into smaller peptides and amino
acids (Steiner et al., 2011a; Picariello et al., 2012). Subsequent mashing process solubilizes the proteins and release them into the wort. During wort boiling, proteins are glycated and coagulated. The flocculated proteins are separated from the wort before fermentation. Due to low pH, the proteins aggregate during fermentation, which allows a separation again. During the whole beer production protein profiles undergo great changes due to degradation, coagulation and modification including glycation, acylation and partial digestion (Iimure & Sato, 2013; Iimure et al, 2015). Enzymatic modifications i.e. glycosylation occur during mashing and malting for instance by proteolytic enzymes. During kilning, malting and wort boiling several complex Maillard reaction products are generated, due to non-enzymatic glycation (Curioni et al., 1995; Lapolla et al., 1993; Leiper et al., 2003a ; Leiper et al., 2003b). Glycated proteins are more stable, resist
proteolysis, survive the malting process and positively impact beer
properties (Lastovickova et al., 2010; Bobalova et al., 2008). Around 3 % to 7 % of glycated proteins contribute to haze formation and 25 % to foam stability (Lastovickova & Bobalova, 2012). As shown in Fig. 1.1, it is clearly visible, that malting and brewing considerably changes the amount and pattern of proteins.
Fig. 1.1: The protein profile changes during the whole brewing process, observed by two-dimensional gel electrophoresis from barley grain to beer [9].
These structural changes, as well as post-translational modification can regulate beer´s flavor, texture and appearance. Gaining knowledge about these changes will help brewers and maltsters to produce better products with desired properties. Since quality and quantity of barley proteins influence beer quality, proteins are extensively studied by the use of proteomics, that allows monitoring changes in protein composition during the whole beer production (Colgrave et al., 2013; Shewry, 1993).
In this review, recent approaches in proteomics in fields of malting and brewing are presented, the most important proteins concerning beer quality are described and specific protein modifications during germination, kiln-drying, mashing, wort boiling and fermentation are elucidated.
2. Proteomics
The genome of barley is about five giga bases large and consists of a high content of repetitive DNA, hence, it is not fully sequenced yet (Colmsee et al., 2015). Recently, Colmsee et al. (2015) developed a web-based application to freely access the developing genomic infrastructure of barley by comparing the physical map of Ariyadasa et al. (2014), the whole genome shotgun assembly from the International Barley Genome Sequencing Consortium (IBGSC, 2012) and the ultra-dense genetic map created by Mascher et al. (2013). These data set of barley genomic sequence will certainly essentially influence the identification of protein species from barley grain, malt, wort and beer. The proteome is even more complicated than the genome, as the proteome is continuously changing through interactions between the genome and the environment (Lastovickova & Bobalova, 2012). Therefore, one single organism exhibits different protein profiles in different organs, at different life cycle stages and under different environmental conditions (Gupta & Lee, 2007).
Proteomics is the study of the proteome - proteins as a whole expressed by the genetic material of an organism, cell or tissue at a specific moment of time under defined circumstances (Wilkins, 2009). Proteomics is divided into three subdivisions. The first division is expression proteomics, that focuses on the analysis of protein expression in different cell types of an organism. The second division deals with structural proteomics. The main objectives are the analysis of protein distribution in sub-cellular compartments of organelles, post-translational modifications of proteins and protein-protein interactions. The last subdivision is functional proteomics. It is used to monitor the relationship between structure and function of proteins (Lastovickova & Bobalova, 2012; Graves & Haysted, 2002).
The proteomic detection of proteins basically consists of a separation and an identification step. Gel-based and gel-free (chromatographic) methods are possible for separating proteins, while also reducing their complexity. Subsequently, the proteins are analyzed intact (top-down) or they are enzymatically digested into peptides (bottom-up) (Lastovickova & Bobalova, 2012). Reliable results for identifying proteins were reached with mass spectrometers, in single and tandem use (MS/MS). Most frequently matrix-assisted laser desorption ionization (MALDI) as ionization source is coupled to time-of-flight (TOF) mass analyzer (Karas & Hillenkamp, 1988; Canas et al., 2006). Alternatively, liquid chromatography (LC) is linked to a mass analyzer by electrospray ionization (ESI). The mass analyzer may also be TOF, a quadrupole or a hybrid of both (Canas et al., 2006 ; Fenn et al., 1989; Wilm, 2011).
2.1. Gel-based Proteomics
Before identifying proteins, a separation of the proteins can be performed by gel-based techniques. Either one- dimensional (1D-) or two-dimensional (2D-) polyacrylamide gel electrophoresis (PAGE) can be applied. For 1D-PAGE sodium dodecyl sulphate (SDS) together with a reducing reagent are used to denature and linearize proteins and overlap their net charge, allowing a separation by molecular weight, solely. Afterwards, the protein spots are automatically detected and cut out of the gel for subsequent MS (Colgrave et al., 2013). After in-gel tryptic digestion of 40 kDa and 7 kDa - 17 kDa bands, 1D-SDS-PAGE followed by tandem mass spectrometry (MS) sequencing revealed 21 proteins in beer and 24 proteins in beer foam. Most of them belong to barley albumins, whereas two are hordein-related (Hao et al., 2006).
2D-PAGE separates proteins according to their isoelectric point (pI) in the first dimension and molecular weight in the second dimension, by coupling isoelectric focusing (IEF) to SDS-PAGE (Colgrave et al., 2013). Typically for IEF, a pH gradient is established to a polyacrylamide gel via immobilines (IPG strips). Applying electric potential to the gel leads to a protein migration in the electrical field according to their specific pI, where their net charge equals zero. When the proteins reach their pI in the gel, they are focused. Following the IEF-PAGE, the SDS-PAGE is performed orthogonally, resulting in a 2D map of protein spots, depending on their specific pI and molecular weight (Colgrave et al., 2013). Bak-Jensen et al. (2004) used 2D-PAGE with pH 6-11 IPG strips to analyze proteins in flour from barley grains and malt. The identification was performed with MALDI-TOF/MS or nano-ESI-MS/MS and unveiled 380 spots in seeds and 500 spots in malt of which most proteins were albumins. Iimure et al. (2012) constructed a wort proteome map that consisted of protein identification on two-dimensional gel electrophoresis images and identified 63 out of 202 protein spots using MALDI-TOF MS, which were categorized into 20 protein species. Iimure et al. (2010) also constructed a beer proteome map by 2D-PAGE with pI range of 4 - 7 and 6 - 9 with peptide mass finger printing using MALDI-TOF/MS hereinafter. They identified 85 spots out of 199 spots and categorized them into 12 protein species including eight species of barley proteins namely protein Z-type serpin (protein Z4), serpin-Z7 (protein Z7), BDAI-1, CMb, LTP1, TAI, BTI-CMe and subtilisin-chymotrypsin inhibitor CI-1B. Perrocheau et al. (2005) used 2D-PAGE with immobilized pH 3 - 10 gradients in the first dimension, followed by silver, colloidal Coomassie Brilliant Blue staining or Western blotting and subsequent LC-MS/MS analysis to identify 30 proteins in beer. Konecna et al. (2012) detected more than 300 spots in beer using 2D-PAGE of which they identified 52 proteins including 25 barley proteins and 20 of yeast origin by using MALDI-MS/MS and LC-MS/MS. Upon this, preparative isoelectric focusing by OFFGEL Fractionator was applied prior to 2D-PAGE to improve the resolution power. This combined approach resulted in a total of 70 beer proteins: 30 proteins from Hordeum vulgare, 31 proteins from Saccharomyces species and nine proteins from other sources. Among these, 37 proteins have not been previously reported in beer samples (Konecna et al., 2012).
2.2. Gel-free Proteomics
Gel-free proteomics are performed by an enzymatic in-solution digestion of complex protein mixtures producing smaller peptides followed by multiple LC separation and MS/MS
characterization (Lastovickova & Bobalova, 2012; Colgrave et al., 2013; Canas et al., 2006). Petry-Podgorska et al. (2010) examined intact proteins and chymotryptic digests by using 2D-HPLC and MALDI-TOF MS to monitor protein glycation during brewing. Their results revealed high amounts of glycated proteins in malt, most likely due to the malting conditions, including high temperatures and availability of glucose and maltose. Colgrave et al. (2012) used tryptic and chymotryptic digestion followed by nano LC-MS/MS to identify 27 proteins in wort and 79 proteins in beer. Amongst these, they also found avenin-like protein-a, in addition to B-hordein, D-hordein and γ-hordein. Gel-free shotgun proteomic analysis revealed 33 gene products in beer, including intact B-hordeins and D-hordeins, as well as 10 proteins from S. spp. (Picariello et al., 2012). In order to detect minor proteins, a pre-fractionation can be applied (Iimure & Sato, 2013). A treatment with combinatorial peptide ligand libraries (CPLL) leads to a saturation of abundant beer proteins e.g. protein Z and LTP1 and a concentration of minor proteins (Iimure & Sato, 2013). Using MS of the recovered fractions after CPLL, Fasoli et al. (2010) categorized such species in 20 different barley protein families, two maize proteins and 40 unique gene products from Saccharomyces cerevisiae, one from S. bayanus and one from S. pastorianus. Picariello et al. (2011) demonstrated that only a few proteins survive the brewing conditions including serpin-Z4 and LTP1. Moreover, they found an avenin-like protein-a in beer foam after silica gel enrichment, SDS-PAGE and nano-ESI MS/MS. 3. Barley Proteins
Nitrogenous compounds including amino acids, peptides, polypeptides, nucleic acids, proteins, and their degradation products affect flavor, foam stability, haze formation, body, texture, color, yeast nutrition and biological stability (Leiper et al., 2003b; Osman et al., 2003; Fontana & Buiatti, 2008). In beer, they mainly originate from barley malt and its adjuncts Fontana & Buiatti, 2008). Metabolic Proteins, that arise during the malting process are important factors in converting barley substances into fermentable substances during malting and brewing (Jin et al., 2014). Depending on the variety, barley consists of 8 % - 13.5 % proteins (Yalcin et al., 2008; Wunderlich & Back, 2008). According to Osborne´s fraction, the storage proteins of barley can be classified by their solubility in different media: albumins are soluble in water, globulins in salt, prolamins in alcoholic solutions and glutelins dilute in alkaline solutions (Fontana & Buiatti, 2008; Dai et al., 2014; Shewry, 1978). The main storage proteins are prolamins (hordeins) with 35 % - 55 % followed by glutelin with 35 % - 40 %. In malt, more albumins and globulines were found than in the barley
grain, while hordeins decreased and glutelins remained the same after malting (Celus et al., 2006; Silva et al., 2008). Water-soluble proteins seem to be prone to proteolysis and heat coagulation. Thus, they survive the whole brewing process nearly intact or somehow modified (Osman et al., 2003). Protein Z, α-amylase and lipid transfer proteins (LTPs) belong to the albumins (Steiner et al., 2011a). They are important concerning beer quality, haze formation and foam stability (Fox & Henry, 1995). In order to form enough haze, an amount of 2 mg L-1 is sufficient (Kaersgaard & Hejgaard, 1979). Foam-positive proteins belong to albumins and hordeins. Among these, foams from albumins are the more stable ones (Kapp & Bamforth, 2002).
Pathogenesis-related proteins (PRs), classified in 17 families, are one of the most important group of proteins for malting and brewing (Gorjanovic, 2010). In contrast to most barley proteins, PRs are resistant to proteolysis and thermal stress. Thus, they are able to resist the harsh conditions during brewing influencing beer quality such as foam and haze (Lastovickova & Bobalova, 2012; Gorjanovic, 2010). Proteomic analysis of barley seed revealed that the barley proteome is dominated by proteins involved in protection against microorganisms, insects, or other stressors (Lastovickova & Bobalova, 2012; Bak-Jensen et al., 2004; Ostergaard et al., 2004). PRs present in the barley genome are glucanase, chitinase, barley wound induced protein (barwin), thaumatin-like protein (TLP), proteinase inhibitors, peroxidase, non-specific lipid-transfer proteins 1 and 2 (ns-LTP1 and ns-LTP2) and HvPR-17. Proteins, such as α-amylase/trypsin inhibitors or CM proteins (CMa, CMb, CMc, CMd and CMe), barley monomeric amylase inhibitor (BMAI), barley dimeric amylase inhibitor (BDAI), the putative trypsin/amylase inhibitor (pUP13), as well as barley α-amylase/subtilisin inhibitor (BASI), subtilisin-chymotrypsin inhibitors (CI-1A, CI-1B) and many spots of barley seed serpins, known as protein Z (Z4, Z7 and Zx) were identified (Bak-Jensen et al., 2004; Ostergaard et al., 2004). Thionins and defensins are also known to be existent in barley seed, however they could not be detected by SDS-PAGE due to their low molecular weight (MW) of about 5 kDa (Shewry, 1993). The most important proteins concerning beer quality are subsequently described in more detail.
3.1. Non-specific lipid transfer proteins (ns-LTP)
Lipid transfer proteins (LTPs) are lipid-binding proteins. These ubiquitous plant proteins catalyze the lipid-transfer between membranes. The non-specific (ns) LTPs are divided into two subfamilies: ns-LTP1 and ns-LTP2 with a molecular mass of 9.7 kDa and 7.0 kDa, respectively (Picariello et al., 2012; Curioni et al., 1995; Gorjanovic, 2007). It is known, that ns-LTPs have remarkable importance on beer quality, foam formation and stabilization, beer gushing and fermentation (van Nierop et al., 2004; Evans & Sheehan, 2002; Jegou et al., 2001; Hippeli & Elstner, 2002; Gorjanovic et al., 2004; van Nierop et al., 2006). Both LTPs have compact structures, that implies heat stability and protease resistance (Gorjanovic, 2009). The temperature of melting of ns-LTP1 is about 100 °C, while ns-LTP2 requires even higher temperatures for denaturation (Gorjanovic, 2009; van Nierop et al., 2004; Perrocheau et al., 2006; Lindorff-Larsen & Winther, 2001). Contradictory, in the final product beer ns-LTP2 was detected less often and to a lesser extent than ns-LTP1 (Picariello et al., 2012; Perrocheau et al., 2005). 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 (Davy et al., 1999). LTP1 owns four lysine residues that can be glycated (Jegou et al., 2001; Lin et al., 2004; Jegou et al., 2000). Jegou et al. (2001) expected that glycation might prevent protein from precipitation on unfolding during the wort boiling step, but proved that glycated LTP1 was unfolded during heat treatment of wort boiling. It is known, that LTP1 also possesses foam forming properties (Hao et al., 2006). More precisely, LTP1 in barley does not possess foaming properties, it is the heat-denatured surface-active modified LTP1 protein in beer that contributes to foam forming (Sorensen et al., 1993). This modification is due to glycation by Maillard reaction during malting, acylation while mashing and structural unfolding at the brewing process (Perrocheau et al., 2006).
3.2. Protease/α-amylase inhibitors
Protease inhibitors are the largest group of proteins, that were identified in all stages of the brewing process. Members of this group are bifunctional barley α-amylase/subtilisin inhibitor (BASI), chymotrypsin/subtilisin inhibitors (CI-1 A, B, C and CI-2A) and trypsin/α-amylase inhibitors (CMa - CMe and BDAI) (Gorjanovic, 2009; Gorjanovic, 2010). Chloroform/methanol (CM) soluble proteins belong to the α-amylase/trypsin inhibitor family with inhibitory activity probably against
enzymes from pathogens and insects (Franco et al., 2002). Silica gel eluate (SE) protein-specific antibodies demonstrated, that a band at 12 kDa SDS-PAGE immunoblot analysis has impact on haze. From cultivars that owned the band, more haze was observed in the produced beer. The 12 kDa band was identified as barley trypsin inhibitor-CMe precursor (BTI-CMe) by combining trypsin treatment, RP-HPLC and N-terminal sequence analysis (Robinson et al., 2004; Robinson et al., 2007a; Robinson et al., 2007b). BTI-CMe was also detected by Jin et al. using 2-DE and MS and by Iimure et al. by PMF using MALDI-TOF MS (Jin et al., 2009a; Iimure et al., 2009). The latter also identified a component of tetrameric α-amylase inhibitor (CMb). CMb and BTI-CMe, both are possible growth factor for haze-active proteins (Robinson et al., 2004; Robinson et al., 2007a; Robinson et al., 2007b; Iimure et al., 2009). As endogenous barley and malt endoprotease inhibitors, CMs are able to affect protein hydrolysis during malting and mashing (Jones & Marinac, 2002; Jones, 2005a; Jones, 2005b). Barley dimeric alpha-amylase inhibitor 1 (BDAI-1) is claimed to be a haze-active and foam-positive protein (Iimure & Sato, 2013; Iimure et al., 2009; Okada et al., 2008; Iimure et al., 2008). Its impact on foam stability was recently demonstrated by Iimure et al. (2015).
Serpins are serine protease inhibitors. Protein Z is the most common protein of this group appearing in malt and beer. Protein Z is known as a major protein contributing to foam quality and beer nutritional value (Hao et al., 2006; Evans & Sheehan, 2002; Iimure et al., 2008). In common barley varieties, it accounts for 1.5 mg - 2.5 mg protein Z per gram seed (Gorjanovic, 2009). Protein Z has at least four antigenically identical molecular forms, ranging in isoelectric point between pI 5.5 and pI 5.8 in barley and pI 5.1 and pI 5.4 in beer. Although, they all possess an equal molecular mass of about 40 kDa (Hejgaard, 1982). Two isoforms of protein Z are were found with molecular weights around 40 kDa in beer, namely serpin-Z4 and serpin-Z7, composing 80 % and 20 %, respectively (Curioni et al., 1995; Colgrave et al., 2013; Silva et al., 2008). Fasoli et al. (2010) and Konecna et al. (2012) also found a third isoform called Protein Zx.
3.3. Other pathogen-related proteins
The β-glucanase activity and the β-glucan content are related to malt and beer quality (Jin et al., 2004; Wang et al., 2004). Predominantly, (1,3;1,4)-β-glucanases are responsible for initial barley degradation, but they are inactivated during kilning and mashing.
The (1,3)-β-glucanases are significantly more heat-stable than (1,3;1,4)-β-glucanases and encounter also harsh conditions of pH and microbial degradative enzymes (Chen et al., 1995). Mixtures of thaumatin-like proteins (TLP) from barley grain inhibit metabolic activities of
S. cerevisiae, causing cell constituent leakage and having a lethal effect on yeasts (Cvetkovic et al., 1997). Therefore they could negatively affect beer quality. However, with temperatures above 75 °C, barley seed TLPs vanish from wort, hence they neither influence the yeast fermentation during brewing, nor the beer quality (Kontogiorgos et al., 2007; Cvetkovic, 1997). Peroxidases are associated with unfavorable effects on flavor, color, texture and nutritional qualities of raw and processed foods (Wichers, 2004). A cultivar variation in barley grain peroxidase isozyme spatio-temporal profiles are assumed to compromise beer quality (Antrobus et al., 1997a; Antrobus et al., 1997b; Laugesen et al., 2007).
3.4. Hordeins
According to their electrophoretic mobility, hordeins can be divided into four groups: B-, C-, D- and γ-hordeins (Picariello et al., 2012; Skerrit & Janes, 1992). The major hordeins are B-hordeins (30 kDa - 45 kDa) and C-hordeins (55 kDa - 70 kDa). The D- (90 kDa - 105 kDa) and γ-hordeins (30 kDa - 40 kDa) are less occurring components (Picariello et al., 2012; Fontana & Buiatti, 2008; Shewry, 1978; Skerrit & Janes, 1992). Hordeins were also classified via size and amino acid composition into A-hordeins (15 kDa - 25 kDa), B-hordeins, C-hordeins and D-hordeins (Shewry, 1978). It is assumed that A-hordeins may be alcohol-soluble albumins or globulins or breakdown products of larger hordeins rather than true hordeins (Baxter, 1981).
During malting and mashing hordeins are degraded into amino acids and low molecular weight polypeptides by proteases. In concentrated beer foam fractions a 23 kDa protein and a 17 kDa protein were found, suggesting that these proteins are foam-active (Sheehan & Skerrit, 1997). Proline and glutamic acid-rich hordeins with molecular masses of 10 kDa up to 30 kDa also initiate the haze development (Asano et al., 1982). B1-, B3-, C-, D- and γ-hordeins were identified in beer with and without enzymatic digestion, but most hordein derived proteins or polypeptides are only low MW molecules (Picariello et al., 2012; Colgrave et al., 2012). B1-, B3-, D- and γ3-hordein have MWs of approximately 32 kDa, 31 kDa, 80 kDa and 33 kDa, respectively. Using multiple reaction monitoring (MRM) MS showed, that hordeins are hydrolyzed to a great extent during the brewing
process (Colgrave et al., 2014). Recently, the 16 kDa - 17 kDa avenin-like protein-a (ALP) was identified in beer, having a partially homologous amino acid sequence as γ-hordein (Picariello et al., 2012; Colgrave et al., 2012; Picariello et al., 2011; Weber et al., 2009). Nevertheless, Iimure et al. (2015) proved that ALP does not influence the foam stability.
4. Protein modifications during beer production
During the whole beer production protein profiles undergo great changes due to degradation, coagulation and modification including glycation, acylation and partial digestion (Iimure & Sato, 2013; Iimure et al, 2015). Around 500 mg L-1 of proteinaceous material with molecular masses varying between 5 kDa and 100 kDa is detectable in beer (Steiner et al., 2011a). The remaining proteins in beer are mainly albumins, globulins, serpins, amylase inhibitors, lipid binding proteins, chaperons and enzymes (Colgrave et al., 2013). Proteins detected in barley grain, partly green malt, malt, partly sweet wort, wort and beer by the use of proteomic techniques are gathered in Table 1.1.
Barley grain describes the unprocessed grain, green malt is germinated, but not kiln-dried and malt is germinated and kilned. Sweet wort is obtained after the mashed malt is separated from insoluble residues, wort is acquired after the boiling process. Green beer is received after the main fermentation, but before maturation. Finally after maturation, the brewing process is completed and the desired beer is produced.
Table 1.1
Summary of barley proteins identified in barley grain, malt, wort and beer
Protein name Sample type Reference
Hordeins
Hordein B wort, beer [1 - 7]
Hordein B1 grain, malt, wort, beer [2 - 4, 6, 7]
Hordein B3 grain, malt, beer [1, 2, 4, 7, 8]
Hordein B3 fragment grain, malt [8]
Hordein C grain, malt, wort, beer [4,6 ,7]
Hordein C fragment beer [2]
Hor1-17 C-hordein wort [4]
Hordein D grain, malt, wort, beer [1, 2, 4 - 7]
Hordein D fragment grain, malt [8]
Hordein γ grain, malt [6]
Hordein γ-1 beer [1, 2, 4, 5]
Hordein γ-3 grain, malt, wort, beer [1 -4, 7 - 9]
Predicted protein, similar to avenin-like protein a
wort, beer [2 - 5]
Lipid transfer proteins
LTP beer [5]
Predicted protein, similar to LTP beer [4, 5]
ns-LTP1 grain, malt, wort, beer [1 - 12]
ns-LTP2 grain, malt, wort, beer [2, 4, 5, 7, 8, 18]
Probable ns-LTP2 grain, malt, wort, beer [10]
ns-LTP6 wort [4]
Lipid transfer protein complex with palmitate grain, malt, wort, beer [10]
Serpins
Protein Z type serpin grain, malt, wort, beer [8, 10, 13 - 15]
Serpin-Z2A beer [4]
Serpin-Z4 wort, beer [1, 2, 4, 5, 8, 9, 11, 16, 17]
Serpin-Z7 wort, beer [1, 2, 4, 5, 9, 11, 12]
Serpin-ZX beer [5, 9]
Protein Z fragment malt [18, 19]
Protein Z malt [1, 7, 19]
alpha-amylase/trypsin inhibitor
BDAI-1 grain, malt, wort, beer [1, 2, 4, 5, 8 - 12]
BMAI-1 grain, malt, wort, beer [4, 5, 8 - 11, 17, 18]
BTICMc grain [8]
BTI-CMe (CMe) grain, malt, wort, beer [2 - 5, 8 - 10, 12, 16, 20, 21]
BTI-CMe2.1 grain, malt, wort, beer [8, 10]
BTI-CMe3.1 sweet wort, beer [11, 12]
CMa grain, malt, wort, beer [2, 4, 5, 7, 8, 10, 11]
CMb grain, malt, wort, beer [2,4, 5, 8, 10 - 12]
Table 1.1 (continued)
CMd grain, malt, wort, beer [1 - 5, 8 - 11]
CMd3 grain, malt, wort, beer [8, 10]
pUP13 grain, malt, beer [1, 2, 5, 8, 11, 12, 22]
pUP38 grain, malt, wort, beer [8, 10, 22]
Other inhibitors
Alpha-amylase inibitor beer [5]
Alpha-amylase/subtilisin inhibitor grain, malt [10]
Alpha-amylase inhibitor/endochitinase grain, malt [10]
Bifunctional alpha-amylase/subtilisin ihibitor grain, malt [10, 19]
Chymotrypsin inhibitor-2 grain, beer [5, 10]
Cystatin Hv-CPI6 beer [5]
Protein synthesis inhibitor 1 malt [19]
Subtilisin-chymotrypsin inhibitor-2A grain, beer [5, 10]
Subtilisin-chymotrypsin inhibitor CI-1A beer [4]
Subtilisin-chymotrypsin inhibitor CI-1B beer [12]
Subtilisin-chymotrypsin inhibitor CI-1C beer [4]
Chaperons, defense and stress response proteins
17 kDa class I small heat shock protein beer [4]
18 Kd heat shock protein beer [5]
Barperm 1 grain, malt, sweet wort,
wort
[10, 11]
Barwin grain, malt, wort, beer [2, 4, 8 - 11]
Hordoindoline-A beer [2, 4, 9]
Hordoindoline-B1 beer [4, 5, 9]
Hordoindoline-B2 wort, beer [4, 9]
Leaf-specific thionin beer [9]
Leaf-specific thionin DB4 beer [9]
Pathogenesis-related protein PR1 malt [10]
Pathogenesis related protein PR-1a (Hv-1a)
malt [10]
Pathogenesis-related protein PRB1-2 malt [10]
Pathogenesis-related protein PRB1-3 malt [10]
Pathogenesis-related protein PR4 malt, wort, beer [5, 7, 11]
Pathogenesis-related protein PR5 grain, malt, sweet wort [10, 19]
Predicted protein, similar to class I heat shock protein
beer [4]
Predicted protein, similar to endosperm transfer cell specific PR60
wort [4]
Predicted protein, similar to heat shock protein
beer [4]
Predicted protein, similar to heat shock protein 17
beer [4]
Predicted protein, similar to late embryogenesis abundant protein
Table 1.1 (continued)
Predicted protein, similar to late embryogenesis abundant protein 3
beer [4, 5]
Predicted protein, similar to late embryogenesis abundant protein B19.1
beer [4]
Thaumatin-like protein 6 grain, malt, sweet wort,
wort
[10, 11]
Thaumatin-like protein 7 grain, malt, sweet wort [10]
Thaumatin-like protein 8 malt, beer [4, 10]
Enzymes
26 kDa endochitinase 1 (precursor) malt [10, 17, 19]
26 kDa endochitinase 2 (precursor) grain, malt, beer [4, 10, 17]
Alanine aminotransferase 2 malt [22]
Aldose reductase grain, malt [10]
Alpha-1,4-glycan-4-glucanohydrolase malt [7]
Alpha-amylase malt, sweet wort [7, 10, 23]
Alpha-amylase 1 (AMY1) green malt [24]
Alpha-amylase 2 (AMY2) green malt, malt [22, 24]
Alpha-galactosidase grain [25]
Arabinoxylan arabinofuranohydrolase (AXAH-I)
malt [22]
Aromatic amino acid aminotransferase malt [22]
Barley grain peroxidase BP1 grain, malt, wort [10, 11, 18, 22, 26]
Beta-amylase malt, grain, wort, beer [1, 2, 4, 7, 10, 15, 19, 23, 27]
Beta-D-xylosidase malt, sweet wort, wort [10, 11, 18]
Beta-glucosidase grain, malt, sweet wort [7, 10]
Calcium-dependent protein kinase beer [5]
Chitinase malt [10, 22]
Crystal Structure of Barley Grain Peroxidase 1, chain A
grain, malt [10, 22]
Cysteine endopeptidase EP-A malt [22]
Endo-(1,3;1,4)-beta-glucanase malt [25]
Fructose-bisphosphate aldolase grain, malt [10, 22]
Glyceraldehyd-3-phosphate-dehydrogenase, cytosolic
grain, malt [10, 22]
Granule-bound starch synthase 1 malt [17]
Nucleoside diphosphate kinase beer [4]
Predicted aconitate hydratase malt [22]
Predicted dienelactone hydrolase malt [22]
Predicted enolase malt [22]
Predicted glutathione reductase malt [22]
Predicted protein, similar to lactoylglutathione lyase
beer [4]
P-type ATPase beer [3]
Putative glutamate decarboxylase beer [3]
Table 1.1 (continued)
Ribulose 1,5-bisphosphate carboxylase malt [14]
S-adenosylmethionine decarboxylase beer [3]
Serine carboxypeptidase grain [18]
Triosephosphate isomerase, cytosolic grain, malt, beer [5, 10]
Other proteins
1,4-beta-D-mannan endohydrolase precursor malt [22]
40S ribosomal protein S7 beer [9]
Alpha-amylase precursor malt [14]
Alpha-amylase type B isozyme malt [10]
Amy2BASI Protein-protein complex alpha-amylase, chain A
grain, malt, sweet wort [10]
Barley peroxidase isozyme BSSP1 green malt [26]
Calmodulin beer [9]
Cold regulated protein 1-fragment grain [8]
Cupin_2 malt [22]
Elongation factor 1-alpha malt [17]
Embryo globulin beer [4]
EP-B 1, precursor malt [22]
Formate-tetrahydrofolate ligase-like malt [22]
Glucose and ribitol dehydrogenase homologue
grain, malt, beer [5, 7, 10]
Glycine-rich RNA-binding protein blt801 beer [9]
Grain softness protein beer [2, 5]
Lichenase II precursor malt [17]
NBS-LRR type resistance protein beer [2]
Pistillata-like protein malt [19]
Predicted protein, 76.9 kDa beer [2]
Predicted protein, potassium ion transmembrane acitivity
beer [2]
Predicted protein, similar to cupin family protein
beer [4]
Putative avenin-like precursor grain [10]
Putative globulin sweet wort [11]
Putative lecitin malt [19]
The numbers used in the table above belong to the following sources:
[1] (Weber et al., 2009); [2] (Picariello et al., 2012); [3] (Picariello et al., 2011); [4] (Colgrave et al., 2012); [5] (Konecna et al., 2012); [6] (Flodrova et al., 2012); [7] (Petry-Podgorska et al., 2010); [8] (Perrocheau et al., 2005); [9] (Fasoli et al., 2010); [10] (Benkovska et al., 2011); [11] (Iimure et al., 2012); [12] (Iimure et al., 2010); [13] (Bobalova et al., 2010); [14] (Ostergaard et al., 2002); [15] (Finnie et al., 2004); [16] (Hao et al., 2006); [17] (Chmelik et al., 2007); [18] (Lastovickova et al., 2010); [19] (Bobalova & Chmelik, 2007); [20] (Robinson et al., 2007a); [21] (Okada et al., 2008); [22] (Jin et al., 2014); [23] (Grabar et al., 1965); [24] (Bak-Jensen et al., 2007); [25] (Bak-Jensen et al., 2004); [26] (Laugesen et al., 2007); [27] (Evans et al., 1997)
4.1. Protein modifications during malting
Malting includes steeping of barley, germination and kiln-drying of the germinated seeds. It is characterized by a great variety of significant metabolic changes especially of polar compounds, while lipid contents are less affected (Bamforth & Barclay, 1993; Frank et al., 2011). For instance, the amount of mono- and disaccharides, e.g. glucose and maltose as well as amino acids are increasing after germination and decreasing after kiln-drying. The increase is due to proteolytic enzyme activity, while the decrease is explainable by the reaction of reducing sugars like fructose and glucose with amino acids forming Maillard products (Frank et al., 2011). The water-soluble protein ratio increases more than 2-fold during malting as a result of the degradation of the water insoluble hordein component of the reserve and due to the releasing of bound (latent) proteins (Osman et al., 2003, Osman et al., 2002; Evans & Hejgaard, 1999).
The malting procedure effects the composition of albumin, hordein, glutelin and globulin, respectively (Dai et al., 2014). Dai et al. (2014) used MALDI-TOF MS to determine the changes of proteins after malting. They monitored a degradation and even disappearance of eight albumin proteins, including LTP1, while three mainly new protein profiles with molecular masses of 4038 Da, 6803 Da and 12166 Da were generated during malting. In general, the proteins with molecular masses below 12 kDa were more affected by malting than the albumins ranging from 12.5 kDa to 16.5 kDa, indicating a part-modification of proteins by enzymatic digestion. The amount of proteins also decreased in the globulin fraction after malting, but the composition was less altered and globulins were more resistant to proteolysis than the other three storage proteins. As against, hordeins were modified quantitatively and five proteins also qualitatively with significant differences regarding the barley variety. The results of glutelins correlate with the hordein results (Dai et al., 2014).
Weiss et al. (1992) used SDS-PAGE and densitometry for qualitative and quantitative determination of changes during malting. They figured out that albumins and globulins are relatively resistant to proteolysis, while hordeins particularly D-hordeins were degraded. The degradation of hordeins into soluble peptides and amino acids happening during malting provides substrates for embryo´s growth (Lastovickova & Bobalova, 2012). Garcia-Villalba et al. (2006) used free-zone capillary electrophoresis to investigate different malting stages. The electropherograms revealed that
amongst B- and C-hordeins the most significant changes occur to C-hordeins. During germination, the amount of C-hordeins increased. During kilning, the pattern of both, B- and C-hordeins changed. On the other hand, Flodrova et al. (2012) could not observe significant qualitative differences in the C-, B- and γ- hordein patterns according to SDS-PAGE results. Moreover, they monitored a 65 % decrease of hordein C by the combination of MS with isobaric labeling (iTRAQ) in the final malt.
Lastovickova et al. (2010) investigated the whole malting process by the linear mode of MALDI-TOF MS. Their results indicate the impact of the malting process on the glycation of certain water-soluble barley proteins, nonspecific lipid transfer protein 1 (LTP1) and protein Z, of which the glycated forms survived the brewing process, implying that glycation may prevent their precipitation. Furthermore, they monitored a disappearance (e.g., 8.80 kDa), an increase (e.g., 4.03 kDa) and a multiplying (e.g., 9.99 kDa) of several MS peaks. Significant changes were observed from the third day of the malting process, but the most marked changes were in the mass spectra of the final kilned malt. Bobalova and Chmelik (2007) combined protein fractionation using convective interaction media and MALDI-TOF/MS in order to detect glycation of protein Z, LTP1 and LTP2 during malting by corresponding molecular weight changes by adducts of 162 Da (hexose units). The glycation of protein Z was detected after two days of the malting process (Bobalova et al., 2010).
4.1.1. Germination
Before germination starts, the barley grains have to be steeped. Adding water to the grains raises the moisture level and activates metabolic processes of the dormant grain (Kramer, 2006). Steeping ensures equally hydrated grains which in turn facilitates uniform germination (Lastovickova & Bobalova, 2012). During germination, three main biochemical processes occur: cytolysis, proteolysis and amylolysis and several enzymes are activated including proteases, amylase and β-glucanase (Iimure & Sato, 2013). Hydrolytic enzymes like endoproteases and carboxypeptidases are released during germination (Steiner et al., 2011a). Thus, storage proteins, particularly hordeins and glutelins, are degraded (Osman et al., 2002). Moreover, the enzymatic
degradation of reserve proteins in the endosperm generates free amino
The germination process also leads to the formation of new proteins: α-amylase, β-D-xylosidase, chitinase and endochitinase were detected in malt, but not in the corresponding grain (Benkovska et al., 2011). Unlike prolamins and glutelins, the amount of albumins and other soluble proteins are more than doubled after germination (Osman et al., 2003; Osman et al., 2002; Evans & Hejgaard, 1999; Baxter & Wainwright, 1979). Protein Z is present in thiol bound forms in the mature seeds which are released during the germination process (Hejgaard, 1982). Protein Z is converted to a heat and protease stable form by proteolytic cleavage in the reactive site loop, thus, it withstands the brewing conditions. Compared to the mature seed, the amount of α-amylase/trypsin inhibitors decreases in germinating grains (Maeda et al., 2004).
4.1.2. Kiln-Drying
After five days of germination, the so-called green-malt is kiln-dried. During kiln-drying the water content of green-malt is reduced to ensure its shelf life. Moreover, at this step aroma and flavor are generated and substantial translations are fixed (Wunderlich & Back, 2008; Wittmann & Eichner, 1989). Kilning is subdivided into two parts: withering and curing. Withering pre-reduces the water content from 45 % to 10 % at low temperatures. Subsequent curing is performed at temperatures between 80 °C and 105 °C. The drying process is completed when the water content reaches 3.5 % - 4 % in pale malts or 1.5 % - 2 % in dark malts (Wunderlich & Back, 2008; Kunze, 2011). During kilning chemical transformations take place at different phases. In the growing phase, growing proceeds until the water content is below 20 % and the temperature is above 40 °C. The enzymatic degradation of the grain starts and is continued in the enzymatic phase at temperatures between 40 °C and 70 °C. The declining water content stops embryo´s growth and enzymatic degradation processes. The degradation products are accumulated. Following this, reduced dispersion and coagulation of colloidal nitrogen substances happen in the chemical phase at temperatures above 70 °C. Temperatures exceeding 95 °C lead to intensive Maillard reaction and the formation of Amadori compounds (Wunderlich & Back, 2008; Wittmann & Eichner, 1989; Kunze, 2011). Free amino groups of peptides and proteins like ε-amino groups of lysine or guanidine groups in arginine react with α-oxoaldehydes, reducing carbohydrates or their derivatives (Lapolla et al., 1993). Glycation predominantly affects lysine residues and to a minor extent arginine residues (Colgrave et al., 2013; Canas et al., 2006; Petry-Podgorska et al., 2010, Bobalova et al., 2010). The product of starch degradation during malting,
particularly D-glucose, is responsible for the glycation of barley proteins, reacting with the free amino residues (Steiner et al., 2011a). Wittmann and Eichner found ten different Amadori compounds that were formed during kiln-drying.
Depending on the kilning conditions the malts have shown different compound amounts and proportions. Kilning leads to modification and reduction of LTP1 and protein Z (Evans & Hejgaard, 1999). Both proteins are glycated during kilning due to Maillard reaction. It is estimated that up to 16 % of the lysine content of the 44 kDa protein (protein Z4) is glycated via Maillard reaction (Hejgaard & Kaersgaard, 1983). The glycation of protein Z causes an improved foam stability (Curioni et al., 1995). LTP1 was also detected in an acylated form in barley and malt (Jegou et al., 2001). Modifications like glycation and acylation could improve the surface activity of LTP1 (Perrocheau et al., 2005). The spots of α-amylase/trypsin inhibitors did not scatter in many spots when processing from barley to malt, suggesting that they are not extensively affected by Maillard reaction during kilning, although they are rich in lysine (Perrocheau et al., 2005). Compared to barley grain, inhibitors like BMAI-1 and CMb disappeared almost completely in beer, which might be due to low or non-existing glycation. Hordeins have a low lysine content. Thus, they are not prone to Maillard reaction. The lack of glycation might explain the almost disappearance of hordeins in beer (Perrocheau et al., 2005).
4.2. Protein modifications during brewing
The brewing process consists of mashing, lautering, wort boiling, fermentation, maturation, storage, filtration and stabilization (Wunderlich & Back, 2008). Thereof, mashing, wort boiling and fermentation have an intense impact on protein patterns.
4.2.1. Mashing
After milling, the grist is mixed with water at 45 °C in a mashing vessel. The two main steps are protein rest (45 °C - 60 °C) and saccharification rest (around 65 °C). Proteins are modified and degraded into amino acids and low molecular weight peptides by proteases during protein rest, providing a nitrogen source for the brewing yeast. The saccharification rest is used for the degradation of starch into maltose by β-amylase, serving as carbon sources during fermentation (Iimure & Sato, 2013). In general, mashing consists of the same enzymatic processes as malting: amylolysis, proteolysis and cytolysis. Amylopectin and amylose, the degradation
products of starch, are further degraded by amylases into sugar and small dextrins. Degrading enzymes of the proteolysis are and exo-peptidases. Inner proteins are decomposed by endo-peptidases and soluble nitrogen content increases. Exo-endo-peptidases react with the end of protein chains, releasing amino acids. Proteolysis occurs most frequent at temperatures around 50 °C. At temperatures between 60 °C - 70 °C medium and high molecular breakdown products arise. Degraded components of hemicelluloses dissolve during cytolysis mainly at temperatures below 50 °C (Wunderlich & Back, 2008; Kunze, 2011). Mashing completes the enzymatic degradation processes, that started during malting and is the first biochemical process step of brewing (Steiner et al., 2011a; Fontana & Buiatti, 2008). Many proteins are degraded by temperature and especially proteolytic activity during mashing, i.e. β-amylase and β-glucosidase. During germination, multiple endoproteinase isoforms emerge that also remain intact after kiln drying, but are quickly inactivated when the temperature exceeds 72 °C (Jones & Marinac, 2002; Osman et al., 2002). During mashing and wort boiling also half of the Amadori compounds are degraded (Wittmann & Eichner, 1989). Glycated ns-LTP1 and ns-LTP1b are recovered during mashing (Gorjanovic, 2007; Jegou et al., 2001). Globulins are diluted, but β-globulin does not precipitate completely, even not after the wort boiling. Thus, it can give rise to haze in beer (Steiner et al., 2011a).
4.2.2. Wort boiling
After the lautering process (the separation of liquid and solid compounds) wort is transferred and boiled in a kettle (Wunderlich & Back, 2008). Wort boiling leads to a formation of flavor and color substances like melanoidins, some proteins are coagulated, denatured, and modified (Iimure & Sato, 2013). Proteins that resisted mashing, but are degraded during wort boiling are for example β-D-xylosidase, barperm1, thaumatin-like proteins and α-amylase (Benkovska et al., 2011). The optimum of coagulated nitrogen is in the range of 15 mg L-1 - 25 mg L-1 ensuring good taste and foam and no turbidity (Wunderlich & Back, 2008). The foam stability is affected by the wort boiling temperature (van Nierop et al., 2004). The higher the wort boiling temperature, the lower the amount of LTP1 in the final beer (van Nierop et al., 2004). During wort boiling LTP1 is unfolded (Gorjanovic et al., 2004; Perrocheau et al., 2006). Only this unfolding gives LTP1 its foam promoting properties (Jegou et al., 2000; Jegou et al., 2001).
Glycation of LTP prevents the precipitation on unfolding that occurs during the wort boiling step (Gorjanovic, 2007). The Glycation of protein Z might also prevent the protein from being denatured during wort boiling (Curioni et al., 1995). Iimure et al. (2012) suggest that protein Z was precipitated by binding with comparatively small size specific fragments derived from sweet wort protein, i.e., barwin during wort boiling. They also monitored that during wort boiling in low molecular weight regions, several protein spots disappeared or decreased. Spot intensities of CMb and α-amylase inhibitor BMAI-1 precursor (BMAI-1) did not change within sweet wort, wort and trub. Spot intensities of α-amylase/trypsin inhibitor CMa precursor (CMa) and trypsin inhibitor BTICMc (CMc) were higher in the trub than in the boiled wort. Vice versa, CMd preprotein (CMd),
BTI-CMe3.1 protein (CMe), chain A, trypsin/amylase inhibitor pUP13 and
non-specific lipid transfer protein 1 (LTP1) were higher in the boiled wort than in the trub. Barley wound induced protein (barwin) and pathogenesis-related protein 4 (PR-4) were observed in sweet wort, but not in boiled wort and trub (Iimure et al., 2012). Before fermentation, hot trub is removed, wort is cooled down and cold trub is separated (Wunderlich & Back, 2008). During these steps a large proportion of protein content is eliminated (Colgrave et al., 2013).
4.2.3. Fermentation
Beer proteins originating from barley also play an import role during fermentation. Degraded to amino acids and peptides, they serve as nitrogen source for the brewer´s yeast (Steiner et al., 2011a). Depending on the proteolysis during malting, malt is modified and free amino nitrogen and peptides are released, that are necessary for yeast growth during fermentation (Agu & Palmer, 1997; Kirsop et al., 1967; Ljungdahl & Sandergren, 1953). Wort contains all physiologically active amino acids, but with differences in their composition between different wort types (Turkia et al., 2015). Following the fermentation, the green beer is stored for further fermentation. The fermentation is completed, when all fermentable sugars are metabolized into alcohol and carbon dioxide by yeast. After fermentation, the beer is filtered, stabilized and filled. During fermentation, a drop in pH causes protein aggregation, allowing them to be separated (Benkovska et al., 2011; Steiner et al., 2011b).
Only one-third of nitrogenous compound can be found in the finished beer, due to mashing and wort boiling. Furthermore, a part of the nitrogenous compounds, namely amino acids, are
assimilated for yeast growth during fermentation (Fontana & Buiatti, 2008; Gorjanovic, 2010). During mashing and wort boiling half of the Amadori compounds were degraded, while no degradation takes place during fermentation (Wittmann & Eichner, 1989). Most barley proteins were proteolytic and thermal degraded, and denatured during malting and brewing. Nevertheless, some proteins are resistant to these extreme conditions and remain in native or modified form in malt and beer, where they positively (e.g. beer foam) or negatively (e.g. beer haze) influence the quality (Gorjanovic, 2007; Evans et al., 2003).
Beer contains around 500 mg L-1 of proteinaceous material with molecular masses varying between 5 kDa and 100 kDa. Most of these polypeptides belong to barley, only a minority of beer proteins originate from yeast (Steiner et al., 2011a). The remaining proteins in beer are mainly albumins, globulins, serpins, amylase inhibitors, lipid binding proteins, chaperons and enzymes (Colgrave et al., 2013). The only present proteins in the final beer, that withstand the brewing conditions with significant amount are protein Z (40 kDa) and LTP1 (9.6 kDa) (Steiner et al., 2011a; Leisegang & Stahl, 2005). In the final beer, 50 mg L-1 - 200 mg L-1 of protein Z and 50 mg L-1 - 90 mg L-1 of LTP1 were detected (Iimure et al., 2010). These proteins are both resistant to proteolysis and heat (Perrocheau et al., 2005).
The reason why protein Z and LTP are not enzymatically degraded during malting and mashing might be due to glycation. The glycation of protein Z was detected after two days of the malting process, LTP1 was significantly modified after three days of malting (Lastovickova et al., 2010; Bobalova et al., 2010). LTP1 becomes a surface-active protein during the malting and brewing processes and is modified during boiling. Only the modified form of LTP influences foam stability (Steiner et al., 2011a). Serpins like protein Z are cleaved in the reactive center at the reactive bond during inhibition, resulting in a drastic conformational change and a formation of an enzyme-inhibitor complex. This complex converts serpins into extremely resistant molecules to further degradation by proteolysis, allowing them to survive the harsh brewing conditions. After proteolysis, the complex dissociates into an active proteinase and an intact serpin (Gorjanovic, 2009). Perrocheau et al. (2005) found several spots of protein Z during all steps of the beer production in their 2D-gels: 10 in barley, 12 in malt and 11 in beer. The amount of spots for protein Z are rather constant, while the spots for LTP1 increase throughout the brewing process. Two spots
were detected in barley, six in malt and eight in beer (Perrocheau et al., 2005). While protein Z raises foam stability, LTP1 contributes to foam formation (Evans & Sheehan, 2002).
In addition to protein Z and LTP1, a group of hordein-derived polypeptides (with sizes ranging from 10 kDa to 30 kDa) seem to survive the brewing process. These polypeptides are involved in haze formation (Asano et al., 1982). Contributors to beer haze formation might be BDAI-1, CMb and CMe, as they are heat and proteolysis resistant during brewing (Jin et al., 2009b). Iimure et al. (2009) suggest that BDAI-1, CMb and CMe are not predominant haze-active proteins, but growth factors of beer colloidal haze. Not only is BDAI-1 claimed to be haze-active, but also foam-positive (Iimure & Sato, 2013; Iimure et al., 2008; Okada et al., 2008; Iimure et al., 2009). Its impact on foam stability was recently verified (Iimure et al, 2015). Avenin-like protein-a was identified in beer, having a partially homologous amino acid sequence as γ-hordein (Picariello et al., 2011; Colgrave et al., 2012; Picariello et al., 2012; Weber et al., 2009). It was assumed to be foam active, but was recently proved to not influence the foam stability (Iimure et al, 2015).
5. Conclusion
Proteomics allows the identification of numerous beer proteins including proteins with complex modifications such as glycation and acylation. Understanding these modifications may improve controlling processes like malting, mashing, wort boiling and fermentation. The data set of barley genome sequence has high impact on the identification of protein species from barley grain, malt, wort and beer. The recently developed web-based application from Colmsee et al. (2015) helps to access the developing genomic infrastructure of barley by comparing the physical map of Ariyadasa et al. (2014), the whole genome shotgun assembly from the International Barley Genome Sequencing Consortium (IBGSC, 2012) and the ultra-dense genetic map created by Mascher et al. (2013). These data set of barley genomic sequence will certainly influence the identification of protein species from barley grain, malt, wort and beer essentially.
Experimental Part: Monitoring protein modifications - Part II. Impact of kiln
temperature on barley (H. vulgare) protein profile.
Authors: Prof. Dr. Leif-Alexander Garbe, Hochschule Neubrandenburg, University of Applied Sciences, Fachbereich Agrarwirtschaft und Lebensmittelwissenschaften, Brodaer Straße 2, 17033 Neubrandenburg, garbe@hs-nb.de
Prof. Dr.-Ing. Heralt Schöne, Hochschule Neubrandenburg, University of Applied Sciences, Fachbereich Agrarwirtschaft und Lebensmittelwissenschaften, Brodaer Straße 2, 17033 Neubrandenburg, schoene@hs-nb.de
Elena Sosna*, Hochschule Neubrandenburg, University of Applied Sciences, Fachbereich Agrarwirtschaft und Lebensmittelwissenschaften, Brodaer Straße 2, 17033 Neubrandenburg, al14009@hs-nb.de
* corresponding author
Descriptors
SDS-PAGE, Barley (Hordeum vulgare), Malt, Kilning, Protein modification Abstract
Barley proteins are crucial for the quality of malt and beer. However, these proteins are degraded and modified by high temperature treatments as during kilning. It is expected that higher kilning temperatures even enhance these processes. Therefore, degradation and modification of different kilned malts were studied by measuring protein concentrations and applying SDS-PAGE. Already after five hours at 120 °C most proteins were degraded. Subsequent congress mashing revealed that only one protein with a molecular mass of around 16 kDa survived these harsh conditions. Neither LTP1, nor protein Z were apparent at the combination of high kilning temperature and enzymatic degradation in higher amounts. No striking differences in malt color between malts further kilned at 80 °C, 100 °C and 120 °C by measuring the CIE L*a*b* parameters were found.
1. Introduction
Proteins originating from barley (Hordeum vulgare L.) highly affect beer properties and quality. Depending on the variety, barley consists of 8 % - 13.5 % proteins (Wunderlich & Back, 2008; (Yalcin et al., 2008). For very bright Pilsener type beers, a protein content of 10 % - 11 % is preferred. Conventional beers are brewed with barley consisting of 11 % - 11.5 %. A higher protein amount of 11 % - 12 % is used for dark malts containing Münchener malt (Wunderlich & Back, 2008). Besides brewing, malting is one of the determining processes concerning protein modifications. Malting consists of steeping, germination and kiln-drying (Wunderlich & Back, 2008; (Wittmann & Eichner, 1989). Kilning is subdivided into two parts: withering and curing. Withering pre-reduces the water content from 45 % to 10 % at low temperatures, while the following curing is performed at temperatures between 80 °C and 105 °C. The drying process is completed when the water content reaches 3.5 % - 4 % in pale malts or 1.5 % - 2 % in dark malts (Wunderlich & Back, 2008; Kunze, 2011). During kilning chemical transformations take place at different phases. In the growing phase, growing proceeds until the water content is below 20 % and the temperature above 40 °C. The enzymatic degradation of the grain starts and is continued in the enzymatic phase at temperatures between 40 °C and 70 °C. The declining water content stops embryo´s growth and enzymatic degradation processes. The degradation products are accumulated. Following this, reduced dispersion and coagulation of colloidal nitrogen substances happen in the chemical phase at temperatures above 70 °C. Temperatures exceeding 95 °C lead to intensive Maillard reaction (Wunderlich & Back, 2008; Kunze, 2011). Final drying of pale malts is performed at 75 °C up to 80 °C. In order to obtain dark malts, moisture reduction is conducted at a slower rate leading to a higher enzymatic release of amino acids and sugars. The final drying temperature of dark malts ranges between 100 °C and 105 °C. Specialty malts like caramel and roasted malts are even exposed to temperatures up to 250 °C (Vandecan et al., 2010). Therefore, dark malts are exposed to higher thermal stress and simultaneously have more reactants for Maillard reaction leading to more browning products.
Maillard reaction products especially melanoidines are generated, due to chemical reactions. The product of starch degradation during malting i.e. D-glucose is responsible for the glycation (non-enzymatic, covalent addition of sugar) of barley proteins (Curioni et al., 1995; Lapolla et al., 1993; Leiper et al., 2003a; Leiper et al., 2003b; Gorjanovic et al., 2007). Reacting with free amino
