Proteome analysis of the wort boiling process

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Abstract

A comprehensive proteome map was constructed of brewers wort. The map consisted of protein identification on two-dimensional gel electrophoresis (2DE) images and identified 63 out of 202 protein spots, which were categorized into 20 protein species. To analyze the modification of protein Z during wort boiling, protein Z spots on 2DE gel of the sweet wort, the boiled wort and the trub were analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI TOF-MS) and then the spectra were compared. The analysis identified several specific signals detected only in the trub, suggesting that specific modification occurred in the precipitated protein Z during wort boiling. The protein Z spot on the 2DE gel of the precipitate was further analyzed by liquid chromatography mass spectrometry/mass spectrometry. The analysis identified low molecular weight fragment (1.3 kDa) derived from wound induced protein (barwin) in the protein Z spot of the trub. These results suggested that protein Z was precipitated by binding with comparatively small size specific fragment(s) derived from sweet wort protein, i.e., barwin during wort boiling. Our results and understandings have application for quality assurance and control in commercial brewing practice, and development of DNA markers for malting barley breeding.

Highlights

► A comprehensive novel sweet wort proteome map was constructed. ► This is the first report of a proteome analysis during wort boiling process. ► CMa and CMc proteins were easily precipitated than other proteins. ► Protein Z was precipitated by binding with small size specific fragment(s).

Introduction

Beer proteins are key factors determining and/or influencing several major beer quality traits. Among these quality characteristics, foam and haze (clarity) are important, visual clues to consumers as to the quality of the beer to be consumed. The quantity and stability of beer foam indicates that the beer is fresh, pleasing to behold and properly carbonated, while consumers judge that beer that displays haze as not being fresh or stale. A group of barley protein Z isoforms have positive relationships with beer foam, conferring high surface viscosity and elasticity (Maeda, Yokoi, Kamada, & Kamimura, 1991). The isoforms in protein Z, protein Z4 and protein Z7 are positively and negatively correlated with beer foam stability, respectively, and markers associated with beer foam stability have been established from the DNA sequences of genes encoding these proteins (Iimure et al., 2011). Barley lipid transfer protein 1 (LTP1) is another factor associated with beer foam stability (Sorensen, Bech, Muldbjerg, Beenfeldt, & Breddam, 1993). Of the proteins in barley grains, hordein and/or its derived polypeptides are the most abundant. It has been suggested that hordein has an important role in haze formation in beer (Asano, Shinagawa, & Hashimoto, 1982). Several older literatures have undeniably identified the presence of significant levels of hordein in beer (Evans et al., 2003, Kauffman et al., 1994, Sheehan and Skerritt, 1997). However Iimure et al. (2010) did not identify hordein in beer, and Fasoli et al., 2010, Perrocheau et al., 2005, Picariello et al., 2011 found only trace amount of γ3- and B-hordeins in beer. Even small amounts of BTI-CMe, CMb component of tetrameric α-amylase inhibitor (CMb) and barley dimeric α-amylase inhibitor-1 (BDAI-1) might affect the level of haze formation (Iimure et al., 2009, Robinson et al., 2007). In addition, some barley malt proteins were suggested to be allergens (García-Casado, Crespo, Rodriguez, & Salcedo, 2001), and modified LTP1 by fungal proteases together with hydrophobic fungal proteins promotes beer gushing (over foaming at the bottle opening) (Hippeli and Hecht, 2009, Laitila et al., 2007).

Barley proteins are modified during the malting and brewing processes. These modifications strongly influence beer quality. LTP1 is modified during wort boiling, and promotes foam stability (Sorensen et al., 1993). On the other hand, van Nierop, Evans, Axcell, Cantrell, and Rautenbach (2004) showed that modified LTP1 does not properly bind to foam negative lipids. They also suggested that wort boiling temperature was a key factor controlling LTP1 modification in beer, since the basic isoelectric point of intact LTP1 is modified during wort boiling by the Maillard reaction, and the resulting glycated LTP1 has an acidic isoelectric point (Hippeli and Hecht, 2009, Jégou et al., 2000). The modification of protein Z, glycation and partial digestion, has been well studied. Protein Z is glycated in the malting process by the Maillard reaction and the resulting glycated protein Z affects beer foam stability (Curioni, Pressi, Furegon, & Dal Belin Peruffo, 1995). Bobálová, Petry-Podgórska, Laštovičková, and Chmelík (2010) detected protein Z modification during malting using matrix-assisted laser desorption ionization time of flight/time of flight mass spectrometry (MALDI TOF/TOF MS). Protein Z is also modified by partial digestion with protease in a reactive site loop (Dahl, Rasmussen, & Hejgaard, 1996), and the end-product is heat and protease stable (Evans, Sheehan, & Stewart, 1999).

More detailed analyses of barley protein content and its modification during malting and brewing processes may improve the control of brewing quality. A set of proteome analyses, e.g., two-dimensional gel electrophoresis, protein identification by mass spectrometry analysis and database searches, is a powerful tool for comprehensively investigating protein content and modification. Using proteome analysis, Iimure et al. (2008, 2009) identified additional beer foam proteins (BDAI-1 and yeast thioredoxin) and haze active proteins (BDAI-1, CMb and CMe). Proteome analyses have been applied to samples from various stages of the malting and brewing processes, such as barley grains (Østergaard, Finnie, Laugesen, Roepstorff, & Svensson, 2004), malt (Bak-Jensen, Laugesen, Roepstorff, & Svensson, 2004), and beer (Fasoli et al., 2010, Iimure et al., 2010, Perrocheau et al., 2005, Picariello et al., 2011). There must be a variation in the proteome among the samples of different stages in the processes of malting and brewing. Similarly some differences between malting barley varieties would also be expected. Investigation of proteome changes during mashing and wort boiling might also be important, since the process might cause protein modification and changing protein content. However, these proteome changes have not been thoroughly investigated.

In this paper, we aimed to construct a wort proteome map using the above proteome analysis system. By comparing 2DE images of the sweet wort, the boiled wort, and the trub, the relationship between proteome changes and beer quality was estimated. The possible improvement of beer quality traits by the control of protein species is also discussed.

Section snippets

Barley and malt samples

A grain sample from the barley cultivar Haruna Nijo produced in Japan in 2002 was used. The malt sample was prepared from 75 kg barley grains. Malting proceeded according to Iimure et al. (2008). The level of ex-steep moisture was controlled at a comparatively low level, approximately 37% to minimize barley protein modification, and to comprehensively investigate the proteome changes during mashing and wort boiling.

Sweet wort, boiled wort, and trub preparation

Sweet wort was prepared according to the methods of the European Brewery

Construction of the sweet wort proteome map

Proteins in the sweet wort of cultivar Haruna Nijo were separated by 2DE (pI 4–7 and 6–9) (Fig. 1). The individual protein spot on the gels was analyzed by MALDI TOF-MS followed by database search to identify protein species. The analysis identified 63 out of 202 spots, which were categorized into 20 protein species (Table 1). Of these, two species hit to the proteins of Oryza sativa and one hit to the protein of Arabidopsis thaliana, in the DNA database. The other 17 species were hit to the

Discussion

There are several reports on the proteome analysis of beer (Fasoli et al., 2010, Perrocheau et al., 2005, Picariello et al., 2011). We developed a beer proteome map using a beer sample produced from the Japanese malting barley cultivar Haruna Nijo (Iimure et al., 2010), which gave us comprehensive information on the beer proteome. The number of protein species was rather limited in the beer proteome map due to changes in protein forms during the brewing process. The proteome may change during

Acknowledgments

We are grateful to T. Yazawa, N. Yatabe, K. Hoshino, K. Ito, and Y. Yamaguchi, the Bioresources Research and Development Department, Sapporo Breweries Ltd. for their technical assistance. We are also grateful to K. Takoi for the malt sample preparation. This study was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences, Japan (PROBRAIN).

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