Several proteome analyses of beer [4
], malt [8
] and beer related processes [6
] have been made, but none seem to have considered the influence of fermentation and brewer’s yeast strains on the beer proteome. To investigate if proteome changes from wort to beer were yeast strain dependent, proteins from wort and beer brewed with two different ale brewer’s yeast strains were separated by 2-DE and identified by MALDI-TOF-MS. It should be noted that the beers in this study are immature, that is beers that have not matured after fermentation. In the following, however, they will be referred to as beer.
The protein content of the beers were 0.29 mg/ml for KVL011 and 0.42 mg/ml for WLP001 (Table ) placing them in the lower end of the range for a normal beer [24
]. The concentration of wort proteins (0.50 mg/ml) is higher than for the brewed beers, indicating that proteins are either degraded proteolytically by the yeast during fermentation and/or precipitate with the yeast slurry.
The most recent proteome studies have identified 20–30 barley proteins in wort and beer [4
]. In our study, nine unique proteins are identified out of 27 distinct protein spots analysed (Table ). Many of the proteins have multiple spots, probably due to different protein modifications taking place during germination of barley grain, killing or wort boiling [11
]. For example, protein Z appears as a dominant diffuse zone in a 2-DE gel probably due to glycosylation of lysine residues by Maillard reactions occurring under the roasting of malt [9
]. All identified barley proteins are reported as protease resistant and heat stable, as most of them are protease inhibitors and have survived a more than one hour long hop boiling (Table ) [7
In the wort proteome, protein Z appears as a cluster of many spots, while in both beer proteomes this cluster is divided into two clusters (Figure ). Division of the protein Z cluster into two in both beers indicates that yeast has an influence on the modifications of protein Z. This, however, remains to be further investigated.
LTP2 is present in two spots in the wort proteome (Figure ; spot A28, A29) but absent in the two beer proteomes, although a faint spot is observed in beer brewed with KVL011 but not identified (Figure ; spot C28). Many studies have shown that denatured and unfolded LTP1 in beer is degraded by yeast-derived proteinase A [27
], which can explain why LTP2 disappears and a decrease in LTP1 intensity is observed in our study. Degradation of LTP1 is not a desired trait in beer production, as LTP1 is a key foam protein and in addition acts as an antioxidant in beer [29
The three high molecular weight proteins, Uth1, Exg1 and Bgl2, found exclusively in beer after fermentation, are identified to be yeast proteins. Uth1 is involved in the cell wall biogenesis, oxidative stress response, and the protein resembles β-glucanases but no activity is reported [31
]. Exg1 and Bgl2 are involved in the modification of the glucan network of the yeast cell wall [33
]. It is reported that Exg1, Bgl2 and Uth1 are anchored to the yeast cell wall by di-sulphide bridges, as they are released from yeast cells upon treatment with reducing agents as DTT [34
]. During wine fermentations, yeast cells release Exg1 and Bgl2 from the cell wall to the wine [36
]. In beer, Fasilo et al.
(2010) identified Exg1, Bgl2 and Uth1 among the 40 protein fragments, originating from S. cerevisiae
], and very recently the presence of these three full-length proteins have also been identified in different commercial beers [5
]. These data correspond well with our findings here.
In addition, we report for the first time that different brewer’s yeast strains render different beer proteomes; i.e. Exg1 and Bgl2 are identified in the KVL011 beers, whereas in the WLP001 beer only Exg1 is identified. These data strongly indicate that changes in the beer proteome are strain dependent.
Identification of released yeast di-sulphide anchored proteins Uth1, Exg1 and Bgl2 in beer indicates the existence of a reducing environment which can be beneficial for the beer quality by reducing and liberating cell wall anchored yeast proteins. Overexpression of β-glucanases, like Exg1 and Blg2, in genetically modified brewer’s yeast strains, have shown positive effects on filtration of beer, due to increased degradation of β-glucans interfering with filtration [37
]. Also in wine fermentations, an elevated production of Exg1 has positive effects on the quality of the end product due to an increased production of volatile products [39
]. Uth1 could be speculated to function as an antioxidant or chelator of transition metals in beer due to its conserved cysteine residue motive with a putative Fe-binding motive [31
]. A controlled release of these cell wall anchored proteins could contribute to improved beer quality.
It should be stressed that our study, using immature beer, only reveals a very limited number of yeast proteins in the beer as compared to the reports of e.g. Fasoli et al
. (2010) and Konecna et al
. (2012). These authors investigate commercial beers that are most likely fully mature and pasteurized [4
], although not specifically stated, thereby explaining the higher number of identified yeast proteins due to cell lysis.