Proteomic and peptidomic characterisation of beer: Immunological and technological implications

Abstract

In this study, the protein and peptide fractions of two commercial Italian barley malt beers, made with different processes by the same producer, have been analysed with a combined immunochemical and mass spectrometry approach. The “gluten” content of beer samples, measured with the R5 monoclonal antibody, was below the caution limit proposed by the Codex Alimentarius for gluten-free foods. The proteomic approach allowed to identify a 17 kDa avenin-like protein partially homologous to hordeins, that was particularly abundant in foam, in addition to the already reported barley albumins (Z₄-barley and ns-LTPs) and to minor amounts of yeast glycolytic enzymes. No intact hordeins were detected, although fragments derived from γ₃- and B-hordein were present. In consideration of the many implications of the protein/peptide pattern, these data provide useful information to improve quality and safety of beer.

Introduction

Malt derived from germinated barley (Hordeum vulgare L.) and, to a minor extent, from other coeliacogenic cereals is the basic ingredient used to produce beer. Through the sequential processes of malting, mashing and brewing, the activated barley endogenous proteinases completely or partially hydrolyse hordeins and other storage proteins. During malting, a complex proteolytic system contributes to hydrolyse both water-soluble and water-insoluble barley proteins, producing soluble polypeptides and free amino acids. Barley and malt endoproteases have recently been reviewed (Jones, 2005). During beer production, several changes to the barley proteins also occur, including glycation by Maillard reactions during malting, acylation during mashing, and structural unfolding during brewing (Perrocheau, Bakan, Boivin, & Marion, 2006).

On average, beer contains 0.2–0.6 g/100 ml of protein/peptide material originated mainly from barley proteins, and to a minor extent from yeasts (Cortacero-Ramírez, Hernáinz-Bermúdez de Castro, Segura-Carretero, Cruces-Blanco, & Fernández-Gutiérrez, 2003). The polypeptide fraction is constituted by either native or chemically modified proteins, and by products of hydrolytic events covering a broad-range of molecular sizes. In particular, the hordein-derived polypeptides were thought to be involved in the mechanism of both haze formation and foam stabilization (Asano et al., 1982, Kaverva et al., 2005a, Kaverva et al., 2005b, Robinson et al., 2007, Sheehan and Skerritt, 1997).

Many of the large barley storage proteins, and in particular hordeins – which are hydrophobic and scarcely soluble in a low alcoholic solution such as beer (ca. 4–7% ethanol) – are precipitated and removed during the mashing and the boiling stages, whereas most of the free amino acids in wort are taken up by yeast during fermentation. In beer, the content of proteins recognised by anti-gliadin antibodies has been estimated to be approximately three orders of magnitude lower than in raw malt (Silva et al., 2008). In several previous investigations, large-sized proteolytic fragments of hordein were reported to be present in beer (Asano et al., 1982, Dostálek et al., 2006, Kaverva et al., 2005a, Kaverva et al., 2005b, Robinson et al., 2007, Sheehan and Skerritt, 1997, Silva et al., 2008). However, data about persistence in beer of hordeins and/or large hordein-derived polypeptides are conflicting (Kaverva et al., 2005a, Kaverva et al., 2005b, Marshall and Williams, 1987, Robinson et al., 2007, Sheehan and Skerritt, 1997, Silva et al., 2008). Perrocheau, Rogniaux, Boivin, and Marion (2005), using a 2D IPG/SDS–PAGE proteomic approach followed by mass spectrometry-based identification, observed very low amounts of an intact γ-hordein, a minor hordein component of 32.8 kDa, in a beer sample. On the contrary, a very recent study identified 85 protein spots in beer, but found no evidence of hordeins (Iimure et al., 2010).

Due to its complexity, no attempt to characterise the low molecular weight peptide fraction of beer has been carried out so far. In particular, the probable presence of hydrolytic peptides of hordeins which could retain or even amplify the immuno-stimulating proprieties of the barley prolamins has not been adequately investigated.

Because of the uncertainties associated to the identification of hordeins and gliadin-like epitopes in this beverage, usually beer is not allowed in the coeliac diet on a “a priori” basis (Ellis, Freedman, & Ciclitira, 1990). The coeliac enteropathy is, in fact, triggered by the exposure of genetically predisposed subjects to the prolamin fraction of cereals such as wheat, barley or rye. However, the “a priori” approach has revealed itself inconsistent for a number of other cereal-derived popular foods that have been proven to be gluten-free when analysed with appropriate protocols (Dostálek et al., 2009, Iametti et al., 2004, Kaukinen et al., 2008).

In a previous report (Iametti, Bonomi, Ferranti, Picariello, & Gabrovska, 2004) the peptide patterns of a number of beer varieties has been traced by matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-TOF MS), demonstrating that the high heterogeneity of the beer peptide fraction does not allow the straightforward identification of epitopes potentially responsible for triggering the intestinal responses in coeliac patients.

In this study, the protein and peptide fractions of two commercial Italian barley malt beers made with different processes by the same producer have been characterised with a proteomic approach. High resolution chromatography and electrophoresis, in combination with MS and tandem MS techniques, were addressed to detect potentially antigenic peptides in either beer samples. A competitive ELISA assay based on the anti-gliadin monoclonal R5 antibody (Ferre, García, & Méndez, 2004) was also used to assess the content of gluten-like epitopes with the aim of producing information useful to evaluate the risk related to beer consumption by coeliac patients.