Objectives

1. Inactivation of Class II hydrophobins as curative method for primary gushing Shokribousjein, Zahra1*., Deckers, Sylvie1., Khalesi, Mohammadreza1., Riveros, David Santiago1., Gebruers, Kurt1., Verachtert, Hubert1., Michiels, Chris1., Martens, Johan2 ., Delcour, Jan1., Derdelinckx, Guy1 1Department of Microbial and Molecular Systems (M²S), CLMT, Leuven Food Science and Nutrition Research Centre (LFoRCe), KasteelparkArenberg 22, bus 2463, BE-3001 Heverlee, Belgium 2Department of Microbial and Molecular Systems (M²S), Centre for Surface Chemistry and Catalysis (COK), KasteelparkArenberg 20, BE-3001 Heverlee, Belgium; *Corresponding author: zahra.shokribousjein@biw.kuleuven.be Malt & Beer Sciences KULeuven-M²S-MBS Introduction: Gushing is a phenomenon observed with many carbonated beverages such as beer, in which without any agitation it vigorously overfoamson opening (Sarlinet al., 2005). The mechanism of gushing is related to the contaminated CO2 gaseous molecules by Class II hydrophobins, which grow and explode on opening of the bottle as the result of the pressure drop (Deckerset al, 2010). Hydrophobins are small surface active proteins which are introduced to the malt by filamentous fungi and cause gushing. There are two classes of hydrophobins, Class I and Class II hydrophobins, which Class II is only reported to cause gushing (Sarlinet al., 2005). Class II hydrophobins includeoneα-helix andtwoβ-hairpin loops (Fig 1, left side). There are eightCysresidues in the amino acid sequences of these proteinswhich make 4 disulfide bonds, andgiverigiditytotheirstructure(Linder et al., 2005). Fig 1.HFB II hydrophobin (Linder et al., 2005) The surface of Class II hydrophobins, is mainlyhydrophilic (yellow in fig1, right) but there are conservedaliphatic side chain amino acids (fig 2) alsoon the surface of these proteinswhich form a flat hydrophobic patch (green in Fig1, right). Thisconfigurationmakesthe molecule amphiphilic (Hakanpaaet al, 2004). Fig 2. Aliphaticamino acids in HFBII in twoorientations (Hakanpaaet al, 2004) Fig3. Contaminated CO2 gaseousmoleculesbyClass II hydrophobins(Deckers et al, 2010) Objectives According to the conservedhydrophobic patch among Class II hydrophobins, it is deducedthatthey can make hydrophobicinteractions. This can bewith free moleculesaddedduring the brewingprocess or absorbentactivesurfaces(Fig. 3) present in filters or in transferredpipes. This is the basis tofind a curativemethodforprimarygushing. 1 2 3 4 Fig 3. self-assembly of hydrophobins on the hydrophobicsurface (e.g. teflon) (Wostenet al, 2000) ThisFigdemonstratemonomers of Class II hydrophobins in a solution (1), self-assembles of hydrophobins in contact with a hydrophobicsurface (2) thissurfacebecomeshydrophilicaftercoveredwith hydrophobins (3), water contact angle shows increase in hydrophilicity of thissurface (4). Therefore, the objectives of this project are: General objective: - Inactivation of hydrophobic patch of Class II hydrophobins Specificobjective: - Investigation of curative methods for primary gushing of carbonated beverages based on exact knowledge Scientificobjective: - Fundamentalunderstanding of inactivation mechanisms - Physico-chemicalrestrictionsandbehaviour of the hydrophobic links Industrial objective: - Make realistic solutions available at industrial scale (antifoams from hops) - Hydrophobicinteractionwithhydrophobicmoleculeslikeantifoamsfrom hops preparedfromemulsion of hop fats andwaxeswithlecithin as emulsifier (sample A) (Ford et al, Barth InnovationsLtd, Paddock Wood, UK) alreadystarted. This product is used as foamcontroler in fermentors. This experiment gave goodresults, showingthatthis product decreasedgushingsignificantly (Shokribousjeinet al, 2012, notpublishedyet) (Table 1). Table 1. Effect of sample A on gushing in presence of HFBI extractedfrom T. reesei Thisstudy is nowcontinuedbystudying the composition of antifoamsfrom hops which affect gushing. Economicobjective:-Prevent economical damages due to beer returned by primary gushing and to propose these solutions for worldwide appliance. • References • Deckers, S.M., Gebruers, K., Baggerman, G., Lorgouilloux, Y., Delcour, J.A., Michiels, C., Derdelinckx, G., Martens, J., Neven, H., 2010. CO2-hydrophobin structures acting as nanobombs in beer. Brew. Sci. 63, 54–61. • Hakanpaa, J., Paananen, A., Askolin, S., Nakari-Setala, T., Parkkinen, T., Penttila, M., Linder, M. B., Rouvinen, J. 2004. Atomic resolutionstructure of the HFBII hydrophobin, a self-assemblingamphiphile. • Linder, M.B., Szilvay, G.R., Nakari-Setälä, T., Penttila, M.E., 2005. Hydrophobins: the protein-amphiphiles of filamentous fungi. FEMS Microbiol. Rev. 29, 877–896. • Sarlin, T., Nakari-Setälä, T., Linder, M., Penttilä, M., Haikara, A., 2005. Fungal hydrophobins as predictors of the gushing activity of malt. Journal of the Institute of Brewing. 111 (2), 105–111. • Ford, YY., Westwood, KT., Gahr, A., RacjaFerreira, A., Wolinska, K., Lad, M. Antifoams from hops. (communicated by Yannick Ford: yannick.ford@barthinnovation.com) • Wosten, H.A.B., de Vocht, M.L.B. 2000. Hydrophobins, the fungal coat unravelled. Biochimica et BiophysicaActa. 1469, 79-86. Acknowledgements: Financial support from Duvel-Moortgat, Orval, Chimay breweries and Cargill Malting are gratefully acknowledged. The authors thank Spadel S.A. company for producing sparkling water. ProMeta is thanked for MALDI-TOF measurements which were carried out there. Barth Innovation Ltd is gratefully thanked for antifoam sample.