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
1
Malt & Beer Sciences
KULeuven-M²S-MBS
Department of Microbial and Molecular Systems (M²S), CLMT, Leuven Food Science and Nutrition Research
Centre (LFoRCe), Kasteelpark Arenberg 22, bus 2463, BE-3001 Heverlee, Belgium
2 Department of Microbial and Molecular Systems (M²S), Centre for Surface Chemistry and Catalysis (COK),
Kasteelpark Arenberg 20, BE-3001 Heverlee, Belgium;
*Corresponding author: zahra.shokribousjein@biw.kuleuven.be
Introduction:
Gushing is a phenomenon observed
with many carbonated beverages such as beer, in which
without any agitation it vigorously overfoams on opening
(Sarlin et 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 (Deckers et 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 (Sarlin et al., 2005).
Class II hydrophobins include one α-helix and two βhairpin loops (Fig 1, left side). There are eight Cys
residues in the amino acid sequences of these proteins
which make 4 disulfide bonds, and give rigidity to their
structure (Linder et al., 2005).
Objectives
According to the conserved hydrophobic patch among Class II hydrophobins, it is deduced
that they can make hydrophobic interactions. This can be with free molecules added during
the brewing process or absorbent active surfaces (Fig. 3) present in filters or in transferred
pipes. This is the basis to find a curative method for primary gushing.
1
2
3
4
Fig 3. self-assembly of hydrophobins on the hydrophobic surface (e.g. teflon) (Wosten et al, 2000)
This Fig demonstrate monomers of Class II hydrophobins in a solution (1), self-assembles of
hydrophobins in contact with a hydrophobic surface (2) this surface becomes hydrophilic after
covered with hydrophobins (3), water contact angle shows increase in hydrophilicity of this
surface (4).
Therefore, the objectives of this project are:
General objective:
- Inactivation of hydrophobic patch of Class II hydrophobins
Specific objective:
Fig 1.HFB II hydrophobin (Linder et al., 2005)
The surface of Class II hydrophobins, is mainly
hydrophilic (yellow in fig 1, right) but there are conserved
aliphatic side chain amino acids (fig 2) also on the surface
of these proteins which form a flat hydrophobic patch
(green in Fig 1, right). This configuration makes the
molecule amphiphilic (Hakanpaa et al, 2004).
- Investigation of curative methods for primary gushing of carbonated beverages based on
exact knowledge
Scientific objective:
- Fundamental understanding of inactivation mechanisms
- Physico-chemical restrictions and behaviour of the hydrophobic links
Industrial objective:
- Make realistic solutions available at industrial scale (antifoams from hops)
- Hydrophobic interaction with hydrophobic molecules like antifoams from hops prepared
from emulsion of hop fats and waxes with lecithin as emulsifier (sample A) (Ford et al,
Barth Innovations Ltd, Paddock Wood, UK) already started. This product is used as foam
controler in fermentors. This experiment gave good results, showing that this product
decreased gushing significantly (Shokribousjein et al, 2012, not published yet) (Table 1).
Table 1. Effect of sample A on gushing in presence of HFBI extracted from T. reesei
sample
200 µl HFBI
50 µl sample A
+ 200 µl HFBI
Overfoaming (g/lit)
77,81
No gushing
Fig 2. Aliphatic amino acids in HFBII in two orientations
(Hakanpaa et al, 2004)
This study is now continued by studying the composition of antifoams from hops which
affect gushing.
Economic objective:
- Prevent economical damages due to beer returned by primary gushing and to propose
these solutions for worldwide appliance.
Fig 3. Contaminated CO2 gaseous molecules by Class II
hydrophobins(Deckers et al, 2010)
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.
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 resolution structure of the HFBII hydrophobin, a self-assembling amphiphile.
 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., Racja Ferreira, 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 Biophysica Acta.
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