Gushing describes the spontaneous over foaming of a carbonated beverage upon opening of a bottle without previous agitation. The phenomenon has been observed in carbonated beverages such as beer (Christian et al., 2011; Fischer, 2001), champagne (Kemp et al., 2015) or sparkling juice drinks (Schuhmacher, 2002). The phenomenon is caused by the presence of high concentrations of hydrophobic condensation nuclei at which dissolved carbon dioxide will instantly turn into the gas phase upon pressure release during opening of the bottle, forming gas-filled bubbles that grow fast and rise upwards, leading to overfoaming of the liquid (Pel- laud, 2002; Casey, 1996).
Gushing is a multicausal phenomenon and two types are commonly distinguished in regard to the causative factors involved (Amaha and Kitabatake 1981; Casey 1996). The term ‘secondary gushing' is used for all technological factors, e.g. dust or other particulate matter, particles leaking from filter materials, or particulate calcium oxalate crystals (Carrington et al., 1972), that cause introduction of nucleation particles into the bottled beverage. Secondary factors can typically be handled by modifying the brewing and filling process to result in exclusion of such particles or surfactants. Primary type gushing is exclusively related to the use of malt or unmalted cereals that have been infected by certain fungi during growth in the field or in the malt house (Gjertsen and Trolle, 1963). Based on assumptions about possible structure/effect relationships, Hippeli and Elstner (2002) were the first to publish speculations on a possible role of hydrophobins as gushing inducers in beer. Authors were obviously unaware of the fact that Haikara et al. (1999) had filed a PCT patent (WO 99/54725) already in 1999 (with priority to a national Finnish patent from 1998) in which hydrophobins were used as indicators for gushing in carbonated beverages (Haikara et al., 1999). Today it has become a generally accepted doctrine that these extremely amphiphilic fungal proteins are responsible for the induction of primary gushing in beer (Sarlin et al., 2005a; Garbe et al., 2011; Specker, 2014).
Hydrophobins have been shown to be produced by a great variety of species within the filamentous fungi so that their production seems to be a general principle in that group (Talbot, 1997). One of their obvious natural functions is to decrease the surface tension of water by self-assembling at water/air interfaces, thus enabling transition of this barrier during the production of aerial mycelia (Wosten et al., 1999; Cox et al., 2007). Another role may be to establish proper contact between fungal cells and host tissue during plant infection (Wosten et al., 1994; Kim et al., 2005). All known proteins of that type share a common pattern of eight cysteines at conserved positions. Sequences between cysteines are however highly variable but the four-domain secondary structure always results in the formation of extremely amphiphilic proteins. Two subgroups of hydrophobins, class 1 and class 2, have been identified according to differences in spacing and sequence between cysteines, hydrophobicity patterns and solubility in organic solvents (Wessels et al., 1994). Among the few hydrophobins available in purified form from transgenic Pichia pastoris cultures only those belonging to class 2 have been shown to induce gushing (Sttibner et al., 2010; Lutterschmid et al., 2011; Niu et al., 2012; Sarlin et al., 2012). Hydrophobins enter the barley-to-beer chain upon use of fungal-contaminated brewing malt (Sarlin et al., 2007). Contaminations with Fusarium spp. such as F. graminearum, F. culmorum or F. poae have been found to be highly correlated with gushing induction in the beer produced (Gjertsen et al., 1965; Niessen et al., 1992; Schwarz et al., 1996; Sarlin et al., 2005b).
The mechanism of action of hydrophobin- induced gushing as well as the way they interact with other promoting or inhibiting factors is still a matter of debate. Currently, a mechanism that is in accordance with the thermodynamic approach of the ‘nano-bomb' theory described by Shokri- bousjein et al. (2011) and refined by Deckers et al. (2013) seems to provide many explanations for the phenomenon observed during primary gushing. The theory is in line with observations about interactions found between hydrophobins and the regular beer foam proteins nsLtp1 and Z4 (Sttibner et al., 2010; Specker et al., 2014) as well as their interaction with lipophilic hop components (Gardner et al., 1973; Lutterschmid et al., 2010; Mtiller et al., 2010; Shokribousjein et al., 2014). Hydrophobin layers fulfil the basic assumptions made in the varying permeability model (for a review see Pellaud, 2002). Simulation of molecular dynamics of carbon dioxide condensation resulted in evidence for a clustering of CO2 molecules at the hydrophobins hydrophobic patch, thus supporting the interaction of CO2 and hydrophobins (Deckers et al., 2012a). According to the nano-bomb theory, small particles of 5-10 nm in diameter represent hydrophobin-coated CO2 micro-bubbles. These develop during yeast fermentation, filling and shaking of bottles. At a critical diameter, the hydrophobic/hydrophilic monolayer hydrophobin film surrounding the bubble becomes impermeable and further shrinkage is prevented according to the varying permeability model. The resulting nano-bubbles were calculated to possess an internal pressure of about 4 bar (Deckers et al., 2010, 2012b). During opening ofthe bottle, the gas-liquid equilibrium between beer and the atmosphere is abruptly misbalanced and nano-bubbles present in the beverage will expand explosively and CO2 from the surrounding liquid phase will diffuse into the bubble leading to uncontrolled bubble growth (Pel- laud, 2002). The rapid expansion of micro bubbles provides the energy which is used to break bonds between CO2 and water molecules in the vicinity of an expanding bubble, hence the name ‘nano bomb. This eventually forces CO2 molecules to transit from the water-soluble state into the gas phase by free diffusion and formation of unstabilized secondary gas bubbles that rise to the surface in masses resulting in gushing (Deckers et al., 2010).
The model recently presented by Specker (2014) is in broad agreement with the nano-bomb model. However, this author demonstrated that addition of purified transgenic nsLtp1 to beer or carbonated water previously mixed with a gushing- inducing concentration of purified transgenic class 2 hydrophobin FcHyd5p from F. culmorum resulted in a significant decrease of gushing volumes compared with a nsLtp1-free control. Results obtained from atomic force microscopy analysis of mixed hydrophobin/nsLtp 1 surface films suggested that nano-bubbles present in gushing beer may in fact be surrounded by mixed layers of amphiphilic proteins which tend to be more prone to disruption than films of either pure protein. Moreover, the author deduced that formation of secondary bubbles during gushing events comes from CO2 nucleation at the hydrophobic inner layers of now exposed fragments of the disrupted bubble skin.