Detection and characterisation of Fusarium hydrophobins inducing gushing in beer: Dissertation

Gushing is a phenomenon in which beer spontaneously, without agitation, vigorously foams out from its container immediately on opening. Gushing has a marked negative effect on the overall image of beer. Numerous factors causing and contributing to gushing have been reported. Two types of gushing can be distinguished based on the origin of gushing-inducing substances. Non-malt related gushing, i.e. secondary gushing, is due to faults in the beer production process or to incorrect treatment of packaged beer. Primary gushing is induced by fungal metabolites, so-called gushing factors, which are present in malt or in other cereal raw materials of beer. Particularly species of the genus Fusarium have been linked to primary gushing. Although gushing factors produced by fungi have been studied for decades, none of them have hitherto been fully characterised. The hypothesis of this dissertation was that small fungal proteins called hydrophobins are one of the gushing factors inducing primary gushing. Hydrophobins are secreted, highly surface active, moderately hydrophobic proteins produced by filamentous fungi. Hydrophobins play key roles in the development and in the interactions of fungi with their environment and other organisms, particularly plants. The aim of this thesis was to isolate and characterise hydrophobins from gushing active fungi, especially from Fusarium species, and to demonstrate that these hydrophobins are able to induce gushing in beer. Currently, there is no practical, reliable and commercially available method for the prediction of beer gushing from large numbers of samples. The main goal of the work was to develop a test for detection of gushing potential of barley and malt by analysing the hydrophobin levels in samples. Moreover, the occurrence and fate of hydrophobins at different stages of the beer production chain were studied. This study revealed numerous effects of Fusarium fungi on the quality of barley grown under Finnish field conditions and of the corresponding malt. In particular, Fusarium infection increased the gushing potential of malt. The results of the study indicated that the extent of the impacts is species-dependent, F. graminearum having more severe detrimental effects on barley and malt quality than F. culmorum and particularly F. poae. We demonstrated that hydrophobins isolated from strains of the genera Fusarium, Nigrospora and Trichoderma induced beer gushing when added to bottled beer. Hydrophobin concentrations at the ppm level were sufficient for gushing induction. The gushing-inducing capabilities of the isolated hydrophobins varied probably due to their structural differences. We generated profile hidden Markov models for the different hydrophobin classes and searched the F. graminearum genome database for predicted proteins with these models. The search revealed five putative hydrophobin genes belonging to both the hydrophobin classes I and II. The best matching sequences and the corresponding genes were isolated from F. graminearum as well as from the related species F. culmorum and F. poae by PCR and were characterized by sequencing. One each of the putative F. graminearum and F. poae hydrophobin genes were expressed in the heterologous host Trichoderma reesei. The proteins corresponding to the genes were purified and identified as hydrophobins and named GzHYD5 and FpHYD5, respectively. Concentrations of 0.003 ppm of these hydrophobins were observed to induce vigorous beer gushing. An enzyme-linked immunosorbent assay (ELISA) was developed for determination of hydrophobin levels in barley and malt. A connection was found between the hydrophobin level and the gushing potential of malt, suggesting that the developed hydrophobin ELISA can be used for prediction of the gushing risk in malt. Fusarium fungi were observed to produce hydrophobins during the growing period of barley in the field as well as during the malting process, especially during the steeping and germination steps. A small portion of hydrophobins originating from Fusarium-infected malt was shown to pass through the brewing process, ending up in the final beer where they induced gushing when present in sufficiently high levels. Addition of a selected antagonistic starter culture, the yeast strain Pichia anomala VTT C-04565, into the steeping water of barley was shown to suppress hydrophobin production in malting, which in turn decreased the gushing potential of the corresponding malt.