Induction of Gushing with Recombinant Class II Hydrophobin FcHyd5p from Fusarium culmorum and the Impact of Hop Compounds on its Gushing Potential

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1 Induction of Gushing with Recombinant Class II Hydrophobin FcHyd5p from Fusarium culmorum and the Impact of Hop Compounds on its Gushing Potential Georg Lutterschmid, Matthias Stübner, Rudi F. Vogel and Ludwig Niessen * ABSTRACT J. Inst. Brew. 116(4), , 2010 Malt-induced gushing is a problem that has been known for many years. Mechanisms and inducing agents are still not fully understood and identified. Hydrophobins produced by various filamentous fungi are currently under discussion as biological gushing-inducing compounds. In the current study the class II hydrophobin FcHyd5p, from the cereal pathogen Fusarium culmorum, was employed in beer and other carbonated beverages for gushing experiments and the influence of hop compounds on gushing potential was examined. It was demonstrated that this protein strongly induces gushing in various carbonated beverages, including beer. It was further demonstrated that the resulting gushing volume is susceptible to certain hop compounds and can be decreased significantly by the addition of these substances. Key words: beer, carbonated water, FcHyd5p, Fusarium culmorum, gushing, hop compounds, hydrophobin. INTRODUCTION The phenomenon of gushing, i.e. the spontaneous overfoaming of beer and other carbonated beverages immediately upon opening of a bottle, is a highly unwanted condition. It causes severe economic losses to breweries and producers of carbonated drinks and is resistant to the unveiling of the mechanisms and inducing agents involved. A first step towards solving this problem was carried out by dividing this unwanted property into two groups, by recognizing its origins in technical failures known as secondary gushing and in the quality of the raw material more precisely in the use of malt produced from mouldinfected grain primary gushing 14. Triggers for secondary gushing were identified as calcium oxalate crystals, metal Lehrstuhl für Technische Mikrobiologie, Technische Universität München, Weihenstephaner Steig 16, Freising, Germany. * Corresponding author. niessen@wzw.tum.de Parts of this paper were presented at the 2nd International Symposium for Young Scientists and Technologists in Malting, Brewing and Distilling, May 19 21, 2010 in Freising Weihenstephan, Germany. Publication no. G The Institute of Brewing & Distilling ions, cleansing agent residues or impurities in bottles 2,8,20,31. These factors can be eliminated by good manufacturing practice and proper handling of brewing equipment. For primary gushing, it is suggested that fungal infections of the brewing malt are responsible for overfoaming of beer. Prentice and Sloey 29 found that from 97 microorganisms isolated from barley, only Fusarium spp. and two other isolates caused gushing. Several other fungi were tested positive in producing uncharacterized, gushing inducing substances 1,11,15,23. Among the fusaria, F. graminearum and F. culmorum have been particularly associated with the occurrence of gushing 13,28,34. Hitherto uncharacterized gushing factors, which were found to be produced in fungal cultures and in cereal malt after fungal infection, were described and comprised peptides, peptidoglycans or surface active proteins. Proteins with such properties were isolated from pure cultures of Nigrospora sp. and also from wheat malt after infection with F. culmorum. They were shown to be water soluble, heat stable and surface active. Addition of these proteins to beer induced strong gushing 22,36. A new class of fungal proteins, the hydrophobins, share many of the properties described above. Hydrophobins are small and extremely surface active proteins secreted by most filamentous fungi. They have come into focus as a gushing factor in recent years 16,24,33. Zapf et al. 40 demonstrated that wort fermented with a class II hydrophobin (FcHyd5p) producing transgenic brewing yeast showed strong gushing. Heterologous expression of the class II hydrophobin FcHyd5p in Pichia pastoris was recently described 35. In that particular communication it was demonstrated that this protein was able to induce gushing in beer and also in carbonated water as a model system. The occurrence of primary gushing strongly depends on the presence of micro bubbles in affected beverages. These micro bubbles serve as condensation nuclei, when carbon dioxide is released from the supersaturated beverage after pressure release, and grow, if their radius exceeds the critical radius according to Laplace 38. Gas bubbles in closed, carbonated beverages normally shrink due to Laplace pressure and surface tension of water-gas interface and dissolve unless they are stabilized. Such stabilization can be achieved by incorporation of surface active molecules into the interface between the aqueous phase and the gas phase 9,25. In non-gushing beer, ca. 2,500 micro VOL. 116, NO. 4,

2 bubbles per ml can be found 6. Those micro bubbles have to be stabilized by the surface active proteins present in the beer 9. In gushing beer, the number of stabilized micro bubbles must thus be increased or the composition of the bubble skin changed. It has also been reported that foaming in beverages is dependent on the size at which the bubbles are stabilized 10. Bubbles with radii greater than the critical radius can grow after supersaturation, and bubbles with smaller radii are not able to grow due to the dominating forces of surface tension and the pressure of the surrounding liquid. This means that in gushing beer, the number of bubbles capable of growth should be increased as compared to non-gushing beer. In addition to the search for the source of gushing, many attempts have been made to find ways to inhibit this phenomenon. A study of the literature reveals that physical approaches can be found, e.g. heating of bottled beer, mechanical percussions or the use of ultrasonication 18,32. Beside these technical methods, a promising possibility to overcome the gushing problem was described by Curtis et al. 7 and Munekata et al. 27, who demonstrated the ability of hops to prevent gushing when applied in sufficient amounts. In the current study, gushing-inducing potential of hydrophobin FcHyd5p in carbonated beverages is further elucidated, as well as the mechanisms leading to overfoaming. The capability of certain hop compounds to reduce or inhibit gushing in beer and other carbonated beverages is examined. MATERIALS AND METHODS Hop compounds and carbonated beverages The following hop compounds were used in gushing experiments: Hop Oil Type Dry (complete range of hop essential oils, main components are myrcene, humulene and caryophyllene, content of linalool ca. 1 2%); Rho 35% (potassium salts of reduced iso-α-acids); Tetra 10% (potassium salts of tetrahydro-iso-α-acids); Iso 30% (isomerized α-acids) (all products above obtained from Hopsteiner, Mainburg, Germany); Linalool 97% (Sigma Aldrich, Steinheim, Germany); bottom fermented beer, different brands, 0.5 L per bottle; carbonated beverages, different brands, black currant juice, 0.5 L per bottle; apple juice, 0.5 L per bottle; elder juice, 0.33 L per bottle; and apple/grape juice, 0.5 L per bottle; carbonated water, Bonaqa, as used for the modified Carlsberg test 30, 0.33 L per bottle. Microbiological growth media For cultivation of P. pastoris X33 from the EasySelect TM Pichia Expression Kit (Invitrogen, Carlsbad, USA) and P. pastoris X33 [ppiczαa-fchyd5p], TMW 3.213, from the strain collection of the Lehrstuhl für Technische Mikrobiologie Weihenstephan, as published by Stübner et al. 35, a buffered glycerol complex medium (BMGY) (1% [w/v] yeast extract; 2% [w/v] peptone; 100 mm potassium phosphate, ph 6.0; 1.34% [w/v] yeast nitrogen base containing ammonium sulphate without amino acids; % [w/v] biotin; 1% [w/v] glycerol) was used. Yeast extract and peptone were mixed in water and autoclaved; remaining ingredients were filter sterilized and added to base medium at room temperature. Expression of hydrophobin FcHyd5p was conducted in a buffered minimal methanol medium (BMM) (100 mm potassium phosphate, ph 6.0; 1.34% [w/v] yeast nitrogen base containing ammonium sulphate without amino acids; % [w/v] biotin; 0.5% [v/v] methanol). All ingredients were filter sterilized and added to sterile deionised water. Heterologous expression and preparation of FcHyd5p Expression of the hydrophobin was performed using P. pastoris strain X33 [ppiczαa-fchyd5p] as described by Stübner et al. 35 The yeast was cultivated in 50 ml BMGY medium overnight at 30 C and 180 rpm to an optical density of OD 600 = 2.0 to 6.0. After removal of the supernatant, cells were transferred into a Fernbach flask, containing 200 ml BMM medium, and grown for 4 days at 30 C. Expression of FcHyd5p was induced by adding 0.05% (v/v) methanol every 24 h. Sufficient aeration was achieved by horizontal shaking at 180 rpm and sealing the flask with a gauze bandage. Following incubation, cells were removed by centrifugation at 6,000 g for 30 min at ambient temperature. Supernatants were dialyzed for 4 days at 4 C against daily changed deionised water, using tubes with a molecular weight cut-off of 3,500 Da (Serva Electrophoresis GmbH, Heidelberg, Germany) and freeze dried after centrifugation at 6,000 g for 15 min at ambient temperature. Wild type strain P. pastoris X33 from the EasySelect TM Pichia Expression Kit (Invitrogen, Darmstadt, Germany) was cultivated and the supernatant processed as described above. This lyophilisate was used as negative control. Induction of gushing and application of hop products Over the period, in which the gushing experiments were performed, five different lots of German lager beer from one brewery were used, in some cases German lager beers from different breweries were tested. Since gushing instability was found in some lots, the gushing tendency of each lot was tested before purchasing greater quantities. This was performed by rotating bottles for 16 h at ambient temperature (28 rpm) and opening after 1 h of rest. Only beer lots that showed no over-foaming after the treatment were used for further experiments. Other carbonated beverages were used without previous examination. For the induction of gushing, bottles were chilled to 0 C in an ice-salt bath to minimize CO 2 loss upon opening. After opening, transgenic hydrophobin FcHyd5p was added and bottles were resealed with sterilized crown caps. Hop products were added at the same step as hydrophobins. The amounts added were based on the recommendations of the manufacturer, in variation experiments higher and lower amounts were added. For comparison with hop oil type dry amounts of linalool were higher than recommended. Treated bottles were rotated for 16 h at ambient temperature (28 rpm) and opened after 1 h of rest. Loss of volume was measured by weighing bottles before and after opening. All tests were conducted in triplicate. 340 JOURNAL OF THE INSTITUTE OF BREWING

3 Ultrasonication treatment of FcHyd5p solution Freeze dried FcHyd5p was dissolved in deionised water to a concentration of 2 mg per ml and subjected to an ultrasonication treatment. This was performed using an ultrasonic probe (Sonoplus, Bandelin, Germany) or an ultrasonic bath (Sonorex, Bandelin, Germany). For the ultrasonic probe, two repetitions of 30 sec each were applied at 70% power and 7 10% cycle, while samples were kept on ice. For the ultrasonic bath, samples were placed on a floater in the bath and treated for 3 min at room temperature with permanent operation. The FcHyd5p solutions were treated immediately prior to addition to the beer or other carbonated beverages. RESULTS Gushing potential of FcHyd5p Hydrophobin FcHyd5p preparations were added to beer and other carbonated beverages, i.e. sparkling mineral water, black currant juice, apple juice, elder juice and apple/grape juice to examine the gushing potential in beer and other less complex beverages. It was found that overfoaming could be induced in each carbonated beverage, whereas all negative controls containing no FcHyd5p could be opened without any loss of content. Figure 1 shows the gushing volumes found with the carbonated drinks. Inoculation of beverages with lyophilisate obtained from culture supernatants of the wild type yeast P. pastoris X33 resulted in beverages without gushing. In beer, a relationship between the amounts of FcHyd5p applied and the resulting loss of liquid volume was found (Fig. 2). A linear correlation existed for the addition of up to 2 mg FcHyd5p per 500 ml bottle, with an average volume loss of 273 ml. Addition of more than 2 mg of the protein resulted in no significant further increase, hence 2 mg of hydrophobin per 500 ml bottle was applied as the standard in all further experiments. When FcHyd5p was added to beer without subsequent rotation, gushing was only observed in samples in which the lyophilisate was added without previous solution in water. Addition of dissolved FcHyd5p without rotation of the bottles did not result in gushing. Bottles, which were rotated prior to opening, always showed gushing, whether the hydrophobin was added in a solid state as lyophilisate or as a solution of FcHyd5p in water. Even so, addition of 1 ml FcHyd5p solution with an amount 2 mg hydrophobin per ml resulted in identical gushing volumes as compared to the addition of 2 mg hydrophobin as lyophilisate. Due to easier handling and higher accurateness of dosing, subsequent experiments were performed with the hydrophobin dissolved in water prior to addition. During the experiments, it was observed that gushing volumes resulting from addition of hydrophobin to beer of different lots from the same brewery, as well as to beer from different brands, were subject to high variation. Beer from a different brewery appeared to be more resistant to hydrophobins and showed no gushing upon addition of 2 mg FcHyd5p. However, addition of higher amounts revealed principally a similar relationship between added quantities and gushing volume. From all samples tested, including beer from different lots and different breweries, the average gushing volume of 115 bottles upon addition of 2 mg hydrophobin was 210 ml ± 57. As opposed to beer, carbonated water proved to be much more sensitive to inoculation with FcHyd5p. Addition of hydrophobin led to an abrupt and uncontrolled push-out of water with a fountain up to 3 m in height. Addition of 0.1 mg hydrophobin to a 0.33 L bottle resulted in a lost volume of 162 ml, while the maximum volume reached with the addition of 2 mg hydrophobin was 179 ml. Addition of FcHyd5p without subsequent rotation led to reduced, but still measurable, gushing vol- Fig. 1. Gushing volumes in percent of the bottle volume upon addition of 2 mg transgenic hydrophobin FcHyd5p to carbonated beverages. 1 = black currant juice, 2 = apple juice, 3 = carbonated water, 4 = elder juice, 5 = apple/grape juice. There was no lost volume on control beverages without added FcHyd5p. VOL. 116, NO. 4,

4 Fig. 2. Relationship between amount of added transgenic hydrophobin FcHyd5p and gushing volume in German lager beer (0.5 L per bottle). Fig. 3. Relative inhibition of gushing volumes after addition of transgenic hydrophobin FcHyd5p and hop oils to German lager beer (0.5 L per bottle). FcHyd5p = gushing induced by transgenic hydrophobin FcHyd5p without inhibition (=100%). HOTD = inhibition of gushing by addition hop oil product hop oil type dry. Linalool = inhibition of gushing by addition of linalool. umes for both FcHyd5p lyophilisate and dissolved hydrophobin. Subsequent experiments with carbonated water were performed with addition of 1 mg of hydrophobin dissolved in deionised water per 0.33 L bottle. The resulting gushing volume obtained from several tests was 174 ml ± 4. Thermal stability of FcHyd5p In order to function as gushing-inducing agents in beer, it is assumed that active compounds must retain their triggering properties even after a thermal treatment such as wort boiling during the production of beer. To examine heat stability, FcHyd5p was boiled in water or synthetic wort in order to simulate wort boiling conditions. Since it is impossible to remove all proteins from conventional wort, synthetic wort containing acid and sugar components, in their characteristic concentrations, was produced according to Zapf 39. During heat treatment, no precipitation of the protein was observed. Addition of FcHyd5p boiled in water, instead of in synthetic wort, into the beer resulted in no change in the gushing potential. In contrast, addition of hydrophobin boiled in synthetic wort resulted in a gushing volume of 123 ml ± 63, which was a reduction of 41% as compared to the volume caused by un- 342 JOURNAL OF THE INSTITUTE OF BREWING

5 Fig. 4. Relative inhibition of gushing volumes after addition of transgenic hydrophobin FcHyd5p and iso-α-acid products to German lager beer (0.5 L per bottle). FcHyd5p = gushing induced by transgenic hydrophobin FcHyd5p without inhibition (=100%). Rho = inhibition of gushing by addition of iso-α-acid product Rho 35%. Tetra = inhibition of gushing by addition of iso-α-acid product Tetra 10%. heated hydrophobin. Addition of hydrophobin boiled in water or synthetic wort did not result in a change of the gushing potential in carbonated water. Ultrasonication treatment of FcHyd5p Since it was observed in the experiments described above that FcHyd5p was able to induce gushing without rotation of the bottles when added as lyophilisate, ultrasonication was applied to FcHyd5p solutions in order to disintegrate assembled hydrophobin molecules acting as condensation nuclei for carbon dioxide. To exclude the influence of a possible introduction of iron derived from ultrasonication equipment, a well-known secondary gushing inducing factor, water was subjected to ultrasonic probe treatment before addition to non-gushing beer. No gushing was detected in these samples. Subsequent gushing experiments showed that ultrasonication treatment of FcHyd5p solutions did not result in a significant change in beer volume loss. As an effect of ultrasonication treatment, it was observed that standard deviations were decreased in samples treated with an ultrasonic probe as compared to samples with addition of untreated hydrophobin. Treatment of hydrophobins in an ultrasonic bath however, showed less reproducible results. Influence of hop oil products on hydrophobin induced gushing of beer Addition of linalool resulted in a strong reduction of over-foaming of beer (Fig. 3). A 2 µl aliquot of linalool reduced the lost beer volume by more than 90%, as compared to the untreated 2 mg hydrophobin containing control. When 10 µl of linalool was added, an almost complete inhibition of gushing was observed. Samples with linalool levels >10 µl showed no gushing. Addition of the hop oil type dry (HOTD) product to beer also had an inhibitory effect on hydrophobin induced gushing (Fig. 3). Adding 1 µl of HOTD reduced the lost volume to 25%, as compared to samples without HOTD. The addition of 3 µl of the product resulted in a further reduction of the gushing volume to 22% of the positive control and with several bottles over-foaming was completely inhibited. Negative controls, with only HOTD or linalool added, showed no gushing. Influence of iso-α-acid products on hydrophobin induced gushing of beer Beside hop oils, three different iso-α-acid products were applied to gushing experiments. It was observed that only the modified iso-α-acids were able to reduce hydrophobin-induced gushing. Reduced iso-α-acid (Rho 35%) and tetrahydro iso-α-acid (Tetra 10%) showed an inhibitory effect on gushing volume, whereas no such property could be detected with unmodified iso-α-acid (Iso 30%). Figure 4 shows the relative decrease of gushing volumes due to the addition of iso-α-acid products. The average reduction of lost volume for both modified iso-α-acids was more than 50%, however, these results were subject to considerable variation. Addition of Rho 35% showed the highest reduction, when 20 µl per bottle was applied, which led to a reduction to 20% as compared to the untreated positive control. Addition of 10 µl or 30 µl of the product resulted in lower reduction values. However, due to the high standard deviations of the results, no optimum value could be determined upon addition of Rho 35%. The gushing volume could also be reduced by addition of the Tetra 10% product. As shown in the figure, addition of 60 µl revealed the highest reduction volumes. However, due to the high standard deviations of the results, there was no significant trend. In contrast to the products containing modified iso-α-acids, addition of unmodified iso- VOL. 116, NO. 4,

6 α-acid led to an increase in the gushing volume. Negative controls containing only iso-α-acids and no hydrophobin resulted in beer without gushing for Rho 35% and Tetra 10%. However, gushing was observed when the Iso 30% product was added to the negative control beer. Influence of hop products on hydrophobin induced gushing in carbonated water The influence of hop compounds on hydrophobin-induced gushing was also tested in carbonated water (Fig. 5). This was conducted to see if the hop compounds used would be able to inhibit hydrophobin gushing by themselves, or if the presence of other substances from beer was necessary for this effect. The amounts of hop product added were identical to the lowest amounts that were added to beer. Addition of hop products to hydrophobintreated carbonated water resulted in an increase in the gushing volume for each hop product, as compared to samples solely inoculated with the hydrophobin. Addition of linalool to hydrophobin-treated carbonated water resulted in the highest increase in water emission. An increase in volume loss of 40% was observed after addition of 1 µl of linalool. Amounts of up to 100 µl of linalool per bottle resulted in the same gushing volume. Addition of 1 µl of HOTD showed no significant increase in the gushing volume. If 3 µl amounts were applied, the increase became significant (data not shown). Addition of the other hop products resulted in gushing volumes over 200 ml, whereas an increase of added hop compound resulted in a slight increase of gushing volume. Negative controls, containing one hop product each, resulted in gushing for the iso-α-products. Addition of the hop oil components did not result in gushing. DISCUSSION Gushing experiments clearly showed that hydrophobin FcHyd5p is capable of inducing gushing when added to beer. Since in practice, a beer with a volume loss of 20 ml would already be termed a gushing beer, concentrations of FcHyd5p sufficient to induce gushing to an extent unwanted by breweries would be even lower than what was observed during these experiments. The exact determination of the hydrophobin content in the lyophilisate was not simple, due to the difficulty in purification and detection of this protein. This may have resulted in an overestimation of the protein concentrations. FcHyd5p is totally resistant to silver staining. Also staining with Coomassie Blue following SDS polyacrylamide gel electrophoresis is only possible when the protein is applied in amounts >10 mg per ml. Comparison of lyophilisate recovery from the FcHyd5p producing yeast with the yield of lyophilisate derived from the wild type strain, resulted in an estimated content of 50 to 75% of hydrophobin in the lyophilisate. The tendency that the gushing volume is dependent on the amount of FcHyd5p added is supported by previous studies 3,5. The authors state that gushing could only be observed from a minimum concentration of gushing inducing substances. In the experiments, gushing was induced by the mere addition of freeze-dried hydrophobin FcHyd5p, without rotating the bottles, and hence without bringing bubbles into the beer mechanically. This indicates that micro bubbles were brought into the beer by the lyophilisate. These micro bubbles could be the result of air, which is retained in the porosity of the lyophilisate and stabilized by hydrophobin molecules upon dissolving in the beverage. This Fig. 5. Effect of hop products on hydrophobin FcHyd5p induced gushing in carbonated water. Results shown as absolute gushing volumes for each hop product after addition of FcHyd5p and hop product. Rho = iso-α-acid product Rho 35%. Tetra = iso-α-acid product Tetra 10%. Iso = iso-α-acid product Iso 30%. HOTD= hop oil product hop oil type dry. Linalool = hop oil component linalool. 344 JOURNAL OF THE INSTITUTE OF BREWING

7 effect disappeared and no gushing occurred when the hydrophobin was dissolved in water prior to addition, and bottles were not rotated. For carbonated water, both cases led to reduced gushing. It was assumed that this effect was caused by the higher sensitivity of that model system as compared to beer. In beer, new bubble interfaces are immediately covered by the surface active substances present as reported by Liger-Belair 26, whereas in carbonated water no such substances are available. Therefore, bubbles in beer, which are formed and introduced during opening and addition of FcHyd5p solution, are covered by hydrophobin molecules and surfaces active molecules from beer. In carbonated water, those bubbles can be stabilized exclusively by the hydrophobin molecules leading to gushing, even after addition of dissolved FcHyd5p. Christian et al. 4 also induced gushing in carbonated water by the addition of a surface active substance called CTAC. They further observed that gushing also occurred without shaking of the bottles. The authors assume that this is caused by the diffusion of CO 2 into the evacuated micelles of CTAC. The observation that carbonated water is much more sensitive to hydrophobin gushing can be explained by the absence of any stabilizing compounds. This is also a proof for the presence of substances in beer that are able to restrict hydrophobin gushing and provide a basic resistance to over-foaming (e.g. the amount and nature of surface active substances in beer). It was observed during the tests that this resistance completely prevented gushing upon addition of FcHyd5p in some bottles of beer from a different brewery. Apparently, there is gushing inhibition in some beers for unknown reasons. This should be investigated in further studies, to determine if there are substances present in some beer that provide resistance and whether gushing resistance can be increased by the choice of raw material or by modification of process parameters during beer production. One such parameter is the addition of hops, which might be a modulating factor of gushing activity in beer. It should be mentioned that the beer used already contained hops, however, responses to the addition of FcHyd5p sometimes differed greatly from lot to lot. This suggests that the gushing inhibiting behaviour of such beer cannot only be ascribed to the presence of hop compounds in these lots. In order to address this question, commercial products containing purified hop components were added to beer after addition of FcHyd5p. In the case of hop oils (linalool and HOTD) as well as with Rho 35% (potassium salts of reduced iso-α-acids) and Tetra 10% (potassium salts of tetrahydro-iso-α-acids), addition led to a reduction of gushing volumes with the highest reduction achieved by the addition of hop oils. This is in accordance with results published previously 12,25. The findings are also supported by the very recent work of Hanke et al. 17 The effects observed could be caused by the foam negative properties of the oil. However, a reduction of gushing volume was also achieved by the addition of the Tetra 10% product, which possesses foam-promoting properties. The gushing-enhancing effect of Iso 30% could have been caused by the mode of addition. It is recommended by the manufacturer that hop products should be added before filtration to ensure any contained particles are removed from the bottled beer. However, filtration could not be performed in the tests. The addition of hop products to hydrophobin treated carbonated water for each product resulted in an increase of the gushing volume. This increase could be explained by a surface-active effect of the products leading to a controlled gushing, in which the throttling property of the bottleneck is reduced. With amounts of hop products as used in beer an increase in the lost volume was found. These findings suggest that none of the hop products are capable of inhibiting gushing on their own. Further substances from beer may be needed to provide gushing inhibiting properties. The high standard deviations found in the gushing experiments may have been caused by factors such as the quality of the inner wall of the beer bottles or of the raw material used. However, this could also be a consequence of the presence or absence of gushing inhibiting compounds. It was demonstrated that the FcHyd5p protein is heat stable and maintains its gushing potential when boiled in water. The reduction of gushing potential in beer, by boiling the hydrophobin in synthetic wort, could be caused by glycosylation of the protein during heat treatment in the presence of wort sugars as was observed for nsltp1 19. In this case, such a glycosylation has to take place in the hydrophobic part of the protein, which would reduce its surface activity and explain the lower gushing potential. This matter has to be elucidated in more detail in the future. Ultrasonication treatment, which was applied as disintegrative conditioning to break down assembled hydrophobin molecules, showed no reducing influence on the gushing volume. This was in accordance with the results of the boiling tests with water dissolved hydrophobin and indicates that monomeric molecules migrate to water-air interfaces, where a concentration takes place and stabilized bubbles are formed. In the experiments, gushing was induced in beer and carbonated water after addition of hydrophobin FcHyd5p. According to Draeger 9 and Krause 25, the existence of micro bubbles is required for the occurrence of gushing. Since no surface active substances are available in carbonated water, gushing-inducing micro bubbles must be formed by hydrophobins alone. Therefore, the mechanism leading to gushing must be associated with the properties of the hydrophobin interface of the bubble. According to the theory as published by Fischer 10 the induction of gushing by substances such as FcHyd5p has to be caused by its ability to stabilize bubbles at sizes that enable them to grow after supersaturation on the one hand. On the other hand, since hydrophobins are able to form highly ordered and stable films in air-water interfaces 21,37, it is suggested that growth of the hydrophobin interface upon pressure release is limited in those bubbles. When a bottle is opened, hydrophobin covered bubbles grow until the surface tension of the hydrophobin layer reaches a point, where either growth is stopped or the bubbles burst providing new condensation nuclei for generation of gushing. In beer, three different types of micro bubbles are probable: bubbles with an interface composition identical to non-gushing beer, bubbles with a homogenous hydrophobin interface, and bubbles with a mixed interface composition containing both. Homogeneous hydrophobin bubbles behave similar to those in carbonated water, whereas VOL. 116, NO. 4,

8 hydrophobin domains in mixed bubbles disintegrate along the domain borders upon bubble growth, due to their limited ability to grow. In this stage they function as new condensation nuclei. CONCLUSIONS The current results show that the class II hydrophobin FcHyd5p is able to induce gushing in beer and other carbonated beverages. FcHyd5p is heat stable and maintains its gushing inducing potential, when boiled in synthetic wort. Also subjecting dissolved FcHyd5p to ultrasonication treatment immediately before addition to beer does not impair its gushing potential. Since the protein is naturally produced as a hyphal surface protein by F. culmorum, a fungus most commonly found on malts leading to primary gushing of beer, the results obtained here give strong experimental evidence that this compound also leads to the induction of beer gushing under natural conditions. It was shown that gushing induced by pure FcHyd5p was susceptible to hop oils and modified iso-αacid products and that the gushing volume can be decreased significantly by the addition of the those substances. ACKNOWLEDGEMENTS This research was supported by Arbeitsgemeinschaft industrieller Forschungsvereinigungen Otto von Guericke e.v. (AiF) (grant no. AiF 14551) and Weihenstephaner Jubiläums- Stiftung We also would like to thank Simon H. Steiner, Hopfen GmbH, Mainburg, Germany, for providing iso-α-acid products and Hop Oil Type Dry as well as Privatbrauerei Erdinger Weißbräu, Werner Brombach GmbH, Germany, for providing Bonaqa as used for the modified Carlsberg test. REFERENCES 1. Amaha, M., Kitabatake, K., Nagakawa, A., Yoshida, J. and Harada, T., Gushing inducers produced by some mould strains. Proceedings of the European Brewery Convention Congress, Salzburg, Elsevier: Amsterdam, 1973, pp Brenner, M. W., Gushing Beer II. Causes and Some Means of Prevention. 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