Fungal profiling and gushing potential

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Fungal profiling and gushing potential Vertti Virkajärvi, Tuija Sarlin & Arja Laitila VTT Technical Research Centre of Finland arja.laitila@vtt.fi EBC Brewing Science Group 10 th Technical Meeting 9-11.9.2014 Vienna, Austria

Outline Beer gushing -background Characterization of fungi in gushing active raw materials Gushing active Fusarium strains Conclusions Fusarium culmorum 2

Beer overfoaming (gushing) A phenomenon in which beer spontaneously over foams out of the container immediately on opening 1. Secondary gushing = non-malt related 2. Primary gushing is induced by fungal infection. Fusarium fungi, also Aspergillus, Nigrospora, Penicillium Sporadic phenomenon, often related to rainy seasons during the harvest period Gushing affects the image of beer negatively significant economical losses Linder et al. 2005 3

Fungal hydrophobins identified as gushing inducers Primary gushing mainly related to Fusarium fungi Several different Fusarium species Genes related to hydrophobin production characterized from F. culmorum, F. graminearum, F. poae, F. verticillioides Similar genes also found in F. oxysporum, F. sporotrichioides, F. equiseti One Fusarium species may produce several different hydrophobins Gushing activity of hydrophobins differs Class II hydrophobins known as gushing active http://www.vtt.fi/publications/ index.jsp References. Sarlin et al. 2005, 2006, 2007, 2012 4

Fungal related gushing is sporadic, but is gushing propensity common and an increasing challenge? Only few publications on actual gushing cases German survey, over 50% of breweries have experienced gushing at least once (Niessen et al. 2007) Belgium 2008: small brewery beer with gushing potential. Announced in the newspaper: beer with champagne effect Gushing potential in malt Finland 1978-1979 (Haikara 1980) Germany 1987-1988 sekä 1998 (Bellmer 1996, Kunert et al. 2001, Winkelmann 2004) North-Europe 1993 (Riis et al. 1995) USA 1993-1998 (Schwarz et al. 1996, Steffenson 1998) Increasing (?) trend in Central Europe from the beginning of 2000 2002 (Aastrup 2003), 2007 exceptionally rainy summer in Central Europe (Gastl et al. 2009, Rath 2009), 2008-2011 (Gastl 2009), Crop 2012 5

Our aims Characterisation of fungi and gushing potential in suspected barley lots Intraspecific differences in gushing active Fusarium-fungi To gain more knowledge about the role of different fungi, especially Fusarium species, in beer gushing. Long-term aim is to develop early-warning systems and alarm limits for Fusarium contamination in brewing rawmaterials 6

Barley and malting 6 barley samples from different geographic locations (B1-B6). B1-B5 had shown gushing risk in small scale studies performed by the maltsters B6 control barley Barley samples were malted in a 1kg scale in specially designed, computer controlled micromalting equipment with a separate drum for each sample. Triplicate samples for analyses 7

Gushing and fungal analyses Method Carlsberg gushing test in beer and carbonated water VTT immunological hydrophobin test Fusarium selective CZID agar (EBC) Multispectral imaging with Videometer Real-time PCR with Fusarium group and species specific primers Fungal community profiling with PCR-DGGE Fungal community profiling with PCR-DHPLC Aim Gushing potential of malt (g) Estimation of hydrophobins, gushing inducers Number of contaminated kernels (%) Visualisation of infected kernels (mould contamination area %) * Performed by the Videometer A/S for some samples Quantification of Fusarium DNA (Fusarium DNA pg/ total DNA ng) Sample specific genetic fingerprits showing predominant fungal groups * PCR fragment separation in denaturing gradient gel PCR fragment separation with automated denaturing high performance liquid chromatography * Identification by sequencing 8

B1 B2 B3 B4 B5 B6 M1 M2 M3 M4 M5 M6 neg. malt pos. malt 1/abs Gushing potential and hydrophobins 8,0 7,0 Gushing in beer (g) 53 3 2 43* 51* 0 0 81 6,0 5,0 4,0 3,0 2,0 1,0 ** 0,0 * Induced gushing also in carbonated water ** 1,7 (1/Abs ) malt value as a risk level for gushing in Carslberg beer test 9

Fusarium contamination assessed with traditional plating on selective agar and multispectral imaging Fusarium CZID (%) Videometer % (barley) Sample Barley Malt Gray moulds Red Fus. B1 67 100 14,9 3,6 B2 50 100 17,8 4,2 B3 56 100 21,1 4,6 B4 94* 100 28,5* 1,9 B5 99* 100 - - B6 4 1 - - - = not determined 10

Fusarium DNA/tot. DNA (pg/ng) Quantification of Fusarium DNA in barley and malt with real-time PCR using group specific and species specific primers 9,0 8,5 8,0 7,5 7,0 6,5 6,0 5,5 5,0 4,5 4,0 3,5 3,0 2,5 2,0 1,5 1,0 0,5 Malt Fusarium sp. F. avenaceum F. graminearum F. culmorum F. poae F. langsethiae* 0,0 M1 M2 M3 M4 M5 M6 11

Fungal community profiling (PCR-DGGE) revealed potential causative species in gushing positive barley samples Pure cultures Gushing in beer and carbonated water M 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. M M: Fungi marker 1. P. anomala (C565) 2. F. graminearum (D470) 3. F. culmorum (D148) 4. F. tricinctum (D600) 5. F. avenaceum (D141) 6. B1 7. B2 8. B3 9. B4 10. B5 11. B6 M: Fungi marker 12

Fluorescence (mv) Fungal community profiles performed with PCR-DHPLC method mv 600 Pichia/Wickerhamomyces sp. (B1, B4 malts) Alternaria/Lewia sp. or related (B1, B2 barleys; B1, B2, B3 malts) F. avenaceum / F. tricinctum (B4, B6 barleys; B4 malt) Alternaria sp. or related (B1, B2 barleys; B1, B2, B3 malts) Cochliobolus sp. or related (B6 malt) 500 400 F. tricinctum / F. avenaceum (B5 malt) 300 200 100 0 B1 malt B2 malt B3 malt B4 malt B5 malt B6 malt 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 mi Time (min) 13

Different Fusarium species and gushing propensity Increased levels of F. culmorum and F. tricinctum detected in this study. F. graminearum in barley The impacts of isolates of the same species from different origin? F. culmorum and F. graminearum species reported as potential gushing inducers Recently, F. tricinctum and Microdochium nivale were associated with an elevated gushing risk (Boivin & Fournier, EBC Luxembourg, 2013) 14

Fusarium strains used in the malting experiments Strain VTT number Acronym Source F. culmorum VTT D-80148 D148 Barley, Finland D-131466 D1466 Barley, Finland D-131460 D1460 Barley, Denmark D-131461 D1461 Wheat, Canada F. graminearum D-82082 D082 Barley, Finland D-131465 D1465 Barley, Finland D-95470 D470 Corn, USA D-051036 D1036 Wheat or barley, USA F. tricinctum D-131523 D1523 Wheat malt, France D-131559 D1559 Barley, Finland malting trials (1kg) Barley heat-treated and artificially inoculated (10 6 / kg) 15

Fusarium sp. DNA/tot.DNA (pg/ng) Growth of different Fusarium strains in malting conditions 9,0 8,0 F. culmorum F. graminearum F. tricinctum 7,0 6,0 5,0 4,0 3,0 2,0 1,0 0,0 Infected barley Malt 16

F. culmorum, F. graminearum and F. tricinctum strains are capable of producing hydrophobin in malting 6,0 5,0 4,0 Vigorous gushing in beer (>30g) * * 3,0 * * 2,0 1,0 0,0 17

Conclusions The predominant Fusarium species in barley was F. avenaceum Competitive interaction between Fusarium and other field fungi especially with Alternaria F. culmorum and F. tricinctum detected in samples causing vigorous gushing F. culmorum, F. graminearum and F. tricinctum are associated with elevated gushing risk differences between the isolates of the same species from different origin targeted control, all Fusaria are not bad 18

Conclusions of methodology Fusarium fungi and their metabolites are not evenly distributed in barley challenge for prediction The CZID method easily overestimates Fusarium contamination VideometerLab results did not correlate with the real-time PCR or CZID results. At the moment, measuring total mouldiness of barley, not Fusaria Real-time PCR perhaps the most potential technique to selectively quantify Fusarium fungi in cereals Fusarium hydrophobins can be detected with the VTT hydrophobin ELISA 19

Future challenge Fusaria and their mycotoxins are considered as a worldwide problem and their occurence in the food and feed chain is expected to increase due to climate change Production of fungal hydrophobins (gushing inducers) linked to variable environmental conditions future? Control means 20

Acknowledgements VTT Technical Research Centre of Finland 21

TECHNOLOGY FOR BUSINESS

Fusarium DNA/tot. DNA (pg/ng) Quantification of Fusarium DNA in barley with real-time PCR using group specific and species specific primers 9,0 8,5 8,0 7,5 7,0 6,5 6,0 5,5 5,0 4,5 4,0 3,5 3,0 2,5 2,0 1,5 1,0 0,5 Fusarium sp. F. avenaceum F. graminearum F. culmorum F. poae F. langsethiae* 0,0 B1 B2 B3 B4 B5 B6 23

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