Investigating a New Cultural Technique for Isolation of Brettanomyces in the Environment

1. Investigating a New Cultural Technique for Isolation of Brettanomyces in the Environment

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3. History - Brettanomyces Beer in Production Breweries Turn of the century Pure yeast culture research and brewing just getting off the ground Ale produced in England have a hard time re-producing flavor of stock ales. 1904 -Yeast is isolated from British stock ales (porters, stouts, pale ales) and termed �Brettanomyces� the British Yeast, and method developed to seed beer twice. Early to Mid-Century Brett is researched more intensely by Belgians, discovered as primary flavor producer in Lambic. Brett discovered in wine as contaminant

4. New Millennium Central plains and West Coast craft and pub breweries Influenced by French/ Belgian brewers, and wild flavors from Brett and other organisms. Began producing American �wild� brews, using partially or 100% wild technique with used wood barrels. Began using Brettanomyces as a �culture� yeast. 2004 Papers presented at the Craft Brewers Conference White Labs releases Brettanomyces strains More American craft brewers began experimentation with �wild ferments� Brett beers begin to surface in market in bottle

5. Brettanomyces Beer in Production Breweries Goose Island Brewing Co goes into first production of �Matilda� Prototype based off of a Pub recipe. Matilda 100% stainless aged with 2 pure yeast cultures, no wood. Belgian strain primary Saccharomyces yeast fermentation Second seeding with Brettanomyces bruxellensis once primary has completed sugar consumption. Posed new issues for a mid-size brewery Propagation Transfer and handling Environmental screening (sanitation validation) Finished goods screening

6. Cultural Techniques for Identification of Brettanomyces For environmental screening; media criteria Need highly selective, differential �enrichment-like� media for presence/absence of high risk organisms Must be easy to use, low cost, easy to interpret. For finished goods; media criteria Need excellent recovery/resuscitation Prefer easy to use, low cost, easy to interpret, and rapid.

7. Cultural Techniques for Identification of Brettanomyces Medias WLN - differential WLN+actidione � differential and selective CuSO4 - selective Lysine � selective Problems with solid medias Each strain grows and develops differently in the lab. Cost limiting; many plates over a serial dilution or many membrane filtrations for large volume. Selectivity not limited to Brettanomyces genera. Dilution required so plate is not over grown. Other methods Non-culture techniques Costly

8. Modified YNB for Brettanomyces isolation 2005 �Paper published on a simple cultural screening media for Brettanomyces in wine* Described a method for the presumptive detection of the yeasts Brettanomyces/Dekkera in wines using a modified yeast nitrogen base. Media had selective and differential agents Presumptive positive cultural method Positive indicated by turbidity and characteristic aroma. * (Couto, J.A., Barbosa, A., T. Hogg. �A simple cultural method for the presumptive detection of the yeasts Brettanomyces/Dekkera in wines�. Letters in Applied Microbiology, 2005, 41. 505-510.)

9. 2005 Couto et. al Media components Difco yeast nitrogen base Glucose Chloramphenicol � Selective - Anti-bacterial p-coumaric acid � Differential Brettanomyces Actidione 20 ppm � Selective - Anti-fungal

10. Differential Media Component

11. GI Changes to Couto Method Followed similar recipe; final composition YNB normal strength 5 g L-1 glucose 20 ppm p-coumaric acid 20 ppm chloramphenicol (antibacterial) 10 ppm cycloheximide (antifungal) 20x strength for dilution with environmental samples. No agitation Incubation anerobically to cut down on mold presence 7 day incubation Monitored for aroma and turbidity at 650 nm at the end of incubation

12. Trial 1 � Pure Culture Recovery Modified YNB Brett. Bruxellensis Yellow � Lab errorYellow � Lab error

13. Trial 2 � Pure Culture Inoculated in Beer Recovery with Modified YNB Blank likely contaminated from open/closeBlank likely contaminated from open/close

14. Pass around sample Positive control with Brettanomyces bruxellensis CAUTION � smell only, do not tilt or spill. Other Brett species do have more acid/horse aromas.

15. Trial 3A � Environmental swabs Multi-Plant Collaborative Study Environmental monitoring vs. finished goods monitoring Issues Environmental swabs will contain competitor organisms, need good recovery. Media is selective, but other wild yeasts and possibly lactics, may be able to overcome hurdles. Potential for false (+) and false (�) results. First challenge was to determine if media exhibits potential for false (-) in a variety of different environments.

16. Multi-Plant Collaborative Study Other breweries were asked to challenge the media Assuming house flora varies in different plants with different internal and external pressures Mostly �Brett beer positive� plants Three potential sites, 3 swabs each site Brett Likely, Unknown, and Brett Negative Brett Likely � Defined as area or surface where a Brett beer residue was left. Unknown � Defined as an area that was post-op Brett Negative � Defined as a recently sanitized site

17. Trial 3A- Multi-Plant Collaborative Study Results A � Possible swabs were not handled correctly or incubated correctly. Others 2 of 3 had good detection of brett in likely places, and 0/3 in sanitized areas A � Possible swabs were not handled correctly or incubated correctly. Others 2 of 3 had good detection of brett in likely places, and 0/3 in sanitized areas

18. Trial 3B- Environmental Swabs �Brett Beer� Pressure Plant Wanted to investigate likelihood of false (+) results. Product contact sites were selected during standard operating conditions (not pre-op) Sites were grouped into three categories Packaging (interior filler during operation) Cellar transfer sites (diverter panels and FV racking arms) Cellar general sites (sanitizer bins, hose ends)

19. Trial 3B- Environmental Swabs Brett Pressured Plant Swabs were conducted alongside MRS duplicates, taken on Friday afternoon. First few replicate weeks we took all swabs in triplicate Triplicates were abandoned after 3 weeks since we experienced 100% positive for all 3 swabs was observed for every presumptive positive. Presumptive (+) swabs were carried through to WLN, WLND, CuSO4 tri-plate medias.

20. Trial 3B- Environmental Swabs Brett Pressured Plant

21. Tri-Plate Examples

22. Trial 3B- Environmental Swabs Brett Pressured Plant Results Modified �YNB Brett� media a good alternative to plated medias for assessing environment for Brettanomyces pressure, and putting a measure for improvement for sanitation. Cheap, easy to use, fast and reproducible.

23. Future Trials Media additions � Could provide benefit with selectivity, but will cost more/swab. Better selectivity More cycloheximide, Oxytetracycline,Copper sulfate, Ethanol? Better differential Bromocresol green for acid production? Continue trials with beer for a less costly alternative to membrane filtration finished goods testing (presence absence) Environmental scan Trials to reduce positive site frequency Comparison to Brettanomyces negative plants

24. Thank You! Thanks to all the collaborative breweries in assisting in this trial. Thanks to Stacia Janes and Jason Karras at Goose Island Beer Quality Lab for all their help with getting this project up and running. Thanks to MBAA for giving me opportunity to share our experience. Bottoms Up!