Brettanomyces and its control Charles G. Edwards Viticulture/Enology Team Washington State University (Pullman) What have we learned about controlling Brettanomyces? Impact of SO 2? SO 2 x Temperature? Removal by filtration? Current directions? Impact of SO 2 A. Can SO 2 cause VBNC? 1. Viable but not culturable. 2. Yeast alive but will not grow on media. 3. If valid yeast could be undetected. B. How to detect VBNC? 1. Measure metabolic activity. 2. Fluorescence microscopy. Green = viable Orange/Red = dead 2012 WAWGG Annual Meeting 1
Population (CFU/mL or cells/ml) Fine Wine-Advanced Enology 2/10/2012 Brettanomyces Active cell Dead cell B. bruxellensis (B1b) 10 8 0.88 mg/l mso 2 10 7 10 2 10 1 CFU/mL 0 5 10 15 20 25 30 35 40 Can SO 2 be used with low temperature to control Brettanomyces? 2012 WAWGG Annual Meeting 2
Culturability (CFU/mL) Culturability (CFU/mL) Fine Wine-Advanced Enology 2/10/2012 Research Focus (SO 2 x Temperature) A. General protocol. 1. Inoculate wine at high populations (> cfu/ml). 2. Experimental design mso 2 = 0 (none), 0.14 (low), 0.38 (medium), or 0.90 (high) mg/l Temperature = 10, 15, 18, or 22 C. Strains = B1b and B5 3. Weekly sample wines for >90 days. Strain B5 (22 C/72 F) No SO 2 Low SO 2 Medium SO 2 High SO 2 80 90 Strain B5 (18 C/64 F) No SO 2 Low SO 2 Medium SO 2 High SO 2 80 90 2012 WAWGG Annual Meeting 3
Culturability (CFU/mL) Culturability (CFU/mL) Fine Wine-Advanced Enology 2/10/2012 Strain B5 (15 C/59 F) No SO 2 Low SO 2 Medium SO 2 High SO 2 80 90 Strain B5 (10 C/50 F) No SO 2 Low SO 2 Medium SO 2 High SO 2 80 90 What about filtration? 2012 WAWGG Annual Meeting 4
Culturability (CFU/mL) Filtration A. Winemakers want to minimize filtration 1. Expensive, quality concerns. B. VBNC cells can exhibit dwarfing. 1. Cells smaller than normal. 2. Acetobacter may (?) become small enough to pass through 0.45 µm membrane. 3. Problem = filtered wines depending on pore size of membrane. Research Focus (Filtration) Red wines (0 or 0.5 mg/l mso 2 ) Treatment Filter Treatment Filter Treatment Filter Replicate Cartridge #1 Cartridge Replicate #2 Cartridge Replicate #3 3 samples (No SO 2 ) 3 samples (SO 2 ) Treatment Replicate = Filter cartridge 3 samples (No SO 2 ) 3 samples (SO 2 ) 3 samples (No SO 2 ) 3 samples (SO 2 ) Multiple ferments incubated >150 days. 10 7 Filtration 1.2 µm (Strain B1b) No SO 2 added Filtration replicates (,, ) >212 days 2012 WAWGG Annual Meeting 5
Culturability (CFU/mL) Culturability (CFU/mL) 10 7 1.2 µm Filtration (Strain F3) No SO 2 added Filtration replicates (,, ) >300 CFU/mL >250 days 0.8 µm Filtration (Strain F3) 10 7 No SO 2 added Filtration replicates (,, ) >250 days Current Ideas? A. What other phenolics can Brettanomyces metabolize? 1. What is present in wine that can help Brettanomyces to grow? B. Other control methods? 1. Use of chitosan 2012 WAWGG Annual Meeting 6
Concentration (mg/l) Log (CFU/mL) Fine Wine-Advanced Enology 2/10/2012 Phenolic Precursors (Brettanomyces) (-) Red Wine (+) Red Wine Phenolic Precursors to Volatile Phenols +tartaric acid CO HC O C O CO Caffeic acid O3 CO Caftaric acid +tartaric acid O3 CO CO Ferulic acid HC O C O CO trans-fetaric acid CO p-coumaric acid +tartaric acid CO HC O C O CO Coutaric acid Strain I1a (Cabernet Sauvignon) 25 8 7 20 6 15 5 4 10 3 5 2 1 0 0 0 2012 WAWGG Annual Meeting 7
A. Structure Research Focus (Chitosan) 1. Polymer of glucosamine units. B. Approved for use by O.I.V. 1. Rate = 10 g/hl (0.8 lb/1000 gal). 2. Approval by TTB pending. C. Mechanism remains unknown 1. Fining-type action? 2. Affect membrane function? Strain B5 ( CFU/mL) Brettanomyces Chitosan 2012 WAWGG Annual Meeting 8
While Brettanomyces can be challenging......there are rational and real approaches Approaches A. Impact of SO 2 on Brettanomyces 1. Fungistatic, not fungicidal ( VBNC ) 2. growth 0.5 mso 2 and 15 C/59 F B1b (more low temperature tolerant) B5 (more SO 2 tolerant) B. Filtration to remove Brettanomyces 1. Porosities of 0.8 µm needed C. Consider applying hurdle technology Initial infection Control Spoilage By Using Multiple Hurdles SO 2 Temperature Filtration Others Spoilage 2012 WAWGG Annual Meeting 9
Sincere Thanks To So Many! WA Wine Advisory Committee WA Wine Industry Washington State University GusmerEnterprises Lallemand Inc. Vinotec Ste Michelle Wine Estates ETS Laboratories Scott Laboratories WAWGG J.T. Larsen Y.C. Huang L.M. Schopp B. Petrova S.L. Jensen J.Q. Sturgeon X. Huajing N.L. Umiker M.J. Semon D. Ansen J.P. Osborne D.R. Webster K.M. Haag B.C. Childs J.C. Bohlscheid K.P. White K.M. Hagen A.D. Morneau J.M. Mills J.M. Zuehlke If SO 2, low temperature, filtration or chitosan does not control Brettanomyces 2012 WAWGG Annual Meeting 10