When Beer Goes Sour: An NMR Investigation ADAM DICAPRIO PROCESS NMR ASSOCIATES 87A SAND PIT ROAD DANBURY, CT 06810 What is Sour Beer? Wikipedia says: “Sour beer is a beer style characterized by an intentionally acidic, tart, sour taste.” “Wild Brews: Beer beyond the Influence of Brewer’s Yeast” Category 17 of the Beer Judge Certification Program Encompasses: Berliner Weisse, Flanders Red Ale, Flanders Brown Ale, Lambic, Fruit Lambic, Gueuze What goes in to a sour beer? Grain Malted Barley, Unmalted Wheat, Specialty Malts Dextrins, Dextrins, Dextrins Sikaru beer (3000 B.C.) – 62.5% Barley Malt + 37.5% Raw Wheat2 Modern Lambic – Brasserie Cantillon recipe – 65% Barley Malt + 35% Raw Wheat2 Hops http://www.newbelgium.com/Community/Blog/12-03-23/Who-wants-more-sour-beer.aspx Aged & Oxidized Aging Vessels – A sour beers home for up to a full century Oak Barrels (French & American) Oak Foudre Stainless Steel Tank http://www.belgianbeermagazine.com/oud-beersel-brewery/ Who goes in to a sour beer? Dozens of organisms2,3 Bacteria Enterobacteriaceae Citrobacter spp., Enterobacter spp., Klebsiella spp., Hafnia spp. Lactobacillaceae Pediococcus spp., Lactobacillus spp., Acetobacter spp. Klebsiella spp. Yeasts Kloeckera apiculata Saccharomyces spp. Brettanomyces spp. Pichia spp. Candida spp. Hansenula spp. Cryptococcus spp. Why is Sour Beer Sour? Straight Lambic, Flanders Ales, Gueuze, Berliner Weisse Lactic, Acetic, Succinic Acid1 85% - 10% - 5% Fruit Lambics1 Cherries, Grapes – Malic Acid Raspberries – Citric Acid Chemistry of Sour Beers Application of Quantitative NMR to Biologically Acidified Mashes Quantitative NMR and Descriptive Chemistry of American Wild Ales and genuine Belgian Lambic Berliner Weisse & Biological Acidification Reinheitsgebot of 1516 Beer can contain only malt, hops & water Unmalted wheat and yeast added in the Provisional Law of 19964 Artificial alteration of pH is illegal5 Development of Biological Acidification/Sour Mashing Utilization of native microbes for pH adjustment6 Perform Hold Cool to Starch Innoculate Temperature ~120°F Conversion (120°F) Temperature Dependence of the Sour Mash Technique Goal: Does the “magic” temperature of 120°F have a chemical significance? Record and quantify sour metabolites & contaminant products as a function of sour mash temperature Determine wt% of metabolites using Maleic Acid internal standard & manual integration Lactic Acid Acetic Acid (contaminant) Succinic Acid Ethanol γ-Amino Butyric Acid (contaminant) Malt-040-H.esp Ethanol Maleic Acid HOD Sugar Anomeric Region Lactic Acid Acetic Acid 0.0015 B-Glucose 0.0010 a-Glucose Lactic Acid Normalized Intensity Normalized 0.0005 Succinic Acid a(1-4) Dextrins 2,3-butanediol Pyruvic Acid 0 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Chemical Shift (ppm) Straight Run of 95°F Sour mash; Assignments from Rodrigues et al. 2010 Malt-025-H.esp HOD 0.11 0.10 Lactic Acid 0.09 0.08 Sugar Anomeric Region 0.07 Maleic Acid Ethanol 0.06 B-Glucose Acetic Acid 0.05 a-Glucose Normalized Intensity Normalized 0.04 0.03 Lactic Acid 2,3-Butanediol 0.02 a(1-4) Dextrins Succinic Acid TMSP 0.01 0 -0.01 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm) Freeze Dried Run of 95°F sour Mash; Assignments from Rodrigues et al. 2010 Absolute Metabolite Proportions 16.00 1.4 Metabolite Weight % vs. Temperature 1.2 Weight % Ethanol 1 14.00 0.8 0.6 0.4 12.00 0.2 Percent by Mass Percent 0 90 100 110 120 130 140 10.00 Temperature (°F) 8.00 6.00 4.00 2.00 0.00 90 95 100 105 110 115 120 125 130 135 140 Lactic Acid Acetic Acid Succinic Acid Poly. (Lactic Acid) Poly. (Acetic Acid) Poly. (Succinic Acid) Relative Proportions Lactic:Acetic Lactic:Succinic 16.00 300.0 14.00 250.0 12.00 R² = 0.9941 200.0 10.00 R² = 0.9676 8.00 150.0 6.00 100.0 WT % LACTIC / WT % ACETIC / WT % LACTIC WT % 4.00 SUCCINIC / WT % LACTIC WT % 50.0 2.00 0.00 0.0 90 100 110 120 130 140 95 105 115 125 135 TEMPERATURE (°F) TEMPERATURE Conclusions 120°F is sub-optimal for acid production Around 120°F Lactic acid reaches a relative maximum Lactic good, Acetic bad Aim is pH adjustment, not flavor adjustment Lambics of Belgium & Lambic-Styles of the USA What’s the difference? Lambic – From the Pajottenland / Senne River Valley Region of Belgium American likenesses styled as “American Wild Ale (AWA)” or “American Coolship Ale (ACA)” Different Microbial Community Follow same general succession ACA involves a more diverse community of Lactic Acid Bacteria and Minority Yeasts3 The Chemistry of Sour Beers Goals Comparative analysis of organic acids using quantitative NMR Manually integrated against a known mass of Maleic Acid Lactic Acid, Acetic Acid, Succinic Acid, Citric Acid, Malic Acid Analyze linear and branched dextrin ratios among multiple styles Utilize multivariate analysis to discriminate multiple styles of sour beer Allagash-014-H.espMaleic Acid HOD Ethanol Ethanol Lactic Acid 0.0010 Acetic Acid 0.0005 Lactic Acid Normalized Intensity Normalized Citric Acid & Malic Acid (if present) Succinic Acid 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 Chemical Shift (ppm) Drie Fonteinen Oude Gueuze Straight Run; Assignments from Rodrigues et al. 2010 Allagash-014-H_FD.esp Sugar Anomeric Region Lactic Acid Lactic Acid Ethanol Maleic Acid Residual Ethanol 2,3-butanediol 0.007 Valine, Leucine, Isoleucine 0.006 Proline 0.005 a(1-4) Maltodextrin Citric Acid & Malic Acid (if present) Normalized Intensity Normalized 0.004 a(1-6) Maltodextrin Aromatic Amino Acids Proline Arginine Residual Phenols 0.003 Benzoic Acids a-Glucose Asparagine Alanine 0.002 Aromatic Amino Acids B-Glucose Residual Phenols Pyruvic Acid 0.001 Benzoic Acids 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 Chemical Shift (ppm) Freeze dried Run of Drie Fonteinen Oude Gueuze; Assignments from Rodrigues et al. 2010, Rodrigues & Gil 2011, & Nord et al. 2004 Allagash-005-H_FD.esp Allagash-006-H_FD.esp Lactic Acid 0.060 0.055 Framboise-Style Kriek-Style 0.030 Lactic Acid 0.050 0.045 0.025 0.040 0.020 0.035 Acetic Acid 0.030 Acetic Acid 0.015 0.025 Normalized Intensity Normalized Normalized Intensity Normalized Citric Acid 0.020 Malic Acid 0.010 Succinic Acid 0.015 Succinic Acid 0.010 0.005 0.005 3.0 2.5 2.0 1.5 3.0 2.5 2.0 1.5 Chemical Shift (ppm) Chemical Shift (ppm) Allagash-004-H_FD.esp Lactic Acid 0.030 Gueuze-Style 0.025 0.020 0.015 Normalized Intensity Normalized 0.010 Acetic Acid Succinic Acid 0.005 No Malic Acid Little to No Citric Acid 3.0 2.5 2.0 1.5 Chemical Shift (ppm) American Geuze-Style American Framboise- Acid Differences Style 3% 20% Beer LA (mg/L) AA (mg/L) SA (mg/L) CA (mg/L) MA (mg/L) 35% 35% American Geuze- 5386.0 1410.0 238.5 0 0 Style American Framboise 3896.7 2972.1 394.6 3890.7 0 3% -Style 77% 27% American 4682.8 1965.7 423.4 0 3777.8 Kriek-Style Lactic Acetic Succinic Citric Boone Lactic Acetic Succinic Mariage 4506.5 488.1 217.0 0 0 Parfait 2009 Belgian Geuze American Kriek-Style Oude Geuze 3497.8 454.1 175.2 0 0 4% Vieille 11% Geuze Fond 6807.8 698.6 218.8 0 0 35% Tradition 43% Drie Fonteinen 5137.6 865.9 234.6 0 0 A Drie Fonteinen 5389.9 917.7 228.9 0 0 4% B 85% 18% Lactic Acetic Succinic Lactic Acetic Succinic Malic Dextrin Ratios Ratio of α(1-4) Maltodextrin to α(1-6) “Degree of Fermentation” Maltodextrin by Style Dependent on: 7 Strain 6) - 6 Style (1 Α 5 / Ingredients 4 3 2 1 MALTODEXTRIN MALTODEXTRIN 0 4) - (1 Α STYLE Multivariate Analysis – Principal Component Analysis Segregate based largely on sugar detail Saiso Saisonsns Belgi Bel.an Foudre Independent of Foudr ingredients e Beer Belgi Lambic an Geuz IPA + es Hybri Lager d IPAs + Lager Beer 100%100% Brett. ACA Ameri Ameri Brett can can Beers Cools Foudr hip Am.e Foudre Beers Beer Conclusions ACAs & Belgian Lambics have differing acid profiles Ratio of linear and branched maltodextrins can be used to differentiate styles Multivariate analysis can differentiate between ACAs, Belgian Lambics and 6 other styles References 1. Sparrow, J. (2005). Wild brews: Beer Beyond the Influence of Brewer’s Yeast, (Strong, G., Ed.), Brewers Publications, Boulder, CO. 2. Guinard, J. X. (1990). Lambic, (Thomas, V., Ed.), Brewers Publications, Boulder, CO. 3. Bokulich, N. A., Bamforth, C. W., Mills, D. A. (2012). Brewhouse-Resident Microbiota Are Responsible for Multi-Stage Fermentation of American Coolship Ale, PLoS One. 7:4, 1-11. 4. Dornbusch, H., Heyse, K. U. (2012) Reinheitsgebot, in the Oxford Companion to Beer, (Oliver, G., Ed.), pp. 691-693, Oxford University Press, Oxford. 5. Narziss, L. (1984) The German Beer Law, J. Inst. Brew. 90, 351-358. 6. Sour Mashing: Techniques, Brew Your Own Magazine. http://byo.com/all-grain- brewing/item/1691-sour-mashing-techniques (last accessed July 2013). 7. Rodrigues, J. E., Erny, G. L., Barros, A. S., et al. (2010). Quantification of organic acids in beer by nuclear magnetic resonance (NMR)-based methods, Anal.