ACS NERM 2013   Sour Beer - NMR Talk
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  • 1. When Beer Goes Sour: An NMR Investigation ADAM DICAPRIO PROCESS NMR ASSOCIATES 87A SAND PIT ROAD DANBURY, CT 06810
  • 2. 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
  • 3. What goes in to a sour beer?  Grain  Malted Barley, Unmalted Wheat, Specialty Malts   Modern Lambic – Brasserie Cantillon recipe – 65% Barley Malt + 35% Raw Wheat2 Hops   Sikaru beer (3000 B.C.) – 62.5% Barley Malt + 37.5% Raw Wheat2   Dextrins, Dextrins, Dextrins 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/
  • 4. 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.
  • 5. 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
  • 6. 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
  • 7. 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  Perform Starch Conversion Utilization of native microbes for pH adjustment6 Cool to ~120°F Innoculate Hold Temperature (120°F)
  • 8. 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)
  • 9. Malt-040-H.esp Ethanol Maleic Acid HOD Sugar Anomeric Region Lactic Acid Acetic Acid B-Glucose Normalized Intensity 0.0015 0.0010 a-Glucose Lactic Acid Succinic Acid 0.0005 a(1-4) Dextrins 2,3-butanediol Pyruvic Acid 0 6.0  5.5 5.0 4.5 4.0 3.5 3.0 Chemical Shift (ppm) 2.5 2.0 Straight Run of 95°F Sour mash; Assignments from Rodrigues et al. 2010 1.5 1.0 0.5
  • 10. Malt-025-H.esp HOD 0.11 Lactic Acid 0.10 0.09 Normalized Intensity 0.08 0.07 Sugar Anomeric Region Maleic Acid 0.06 Ethanol B-Glucose Acetic Acid 0.05 a-Glucose 0.04 0.03 Lactic Acid 0.02 a(1-4) Dextrins 2,3-Butanediol Succinic Acid TMSP 0.01 0 -0.01 6.0  5.5 5.0 4.5 4.0 3.5 3.0 2.5 Chemical Shift (ppm) 2.0 1.5 1.0 Freeze Dried Run of 95°F sour Mash; Assignments from Rodrigues et al. 2010 0.5 0
  • 11. Absolute Metabolite Proportions Metabolite Weight % vs. Temperature Percent by Mass 16.00 14.00 12.00 1.4 1.2 1 0.8 0.6 0.4 0.2 0 Weight % Ethanol 90 10.00 100 110 120 130 Temperature (°F) 140 8.00 6.00 4.00 2.00 0.00 90 95 Lactic Acid 100 Acetic Acid 105 110 Succinic Acid 115 Poly. (Lactic Acid) 120 125 Poly. (Acetic Acid) 130 135 Poly. (Succinic Acid) 140
  • 12. Relative Proportions Lactic:Acetic Lactic:Succinic 16.00 300.0 250.0 12.00 WT % LACTIC / WT % SUCCINIC WT % LACTIC / WT % ACETIC 14.00 R² = 0.9941 10.00 8.00 6.00 4.00 200.0 R² = 0.9676 150.0 100.0 50.0 2.00 0.00 0.0 90 100 110 120 TEMPERATURE (°F) 130 140 95 105 115 TEMPERATURE 125 135
  • 13. 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
  • 14. 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
  • 15. 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
  • 16. Allagash-014-H.esp Maleic Acid HOD Ethanol Ethanol Lactic Acid Normalized Intensity 0.0010 Acetic Acid 0.0005 Lactic Acid Citric Acid & Malic Acid (if present) Succinic Acid 0 6.5  6.0 5.5 5.0 4.5 4.0 3.5 Chemical Shift (ppm) 3.0 2.5 2.0 Drie Fonteinen Oude Gueuze Straight Run; Assignments from Rodrigues et al. 2010 1.5 1.0
  • 17. Allagash-014-H_FD.esp Sugar Anomeric Region Lactic Acid Lactic Acid Normalized Intensity Maleic Acid Residual Ethanol 2,3-butanediol Valine, Leucine, Isoleucine 0.007 0.006 Proline 0.005 a(1-4) Maltodextrin 0.004 0.003 8.5 8.0 7.5 Proline a-Glucose Aromatic Amino Acids Residual Phenols Benzoic Acids 0.001 Citric Acid & Malic Acid (if present) a(1-6) Maltodextrin Aromatic Amino Acids Residual Phenols Benzoic Acids 0.002  Ethanol 7.0 6.5 6.0 Asparagine B-Glucose 5.5 5.0 4.5 4.0 Chemical Shift (ppm) Arginine Alanine Pyruvic Acid 3.5 3.0 2.5 2.0 1.5 1.0 Freeze dried Run of Drie Fonteinen Oude Gueuze; Assignments from Rodrigues et al. 2010, Rodrigues & Gil 2011, & Nord et al. 2004
  • 18. Allagash-006-H_FD.esp Allagash-005-H_FD.esp 0.060 Framboise-Style 0.055 Kriek-Style 0.030 Lactic Acid Lactic Acid 0.050 0.025 0.045 Normalized Intensity 0.035 0.030 Acetic Acid 0.025 0.020 Acetic Acid 0.015 Citric Acid 0.020 Malic Acid 0.010 0.015 Succinic Acid Succinic Acid 0.010 0.005 0.005 3.0 2.5 2.0 3.0 1.5 2.5 Chemical Shift (ppm) Chemical Shift (ppm) Allagash-004-H_FD.esp Lactic Acid 0.030 Gueuze-Style 0.025 Normalized Intensity Normalized Intensity 0.040 0.020 0.015 0.010 Acetic Acid Succinic Acid 0.005 No Malic Acid Little to No Citric Acid 3.0 2.5 2.0 Chemical Shift (ppm) 1.5 2.0 1.5
  • 19. Acid Differences American Geuze-Style American FramboiseStyle 3% Beer American GeuzeStyle American Framboise -Style American Kriek-Style Boone Mariage Parfait 2009 Oude Geuze Vieille Geuze Fond Tradition Drie Fonteinen A Drie Fonteinen B LA (mg/L) AA (mg/L) SA (mg/L) CA (mg/L) MA (mg/L) 5386.0 1410.0 238.5 0 2972.1 394.6 3890.7 0 4682.8 1965.7 423.4 0 35% 35% 0 3896.7 20% 3777.8 3% 77% 27% Lactic 4506.5 488.1 217.0 0 0 3497.8 454.1 175.2 0 0 6807.8 698.6 218.8 0 Acetic Succinic 0 Belgian Geuze 11% Lactic Acetic Succinic Citric American Kriek-Style 4% 35% 43% 5137.6 865.9 234.6 0 0 5389.9 917.7 228.9 0 0 4% 85% Lactic Acetic Succinic 18% Lactic Acetic Succinic Malic
  • 20. Dextrin Ratios Α(1-4) MALTODEXTRIN / Α(1-6) MALTODEXTRIN Ratio of α(1-4) Maltodextrin to α(1-6) Maltodextrin by Style 7  “Degree of Fermentation”  Dependent on:   5 4 3 2 1 0 STYLE Style  6 Strain Ingredients
  • 21. Multivariate Analysis – Principal Component Analysis  Saiso Saisons ns Lambic Belgi an Geuz es ACA Ameri can Cools hip Beers Belgi Bel. Foudre an Foudr e Beer Hybri d Beer Ameri can Foudr e Beer IPA + Lager s IPA + Lager 100% 100% Brett. Brett Beers Am. Foudre Segregate based largely on sugar detail  Independent of ingredients
  • 22. 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
  • 23. 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-grainbrewing/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. Chim. Acta. 674(2):166175. 8. Rodrigues, J.E., Gil, A. M. (2012). NMR methods for beer characterization and quality control, Magn. Reson. Chem. 49: S37-S45. 9. Nord, L. I., Vaag, P., Duus, J. O. (2004). Quantification of Organic and Amino Acids in Beer by 1H NMR Spectroscopy, Anal. Chem. 76(16):4790-4798.