Practical Brewing Enzymology

1. Practical Brewing Enzymology Tobin L. Eppard Coors Brewing Company www.coors.com Rocky Mountain District - MBAA May 11, 2005 MBAA – Rocky Mountain District

2. What is in (and out) the box for today A Brief Primer on Enzymes Malting processes Mashing processes Practical Brewing Implications Q&A Session MBAA – Rocky Mountain District

3. Historically speaking.. • 1783 – Spallanzani noted a non cellular breakdown of meat by gastric juices • 1814 – Kirchoff, wheat paste converted starch to sugars… • 1878 – Kuhne suggested Enzyme = “In Yeast” • Catalytic Activity Substrate Enzyme Enzyme/Substrate Product(s) MBAA – Rocky Mountain District

4. What are Enzymes? • Protein • Structure is specific to function • How is shape determined? • Functional groups • Amino acids…. MBAA – Rocky Mountain District

5. What are Enzymes? • Primary Structure • Secondary Structure • Tertiary Structure • Quaternary Structure MBAA – Rocky Mountain District

6. What do they do? • “Spontaneous” Chemical Reactions • Energy Barriers, EA - Energy of Activation • Reduction of EA (G) by as much as 1015 Energy Level B2 B1 A EA of Enzyme Catalyzed Reaction C MBAA – Rocky Mountain District

7. Enzyme Biochemistry • Lock and Key Model Product(s) Substrate Enzyme/Substrate Complex Enzyme Enzyme MBAA – Rocky Mountain District

8. Enzyme Biochemistry • Enzyme “Process” in Biologic Sense NEED Energy RNA Inhibition AMINO ACIDS DNA Ribosome Yeast 4 o Sequence A-T-G-A-C-G Functional Protein (Enzyme?) 3 o Sequence 2 o Sequence 1 o Sequence MBAA – Rocky Mountain District

9. Enzyme Biochemistry • Factors Influencing Enzyme Rates: • Endo vs Exo enzymes • pH • Temperature • Products/Inhibition • Kinetics MBAA – Rocky Mountain District

10. Enzymes in Malting What’s going on in barley? Thousands of enzymes… Cellulases Proteases Amylases MBAA – Rocky Mountain District

11. Enzymes in Malting Barley Modification Pictures: David A. Thomas, Coors Brewing Company and Kunze, Technology Brewing and Malting MBAA – Rocky Mountain District

12. Enzymes in Mashing Mashing • Solubilize starches and Amino Acids/peptides • Starches to reducing sugars • Denature protein MBAA – Rocky Mountain District

13. MBAA – Rocky Mountain District

14. Brewing Enzymes Enzyme Action Opt. Temp pH • Amylase  67C 5.2 • Amylase  62C 5.5 Proteases < 52C 5.5  Glucanase 40 – 52C 6.0 Glycosidic -1,4 Endoamylase Glycosidic -1,4 (Non-reducing end C-4) Exoamylase Solubilize Proteins and Polypeptides (FAN) Endo and Exoproteinases Glycosidic β-1,4 and β-1,3 Gum/Cell walls, reduction in wort viscosity Endoglucanase MBAA – Rocky Mountain District

15. *Exogenous Brewing Enzymes Enzyme Action Opt. Temp pH • Amylase (HS) ~ 67C 5.2 Amyloglucosidase ~ 60C  5.5 (AMG) • Glucanase 40 – 52C 6.0 Pullulanase - - Glycosidic -1,4 Endoamylase Glycosidic -1,4 Endoamylase Glycosidic β-1,4 and 1,3 Gum/Cell walls, reduction in wort viscosity Endoglucanase Glycosidic -1,6 (Random) Endoamylase * - Not an exhaustive list, contact Novo or Genencor for more information MBAA – Rocky Mountain District

16. Practical Aspects of Brewing Enzymes… MBAA – Rocky Mountain District

17. First Aide Kit Extract Production • Starch Gelatinization Temperatures • Well Modified Malt(s) • High DP malt OR • Heat stable Alpha Amylase and Beta Amylase • Adjunct usage rates • Dilution of DP.. MBAA – Rocky Mountain District

18. First Aide Kit Extract Separation • Maximize Beta Glucanase (what little is there) • Consider, lowering your first mash-in temperature • Exogenous Beta Glucanase • Minimize shear forces MBAA – Rocky Mountain District

19. First Aide Kit Wort Fermentability (more or less?) • Exogenous Alpha Amylase, Beta Amylase • Amyloglucosidase, Pullulanase • Slightly lower conversion hold (64-65C) • Slower ramp from conversion hold to mash off (2ºC/min vs. 1ºC/min) • Consider the use of kettle adjuncts like HFCS… @DE of 95% MBAA – Rocky Mountain District

20. First Aide Kit Beer Filterability • Exogenous Beta Glucanase • Maximize Trub Formation (finings, boil) • Maximize cold maturation (if possible) • Add DE filtration first, before sheet filtration (if possible) • Reduce shear forces • Yeast flocculation issues • Secondary finings (isinglass) and hydrogels/xerogels… MBAA – Rocky Mountain District Pitures: Bill Simpson (Cara Technologies)

21. First Aide Kit Beer Foam • Maximize solubilization of HMW Proteins • Consider raising your initial mash in temp (60-62C) • Hops + • Viscosity (higher PG)+ • Gas + • Yeast Enzymes • Stress and high gravity – hydrophobic polypeptide reduction MBAA – Rocky Mountain District

22. Quality System • Most important of all… Set Malt Quality parameters and hold supplier accountable to meet them • Extract - S/T or Kohlbach • Viscosity - Total Protein • Color - Friability • DP MBAA – Rocky Mountain District

23. Final Notes on Exogenous Enzymes: Safety Concerns (handling) It’s a Soup of a larger cast of characters Side Reactions MBAA – Rocky Mountain District

24. Discussion? Pictures and Acknoledgements: David A Thomas – Barley grain modification SEM(s) Dr. Bill Simpson – CaraTechnologies, UK Mathewson, Paul R. Enzymes – Practical Guides for the Food Industry Eagan Press Handbook Series, St. Paul Minnesota, USA 1998 (sponsored by the American Society of Brewing Chemists)