The Art of Brewing and The Biology of Lager Yeast

1. The Art of Brewing and The Biology of Lager Yeast Tom Pugh Miller Brewing Company

2. Purpose • Provide a better understanding of... • The brewing process • Types of brewing yeasts • Attributes important to the brewer Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

3. The Art of Brewing Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

4. Definition of Beer • An alcoholic beverage produced by the fermentation of sugar-rich extracts derived from cereal grains or other starchy materials. Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

5. History of Brewing • Man has been making beer since the dawn of civilization. • Where grain was grown, beer was made. • Sumaria (4000 BC) Sikaru • Egypt (3000 BC) Zythum • India (2000 BC) Sura • China (2000 BC) Kiu Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

6. History of Brewing • Sumarian beer recipe • 3000 BC • Resembled liquid bread: • Barley and Emmer • Spices / fruits • No Hops • Safe, nutritious, and exhilarating beverage. Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

7. The Role of Yeast in Brewing • Unwittingly, ancient brewers domesticated yeast. • Selected yeast that made good beer. • Deduced that yeast was important to make beer. • Collect the creamy foam or sediment from one brew. • Use it to pitch the next brew. Did not know what yeast was. Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

8. The Role of Yeast in Brewing • 1680 Antonie van Leeuwenhoek • Observed yeast in beer. • 1837 - Cagniard Latour • Microbe is responsible for alcoholic fermentation. • 1839 -Justus von Liebig and Friedrich Wohler • Alcohol is produced by a chemical process in which dead and decaying yeast participated. • Satired Latour’s theory in Annalen der Chemie . . . Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

9. ….small animal which sips sugar through its snout, and excretes alcohol from its gut and carbonic acid from its urinary organ. Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

10. The Role of Yeast in Brewing • 1866 - Louis Pasteur • Yeast was responsible for alcoholic fermentation. • 1883 - Emil Christian Hansen • Developed pure culture technique • Isolated pure cultures of brewing yeasts Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

11. Brewing Yeasts Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

12. Types of Brewing Yeasts • Two types of brewing yeasts, originally classified on flocculation behavior… • Top-fermenting • Ale yeast • Weiss yeast • Bottom-fermenting • Lager yeast Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

13. Ale Weiss Lab Lager Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

14. Ale Yeast • Predominant brewing yeast prior to the mid-1800s. • Displaced by lager yeast • Strains are genetically more diverse - several origins • Warm fermentation temperatures: 65 to 72 °F. Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

15. Weiss Yeast • Bavarian origins - closely related. • Produces beer that has spicy, clove, vanilla, and nutmeg flavor notes - POF. • PAD1 gene phenylacrylic acid decarboxylase • Decarboxylation of ferulic acid forms 4-vinyl-guaiacol, which gives the characteristic clove flavor. • Warm fermentation temperatures: 65 to 72 °F. Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

16. Lager Yeast • Bavarian origin. • 1400s in Munich - cool fermentations (selective pressure) • Taken to Pilsen and Copenhagen in 1840s • Pale malt, soft water, aromatic hops • Became very popular - displaced ale yeast • Popularity fueled by advances of Industrial Revolution • Steam power, refrigeration, railroads, pasteurization and filtration technology • Strains are closely related - common origins • Cool fermentation temperatures: 42 to 52 °F • Beers are more delicate, clean, drinkable, and less aromatic. Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

17. Taxonomy • Ale and Weiss yeasts - Saccharomyces cerevisiae • Polyploid and probably aneuploid. • Non-mating • Sporulates poorly and poor spore viability • Lager yeast - Saccharomyces pastorianus • S. cerevisiae • S. carlsbergensis • S. uvarum • Sporulates very poorly - poor spore viability Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

18. Distinguishing Characteristics • Colony morphology • Microscopic appearance • Chain formation • Fermentation characteristics • Flocculation behavior / flavor compound profiles • Growth at 37 °C • Melibiase • Electrophoretic karyotyping Yeast 37 °C Melibiase POF Lager - + - Ale + - - Weiss + - + Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

19. Distinguishing Characteristics • Difficult to distinguish between different lager yeasts using conventional techniques • Colony and cell morphologies similar • Fermentation characteristics • PCR - limited success • Electrophoretic karyotyping Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

20. Genome Structure - Lager Yeast • Allopolyploid and probably aneuploid. • Tetraploid • Natural hybrid • S. cerevisiae and S. bayanus • S. cerevisiae and S. monacensis • Contains two types of chromosomes • S. cerevisiae type • S. bayanus type Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

21. Genome Structure • Gene order and function highly conserved • Single chromosome transfer experiments • Gene length similar, but nucleotide divergence. • Low levels of recombination between homeologues AA. Identity Nt. Identity Gene ILV1 86 % 96 % ILV2 85 92 MET2 84 94 URA3 79 93 Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

22. Electrophoretic Karyotypes parad. cerev. Ale Lager Lab Weiss Ale Lab bayan. Lager pastor. XII XII IV IV XV, VII XV, VII XVI, XIII XVI, XIII II, XIV X II, XIV XI X V XI VIII V, VIII IX IX III VI III VI I I C C T Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

23. The Brewing Process Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

24. Ingredients • Malted barley • Cereal Adjunct • Hops • Water Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

25. Malted Barley • Two types of barley • 2-rowed • 6-rowed • Provides fermentable sugars, flavor, and color. • Malting process: • Steeping • Germination • Kilning • Purpose: • Activate enzyme systems • Preserve for brewhouse Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

26. Steeping • Soak, aerate, drain. • 2 days Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

27. Germination • Ventilated to remove CO2 • Repeated turning • 4 to 5 days Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

28. Cereal Adjuncts • Types of adjuncts commonly used: • Corn grits • Rice • Corn syrups (high maltose and dextrose) • Purpose: • Additional source of fermentable sugars • Lighter body Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

29. Hops • Spice of beer • Provides aroma and bitterness • Flower (cone) of a vine-growing plant • Humulus lupulus • Female triploid • Used as: • Whole cones • Pellets • Extracts Lupulin Glands Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

30. Hops Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

31. The Brewing Process Purpose Step Starch Brewhouse Sugars Wort production Ethanol Sugars Fermentation Flavor production Carbonation Flavor maturation Lagering Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

32. The Brewing Process Malt Mill Cereal Cooker Mash Tun Brink Fermentation Lauter Tun Brew Kettle Hops Aeration Lagering Hot Wort Receiver Wort Cooler Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

33. Mash Tun / Cereal Cooker • Activate malt enzymes • Convert starch to fermentable sugars Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

34. Lauter Tun • Strainer Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

35. Brew Kettle • Sterilization • Protein coagulation • Hop extraction • Volatile removal Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

36. The Brewing Process Malt Mill Cereal Cooker Mash Tun Brink Fermentation Lauter Tun Brew Kettle Hops Aeration Lagering Hot Wort Receiver Wort Cooler Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

37. Wort CompositionCarbohydrates 73% Fermentable Non-fermentable Fermentable Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

38. Wort CompositionFermentable Sugars** need to adjust to normal wort Maltose Maltotriose Glucose Fructose Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

39. Wort CompositionAmino Acids (** adjust to normal wort) Arg Phe Asp Met Leu Gln Asn Thr Tyr Pro Lys Val Ala Ser Glu Gly His Ile Not included: Cys (2 ppm) and Trp (50 ppm) Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

40. The Brewing Process Malt Mill Cereal Cooker Mash Tun Brink Fermentation Lauter Tun Brew Kettle Hops Aeration Lagering Hot Wort Receiver Wort Cooler Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

41. Yeast growth Alcohol and CO2 Flavor compounds Large - 600,000 L Fermentation Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

42. Carbonation Off-flavor reduction Lagering Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

43. The Brewing Process Malt Mill Cereal Cooker Mash Tun Brink Fermentation Lauter Tun Brew Kettle Hops Aeration Lagering Hot Wort Receiver Wort Cooler Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

44. Balanced Growth • Yeast growth affects beer flavor. • Need balance between yeast growth and beer flavor. • The brewer needs... • Desired flavor profile in desired time. • Sufficient yeast crop for subsequent fermentations. • Oxygen is growth limiting nutrient. • Control point Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

45. Yeast Metabolism During Fermentation Sugars Oxygen Membranes Glucose CO2 Energy Unsaturated Fatty Acids Sterols Esters Pyruvate Ethanol Higher Alcohols TCA Cycle Amino Acids Acetaldehyde VDK Organic Acids Sulfur Volatiles Amino Acids Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

46. Higher Alcohols • Formed by the decarboxylation and reduction of a-keto acids. • From amino acid anabolism and catabolism. Alcohol Amino Acid a-keto acid Isoamyl Leucine a-keto-isocaproate Amyl Isoleucine a-keto-3-methylvalerate Isobutanol Valine a-keto-isovalerate Propanol Threonine a-keto-butyrate Alcoholic, solventy, and fruity flavor notes Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

47. Esters • Closely linked to lipid metabolism - growth. • Reaction of an alcohol and fatty acid intermediate • Acetate esters • Ethyl acetate solventy, fruity, sweet • Isoamyl acetate banana • Phenethyl acetate roses, honey, apple • Fatty acid esters • Ethyl caproate apple, aniseed • Ethyl caprylate apple • Isoamyl decanoate tropical fruits Fruity flavor notes Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

48. Vicinal Diketones Pentanedione Threonine a-acetohydroxybutyrate a-ketobutyrate Isoleucine Valine a-acetolactate pyruvate Diacetyl Buttery, butterscotch flavor Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

49. Thanks to David Ryder of Miller Brewing Companyand Tom Pugh, formerly of Miller Brewing Company,for providing this presentation to the Saccharomyces Genome Databasefor dissemination to the yeast community.