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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Wort Composition Carbohydrates 73% Fermentable Fermentable Non-fermentable Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

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

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

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

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

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

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

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

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

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

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

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

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