Tom Pugh
Miller Brewing Company

• 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

• An alcoholic beverage produced by the fermentation of sugar-rich extracts derived from cereal grains or other starchy materials.
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• 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
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• Sumarian beer recipe
– 3000 BC
• Resembled liquid bread:
– Barley and Emmer
– Spices / fruits
– No Hops
• Safe, nutritious, and exhilarating beverage.
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• 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.
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• 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 . . .
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….small animal which sips sugar through its snout, and excretes alcohol from its gut and carbonic acid from its urinary organ.
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• 1866 - Louis Pasteur
– Yeast was responsible for alcoholic fermentation.
• 1883 - Emil Christian Hansen
– Developed pure culture technique
– Isolated pure cultures of brewing yeasts
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• Two types of brewing yeasts, originally classified on flocculation behavior…
• Top-fermenting
– Ale yeast
– Weiss yeast
• Bottom-fermenting
– Lager yeast
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Weiss
Lager
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Ale
Lab

• 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.
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• 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.
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• 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.
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• 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
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• Colony morphology
• Microscopic appearance
– Chain formation
• Fermentation characteristics
– Flocculation behavior / flavor compound profiles
• Growth at 37 °C
• Melibiase
• Electrophoretic karyotyping
Yeast
Lager
Ale
Weiss
37 °C Melibiase POF
-
+
+ -
-
+
-
-
+
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• Difficult to distinguish between different lager yeasts using conventional techniques
– Colony and cell morphologies similar
– Fermentation characteristics
• PCR - limited success
• Electrophoretic karyotyping
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• 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
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• 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
ILV2
MET2
URA3
86 % 96 %
85 92
84
79
94
93
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XII
IV
Lager
Lab
XV, VII
XVI, XIII
II, XIV
X
XI
V, VIII
Ale
Lager cerev .
Ale .
parad .
IX
III
VI
I
T C C
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Lab
IX
III
VI
I
XII
IV
XV, VII
XVI, XIII
II, XIV
X
XI
V
VIII

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• Malted barley
• Cereal Adjunct
• Hops
• Water
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• 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
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• Soak, aerate, drain.
• 2 days
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• Ventilated to remove CO
2
• Repeated turning
• 4 to 5 days
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• Types of adjuncts commonly used:
– Corn grits
– Rice
– Corn syrups (high maltose and dextrose)
• Purpose:
– Additional source of fermentable sugars
– Lighter body
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• Spice of beer
– Provides aroma and bitterness
• Flower (cone) of a vine-growing plant
–
Humulus lupulus
– Female triploid
• Used as:
– Whole cones
– Pellets
– Extracts
Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company
Lupulin Glands

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

Malt Mill
Mash Tun
Cereal
Cooker 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

• Activate malt enzymes
• Convert starch to fermentable sugars
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• Strainer
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• Sterilization
• Protein coagulation
• Hop extraction
• Volatile removal
Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

Malt Mill
Mash Tun
Cereal
Cooker 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

20
15
10
5
0
Carbohydrates
73% Fermentable
11.77
4.43
Fermentable Non-fermentable
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40
20
0
100
80
60
Fermentable Sugars
** need to adjust to normal wort
52.9
Maltose
28.4
Glucose
16.1
Maltotriose
2.6
Fructose
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Amino Acids (** adjust to normal wort)
300
250
200
150
107
132
100
50
65
53
30
49
63
151
89
31
126
0
269
56
93
105110
17
68
Not included: Cys (2 ppm) and Trp (50 ppm)
Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

Malt Mill
Mash Tun
Cereal
Cooker 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

• Yeast growth
• Alcohol and CO
2
•
Flavor compounds
• Large - 600,000 L
Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

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

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

• 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

CO
2
Ethanol
Acetaldehyde
Organic Acids
Sugars Oxygen
Glucose
Pyruvate
Energy
TCA
Cycle
Membranes
Unsaturated Fatty Acids
Sterols
Amino Acids
Esters
Higher
Alcohols
VDK
Sulfur
Volatiles
Amino Acids
Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company

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

• Closely linked to lipid metabolism - growth.
• Reaction of an alcohol and fatty acid intermediate
• Acetate esters
– Ethyl acetate
– Isoamyl acetate
– Phenethyl acetate solventy, fruity, sweet banana 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

Pentanedione
Threonine a
-ketobutyrate a
-acetohydroxybutyrate pyruvate a
-acetolactate
Diacetyl
Buttery, butterscotch flavor
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Isoleucine
Valine

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.