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

The Art of Brewing
and
The Biology of Lager Yeast
Tom Pugh
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
The Art of Brewing
Definition of Beer
An alcoholic beverage produced by the
fermentation of sugar-rich extracts derived
from cereal grains or other starchy
materials.
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
History of Brewing
Sumarian beer recipe

3000 BC
Resembled liquid bread:

Barley and Emmer
Spices / fruits
No Hops
Safe, nutritious, and

exhilarating beverage.
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.


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 Latours theory in Annalen der Chemie . . .
.small animal which sips sugar through its snout, and

excretes alcohol from its gut and carbonic acid from its
urinary organ.

1866 - Louis Pasteur
Yeast was responsible for alcoholic
fermentation.
1883 - Emil Christian Hansen

Developed pure culture technique
Isolated pure cultures of brewing yeasts
Brewing Yeasts
Types of Brewing Yeasts

Two types of brewing yeasts, originally classified
on flocculation behavior
Top-fermenting
Ale yeast
Weiss yeast
Bottom-fermenting
Lager yeast
Weiss
Ale
Lager
Lab
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.
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.
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.
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
Distinguishing Characteristics
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
+
+
+
+
Distinguishing Characteristics
Difficult to distinguish between different lager yeasts
using conventional techniques
Colony and cell morphologies similar
Fermentation characteristics
PCR - limited success

Electrophoretic karyotyping
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
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
ILV2
MET2
URA3
86 %
96 %
85
92
84
94
79
93
Electrophoretic Karyotypes
cerev.
Ale
Lager
Lager
Lab
parad.
Weiss
bayan.
pastor.
Ale
XII
Lab
XII
IV
IV
XV, VII
XV, VII
XVI, XIII
XVI, XIII
II, XIV
X
XI
II, XIV
X
XI
V
VIII
V, VIII
IX
IX
III
VI
III
VI
I
The Brewing Process
Ingredients
Malted barley
Cereal Adjunct
Hops
Water
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
Steeping
Soak, aerate, drain.
2 days
Germination
Ventilated to remove CO2

Repeated turning
4 to 5 days
Cereal Adjuncts
Types of adjuncts commonly used:
Corn grits
Rice
Corn syrups (high maltose and dextrose)
Purpose:
Additional source of fermentable sugars
Lighter body
Spice of beer
Hops
Provides aroma
and bitterness
Flower (cone) of a
vine-growing plant
Humulus lupulus
Female triploid
Used as:
Whole cones
Pellets
Extracts
Lupulin Glands
Hops
The Brewing Process

Step
Purpose
Brewhouse
Starch
Sugars
Wort production
Fermentation
Lagering
Ethanol
Sugars
Flavor production
Carbonation
Flavor maturation
The Brewing Process

Malt Mill
Mash Tun
Cereal
Cooker
Brink
Hops
Aeration
Fermentation
Lauter Tun
Brew
Kettle
Hot Wort
Receiver
Lagering
Wort
Cooler
Mash Tun / Cereal Cooker

Activate malt enzymes
Convert starch to
fermentable sugars
Lauter Tun
Strainer
Brew Kettle
Sterilization
Protein coagulation
Hop extraction
Volatile removal
The Brewing Process

Malt Mill
Mash Tun
Cereal
Cooker
Brink
Hops
Aeration
Fermentation
Lauter Tun
Brew
Kettle
Hot Wort
Receiver
Lagering
Wort
Cooler
Wort Composition
Carbohydrates
20
73% Fermentable
CHO (% w/v)
15
11.77
10
4.43
Fermentable
Non-fermentable
Wort Composition
Fermentable Sugars
** need to adjust to normal wort
100
Percent (% w/v)
80
60
52.9
40
28.4
16.1
20
2.6
0
Maltose
Glucose
Maltotriose
Fructose
Wort Composition
Amino Acids (** adjust to normal wort)
300
269
250
151
105110
68
56
Not included: Cys (2 ppm) and Trp (50 ppm)

Ser
Asn
Val
Tyr
Thr
Pro
17
Phe
Met
Lys
31
Leu
30
49
63
Ile
Asp
Arg
53
His
65
50
0
93
89
Gly
100
126
Gln
132
107
Glu
150
Ala
PPM
200
The Brewing Process

Malt Mill
Mash Tun
Cereal
Cooker
Brink
Hops
Aeration
Fermentation
Lauter Tun
Brew
Kettle
Hot Wort
Receiver
Lagering
Wort
Cooler
Fermentation
Yeast growth
Alcohol and CO2
Flavor compounds
Large - 600,000 L
Lagering
Carbonation
Off-flavor reduction
The Brewing Process

Malt Mill
Mash Tun
Cereal
Cooker
Brink
Hops
Aeration
Fermentation
Lauter Tun
Brew
Kettle
Hot Wort
Receiver
Lagering
Wort
Cooler
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
Yeast Metabolism During Fermentation

Sugars
Oxygen
Membranes
Glucose
CO2
Ethanol
Acetaldehyde
Energy
Pyruvate
TCA
Cycle
Unsaturated Fatty Acids

Sterols
Amino Acids
Esters
Higher
Alcohols
VDK
Organic Acids
Amino Acids
Sulfur
Volatiles
Higher Alcohols
Formed by the decarboxylation and reduction of
-keto acids.
From amino acid anabolism and catabolism.
Alcohol
Isoamyl
Amino Acid
Leucine
-keto acid
-keto-isocaproate
Amyl
Isoleucine
-keto-3-methylvalerate
Isobutanol Valine
-keto-isovalerate
Propanol Threonine
-keto-butyrate
Alcoholic, solventy, and fruity flavor notes

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 decanoatetropical fruits
Fruity flavor notes
Vicinal Diketones
Threonine
Pentanedione
-ketobutyrate
-acetohydroxybutyrate
pyruvate
-acetolactate
Diacetyl
Buttery, butterscotch flavor

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.

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

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The Art of Brewing

The Art of Brewing

Sumaria (4000 BC) Sikaru

Sumarian beer recipe

Resembled liquid bread:

Safe, nutritious, and

The Role of Yeast in Brewing

Deduced that yeast was important to make beer.

Did not know what yeast was.

The Role of Yeast in Brewing

1837 - Cagniard Latour

1839 -Justus von Liebig and Friedrich Wohler

.small animal which sips sugar through its snout, and

The Role of Yeast in Brewing

1883 - Emil Christian Hansen

Types of Brewing Yeasts

Strains are genetically more diverse - several origins

Warm fermentation temperatures: 65 to 72 F.

Became very popular - displaced ale yeast

Strains are closely related - common origins

Lager yeast - Saccharomyces pastorianus

PCR - limited success

Genome Structure - Lager Yeast

Contains two types of chromosomes

Gene length similar, but nucleotide divergence.

Nt. Identity AA. Identity

The Brewing Process

Provides fermentable sugars,

Ventilated to remove CO2

The Brewing Process

The Brewing Process

Mash Tun / Cereal Cooker

The Brewing Process

Not included: Cys (2 ppm) and Trp (50 ppm)

The Brewing Process

The Brewing Process

The brewer needs...

Oxygen is growth limiting nutrient.

Yeast Metabolism During Fermentation

Unsaturated Fatty Acids

Alcoholic, solventy, and fruity flavor notes

Fatty acid esters

Buttery, butterscotch flavor

Thanks to David Ryder of Miller Brewing Company

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