Transcript Art of Brewing - Saccharomyces Genome Database

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
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The Art of Brewing
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Definition of Beer
• An alcoholic beverage produced by the
fermentation of sugar-rich extracts derived
from cereal grains or other starchy
materials.
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History of Brewing
• Man has been making beer since the dawn of
civilization.
– Where grain was grown, beer was made.
•
•
•
•
Sumaria (4000 BC)
Egypt (3000 BC)
India (2000 BC)
China (2000 BC)
Sikaru
Zythum
Sura
Kiu
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History of Brewing
• Sumarian beer recipe
– 3000 BC
• Resembled liquid bread:
– Barley and Emmer
– Spices / fruits
– No Hops
• Safe, nutritious, and
exhilarating beverage.
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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.
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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 . . .
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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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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
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Brewing Yeasts
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Types of Brewing Yeasts
• 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
Ale
Lager
Lab
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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.
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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.
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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.
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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
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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 °CMelibiase
+
+
+
-
POF
+
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Distinguishing Characteristics
• 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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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
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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
ILV1
ILV2
MET2
URA3
Nt. Identity AA. Identity
86 %
85
84
79
96 %
92
94
93
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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
I
T
C
C
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The Brewing Process
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Ingredients
• Malted barley
• Cereal Adjunct
• Hops
• Water
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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
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Steeping
• Soak, aerate, drain.
• 2 days
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Germination
• Ventilated to remove CO2
• Repeated turning
• 4 to 5 days
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Cereal Adjuncts
• 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
Hops
– Provides aroma
and bitterness
• Flower (cone) of a
vine-growing plant
– Humulus lupulus
– Female triploid
• Used as:
– Whole cones
– Pellets
– Extracts
Lupulin Glands
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Hops
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The Brewing Process
Step
Purpose
Brewhouse
Starch
Sugars
Wort production
Fermentation
Lagering
Ethanol
Sugars
Flavor production
Carbonation
Flavor maturation
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The Brewing Process
Malt Mill
Mash Tun
Cereal
Cooker
Brink
Hops
Aeration
Fermentation
Lauter Tun
Brew
Kettle
Hot Wort
Receiver
Lagering
Wort
Cooler
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Mash Tun / Cereal Cooker
• Activate malt enzymes
• Convert starch to
fermentable sugars
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Lauter Tun
• Strainer
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Brew Kettle
• Sterilization
• Protein coagulation
• Hop extraction
• Volatile removal
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The Brewing Process
Malt Mill
Mash Tun
Cereal
Cooker
Brink
Hops
Aeration
Fermentation
Lauter Tun
Brew
Kettle
Hot Wort
Receiver
Lagering
Wort
Cooler
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Wort Composition
Carbohydrates
20
73% Fermentable
CHO (% w/v)
15
11.77
10
4.43
5
0
Fermentable
Non-fermentable
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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
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Wort Composition
Amino Acids (** adjust to normal wort)
300
269
250
151
126
68
56
Not included: Cys (2 ppm) and Trp (50 ppm)
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Asn
Val
Tyr
Thr
Pro
17
Phe
Met
Lys
31
Leu
Gly
Glu
Asp
Arg
30
49
63
Ile
53
His
65
50
0
93
89
100
105 110
Ser
107
Gln
132
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
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Fermentation
• Yeast growth
• Alcohol and CO2
• Flavor compounds
• Large - 600,000 L
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Lagering
• Carbonation
• Off-flavor reduction
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The Brewing Process
Malt Mill
Mash Tun
Cereal
Cooker
Brink
Hops
Aeration
Fermentation
Lauter Tun
Brew
Kettle
Hot Wort
Receiver
Lagering
Wort
Cooler
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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
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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
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Sulfur
Volatiles
Higher Alcohols
• Formed by the decarboxylation and reduction of
a-keto acids.
– From amino acid anabolism and catabolism.
Alcohol
Isoamyl
Amino Acid
Leucine
a-keto acid
Amyl
Isoleucine
a-keto-3-methylvalerate
Isobutanol
Valine
a-keto-isovalerate
Propanol
Threonine
a-keto-butyrate
a-keto-isocaproate
Alcoholic, solventy, and fruity flavor notes
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Esters
• 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
– Ethyl caprylate
– Isoamyl decanoate
apple, aniseed
apple
tropical fruits
Fruity flavor notes
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Vicinal Diketones
Threonine
Pentanedione
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.