Art of Brewing - Saccharomyces Genome Database

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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

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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)

Sikaru

• Egypt (3000 BC) Zythum

• India (2000 BC)

Sura

• China (2000 BC) 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

Lager

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Ale

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.

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.

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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

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

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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 Nt. Identity AA. Identity

ILV1

ILV2

MET2

URA3

86 % 96 %

85 92

84

79

94

93

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Electrophoretic Karyotypes

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

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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• Soak, aerate, drain.

• 2 days

Steeping

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Germination

• Ventilated to remove CO

2

• 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

– Provides aroma and bitterness

• Flower (cone) of a vine-growing plant

Humulus lupulus

– Female triploid

• Used as:

– Whole cones

– Pellets

– Extracts

Hops

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Lupulin Glands

Hops

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Step

The Brewing Process

Purpose

Brewhouse Starch Sugars

Wort production

Fermentation Sugars Ethanol

Flavor production

Lagering

Carbonation

Flavor maturation

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The Brewing Process

Malt Mill

Mash Tun

Cereal

Cooker Brink Fermentation

Lauter Tun

Brew

Kettle

Hops

Aeration

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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• Strainer

Lauter Tun

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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 Fermentation

Lauter Tun

Brew

Kettle

Hops

Aeration

Hot Wort

Receiver

Lagering

Wort

Cooler

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10

5

0

20

15

Wort Composition

Carbohydrates

73% Fermentable

11.77

4.43

Fermentable Non-fermentable

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100

80

60

40

20

0

Wort Composition

Fermentable Sugars

** need to adjust to normal wort

52.9

Maltose

28.4

Glucose

16.1

Maltotriose

2.6

Fructose

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Wort Composition

Amino Acids (** adjust to normal wort)

300

250

200

150

107

132

100

50

65

53

30

49

63

151

89

31

126

269

56

93

105110

17

68

0

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

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The Brewing Process

Malt Mill

Mash Tun

Cereal

Cooker Brink Fermentation

Lauter Tun

Brew

Kettle

Hops

Aeration

Hot Wort

Receiver

Lagering

Wort

Cooler

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• Yeast growth

• Alcohol and CO

2

Fermentation

Flavor compounds

• Large - 600,000 L

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• Carbonation

Lagering

• Off-flavor reduction

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The Brewing Process

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

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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CO

2

Ethanol

Acetaldehyde

Organic Acids

Yeast Metabolism During Fermentation

Sugars Oxygen

Glucose

Pyruvate

Energy

TCA

Cycle

Membranes

Unsaturated Fatty Acids

Sterols

Amino Acids

Esters

Higher

Alcohols

VDK

Sulfur

Volatiles

Amino Acids

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Higher Alcohols

• 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

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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 apple, aniseed

– Ethyl caprylate apple

– Isoamyl decanoate tropical fruits

Fruity flavor notes

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Vicinal Diketones

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.

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