The science of malting, brewing, and fermenting beer

Oct 6, 2010

DNA

RNAtranscription translation

Protein: a chain made up of 20 different amino acids from a few to as many as 34,350 residues

α-amylase

Temperature effects on biology and chemistry

Water is the universal solvent of life

The Structure and Properties of WaterD. Eisenberg and W. Kauzmann 308 pagesOxford Press1969

O

H H

OHH

H

O-

H

+::

:

:

:

:

Overview of metabolism• Most efficient way of getting energy is by combining

reduced carbon with oxygen. The more reduced the carbon, the more energy it has, the more oxygen added, the more energy released.

• In the absence of oxygen, fermentation is a suitable alternative.

O

HO

O

fatty acid

pyruvate

glucose and other sugars

lactic acid (-198 kJ/mol)

ethanol + CO2

(-235 kJ/mol)

(-2880 kJ/mol)

HO

O

HO

OH

OH

OH

OH

O

CO

OHO

O

OH

HO

Overview of metabolism

HO

OH

OH

OH

OH

O

H HH

H

H

H

H

HO

OH

OH

OH

OH

O

=

H OH

HO H

O H

H OH

OHH

OH

H

H

=

O

OH

HOHO

OH

OHO

OH

HOHO

OHOH

O

OH

OH

HO OH

OH

=

= =

βα

Overview of metabolismR = some chemical group

+H3N

O

O-

R

+H3N

O

O-

CH2

CH2

S

CH3

Overview of metabolism

Barley:

A member of the grass family. It is a self-pollinating, diploid species with 14 chromosomes. The wild ancestor of domesticated barley, Hordeum vulgare subsp. spontaneum, is abundant in grasslands and woodlands throughout the Fertile Crescent and has been cultivated for millennia.

Malting barley is usually lower protein which leads to more uniform germination, with shorter steeping.

The lower protein content also reduces the haze that results from precipitated protein.

Two-row barley generally has a lower protein content compared to six-row.

-A single mutation is responsible for the difference between two-row and six-row barley.-Two mutations of wild barley prevent the spike from shattering.

Traditionally barley was classified by morphological differences and were considered to be different species. -Two-rowed barley with shattering spikes (wild barley) is classified as Hordeum spontaneum K.Koch. -Two-rowed barley with non-shattering spikes is classified as H. distichum L.-Six-rowed barley with non-shattering spikes as H. vulgare L. (or H. hexastichum L.). -Six-rowed with shattering spikes as H. agriocrithon Åberg.

Recent cytological and molecular evidence has led most recent classifications to consider all forms as a single species, H. vulgare L.

Maris Otter is a 2-row, "winter" variety bred by researchers at Cambridge and introduced in 1966 possessing low nitrogen (protein) and superior malting characteristics.It is a cross of Proctor and Pioneer.

http://plantphys.info/plant_physiology/gibberellin.shtml

Germinating/maltingbreaking down chains of stuff

Drying and KilningDuring drying and or kilning, some enzymes become denatured. Generally darker grains are roasted longer and at higher temperatures and thus have less active enzymes then pale malt. Crystal malt is kilned without drying which denatures all enzymes.

Importantly, during the drying phase, most lipase and lipoxygenase enzymes are destroyed. These enzymes are implicated in the formation of off flavors in beer as it ages.

Kilning also roasts the grain. During the roasting process a glorious reaction called the Maillard reaction occurs.

Modification: the degree of breakdown of the starch-protein matrix during the malting, drying, and kilning process

O

O

OH

O

CHO

The Maillard reaction A general reaction between an amino acid and a reducing sugar and contributes to the color and flavor of browned bread, chocolate, seared meat, caramel and deep fried death.

O

melanoidins (dark color and toasty aroma)pyrazinesthiophenes pyrrolesfuransisobutyraldehyde (wet cereal/straw)

Strecker degradation

amino acid

sugar

O

O

NH2

O

R1

N

R2

N

O

http://blog.targethealth.com/?p=9586

The modern history of enzymes began in 1833 when French chemists described the isolation of an amylase complex from germinating barley and named it diastase.

Sugar enzymology I

Mashing and restingbreaking down more chains of stuff

Temp °C Temp °F Enzyme Breaks down

40 °C 104.0 °F β-Glucanase Cellulose (β-Glucan)

50 °C 122.0 °F Protease Protein

62 °C 143.6 °F β-Amylase Starch

72 °C 161.6 °F α-Amylase Starch

β-1,4 glycosidic linkage

Generally only needed when using >25% unmalted wheat or barley, corn, or rye

cellulose

Temp °C Temp °F Enzyme Breaks down

40 °C 104.0 °F β-Glucanase Cellulose (β-Glucan)

50 °C 122.0 °F Protease Protein

62 °C 143.6 °F β-Amylase Starch

72 °C 161.6 °F α-Amylase Starch

Protein rest: lower temperatures (122ºF (50ºC) ) yield shorter peptides and single amino acids (free amino nitrogen aka FAN) which aren’t as good for head retention.

Temperatures closer to 133ºF (55ºC) leave longer peptide chains and more available amino acids for yeast.

Some recent research suggests that most proteases are destroyed during kilning and that there is no significant reduction in the molecular weight spectrum of the mash.

~122 °F

~133 °F

Generally only needed when using minimally modified malt or a lot (>25%) of adjuncts

.

O O O O O O O O

O

O

O

O

O…

O

O

O

OO

OO

O

O

O

O

O OO

OO

O

O

O

amylopectin

amylose

O

O

O

O

O

O

O

limit dextrinase (debranching enzyme)

Sugar enzymology II

.β

.

α

.

.

.β

α

α β

Temp °C Temp °F Enzyme Breaks down

40 °C 104.0 °F β-Glucanase Cellulose (β-Glucan)

50 °C 122.0 °F Protease Protein

62 °C 143.6 °F β-Amylase Starch

72 °C 161.6 °F α-Amylase Starch

Sugar enzymology II

The thicker the mash, the more active the enzymes.

KM

reac

tion

rate

([p

rodu

cet]

/sec

ond)

Maximum rate possible

[Substrate]

General enzymologyEnzyme activity is affected mainly by temperature, but also pH,

presence of metals or cofactors, substrate concentration, viscosity, etc

Humulus lupulus

Lupulin16% Soft Resins 13%

Alpha Acids 8%Beta Acids 4%Other Soft Resins 1%

Hard Resins 2%Essential Oils 1%

Hydrocarbons 0.75%Oxidation Products 0.2%Sulphur containing compounds 0.05%

Vegative Matter 84%

lupulones

MyrceneCaryophyleneFareseneSelinene

Linalool (spicy)Geraniol

OH O

R

HO O

ΔThumulone

isohumulone

R

OO

HO OH

O

OH O

R

HO O

HO

trans-isohumulone

cis-isohumulone

R

OO

HO OH

O

R

OO

HO OH

O

D-form amino acids tend to taste sweet, L-form amino acids are generally tasteless.

Proteins use L-amino acidsMost sugars we digest and incorporate are D

Further adventures in stereochemistry

thalidomide

carvone

spearmint caraway

N

O

O

NH

O

O

O

H

O

(R)

O

H(S)

R

OHO

O

O

O

Antibiotic properties of hopsHop compounds act as ionophores that exchange protons for cellular divalent cations. This decreases the intracellular pH and dissipates the transmembrane proton gradient (ΔpH) and the proton motive force (pmf). Bacteria have evolved a number of ways to resist killing by hops.

HorA (a) and probably also by a pmf-dependent transporter (b)overexpressed H+-ATPase increases the pumping of protons released from the hop compounds (c) Galactosylated glycerol teichoic acid in the cell wall and a changed lipid composition of the cytoplasmic membrane of beer spoilage lactic acid bacteria may increase the barrier to hop compounds.

trans-isohumulone

antibacterial form

R

OHO

O

OH

O

-

+ H+

Saccharomyces cerevisiaea-Single-celled fungus from the phylum Ascomycota-One of the most well characterized organisms-Genome sequenced in 1996 -Capable of sexual and asexual reproduction-Found in wild on fruit surfaces

Lager yeast is more complex. First called S. carlsbergensis or S. pastorianus, then considered to be S. cerevisiae, are now recognized as a hybrid of S. cerevisiae and S. bayanus

S. cerevisiae life cycle

Time

Brejning et al. J Appl Microbiol. 2005.

Gene expression in lag phase and early log phase

If fermentation is anaerobic why is so much oxygen needed when pitching?

Fermentation profiles with various sugar supplements

Piddocke et al. Applied Microbiology and Biotechnology 2009

S. cerevisiae life cycle

S. cerevisiae life cycle

Gray et al. Microbiology and Molecular Biology Reviews. 2004.

Stationary phase is more complex than it seems

Extending healthy life span--from yeast to humans.Fontana L, Partridge L, Longo VD.Science. 2010 Apr 16;328(5976):321-6. Review.

Insulin/IGF-I and related signaling pathways regulate aging in nondividing cells: from yeast to the mammalian brain.Parrella E, Longo VD.ScientificWorldJournal. 2010 Jan 21;10:161-77. Review.Genetic links between diet and lifespan: shared mechanisms from yeast to humans.Bishop NA, Guarente L.Nat Rev Genet. 2007 Nov;8(11):835-44. Review.

Yeast metabolism

Why is S. cerevisiae so good at making beer?

“Make-accumulate-consume”Yeast can suppress respiration in the presence of glucose and oxygen

Yeast settling to the bottom is not a passive process

diacetyl rest:yeast convert acetolactic acid into valine instead of diacetyl (butanedione) and converts any butanedione into butanediol which is neutral as far as beer flavoring

Lagering: beer stored at 34-40 F for a few weeks. levels of diacetyl, acetaldehyde and sulfur compounds decrease.

What goes wrong when beer goes bad?

The dynamics of the Saccharomyces carlsbergensis brewing yeast transcriptome during a production-scale lager beer fermentation.Olesen K, Felding T, Gjermansen C, Hansen J.FEMS Yeast Res. 2002 Dec;2(4):563-73.

Two-dimensional gel analysis of the proteome of lager brewing yeasts.Joubert R, Brignon P, Lehmann C, Monribot C, Gendre F, Boucherie H.Yeast. 2000 Apr;16(6):511-22.

O

O-O O

O

O

OH

HO

-O

O

OO

HO

pyruvateacetoin diacetyl

valine

acetolactate

ethanol

acetaldehyde

O-

O+H3N

butanediol

OH

OH

Flavor in beer Organoleptic threshold (ppm)

Concentration in Japanese beer (ppm)

Higher alcohols Propan-1-ol (n-propanol) Alcohol 800 8–15

2-Methyl propanol (isobutyl alcohol) Alcohol 200 7–14

2-Methyl butanol (active amyl alcohol)

Alcohol, banana, medicinal, solvent 65 46–71

3-Methyl butanol (isoamyl alcohol)

Alcohol, banana, sweetish, aromatic 70

2-Phenyl ethanol Roses, sweetish, perfumed 125 20–27

Esters

Ethyl acetate Solvent, fruity, sweetish 30 10–20

Isoamyl acetate Banana, apple, solvent, estery 1.2 1.3–2.5

2-Phenylethyl acetate Roses, honey, apple, sweetish 3.8 0.4–1.3

Ethyl caproate Sour apple 0.21 Ethyl caprylate Sour apple 0.9

Carbonyl compounds Acetaldehyde Green leaves, fruity 25 2.9–3.4 2,3-Butanedione (diacetyl) Butter-scotch 0.15 <0.01–0.06

Kobayashi et al. J. Biosci. and Bioengr. 2008.

Kobayashi et al. J. Biosci. and Bioengr. 2008.

Parameter Production of higher alcohols and esters

Amino acid Leu Addition promoted (amyl alcohols and esters) and no effect on isobutyl alcohol

Val Addition promoted (only isobutyl alcohol production)

Ile Addition promoted (only amyl alcohol production)

Asp Addition promoted (ethyl acetate and n-propanol) and repressed (isobutyl alcohol)

Metal Zn Addition promoted (higher fermentation rates was obtained.)

Lees oil Addition promoted (isoamyl acetate) and no effect on ethyl acetate

EDTA Addition did not significantly promoteFatty acid C18:2 Addition repressed (only acetate esters)

Gravity Promoted (from 12 to 20° Plato media) and repressed in higher-gravity media

Temperature Promoted as temperature increasedTop pressure Repressed as top pressure increasedOxygen Repressed during aeration prior to pitching

pH (4.9–8.5) Promoted (isoamyl alcohol and isoamyl acetate) and repressed (ethyl acetate)

Serial repitching Promoted as the number of repitchings increased

Kobayashi et al. J. Biosci. and Bioengr. 2008.

Compounds Flavor note Odor threshold in beer (ppb)a Probable precursor Concentration in

finished beer (ppb)Sulfur dioxide (SO2) Burnt matches 25 ppm Sulfate/sulfite 200

ThiolsHydrogen sulfide (H2S) Pungent, rotten eggs 5–10 Sulfate, cysteine 0.5–20Methanethiol (MTL) Cooked cabbage; putrid 2 Methionine NdPolyfunctional thiols

3-Methyl-2-butene-1-thiol Onion, leek, skunky flavor 1–100 pptHop (isohumulone) + cysteine + riboflavine + light

Nd

2-Mercaptoethanol rotten eggs Cysteine Nd3-Mercaptopropanol Homocysteine NdSulfides

Dimethyl sulfide Cabbage, corn, onion, blackcurrant 30 SMM, dimethylsulfoxide 5–90

Dimethyl disulfide Cooked cabbage, onion 3–50 MTL 0.3–1.5

Dimethyl trisulfide Fresh onion, cooked vegetables 0.1

MTL, H2S, 3-MTP, S-methylcysteinesulfoxide

0.1–1.8

Dimethyl tetrasulfide Onion, cooked vegetables 1.2 Unknown 0.2ThioestersS-Methylthioacetate Cheese, cooked vegetables >100 MTL and acetyl–CoA 3–8S-Ethylthioacetate Ripened cheese, cabbage 0.8–3.5 Unknown 40Alkyl thio derivativesMethional Soap, potato 250 < 0.1b Methionine NdMethionol Cauliflower 2,000 Methionine NdSulfured terpens1,2-Epithiohumulene Cardboard, musty 200 hop 10–9,300 ppm

nd Not determined, SMM S-methyl methionine, and 3-MTP 3-methylthiopropionaldehyde aUnless stated otherwise, odor threshold values were determined in beer. bIn alcohol-free beer

Landaud et al. App. Microbiol. Biotechnol. 2008

Landaud et al. App. Microbiol. Biotechnol. 2008

Breeding an Amylolytic Yeast Strain for Alcoholic Beverage Production.Cheng MC, Chang RC, Dent DF, Hsieh PC.Appl Biochem Biotechnol. 2010 Sep 5. [Epub ahead of print]

How can we make even better beer?

Genetic improvement of brewer's yeast: current state, perspectives and limits.Saerens SM, Duong CT, Nevoigt E.Appl Microbiol Biotechnol. 2010 May;86(5):1195-212. Epub 2010 Mar 2. Review.

Improvement of Saccharomyces yeast strains used in brewing, wine making and baking.Donalies UE, Nguyen HT, Stahl U, Nevoigt E.Adv Biochem Eng Biotechnol. 2008;111:67-98. Review.

The potential of genetic engineering for improving brewing, wine-making and baking yeasts.Dequin S.Appl Microbiol Biotechnol. 2001 Sep;56(5-6):577-88. Review.

Multiobjective optimization and multivariable control of the beer fermentation process with the use of evolutionary algorithms.Andrés-Toro B, Girón-Sierra JM, Fernández-Blanco P, López-Orozco JA, Besada-Portas E.J Zhejiang Univ Sci. 2004 Apr;5(4):378-89.

Use of a modified alcohol dehydrogenase, ADH1, promoter in construction of diacetyl non-producing brewer's yeast.Onnela ML, Suihko ML, Penttilä M, Keränen S.J Biotechnol. 1996 Aug 20;49(1-3):101-9.

Study Strains and conditions investigated Purpose of investigation Level of global analysis (method applied)

Blieck et al. (2007) Lager yeast strain (CMBS33) and an UV-induced mutant of this strain showing improved fermentation performance in high-gravity wort (23 °P, 2 l scale, tall tube vessels)

Strain improvement via inverse engineering Transcriptome (microarray, S.c. gene probeset)

Olesen et al. (2002) Industrial lager yeast strain in 5,000 hl 14 °P wort in cylindroconical fermentation tanks Dynamics of brewing fermentation Transcriptome (microarray, S.c. gene probeset)

Gibson et al. (2008) Lager yeast strain (CB11) in cylindroconical fermentation tanks (3,275 hl scale, 17 °P wort)

Study of the response of lager brewing yeast to changes in wort fermentable carbohydrate concentration and composition

Transcriptome (microarray, S.c. gene probeset)

James et al. (2003) Two bottom-fermenting lager strains (Guinness 6701 and 7012) in 2 l 15 °P wort in European Brewery Convention (EBC) tall cylindroconical fermentation vessels

Dynamics of brewing fermentation Transcriptome (microarray, S.c. gene probeset)

Higgins et al (2003) Industrial lager yeast in 20 l 12 °P wort in industrial fermentation vessels Study of the stress response during an industrial lager fermentation Transcriptome (microarray, S.c. gene probeset)

Mizuno et al. (2006) Top-fermenting brewer’s yeast strain (NCYC1245) and a 2-deoxyglucose-resistant mutant of this strain with low acetic acid and high ethanol productivities (100 ml scale, 13 °P wort)

Identification of the genes involved in the low acetic acid/high ethanol phenotype Transcriptome (microarray, S.c. gene probeset)

Bond et al. (2004) Two bottom-fermenting lager yeast strains (CMBS33 and Guinness 6701) in comparison with the haploid laboratory strain S-150

Aneuploidy and copy number breakpoints in lager yeast strains Genome (CGH, S.c. gene probeset)

Pope et al. (2007) Two ale brewer’s strains, six lager brewer’s strains and one type strain of S. cerevisiae in complex medium (YM)

Differentiation between industrially used brewer’s strains

Genome (CGH, S.c. gene probeset) Exometabolome (DIMS, GC-TOF-MS)

Joubert et al. (2001) Lager brewer’s yeast strain (K11) in minimal medium (YNB, 2% glucose) Identification of proteins which do not co-migrate with the known proteins of S.c.

Proteome (2D gel electrophoresis, MALDI–MS and MS/MS)

Joubert et al. (2000) Seven lager brewer’s yeast strains, type strains of S. cerevisiae, S. bayanus, S. carlsbergensis, S. monascensis, S. pastorianus and S. uvarum in minimal medium (YNB, 2% glucose)

Obtain information about the identity of the ancestors of lager brewer’s yeast

Proteome (2D gel electrophoresis, gas–liquid phase sequencing)

Brejning et al. (2005) Lager brewer’s yeast strain (KVL001) in minimal medium (YNB, 0.5% glucose)

Identify proteins whose expression is induced in lager brewing during lag phase and early exponential growth

Proteome (2D gel electrophoresis, MALDI–MS and MS/MS)

Kobi et al. (2004) Ale yeast strain (A38) in complex medium (YPD) and brewer’s wort

Comparison of an ale, a lager and a laboratory yeast strain Proteome (2D gel electrophoresis, MALDI–MS)Lager brewers’ yeast strain (K11) in YPD medium

Laboratory yeast (S288c) in complex medium (YPD)

Minato et al. (2009) Lager brewers’ yeast strain (KBY011), S. cerevisiae laboratory strain and S. pastorianus in complex medium (YPD)

Expression of S.c-type and non-S.c.-type genes in a lager brewer’s yeast

Transcriptome (microarray, S.c. gene and non-S.c. EST probes)

Yoshida et al. (2008) One lager brewer’s yeast (KBY011) and one baker’s yeast (S288c) showing significant differences in sulphite production (SD10 medium lacking amino acids, 2 l scale, anaerobic conditions)

Strain improvement via inverse engineering (increase of sulphite production)

Transcriptome (microarray with S.c. gene and non-S.c. EST probes)

Endometabolome (CE–ESI–MS)

Dunn and Sherlock (2008) 17 lager brewers strains and 3 ale strains Differentiation between brewer’s yeast strains and identification of the ancestors of S. Pastorianus

Genome (CGH, “two-species array” with probes for genes from S.c. and S. bayanus var. uvarum)

Duong Cam et al. (in preparation) Three lager brewer’s yeast strains which show significant differences in diacetyl production analysed in wort under conditions relevant in brewing

Strain improvement via inverse engineering (reduction of diacetyl formation)

Genome (whole-genome array CGH with S.c. gene and non-S.c. probesets) Transcriptome (whole-genome array with S.c. gene and non-S.c. probesets) Proteome (2D gel electrophoresis, MALDI–MS)

Nakao et al. (2009) Lager brewer’s yeast strain (Weihenstephan 34/70) Identify the complete genomic sequence of a commonly used lager yeast strain Genome (whole genome sequencing)

Saerens et al. Appl Microbiol Biotechnol. 2010.

Yeast and human healthTumor cell energy metabolism and its common features with yeast metabolism.Diaz-Ruiz R, Uribe-Carvajal S, Devin A, Rigoulet M.Biochim Biophys Acta. 2009 Dec;1796(2):252-65. Epub 2009 Aug 12. Review. Brewer's/baker's yeast (Saccharomyces

cerevisiae) and preventive medicine: Part II.Moyad MA.Urol Nurs. 2008 Feb;28(1):73-5. Review.

Combined yeast-derived beta-glucan with anti-tumor monoclonal antibody for cancer immunotherapy.Liu J, Gunn L, Hansen R, Yan J.Exp Mol Pathol. 2009 Jun;86(3):208-14. Epub 2009 Jan 21. Review.

Yeast cell wall polysaccharides as antioxidants and antimutagens: can they fight cancer?Kogan G, Pajtinka M, Babincova M, Miadokova E, Rauko P, Slamenova D, Korolenko TA.Neoplasma. 2008;55(5):387-93. Review.

Protein folding diseases and neurodegeneration: lessons learned from yeast.Winderickx J, Delay C, De Vos A, Klinger H, Pellens K, Vanhelmont T, Van Leuven F, Zabrocki P.Biochim Biophys Acta. 2008 Jul;1783(7):1381-95. Epub 2008 Feb 11. Review.

Saccharomyces cerevisiae: a useful model host to study fundamental biology of viral replication.Alves-Rodrigues I, Galão RP, Meyerhans A, Díez J.Virus Res. 2006 Sep;120(1-2):49-56. Epub 2006 May 15. Review.

References and further reading

http://www.brewingtechniques.com/

National Institutes of Health digital archive of biomedical and life sciences journal literaturehttp://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed

http://homebrewandchemistry.blogspot.com/

Site of Brew Your Own magazinehttp://www.byo.com/

http://forum.northernbrewer.com/

http://www.wikipedia.org

Site of UC-Davis Anheuser-Busch Endowed Professor of Brewing Science Charles Bamforthhttp://foodscience.ucdavis.edu/bamforth/

Rensselaer Polytechnic Institute brewing classhttp://www.rpi.edu/dept/chem-eng/Biotech-Environ/beer/index1.htm

Gas chromatograph (GC)

High performance liquid chromatograph (HPLC)

Electrospray ionization mmass spectrometer (ESI-MS)

Tools of the trade

Fermenter