The science of malting, brewing, and fermenting beer

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The science of malting, brewing, and fermenting
beer

• Oct 6, 2010

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Protein a chain made up of 20 different amino
acids from a few to as many as 34,350 residues
a-amylase
DNA
transcription
translation
RNA
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Temperature effects on biology and chemistry
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Water is the universal solvent of life
The Structure and Properties of Water D.
Eisenberg and W. Kauzmann 308 pages Oxford
Press 1969







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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.

glucose and other sugars
fatty acid
(-2880 kJ/mol)
lactic acid (-198 kJ/mol)
ethanol CO2 (-235 kJ/mol)
pyruvate
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Overview of metabolism





ß
a
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Overview of metabolism
R some chemical group
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Overview of metabolism
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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.
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-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.
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Germinating/maltingbreaking down chains of stuff
http//plantphys.info/plant_physiology/gibberellin
.shtml
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Drying and Kilning
During 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
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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.
sugar
amino acid
Strecker degradation
melanoidins (dark color and toasty
aroma) pyrazines thiophenes pyrroles furans isobu
tyraldehyde (wet cereal/straw)
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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
http//blog.targethealth.com/?p9586
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Mashing and resting breaking down more chains of
stuff
Temp C Temp F Enzyme Breaks down
40 C 104.0 F ß-Glucanase Cellulose (ß-Glucan)
50 C 122.0 F Protease Protein
62 C 143.6 F ß-Amylase Starch
72 C 161.6 F a-Amylase Starch
Generally only needed when using gt25 unmalted
wheat or barley, corn, or rye
ß-1,4 glycosidic linkage
cellulose
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Temp C Temp F Enzyme Breaks down
40 C 104.0 F ß-Glucanase Cellulose (ß-Glucan)
50 C 122.0 F Protease Protein
62 C 143.6 F ß-Amylase Starch
72 C 161.6 F a-Amylase Starch
Protein rest lower temperatures (122ºF (50ºC) )
yield shorter peptides and single amino acids
(free amino nitrogen aka FAN) which arent as
good for head retention. Temperatures closer
to 133ºF (55ºC) leave longer peptide chains and
more available amino acids for yeast.
.
Generally only needed when using minimally
modified malt or a lot (gt25) of adjuncts
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.
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Sugar enzymology II
amylopectin
amylose
ß
O
O
O
O
limit dextrinase (debranching enzyme)
O
.
O
O
O
O
O
O
ß
O
a
O
O
O
O
O
O
O
a
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Sugar enzymology II
a
ß
Temp C Temp F Enzyme Breaks down
40 C 104.0 F ß-Glucanase Cellulose (ß-Glucan)
50 C 122.0 F Protease Protein
62 C 143.6 F ß-Amylase Starch
72 C 161.6 F a-Amylase Starch
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General enzymology Enzyme activity is affected
mainly by temperature, but also pH, presence of
metals or cofactors, substrate concentration,
viscosity, etc
The thicker the mash, the more active the enzymes.
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Humulus lupulus
?T
humulone
isohumulone
lupulones
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
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Linalool (spicy)Geraniol
MyrceneCaryophyleneFareseneSelinene
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trans-isohumulone
cis-isohumulone
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Further adventures in stereochemistry
carvone
thalidomide
D-form amino acids tend to taste sweet, L-form
amino acids are generally tasteless. Proteins
use L-amino acids Most sugars we digest and
incorporate are D
(R)
(S)
caraway
spearmint
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Antibiotic properties of hops
Hop compounds act as ionophores that exchange
protons for cellular divalent cations. This
decreases the intracellular pH and dissipates the
transmembrane proton gradient (?pH) 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.
-
H
antibacterial form
trans-isohumulone
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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
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S. cerevisiae life cycle
Gene expression in lag phase and early log phase
Time
Brejning et al. J Appl Microbiol. 2005.
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If fermentation is anaerobic why is so much
oxygen needed when pitching?
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S. cerevisiae life cycle
Fermentation profiles with various sugar
supplements
Piddocke et al. Applied Microbiology and
Biotechnology 2009
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S. cerevisiae life cycle
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 16328(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
2110161-77. Review.
Genetic links between diet and lifespan shared
mechanisms from yeast to humans. Bishop NA,
Guarente L. Nat Rev Genet. 2007 Nov8(11)835-44.
Review.
Gray et al. Microbiology and Molecular Biology
Reviews. 2004.
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Yeast metabolism
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Why is S. cerevisiae so good at making beer?
Make-accumulate-consume Yeast can suppress
respiration in the presence of glucose and oxygen
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Yeast settling to the bottom is not a passive
process
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What goes wrong when beer goes bad?
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.
ethanol
valine
butanediol
acetaldehyde
pyruvate
acetoin
diacetyl
acetolactate
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 Dec2(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 Apr16(6)511-22.
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Flavor in beer Organoleptic threshold (ppm) Concentration in Japanese beer (ppm)
Higher alcohols
 Propan-1-ol (n-propanol) Alcohol 800 815
 2-Methyl propanol (isobutyl alcohol) Alcohol 200 714
 2-Methyl butanol (active amyl alcohol) Alcohol, banana, medicinal, solvent 65 4671
 3-Methyl butanol (isoamyl alcohol) Alcohol, banana, sweetish, aromatic 70
 2-Phenyl ethanol Roses, sweetish, perfumed 125 2027
Esters
 Ethyl acetate Solvent, fruity, sweetish 30 1020
 Isoamyl acetate Banana, apple, solvent, estery 1.2 1.32.5
 2-Phenylethyl acetate Roses, honey, apple, sweetish 3.8 0.41.3
 Ethyl caproate Sour apple 0.21
 Ethyl caprylate Sour apple 0.9
Carbonyl compounds Carbonyl compounds
 Acetaldehyde Green leaves, fruity 25 2.93.4
 2,3-Butanedione (diacetyl) Butter-scotch 0.15 lt0.010.06
Kobayashi et al. J. Biosci. and Bioengr. 2008.
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Kobayashi et al. J. Biosci. and Bioengr. 2008.
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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 promote
Fatty acid C182 Addition repressed (only acetate esters)
Gravity Promoted (from 12 to 20 Plato media) and repressed in higher-gravity media
Temperature Promoted as temperature increased
Top pressure Repressed as top pressure increased
Oxygen Repressed during aeration prior to pitching
pH (4.98.5) 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.
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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
Thiols Thiols Thiols Thiols Thiols
Hydrogen sulfide (H2S) Pungent, rotten eggs 510 Sulfate, cysteine 0.520
Methanethiol (MTL) Cooked cabbage putrid 2 Methionine Nd
Polyfunctional thiols Polyfunctional thiols Polyfunctional thiols Polyfunctional thiols Polyfunctional thiols
3-Methyl-2-butene-1-thiol Onion, leek, skunky flavor 1100 ppt Hop (isohumulone)  cysteine  riboflavine  light Nd
2-Mercaptoethanol  rotten eggs   Cysteine Nd
3-Mercaptopropanol     Homocysteine Nd
Sulfides Sulfides Sulfides Sulfides Sulfides
Dimethyl sulfide Cabbage, corn, onion, blackcurrant 30 SMM, dimethylsulfoxide 590
Dimethyl disulfide Cooked cabbage, onion 350 MTL 0.31.5
Dimethyl trisulfide Fresh onion, cooked vegetables 0.1 MTL, H2S, 3-MTP, S-methylcysteinesulfoxide 0.11.8
Dimethyl tetrasulfide Onion, cooked vegetables 1.2 Unknown 0.2
Thioesters Thioesters Thioesters Thioesters Thioesters
S-Methylthioacetate Cheese, cooked vegetables gt100 MTL and acetylCoA 38
S-Ethylthioacetate Ripened cheese, cabbage 0.83.5 Unknown 40
Alkyl thio derivatives Alkyl thio derivatives Alkyl thio derivatives Alkyl thio derivatives Alkyl thio derivatives
Methional Soap, potato 250 lt?0.1b Methionine Nd
Methionol Cauliflower 2,000 Methionine Nd
Sulfured terpens Sulfured terpens Sulfured terpens Sulfured terpens Sulfured terpens
1,2-Epithiohumulene Cardboard, musty 200 hop 109,300 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
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Landaud et al. App. Microbiol. Biotechnol. 2008
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How can we make even better beer?
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
The potential of genetic engineering for
improving brewing, wine-making and baking
yeasts. Dequin S. Appl Microbiol Biotechnol. 2001
Sep56(5-6)577-88. 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. 200811167-98. Review.
Genetic improvement of brewer's yeast current
state, perspectives and limits. Saerens SM, Duong
CT, Nevoigt E. Appl Microbiol Biotechnol. 2010
May86(5)1195-212. Epub 2010 Mar 2. 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
Apr5(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
2049(1-3)101-9.
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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 brewers 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 brewers strains, six lager brewers strains and one type strain of S. cerevisiae in complex medium (YM) Differentiation between industrially used brewers strains Genome (CGH, S.c. gene probeset)
Pope et al. (2007) Two ale brewers strains, six lager brewers strains and one type strain of S. cerevisiae in complex medium (YM) Differentiation between industrially used brewers strains Exometabolome (DIMS, GC-TOF-MS)
Joubert et al. (2001) Lager brewers 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, MALDIMS and MS/MS)
Joubert et al. (2000) Seven lager brewers 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 brewers yeast Proteome (2D gel electrophoresis, gasliquid phase sequencing)
Brejning et al. (2005) Lager brewers 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, MALDIMS and MS/MS)
Kobi et al. (2004) Ale yeast strain (A38) in complex medium (YPD) and brewers wort Comparison of an ale, a lager and a laboratory yeast strain Proteome (2D gel electrophoresis, MALDIMS)
Kobi et al. (2004) Lager brewers yeast strain (K11) in YPD medium Comparison of an ale, a lager and a laboratory yeast strain Proteome (2D gel electrophoresis, MALDIMS)
Kobi et al. (2004) Laboratory yeast (S288c) in complex medium (YPD) Comparison of an ale, a lager and a laboratory yeast strain Proteome (2D gel electrophoresis, MALDIMS)
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 brewers yeast Transcriptome (microarray, S.c. gene and non-S.c. EST probes)
Yoshida et al. (2008) One lager brewers yeast (KBY011) and one bakers 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)
Yoshida et al. (2008) One lager brewers yeast (KBY011) and one bakers 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) Endometabolome (CEESIMS)
Dunn and Sherlock (2008) 17 lager brewers strains and 3 ale strains Differentiation between brewers 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 brewers 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)
Duong Cam et al. (in preparation) Three lager brewers 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) Transcriptome (whole-genome array with S.c. gene and non-S.c. probesets)
Duong Cam et al. (in preparation) Three lager brewers 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) Proteome (2D gel electrophoresis, MALDIMS)
Nakao et al. (2009) Lager brewers 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.
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Yeast and human health
Tumor cell energy metabolism and its common
features with yeast metabolism. Diaz-Ruiz R,
Uribe-Carvajal S, Devin A, Rigoulet M. Biochim
Biophys Acta. 2009 Dec1796(2)252-65. Epub 2009
Aug 12. 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.
200855(5)387-93. Review.
Brewer's/baker's yeast (Saccharomyces cerevisiae)
and preventive medicine Part II. Moyad MA. Urol
Nurs. 2008 Feb28(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 Jun86(3)208-14. Epub
2009 Jan 21. 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 Jul1783(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
Sep120(1-2)49-56. Epub 2006 May 15. Review.
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References and further reading
National Institutes of Health digital archive of
biomedical and life sciences journal
literature http//www.ncbi.nlm.nih.gov/sites/entr
ez?dbpubmed
http//www.wikipedia.org
http//homebrewandchemistry.blogspot.com/
Site of Brew Your Own magazine http//www.byo.com
/
http//forum.northernbrewer.com/
http//www.brewingtechniques.com/
Site of UC-Davis Anheuser-Busch Endowed Professor
of Brewing Science Charles Bamforth http//foodsc
ience.ucdavis.edu/bamforth/
Rensselaer Polytechnic Institute brewing
class http//www.rpi.edu/dept/chem-eng/Biotech-En
viron/beer/index1.htm
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Tools of the trade
High performance liquid chromatograph (HPLC)
Gas chromatograph (GC)
Fermenter
Electrospray ionization m mass spectrometer
(ESI-MS)