Presentation Slides - Society of Barley Engineers

Report
The science of malting, brewing,
and fermenting beer
Oct 6, 2010
Protein: a chain made up of 20
different amino acids from a few to
as many as 34,350 residues
α-amylase
DNA
transcription
translation
RNA
Temperature effects on biology and
chemistry
Water is the universal solvent of life
:
The Structure and Properties of Water
D. Eisenberg and W. Kauzmann
308 pages
Oxford Press
1969
+
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)
pyruvate
lactic acid (-198 kJ/mol)
ethanol + CO2
(-235 kJ/mol)
Overview of metabolism
=
=
=
=
=
α
β
Overview of metabolism
R = some chemical group
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.
Germinating/malting
breaking down chains of stuff
http://plantphys.info/plant_physiology/gibberellin.shtml
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
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
isobutyraldehyde (wet cereal/straw)
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/?p=9586
Mashing and resting
breaking 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
Generally only needed
when using >25%
unmalted wheat or
barley, corn, or rye
β-1,4 glycosidic linkage
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
~122 °F
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.
~133 °F
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.
Generally only needed when using
minimally modified malt or a lot
(>25%) of adjuncts
Sugar enzymology II
amylopectin
amylose
limit dextrinase
(debranching enzyme)
O
β
O
O…
O
O
O
O
O
O
O
O
O
O
O
O
O
Sugar enzymology II
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
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.
reaction rate
([producet]/second)
Maximum rate
possible
KM
[Substrate]
Humulus lupulus
ΔT
humulone
isohumulone
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
Linalool (spicy)
Geraniol
Myrcene
Caryophylene
Faresene
Selinene
trans-isohumulone
cis-isohumulone
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)
spearmint
(S)
caraway
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 (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.
+ H+
antibacterial form
trans-isohumulone
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
Gene expression in lag phase and early
log phase
Time
Brejning et al. J Appl Microbiol. 2005.
If fermentation is anaerobic why is so
much oxygen needed when pitching?
S. cerevisiae life cycle
Fermentation profiles with various sugar supplements
Piddocke et al. Applied Microbiology and Biotechnology 2009
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 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.
Gray et al. Microbiology and Molecular Biology Reviews. 2004.
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
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
ethanol
valine
butanediol
pyruvate
Lagering: beer stored at 34-40 F for a
few weeks. levels of diacetyl,
acetaldehyde and sulfur compounds
decrease.
acetaldehyde
acetolactate
acetoin
The dynamics of the Saccharomyces carlsbergensis
brewing yeast transcriptome during a productionscale lager beer fermentation.
Olesen K, Felding T, Gjermansen C, Hansen J.
FEMS Yeast Res. 2002 Dec;2(4):563-73.
diacetyl
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.
Flavor in beer
Organoleptic
threshold (ppm)
Concentration in
Japanese beer
(ppm)
Alcohol
800
8–15
Alcohol
200
7–14
65
46–71
Higher alcohols
Propan-1-ol (npropanol)
2-Methyl propanol
(isobutyl alcohol)
2-Methyl butanol
(active amyl alcohol)
3-Methyl butanol
(isoamyl alcohol)
2-Phenyl ethanol
Alcohol, banana,
medicinal, solvent
Alcohol, banana,
sweetish, aromatic
Roses, sweetish,
perfumed
70
125
20–27
30
10–20
1.2
1.3–2.5
3.8
0.4–1.3
Esters
Solvent, fruity,
sweetish
Banana, apple, solvent,
Isoamyl acetate
estery
Roses, honey, apple,
2-Phenylethyl acetate
sweetish
Ethyl caproate
Sour apple
Ethyl caprylate
Sour apple
Ethyl acetate
0.21
0.9
Carbonyl compounds
Acetaldehyde
2,3-Butanedione
(diacetyl)
Green leaves, fruity
25
2.9–3.4
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
Amino acid
Metal
Lees oil
EDTA
Fatty acid
Gravity
Temperature
Top pressure
Oxygen
Production of higher alcohols and esters
Addition promoted (amyl alcohols and esters) and
no effect on isobutyl alcohol
Addition promoted (only isobutyl alcohol
Val
production)
Ile
Addition promoted (only amyl alcohol production)
Addition promoted (ethyl acetate and n-propanol)
Asp
and repressed (isobutyl alcohol)
Addition promoted (higher fermentation rates was
Zn
obtained.)
Addition promoted (isoamyl acetate) and no effect
on ethyl acetate
Addition did not significantly promote
C18:2 Addition repressed (only acetate esters)
Promoted (from 12 to 20° Plato media) and
repressed in higher-gravity media
Promoted as temperature increased
Repressed as top pressure increased
Repressed during aeration prior to pitching
Leu
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
Probable precursor
(ppb)a
Concentration in
finished beer (ppb)
Sulfur dioxide (SO2)
Burnt matches
25 ppm
Sulfate/sulfite
200
Pungent, rotten eggs
Cooked cabbage; putrid
5–10
2
Sulfate, cysteine
Methionine
0.5–20
Nd
3-Methyl-2-butene-1-thiol
Onion, leek, skunky flavor
1–100 ppt
2-Mercaptoethanol
rotten eggs
Thiols
Hydrogen sulfide (H2S)
Methanethiol (MTL)
Polyfunctional thiols
3-Mercaptopropanol
Hop
(isohumulone) + cysteine + ri Nd
boflavine + light
Cysteine
Nd
Homocysteine
Nd
30
SMM, dimethylsulfoxide
5–90
3–50
0.3–1.5
1.2
MTL
MTL, H2S, 3-MTP, Smethylcysteinesulfoxide
Unknown
Cheese, cooked vegetables
Ripened cheese, cabbage
>100
0.8–3.5
MTL and acetyl–CoA
Unknown
3–8
40
Soap, potato
Cauliflower
250 < 0.1b
2,000
Methionine
Methionine
Nd
Nd
Cardboard, musty
200
hop
10–9,300 ppm
Sulfides
Dimethyl sulfide
Dimethyl disulfide
Dimethyl trisulfide
Dimethyl tetrasulfide
Cabbage, corn, onion,
blackcurrant
Cooked cabbage, onion
Fresh onion, cooked
vegetables
Onion, cooked vegetables
0.1
0.1–1.8
0.2
Thioesters
S-Methylthioacetate
S-Ethylthioacetate
Alkyl thio derivatives
Methional
Methionol
Sulfured terpens
1,2-Epithiohumulene
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
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]
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.
Multiobjective optimization and multivariable
control of the beer fermentation process with
the use of evolutionary algorithms.
Andrés-Toro B, Girón-Sierra JM, FernándezBlanco P, López-Orozco JA, Besada-Portas E.
J Zhejiang Univ Sci. 2004 Apr;5(4):378-89.
The potential of genetic engineering for
improving brewing, wine-making and baking
yeasts.
Dequin S.
Appl Microbiol Biotechnol. 2001 Sep;56(56):577-88. Review.
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.
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
Blieck et al. (2007)
Olesen et al. (2002)
Gibson et al. (2008)
James et al. (2003)
Higgins et al (2003)
Mizuno et al. (2006)
Bond et al. (2004)
Pope et al. (2007)
Joubert et al. (2001)
Joubert et al. (2000)
Brejning et al. (2005)
Kobi et al. (2004)
Minato et al. (2009)
Yoshida et al. (2008)
Strains and conditions investigated
Purpose of investigation
Level of global analysis
(method applied)
Lager yeast strain (CMBS33) and an UV-induced mutant of this strain
showing improved fermentation performance in high-gravity wort (23 °P, 2 Strain improvement via inverse engineering
Transcriptome (microarray, S.c. gene probeset)
l scale, tall tube vessels)
Industrial lager yeast strain in 5,000 hl 14 °P wort in cylindroconical
Dynamics of brewing fermentation
Transcriptome (microarray, S.c. gene probeset)
fermentation tanks
Study of the response of lager brewing yeast to
Lager yeast strain (CB11) in cylindroconical fermentation tanks (3,275 hl
changes in wort fermentable carbohydrate
Transcriptome (microarray, S.c. gene probeset)
scale, 17 °P wort)
concentration and composition
Two bottom-fermenting lager strains (Guinness 6701 and 7012) in 2 l 15 °P
wort in European Brewery Convention (EBC) tall cylindroconical
Dynamics of brewing fermentation
Transcriptome (microarray, S.c. gene probeset)
fermentation vessels
Study of the stress response during an industrial lager
Industrial lager yeast in 20 l 12 °P wort in industrial fermentation vessels
Transcriptome (microarray, S.c. gene probeset)
fermentation
Top-fermenting brewer’s yeast strain (NCYC1245) and a 2-deoxyglucoseIdentification of the genes involved in the low acetic
resistant mutant of this strain with low acetic acid and high ethanol
Transcriptome (microarray, S.c. gene probeset)
acid/high ethanol phenotype
productivities (100 ml scale, 13 °P wort)
Two bottom-fermenting lager yeast strains (CMBS33 and Guinness 6701) in Aneuploidy and copy number breakpoints in lager
Genome (CGH, S.c. gene probeset)
comparison with the haploid laboratory strain S-150
yeast strains
Genome (CGH, S.c. gene probeset)
Two ale brewer’s strains, six lager brewer’s strains and one type strain of S. Differentiation between industrially used brewer’s
cerevisiae in complex medium (YM)
strains
Exometabolome (DIMS, GC-TOF-MS)
Identification of proteins which do not co-migrate with Proteome (2D gel electrophoresis, MALDI–MS and
Lager brewer’s yeast strain (K11) in minimal medium (YNB, 2% glucose)
the known proteins of S.c.
MS/MS)
Seven lager brewer’s yeast strains, type strains of S. cerevisiae, S. bayanus,
Obtain information about the identity of the ancestors Proteome (2D gel electrophoresis, gas–liquid phase
S. carlsbergensis, S. monascensis, S. pastorianus and S. uvarum in minimal
of lager brewer’s yeast
sequencing)
medium (YNB, 2% glucose)
Identify proteins whose expression is induced in lager
Lager brewer’s yeast strain (KVL001) in minimal medium (YNB, 0.5%
Proteome (2D gel electrophoresis, MALDI–MS and
brewing during lag phase and early exponential
glucose)
MS/MS)
growth
Ale yeast strain (A38) in complex medium (YPD) and brewer’s wort
Comparison of an ale, a lager and a laboratory yeast
Lager brewers’ yeast strain (K11) in YPD medium
Proteome (2D gel electrophoresis, MALDI–MS)
strain
Laboratory yeast (S288c) in complex medium (YPD)
Lager brewers’ yeast strain (KBY011), S. cerevisiae laboratory strain and S. Expression of S.c-type and non-S.c.-type genes in a
Transcriptome (microarray, S.c. gene and non-S.c. EST
pastorianus in complex medium (YPD)
lager brewer’s yeast
probes)
Transcriptome (microarray with S.c. gene and non-S.c.
One lager brewer’s yeast (KBY011) and one baker’s yeast (S288c) showing
Strain improvement via inverse engineering (increase EST probes)
significant differences in sulphite production (SD10 medium lacking amino
of sulphite production)
acids, 2 l scale, anaerobic conditions)
Endometabolome (CE–ESI–MS)
Genome (CGH, “two-species array” with probes for
genes from S.c. and S. bayanus var. uvarum)
Genome (whole-genome array CGH with S.c. gene and
non-S.c. probesets)
Three lager brewer’s yeast strains which show significant differences in
Strain improvement via inverse engineering (reduction
Duong Cam et al. (in preparation)
Transcriptome (whole-genome array with S.c. gene
diacetyl production analysed in wort under conditions relevant in brewing of diacetyl formation)
and non-S.c. probesets)
Proteome (2D gel electrophoresis, MALDI–MS)
Identify the complete genomic sequence of a
Nakao et al. (2009)
Lager brewer’s yeast strain (Weihenstephan 34/70)
Genome (whole genome sequencing)
commonly used lager yeast strain
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
Saerens et al. Appl Microbiol Biotechnol. 2010.
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 Dec;1796(2):25265. 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. 2008;55(5):387-93. 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.
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):138195. Epub 2008 Feb 11. Review.
Brewer's/baker's yeast (Saccharomyces
Combined yeast-derived beta-glucan with anti- cerevisiae) and preventive medicine: Part II.
Moyad MA.
tumor monoclonal antibody for cancer
Urol Nurs. 2008 Feb;28(1):73-5. Review.
immunotherapy.
Liu J, Gunn L, Hansen R, Yan J.
Protein folding diseases and
Exp Mol Pathol. 2009 Jun;86(3):208-14. Epub
neurodegeneration: lessons learned from
2009 Jan 21. Review.
yeast.
References and further reading
National Institutes of Health digital archive of biomedical and life sciences journal literature
http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed
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://foodscience.ucdavis.edu/bamforth/
Rensselaer Polytechnic Institute brewing class
http://www.rpi.edu/dept/chem-eng/Biotech-Environ/beer/index1.htm
Tools of the trade
High performance
liquid chromatograph
(HPLC)
Gas chromatograph (GC)
Fermenter
Electrospray ionization m
mass spectrometer (ESI-MS)

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