Petite Mutations and their Impact of Beer Flavours Maria Josey and Alex Speers ICBD, Heriot Watt University IBD Asia Pacific Meeting March 2016
Petite Mutations and their
Impact of Beer Flavours
Maria Josey and Alex Speers
ICBD, Heriot Watt University
IBD Asia Pacific Meeting March 2016
IntroductionExperimental
DesignResults Discussion Conclusion
Table of Contents
What Are They?
• No or reduced
mitochondrial function
• Spontaneous genetic
mutation
• Also called respiratory
deficient (or RD) cells
Experimental Design
Results Discussion ConclusionIntroduction
Figure 1. TTC Overlay Technique with Petite
Mutations Noted with an Arrow
Petites in the Industry
Exist in frequencies of:
0.1% to 0.5%
1% to 4%
Strain Dependent
Higher if yeast is abused
IntroductionExperimental
DesignResults Discussion Conclusion
“The RD mutant produced
considerably higher
amounts of isobutanol and
isoamyl alcohol”
Ernandes, 1993
Isoamyl alcohol has a pear drop
flavour
Isobutanol has a solvent-like
aroma
Experimental Design
Results Discussion ConclusionIntroduction
Previous Research:
End ofFermentation
Method for Fermentation (ASBC Yeast-14)
IntroductionExperimental
DesignResults Discussion Conclusion
Density and
Absorbance
Measurements
Flavour
Analysis
ThroughoutFermentation
Ethidium bromide
to produce 3.66,
5.10, 8.67, and
10.77% RD’s
measured by TTC
Overlay method
Mini-fermentation Assay
Originated for flocculation determination
Use 12.6 oP wort plus 4% glucose & 3 reps,
Turbidity/density measurements taken 10
x’s @ 21oC over 75 h,
Model sugar consumption and yeast in
suspension,
Standard method ASBC Yeast-14
7Introduction
Experimental Design
Results Discussion Conclusion
24 48 72
1
3
4
5
Time (hr)
Ab
sorb
ance
at
60
0n
m
Tilted Gaussian fit[1]
A – Amplitude µ – Meanσ – Standard Deviation R– Rotation (slope term)Height of peak, P = Rt+A
σ
A
µ
F(t)= Rt + Ae-1
2
t-æ
èç
ö
ø÷
2
P
2
Mini-fermentation Assay
Key to assay is the analysis
of both change in oP/SG
and A600
These non-linear curves
can be fit with Excel and
statistically compared with
Prism™
Yeast in Suspension Trends For All Four
Fermentations
IntroductionExperimental
DesignResults Discussion Conclusion
0 20 40 60 80 1000
1
2
3
Time (hr)
Ab
so
rban
ce (
600n
m)
3.66%
5.10%
8.39%
10.77%
Time (hr)
Ab
sorb
an
ce
(600n
m)
Figure 2. Yeast in suspension trends with
fermentations containing varying levels of petites
No significant (p>0.05) difference for
yeast in suspension characteristics as the
petite mutations present in the
fermentation increased
End ofFermentation
Method for Fermentation (ASBC Yeast-14)
IntroductionExperimental
DesignResults Discussion Conclusion
Density and
Absorbance
Measurements
Flavour
Analysis
ThroughoutFermentation
Flavours
Analysed
“Significant” means the
flavour compounds
present in the beer
changed as the
percentage of petites
changed
No significant difference in
all higher alcohols
analysed and acetone
Flavour Compound Significant (p>0.05)
Acetaldehyde Yes
Butanedione Yes
Ethyl acetate Yes
Isobutyl acetate Yes
Ethyl butyrate Yes
Pentanedione Yes
Ethyl Octanoate Yes
Iso amyl acetate Yes
Ethyl hexanoate Yes
3-Methyl butanol No
2-Methyl butanol No
Propan-1-ol No
Isobutanol No
Acetone No
Table 1. Results showing if there was a significant change in
flavour compound levels post beer fermentation as the
percentage of petites present increased up to 11%
IntroductionExperimental
DesignResults Discussion Conclusion
Acetaldehyde
Levels Post
Fermentation
Linear increase in acetaldehyde
level post fermentation with
increasing levels of petite
mutations
Same trends found with other eight
flavour compounds
% Petite Mutations
Ac
eta
lde
hyd
e (
mg
/L)
R2=0.9431p>0.0001
Figure 4. Acetaldehyde levels in the beer post fermentation as
the percentage of mutated cells increased in the population
IntroductionExperimental
DesignResults Discussion Conclusion
Flavours
Analysed
Flavours outlined showed a
significant change in flavour
levels as the petite mutations in
the pitching yeast changed.
Flavour Compound Significant (p>0.05)
Acetaldehyde Yes
Butanedione Yes
Ethyl acetate Yes
Isobutyl acetate Yes
Ethyl butyrate Yes
Pentanedione Yes
Ethyl Octanoate Yes
Iso amyl acetate Yes
Ethyl hexanoate Yes
3-Methyl butanol No
2-Methyl butanol No
Propan-1-ol No
Isobutanol No
Acetone No
Table 1. Results showing if there was a significant change in
flavour compound levels post beer fermentation as the
percentage of petites present increased up to 11%
IntroductionExperimental
DesignResults Discussion Conclusion
Does increase in flavour compounds
make noticeable differences?
Acetaldehyde
Flavour Threshold – 5mg/L
From experimental values, a 10% increase
in petites would result in a 1.44mg/L
increase in acetaldehyde
Isoamyl acetate
Flavour Threshold – 1.1mg/L
10% increase in petites would result in
0.58mg/L increase in isoamyl acetate
IntroductionExperimental
DesignResults Discussion Conclusion
Does increase in flavour compounds
make noticeable differences?
Butanedione
Flavour threshold – 0.01mg/L
10% increase resulted in 0.05mg/L
increase in butanedione
IntroductionExperimental
DesignResults Discussion Conclusion
Ethyl Acetate
Flavour threshold - 5mg/L
With 10% increase in petites, 5.6 mg/L
increase of ethyl acetate
Possible Index Compounds?
Conclusions from Petite Mutation Experiment
Nine of the flavour compounds analysed increased with increasing
petite mutations in the population
Eth
yl o
cta
no
ate
(mg
/L)
% Petite Mutations
At low levels of petites, flocculation characteristics didn’t
change
IntroductionExperimental
DesignResults Discussion Conclusion
Conclusions continued
Higher alcohols or acetone did not change with increasing
levels of petites
Based on results, it’s likely ethyl acetate and butanedione
will be the first noticeable flavour changes in beer if a high
number of petites are present
IntroductionExperimental
DesignResults Discussion Conclusion
Thank you for listening,
Questions?
Acknowledgements
IBD Ph.D. Studentship – M Josey
Alltech ICBD donation
References
American Society of Brewing Chemists. Methods of Analysis, 2012, 12th ed, Yeast-14 Miniature Fermentation Assay. The Society, St. Paul, MN.
Ernandes, J.R., et al., Respiratory Deficiency in Brewing Yeast Strains—Effects on Fermentation, Flocculation, and Beer Flavor Components. Journal of the American Society of Brewing Chemists, 1993. 51(1): p. 16-20.
Lawrence, S.J., et al., The Relationship Between Yeast Cell Age, Fermenter Cone Environment, and Petite Mutant Formation in Lager Fermentations. Journal of the American Society of Brewing Chemists, 2013. 71(2): p. 90-96.
Morrison, K.B. and A. Suggett. Yeast handling, petite mutants, and lager flavour. in European Brewery Convention. 1983. London, UK: IRL Press Limited.
Pires, E.J., et al., Yeast: the soul of beer's aroma - a review of flavour-active esters and higher alcohols produced by the brewing yeast. Applied Microbiology and Biotechnology, 2014. 98: p. 1937-1949.
Quilter, M.G., et al., The Production of Isoamyl Acetate from Amyl Alcohol by Saccharomyces cerevisiae. Journal of the Institute of Brewing, 2003. 109(1): p. 34-40.
Slonimski, P.P., G. Perrodin, and J.H. Croft, Ethidium bromide induced mutation of yeast mitochondria: Complete transformation of cells into respiratory deficient non-chromosomal “petites”. Biochemical and Biophysical Research Communications, 1968. 30(3): p. 232-239.
Propan-1-ol
Levels Post
Fermentation
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
Pro
pa
n-1
-ol (m
g/L
)
4% Petites 5% Petites 8% Petites 11% Petites
IntroductionExperimental
DesignResults Discussion Conclusion
Increasing levels of petite
mutations showed no significant
(p>0.05) in propan-1-ol
Same trends found with other
higher alcohols and acetone
Figure 3. Propan-1-ol (mg/L) in beer fermented with
varying levels of petite mutated yeasts
Flavour compounds present in beer
increase as the percentage of mutated
cells increase
% Petite Mutations
Iso
am
yl a
ce
tate
(m
g/L
)
R2=0.844p>0.0001
Eth
yl h
exa
no
ate
(mg
/L)
% Petite Mutations
R2=0.8291p>0.0001
Eth
yl o
cta
no
ate
(mg
/L)
% Petite Mutations
R2=0.9449p>0.0001
Higher Alcohol Formation
Fig. 1
“The Ehrlich pathway and the main genes involved in the synthesis of enzymes catalyzing each reaction. The
reversible transamination reaction uses different BAT-encrypted enzymes—while BAT2 catalyses the transfer of the
amino group from the amino acid to α-ketoglutarate (AKG), BAT1 is usually required on the reverse transamination for
amino acid biosynthesis” (Pires, Teixeira et al. 2014)
Taken from Pires, Teixeira et al.
2014
Acetate Esters and MCFA Ethyl Ester
Formation
Fig. 3
“A scheme of the chemical reactions involving the biosynthesis of acetate esters (a) and medium-chain fatty acid ethyl
esters (b). The main genes involved in each reaction are presented above the reaction arrows” (Pires, Teixeira et al.
2014)
Taken from Pires, Teixeira et al.
2014