petite mutations and their impact of beer...

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



“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)



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



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)



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%



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



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%



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



Does increase in flavour compounds

make noticeable differences?

Butanedione

Flavour threshold – 0.01mg/L

10% increase resulted in 0.05mg/L

increase in butanedione



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



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



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



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

petite mutations and their impact of beer...

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