Maria Josey

International Centre for Brewing and Distilling

Heriot-Watt University, U.K.

Serial Re-Pitching:

Two Case Studies

What is serial re-pitching?

Ye

ast B

rin

k

Fresh Wort

OR

Lager

fermentations

Ale

fermentations

Completed Primary Fermentation

Flocculated

yeast

Flocculated yeast

Yeast

Propagation

1st time only

Benefits of serial re-pitching

Saves time propagating yeast

Takes multiple days

Cost saving

Reduces first crop yeast use

New yeast crops exhibit abnormal fermentation profiles

• Are breweries maximizing their

crop number?

CASE STUDY:

RE-PITCHED LAGER FERMENTATIONS

Cold

Cropped

Stored in Cornelius Keg

at 11 °C and 4.3% ABV

Cells Quantified Pitched into

12 P Wort*

Industrial Lager

Crop Collected

(3rd crop)

1st time only

Density Sampled

Flavours quantified

*Malt bill: Lager malt (73%),

rice (25%), Crystal (or

Munich) (2%)

Lager Case Study: Experimental Design

1.6 hL

Fermentation

Nonlinear Regression Analysis

0 15 30 45 60 75

15

12

9

6

3

Pi

M

B

Pe

Time (hr)

Den

sity

(°P

)

)(1 MtB

eiet

e

PPPP

Figure 1. Nonlinear

logistic describing

density attenuation

during fermentation

(ASBC Yeast-14)

Lag Period

0 1 0 0 2 0 0 3 0 0 4 0 0

0

5

1 0

1 5

3

2

1

9

8

7

6

5

4

Figure 2. Modelled density attenuation curve for nine re-

pitched fermentations, the re-pitched number is noted by

individual colours.

0 1 0 0 2 0 0 3 0 0 4 0 0

0

5

1 0

1 5

3

2

1

9

8

7

6

5

4

0 1 0 0 2 0 0 3 0 0 4 0 0

0

5

1 0

1 5

3

2

1

9

8

7

6

5

4

Time (hr)

Density (

P)

Serial Re-Pitched Lager Fermentations

Density Attenuation Significance of density attenuation

curves?

Fermentations

containing 2%

Crystal malt

noted in orange

Fermentations

containing 2%

Munich malt

noted in black

4 &5: Identical

curves

(p>0.05)

1&9: Identical

curves

(p>0.05)

0 2 4 6 8 1 0

0

2 0

4 0

6 0

8 0

C r o p N u m b e r

Mi

dp

oi

nt

(

hr

)

Figure 3. Midpoint (M) for each serial re-pitched

lager fermentation

Did the length of the fermentation

change between crop number?

An F-Test on the slope showed

the crop number had no

significant (p>0.05) correlations

to on the midpoint

Ester levels post fermentation with

respect to crop number

Figure 4. Ethyl ester levels in the beer at hour 122

in fermentation for each fermentation with an

increasing crop number

(Beer Biochemistry Text Book)

Flavour thresholds (mg/L) -

Ethyl acetate: 15-30

Butanedione: 0.1-0.15

Ethyl octanoate: 0.9-1

n-propanol: 600

Iso amyl acetate: 1.2-2

Ethyl hexanoate: 0.2-0.23

Ethyl Octanoate: 0.9-1.0

1 2 3 4 5 6 7 8 9

0 . 0

0 . 2

0 . 4

0 . 6

0 . 8

1 . 0

C r o p N u m b e r

mg

/L

E t h y l h e x a n o a t e

E t h y l O c t a n o a t e

Ethyl hexanoate

flavour threshold

Ethyl octanoate

flavour threshold

1 2 3 4 5 6 7 8 9

0

2 0

4 0

6 0

C r o p N u m b e r

mg

/L

I s o a m y l a l c o h o l

Iso amyl alcohol flavour

threshold range

Figure 5. Iso amyl alcohol levels in the beer at hour 122

in fermentation for each fermentation with an increasing

crop number

Iso amyl alcohol levels compared to

crop number

Was the crop number maximized?

• Density attenuation had no effect on crop number

• up to crop number 9

• Flavour compound levels not correlated to crop number with the

exception of ethyl octanoate, which was below flavour threshold

Future work

• Wort analysis along with fermentation monitoring

CASE STUDY:

RE-PITCHED ALE FERMENTATIONS

Ale Case Study: Experimental Design

152 hL

Fermentation

Yeast

Collected

Stored in

Yeast Brink

Pitched

Yeast

Propagated

Fermentation Monitoring

• Density monitored

• Sugar analysis t=0 and

95% complete

• Flavour analysis t=0 and

95% complete

• Initial absorbance (600nm)

Look at concentration of

sugars to see if the yeast

changed sugar uptake

during fermentation as they

were repitched

Acid

Washed

Nonlinear Regression Analysis

0 15 30 45 60 75

15

12

9

6

3

Pi

M

B

Pe

Time (hr)

Den

sity

(°P

)

)(1 MtB

eiet

e

PPPP

Figure 1. Nonlinear

logistic describing

density attenuation

during fermentation

(ASBC Yeast-14)

Lag Period = 𝑴−𝟏

𝑩+𝑷𝟎 − 𝑨

𝑩𝑫𝒆

0 2 0 4 0 6 0 8 0 1 0 0

0

1 0

2 0

3 0

4 0

5 0

T i m e ( h )

Sp

ec

if

ic

G

ra

vi

ty

1

2

3

3

4

4

5

5

6

7

7

8

8

9

All Density Attenuation Curves for Ale

Fermentations

7&7: Identical

curves

(p>0.05)

2,3,3 & 9:

Identical curves

(p>0.05)

0 2 0 4 0 6 0 8 0 1 0 0

0

1 0

2 0

3 0

4 0

5 0

T i m e ( h )

Sp

ec

if

ic

G

ra

vi

ty

1

2

3

3

4

4

5

5

6

7

7

8

8

9

Figure 6. Specific gravity attenuation data with a yeast crop that

was re-pitched up to 9 times that was modelled using a four

parameter nonlinear regression.

Significance of density attenuation

curves?

Figure 7. Density attenuation for 9 serial re-pitched

ale fermentations highlighting the two abnormal

fermentations in red

Two abnormal fermentations

0 2 0 4 0 6 0 8 0 1 0 0

0

1 0

2 0

3 0

4 0

5 0

T i m e ( h )

Sp

ec

if

ic

G

ra

vi

ty

1

2

3

3

4

4

5

5

6

7

7

8

8

9

0 1 0 2 0 3 0 4 0 5 0

0 . 0

0 . 5

1 . 0

1 . 5

M i d p o i n t ( h r )

Ab

so

rb

an

ce

(

60

0n

m)

Figure 8. The midpoint of the

fermentation compared to the

initial absorbance relating to

the quantity of yeast at the

start of fermentation. The two

data points in red are the

noted abnormal

fermentations

Abnormal fermentations a result of

under pitching

0 2 4 6 8 1 0

0

1 0

2 0

3 0

4 0

5 0

C r o p N u m b e r

Mi

dp

oi

nt

(

hr

)Midpoint (hr) for each serially

re-pitched fermentation

Figure 9. The midpoint for each

re-pitched fermentation with the

two abnormal fermentations in

red.

An F-Test on the slope showed

the crop number had no

significant (p>0.05) correlations

to on the midpoint

2 3 3 4 4 5 6 7 7

0 . 0

0 . 2

0 . 4

0 . 6

0 . 8

1 . 0

C r o p N u m b e r

mg

/L

E t h y l o c t a n o a t e

E t h y l h e x a n o a t e

Ester levels post fermentation with

respect to crop number

Figure 10. Ethyl ester levels in the beer at 95% complete

fermentations as the crop number increased.

Ethyl hexanoate flavour threshold

Ethyl octanoate flavour threshold

2 3 3 4 4 5 6 7 7

0

2 0

4 0

6 0

C r o p N u m b e r

mg

/L

I s o a m y l a l c o h o l

Figure 11. Iso amyl alcohol levels in the beer at 95%

complete fermentations as the crop number increased.

Iso amyl alcohol levels with respect to

crop number

Iso amyl alcohol flavour

threshold range

• As the lag time increased,

the maltotriose consumed

by 95% complete

fermentations decreased

Figure 12. Linear correlations between maltotriose

consumed and the lag time of the fermentation

Trends in maltotriose consumption with

lag period

0 1 0 2 0 3 0 4 0

0

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

L a g T i m e ( h r )

Ma

lt

ot

ri

os

e C

on

su

me

d (

mg

/L

)

R2

= 0 . 8 5 4 7

Was the crop number maximized?

• Same as the lager case study, the density attenuation not

indicative of drifting in one direction based on crop number up to

crop number 9

• Flavour compound levels were not correlated to the crop number

Future work

• Maltotriose deserves further investigation

Summary

• With both case studies, there was no indicators that the changes in

density attenuation or flavour profiles were due to the crop number

• Potential to investigate extending the crop number

Literature

• Bühligen, F., P. Lindner, I. Fetzer, F. Stahl, T. Scheper, H. Harms and S. Müller (2014). "Analysis of aging in lager brewing

yeast during serial repitching." Journal of Biotechnology 187: 60-70.

• Bühligen, F., P. Rüdinger, I. Fetzer, F. Stahl, T. Scheper, H. Harms and S. Müller (2013). "Sustainability of industrial yeast

serial repitching practice studied by gene expression and correlation analysis." Journal of Biotechnology 168: 718-728.

• Cutaia, A. J., A.-J. Reid and R. A. Speers (2009). "Examination of the Relationships Between Original, Real and Apparent

Extracts, and Alcohol in Pilot Plant and Commercially Produced Beers." Journal of the Institute of Brewing 115(4): 318-327.

• Jenkins, C. L., A. I. Kennedy, J. A. Hodgson, P. Thurston and K. A. Smart (2003). "Impact of Serial Repitching on Lager

Brewing Yeast Quality." Journal of the American Society of Brewing Chemists 61(1): 1-9.

• Miller, K. J., W. G. Box, D. M. Jenkins, C. Boulton, R. S. T. Linforth and K. A. Smart (2013). "Does Generation Number

Matter? The Impact of Repitching on Wort Utilization." Journal of the American Society of Brewing Chemists 71(4): 233-241.

• Powell, C. and T. Fischborn (2010). "Serial Repitching of Dried Lager Yeast." Journal of the American Society of Brewing

Chemists 68(1): 48-56.

• Powell, C. D. and A. N. Diacetis (2007). "Long Term Serial Repitching and the Genetic and Phenotypic Stability of Brewer’s

Yeast." Journal of the Institute of Brewing 113(1): 67-74.

• Smart, K. A. and S. Whisker (1996). "Effect of Serial Repitching on the Fermentation Properties and Condition of Brewing

Yeast." Journal of the American Society of Brewing Chemists 54(1): 41-44.

• Somani, A., F. Bealin-Kelly, B. Axcell and K. A. Smart (2012). "Impact of Storage Temperature on Lager Brewing Yeast

Viability, Glycogen, Trehalose, and Fatty Acid Content." Journal of the American Society of Brewing Chemists 70(2): 123-130.

• Speers, R. A., P. Rogers and B. Smith (2003). "Non-Linear Modelling of Industrial Brewing Fermentations." Journal of the

Institute of Brewing 109(3): 229-235.

• Speers, R. A. and S. Stokes (2009). "Effects of Vessel Geometry, Fermenting Volume and Yeast Repitching on Fermenting

Beer." Journal of the Institute of Brewing 115: 148-150.

• Speers, R. A., Y.-Q. Wan, Y.-L. Jin and R. J. Stewart (2006). "Effects of Fermentation Parameters and Cell Wall Properties on

Yeast Flocculation." Journal of the Institute of Brewing 112(3): 246-254.

Thanks and acknowledgments

• International Centre for Brewing and Distilling (ICBD)

• Alex Speers (ICBD and Dalhousie)

• Dawn Maskell

• James Bryce

• Annie Hill

• Matthew Pauley

• Margaux Huismann

• Scottish breweries

• Production crew and technical staff

• Institute of Brewing and Distilling

Thank you for listening!

Maria Josey

ICBD, Heriot-Watt University

Edinburgh, U.K.

Email: mej2@hw.ac.uk

1 2 3 4 5 6 7 8 9

0 . 0

0 . 5

1 . 0

C r o p N u m b e r

mg

/L

B u t a n e d i o n e

P e n t a n e d i o n e

Iso amyl acetate flavour threshold range

Pentanedione flavour threshold

Figure 5. Vicinal diketone levels in the beer at hour 122

in fermentation for each fermentation with an increasing

crop number

Vicinal diketone levels post fermentation

with respect to crop number

(Lager Fermentations)

Figure 11. Vicinal diketone levels in the beer at 95%

complete fermentations as the crop number increased.

Vicinal diketones present post

fermentation with respect to crop number

(Ale Fermentations)

2 3 3 4 4 5 6 7 7

0 . 0

0 . 5

1 . 0

C r o p N u m b e r

mg

/L

B u t a n e d i o n e

P e n t a n e d i o n e

Butanedione flavour threshold

Pentanedione flavour threshold

0 2 4 6 8 1 0

0

5

1 0

1 5

2 0

E t h y l A c e t a t e a n d I s o A m y l A c e t a t e

C r o p N u m b e r

mg

/L

I s o a m y l a c e t a t e

E t h y l a c e t a t e

0 2 4 6 8 1 0

0 . 0 0

0 . 0 5

0 . 1 0

I s o b u t y l A c e t a t e a n d E t h y l b u t y r a t e

C r o p N u m b e r

mg

/L

E t h y l b u t y r a t e

I s o b u t y l a c e t a t e

0 2 4 6 8 1 0

0

1 0

2 0

3 0

4 0

5 0

H i g h e r A l c o h o l s

C r o p N u m b e r

mg

/L

2 - M e t h y l b u t a n o l

I s o b u t a n o l

P r o p a n - 1 - o l

3 - M e t h y l b u t a n o l

0 2 4 6 8 1 0

0 . 0

0 . 1

0 . 2

0 . 3

0 . 4

0 . 5

E t h y l O c t a n o a t e a n d E t h y l H e x a n o a t e

C r o p N u m b e r

mg

/L

E t h y l h e x a n o a t e

E t h y l o c t a n o a t e

Flavour thresholds (mg/L) -

Ethyl acetate: 15-30

Butanedione: 0.1-0.15

Ethyl octanoate: 0.9-1

n-propanol: 600

Iso amyl acetate: 1.2-2

Ethyl hexanoate: 0.2-0.23

Ethyl Octanoate: 0.9-1.0

Esters and Higher Alcohols Analysed in the Serial Re-

Pitching with Lager Yeast Case Study