Impact of Brewing Yeast Practice On Yeast Performanceyoungscientistssymposium.org/YSS2016/pdf/Smart.pdf · 2021. 3. 15. · Impact of Brewing Yeast Practice On Yeast Performance Professor

Impact of Brewing Yeast Practice On Yeast Performance

Professor Katherine Smart

Group Chief Brewer

© SABMiller 2014

Typical Brewing Yeast Supply Chain

2

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© SABMiller 2014 3

Serial Repitching

Spent Yeast

© SABMiller 2014

Fermentation• Key Challenges

• High gravity worts

• Initial and Final DO levels

• Multi brew filling

• Low FAN, FAN not in balance

• High acetaldehyde and ethanol

• Temperature downshift

• Cold storage, nutrient limitation, low pH

© SABMiller 2014 5

Impact of Serial Repitching on Viability

0

50

100

150

Prop Crop G5 Crop G7

%

Via

bilit

y

Viability of Serially Repitched SCB4

MVC

MgANS

PC

C. L. Jenkins, A. I. Kennedy, P. Thurston, J. A. Hodgson

and Smart, K. A. (2003). Journal of the American Society

of Brewing Chemists. 61 (1) 1-9.

© SABMiller 2014 6

Cell Death By Natural Causes

Smart, K. A. (2000). The Death of the Yeast Cell. In Brewing Yeast

Fermentation Performance. Edited by K. A. Smart. Blackwell Science.

© SABMiller 2014 7

Viability and ethanol profiles during fermentation of S. cerevisiae LAL7 under

different glucose concentrations. Pornpukdeewattana and Smart, unpublished data

Causes of Lethal Stress

Glucose 120 g/l50

70

90

0 10 20 30 40 50 60 70

Time (hours)

Per

cent

age

viab

ility

0

10

20

30

40

50

60

Etha

nol (

g/l)

viability (%)

ethanol (g/l)

© SABMiller 2014 8

Osmotolerance Is Strain Dependent

0

10

20

30

40

50

60

70

80

90

100

0 5 10 15 20 25 30

Sorbitol (%w/v)

Per

cen

tag

e V

iab

ility

SCB1

SCB2

SCB3

SCB4

SCB5

SCB6

SCB7

SCB8

S288C

Mortality profiles of industrial strains of Saccharomyces species to sorbitol induced osmotic stress. Stationary

phase cells were incubated in sorbitol (0-30% [w/v]) and incubated at 25°C for 48 hours (White and Smart,

unpublished data)

© SABMiller 2014

0

20

40

60

80

100

0 8 16 24 32 40 48 56 64 72 80 88 96

Pe

rce

nta

ge

via

bil

ity

Time(hr)

Tolerance of strains when exposed to 10%(v/v) ethanol for 96 hours in anaerobic conditions

Ethanol Tolerance is Strain Dependent

Cheung, A.W.Y., Brosnan, J.M., Phister, T. and Smart, K. A. (2012). Impact of dried, creamed

and cake supply formats on the genetic variation and ethanol tolerance of three Saccharomyces

cerevisiae distilling strains. Journal of the Institute of Brewing and Distilling, 118(2), 152-162.

© SABMiller 2014

Viability as a Function of Gravity and Pitching rate

• In general, cell viability decreased with the increased wort density.

• Lager strains showed a more moderate decrease of cell viability than ale strains in the

study.

40

50

60

70

80

90

100

0 20 40 60 80 100 120

Viab

ility

(%)

Time (hours)

40

50

60

70

80

90

100

0 20 40 60 80 100 120

Viab

ility

(%)

Time (hours)

40

50

60

70

80

90

100

0 20 40 60 80 100 120

Viab

ility

(%)

Time (hours)

40

50

60

70

80

90

100

0 20 40 60 80 100 120

Viab

ility

(%)

Time (hours)

40

50

60

70

80

90

100

0.0 3.0 13.6 24.1 48.0 62.5 90.0 120.0

Viab

ility

(%)

Time (hours)

Lager1

13P15M 18P15M 18P18M 24P15M 24P24M

Lager1 W34/70 NCYC1332 M2

Zhuang, Smart and Powell, manuscript in preparation

© SABMiller 2014

Some of the Impacts of Acetaldehyde and Ethanol Damage are Sub-lethal - Spontaneous Mutant Isolation

Professor K. A. Smart 11

Petite and wild type colony

appearance in TTC overlaid-

plates. Yeast colonies were

grown in YPD plates for 3

days at 25oC before overlay

with TTC agar. Wild type

colonies changed colour to red

(red arrows) while “white”

colonies indicated petite

mutants (white arrows).

© SABMiller 2014 12

Evidence of Accumulation of Damage

0

2

4

6

8

10

12

14

0 1 2 3 4 5 6

Incidence of Petites% Petites

Crop Generation Number

Jenkins, Lawrence, Kennedy, Thurston, Hodgson, and

Smart Journal of the American Society of Brewing

Chemists, 67 (2) 72-80

© SABMiller 2014

Pre-disposition to Ethanol Sub-Lethal Damage is Strain Dependent


Mean of percentage petite mutant frequency

from CB11 (a), W34/70 (b), NCYC2593 (c)

and BY4741 (d) after exposure to ethidium

bromide (EtBr). Prior to exposure to EtBr,

yeast strains were stressed with 0% ,2%,

4%,6% and 8% for 2 days at 40C. Petite

mutant frequencies were determined by TTC

overlay assay. The values represented the

means of triplicate analysis and the standard

deviations were shown as error bars (Pham,

Nicholls and Smart, unpublished data).

© SABMiller 2014

Sub-Lethal Damage Increases The Risk of Cell Death Petite Cell Death Occurs in the Presence of Ethanol


Viability of wild type and petites from the strain W34/70 during exposure to 0% ethanol (a) and 8% ethanol (b) at

10oC. Wild type colonies were isolated from the cultures treated with 0% ethanol (0% WT) and with 8% ethanol

(8%WT). Two types of petites tested were spontaneous petites from 0% ethanol (0%P) and from 8%-ethanol-

induced petite (8%P). The values were the means of three independent analyses with standard deviations (Pham,

Nicholls and Smart, unpublished data).

In An Ideal World We Would Select Strains For Fermentation Robustness

© SABMiller 2014

Yeast cell growth

and metabolism

NADHTV

ColourlessTV

ColourTV = Tetrazolium violet

Screening of Strains For Tolerance To These

Unintended Outcomes:

• Confirmation of Assay Efficacy (Macrolog)

• Novel Plates For Assaying

© SABMiller 2014

Sorbitol induced-osmotic stress

20% SORBITOL

D2

F2

F5

NCYC2592

20% SORBITOL AN

F4

D2F5

F2NCYC2592

F4

F2 D2F5NCYC2592

Parental strains – (89)

Parental Saccharomyces sensu stricto strains tolerance to osmotic

stress in the presence of sorbitol (A) 20% and (B) 30% at 24 hours

incubation at 300C in anaerobic conditions. Inset is the data for the

entire incubation period for NCYC2592 (Control) and the more

efficient sugar utilising parental strain (D2) in anaerobic condition.

Spot plates for exemplar strains is also shown (C).

106 105 104 103 102

A B

C

© SABMiller 2014

Ethanol stress

106 105 104 103 102

Parental Saccharomyces sensu stricto strain tolerance to ethanol

stress in the presence of ethanol (A) 5% (B) 10% and (C) 15% at 24

hours incubation at 300C in anaerobic conditions. Insert is the data for

the entire incubation period for NCYC2592 (Control) and the more

efficient sugar utilising parental strains (D2 and F5) in anaerobic

condition. Spot plates for exemplar strains is also shown (D).

BF4

D2F5

F2

C7

NCYC2592

F4

D2

F5

F2

C7

F

4D2 F

5

F

2

Parental strains – (89)

A

C D

Older Generations Might Be Impaired But Limited RepitchingImproves Performance

19

0

50

100

150

Prop Crop G5 Crop G7%

V

iab

ilit

y

Viability of Serially Repitched SCB4

MVC

MgANS

PC


Laboratory Fermentations

© SABMiller 2014 Professor K. A. Smart 21

Cell Division During Fermentation

0.0

2.0

4.0

6.0

8.0

10.0

12.0

0 20 40 60 80 100 120 140 160 180

Time (h)

Via

ble

cel

l co

un

t (x

107 ce

lls/m

l)

0

10

20

30

40

50

60

70

80

90

100

Bud

ding

Inde

x (%

)

a

0

2

4

6

8

10

12

0 20 40 60 80 100 120 140 160 180

Time (h)

Via

ble

cell c

ou

nt

(x10

7 c

ells/m

l)

0

10

20

30

40

50

60

70

80

90

100

Bu

dd

ing

In

dex (

%)

b

Lag phase duration longer for generation 0 fermentations (Blue Line), Initial budding index higher for generation 0 fermentations

Miller, K.L., Box, W.G., Boulton, C.A., and Smart, K.A. (2012). Cell Cycle Synchrony of Propagated and Recycled Lager

Yeast and Its Impact on Lag Phase in Fermenter. Journal of the American Society of Brewing Chemists, 70 (1), 1-9.

GENERATION 0 GENERATION 1

© SABMiller 2014

Analysis of Data


Generation 0 Generation 1

Time of initial increase in cell density (h) 7 6

Budding index of pitched yeast (%) 7 ± 0.9 0

Time of initial budding index increase (h) 3 3

Time of peak budding index (h) 8 7.5

Peak budding index (%) 89 ± 0.6 96 ± 0.6

Rate of budding index increase to peak (% h-1) 16.3 23.9

Comparison of viable cell density and budding index profiles for generation 0 and generation 1 fermentations with the

lager strain. Values are the mean of triplicate measurements. Standard error of the mean is shown.

Miller, K.L., Box, W.G., Boulton, C.A., and Smart, K.A. (2012). Cell Cycle Synchrony of Propagated and Recycled Lager Yeast and

Its Impact on Lag Phase in Fermenter. Journal of the American Society of Brewing Chemists, 70 (1), 1-9.


To Understand This Better

G1 S

G2

M

G0

START

G1 S(White and Smart, Unpublished Data)


Generation 1 DNA content

13 h

23 h

167 h

1 h1 h

1.5 h1.5 h

2 h2 h

2.5 h2.5 h

3 h3 h

3.5 h3.5 h

4 h4 h

4.5 h4.5 h

5 h5 h

5.5 h5.5 h

6 h6 h

6.5 h6.5 h

7 h7 h

7.5 h7.5 h

8 h8 h

8.5 h8.5 h

9 h9 h

9.5 h9.5 h

10 h10 h

10.5 h10.5 h

11 h11 h

11.5 h11.5 h

12 h12 h

0 h0 h




DNA Synthesis

0

10

20

30

40

50

60

70

80

90

100

0 2 4 6 8 10 12

Time (h)

Pro

port

ion

of c

ells

(%

)

a

0

10

20

30

40

50

60

70

80

90

100

0 2 4 6 8 10 12

Time (h)

Pro

port

ion

of c

ells

(%

)

b

GENERATION 0 GENERATION 1

• Proportion of cells with greater than one copy DNA determined at pitching

• Rate of DNA synthesis is slower in generation 0 fermentations




Cellular DNA Content After Propagation

Flow cytometric analysis of DNA content of yeast samples collected from a 140 hl propagation vessel.

Miller, Box, Boulton and Smart (in preparation)

26


Cellular DNA Content After Storage

27

Flow cytometric analysis of DNA content of yeast samples collected

from 50 hl YCVs. Miller, Box, Boulton and Smart (in preparation)


Viability Is Not Affected

Viability (%) of yeast samples collected from a 140 hl propagation

vessel and 50 hl storage tanks (Miller, Box, Boulton and Smart, in

press).

© SABMiller 2014

Gravity Decline


0

2

4

6

8

10

12

14

16

0 20 40 60 80 100 120 140 160 180

Gra

vit

y (

oP

)

Time (h)

Changes in wort gravity during generation 0 (open

square) and generation 1 (closed square)

fermentations with the lager strain CB11. Values

are the means of measurement of samples

collected from three independent fermenters.

Error bars indicate the standard error of the mean.

Miller, K.L., Box, W.G., Boulton, C.A., and Smart, K.A. (2012). Cell Cycle Synchrony of Propagated and Recycled

Lager Yeast and Its Impact on Lag Phase in Fermenter. Journal of the American Society of Brewing Chemists, 70 (1),

1-9.


Sugar UtilisationSucrose (∆), Fructose ()and

Glucose (□) concentrations in

wort sampled from generation 0

(A) and generation 1 (B)

fermentations with the lager

strain CB11. Concentrations

determined using High

Pressure Liquid

Chromatography (HPLC).

Values are the means of

measurement of samples

collected from three

independent fermenters. Error

bars indicate the standard error

of the mean.

Miller, K.L., Box, W.G., Boulton,

C.A., and Smart, K.A. (2012). Cell

Cycle Synchrony of Propagated

and Recycled Lager Yeast and Its

Impact on Lag Phase in

Fermenter. Journal of the

American Society of Brewing

Chemists, 70 (1), 1-9.


Sugar UtilisationSucrose (∆), Fructose ()and

Glucose (□) concentrations in

wort sampled from generation 0

(A) and generation 1 (B)

fermentations with the lager

strain CB11. Concentrations

determined using High

Pressure Liquid

Chromatography (HPLC).

Values are the means of

measurement of samples

collected from three

independent fermenters. Error

bars indicate the standard error

of the mean.

Miller, K.L., Box, W.G., Boulton,

C.A., and Smart, K.A. (2012). Cell

Cycle Synchrony of Propagated

and Recycled Lager Yeast and Its




Chemists, 70 (1), 1-9.

© SABMiller 2014

Sugar Utilisation


Maltose (■) and maltotriose (▲) concentrations in

wort sampled from a generation 0 (A) and

generation 1 (B) fermentations with the lager

strain CB11. Concentrations determined using

high pressure liquid chromatography (HPLC).

Values are the means of measurement of samples

collected from three independent fermenters.

Error bars indicate the standard error of the mean.

Miller, K.L., Box, W.G., Boulton, C.A., and Smart, K.A.

(2012). Cell Cycle Synchrony of Propagated and

Recycled Lager Yeast and Its Impact on Lag Phase in

Fermenter. Journal of the American Society of

Brewing Chemists, 70 (1), 1-9.


FAN Levels

0

50

100

150

200

250

300

Generation 0 Generation 1

Co

nce

ntr

ati

on

(m

g l

-1)

Differences in free amino nitrogen concentration (mg l-1) in generation 0 and generation 1 fermentations with the

lager strain CB11 at 0h (Dark Bar) and 167 h (Blue Bar) after pitching. Values are the means of measurement of

samples collected from three independent fermenters. Error bars indicate the standard error of the mean.

© SABMiller 2014

Amino Acid Utilisation Profile


Amino acid Initial concentration (mmol l-1) Final concentration (mmol l-1) Percentage assimilated

Gen 0 Gen 1 Gen 0 Gen 1 Gen 0 Gen 1

Aspartic acid 27.1 35.9 0.9 1.2 96.6 96.7

Asparagine 56.9 68.4 1.9 2.3 96.7 96.7

Glutamic acid 24.5 34.2 2.2 4.4 91.2 87.3

Lysine 31.1 39.8 0.2 0.2 99.4 99.4

Serine 75.7 86.1 1.8 2.4 97.6 97.2

Threonine 21.9 22.2 0.2 0.1 99.2 99.6

Histidine 26.7 31.0 0.3 2.0 99.0 93.5

Isoleucine 19.9 21.2 0.6 2.3 97.2 89.4

Leucine 48.7 53.9 1.1 3.3 97.8 93.9

Methionine 6.7 6.1 0.1 0.1 98.1 97.7

Valine 36.4 39.0 4.2 12.4 88.5 68.2

Alanine 45.6 51.3 12.9 27.9 71.7 45.5

Glycine 16.8 17.1 4.2 8.2 75.1 52.0

Phenylalanine 30.3 31.6 1.9 6.7 93.7 78.7

Tryptophan 9.9 10.1 2.5 4.2 74.6 58.1

Tyrosine 22.4 23.9 5.0 10.6 77.8 55.8

Proline 136.7 160.9 136.1 147.8 0.4 8.1

Initial and final

concentrations of wort

amino acids and their

percentage depletion in

generation 0 (Gen 0) and 1

(Gen 1) fermentations with

the lager strain CB11.

Concentrations determined

using gas chromatography

mass spectrometry

(GCMS). Values are the

means of measurement of

samples collected from

three independent

fermenters.

Miller, K.L., Box, W.G.,

Boulton, C.A., and Smart,

K.A. (2012). Cell Cycle

Synchrony of Propagated and

Recycled Lager Yeast and Its




Chemists, 70 (1), 1-9.

© SABMiller 2014

Amino Acid Utilisation Profile


Amino acid Initial concentration (mmol l-1) Final concentration (mmol l-1) Percentage assimilated

Gen 0 Gen 1 Gen 0 Gen 1 Gen 0 Gen 1

Aspartic acid 27.1 35.9 0.9 1.2 96.6 96.7

Asparagine 56.9 68.4 1.9 2.3 96.7 96.7

Glutamic acid 24.5 34.2 2.2 4.4 91.2 87.3

Lysine 31.1 39.8 0.2 0.2 99.4 99.4

Serine 75.7 86.1 1.8 2.4 97.6 97.2

Threonine 21.9 22.2 0.2 0.1 99.2 99.6

Histidine 26.7 31.0 0.3 2.0 99.0 93.5

Isoleucine 19.9 21.2 0.6 2.3 97.2 89.4

Leucine 48.7 53.9 1.1 3.3 97.8 93.9

Methionine 6.7 6.1 0.1 0.1 98.1 97.7

Valine 36.4 39.0 4.2 12.4 88.5 68.2

Alanine 45.6 51.3 12.9 27.9 71.7 45.5

Glycine 16.8 17.1 4.2 8.2 75.1 52.0

Phenylalanine 30.3 31.6 1.9 6.7 93.7 78.7

Tryptophan 9.9 10.1 2.5 4.2 74.6 58.1

Tyrosine 22.4 23.9 5.0 10.6 77.8 55.8

Proline 136.7 160.9 136.1 147.8 0.4 8.1

Initial and final

concentrations of wort

amino acids and their

percentage depletion in

generation 0 (Gen 0) and 1

(Gen 1) fermentations with

the lager strain CB11.

Concentrations determined

using gas chromatography

mass spectrometry

(GCMS). Values are the

means of measurement of

samples collected from

three independent

fermenters.

Miller, K.L., Box, W.G.,

Boulton, C.A., and Smart,

K.A. (2012). Cell Cycle

Synchrony of Propagated and

Recycled Lager Yeast and Its




Chemists, 70 (1), 1-9.

© SABMiller 2014

Full Scale Amino acid uptakePropagation Vs. Fermentation

0

1

2

3

4

5

0 20 40 60 80 100

Time (hours)

Am

ino

acid

s (m

mol

L-1

)

Alanine

Glycine

Valine

Leucine

allo-leucine

Isoleucine

Threonine

Serine

Proline

Asparagine

Aspartic acid

Methionine

Glutamic acid

Phenylalanine

Glutamine

Lysine

Histidine

Tyrosine

Tryptophan


0

2

4

6

8

10

0 10 20 30

Time (hours)

Am

ino

acid

s (m

mol

L-1

)

Alanine

Glycine

Valine

Leucine

allo-leucine

Isoleucine

Threonine

Serine

Proline

Asparagine

Aspartic acid

Methionine

Glutamic acid

Phenylalanine

Glutamine

Lysine

Histidine

Tyrosine

Tryptophan

(Gibson, Boulton, Box, Graham, Lawrence,

Linforth and Smart (2010) Journal of the

American Society of Brewing Chemists

doi:10.1094 /ASBCJ-2009-1123-01)

Gibson, Boulton, Box, Graham, Lawrence, Linforth and

Smart (2009). Amino acid uptake and yeast gene

transcription during industrial brewery fermentation.

Journal of the American Society of Brewing Chemists,

67 (3): 157-165.

© SABMiller 2014

ConclusionsMaximum Generation Number is Dependent on

Strain and process conditions

New high-throughput selection methods for strains with improved characteristics

Knowing how to manage process conditions to alleviate stress is critical

Yeast From Storage Tank

Is more synchronous

Doesn’t deplete Group B and C amino acids in the next fermentation

Freshly Propagated Yeast Behaves Differently To Generation One Yeast

Delay in glucose exhaustion

Delay in next cell division


© SABMiller 2014

Acknowledgements

SABMiller PLC,Coors & Bass Scottish Courage

Professor Chris Boulton Dr David Brown, Hilary Jones

Dr Alan Kennedy

University of Nottingham University of Oxford Brookes

Mrs Wendy Box Dr Cheryl Jenkins

Dr Chris Powell Dr Phil White

Dr Brian Gibson

Dr Annie Cheung

Dr Stephen Lawrence

Dr Katherine Miller

Shiwen Zhuang

38

http://www.coorsbrewers.com/index.html

http://www.coorsbrewers.com/index.html

http://www.bbsrc.ac.uk/

http://www.bbsrc.ac.uk/

Thank you for your kind attention

Documents

Impact of Brewing Yeast Practice On Yeast Performanceyoungscientistssymposium.org/YSS2016/pdf/Smart.pdf · 2021. 3. 15. · Impact of Brewing Yeast Practice On Yeast Performance Professor