○Hiroyuki Yoshimoto, Hiroki Fujiwara,

Aya Takahashi, Shigehiro Yoshizaki

Research Laboratories for Alcoholic Beverage Technologies,

Kirin Company, Limited, Japan

More Efficient and Flexible Culture

for Yeast Propagation

Asia Pacific Convention 2018 | Wellington, New Zealand Institute of Brewing and Distilling Asia Pacific Section 1

Asia Pacific Convention 2018 | Wellington, New Zealand Institute of Brewing and Distilling Asia Pacific Section 2

1. Limiting factor for propagation

(1) Importance of Nutrient balance

(2) Zinc

(3) Fed-batch culture

2. Acclimation from propagation to

fermentation

Agenda

2

YPD10

10% Glucose (Glc)

1% Yeast extract (YE)

2% Peptone (PE)

KBY011, 20℃, 4 days

Importance of Nutrient Balance

CO2 ＋ - - ＋

Dead c

ell r

atio (

%)

Sugar induced cell

death=SICD ⇒Lack of Non-sugar

Nutrients induces SICD

3

Nutrient Balance between Sugar and Non-Sugar

Nutrients

Constant

of Sugar

conc. Reduction

of non-sugar

Nutrients

Conc.

Reduction of non-sugar Nutrients conc. induces reduction of propagation and

fermentation performance and cell death.

4

Autophagy: Nutrient Recycling System

Resource: Front. Biol. 2015, 10 : 154-164 5

Cell Growth, Autophagy, SICD

Nutrient balance between Sugar and non-Sugar nutrients controls

Autophagy and SICD.

Cell Growth

(Fermentation)Autophagy SICD

deathHigh Vitality

Worst

Propagation

(Ferment

ation)

Good propagation

(Fermentation)

Low vitality

Worse

Propagation(Fer

mentation)

6

What is the Limiting Factor for Propagation?

Nutrient

from Wort

Limiting factor:

Mineral (Zinc)

Amino acids

Vitamin etc.7

Asia Pacific Convention 2018 | Wellington, New Zealand Institute of Brewing and Distilling Asia Pacific Section 8

1. Limiting factor for propagation

(1) Importance of Nutrient balance

(2) Zinc

(3) Fed-batch culture

2. Acclimation from propagation to

fermentation

Agenda

8

Zinc is one of Limiting Factors

Zinc is an essential mineral that stimulates the activity of about 100 enzymes

such as alcohol dehydrogenase and transcriptional factor with zinc finger.

Fig u re Stru ctu re o f a lcoh o l

d eh yd rog en a seResou rce :

h ttp : //p d b 1 0 1 . r csb . o rg /m o tm /1 3

Fig u re Stru ctu re o f

Tra n cr ip tio n a l fa cto r

w ith z in c f in g er

Resou rce : h ttp : //w w w . w eb -

b ook s. com /M oBio /Free/Ch 4 F2 . h tm9

Relationship between Zinc in Wort and

Intracellular Zinc in Yeast

No zinc supply from wort

Poor zinc con. in wort ( more than 1-fold ) Zinc

concentration in cell

Rich zinc conc. in wort

( 0.5-fold ) Zinc

concentration in cell

(0.25-fold ) Zinc

concentration in cell

Zinc supply from wort

Nutrient

from Wort

Nutrient

from Wort

Nutrient

from Wort

10

Zinc is the Limiting Factor in Propagation

Zinc in yeast cell decreased in case of over-propagation. Usual wort zinc

content is only about 0.1mg/L and totally taken up by propagated yeast.

Figure Yeast zinc content change during the propagation process.

0

50

100

150

200

0 10 20 30 40 50

Time (h)

Zin

c (

pp

m y

ea

st)

Ce

ll c

ou

nt

(10

6/M

L)

0

5

10

15

20

Ex

tra

ct

(°P

)

Zinc in Yeast

Cell countExtract

11

Figure Wort zinc concentration and yeast propagation.

0

100

200

300

0 10 20 30

Propagation time (h)

Ce

ll c

ou

nt

(10

6/M

L)

Zn 0.60mg/L

Zn 0.30mg/L

Zn 0.15mg/L

Zn 0.05mg/L

Addition of Zinc Improved Propagation

Addition of zinc ion in wort increased the maximum cell count during

propagation.

12

Young beer (168h) flavors

analysis

Fermentation Characteristics of Propagated Yeast

Fermentation profile of yeast

propagated with high zinc wort.

Time (h)

Ex

tra

ct

(0P

)

13

It showed a faster fermentation compared to yeast propagated with

normal wort (Control) and off-flavors such as acetaldehyde was reduced.

Asia Pacific Convention 2018 | Wellington, New Zealand Institute of Brewing and Distilling Asia Pacific Section 14

1. Limiting factor for propagation

(1) Importance of Nutrient balance

(2) Zinc

(3) Fed-batch culture

2. Acclimation from propagation to

fermentation

Agenda

14

COPY SLIDE OPTION 1 Fed-batch Culture for Brewing Yeast

Nutrient balance between sugar and non-sugar nutrients is important and zinc is

one of the Limiting factor. Taking account for these two theories, we have

developed more efficient and flexible Fed-batch culture system for brewing yeast.

Cell growth

Ethanol

production

Limited

Glucose

Cell Growth

inhibition

Sugar concentration is kept

very low (<<0.1 Plato).

15

COPY SLIDE OPTION 1 Comparison with Conventional Culture Method

Fed-batch culture improved yield and maximum cell count

compared to conventional batch culture.

Table Major technical data in the

brewing yeast culture

Condition: 10L scale propagator, 20℃,

5L/min air, 800rpm stir

Table Fed-batch culture medium

Com p on en ts

Feed Sug ar

Culture

m edium

Yeast extract

Am m on ium su lfate

Am m on ium phosphate

Other Salts,

Minera ls(Zinc) and

Vitam ins

Con ven tio n a l

b a tch cu ltu re

Fed -b a tch

cu ltu re

Med ium W ort Synthetic

m ed ium

Yield (m illion

cells/m l per Plato)34 . 8 105 . 2

Maxim um cell count

(m illion cells/m l)120 989

Viab lility ( % ) 99 99

16

COPY SLIDE OPTION 1 Advantages of the Fed-batch Culture Method

Advantages of fed-batch culture are

(1) Maximum cell count is very high and the number of scale-up steps can be

drastically reduced.

(2) Yeast culture schedule is independent of wort production.

(1)

Wort production

(2)

Use synthetic medium

instead of wort 17

Asia Pacific Convention 2018 | Wellington, New Zealand Institute of Brewing and Distilling Asia Pacific Section 18

1. Limiting factor for propagation

(1) Importance of Nutrient balance

(2) Zinc

(3) Fed-batch culture

2. Acclimation from propagation to

fermentation

Agenda

18

Acclimation between Respiration and

Fermentation Phase shift occurs between Respiration and fermentation. Acclimation is very

important for the transition from propagation to fermentation.

Respiration

(Propagation)Fermentation

Acclimation

(adaptation)

Phase shift

Aerobic Anaerobic

19

COPY SLIDE OPTION 1 Acclimation Experimental Outline

20

1 Fermentation Profiles and Flavor contents of

Young Beer

21

Principal Component Analyses

of the Metabolites in Yeast

22

COPY SLIDE OPTION 1 Conclusions

1. Nutrient balance between sugar and non-sugar nutrients is

important for yeast growth during propagation and

fermentation.

2. Zinc is one of the Limiting Factor in Propagation. Conditions

need be maintained to ensure that there is no limiting factor.

3. We have developed a fed-batch culture that allows brewing

yeast to reproducibly reach densities of 1000 million cells/ml.

4. Acclimation is very important for the transition from

propagation to fermentation, because acclimation facilitates

a phase shift from respiration to fermentation.

23

IBD PPT – Final Slide

Thank you for your

attention!