0.5cm

Stress, Viability and Active Dried Yeast

Chris Powell The University of Nottingham, UK

0.5cm

Introduction

• Good quality brewers yeast • How does stress influence yeast cultures

• What stresses are yeast subjected to during brewing • The effect of stress on vitality/viability/damage

• Desiccation as a brewing yeast stress • Dehydration/rehydration

• Prevention of stress by desiccation • Anti stress compounds

What is Good Quality Brewers Yeast ?

Good quality yeast slurry

• Pure and free of contaminants • Standard cycle time for fermentation

• Attenuation • Diacetyl reduction

• Standard product from fermentation • Flavor profile

• Cells of consistent physiological condition • Homogenous culture • Highly viable/vital • Can be used for repitching

Specific Gravity Diacetyl

Fermentation Progression

Yeast in the brewery

• Yeast used in breweries is unique • Reused (Serial repitched)

• Propagated prior to use in fermentation • Yeast re-used between 5 to >20 times

• Brewery yeast is not perfect • First fermentation often slow with long diacetyl rest • Time to ferment can vary over serial repitching • Selection of sub-populations • Stress accumulation

Yeast stress in brewing

• Cold • Storage

• Osmotic • Storage, fermentation

• Ethanol • Storage, fermentation

• Starvation • Storage

Repeated exposure to stress leads to a reduction in yeast

quality

• Oxidative • Fermentation

• Shear forces • Handling

• Desiccation • Dry yeast production

Stress and dried yeast

• Dried yeast growing in popularity • Used by breweries of all sizes

• Convenient size packets • Easy to handle/transport/store

• Stable for extended periods of time • Up to 2 years at 4-8°C

• Ready to use in several hours • Flexible – Brew on demand

• Every package guaranteed to be identical • Can be used for fermentation with predictable results

• Versatile • Primary fermentations/propagation/bottle conditioning

Stress and dried yeast

• Huge improvements in dried yeast quality as a result of a more complete understanding of yeast stress factors occurring during production

• Intracellular water is removed during drying • Stressful and can result in cell death

• Analysis of cell components to determine location of stress

• Adjustments to production process • Prevent or minimize stress

Dried ADY

DilutionBeetCane SterilizationSterilizationCentrifuge

CentrifugeLiquidYeastTankAeration

HeatExchanger

Fed Batch

Aeration

HeatExchanger

Fed Batch

2.Yeast Propagation

SterileMolasses

Tank1.Molasses

NutrientsMinerals

BatchSystem

LaboratoryScale-up

RVF

3.Drying

FluidizedBedDrier

FluidizedBedDrier

Warehouse

10Kg

500g

11g

10Kg

500g

11gExtruderExtruder

• Air volume • Too low: no fluid bed • Too high: Product is

‘blown out of chamber’ • Air temperature

• Product temperature maintained at 35°C

• Final dry weight 93-95%

Fluidized bed drying

Air

Dried Product

ExtrudedYeast

Dust

ExhaustAir

Cyclone

Filter

Filter

YeastFeed

Dehumidifier& Heat

Fluidized BedMembrane Air

Dried Product

ExtrudedYeast

Dust

ExhaustAir

Cyclone

Filter

Filter

YeastFeed

Dehumidifier& Heat

Fluidized BedMembrane

Fluidized Bed Drier

Analysis of viability during drying

Viability loss during drying of lager yeast (no preconditioning)

40

50

60

70

80

90

100

0 5 10 15 20 25

Viab

ility

(%)

Time (Minutes)

Viability (M Blue) Linear (Viability (M Blue))

Viability and dry weight

Viability loss occurs as the last water is removed from cells

40

50

60

70

80

90

100

30 40 50 60 70 80 90 100

Viab

ility

(%)

Dry Weight (%)

Methylene Blue Slide Count

Viability and dry weight

Viability loss occurs as the last water is removed from cells

40

45

50

55

60

65

70

75

90 90.5 91 91.5 92 92.5 93

Viab

ility

(%)

Dry Weight (%)

Methylene Blue Slide Count

Potential routes for loss of viability

• Yeast lose viability during drying and the final stages of water removal in particular • External water is removed during the initial stages of

drying • The final 2-3% of water corresponds to cellular water • Desiccation

• Several cell components are vulnerable to desiccation • DNA • Plasma membrane

Yeast TEM

Damage at the cell membrane

• Evidence to suggest that the main reason for viability loss is damage at the cell membrane • Aquaporins and water channels allow water movement • Rapid removal and entry of water may cause damage

• Damage by dehydration or rehydration ? • The two are closely linked • Rehydration has been shown to be important in

maintaining dried yeast quality • Following the rehydration guidelines can help to

optimize yeast condition • Time, temperature (lager/ale), rehydration media (NOT

wort), NO vigorous agitation • Difficult to impose in breweries

Provided by Tobias Fischborn, Lallemand

The benefit of correct rehydration

6.0

7.0

8.0

9.0

10.0

11.0

12.0

13.0

0 1 2 3 4 5 6 7Time (days)

Extr

act [

ºBrix

]

30 ºC wort-water mix 15 ºC wort-water mix

20 ºC wort 30 ºC wort

direct pitching in wort

Why is correct rehydration so important ?

• Cell membrane is fragile during rehydration − Gentle rehydration prevents membrane damage

Gel Phase Liquid Phase

Rehydration

• The interior of a lipid bi-layer is normally highly fluid • When rehydrated at low temperatures cell membranes can

undergo a ‘gel’ to ‘liquid crystal’ phase transition • Fatty acid tails become rigid and packed tightly together • van der Waals interactions between adjacent chains become

stronger resulting in a loss of fluidity • Leakage of cytoplasmic components and cell death

Rehydrating dried yeast

Rehydration (1)

Phase Transition Temperature

1 Rehydrating cells at a lower temperature will result in

membrane phase transition (damage)

Tem

pera

ture

Aw

2 Rehydrating cells at a higher

temperature will result in damage to cellular components

Rehydration Temperature

Aw 0.117 Tm = 60°C

Rehydration (2)

Phase Transition Temperature

Lowering the Phase Transition Temperature means that

yeast can be rehydrated at lower temperatures

Tem

pera

ture

Aw

Rehydration Temperature

How can the phase transition temperature be lowered?

• Unsaturated fatty acids • Kinks in fatty acid chains, due to cis double bonds,

interfere with packing in the crystalline state, and lower the phase transition temperature

• Sterols (ergosterol) • Trehalose and other sugars

• Interactions with membranes can lower the phase transition temperature from 60ºC to <40 ºC

Trehalose (other sugars/sterols)

Hydrogen bonds bind water to the polar phosphate heads of the membrane phospholipids.

The hydrogen bonding increases the distance between adjacent phospholipids lowering Van-der-

Waals forces between the acyl chain tails

Without water to space the phospholipids, the lipid bilayer fuses and the membrane becomes rigid

losing its vital fluidity.

By forming hydrogen bonds with the polar heads of the phospholipids trehalose is able to maintain the spacing of the acyl groups in the membrane tails.

This stabilizes the lipids in a fluid phase and inhibits membrane fusion.

1

2

3

Trehalose in dried yeast

• Trehalose naturally accumulated during aerobic growth (approx 2%)

• Dried yeast contains high levels of trehalose • Lager: 10-15% • Ale: 15-20% • Bakers yeast: 16-20%

• Strain dependent

0

10

20

30

40

50

60

70

80

90

100

Viab

ility

[%]

0

10

20

30

40

50

60

70

80

90

100

Viab

ility

[%]

Ale Strains Lager Strains

How does trehalose get into cells ?

• Taken up from media • Trehalose can enter the cell using a low affinity

transport system • Facilitated diffusion (permeases)

• Trehalose principally enters the cell using a high affinity trehalose-H+ symporter • Transported against a concentration gradient

• Can be metabolized internally or externally • Genetically regulated

Outside Cell

Cell Wall

Cell Membrane

Inside Cell

Cell Glucose

Cell Trehalose

Trehalose Glucose

Vacuole

Trehalose

Trehalose Glucose

Outside Cell

Cell Wall

Cell Membrane

Inside Cell Mal

Cell Trehalose

Cell Glucose

Vacuole

Trehalose

Trehalose

Trehalose Glucose

H+

Outside Cell

Cell Wall

Cell Membrane

Inside Cell

Agt1p

Cell Trehalose

Mal

Cell Glucose

Vacuole

Trehalose

Trehalose

Trehalose

Glucose

Glucose

H+

Outside Cell

Cell Wall

Cell Membrane

Inside Cell

Ath1p

Agt1p

Ath1p

Cell Trehalose

Mal

Mal

Cell Glucose

Nth1p

Vacuole

Ath1p

Trehalose

Trehalose

Trehalose

Glucose

Glucose

H+

Outside Cell

Cell Wall

Cell Membrane

Inside Cell

Ath1p

Agt1p

Ath1p

Cell Trehalose

Mal

Mal

Cell Glucose

Nth1p

Nth2p

Nth2p

Nth2p

Vacuole

Ath1p

Suntory brewery

Provided by Takaaki Izumi, Suntory

Suntory drying process

Provided by Takaaki Izumi, Suntory

Standard drying procedure

Cells soaked in glycerol to repress trehalase action prior to drying

Trehalose

Trehalose

Trehalose

Glucose

Glucose

H+

Outside Cell

Cell Wall

Cell Membrane

Inside Cell

Ath1p

Agt1p

Ath1p

Cell Trehalose

Mal

Mal

Cell Glucose

Nth2p

Nth2p Glycolysis

Glucose-6-P

Fructose-6-P

Pyruvate

Nth1p Nth2p

Vacuole

Ath1p

Trehalose

Trehalose

Trehalose

Glucose

Glucose

H+

Outside Cell

Cell Wall

Cell Membrane

Inside Cell

Ath1p

Agt1p

Ath1p

Cell Trehalose

Mal

Mal

Cell Glucose

Nth2p

Nth2p Glycolysis

Glucose-6-P

Fructose-6-P

Pyruvate

Tps2p Tps1p

Tsl1p Tps3p

TPS Enzyme complex

Tps1p Tps2p

Trehalose-6-P

Nth1p Nth2p

Vacuole

Ath1p

Genetic regulation of trehalose production

• Genes responsible for cellular production of trehalose • TPS1, TPS2, TPS3

• Activated in response to a number of stresses as part of the Global STress Response Element in yeast

• STRE identified in upstream region of TPS2 • Heat (above 28oC) and cold • Starvation • Osmotic stress • Free radicals • Heavy metals • Etc.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

AGT1 TPS1 TPS2 NTH1 NTH2 ATH1

1000

200

800700600

500

400

300

100

S D NS D N

S D N S D N S D N S D N

Inducing trehalose production

Feeding Rate

Temperature Profile

0 6 34 36

28ºC32ºC

Yeast Growth

Sugar Concentration

Time (Hours)

Feeding Rate

Temperature Profile

0 6 34 36

28ºC32ºC

Yeast Growth

Sugar Concentration

Time (Hours)

• Yeast preconditioning at the end of propagation • Nutrient feeding is stopped to arrest cell division

• Budding individuals may be more susceptible to damage

• At the same time mild heat shock is induced • STRE is activated • Cells produce trehalose

which benefits the yeast during drying and rehydration

Trehalose during yeast production

Lager yeast

Standard propagation and drying process

83.5% Viable 19% Trehalose

0

20

40

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100

120

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160

180

200

Batch Fed Batch Preconditioning

Cream Extruded Dried

Tre

halo

se a

nd G

lyco

gen

20

22

24

26

28

30

32

34

Tem

pera

ture

Trehalose Glycogen Temperature

Typical dried yeast viabilities

Viability determined by methylene blue staining. Results refer to a range of commercially available strains

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90

100Vi

abili

ty [%

]

0

10

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90

100Vi

abili

ty [%

]

Ale Strains Lager Strains

Conclusions

• All brewing yeast cultures are subject to a variety of stress factors • Stress can affect fermentation performance

• Desiccation is a stress primarily associated with dried yeast • Cells are particularly affected when cellular water is removed • Yeast can be preconditioned to increase survival during drying

• Viability typically 70-80% for lager, 80-90% for ale strains

• The impact of desiccation stress is ONLY observed during the first rehydration step • Once dried yeast has been converted to a wet slurry it can be

used for serial repitching in exactly the same way as propagated yeast

University of Nottingham

Brewing Research Team

Lallemand Inc

Tobias Fischborn

Acknowledgements