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Stress, Viability and Active Dried Yeast
Chris Powell The University of Nottingham, UK
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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
60
80
100
120
140
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
0
10
20
30
40
50
60
70
80
90
100Vi
abili
ty [%
]
0
10
20
30
40
50
60
70
80
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