Wine Yeast Population Dynamics During Inoculated...
Transcript of Wine Yeast Population Dynamics During Inoculated...
MSc Candidate: Jessica Lange
Supervisor: Dr. Daniel Durall
July 17th, 2012
Wine Yeast Population DynamicsDuring Inoculated and Spontaneous
Fermentations in Three BritishColumbia Wineries
Please note:
Darryl Brooker’s presentation titled “S.cerevisiae Population Dynamics: A PracticalApplication” is included at the end of thisPowerPoint
CONTACT
Jessica Lange, BSc (Microbiology)
UBCO MSc Candidate
e.mail: [email protected]
phone: 250.869.5840
Supervisor e.mail: [email protected]
Research Objective
To determine the population dynamics of wineyeast species and strains in inoculated andspontaneous fermentations of three Okanaganwineries
Wineries of Study
Four tanks sampled in each winery (2010 vintage):
3 Inoculated fermentation tanks
1 Spontaneous fermentation tank
Vitis vinifera L. var Pinot noir
Quails’ Gate Cedar Creek Road13
Fermentation Stages Sampled
20-25 ºBrix
CS10-15 ºBrix
M
15-20 ºBrix
ER
< 5 ºBrix
F
DNA Extraction and Amplification
Target RegionTarget Region
Non-Saccharomyces isolates:• Amplification of the ITS and the D1/D2 regions of rDNA• Primers: ITS1/ITS4 and NL1/NL4• DNA Sequencing Species level discrimination
S. cerevisiae isolates:• Amplification of six loci specific to S. cerevisiae genome• Primers: C4, C5, C11, YPL, SCAAT1c, SCY009c (Legras et al. 2005)• DNA Fingerprinting Strain level discrimination
What is ‘Microsatellite Fingerprinting’?
After amplifying the six different loci of the S. cerevisiae genome(DNA), it looks like this:
The peaks differ between strains by shifting left or right
The red, green, black, and blue peak pattern is the ‘fingerprint’
Fingerprints like this were made for all the commercial strainsreported utilized within the wineries of study
I compared the fingerprints of the yeast I obtained from the tanks tothe commercial fingerprints I constructed achieved S. cerevisiaestrain identity
Fingerprints are very strain specific
C5
C11
SCA
C4
SCY
YPL
Winery Information
Number of different strains used in the wineries:
Cedar Creek: 32 commercial S. cerevisiae strains
Quails’ Gate: 13 commercial S. cerevisiae strains
Road13: 7 commercial S. cerevisiae strains
Age of the wineries:
Cedar Creek: 1983 (29 years)
Quails’ Gate: 1989 (23 years)
Road13: 1998 (14 years)
0
0.25
0.5
0.75
1
CS ER M F
Spontaneous
0
0.25
0.5
0.75
1
CS ER M F
Specie
s/S
train
Fre
quency
ofO
ccurr
ence
0
0.25
0.5
0.75
1
CS ER M F
0
0.25
0.5
0.75
1
CS ER M F
Spec
ies/
stra
inFre
quen
cyofO
ccurr
ence
Tank 1 (Lalvin RC212) Tank 2 (Lalvin RC212)
Tank 3 (Lalvin RC212)
H. uvarum
Lalvin RC212
Lalvin ICV-D254
Lalvin A. Plus
Lalvin CY3079
RS P. Cuvee
QG-UN1
QG-UN2
QG-UN3
QG-UN4
QG-UN5
QG-UN6
Quails’ Gate Results
Spontaneous
0
0.25
0.5
0.75
1
CS ER M F
Tank 1 (L2TD 7013/7035) Tank 2 (Enoferm AMH)
Tank 3 (Lalvin RC212)
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0.25
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0.75
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CS ER M F
Spec
ies/Strain
Fre
quen
cyof
Occ
urren
ce
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CS ER M F
Spe
cies
/Strain
Freq
uenc
yof
Occ
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CS ER M F
Occ
urren
ce
H. uvarum
S. uvarum
Tolurospora
Metschnikowia
Wickerhamomyces
Lalvin ICV-D254
Lalvin RC212
Enoferm AMH
Zymaflore VL1
Zymaflore FX10
Zymaflore X5
LallemandEC118
LallemandR2056
LallemandD47
CC-UN1
Cedar Creek Results
Spontaneous
0
0.25
0.5
0.75
1
CS ER M F
Tank 1 (Lalvin RC212) Tank 2 (Lalvin RC212)
Tank 3 (Lalvin RC212)
0
0.25
0.5
0.75
1
CS ER M F
Specie
s/S
train
Fre
quency
ofO
ccurr
ence
0
0.25
0.5
0.75
1
CS ER M F
Specie
s/S
train
Fre
quency
ofO
ccurr
ence
0
0.25
0.5
0.75
1
CS ER M F
H. uvarum
S. uvarum
Tolurospora
Metschnikowia
Wickerhamomyces
Pichia
S. bayanus
Lalvin RC212
Fermol Chardonnay
R13-UN1
R13-UN2
R13-UN3
R13-UN4
R13-UN5
R13-UN6
R13-UN7
R13-UN8
R13-UN9
R13-UN10
R13-UN11
R13-UN12
R13-UN13
R13-UN14
R13-UN15
Road13 Results
Future Studies
Our lab is currently observing the interactions andmetabolomics of Lalvin RC212 and ICV-D254through co-inoculations at varying ratios and itseffects on Pinot noir sensorial attributes
Cell density during fermentation (CFUs/mL)Fermentation duration (days)
Acknowledgements
Winery Establishments & Pinot noir samples provided by:
Grant Stanley of Quail’s Gate
Darryl Brooker of Cedar Creek
Michael Bartier & Bailey Williamson
of Road13 (present during the 2010 year)
Lallemand, Lalvin, AEB Fermol, Anchor, Laffort companies
Commercial S. cerevisiae donations
Supervisor: Dr. Daniel Durall
Assistance in the lab: Hanna Pawluck & Liz Halverson
UBCO FADSS technician: Sheri Maxwell
Funding: NSERC & Quail’s Gate Estate Winery
Collaborative Research Development (CRD) Grant
S. CEREVISIAEPOPULATION DYNAMICS
A PRACTICAL APPLICATIONDARRYL BROOKER
PROCESSING
Each block was hand harvested into 700lb bins anddelivered to the winery within 2hrs of picking
Both blocks were destemmed with minimal crushing (norollers)
40ppm SO2 was added to each tank
Cooling applied to each tank + dry ice and temperature waslowered to <8oC
Tanks were warmed to approx. 15oC for ferment and yeastwas added (where indicated) at 250ppm followingmanufacturers rehydration protocol
All tanks were pumped over during cold soak and handplunged during fermentation/post ferment maceration
SPONTANEOUS TANK (10PNCC101) -Population Dynamics
This was a spontaneous fermentation, S. uvarum was dominant inthe cold soak
D254, which was obviously not added, was the dominant yeast inthe EARLY stage – occurring 6 days after destemming
D254 continued to dominate the ferment through mid and end, withthree other yeasts present (X5, Rhone 2056 and RC212)
Even though this tank was a spontaneous fermentation, it appearsthat common wine yeasts used in the winery were responsible forthe majority of alcoholic fermentation
This tank fermented to dryness (3.22g/L) with a relatively lowVolatile Acidity (0.44g/L) on pressing off
TANK 1 (10PNCC102) - Population Dynamics
This was a double inoculant fermentation – T. delbrueckii (a knownvineyard yeast) followed by S. cerevisiae
Very similar yeast population during cold soak as batch 101
Much greater yeast diversity during the early, mid and end
Great to see that T. delbrueckii was present during the early, givenit was added just before this sample was taken
T. delbrueckii was not present during the mid and end stage, whichsupports the notion that it is not an alcohol tolerant yeast
I found it interesting that three non-saccharomyces yeast werepresent during early ferment and as expected all yeast were S.cerevisiae by mid and end ferment
This tank fermented to dryness (3.46g/L) with a slightly higherVolatile Acidity (0.48g/L vs 0.44g/L) than batch 101, however it didtake longer (20 days vs 16 days for 101)
TANK 3 (10PNCC401) - Population Dynamics
This tank was inoculated with RC212
Very similar yeast population during cold soak as previous batches
This tank fermented warmer than batch 101 and 102 (27oC) andthe fastest to ferment to dryness (15 days)
RC212 remained relatively dominant in the early, mid and endstages, even in the presence of D254
Presence of ‘white yeast’ in this ferment, being X5 and D47 whichwere both used in a nearby white wine cellar
This tank fermented to dryness (2.14g/L) with the lowest VolatileAcidity (0.38g/L)
TANK 2 (10PNCC402) - Population Dynamics
This tank was inoculated with AMH, known as a weak fermenter
Very similar yeast population during cold soak as previous batches
AMH was detected at a low rate in the early stage of ferment andwas not present in the mid and end stages
This tank was dominated by RC212 in the early stage and D254 inthe latter stages of ferment – it appears that AMH had little effecton ferment
This tank had the highest ferment temperature of 28oC
This tank fermented to dryness (2.39g/L) with a relatively lowVolatile Acidity (0.41g/L)
My ‘take home’ points
The known vineyard yeast H. uvarum is relatively resistant to SO2and dominated all cold soaks after the addition of 40ppm SO2
S. cerevisiae was only present in small amounts during the coldsoak – likely due to the high dose of SO2
Strong yeast such as D254 and RC212 present in the winerydominated most of the Pinot Noir ferments by mid/end stage
Yeast dispersion by air was obvious as ‘white’ yeasts used in adifferent part of the cellar were present in all ferments
My ‘take home’ points continued
The spontaneous fermentation showed the lowest diversity in yeastpopulation
The two stage inoculation using T. delbrueckii showed the greatestdiversity in yeast population
Weaker yeasts (AMH) may struggle to survive in the presence ofstrong fermenting yeast, even if the strong fermenting yeast are notthe inoculum
Yeast competition was obvious in all tanks and diversity/populationmay be able to be manipulated through SO2 additions, cold soakduration, ferment temperature and inoculant choice