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)


0

0.25

0.5

0.75

1

CS ER M F

Spec

ies/Strain

Fre

quen

cyof

Occ

urren

ce

0

0.25

0.5

0.75

1

CS ER M F

Spe

cies

/Strain

Freq

uenc

yof

Occ

urre

nce

0

0.25

0.5

0.75

1

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)


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)


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)


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

Wine Yeast Population Dynamics During Inoculated...

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