Yeast Nutrition

& stimulation to reveal aromas :New knowledge!

Key points for a successful fermentation & an optimum wine quality

Sigrid Gertsen-Schibbye

Scandinavia and UK

VitiNord 2018

→ Objective n°1, 30 years ago : limit the risk of stuck or sluggish fermentation

• Nitrogen (YAN) is a key compound:- defines biomass (Xmax)- direct impact on the fermentation rate (Vmax)- on the yeast fermentative activity

Yeast nutrition management in alcoholic fermentation : evolution over the years

→ Objective n°2, 10 years ago : show the importance of nitrogen source

• Organic Nitrogen (aa) better than DAP (NH4+):- more efficient for same YAN- amino acids impact directly esters synthesis in yeast metabolism- organic YAN = more flavour in wines

Yeast nutrition management in alcoholic fermentation

→ Objective n°3, 3 years ago : need for well-balanced nutrition

• Organic nitrogen YES, but not only …:- vitamins, minerals, sterols- limit the risk of nutritional imbalancesresponsible for : loss of viability, activity

H2S overproductionloss of aromas release

Yeast nutrition management in alcoholic fermentation

Normal Fermentation Curve

2-4 million

CFU/mL

>100-150

million

CFU/mL

2-4 million

CFU/mLTime

Po

pu

lati

on

Survival factors are important to ensuring the proper working of the cellular membrane: poly-unsaturated

fatty acids and sterols

4-8 million

CFU/mL

Higher yeast inoculation rate

lowers dilution of the initial yeast

cells survival factors

Brix

0

0,2

0,4

0,6

0,8

1

1,2

1,4

0 100 200 300 400 500 600

dC

O2d

t-1(g

L-1h

-1)

Time (h)

Fermentation kinetic

Low nitrogen

High nitrogen/ Low oleic acid

High nitrogen/ Low ergosterol

High nitrogen/ Low pantothenic acid

High nitrogen/ Low nicotinic acid

Nutritional imbalances: impact on fermentation kinetics

High nitrogen with- Low oleic acid- Low ergosterol- Low pantothenate- Low nicotinic acid

57,2 g/l residual sugars63,7 g/l residual sugars

Last results from Camille DUC ongoing thesisLallemand - Supagro

Nutritional imbalances : Nitrogen & lipid(ergosterol) Impact on yeast viability

Blondin et Tesnières., 2013, PlosOne : 8, e1645

• High lipids content• 3 levels of nitrogen : from 70 to 425 mg/l

YAN)

• Lipids deficiency• 3 levels of nitrogen : from 70 to 425 mg/l

YAN)

CO2 production (g/l)

Via

bili

ty(%

)

Lipids deficiency :

Mortality rate modulated by nitrogen concentration:

the highest YAN with low lipids, the highest mortality.

8

0

10

20

30

40

50

60

70

80

90

100

0 20 40 60 80 100

Via

ble

cells (

%)

CO2 produced (g/L)

MS71,Low oleic acid, Fermaid O20g/hL

MS71, Low erosterol, Fermaid O20g/hL

MS71, Low pantothenic acid,Fermaid O 20g/hL

MS71,Low nicotinic acid, FermaidO 20g/hL

0

10

20

30

40

50

60

70

80

90

100

0 20 40 60 80 100

Via

ble

cells (

%)

CO2 produced (g/L)

MS71, low oleic acid + 354mg/lYAN eq NH4Cl

MS71, low ergsoterol + 354mg/lYAN eq NH4Cl

MS71, low pantothenic acid +354mg/l YAN eq NH4Cl

MS71, low nicotinic acid +354mg/l YAN eq NH4Cl

+ DAP

+ Fermaid O

Nutritional imbalances: addition of DAP vs additon of Femaid O : Impact on viability

→ Different micronutrient starvation in condition of high nitrogen leads to yeast death

→ Different impact of nitrogen sources (amino acids) on viability & fermentation

→ Stuck fermentation with some vitamin starvation

→ limit some varietal aromas revelation, H2S...

Management of nutritional imbalance & nutrientsinteractions/ synergy

Nutritional imbalances :

Today : Yeast nutrition management: 1. Focus on nutrients interactions and balance

2. Optimize yeast metabolism

STIMULATION of yeast metabolism :

- Specific composition (interactions of vitamins, sterols, YAN…)

- Timing of addition- Target on aromas depending on the grape matrix

(precursors)

Goal : optimize the aromatic potential of the grape

Yeast PROTECTION is essential

&

Yeast NUTRITION is vital.

Fermentative aromas : Esters

• Higher alcohols : 10 – 500 mg/L:< 300 mg/l , impact positively the aromatic complexity and fruity character of

the wine (Swiegers et al., 2007)

• Acetate esters 0.01 – 10 mg/L

• Ethyl esters 3 mg/L

Leucine Isoamyl alcool Valine IsobutanolIsoleucine Amyl alcool Phenylalanine Phenyl ethanol

Amino acids α-cétoacid aldehyde alcoholCO2NH4

Isoamyl acetateIsobutyl acetateAmyl acétatePhenyl ethyl acetate

alcohol + acetyl-CoA acetyl ester

Ethyl acetate acetyl-CoAEthyl butyrate butytyl-CoAEthyl hexanoate hexanoyl-CoAEthyl octanoate octanoyl-CoAEthyl décanoate décanoyl-CoA

ethanol + fatty acids-CoA Ethyl ester

Kinetic profile of the fermentative aromas synthesis

Fermentative aromas are produced all along the fermentation

0.0

0.5

1.0

0.0

0.4

0.8

0 50 100 150

cC

O2/d

t (g

/L.h

)

Octa

no

ate

d'é

thyle

(m

g/L

)

Sucre consommé (g/L)

Moût synthétique

2 phases of linear synthesis in function of the sugarconsumption

Yield of production of the 2nd

phase always higher(Mouret et al., 2014)

Esters synthesis : Impact of timing of nutrientaddition (Stimula Chardonnay)

• Nitrogen addition impacts on the esters synthesis

• Addition @ 1/3 : higher efficiency: +40% compared to addition at t=0

• Stimula Chardonnay: more efficient than DAP

Isoamyl acetate : Addition @1/3 of AF

Isoamyl acetate : Addition @ T0

0

0,1

0,2

0,3

0 50 100 150

C°

(mg

/L)

sucre consommé (g/L)

Isobutyle acetate

témoin

Nutriment complexeorganique

Control

Ne

0

1

2

3

4

5

0 50 100 150

C°

(mg

/L)

sucre consommé (g/L)

Isoamyl acetate

témoin

Nutriment complexeorganique

Control

Ne

Impact of Stimula Chardonnayadded @ 1/3 on esters synthesis

Extreme conditions : ViognierYAN = 37 mg/l Turbidity : 70 NTU

0

0,1

0,2

0,3

0,4

0,5

0,6

0 50 100 150 200 250 300 350 400 450

dC

O2/D

T (

g/L

.h-1

)

Time (h)

Nutrition trialViognier ; 20ºC ; QA23: 25g/hL ; Goferm Protect Evolution 30g/hL

YAN: 37mg/L ; Sugar 200g/L ; turbidity: 70 NTU ; pH: 3,46 ; TA; 2,73

Control

Fermaid O 20g/hlT=0 + 20g/hL1/3AF

NE 40g/hL 1/3AFStimula Chardonnay 40g/hl 1/3 AF

+27%

+14%

+43%

+21%

+23%

+46%

+12%

Significant increase of ester synthesis withStimula Chardonnay addition at 1/3 of AF

From 12% to 46% depending on aromas

→

to stimulate the yeast estersynthesis metabolism

When added @ 1/3 (afterthe exponential phase)

When volatile thiols are produced?

→ During yeast growth phase:

- Cells multiplication at maximum

- Uptake of the assimilable nitrogen

- High enzymatic activity

0

50

100

150

200

250

300

350

400

450

500

3MH A-3MH

Témoin

Ajout de DAP

0

1000

2000

3000

4000

5000

6000

7000

3MH A-3MH

Témoin

Ajout de DAP

Sauvignon blanc

Languedoc 2004

Sauvignon blanc

Gers 2006

Ammonium addition in excess at thebeginning of the fermentation can lead to aretro-inhibition limiting the precursor entryinto yeast cell and consequently the varietalthiols production by yeast.

NH4+ en excès

Gap1p

Acides aminés

Cys-3MH

NCR nitrogencatabolic

repression

Subileau & Salmon, FEMS Yeast Res 8 (2008) 771-780

Schneider 2008

The role of yeast nutrition on thiol precursors uptake and conversion

ADY 1

ADY 2

ADY 1

ADY 2

Subileau thesis

Consumption of Cys-4MMP and production of 4MMP by 2 wine yeasts on Sauvignon Blanc

Complemented in Cys-4MMP (35µg/l)

Zoom on growth phase & beginning of stationnary phase (0-80h)

4MMP release: conclusions

• All precursors uptake during lag + growth phase (no residualprecursors)

• No yeast difference on the consumption : system of transport identical

• Yeast impact on the precursors conversion into volatile thiols: R2 produces 5 times more 4MMP with the same uptake ability

• Stimula Sauvignon blanc improves yeast conversion yield

• Stimula SVG impact is yeast related : Higher increase with QA23

3MH release: conclusions

• Precursors uptake during lag + growth phase BUT not the total level is consumed (Residual : 40ng/l R2 60ng/l QA23)

• Contrary to 4MMP precursors : The uptake of these 3MH precursors is yeast dependant

• Less yeast difference on the conversion of precursors

• Stimula Sauvignon blanc improves yeast conversion yieldStimula Sauvignon blanc : higher increase of 3MH with QA23 suggesting that « stimulation » is yeast related

→

to stimulate the yeast uptake of varietalthiol precursors

to add @ t=0 when yeast consumenitrogen sources & when its enzymaticactivity is at its maximum

Well balance organic nutrition:

the key to assure yeast viability& optimize the aromas biosynthesisand release

Nitrogen

Organic nitrogen : YES….but not only