Alcoholic fermentation management The yeasts life from rehydratation

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Alcoholic fermentation management The yeast’s life: from rehydratation to the completion of fermentation

Alcoholic fermentation management The yeast’s life: from rehydratation to the completion of fermentation

Alcoholic Fermentation scenario I II IV V Latency phase: enzymatic and physiological adaptation Growth

Alcoholic Fermentation scenario I II IV V Latency phase: enzymatic and physiological adaptation Growth phase: first third of the alcoholic fermentation. Good physiological status, low population Growth slowing phase Stable phase: the population has reached its’ maximum level. Second third of the alcoholic fermentation. Good population, average physiological state. Decline phase: Damaged cells in population and declining physiological state. IV V II

THREATS OBJECTIVES SOLUTIONS The yeast’s life during the alcoholic fermentation III II I IV

THREATS OBJECTIVES SOLUTIONS The yeast’s life during the alcoholic fermentation III II I IV V

I. Latency period

I. Latency period

THREATS OBJECTIVES SOLUTIONS • Inhospitable conditions in the must (p. H, sugars, SO 2,

THREATS OBJECTIVES SOLUTIONS • Inhospitable conditions in the must (p. H, sugars, SO 2, toxins, temperature) • Good physiological state with no population loss • Exercise good practises in the rehydration phase • Compition from indigenous flora • Short latency period • Choice of the yeast • Yeast dose: at least 20 g/h. L III II I Latency phase IV V

Key points for the rehydration -DO NOT USE MUST at the beginning of the

Key points for the rehydration -DO NOT USE MUST at the beginning of the rehydration. - WATCH THE TEMPERATURE of the inoculum and the must. Yeasts are very sensitive to variations in temperature. It is essential to use a thermometer for every step of the rehydration. -ADAPT THE DOSE of yeasts to the must you want to ferment. Increase the amount in the case of ; -the presence of Brettanomyces or other indigenous flora - Baume > 13 % vol. -Minimum dosage: 20 g/h. L. - IF NECESSARY, USE A YEAST PROTECTOR - (We will come back to this later) - INOCULATE AS SOON AS POSSIBLE AFTER CRUSHING / CLARIFYING

Inoculation step by step Correct rehydration is a crucial step in the life and

Inoculation step by step Correct rehydration is a crucial step in the life and efficiency of the yeast Reliable inoculation 0 Mix the yeast in 10 times its’ weight in water at 37°C (dose: 20 g/h. L) No link between the presence of foam and the yeast’s effectiveness Wait for 15 minutes Acclimatise the inoculum to the temperature of the must by adding the must progressively Objective: not more than 10°C between the inoculum temperature and the must temperature Total rehydration time (step 1 and 2) must never exceed 45 minutes Incorporate the acclimatised inoculum into the must and homogenise Add ECOBIOL PDC (water: 43°C; Volume: 20 times the yeast weight) and then go to step 1

How to choose the best yeast ? • Technological criteria: Choose a strain adapted

How to choose the best yeast ? • Technological criteria: Choose a strain adapted to the potential alcohol and temperature conditions. • Qualitative criteria: grape variety, wine style, aroma potential, mouth feel etc. . .

Manage the inoculation rate Impact on the latency phase Length of the latency phase

Manage the inoculation rate Impact on the latency phase Length of the latency phase 70 Time (hours) 60 50 10 g/h. L rep 1 10 g/h. L rep 2 25 g/h. L rep 1 25 g/h. L rep 2 40 30 20 10 0 14°C/ 15 NTU 28°C/220 NTU Echelle labo (15, 4 % vol) 14°C/11 NTU 28°C/193 NTU Echelle pilote (14, 3 % vol)

Manage the inoculation rate Impact on the end of the alcoholic fermentation Residual sugars

Manage the inoculation rate Impact on the end of the alcoholic fermentation Residual sugars at the end of the alcoholic fermentation

II. GROWTH PHASE AND SLOWING DOWN PHASE

II. GROWTH PHASE AND SLOWING DOWN PHASE

THREATS OBJECTIVES • Must nutritional deficiency • Biomass too high • Over clarification (lack

THREATS OBJECTIVES • Must nutritional deficiency • Biomass too high • Over clarification (lack of sterols) III II I Growth phase and slowing down phase SOLUTIONS • Get a reasonable population without degrading the physiological state of each individual. • Complement nutrients at the right time and only if necessary • Limit the supply of ammoniacal nitrogen • Ensure a good level of sterols in the yeast culture • Management of the oxygen and yeast protection IV V

Complement only if necessary (1 of 2) Nitrogen Henschke P. A. and V. Jiranek,

Complement only if necessary (1 of 2) Nitrogen Henschke P. A. and V. Jiranek, Int. Symp. on Nitrogen in Grapes and wines, 1991, 172 -184 SO 2 nitrogenous group Accumulation of sulphured components (H 2 S…) S 2 méthionine bit i h n ion -i ro Rét Impact of nitrogen deficiency on the production of components sulphured by the yeast.

Complement only if necessary (2 of 2) Variability of the musts nitrogen composition The

Complement only if necessary (2 of 2) Variability of the musts nitrogen composition The deficiency line varies according to: - sugars - temperature -Yeast strain Seuil de carence moyen High maturity= high [Sugar] and low [N]

Add nutrition at the RIGHT time Control Add at the beginning of fermentation Add

Add nutrition at the RIGHT time Control Add at the beginning of fermentation Add around 1040 Sablayrolles, INRA The best moment for nitrogen supply is at the end of the growth phase, right after the yeast population has reached its highest level. For practical purposes, it is often necessary to add a little bit more in the beginning of the alcoholic fermentation, in order to not give too much nitrogen in the first third of the alcoholic fermentation (thermal peak risks).

Limit the nitrogen from ammonium salts (1 of 5) Assimilable nitrogen sugars The organic

Limit the nitrogen from ammonium salts (1 of 5) Assimilable nitrogen sugars The organic nitrogen (amino) has a slower and more regulated assimilation than ammonia nitrogen Amino nitrogen Ammonium time

Limit the nitrogen from ammonium salts (2 of 5) Nitrogen: auto-consumption of the yeast’s

Limit the nitrogen from ammonium salts (2 of 5) Nitrogen: auto-consumption of the yeast’s amino acids and consequences Ammonium add Fast consumption of ammonium by the yeasts. Fast yeast growth Large biomass Formation of H 2 S Liberation of radicals S 2 - Fast impoverishment of the environment Consumption of its own amino acids (even sulphured) by the yeast Inferred nitrogen deficiency Difficulties with the Malo-lactic ferment

Limit the nitrogen from ammonium salts (3 of 5) 10 am mo n Jiranek,

Limit the nitrogen from ammonium salts (3 of 5) 10 am mo n Jiranek, 2000 iu m niu mo 5 m am H 2 S liberation speed (mmol/g weight sec/h) 15 heures 0 12 15 18 21 The addition of DAP leads to more biomass, giving an environmental deficiency, so yeasts autoconsume their amino acids, liberating radicals S 2 and produces even more H 2 S

Limit the nitrogen from ammonium salts (4 of 5) Perceived olfactory intensity Intensity of

Limit the nitrogen from ammonium salts (4 of 5) Perceived olfactory intensity Intensity of the perception of sulphidic smells ICV, dept. R&D Chardonnay must (NFA : 140 mg/l) completed by 20 g/hl of « complexe nutriment » or 7. 5 g/hl of DAP

Limit the nitrogen from ammonium salts (5 of 5) Role of the wine production

Limit the nitrogen from ammonium salts (5 of 5) Role of the wine production on positive aromas The assimilation of the ammonia nitrogen by the yeast is made to the detriment of : – The amino acids that are likely to intercede in the production of fermentation aromas. – The varietal thiol precursors (grapefruit, passion fruits aromas)

Conversion of the thiols precursors: the nutrition role Subileau & Salmon, FEMS Yeast Res

Conversion of the thiols precursors: the nutrition role Subileau & Salmon, FEMS Yeast Res 8 (2008) 771780 500 Sauvignon blanc Languedoc 2004 450 400 350 300 250 200 150 100 50 0 Témoin Ajout de DAP 3 MH A-3 MH Sauvignon blanc Gers 2006 7000 6000 Too much ammonium added at the beginning of the fermentation can limit the entrance of the precursor into the yeast and then limit the thiols conversion. 5000 4000 Témoin 3000 Ajout de DAP 2000 1000 0 3 MH A-3 MH

buis, genêt pamplemousse, fruit de labuis, passion fruit de la passion Conversion of the

buis, genêt pamplemousse, fruit de labuis, passion fruit de la passion Conversion of the thiol precursors: the nutrition role Wine composition of volatile thiols (sauvignon 2010, Sancerre) DAP Nutribio A-3 MH: > seuil 3 MH*: > seuil 4 MMP* 0% 20% 40% 60% 80% 100% 120% Indice de concentration indexé (Témoin = 100%) Trials conducted in partnership with the SICAVAC 140% 160%

Aromatic expressions: the yeast nutrition effect 70. 0 Wine positive aromatic composition (Sauvignon 2010,

Aromatic expressions: the yeast nutrition effect 70. 0 Wine positive aromatic composition (Sauvignon 2010, Sancerre) Organique Complexe Minéral 60. 0 Somme des concentrations (mg/L) 50. 0 40. 0 30. 0 20. 0 10. 0 Total esters x 10 (acétate et esters éthyliques) Total autres arômes positifs (2 -phényléthanol, 4 -vinylguaiacol, beta-damascénone) Trials conducted in partnership with the SICAVAC

YEAST PROTECTION

YEAST PROTECTION

Two good reasons to protect your yeast • Avoid stopping the alcoholic fermentation •

Two good reasons to protect your yeast • Avoid stopping the alcoholic fermentation • Limit the stress of the yeast and therefore production of inhibitory toxins

What is the PROTECTION? What is the difference between protection and nutrition ?

What is the PROTECTION? What is the difference between protection and nutrition ?

NUTRITION PROTECTION

NUTRITION PROTECTION

Yeast protection How to manage the membrane integrity of the yeast ? the central

Yeast protection How to manage the membrane integrity of the yeast ? the central role of the sterols

The membrane: an interface acidity pump Sterols acidity energy Sugar transportation proteins Alcoholic Fermentation

The membrane: an interface acidity pump Sterols acidity energy Sugar transportation proteins Alcoholic Fermentation [Acidity] p. H int. = 5 -6 [Acidity] p. H ext. = 3 -4

Effect of a sterols deficiency acidity acidity acidity energy acidity acidity Ethanol p. H

Effect of a sterols deficiency acidity acidity acidity energy acidity acidity Ethanol p. H Int. Alcoholic Fermentation

Importance of the time when oxygen is added 1 : initial addition 2 :

Importance of the time when oxygen is added 1 : initial addition 2 : addition at the end of the growth phase -Best moment= end of the growth -Best moment considering the oxidation risk

Yeast protection Dilution of sterols during growth phase at the end of this phase

Yeast protection Dilution of sterols during growth phase at the end of this phase : low level potentialy weak yeast Protector Oxygen

Yeast protection What is a yeast protector ? Specific Sterols Protector. NATSTEP® (Inactivated specific

Yeast protection What is a yeast protector ? Specific Sterols Protector. NATSTEP® (Inactivated specific yeast) Rehydratation water Microprotectants Micellar structure

Yeast protection Protector Oxygen

Yeast protection Protector Oxygen

Non protected yeasts Yeasts protected by Bioprotect®

Non protected yeasts Yeasts protected by Bioprotect®

Effect of the protection on the viability of the yeast Control Specific sterols added

Effect of the protection on the viability of the yeast Control Specific sterols added during REHYDRATATION 70% 90% Viability

The effect of the protection on the fermentation security Straight end of the fermentation

The effect of the protection on the fermentation security Straight end of the fermentation Time after inoculation (days))

IV. STABLE PHASE

IV. STABLE PHASE

THREATS OBJECTIVES • Increasing alcohol and temperature • Organoleptic risks III SOLUTIONS • Control

THREATS OBJECTIVES • Increasing alcohol and temperature • Organoleptic risks III SOLUTIONS • Control of temperature • Avoid excess ammonium salts or supplies that lead to temperature peaks and, in a general way, avoid excess nitrogen • Avoid changes in fermentation rate and control them • Limit oxygen supply IV V II Stable phase I The yeast still can assimilate nutrients (nitrogen and oxygen also) at this step but with much less efficiency.

V. DECLINE PHASE

V. DECLINE PHASE

THREATS OBJECTIVES SOLUTIONS • Maximum hostility of the environment (alcohol, temperature, toxins, flora development)

THREATS OBJECTIVES SOLUTIONS • Maximum hostility of the environment (alcohol, temperature, toxins, flora development) • Limit the toxicity of the environment • Detoxification • Control of the temperature • Risks of languishing alcoholic fermentation or stopping altogether • Optimise the physiological state of the surviving yeasts • Everything hangs on getting the beginning of the alcoholic fermentation correct: nitrogen, oxygen, sterols III II I Decline phase IV V The yeast still can assimilate nutrients (nitrogen and oxygen also) at this step but with less and less efficiency.

Conclusion • Everything takes place during the first phases : – Choosing the yeast

Conclusion • Everything takes place during the first phases : – Choosing the yeast – Rehydration (temperature, time, protection, etc. . . ) – Inoculation (Inoculation rate, temperature, . . . ) – Growth phase and beginning of the stable phase: nutrition, oxygen supply

Optimisation of the alcoholic fermentation conditions (1 of 2) Aggressiveness of the environment Alcohol

Optimisation of the alcoholic fermentation conditions (1 of 2) Aggressiveness of the environment Alcohol >13, 5 % vol and/or Turbidity < 80 NTU and/or T°<15°C or >28°C Extreme nitrogen deficiency Assimilable nitrogen <100 mg/L BIOPROTECT at rehydration + BIOCLEAN 2/3 through fermentation If combining both factors • beginning of fermentation: VITISTART (whites/rosés) or ACTIVIT (reds) 20 g/h. L • 1/3 FA: ACTIVIT 20 g/h. L + DAP 15 g/h. L if alcohol degree below 12. 5% If the yeast needs a lot of nitrogen, calculate as if there was one more degree of alcohol. If it doesn’t need a lot of nitrogen, one degree of alcohol less.

Optimisation of the alcoholic fermentation conditions (2 of 2) Medium nitrogen deficiancy assimilable nitrogen

Optimisation of the alcoholic fermentation conditions (2 of 2) Medium nitrogen deficiancy assimilable nitrogen between 100 and 140 mg/L • Beginning of fermentation: NUTRIBIO 20 g/h. L + thiamine • 1/3 through fermentation: VITISTART (whites/rosés) or ACTIVIT (reds) 20 g/h. L + DAP 15 g/h. L if degree of alcohol is below 12. 5% Light nitrogen deficiency assimilable nitrogen between 140 and 220 mg/L • Beginning of FA: thiamine • 1/3 FA: NUTRIBIO 40 g/h. L + DAP 15 g/h. L if degree of alcohol is below 13. 5% If the yeast needs a lot of nitrogen, calculate as if there was one more degree of alcohol. If it doesn’t need a lot of nitrogen, one degree of alcohol less