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

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

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

I. Latency period

IV III V II I Latency phase THREATS OBJECTIVES SOLUTIONS Inhospitable conditions in the must (pH, sugars, SO2, toxins, temperature) Compition from indigenous flora Good physiological state with no population loss Short latency period Exercise good practises in the rehydration phase Choice of the yeast Yeast dose: at least 20 g/hL I II III IV V Latency phase

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/hL. 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 efficiency of the yeast Reliable inoculation Add ECOBIOL PDC (water: 43°C; Volume: 20 times the yeast weight) and then go to step 1 Mix the yeast in 10 times its’ weight in water at 37°C (dose: 20g/hL) 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

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

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

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

Complement only if necessary (1of2) Henschke P.A. and V. Jiranek, Int. Symp. on Nitrogen in Grapes and wines,1991, 172-184 Nitrogen SO2 nitrogenous group Accumulation of sulphured components (H2S…) S2- Rétro-inhibition méthionine Impact of nitrogen deficiency on the production of components sulphured by the yeast.

Complement only if necessary (2of2) 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 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 (1of 5) Assimilable nitrogen sugars The organic nitrogen (amino) has a slower and more regulated assimilation than ammonia nitrogen Amino nitrogen Ammonium time

Nitrogen: auto-consumption of the yeast’s amino acids and consequences 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 H2S Fast impoverishment of the environment Liberation of radicals S2- 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 (3of5) 5 10 15 H2S liberation speed (mmol/g weight sec/h) 18 12 ammonium 21 Jiranek, 2000 heures The addition of DAP leads to more biomass, giving an environmental deficiency,so yeasts autoconsume their amino acids, liberating radicals S2- and produces even more H2S

Limit the nitrogen from ammonium salts (4of5) Intensity of the perception of sulphidic smells Perceived olfactory intensity 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 (5of5) 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 8 (2008) 771-780 Sauvignon blanc Languedoc 2004 Sauvignon blanc Gers 2006 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.

Conversion of the thiol precursors: the nutrition role Trials conducted in partnership with the SICAVAC

Aromatic expressions: the yeast nutrition effect Trials conducted in partnership with the SICAVAC

YEAST PROTECTION

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 ?

PROTECTION NUTRITION

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

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

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

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

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

Yeast protection Protector Oxygen

Yeasts protected by Bioprotect® Non protected yeasts Yeasts protected by Bioprotect®

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

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

IV. STABLE PHASE

IV III V II Stable phase I THREATS OBJECTIVES SOLUTIONS Increasing alcohol and temperature Organoleptic risks Avoid excess ammonium salts or supplies that lead to temperature peaks and, in a general way, avoid excess nitrogen Limit oxygen supply Control of temperature Avoid changes in fermentation rate and control them IV III 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

IV Decline phase III V II I THREATS OBJECTIVES SOLUTIONS Maximum hostility of the environment (alcohol, temperature, toxins, flora development) Risks of languishing alcoholic fermentation or stopping altogether Limit the toxicity of the environment Optimise the physiological state of the surviving yeasts Detoxification Control of the temperature Everything hangs on getting the beginning of the alcoholic fermentation correct: nitrogen, oxygen, sterols IV Decline phase III V II I 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 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 (1of2) Aggressiveness of the environment Alcohol >13,5 % vol and/or Turbidity < 80 NTU and/or T°<15°C or >28°C BIOPROTECT at rehydration + BIOCLEAN 2/3 through fermentation If combining both factors beginning of fermentation: VITISTART (whites/rosés) or ACTIVIT (reds) 20 g/hL 1/3 FA: ACTIVIT 20 g/hL + DAP 15 g/hL if alcohol degree below 12.5% Extreme nitrogen deficiency Assimilable nitrogen <100 mg/L 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 (2of2) Medium nitrogen deficiancy assimilable nitrogen between 100 and 140 mg/L Beginning of fermentation: NUTRIBIO 20 g/hL + thiamine 1/3 through fermentation: VITISTART (whites/rosés) or ACTIVIT (reds) 20 g/hL + DAP 15 g/hL 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/hL + DAP 15 g/hL 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