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

2. 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

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

4. I. Latency period

5. 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

6. 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

7. 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

8. 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...

9. Manage the inoculation rate
Impact on the latency phase

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

11. II. GROWTH PHASE AND SLOWING DOWN PHASE

12. 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

13. Complement only if necessary (1of2)
Henschke P.A. and V. Jiranek, Int. Symp. on Nitrogen in Grapes and wines,1991,
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.

14. 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]

15. 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).

16. 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

17. 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

18. 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

19. 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

20. 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)

21. Conversion of the thiols precursors: the nutrition role
Subileau & Salmon, FEMS Yeast Res 8 (2008)
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.

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

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

24. YEAST PROTECTION

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

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

27. PROTECTION
NUTRITION

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

29. 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]

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

31. 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

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

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

34. Yeast protection
Protector
Oxygen

35. Yeasts protected by Bioprotect®
Non protected yeasts
Yeasts protected by Bioprotect®

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

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

38. IV. STABLE PHASE

39. 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.

40. V. DECLINE PHASE

41. 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.

42. 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

43. 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.

44. 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