1. Creating Character The importance of knowing juice composition Nutrient assimilation, methods of analyses and interpretation as a guide to what additions should be made March, the 18 th 2011 University of California, Davis

2. Macro and micronutrients – Related effects Reminder: Must nutrients TypeCategoryNatureEffects Macronutrients (Cell material renewal) CarbonGlu/Fru, SucroseEnergy sources (glycolytic pathway) NitrogenAmino acids, ammonia, nucleotides, peptides Protein synthesis : Production of biomass, Fermentation rate-time-flavors Phosphate* / Sulfur Inorganic and organic P/S compounds Cell growth (biomass)  fermentation rate S-volatiles flavors Survival factors Oxygen Fatty acids Sterols (ergosterols) Yeast growth: Energy, fermentation rate Glycogen and threalose (stress protecting factors) high content maintaining Stimulate lipid biosynthesis  Strengthen yeast membrane (integrity, permeability)  viability Decrease production of toxic medium chain fatty acids Micronutrients (Biochemical reactions catalysts) Vitamins*Most important: Biotin, Thiamine, Pantothenate Growth factors, Co-factors in enzymatic conversions Minerals*Most important: Mg, K, Mn, Zn, Fe, Cu Co-factors for glycolytic and other enzymatic reactions * Generally sufficient in grape musts

3. Focus on Nitrogen YAN: Yeast Assimilable or Available Nitrogen  Nitrogen that will be taken up and used by the yeast for its metabolism: Growth and fermentative power = Primary or alpha amino acids FAN (Free Amino Nitrogen) without proline + Ammonium ions FAN! Free Amino Nitrogen or Free Assimilable/Available Nitrogen YAN definition

4. Must composition in yeast available nitrogen compounds Focus on Nitrogen Ammonium ions Up to 30% of YAN Amino acids (AA) Most prevalent form in must  Up to 90% of YAN Major sources: Proline and arginine (30 to 65% of total AA content), located mostly in grapes skin  Importance of grape processing practices Alanine, glutamine (increased with fertilization), serine and threonine YAN measurements directly on juice sample at inoculation  Avoid over estimation (processing losses)  Juice samples taken form grape musts can underestimate total berry YAN (important grape skin aa content)

5. Nitrogen compounds use Focus on Nitrogen Ammonium ions Preferred nitrogen source as small and directly available Converted into amino acids with energy Amino acids Second nitrogen source: protein building blocks Incorporated as is, Transformed into a different AA (transamination based on key compound: glutamate) Broken down as a source of nitrogen or sulfur when ammonia nitrogen source is limiting. Storage inside the cell (vacuole, cytoplasm) for later usage in protein synthesis Uptake of glutamine first (easier and break down to glutamate and ammonia) Asparagine, second preferred N source.

6. Yeast assimilation process Focus on Nitrogen Assimilation mechanism: Every AA uptake  H + uptake [EtOH] increase  Membrane permeabilization  Difficult pH maintaining  Shut down of AA uptake first then NH 4 + Source Salmon (1998) Plasma membrane not freely permeable to N compounds  1 st step: Transport AA: 2 mechanisms General amino acid permases but not proline Adaptative uptake systems under stress conditions Ammonia: easy uptake

7. Assimilation particularities Focus on Nitrogen NH 4 + assimilation consequences NH 4 + reduce catabolic enzyme levels and transport activities for non preferred N sources. Alternative N-assimilatory pathways not expressed when NH 4 + is present. As NH 4 + is consumed, amino acids are taken up relatively to cell needs (concentration gradient) Particular AA Proline: No assimilation during fermentation  Uptake inhibition by other aa and oxygen needed Arginine: Less readily utilized source of N  Uptake during active fermentation and stationary phase 3 of its 4 N atoms assimilated, 4 th N incorporated into proline Breakdown results in the formation of urea and ammonia.  possible ethyl carbamate production

8. Different measurements  Different indications YAN measurement assays AssayBase principleN compounds measured Advantages / Disadvantages Kjeldahl method Heat mineralization in acidic medium All N form transformed in NH3. Radical and fast Take into account all N compounds and not only available N compounds Formol titration Amino group and NH 4 + blocked by formol addition. Resulted acid dosed by NaOH. Amino acids, peptides and NH 4 + Fast global YAN analysis Carcinogenic and bronchial irritant agent (well trained analyst and suitable lab) Adjustment of formaldehyde pH critical to method consistency ~17% of proline and ~85% of NH 4 + recovery HPLCLiquid ChromatographyEach single amino acids Very accurate but expensive and too slow For research purpose

9. Different measurements  Different indications YAN measurement assays AssayBase principleN compounds measured Advantages / Disadvantages Mid-infrared /IRTF Spectrophotometry illustrating organic links absorption in near and mid IR Amino acids and NH 4 + separately Very fast and accurate (N compounds in isolated spectrum area) Difficulty of calibration (mastering reference data base, well trained analyst) Ammonia selective gas sensing electrode NH 3 (aq) and NH 4 +  NH 3 (aq) by raising pH >11 with a strong base. NH 3 (aq) diffuses through membrane and changes internal solution pH sensed by a pH electrode. NH 4 + onlyHigh degree of accuracy and low limit of detection Inexpensive, rapid Calibration and interference

10. Measurement methods of choice YAN measurement assays AssayBase principleN compounds measured Advantages / Disadvantages NOPADerivatization of primary amino groups with o-phthaldialdehyde/N-acetyl-L-cysteine reagent to form an isoindole derivative which can be conveniently measured at the near ultraviolet wavelength of 335nm Yeast available free amino acids Accurate and fast (well correlated with HPLC) Insensitive to proline and 3,5% NH 4 + recovery  real Free Amino Nitrogen Low toxic reagents Necessary reagent blank as flavonoids absorb at 335nm Enzymatic NH 4 + Ammonia reacts with α-ketoglutarate and NADH in the presence of glutamate dehydrogenase (GlDH) to form L-glutamate and NAD. Amount of NADH consumed measured at 340 nm and related to the amount of NH 4 + present. NH 4 + onlyFast and specific Best association for YAN measurement!

11. YAN interpretation and other important factors Is YAN enough? Minimum YAN is considered to be about 140-150 mg N/L (ppm) Optimum/maximum fermentation rate: 800-900ppm (only 400-500 ppm assimilated) Adding a standard number is NOT the best solution As Overaddition of N could be detrimental in terms of N uptake, fermentation progress and flavors N extra addition function of potential EtOH yield increase  More functional proteins and more resistant cell wall necessary But first of all Differences in assimilable nitrogen and oxygen demands account for most of the differences between yeast strains

12. Yeast specific N requirements Is YAN enough? Commercial strain 1 Commercial strain 2

13. Other important factors Is YAN enough? Low YAN means low nutrient content Yes, BUT pH (too low: cell viability, too high: microbiological spoilage) Low turbidity  Low nutrient level, low lipid supply and low nucleation sites for decreasing concentration of dissolved CO2 Temperature management is always critical (too low: activity/growth, too high: EtoH toxicity) and affects yeast nitrogen requirements in terms for both quantities and quality O 2 required at the end of the growth phase  To synthesize sterols and safe fermentation  membrane strength  yeast viability  To avoid toxic medium chain fatty acid production

14. Speed or Dryness Importance of assuring completion Vmax  N requirements Ethanol inhibition Viability = O 2 Combination N/O 2 to speed but also secure fermentation! Source Sablayrolles (2010)

15. Crucial timing of N addition Inoculation time  yeast growth Start of the stationnary phase, existing yeasts reactivation while increasing consumption rate of sugar  3-5% alcohol : best addition time as No cellular growth Fermentative activity increase (increase of hexose transporter quantity) Constant addition effect But not everything at the same time  N assimilation processes! Timing of N addition O2O2 N Source Sablayrolles (2010)

16. Dangers of under/over N addition Not enough production of yeast biomass Decrease of fermentation rate and slow down of fermentation time Production of off-flavors H 2 S accumulation and production Too much higher alcohol production YAN Limitation

17. Dangers of under/over N addition At fermentation start: too much yeast growth Increase in overall N demand later in fermentation Heat peak  cell damages, loss volatile aromas Over addition of ammonium ions Prevention of aa and cysteine conjugates uptake If DAP used: acidification and salty taste because of phosphate excess Presence of non assimilated N at the end of AF Brettanomyces growth Possible health risk ethyl carbamate production Possible biogenic amines production by bacteria Over production of ethyl acetate, acetic acid (VA) and succinic acid YAN Excess

18. Importance of selected yeast strains Addition guidelines Yeast choice: Type of wines/ alcohol and temperature resistance Function of Selected Yeast Bonus: Sterol source

19. Addition guidelines Example: Round and fruity Pinot Noir Initial YAN: Function of Must Sequential additions: No detrimental effect Importance of selected nutrients Not after the middle of fermentation: No uptake

20. Addition guidelines Nutrient addition calculation guidelines PA limit for extra nutrient addition: Function of Selected Yeast! Ex: CK S102 limit

21. Questions?

22. For further information: Etienne DORIGNAC Fermentis Division of S.I.Lesaffre Cell : + 33 6 26 65 17 90 E-mail : edorignac.fermentis@lesaffre.fredorignac.fermentis@lesaffre.fr Homepage : http://www.fermentis.comhttp://www.fermentis.com Thank you for your attention