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 18th 2011University of California, Davis

Macro and micronutrients – Related effects

Reminder: Must nutrients

Type Category Nature Effects

Macronutrients(Cell material renewal)

Carbon Glu/Fru, Sucrose Energy sources (glycolytic pathway)

Nitrogen Amino 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

OxygenFatty acidsSterols (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

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

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)

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.

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 NH4

+

Source Salmon (1998)

Plasma membrane not freely permeable to N compounds 1st step: Transport

AA: 2 mechanisms• General amino acid permases but not proline• Adaptative uptake systems under stress conditions

Ammonia: easy uptake

Assimilation particularities

Focus on Nitrogen

NH4+ assimilation consequences

• NH4+ reduce catabolic enzyme levels and transport activities for non

preferred N sources. • Alternative N-assimilatory pathways not expressed when NH4

+ is present. • As NH4

+ 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, 4th N incorporated into proline• Breakdown results in the formation of urea and ammonia. possible ethyl carbamate production

Different measurements Different indications

YAN measurement assays

Assay Base principle N compounds measured

Advantages / Disadvantages

Kjeldahl method

Heat mineralization in acidic medium

All N form transformed in NH3.

Radical and fastTake into account all N compounds and not only available N compounds

Formol titration

Amino group and NH4+ blocked by

formol addition. Resulted acid dosed by NaOH.

Amino acids, peptides and NH4

+

Fast global YAN analysisCarcinogenic and bronchial irritant agent (well trained analyst and suitable lab)Adjustment of formaldehyde pH critical to method consistency

~17% of proline and ~85% of NH4+ recovery

HPLC Liquid Chromatography Each single amino acids

Very accurate but expensive and too slowFor research purpose

Different measurements Different indications

YAN measurement assays

Assay Base principle N compounds measured

Advantages / Disadvantages

Mid-infrared/IRTF

Spectrophotometry illustrating organic links absorption in near and mid IR

Amino acids and NH4

+ 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

NH3(aq) and NH4+ NH3(aq) by raising pH >11 with a strong base. NH3(aq) diffuses through membrane and changes internal solution pH sensed by a pH electrode.

NH4+ only High degree of accuracy and low limit of

detectionInexpensive, rapid Calibration and interference

Measurement methods of choice

YAN measurement assays

Assay Base principle N compounds measured

Advantages / Disadvantages

NOPA Derivatization 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% NH4+

recovery real Free Amino NitrogenLow toxic reagentsNecessary reagent blank as flavonoids absorb at 335nm

Enzymatic NH4

+

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 NH4

+ present.

NH4+ only Fast and specific

Best association for YAN measurement!

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 allDifferences in assimilable nitrogen and oxygen demands account for

most of the differences between yeast strains

Yeast specific N requirements

Is YAN enough?

0

1

2

3

4

5

6

7

8

0 2 4 6 8 10 12 14 16

Time (days)

We

igh

t lo

ss

(g

)

<N1200-37°C>

<N250-37°C>

<N150-37°C>

0

1

2

3

4

5

6

7

8

0 2 4 6 8 10 12 14 16

Time (days)

Wei

ght l

oss

(g)

<N1200-37°C>

<N250-37°C>

<N150-37°C>

Commercial strain 1

Commercial strain 2

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

• O2 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

Commercial strain 3

0

1

2

3

4

5

6

7

8

0 5 10 15 20 25 30 35

<N150S250 24°C>

<N1200S250 24°C>

<N1200S250 17°C>

<N150S250 17°C>

Speed or Dryness

Importance of assuring completion

Vmax N requirements Ethanol inhibition

Viability = O2

Combination N/O2 to speed but also secure fermentation!

Source Sablayrolles (2010)

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

O2

N

Source Sablayrolles (2010)

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• H2S accumulation and production• Too much higher alcohol production

YAN Limitation

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

Importance of selected yeast strains

Addition guidelines

Yeast choice: Type of wines/ alcohol and temperature resistance

Function of Selected Yeast

Bonus: Sterol source

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

Addition guidelinesNutrient addition calculation guidelines

PA limit for extra nutrient

addition:Function of

Selected Yeast!

Ex: CK S102 limit

Questions?

For further information:

Etienne DORIGNACFermentis Division of S.I.LesaffreCell : + 33 6 26 65 17 90E-mail : edorignac.fermentis@lesaffre.frHomepage : http://www.fermentis.com

Thank you for your attention