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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 2011
University of California, Davis
Creating Character
Reminder: Must nutrients
Macro and micronutrients – Related effects
Type
Macronutrients
(Cell material
renewal)
Micronutrients
(Biochemical
reactions
catalysts)
Category
Nature
Effects
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
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
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 definition
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
Focus on Nitrogen
Must composition in yeast available nitrogen compounds
• 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)
Focus on Nitrogen
Nitrogen compounds use
• 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.
Focus on Nitrogen
Yeast assimilation process
Plasma membrane not freely permeable to N compounds  1st step: Transport
Ammonia: easy uptake
AA: 2 mechanisms
• General amino acid permases but not proline
• Adaptative uptake systems under stress conditions
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)
Focus on Nitrogen
Assimilation particularities
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
YAN measurement assays
Different measurements  Different indications
Assay
Base principle
N 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 NH4+ blocked by
formol addition. Resulted acid
dosed by NaOH.
Amino acids,
peptides and
NH4+
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 NH4+ recovery
HPLC
Liquid Chromatography
Each single
amino acids
Very accurate but expensive and too slow
For research purpose
YAN measurement assays
Different measurements  Different indications
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
detection
Inexpensive, rapid
Calibration and interference
YAN measurement assays
Measurement methods of choice
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 Nitrogen
Low toxic reagents
Necessary 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!
Is YAN enough?
YAN interpretation and other important factors
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
Is YAN enough?
Yeast specific N requirements
8
7
6
<N1200-37°C>
Commercial strain 1
<N150-37°C>
4
3
2
8
1
7
0
0
2
4
6
8
10
Time (days)
Commercial strain 2
12
146
Weight loss (g)
Weight loss (g)
<N250-37°C>
5
<N1200-37°C>
16
<N250-37°C>
5
<N150-37°C>
4
3
2
1
0
0
2
4
6
8
Time (days)
10
12
14
16
Is YAN enough?
Other important factors
Low YAN means low nutrient content
Commercial strain 3
8
Yes, BUT
<N150S250 24°C>
7
<N1200S250 24°C>
6
• pH (too low: cell viability, too high: microbiological
spoilage)
<N1200S250 17°C>
<N150S250 17°C>
5
4
• Low turbidity  Low nutrient level, low
lipid supply and low nucleation sites for
decreasing concentration of dissolved CO2
3
2
• Temperature management is always 1critical (too low: activity/growth, too high: EtoH
toxicity) and affects yeast nitrogen requirements in terms for both quantities and quality
0
0
5
10
15
20
25
30
35
• 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
Importance of assuring completion
Speed or Dryness
Vmax  N requirements
Ethanol inhibition
Viability = O2
Combination N/O2 to speed but also
secure fermentation!
Source Sablayrolles (2010)
Timing of N addition
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
O2
N
Source Sablayrolles (2010)
But not everything at the same time  N assimilation processes!
Dangers of under/over N addition
YAN Limitation
• 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
Dangers of under/over N addition
YAN Excess
• 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
Addition guidelines
Importance of selected yeast strains
Yeast choice:
Type of wines/ alcohol and temperature resistance
Function of Selected
Yeast
Bonus: Sterol
source
Addition guidelines
Importance of selected nutrients
•
Example: Round and fruity Pinot Noir
Initial YAN:
Function of Must
Sequential
additions:
No
detrimental
effect
Not after the
middle of
fermentation:
No uptake
Addition guidelines
Nutrient addition calculation guidelines
PA limit for
extra nutrient
addition:
Function of
Selected Yeast!
Ex: CK S102 limit
Questions?
Thank you for your attention
For further information:
Etienne DORIGNAC
Fermentis Division of S.I.Lesaffre
Cell : + 33 6 26 65 17 90
E-mail : [email protected]
Homepage : http://www.fermentis.com