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Kinetic analysis and gas-liquid balances of the
production of fermentative aromas during winemaking fermentations
Jean-Roch Mouret, Carole Camarasa, Evelyne Aguera, Stéphanie Rollero, Marc Perez, Vincent Farines, Jean-Marie Sablayrolles
To cite this version:
Jean-Roch Mouret, Carole Camarasa, Evelyne Aguera, Stéphanie Rollero, Marc Perez, et al.. Ki- netic analysis and gas-liquid balances of the production of fermentative aromas during winemaking fermentations. ISSY 31, Oct 2014, Vipana et Nova Garica, Slovenia. �hal-01837716�
Kinetic analysis and gas-liquid balances of the production of fermentative aromas
during winemaking fermentations
Mouret J.R., Camarasa C., Aguera E., Rollero S., Perez M., Farines V., Sablayrolles J.M.
Introduction
Alcoholic fermentation in winemaking conditions
Key step of the winemaking process
Optimization of product quality
Quality markers = Aroma compounds
Grape must Alcoholic Wine
fermentation
Introduction
Wine aromas
Complex mixture of volatile molecules of different origins Grapes
Must
Young wines
Wines
Varietal aromas: representative of grape varieties
Fermentative aromas:
Secondary metabolites synthetized by yeast during winemaking fermentation
Most abundant : higher alcohols and esters
Post-fermentative aromas: post-fermentative chemical reactions, produced during aging
Introduction
Fermentative aromas
Metabolic pathways well known but:
Synthetic pathways highly connected and
interdependent
(involvement of C and N metabolisms)
Complex regulations
Development of an innovative monitoring
system to access to the kinetic parameters
Glucose Glycerol
Pyruvate Acetaldehyde
Acetic acid Acetyl-CoA
TCA
cycle α-ketoacids Amino
acids
Ethanol
Acyl-CoA
Acetate esters
Aldehydes
Higher alcohols Succinic acid
Fatty acids Ethyl esters
Need for new approaches to better describe, understand and control
Online monitoring of fermentative aromas
Heated transfer lines
Valve selector
Cold trap - GC
16 carbon compounds analysed
Maximum analysis frequency: 1h (with 1 tank)
Calculation of kinetic parameters
Gas-liquid balances of volatile compound production
Estimation of the gas losses
Calculation of the liquid concentrations
Calculation of the total productions (sum of liquid accumulation and gas losses)
Morakul et al., 2011 Mouret et al., 2014
Losses of volatile compounds
18°C 24°C 30°C
Propanol 0.4% 0.6% 0.9%
Isobutanol 0.4% 0.6% 1.0%
Isoamyl alcohol 0.5% 0.7% 1.4%
Isoamyl acetate 14% 21% 33%
Ethyl hexanoate 29% 42% 50%
Ethyl octanoate 26% 37% 49%
Losses:
Dependent on the studied compound
Negligible for higher alcohols
High for esters
Mouret et al., 2014
Gas-liquid balance
I
mportance of the gas-liquid balance for esters:
Overestimation of T° effect on ester synthesis if considering only C°liq
0.0 0.2 0.4
0 50 100 150
Liquid content of ethyl hexanoate (mg/L)
Consumed sugar (g/L)
18°C 24°C
30°C 0.0
0.2 0.4 0.6
0 50 100 150
Total production of ethyl hexanoate (mg/L)
Consumed sugar (g/L)
18°C 24°C 30°C
Mouret et al., 2014
Experimental plan
Impact of T° and initial nitrogen content
1 2 3
4 5 6
7 8 9
1 2 3
4 5 6
7 8 9
Focus on 6 fermentative aromas:
3 higher alcohols: propanol, isobutanol, isoamyl alcohol
1 acetate ester: isoamyl acetate
2 ethyl esters: ethyl
hexanoate, ethyl octanoate
Study focused on the total production of these volatile compounds:
representative of the true capacity of the yeast to
synthesize these molecules
Impact of T° and initial nitrogen content
The synthesis profiles are mostly dependent on initial YAN
0.0E+00 5.0E+02 1.0E+03 1.5E+03 2.0E+03 2.5E+03
0 50 100 150
Rate of total production of isamyl alcohol (µg l-1h-1)
Consumed sugar (g l-1)
Different YAN, same T° MS70
MS250 MS430
Mouret et al., 2014
T° mainly acts on the maximal production rate
Interaction nitrogen / T° on the regulation of yeast metabolism
0.0E+00 1.0E+03 2.0E+03 3.0E+03
0 50 100 150
Rate of total production of propanol (µg/L.h)
Consumed sugar (g/L)
Different T°, same YAN
18 24 30
Kinetics of production of propanol
Synthesis exclusively during the nitrogen consumption phase
Amount proportional to the initial nitrogen concentration
Propanol identified as a quantitative, metabolic marker of nitrogen availability
0.0E+00 5.0E+07 1.0E+08
0 200 400
0 50 100 150
Cell count (nb cell ml-1 ) Production of propanol (*10 mg l-1 ), residual nitrogen content (mgN l-1 )
Consumed sugar (g l-1)
propanol Nitrogen Biomass
0 10 20 30 40 50
0 100 200 300 400
Final production of propanol (mg l-1)
Initial nitrogen content (mgN l-1)
Mouret et al., 2014
Valine
α-KG Glutamate
α-ketoisovalerate
Isovaleraldehyde
Isobutanol
Leucine
α-ketoisocaproate
Isoamylaldehyde
Isoamyl alcohol α-KG Glutamate BAT1, BAT2
PDC1,5,6
ADH1/5, SFA1 NADH
NAD+
BAT1, BAT2
THI3, ARO10
ADH1/5, SFA1 NADH NAD+
Isobutyl acetate Isoamyl acetate
ATF1, ATF2 Acetyl-CoA ATF1, ATF2 Acetyl-CoA
2-isopropylmalate
Acetyl-CoA CoA LEU4, LEU9
3-isopropylmalate
LEU1
NAD+ NADH LEU2
Glucose
Pyruvate
Aspartate
Methylacetaldehyde
Propanol
TCA cycle
Threonine
α-KG Glutamate
α-ketobutyrate
NADH NAD+ BAT1, BAT2
PDC1,5,6
ADH1/5, SFA1
PYC1, PYC2
α-KG Glutamate AAT1, AAT2
Threonine
Kinetics of production of propanol
Exclusively generated by direct deamination of exogenous threonine or interconversion between other amino acids and threonine
Kinetics of production of the other aromas
Isobutanol, isoamyl alcohol, isoamyl acetate and the 2 ethyl esters synthetized throughout the fermentation process
Production linked to carbon metabolism
Mouret et al., 2014
0.E+00 5.E+07 1.E+08
0.0 0.4 0.8
0 50 100 150
Cell count (nb cells/mL)
Total production of ethyl octanoate (mg/L)
Consumed sugar (g/L) 0.E+00
5.E+07 1.E+08
0 50 100 150
0 50 100 150
Cell count (nb cells/mL)
Total production of isoamyl alcohol (mg/L)
Consumed sugar (g/L)
Kinetics of production of the other aromas
0.E+00 5.E+07 1.E+08
0 50 100 150
0 50 100 150
Cell count (nb cells/mL)
Total production of isoamyl alcohol (mg/L)
Consumed sugar (g/L)
0.E+00 5.E+07 1.E+08
0.0 0.4 0.8
0 50 100 150
Cell count (nb cells/mL)
Total production of ethyl octanoate (mg/L)
Consumed sugar (g/L)
2 successive linear production phases from sugar (with yields dependent on initial nitrogen content and temperature)
Higher alcohols and acetate ester: transition simultaneous to the end of the growth phase
Ethyl esters: transition at the same sugar content whatever the YAN and T° values
Mouret et al., 2014
Isobutanol and Isoamyl alcohol
0 200
400 20
25 30 0.5
1 1.5
T (°C) A
N0 (mg/L) Y1 Isoamyl alcohol (mg/g)
0 100 200 300 400 20
25 30
0.8 1 1.2 1.4 1.6 1.8
T (°C) N0 (mg/L)
B
Y2 Isoamyl alcohol (mg/g)
0 200
400 20
25 30 0.2
0.4 0.6
T (°C) A
N0 (mg/L) Y1 Isobutanol (mg/g)
0 200
400 20
25 30 0
0.2 0.4 0.6
T (°C) B
N0 (mg/L) Y2 Isobutanol (mg/g)
Isoamyl alcohol
Isobutanol
Production yields of isobutanol and
isoamyl alcohol
differently impacted by the environmental parameters
phase 1 phase 2
Kinetics of production of higher alcohols
Isobutanol and isoamyl alcohol
Differences in the rates and specific rates of production
Major differences in the regulation of synthesis of isobutanol and isoamyl alcohol, despite their partially shared metabolic pathways
0.0E+00 2.0E+03 4.0E+03
0.0E+00 8.0E+02 1.6E+03
0 50 100 150
Rate of total production of isoamyl alcohol (µg l-1h-1) Rate of total production of isobutanol (µg l-1h-1)
Consumed sugar (g l-1)
isobutanol isoamyl alcohol
0.0E+00 5.0E-08 1.0E-07
0.0E+00 4.0E-08 8.0E-08
0 50 100 150
Specific rate of total production of isoamyl alcohol (µg l-1h-1) Specific rate of total production of isobutanol (µg l-1h-1)
Consumed sugar (g l-1)
isobutanol isoamyl alcohol
Mouret et al., 2014
Valine
α-KG Glutamate
α-ketoisovalerate
Isovaleraldehyde
Isobutanol
Leucine
α-ketoisocaproate
Isoamylaldehyde
Isoamyl alcohol
α-KG Glutamate
BAT1, BAT2
PDC1,5,6
ADH1/5, SFA1
NADH NAD+
BAT1, BAT2
THI3, ARO10
ADH1/5, SFA1 NADH NAD+
Isobutyl acetate
Isoamyl acetate
ATF1, ATF2 Acetyl-CoA ATF1, ATF2 Acetyl-CoA
2-isopropylmalate
Acetyl-CoA CoA
LEU4, LEU9
3-isopropylmalate
LEU1
NAD+ NADH
LEU2
Glucose
Pyruvate
Aspartate
Methylacetaldehyde
Propanol TCA cycle
Threonine α-KG Glutamate
α-ketobutyrate
NADH NAD+
BAT1, BAT2
PDC1,5,6
ADH1/5, SFA1
PYC1, PYC2
α-KG Glutamate
AAT1, AAT2
Threonine
Kinetics of production of higher alcohols
Isobutanol and isoamyl alcohol
Different management of the pools of -keto-acids
Importance of the gas-liquide balance:
Differentiation of biological and physical consequences of T° modification
Impact of T° on ester synthesis overestimated if only liquid content was considered
Production of fermentative aromas closely linked to central carbon metabolism, except for propanol
Two successive linear production phases (with yields dependent on initial nitrogen and T°) from sugar
Differences in the regulation of the synthesis of isobutanol and isoamyl alcohol, despite their partially shared metabolic pathways
Propanol identified as a quantitative, metabolic marker of assimilable nitrogen
Conclusions
Genericity of the results
Experiments in other media, especially natural musts
Study of other yeast strains
