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Improving acid tolerance in Oenococcus oeni by Adaptive Evolution
Frédérique Julliat, Stéphane Guyot, Herve Alexandre, Cosette Grandvalet
To cite this version:
Frédérique Julliat, Stéphane Guyot, Herve Alexandre, Cosette Grandvalet. Improving acid tolerance in Oenococcus oeni by Adaptive Evolution. 12th International Symposium on Lactic Acid Bacteria (LAB), Aug 2017, Egmond aan Zee, Netherlands. �hal-03150712�
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Parental E1 E2 E3 E4 E5 E6
Permeabilizedcells(%)
Improving acid tolerance in Oenococcus oeni by Adaptive Evolution
Julliat F*. , Guyot S., Alexandre H. and Grandvalet C.
Université de Bourgogne Franche-Comté, AgroSup Dijon, UMR Procédés Alimentaires et Microbiologiques (UMR PAM), Dijon, France
References
[1] Bartowsky, E.J. (2005). Oenococcus oeni and malolactic fermentation – moving into the molecular arena. Aust. J. Grape Wine Res. 11, 174–187.
[2] Bauer, R., and Dicks, L.M.T. (2004). Control of malolactic fermentation in wine. A review. Afr J Enol Vitic 25, 74–88.
Oenococcus oeni
Saccharomyces cerevisiae
Alcoholic fermentation
Malolactic fermentation
Winemak ing
Oenococcus oeni is a lactic acid bacterium (LAB) mainly responsible for the malolactic fermentation (MLF) in wine. MLF plays an important role in determining the final quality of wines
[1]. Even though this LAB is naturally present in musts, wines and oenological environment, spontaneous MLF are usually unpredictable because of the stressful conditions and especially due to acidity
[2]. The consequence of the mismanagement of this step might lead to the depreciation of wine quality.
To obtain a clone more tolerant to acidity, we undertaken to replicate O. oeni in a temporally varying environment (pH 5.3 to 3.2) to improve acid tolerance. To discriminate stress tolerance of evaluated populations versus parental strain an ethanol or acid stress were improved to both population.
Conclusions & Perspectives Background
Strategy
0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8
0 10 20 30 40 50 60 70
Optical density(600 nm)
Time (hours)
Parental Evolved
Oenococcus oeni
ATCC BAA-1163
6 Independent populations Propagated until they reached the stationary phase
Growth of evoluated populations in ethanol stress condition
First Results
The growth of evolved and parental strains was monitored in ethanol supplemented medium. Biomass of evolved strains increase in medium containing ethanol and especially for two clones where biomass has doubled compared with the parental strain
Growth follow up of evolved and parental strains in ethanol supplemented medium. Cells were cultivated at 28°C in FT80m medium (pH 5.3 for parental strain and 3.2 for evolved strain) until mid-exponential growth phase (OD600nm=0.8 for parental strain and OD600nm=0.3 for evolved strain) and inoculated in FT80m medium at pH 5.3 supplemented by 8% (v/v) ethanol.
• Two clones appears discriminant by increase biomass in ethanol supplemented medium compared with the parental strain.
• An adaptation based on improve acidity tolerance enabled ethanol tolerance for two clones.
• Further works will focus on the origin of this phenotype by an exploration of the genome and transcriptome.
• It would be necessary to test evolved clones in oenological condition to perform MLF.
PARENTAL STRAIN
Parental E2
E4
E5E1 E6 E3
The membrane permeability was measured on the evolved and parental strains during ethanol or acid stress.
Evolved strains maintained better membrane integrity during acid stress than the parental strain.
Modification of membrane fluidity of evoluated populations during stress
Membrane permeability during stress conditions. Cells were cultivated at 28 °C in FT80m medium (pH 5.3 for parental strain and 3.2 for evolved strain) until mid-exponential growth phase (OD600nm= 0.8 for parental strain and OD600nm= 0.3 for evolved strain). Cells were transferred into (A) medium supplemented by 8% ethanol (v/v) or into (B) medium at pH 2.4 and 90 min after transfer membrane permeability was measured by flow cytometry using propidium iodide as probe.
x6
FT80m pH 5.3
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Parental E1 E2 E3 E4 E5 E6
Permeabilizedcells(%)
A
B
***
*** *** *** *** ***
Nervertheless, no significant difference between parental and evolved strains during ethanol stress
PARENTAL STRAIN
250
generation s
From pH 5.3 to 3.2
6 EVOLVED
STRAINS (E1 to E6) FT80m x6
pH 4.5 FT80m
pH 5.3
FT80m pH 4
FT80m pH 3.7
FT80m pH 3.5
FT80m
pH 3.2
vs
Membrane permeability and growth in stress
conditions
