MLF tool to reduce acidity and improve aroma under cool climate conditions Maret du Toit Lynn Engelbrecht, Elda Lerm, Doris Rauhut, Caroline Knoll and Sibylle Krieger-Weber
Malolactic fermentation l Deacidification of wine l Microbial stability l Improvement of aroma and flavour profile more complex, better structured creamier and fuller palate (ethyl lactate) more butteriness (diacetyl) reduced vegetative aromas enhanced fruity notes (esters) l ph l Sulfur dioxide l Ethanol l Malic acid concentration l Temperature of wine l Interaction with yeast l Nutrients l MLF inoculation scenario l Fungicide residues l Inhibitors that will influence growth Phenolic acids Lysozyme
MLF starter cultures used l O. oeni Tolerates wine ph 2.9-4 Tolerant up 16% ethanol Conduct MLF at low temperatures Total SO 2 50 mg/l Minimal increase in VA Enhance aroma profile of wine Produces no off-flavours l L. plantarum Prefers wine ph >3.5 Survive up 8-10% ethanol Grow best at 20 C Total SO 2 40 mg/l Produces no acetic acid Enhance aroma profile of wine Produces bacteriocins
Time of inoculation l After alcoholic fermentation Advantages: Easy to control MLF as alcoholic fermentation is finished Risks: Stuck MLF due to high [alcohol] Nutrients depleted l Simultaneous or Co-inoculation Advantages: No impact of alcohol Enough nutrients available MLF finish earlier and wine can be stabilized after alcoholic fermentation
Impact of malic acid [ ] Factors that impact MLF Initial level of malic acid (g/l) Easy Moderate Difficult Extreme 2-4 4-5 or 1-2 5-7 or 0.5-1 >7 or <0.5 Chardonnay 12.5%v/v ph - 3.25 Malic acid - 2.6g/L Temp. - 16 C 35 28 21 14 Time (days) Time to achieve 90% of MLF malate : 0.75 malate : 1.35 malate : 2,6 malate: 5,2 Duration of MLF increases with malic acid content. Speed of malic acid degradation increases with malic acid content. 7 0 Expertise S Lalvin 31 Alpha 49A1
Impact of lactic acid [ ] Chardonnay 12.5%v/v ph - 3.25 Temp.= 16 C Time to achieve 50% of MLF Initial L-malic acid=3g/l 1,0E+07 Bacteria population - Alpha Time (days) 110 100 90 80 70 60 50 40 30 20 10 0 Expertise S Lalvin 31 Alpha 49A1 Lactate : 0 Lactate : 1.5 g/l Lactate: 3.0 g/l log cfu/ml 1,0E+06 1,0E+05 Lactic acid 0 Lactic acid 1.5 Lactic acid 3.0 1,0E+04 0 1 2 3 4 5 6 7 Time (days) Addition of 1.5 g/l highly increases the time to achieve MLF. Addition of 3g/L induces a high loss of viability which leads to stuck MLF.
Impact of wine yeast on MLF Longer lag phase Neutral towards MLF Yeast 1 Yeast 2 Mix Mix Yeast 3 Mix Stimulatory towards MLF
Impact of yeast strain on MLF l Chardonnay Bacteria 1 ph 3.3 TA 9.3 Malic acid (g/l) 5.0 4.0 3.0 2.0 1.0 VIN 2000 VIN 13 Fermicru LVCB Exo<cs Yeast Bacteria combinations are crucial 0.0 5.0 Malic acid (g/l) 4.0 3.0 2.0 1.0 0 3 6 9 12 15 18 21 28 35 42 Days a0er inocula6on Bacteria 2 Control (Exo<cs) VIN 2000 VIN 13 Fermicru LVCB Exo<cs 0.0 0 21 28 35 42 Control (Exo<cs) Days a0er incocula6on
Impact of wine yeast - Esters 250 Merlot 2011 225 200 Concentra6on (mg/l) 175 150 125 100 75 50 Mix O. oeni 2 O. oeni 1 Control yeast
Impact of ph and ethanol
Experimental design Grape/Must Alcoholic fermentation (AG) MLF Chemical Analyses Riesling Rheingau - Deutschland Chardonn ay Chardonnay Stellenbosch - Südafrika ph 3,2; 3,6; 3,8 EtOH 12,5 & 15 % (v/v) 4 Inoculation times: 24 h AF (218 g/l RZ) 40 % AF (125 g/l RZ) 60 % AF (109 g/l RZ) Sequential (1,5 g/l RZ) RZ sugar left
Impact of ph and ethanol 15 O.oeni R1105 12. 5 O.oeni R1106 3.2 3.6 3.8
Impact on aroma profile - O. oeni R1105 Relative Change in Total Ester Concentration [%] 97% 93 % Control 12,5 % ph 3,2 95 % Malic acid degrada6on in % 90 % 12,5 % ph 3,6 100 % 99 % 12,5 % ph 3,8 > 10 3 cfu/ml 24 % > 10 2 cfu/ml 52 % 15 % ph 3,2 90 % 66 % 15 % ph 3,6 97 % 100 % 15 % ph 3,8
Impact of ph and alcohol on aroma 15 % (v/v) Acetic acid-3- ph 3,8 methylbuthylester (banana) Long chain fatty acid esters (green apple, floral, soapy) ph 3,6 Diethylester (fruity) ph 3,2 12,5 % (v/v) Hexanol (grassy)
MLF and Riesling wine
MLF and low ph wine Simultaneous Time safed: 25 50 Days relative Sugar concentration (%) 100 50 24 h 40 % AF 3,1 2,9 3,0 60 % AF O. oeni R1105 O. oeni R1124 Sequen6al 3,1 Duration MLF [days] 0 Winemaking AF alcoholic fermentation
Co-Inoculation vs. Sequential 1400 1200 a 24 h 24h Sequen6al AFEnd 1000 Concentra6on [µg/l] 800 600 400 200 b a b a b a b a b 0 Acetic Essigsäure-3-methylacid-3-methyl- Acetic Essigsäure-2-phenylacid-2-phenylbutylester ethylester butylester ethylester Bernsteinsäure- Succinic aciddiethylester diethylester Acetic Essigsäureacid - hexylester hexylester BuTersäure- Butyric acid - ethylester ethylester (Banana) (floral) (fruity) (fruity / floral) (Apple)
Impact of MLF scenario on Riesling aroma 24h Acetate-3- methylbuthylester (Banana) No MLF Sequential MLF Acetate ethylester (Apple, acetone) Other Ethylester 60% F AF (fruity) 40% AF Succinic acid Diethylester (fruity) 24h Co-inoculation
Impact of LAB blends low ph Aroma6c profile (Tempranillo wine, Spain) Aroma intensity 3 2 Green 1 Red fruit 0 Taste profile (Tempranillo wine, Spain) 3.5 Dried fruit Dark fruit 3.4 3.3 BLEND OF BACTERIA HIGH ph BLEND OF 3.2 BACTERIA LOW ph 3.1 3.0 2.9 2.8 2.7 2.6 2.5 Tannin intensity BLEND OF BACTERIA HIGH ph Astringency BLEND OF BACTERIA LOW ph
Summary of MLF aroma impact Strain & <me of inocula<on Dependent on: chemical composi<on Inocula<on <me Strain Cul<var BuTer MLF rate, ph & EtOH-dependent fruity / chemical solvent fruity Banana floral fruity
Acknowledgements l MLF research groups Stellenbosch and Geisenheim Research Center l Chemical Analytical laboratories l Sensory laboratories l Financial support
Next time you open a fermented product, spare a thought for those marvelous microorganisms that helped create such a complex product Cheers to these little creators of diversity