1. Impact of Malolactic Fermentation Strain on Wine Composition Lucy Joseph U.C. Davis Department of Viticulture and Enology

2. Outline Introduction to malolactic fermentation (MLF) Lactic acid bacteria metabolism Commercial inoculum Wine matrix effects –Interaction with oak Timing of inoculation

3. Malolactic Fermentation Any wine containing malic acid could be considered unstable. Certain indigenous bacteria can metabolize malic acid as a very poor carbon and energy source in the fermenter and in the bottle. Conversion of malic to lactic acid by a controlled malolactic fermentation prior to bottling eliminates the instability. It can be a problem to start, very slow activity, long time to finish and can start and stop.

4. MALOLACTIC BACTERIA What are they? –“bacteria”: single-celled non-nucleated microorganisms –“lactic acid bacteria”: produce lactic acid from sugar Acid loving Nutritional fastidious Carry out many food fermentations

5. Malolactic bacteria are lactic acid bacteria which can convert malic acid to lactic acid: stronger acid weaker acid 2 carboxyl groups 1 carboxyl group Malic Acid CO 2 Lactic Acid + Carbon Dioxide

6. Oenococcus oeni Only two species Oenoccocus oeni (formerly Leuconostoc oenos) are only found in wine Oenococcus kitaharae was ‘discovered’ in 2006 in the spoiled remains of a sake mash (shochu, high pH) and lacks the gene for malolactic fermentation

7. What happens during a malolactic fermentation Deacidification –Each gram per liter of malic converted to lactic creates a loss of 7.46 mM/L of titratable H+ ions, or 1.12 grams/L as tartaric measured by titration (TA) pH changes Micronutrients are sequestered Secondary metabolites can contribute to the flavor profile

8. THE COURSE OF THE MLF Bacterial growth is finished several days after the conversion of malate to lactate. Full bacteria growth in wine is only 10 7 cells/mL, and the malate has usually disappeared at 10 6 cells/mL. (Diacetyl may be being formed at that time.)

9. BacteriaPositivesNegatives Oenococcus oeniReduction of total acidity Increase in volatile acidity (high pH and residual sugar) Increase microbial stability Production of biogenic amines and ethyl carbamate Reduction of ketone and aldehyde equivalents (reduces SO 2 use) Spoilage aromas (mousy, sweat, sulfur) Reduction of grassy, vegetative notes Loss of varietal aromas and fruity esters Enhanced mouthfeel Increase of diacetyl and other aroma and flavor compounds Excess diacetyl production Out-competes other bacteria Enhanced color stability (co-pigmentation)Loss of color (high pH) Lactobacillus plantarumReduction in total acidity Sensitive to low pH and high alcohol No acetic acid productionSluggish fermentation Production of spoilage aromas

10. Relevant Metabolic Activity in Oenococcus oeni

12. ML Metabolism

13. Buttery Character Diacetyl

14. Acetaldehyde Conversion J.P. Osborne et al. /FEMS Microbiology Letters 191 (2000)

15. Mousy Character

16. Commercial Malolactic Strains Oenococcus oeni Lactobacillus plantarum

17. Commercial Strains - Inoculation Direct inoculation Bacteria is pre-adapted and can be added directly to the fermentation One step strains Bacteria need to be rehydrated and grown for 24 hours prior to addition Traditional Requires growth and build up of inoculum prior to addition

18. Commercial Strains-Selection Criteria  pH tolerance  Alcohol tolerance  SO 2 tolerance  Temperature range  Competitive ability  Stuck MLF  Biogenic amine production

19. Commercial Strains-Sensory Diacetyl production Color stability Mouthfeel Varietal enhancement Avoidance of defects, i.e. vegetative, sulfur Interaction with oak

20. Wine Matrix Effects

21. Cultivar & Strain Influence

22. Compounds found in MLF wines by GC-MS Purge and trap system of Montrachet wines 4-Methyl-3-pentenoic acid Methyl acetate (Sweet, solvent-like) Ethyl hexanoate (Fruity, rum-like) Hexyl acetate (Fruity) Freon 114 extraction of Epernay 2 wines 1,12-Tridecadiene* Hexadecanoic acid (mild waxy)* 1,2-Benzene dicarboxylic acid (mild ester)* Farnesol (floral)* *Spectral fit < 900 Am. J. Enol. Vitic., Vol. 43, No. 3, 1992 R. M. AVEDOVECH, M. R. McDANIEL, B. T. WATSON, and W. E. SANDINE

23. Other Reported Flavor Enhancers 1-hexanol - fruity ethyl acetate - fruity ethyl lactate - buttery diethyl succinate - brandy butyrolactone – aroma enhancer glycoaldehyde – aroma complexity, browning

24. MLF in Oak Bacteria can breakdown glycosides in solution to release the aglycone

25. Breakdown of Glycosides Glycosidic activities for a selection of Oenococcus oeni strains on four substrates: p-nitrophenyl-β-D- glucoside, p-nitrophenyl-α-L-arabinofuranoside, p-nitrophenyl-α-L-rhamnopyranoside, p-nitrophenyl- β-D-xyloside

26. Tannat Wines- Different MLF-Before Aging Control (gray filled square), MLF with DSM 7008 (black filled square)and D-11 (open square) J. Agric. Food Chem. 2009, 57, 6271–6278. E. Boido, K Medina, L. Farina, F. Carrau, G. Versini, E. Dellacassa

27. Timing of Inoculation InoculationAdvantageDisadvantage Pre- fermentationMLF completionReduced nutrients for AF Production of yeast inhibitors such as acetic acid Early- FermentationSimple-shorter production timeIncreased acetic acid production Tends to avoid MLF failure Incompatibility of yeast and bacteria Allows optimization of management throughout the fermentation Mid- Fermentation Better domination of the MLF by inoculated strainIncompatibility issues More traditional, allowing AF to complete before MLF completion Post- fermentation MLF occurs after AF is complete allowing better control of temperature and SO 2 levelsSome compatibility issues MLF can be done in barrelsNutrients are depleted Less color lossInhibitors may be high i.e. alcohol

28. Summary Reduction in acid Production of desirable compounds (diacetyl) Production of other flavor compounds during growth (1-hexanol, ethyl acetate, ethyl lactate, diethyl succinate, butyrolactone, glycoaldehyde, glyoxal, 2,3- butanediol, caprylic acid, hydroxycinnamic acid) Release of aglycones from glycosides in the wine