Impact of Malolactic Fermentation Strain on

Wine Composition

Lucy JosephU.C. Davis Department of Viticulture

and Enology

Outline

• Introduction to malolactic fermentation (MLF)• Lactic acid bacteria metabolism• Commercial inoculum• Wine matrix effects

– Interaction with oak• Timing of inoculation

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.

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

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

CO2

Lactic Acid + Carbon Dioxide

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

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

THE COURSE OF THE MLFBacterial growth is finished several days after the conversion of malate to lactate. Full bacteria growth in wine is only 107 cells/mL, and the malate has usually disappeared at 106 cells/mL. (Diacetyl may be being formed at that time.)

Bacteria Positives NegativesOenococcus

oeni Reduction of total acidityIncrease in volatile acidity

(high pH and residual sugar)

Increase microbial stabilityProduction of biogenic amines

and ethyl carbamate

Reduction of ketone and aldehyde

equivalents (reduces SO2 use)Spoilage aromas (mousy,

sweat, sulfur)

Reduction of grassy, vegetative notesLoss of varietal aromas and

fruity esters

Enhanced mouthfeel

Increase of diacetyl and other aroma

and flavor compoundsExcess diacetyl production

Out-competes other bacteria

Enhanced color stability

(co-pigmentation) Loss of color (high pH)Lactobacillus

plantarum Reduction in total aciditySensitive to low pH and high

alcohol

No acetic acid production Sluggish fermentation

Production of spoilage aromas

Relevant Metabolic Activity in Oenococcus oeni

ML Metabolism

Buttery Characte

r Diacetyl

Acetaldehyde Conversion

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

Mousy Character

Commercial Malolactic Strains

Oenococcus oeniLactobacillus plantarum

Commercial Strains - Inoculation

• Direct inoculationBacteria 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

• TraditionalRequires growth and build up of inoculum

prior to addition

Commercial Strains-Selection Criteria

pH tolerance Alcohol tolerance SO2 tolerance Temperature range Competitive ability

Stuck MLF

Biogenic amine production

Commercial Strains-Sensory

• Diacetyl production• Color stability• Mouthfeel• Varietal enhancement• Avoidance of defects, i.e. vegetative, sulfur• Interaction with oak

Wine Matrix Effects

Cultivar & Strain Influence

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 < 900Am. J. Enol. Vitic., Vol. 43, No. 3, 1992R. M. AVEDOVECH, M. R. McDANIEL, B. T. WATSON, and W. E. SANDINE

Other Reported Flavor Enhancers

• 1-hexanol - fruity• ethyl acetate - fruity• ethyl lactate - buttery • diethyl succinate - brandy• butyrolactone – aroma enhancer• glycoaldehyde – aroma complexity,

browning

MLF in Oak

Bacteria can breakdown glycosides in solution to release the aglycone

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

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

Timing of InoculationInoculation Advantage Disadvantage

Pre-fermentation MLF completion Reduced nutrients for AF

Production of yeast inhibitors such

as acetic acidEarly-

Fermentation Simple-shorter production time Increased acetic acid production

Tends to avoid MLF failureIncompatibility of yeast and

bacteria

Allows optimization of management

throughout the fermentation Mid-

FermentationBetter domination of the MLF by inoculated

strain Incompatibility issues

More traditional, allowing AF to complete

before MLF completion Post-

fermentationMLF occurs after AF is complete allowing

better control of temperature and SO2 levels Some compatibility issues MLF can be done in barrels Nutrients are depleted Less color loss Inhibitors may be high i.e. alcohol

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