The malolactic fermentation of wine

In recent posts I have discussed malic acid and lactic acid bacteria (LAB), two of the major actors in the malolactic fermentation (MLF) drama. In this post I will elaborate on the arena within which this drama unfolds and how the interaction of malic acid and other minor players with LAB results in lactic acid and other metabolites. Let us begin by defining MLF.

According to Sauvageot and Vivier (Effects of Malolactic Fermentation on Sensory Properties of Four Burgundy Wines, AJEV 48(2), 1997), MLF is a bacterial conversion -- most commonly performed by Leuconostoc strains due to their tolerance of the high acid and alcohol content associated with wine -- of L-malic acid to L-lactic acid and CO₂. The MLF process can be represented thusly (Lerm et al., Malolactic Fermentation: The ABCs of MLF, S. Afr. J. Enol. Vitic. 31(2), 2010):

L-malic acid  + LAB  →    L-lactic acid          + CO₂
(dicarboxylic)                  (monocarboxylic)

wherein a carboxyl group (C(O)OH) is removed from the dicarboxylic L-malic acid. The reaction is catalyzed by the LAB along one of three pathways (Lerm et al., Bauer and Dicks, Control of Malolactic Fermentation in Wine: A Review, S. Afr. J. Enol. Vitic. 25(2), 2004):

1. Direct conversion of malic acid to lactic acid via malate decarboxylase (the preferred pathway for wine LAB)
2. L.casei and Enterococcus faecales possess a malic enzyme that converts L-malic to pyruvic acid which is in turn reduced to lactic acid by L-lactate dehydrogenase
3. Via L. fermentum,  malate is reduced by malate dehydrogenase to oxaloacetate, followed by decarboxylation to pyruvate which is further reduced to lactic acid.

The main effects of MLF on wine are (i) a reduction in titratable acidity (by 0.1 to 0.3%) and an increase in pH (0.15 to 0.30). In addition, dramatic organoleptic changes to the wine are evidenced (Lonvaud-Funel, Microbiology of the Malolactic Fermentation: Molecular Aspects, FEMS Microbiology Letters):

• The specific taste of malic acid disappears
• Sugars are catabolized to produce mainly lactic and acetic acid
• Citric acid is transformed into acetic acid and carbonyl compounds, notably the butter-flavored diacetyl
• Wine taste and color are modified due to the metabolic activity of bacteria on phenolic compounds (tannins, anthocyannins).

By synthesizing anti-bacterial compounds and depriving the wine of nutrients, MLF also contributes to its microbial stability (Lonvaud-Funel).

In practice, most red wines, and selected white wines, undergo MLF. The process is encouraged (Bauer and Dicks, Control of Malolactic Fermentation in Wine, S. Afr. J. Enol. Vitic. 25(2), 2004): in cooler areas where grapes have high malic acid content; in cases where the wine is aged in oak barrels; and when the wine style calls for long-term aging in bottle. The practice is sometimes forsworn in warmer, lower-acid areas and in the cases where undesirable organoleptic changes or the production of biogenic amines result.

MLF is initiated either naturally or through inoculation of the wine with an LAB strain. In the case of indigenous initiation, upon the completion of alcoholic fermentation, and following a lag phase, the surviving LAB begin to multiply rapidly. MLF begins when their numbers approach 10⁶ cells/ml (Savageot and Vivier; Wibowo et al.; Lonvaud-Funel; Lerm et al.). Lafon-Lafourcade et al., posit that this growth originates from winery equipment which serve as incubators for the LAB. Oenococcus. oeni is the main species that develops here but Lactobacillus and Pediococcus spp. may proliferate and conduct the MLF if the wine pH approaches 4.0. If MLF is not desired, clarification of must or newly fermented wine will remove the majority of the LAB and, if excessive, its potential nutrient sources, and reduce the possibility of indigenous inoculation (Wibowo et al.). In addition, wines that have undergone thermovinification (rapid heating of the must to near boiling point in order to extract anthocyanins and tannins) are less susceptible to MLF (Wibowo et al.).

During the time between the end of AF and the initiation of MLF, no SO₂ can be applied to the wine because of its toxic effect on the LAB. During this time, then, the wine is exposed to the potential of oxidation and attack by spoilage organisms. The use of starter cultures reduces this risk by shortening the time between the end of AF and the initiation of MLF and by ensuring a rapid onset of MLF with a very high population of viable bacteria (on the order of 10¹¹ cells/g, according to Lerm et al.). Given the environment within which the LAB has to operate, a starter culture should have the following characteristics (Lerm et al.):

• Tolerance to low pH, high ethanol, and SO₂
• Good growth characteristics under winemaking conditions
• Compatibility with the S. cerevisiae strain(s) being used for alcoholic fermentation
• Ability to survive the production environment
• Does not produce biogenic amines
• Does not produce off-flavors or off-odors
• Production of aroma compounds that will favorably impact the wine’s aroma profile.

There are a number of factors that affect the development of LAB and, as a result, the activation and effectiveness of MLF. For example, wine pH affects (Wibowo et al.):

• LAB growth rate
• The LAB species that proliferate
• The metabolic behavior of the species that grow
• The survival of LAB.

Temperature is synergistic with ethanol levels as it relates to inhibiting LAB (Lerm et al.):

• The optimal growth temperature of LAB decreases at high ethanol concentrations
• Elevated temperatures lower the ability of LAB to withstand increased ethanol concentration
• Temperatures of 25℃ and above, combined with ethanol levels of 10 -14%, almost completely inhibits LAB growth.

A listing of the factors beyond pH and temperature that affect LAB growth and development are provided in the tables below.

TABLE 1. The influence of different winemaking practices on LAB growth

Practice	Influence
Degree of must clarification	Significant impact on bacterial growth. Yeast produce more medium chain fatty acids in highly clarified must
Skin contact prior to AF	Direct effect on extraction of nitrogenous and other macromolecules stimulate LAB growth and malolactic activity
Choice of yeast strain	Inhibitory and stimulatory effects differ between strains
Aging of wine on yeast lees	Yeast autolysis release nutrients that stimulate LAB growth and malolactic activity

Source: Lerm et al., TABLE 2

TABLE 2. Yeast activity inhibiting LAB via the production of yeast metabolites

Yeast Metabolite	Effect on LAB and/or MLF
Ethanol	Affects growth ability
SO2	AF with SO2 producing yeast strain results in wine inhibitory to MLF
Medium chain fatty acids	Affect LAB growth and reduce ability to metabolise malic acid. Combination of fatty acids (hexanoic, octanoic and decanoic acid) cause greater inhibition than individual compounds.
Metabolites of protein nature	Peptide produced by S. cerevisiae during AF: inhibit O. oeni by disruption of cell membrane; inhibition dependent on SO2

Source: Lerm et al., TABLE 3

According to Boulton et al. (Principles and Practices of Winemaking, Chapman and Hall, 1996), there is a difference in perception as to what constitutes the MLF period, depending on whether you are a microbiologist or a winemaker. The microbiologist measures the MLF from the “introduction of viable bacteria into the wine or must” and it ends “when the bacteria have gone through the growth phase and have re-entered their final resting or stationary phase.” The winemaker, on the other hand, will equate the start of fermentation with a noticeable drop in malic acid levels (grape juice contains between 1 and 8 g/l malic acid) and as completed when the malic acid has finally disappeared (vintessential.com.au (Malolactic Fermentation Monitoring -- Resources for Winemakers) recommends a figure of < 0.05 g/l as safe for declaring the end of MLF while Katherine Mansfield (Monitoring Malolactic Fermentation 3 Ways, Cornell Cooperative Extension) cites the number 30 g/ml. That discrepancy in the metric notwithstanding, Boulton et al., see the “measurement of the disappearance of malic acid as the accepted means for determining whether the malolactic fermentation has occurred.”

Measurement of the degradation of malic acid is one of the key aspects of monitoring MLF, the topic of my next post

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