Malolactic fermentation in red wine.|| MLF/Fermentation || Wine Making

 

Malolactic fermentation (MLF) is a secondary bacterial fermentation that occurs in most red wines. Oenococcus oeni, a member of the lactic acid bacteria (LAB) family, is the primary bacterium responsible for MLF owing to its capacity to withstand the severe circumstances of wine (high alcohol, low pH, and low nutrition) and produce good wine sensory qualities.

One key purpose of MLF is to provide microbiological stability for the subsequent metabolism of L-malic acid. MLF specifically eliminates L-malic acid from wine, which can serve as a carbon source for yeast and bacterial development, potentially resulting in spoilage, spritz, and unpleasant flavours. MLF can also be used in select wines to alter their character.

MLF can be generated by inoculating a specific bacterial strain or happens spontaneously during primary fermentation. Because natural or 'wild' MLF can be unpredictable in terms of onset time and influence on wine quality, malolactic starter cultures are widely employed.

This article provides practical information for induction of MLF in red wine. Achieving effective malolactic fermentation provides further practical instructions for MLF induction, monitoring, and maintenance.

Essential parameters for a successful MLF in red wine.

• Composition of Red Wine or Must.

Alcohol, pH, temperature, and SO2 concentration are key wine compositional parameters influencing MLF performance. Before beginning with MLF inoculation, it is advised to test these parameters and make modifications as needed. Each of these parameters has a range within which MLF is beneficial. As one or more of these criteria grow unfavourable, the MLF will become more challenging (the variables are cumulative). The table below summarises favourable and unfavourable circumstances for MLF in red wine and provides further explanation.

Parameter	Favourable	Unfavourable
Ethanol (%v/v)	<14	>16
Temperature (℃)	18 -22	>16 - <25
pH	3.3-3.5	<3.3
Total SO2(mg/L)	<30	>40

• Alcohol

The possible alcohol concentration of red wine is an important factor to consider while inducing MLF. It is advised that wines with a potential ethanol level more than 15-16% v/v utilize an ethanol-tolerant strain of malolactic starting culture. Furthermore, co-inoculation should be explored since it may help the bacteria culture adapt to increased ethanol concentrations during fermentation. Consider pre-adapting the starting culture to high ethanol conditions.

• Temperature

The optimal growth temperature for LAB in grape juice is approximately 30°C. However, when ethanol content increases, the optimal temperature decreases due to the harmful effects of ethanol on bacteria at higher temperatures. Temperature should be 15-25°C (ideally 18-22°C when other factors are unfavourable and alcohol concentration is about 10% v/v). Inoculation temperature is crucial as it is the development stage that is most vulnerable to suboptimal temperatures. Temperatures over 25°C slow down MLF, increasing the danger of bacterial deterioration and volatile acidity. When using co-inoculation, it's important to prevent high temperatures throughout the alcoholic fermentation process.

• pH

Higher pH levels, especially over 3.5, promote growth of MLF bacteria and other spoilage germs such as Pediococcus and Lactobacillus strains. To prevent the formation of spoilage bacteria in red must and wines with pH levels above 3.5, consider co-inoculation and post-MLF stabilisation.

• SO2 concentration

Lactic acid bacteria, particularly Oenococcus oeni, are extremely sensitive to the molecular form of SO2. To prevent the harmful effects of molecular SO2 on malolactic bacteria, it is recommended that must/wines for MLF induction have no detectable free or molecular SO2. It is worth noting that traditional SO2 measurement methods in red wines, such as aeration-oxidation, can overestimate free and molecular SO2 concentrations. Total SO2 content is a good indicator of a wine's possible influence on malolactic bacteria and MLF, since bound SO2 may hinder them.

Adding a maximum of 50 mg/L total SO2 to grapes before crushing is not expected to negatively impact MLF. However, due to the possibility of SO2 buildup from various extrinsic (e.g., grape harvesting and shipping) and intrinsic (e.g., yeast strains employed in alcoholic fermentation) sources, it is advised that an accurate total SO2 measurement be performed prior to MLF induction.

For ideal MLF conditions, young red wine should have a total SO2 level of less than 30 mg/L. Total SO2 concentrations above 40 mg/L might delay or totally inhibit malolactic fermentation (MLF), depending on the malolactic bacteria strain and wine conditions. Concentrations above 50-60 mg/L may completely inhibit MLF.

• Other inhibitory factors

Pesticide residues, elevated copper levels in the vineyard, and yeast-derived medium-chain fatty acids can all impede MLF.

MLF strain selection criteria for red wine.

• Bacteria strain tolerance

When selecting a malolactic starter culture, it's important to consider the strain's tolerance to the physical and chemical conditions of the wine. This is especially true when selecting a strain for red must/wine, where, for example, in wines with an ethanol level of 15-16% v/v or more, the strains available are limited to those with high alcohol tolerance.

• Malolactic starter cultures for red wine: Oenococcus oeni and Lactobacillus plantarum.

Commercial malolactic starter cultures consist of many Oenococcus oeni strains, each with unique sensitivities to various winemaking conditions and uses. However, starter cultures of Lactobacillus plantarum, another member of the wine lactic acid bacteria family, are becoming accessible for use in red winemaking. Winemakers can use L. plantarum strains' specific physiological features to induce MLF in red wines.

• Yeast-bacteria compatibility

The compatibility of primary fermentation yeast with malolactic bacteria is a significant determinant in the success or failure of MLF. Certain yeast strains may block MLF by producing metabolites such as SO2 and certain fatty acids. Using a yeast strain with high nitrogen demand, especially under low YAN must circumstances, might deplete the nitrogen pool for malolactic bacteria. To lessen yeast inhibitory effects on MLF in red wines, choose an appropriate yeast/bacteria mix (according to supplier/manufacturer recommendations).

• Period of inoculation

Bacteria can be inoculated at various phases of the winemaking process, with sequential and co-inoculation being the most prevalent ways. Timing decisions may be influenced by processing concerns and the type of starting culture employed.

Co-inoculation, which involves inoculating malolactic bacteria at the beginning of alcoholic fermentation (18-24 hours after yeast inoculation), is becoming increasingly popular. Importantly, especially in red winemaking, co-inoculation may potentially facilitate:

• A shorter overall fermentation time, which can lower the risk of spoilage by other microorganisms including Brettanomyces.
• Overcoming MLF problems associated with high ethanol levels and reduced nitrogen content at the end of primary ferment.
• Co-inoculation can improve the usage of particular Lactobacillus plantarum strains for MLF in red winemaking.

Lactic acid bacteria are sensitive to SO2, hence when co-inoculation is performed, bacteria should be added after yeast activity has been detected. After adding SO2, wait at least 18-24 hours after inoculation to allow yeast to bind the free SO2. Co-inoculation requires temperature management, especially in red wines where high fermentation temperatures can harm both bacteria and yeast.

There may be some risks associated with co-inoculation, including:

• Inhibition by high SO2 added during harvest/crushing
• Competition with yeast growth
• Antagonistic yeast/bacteria relationships (MLF strain compatibility is thus important.)
• Stuck primary ferments causing possible production of acetic acid from LAB.

In some red winemaking applications, several timing choices might be employed. Delaying the beginning of MLF, for example, may help to retain colour in lighter-colored red wine varietals from colder winemaking locations. Furthermore, for certain commercial preparations of Lactobacillus plantarum, the producer may prescribe inoculation of a specific starter culture prior to the start of alcoholic fermentation.

Role of Monitoring MLF in quality control parameter in red winemaking.

Monitoring MLF completion is crucial for ensuring quality control in red wine production. Regular assessment of L-malic acid content allows for correct commencement and completion of MLF, as well as efficient post-MLF wine stabilisation (e.g., SO2 addition and pH adjustment).

Controlling MLF with a chosen malolactic strain requires determining its onset. Regular monitoring can easily establish the incidence of delayed or stalled MLF, and if necessary, a rescue starting culture may be immediately deployed. This avoids the dangers associated with uncontrolled proliferation of spoiling bacteria.

To determine MLF completion, strive for a 'not found' malic acid result (<0.05 g/L by enzymatic assay). However, a result of 0.1 g/L or fewer is low enough for the MLF to be regarded nearly complete and to reduce the danger of MLF spoiling after bottling. Precise monitoring of MLF completion reduces the possibility of uncontrolled growth of other wine microorganisms, as well as spoiling. Delays in post-MLF wine stabilisation, in particular, can result in increases in volatile acidity and other spoiling events. Wines are susceptible to oxidation and spoiling from microbes including acetic acid bacteria and Brettanomyces. Accurate identification of MLF completion is therefore critical in preventing such spoilage and quality degradation.

Co-inoculation can lead to MLF completion before alcoholic fermentation. In such instances, it is advised that post-MLF wine stabilisation be carried out once alcoholic fermentation is complete.

References

• Howe, P.A., Worobo, R., Sacks, G.L. 2018. Conventional measurements of sulfur dioxide (SO2) in red wine overestimate SO2 antimicrobial activity. Am. J. Enol. Vitic. 69: 210-220.
• Nordestgaard, S. 2019. AWRI Vineyard & Winery Practices Survey.
• Coelho, JM., Howe, P.A., Sacks, G.L. 2015. A headspace gas detection tube method to measure SO2 in wine without disrupting SO2 equilibria. Am J. Enol. Vitic. 66: 257-265.

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