Novel lactic acid bacteria for use as MLF starter cultures

Nuevas bacterias de ácido láctico para su uso como iniciadores
de la fermentación maloláctica

Maret du Toit
Institute for Wine Biotechnology, Department of Viticulture and Oenology
Stellenbosch University, Matieland, South Africa

Which cultures are suitable as MLF starter?

Lactic acid bacteria (LAB) is responsible for the reaction were L-malic acid is converted to L-lactic acid which is known as malolactic fermentation (MLF). Lactic acid bacteria that can survive the harsh wine conditions and also possess the malolactic enzyme (mle gene) have the ability to be used as MLF starter cultures. The result of MLF is the deacidification of wine with an increase in pH of 0.1-0.3 units, microbial stabilization through the depletion of important nutrients and the improvement of sensorial quality by producing aroma active compounds. The four genera associated with spontaneous MLF are Oenococcus, Leuconostoc, Lactobacillus and Pediococcus (Du Toit et al. 2011; Lerm et al. 2010).

There is only two Leuconostoc species, namely mesenteroides and paramesenteroides (now of the genus Weissella) that is involved in winemaking and they are more known for spoilage through the production of exopolysaccharides causing ropiness and bitterness by degrading glycerol (Du Toit & Pretorius 2001), but it was shown by Mtshali et al. (2011) that all strains contained the mle gene and that they also have genes that might enhance the aroma profile of wine. It was seen that they do not contain the citric acid pathway genes which will lead to low levels of diacetyl (buttery character) which for certain wine styles could be desirable (Mtshali et al. 2011). Therefore certain strains that do not have the ability to produce ropiness can be selected for specific sensory attributes that might contribute to wine quality. This genus is currently not being used commercially as MLF cultures.

The genus Pediococcus has four species that play a role in winemaking viz. damnosus, inopinatus, parvulus and pentosaceus (Dicks & Endo 2009). Pediococci is seen as a serious threat to wine quality, especially after MLF and in high pH (>3.6) wines, contributing to biogenic amines and ropiness (Du Toit & Pretorius 2001). It has been shown that certain pediococci possess enzymes of interest to wine aroma and quality (Matthews et al. 2004), therefore certain strains might have the potential to be used as MLF starter cultures.

Lactobacillus is the genus that has the most species associated with winemaking, namely 18 species. The species can be divided in to two groups those that are mainly found in must and are not tolerant to alcohol and the second group that is alcohol tolerant and can participate in after alcoholic fermentation MLF (Du Toit et al. 2011). The species that are most prominent in wine and have the ability to survive alcoholic fermentation are brevis, buchneri, collinoides, fermentum, fructivorans, hilgardii, kunkeei, mali, nagelii, plantarum and vini. The two species that have received the most attention to evaluate their potential as MLF starter cultures are Lb. plantarum and Lb. hilgardii (Du Toit et al. 2011; Torriani et al. 2011). In 1988 the potential of Lb. plantarum was realised by Prahl (1988) with the first freeze-dried culture being released. Today there are a few Lb. plantarum strains commercially available as MLF starter cultures (Fumi et al. 2010; Lerm et al. 2011). The characteristics that make Lb. plantarum the possible next generation MLF starter culture, is its ability to function well in high pH conditions, it can tolerate ethanol of up to 14%, it performs well at 18-20°C, it has similar SO2 tolerance than O. oeni, it does not produce acetic acid from carbohydrates, it has a more diverse array of enzymes that could lead to more aroma compounds being produced and they can also produce plantaricins that can reduce the participation of other LAB during MLF (Fumi et al. 2010; Du Toit et al. 2011; Lerm et al. 2011; Mtshali et al. 2010; Spano et al. 2005).

Oenococcus oeni is the best studied malolactic bacteria that can survive the harsh winemaking conditions, such as low pH, high ethanol, SO2 used, survive at low temperatures, resistant to fatty acids, tannins and lysozyme. O. oeni contribute small amounts of acetic acid to wine and produce positive aroma compounds (Lerm et al. 2010). Genomics and transcriptomics studies have shown that the species is highly diverse and therefore strains can be selected with very specific traits related to specific needs of the winemaker (Borneman et al. 2010; Torriani et al. 2011).

Selecting novel strains….

Any of the above mentioned species have the potential to be used for MLF if they have been selected to contribute positively to wine quality and to ensure that they do not produce negative compounds. Therefore the selection criteria used in many studies can be divided into three main groups, namely (i) technological challenges (resistance to the main wine parameters, yeast compatibility and withstanding the production processes), (ii) malolactic performance and flavour production (malic acid degradation; production of positive aromas, such as esters and carbonyls; no negative contribution to wine aroma, such as volatile sulphur compounds or phenols, acetic acid) and (iii) ensure enhancement of the wholesomeness of wine (no production of biogenic amines and ethyl carbamate).

There are currently two main inoculation strategies that are used by winemakers each with their own challenges when selecting strains. The traditional MLF inoculation scenario is to inoculate after alcoholic fermentation, where the biggest pressure on the strains is a high alcohol contents in warmer climate regions, low nutrient status and a high pH with a higher natural LAB population participating in MLF (Capozzi et al. 2010; Du Toit et al. 2011; Lerm et al. 2010; Solieri et al. 2010; Torriani et al. 2011). In this scenario, O. oeni strains still fair the best, but Lb. plantarum and hilgardii have shown that they can also perform MLF just as well (Fig. 1) (Du Toit et al. 2011; Lerm et al. 2011).

Figura 1

Figure 1: Kinetics of malic acid degradation in a 2010 Merlot (South Africa). Inoculation post alcoholic fermentation [pH 3.5 / 22.9º Balling / 13.6 % (v/v)] (Du Toit et al. 2011).

The second inoculation strategy that is becoming more popular, is co-inoculation, especially in warmer climate regions where alcohol can be by-passed as the major factor that is responsible for problematic MLF. Therefore Lb. plantarum is earmarked for co-inoculation in high pH and sugar wines, where alcohol will not be such a major factor. Lb. plantarum V22 from Lallemand was selected for alcohol tolerance and have shown that they can be just as effective as O. oeni when used to induce MLF after alcoholic fermentation (Figs. 2 & 3).

Figura 2

Figure 2: Kinetics of malic acid degradation in a 2009 Sangiovese (Tuscany, Italy). Co-inoculation 24 hrs after yeast [pH 3.6 / 25.8 Brix / 14.3 % (v/v)] (Du Toit et al. 2011).


Figura 3

Figure 3: Malic acid concentration and viable cell counts during a co-inoculated malolactic fermentation in Shiraz with a commercial Oenococcus oeni starter culture and two Lactobacillus hilgardii strains (Du Toit et al. 2011).

Co-inoculation, where alcohol is not the biggest factor that will impact on the functioning of a strain, will definitely open the door for other species being used for MLF other than the three that are currently available today. Therefore many of the studies that have looked at the natural isolates of a country or a region or a cultivar might deliver novel strains with interesting characteristics to be used for MLF in future. Spain has many studies that have looked at the natural isolates of specific regions or cultivars and therefore have a large collection of potential MLF strains as seen in the studies for example done by Lopez et al. (2008) and Ruiz et al. (2010). Apart from Lb. plantarum in South Africa, the other species that have been evaluated is Lb. hilgardii in both alcoholic and co-inoculation scenarios. Results showed that the selected strains could complete MLF in a similar time as O. oeni (unpublished data).


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