A Technical Guide for Wine Producers

RECENT ARTICLES   |   WYNBOER HOME Lactoenos 450 PreAc: A new type of malolactic starter Emmanuel Gindreau1,2 and Charlotte Augustin1 1. SARCO Laboratory, Laffort Oenologie, BP 17, 33015 Bordeaux, France, www.laffort.com. 2. Faculté d'Oenologie, Université Victor Segalen Bordeaux2, 351, cours de la liberation, 33405 Talence, France. Introduction The process of alcoholic fermentation is relatively well understood and therefore controlled in the commercial environment, thanks in part to an ongoing active research programme by various global facilities. This has instilled a high level of confidence in winemakers regarding the efficient management of fermentations in order to produce the desired characteristics in the final wine (with respect to chemical parameters, varietal aromatic profiles, mouthfeel, tannin structure etc.) in addition to the simple logistics of running a winery at maximum efficiency whilst maintaining appropriate wine quality. In comparison, malolactic fermentation (the conversion of malic acid to lactic acid by bacteria) remains more difficult for winemakers to manage, in part because it is less well understood and in part because bacteria are more difficult micro-organisms to control than yeast. Laffort Oenologie has developed a new bacterial strain coupled with a new production process to provide a simple yet highly successful method of inoculating wine for malolactic fermentation in order to address this imbalance and to hand control back to the winemaker. The advantage of controlling malolactic fermentation As for alcoholic fermentations, the control of malolactic fermentation (MLF) is essential for sensory, sanitary and economic reasons. By controlling the lactic flora responsible for MLF, it is possible to avoid microbial contaminations causing sensory (volatile phenols, piqure lactique, mousiness etc.) and health issues (biogenic amines). Moreover, the correct management of MLF allows a significant reduction in wine heating costs. Finally, early stabilisation of the wine is crucial for Primeur wines, or for the submission of wines for regulatory and export controls. FIG 1 Malolactic activity of whole cells. This measurement shows the degradation of malic acid (in mg) per hour per mg of cells. The measurement was measured in buffer solution at 25°C. FIG 2 Volatile acidity production during MLF. Ind = MLF by indigenous flora. The numbers represent the difference between the volatile acidity values obtained before MLF and after post-MLF sulphiting (average of 3 Merlot wines shown; MLF in barrels). Note that levels are quoted as g/L H2SO4 equivalents; for conversion to acetic acid equivalents multiply by 0.61. FIG 3 Increase in survival 48 hours after inoculation. The survival of 3 strains used as direct inoculation and PreAc preparations was measured. The graph represents the increase measured (in %) for the PreAc preparations in comparison with the direct inoculations (Cabernet Sauvignon, 12.5% Alc. (v/v), titratable acidity 7.41 g/L (as tartaric acid), pH 3.62, total SO2 18 mg/L). The 2005 vintage in Aquitaine The 2005 vintage is a perfect example to illustrate the advantage of MLF management. In Aquitaine (the Bordeaux region and surrounds), in many cellars MLF had not yet occurred in Spring (March 2006). In these wines, the population of Brettanomyces was frequently significant, and the levels of volatile phenols were above the sensory thresholds. Often, these difficult malolactic fermentations followed on from difficult alcoholic fermentations (AF). At the end of AF, the measured Brettanomyces cell numbers were significant. The presence of these yeasts, along with the temperature decrease caused by the onset of winter, affected or sometimes completely inhibited the growth of lactic flora, and thus hindered the start of MLF despite curative inocula with most commercial starters. Parameters to be controlled The 2005 vintage emphasises that in order to properly manage MLF it is necessary to control the entire fermentation process, including alcoholic fermentation. After AF, there is always a lag phase during which the lactic flora grow (Lonvaud-Funel, 1992). Once the lactic flora cell concentration reaches a value of approximately 106 cfu/mL, MLF starts. The duration of this lag phase is highly variable, and essentially depends on the initial population of lactic acid bacteria and their growth conditions. MLF is affected by different factors, among which are the wine's pH, SO2 levels, alcohol concentration and temperature (Bauer et Dicks, 2004; Reguant et al., 2005; Gockowiak et Henschke, 2003). If MLF is to be carried out under optimal conditions, it is crucial for the winemaker to know the value of these parameters precisely. They govern directly the survival and growth of lactic acid bacteria, and thus the duration of the lag phase and the kinetics of the MLF. It is difficult to assess the precise threshold values of these parameters above or below which MLF would be hindered. Indeed, these factors act in a synergistic manner, and others influence the course of MLF. Among these factors, the interactions between different micro organisms play a major role. It was shown that yeasts and lactic acid bacteria interact (Alexandre et al., 2004) through competition for common nutrients (Beelman et al., 1982) and the production of inhibitors (Lonvaud-Funel et al., 1988; Comitini et al., 2005). Medium-chain fatty acids of yeast origin affect the bacterial growth and therefore malolactic activity. The same is true for certain organic acids (fumaric, succinic). In addition, certain yeast strains produce significant quantities of SO2 during AF, which can result in a difficult MLF. The results of the trials carried out during the 2005 vintage suggest the presence of other types of interactions, specifically between lactic acid bacteria and Brettanomyces yeasts (Renouf et al., 2005). Interactions amongst lactic acid bacteria have been demonstrated as well (Lonvaud-Funel and Joyeux, 1993; Navarro et al., 2000), and O. oeni bacteriophages were also suggested as being responsible for failed MLF (Sozzi et al., 1982). In summary, specific easily controlled parameters are known for their ability to affect bacterial growth, and thus to influence the course of MLF. It is essential for the winemaker to identify and measure these parameters. Other factors are also known to influence MLF, mainly microbial interactions. Good management of MLF also requires the implementation of a microbial assessment (Murat et al., 2006). FIG 4 Effect of Energizer utilization on the lactic acid bacteria populations after 24 hours of acclimatisation. The bacterial populations for Energizer are compared with one treatment without nutrient addition (0), and one treatment using a MLF activator. The bacterial populations were measured by epifluorescence. FIG 5 Example of the course of malic acid degradation. A Merlot wine (14.7% alc. (v/v), titratable acidity 6.09 g/L (as tartaric acid), pH 3.47, total SO2 0 mg/L) in new barrels after alcoholic fermentation was inoculated with strain A (direct inoculation) and with strain 450 PreAc. A non-inoculated control (Ind) was also monitored. The wine temperature was maintained at 20°C. FIG 6 Inoculation in a wine in which MLF had not yet started in Spring (March, northern hemisphere) 2006. The wine was inoculated in April 2006. The non-Saccharomyces yeast population had then reached a value of 1.2 x 105 cell/mL (Syrah wine, 12.55% alc. (v/v), titratable acidity 7.60 g/L (as tartaric acid), pH 3.24, total SO2 6 mg/L). Malolactic starter selection Until now only two types of bacterial preparations were available on the market: the preparations for direct inoculation and the "Standard" preparations, which have to be acclimatised in the cellar following a strict protocol. The choice between these two types of starters is mainly determined by the characteristics of the wine to be inoculated. Starters for direct inoculation are very easy to use: they only have to be rehydrated in water for a few minutes. They can be used for all types of wines, as long as none of the parameters is critical for bacterial survival. These preparations are acclimatised during their production, which allows them to endure the stress of direct inoculation into the wine without particular damage. If the wine to be inoculated presents a difficult analytical profile (for example, very low pH or very high alcohol concentration), a Standard starter should be favoured. The application of these preparations requires the build up of two consecutive bacterial starters in increasingly more concentrated wine. These steps permit the bacterial biomass to multiply, and also a progressive acclimatisation to the physiochemical conditions of the wine. While the performance of the resulting starter is undeniable, its preparation remains delicate as each step has to be performed carefully and methodically. The concentration of residual malic acid has to be measured daily. Sometimes, the entire preparation may take a whole week, during which all relevant hygienic precautions have to be adhered to in order to avoid microbial defects, which could compromise the inoculation success. The alternative solution: Lactoenos 450 PreAc Now there is a third inoculation type available. This option combines a specific production process and a strain selected for its high malolactic efficiency. One of the main advantages of this bacterial alternative is based on the implementation of a final, simplified (one step only) acclimatisation phase in the cellar, during which the strain is adapted to the specific conditions of the inoculated wine. This phase lasts 24 hours and is performed in half-diluted wine in the presence of "Energizer", a bacterial nutrient specifically developed for Lactoenos 450 PreAc. During this stage, bacterial acclimatisation is completed. This process combines the simplicity of use of the starter with a bacterial preparation specifically adapted to the wine to be inoculated. The PreAc process demonstrates its efficiency on two levels: improvement of bacterial survival in the specific wine to be inoculated, and faster MLF kinetics as a result of this increased survival rate. The specificities of the new preparation In contrast to other new malolactic preparations, Lactoenos 450 PreAc associates the new PreAc process to a strain specifically selected for this application. The 450 strain is characterised by its remarkable technological capacities and by its behaviour, which is particularly adapted to the production conditions of the PreAc process. The O. oeni strain 450 was selected from a collection of over 600 strains, which were isolated from wines during spontaneous MLF. In order to internally classify this collection, particular attention was given to the malolactic performance of each strain: micro-vinifications of red wines with different analytical profiles (with regards to alcohol level, pH and SO2 levels) demonstrated the broad application and the elevated malolactic efficiency of this strain. The strain 450 displayed strong malolactic activity in laboratory trials. This partly explains its good performance in wine (Figure 1). The absence of biogenic amine production was also verified, which should be considered in the future for wines destined for export to Europe. Moreover, the strain produced little volatile acidity during MLF (Figure 2). The suitability of strain 450 for the PreAc process was compared with the suitability of other bacterial strains. For this comparison, 3 different O. oeni strains were produced according to two processes, as both PreAc and direct inoculation preparations. Two days after inoculation into the wine, the survival rates were calculated for the different preparations. The results are shown in Figure 3. Regardless of the strain, it was observed that the PreAc preparation process improved bacterial survival after 48 hours, compared with the direct inoculation preparation. The increase of the survival rate was 5 and 17% for the strains B and A, respectively. However, strain 450 clearly demonstrated a superior survival rate (75%). Thus, it appears that depending on the strain considered, the response to the PreAc process differs. Amongst the strains tested, strain 450 appears to be best suited to this type of utilisation. Energizer: A specific nutrient A specific nutrient was developed for the acclimatisation step in the cellar. The efficiency of this nutrient was validated during comparative experiments. For this, the 450 PreAc preparation was subjected to the conditions of the final acclimatisation step over a period of 24 hours at 20°C, in half-diluted wine according to several treatments: absence of nutrients, traditional MLF activator, and Energizer. The treatment with Energizer showed the highest bacterial population after the final acclimatisation step. Specifically, the application of Energizer led to 20% more biomass compared with the sample containing activator. The results of these experiments are presented in Figure 4. These trials show that the presence of nutrients during the acclimatisation phase is important for the preparation of the biomass before inoculation. The trials also demonstrate that the quality of the nutrients is important and directly determines the active biomass concentration. The specific formulation of Energizer allowed attainment of the most concentrated biomass. In practice, the 450 PreAc preparation combines the selected and freeze dried O. oeni strain, and a sachet containing specific nutrients (the Energizer). The bacteria are rehydrated in half-diluted wine, which is enriched with Energizer, over a period of 24 hours at a temperature of 20°C. This step allows the bacteria to multiply and to acclimatise to the conditions of the specific wine to be inoculated. The completed preparation so obtained is inoculated into the wine, which is maintained at 20°C. The growth of the specifically adapted biomass allows great improvement in the survival rate of the bacteria after inoculation, and therefore the kinetics of the MLF. Effect on the kinetics of MLF The greater survival of the 450 PreAc preparation in the wine after inoculation favours the presence of a more concentrated active bacterial biomass, which leads to better MLF kinetics. Figure 5 shows the case of a Merlot inoculated with 450 PreAc and another commercial preparation for direct inoculation. A non-inoculated control was also examined. In the wine inoculated with the 450 PreAc preparation, MLF started earlier and was achieved after 26 days, compared with over 51 days for the wine inoculated with the other MLF starter, or with the control wine. The lactic acid bacteria population of these wines was determined 5 days after inoculation. In the PreAc treatment, 5 x 105 cfu/mL of lactic acid bacteria were measured compared with only 1.3 x 104 cfu/mL in the wine inoculated with the other starter, and 1.3 x 103 cfu/mL in the non-inoculated wine. Firstly, these values reflect the difficulties caused by the wine conditions. The cell concentrations after 5 days, which were below the inoculated quantity, show that the bacteria suffered after inoculation into the wine. However, the response of the bacteria to this stress varied according to the preparation: Lactoenos 450 PreAc resisted better, and approximately 40 times more bacteria were present compared with the treatment with the other starter. This higher concentration of viable cells allowed Lactoenos 450 PreAc to start and complete MLF more rapidly. Advantages in stuck wines While Lactoenos 450 PreAc appears to be one of the most efficient tools for the efficient management of MLF, the 2005 Aquitaine vintage also demonstrated the advantage of using it for curative treatment (i.e. stuck MLF). Indeed, numerous wines had not undergone MLF in Spring (March) 2006, and in some cases the microbial status of these wines was risky: low levels of lactic flora, and the presence of Brettanomyces. The analytical assessment of these wines did not allow a determination of the precise causes for the inhibition of MLF, however the low malolactic fermentability of the bacteria was certain since MLF had not started spontaneously. In these difficult cases, the utilisation of 450 PreAc was highly efficient. Even in certain highly Brettanomyces-contaminated wines, MLF could be started using this preparation, even though the indigenous lactic flora were unable to multiply under the same conditions. Figure 6 shows one of these cases: a particularly acidic (pH 3.24) and highly Brettanomyces-contaminated (1.2 x 105 cfu/mL) Syrah wine from the Coteaux d'Aix area was inoculated in March 2006 (Spring in the northern hemisphere) with the 450 PreAc preparation. MLF was completed after 17 days, whereas it had not started in the non-inoculated wine. Conclusion The results presented clearly demonstrate that the Lactoenos 450 PreAc preparation provides an efficient solution for both the preventive and curative management of MLF. Lactoenos 450 PreAc is not only easier to use than the standard starters, it is also more reliable than the preparations for direct inoculation. Its efficiency relies on the new process of PreAc production in combination with the high performance of the 450 strain. The experience gained in the 2005 (northern hemisphere) vintage shows how difficult the management of a malolactic fermentation can be. Indeed, the likelihood of the onset of spontaneous MLF remains uncertain. However, long lag phases before the onset of MLF result in microbiologically unstable wines, in which the potential for the growth of spoilage micro organisms is greatly increased. These contaminations then prevent the growth of lactic acid bacteria and further delay the onset of MLF. The winemaker often has no other choice than to remove these spoilage micro organisms with heavy treatments which can impact negatively on wine quality (such as flash pasteurisation, sterile filtration) and to inoculate the wine afterwards with preparations of lactic acid bacteria. Even in these particularly complex situations, Lactoenos 450 PreAc provides a new solution for the management of malolactic fermentation. Emmanuel Gindreau is the Laffort Oenologie Product Manager for Bacteria. Charlotte Augustin is the Laffort Oenologie Product Manager for Yeast. Queries from the South African market should be directed via Grettchen Visagie in Laffort Oenologie South Africa's office: phone (021) 882-8106, mobile 082 895 5695, grettchen.visagie@laffort.com. Bibliography Alexandre H., Costello P.J., Remize F., Guzzo J. et Guilloux-Benatier M. 2004. Int J. Food Microbiol. 93(2), 141 - 154. Bauer R. et Dicks L.M.T. 2004. Control of malolactic fermentation in wine. A review. S. Afr. J. Enol. Vitic. 25 (2) 74 - 87. Beelman R.B., Keen R.M., Banner M.J. et King S.W. 1982. Interactions between wine yeast and malolactic bacteria under wine conditions. Develop. Indust. Microbiol. 23, 107 - 121. Comitini F., Ferreti R., Clementi F., Mannazzu I. et Ciani M. 2005. Interaction between Saccharomyces cerevisiae and malolactic bacteria: preliminary characterization of a yeast proteinaceous compound(s) active against Énococcus Éni. J. Appl. Microbiol. 99, 105 - 111. Gockowiak H et Henschke P.A. 2003. Interaction of pH, ethanol concentration and wine matrix on induction of malolactic fermentation with commercial ‘direct inoculation’ starter cultures. Aust. J. Grape Wine Res. 9, 200 - 209. Lonvaud-Funel A. 1992. Les acquisitions récentes en microbiologie du vin. Tec&Doc, Lavoisiser, Paris. Lonvaud-Funel A., Joyeux A. et Dessens C. 1988. Inhibition of malolactic fermentation of wines by products of yeast metabolism. J. Sci. Food Agric. 44, 183 - 191. Lonvaud-Funel A. et Joyeux A. 1993. Antagonism between lactic acid bacteria of wines: Inhibition of Leuconostoc ænos by Lactobacillus plantarum and Pediococcus pentosaceus. Food Microbiol. 10, 411 - 419. Murat M.L., Gindreau E., Costella F. et Dumeau F. 2006. Prévention du risque Brettanomyces par l’utlisation d’outils analytiques innovants et la gestion rigoureuse du process d’élaboration. Rev ænologues, Septembre. Navarro L., Zarazaga M., Saenz J., Ruiz-Larrea et Torres C. 2000. Bacteriocin production by lactic acid bacteria isolated from Rioja red wines. J. Appl. Microbiol. 88, 44 - 51. Reguant C., Carreté R., Constanti M. et Bordons A. 2005. Population dynamics of ænococcus æni strains in a new winery and the effect of SO2 and yeast strain. FEMS Microbiol Lett. 246 (1) 111 - 117. Renouf V., Gindreau E., Claisse O. et Lonvaud-Funel A. 2005. Modification de l’écosystème microbien pendant la fermentation malolactique lors de l’élaboration des vins rouges. J. Int. Sci. Vigne Vin. 39(4), 179 - 190. Sozzi T., Gnaegi F., D’Amico N. et Hose H. 1982. Difficulté de fermentation malolactique du vin dues à des bactériophages de . Rev. Suisse Vitic Arboric Hortic. 14, 17 - 23.

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