By Dr. Molly Kelly, Enology Extension Educator, Department of Food Science
As harvest comes to a close we have planned which wines will be going through malolactic fermentation (MLF). This article provides some information to assist you in dealing with a potentially difficult MLF.
Malolactic fermentation (MLF) is a process of chemical change in wine in which L-malic acid is converted to L-lactic acid and carbon dioxide. This process is normally conducted by lactic acid bacteria (LAB) including Oenococcus oeni, Lactobacillus spp. and Pediococcus spp. O.oeni is the organism typically used to conduct MLF due to its tolerance to low pH, high ethanol and SO2. Most commercial strains are designed to produce favorable flavor profiles.
Although inoculation with a commercial starter is recommended, MLF may occur spontaneously. The lag phase associated with spontaneous MLF may increase the risk of spoilage organisms as well as the production of volatile acidity. Inoculation with a LAB culture can help avoid these problems by providing the cell population needed to successfully conduct MLF (more than 2×106 cells/mL). The compatibility of yeast and LAB should be taken into account since failed MLF may be due to incompatibility between these two organisms.
The key to a successful MLF is to manage the process and to monitor the progress. Although there has been extensive research on the MLF process, it may still be difficult to initiate at times. The possible causes of difficult MLF have been studied less extensively than those of stuck/sluggish alcoholic fermentation. In this article, factors that may influence the start and successful completion of MLF will be discussed.
The main chemical properties that influence MLF are well known: pH, temperature, ethanol and SO2 concentration. A study by Vaillant et al (1995) investigating the effects of 11 physico-chemical parameters, identified ethanol, pH and SO2 as having the greatest inhibitory effect on the growth of LAB in wine.
Generally, LAB prefer increased pH’s and usually, minimal growth occurs at pH 3.0. Under winemaking conditions, pH’s above 3.2 are advised. The pH will determine the dominant species of LAB in the must or wine. At a low pH (3.2 to 3.4) O. oeni is the most abundant LAB species, while at higher pH (3.5 to 4.0), Lactobacillus and Pediococcus will out-number Oenococcus.
MLF is generally inhibited by low temperatures. Research demonstrates that MLF occurs faster at temperatures of 200 C (68˚F) and above versus 150C (59˚F) and below. In the absence of SO2 the optimum temperature range for MLF is 23-250C (73.4˚F-77˚F) with maximum malic acid conversion taking place at 20-250C (68˚F-77˚F). However, with increasing SO2 levels, these temperatures decrease and 200C (68˚F) may be more acceptable.
LAB are ethanol-sensitive with slow or no growth occurring at approximately 13.5%. Commercial O. oeni strains are preferred starter cultures due to tolerance to ethanol. The fatty acid composition of the cell membrane of LAB can be impacted by ethanol content.
LAB may be inhibited by the SO2 produced by yeast during alcoholic fermentation. A total SO2 concentration of more than 50 ppm generally limits LAB growth, especially at lower pH where a larger portion of SO2 is in the antimicrobial form. Generally, it is not recommended to add SO2 after alcoholic fermentation if MLF is desired.
Some of the lesser known factors impacting MLF are discussed below.
MLF can be inhibited by medium chain fatty acids (octanoic and decanoic acids) produced by yeast. It is difficult to finish MLF when octanoic acid content is over 25 mg/L and/or decanoic acid is over 5 mg/L. Bacterial strains that tolerate high concentrations of octanoic and decanoic acids may be important in successful MLF. It is important to check your supplier regarding strain specifications. Yeast hulls may be added before the bacteria are inoculated (0.2g/L) to bind fatty acids. Yeast hulls may also supply unsaturated fatty acids, amino acids and assist with CO2 release.
Some fungicide and pesticide residues may negatively impact malolactic bacteria. Residues of systemic pesticides used in humid years to control botrytis can be most detrimental. Care should be taken in harvest years with high incidence of botrytis. Winegrowers should be familiar with sprays used on incoming fruit and also adhere to pre-harvest intervals.
Lees found at the bottom of a tank can become compacted due to hydrostatic pressure, resulting in yeast, bacteria and nutrients being confined to the point that they cannot function properly. Larger tank sizes may contribute to increased delays in the start of MLF. This inhibition of the start of MLF can be remedied by pumping over either on the day of inoculation or on the second day after inoculation of the bacteria.
Alternatively, contact with yeast lees can have a stimulating effect on MLF. Yeast autolysis releases amino acids and vitamins which may serve as nutrients for LAB. Yeast polysaccharides may also detoxify the medium by adsorbing inhibitory compounds. A general recommendation is to stir lees at least weekly to keep LAB and nutrients in suspension.
Residual levels of lysozyme may impact MLF. Follow the supplier’s recommendations regarding the required time delay between lysozyme additions and the inoculation of the commercial MLF culture. Strains of O. oeni are more sensitive to the effects of lysozyme compared to strains of Lactobacillus or Pediococcus.
Malic acid concentration
Malic acid concentrations vary between grape cultivars and may also differ from year to year in the same grape cultivar. MLF becomes increasingly difficult in wines with levels of malic acid below 0.8g/L. In this case a ML starter culture with high malate permease activity or a short activation protocol is recommended. Check with your supplier to ensure that the chosen strain has these attributes if needed.
Wines with levels above 5 g/L malic acid may start MLF, but may not go to completion. This may be due to inhibition of the bacteria by increasing concentrations of L-lactic acid derived from the MLF itself.
Difficult MLF can result from insufficient nutrients necessary for LAB growth. Since yeast can reduce available nutrients for LAB, time of inoculation is important to avoid competition for nutrients. The addition of nutrients when inoculating for MLF is especially important if the must and wine has low nutrient status or if yeast strains with high nutritional requirements are used. The addition of bacterial nutrients can help ensure a rapid start and successful completion of MLF.
Research demonstrates that the longer it takes to initiate MLF, there is a greater risk for Brettanomyces growth. Some inoculate during alcoholic fermentation (AF) to avoid this problem. Co-inoculation involves adding malolactic starter 24 hours after AF starts. By controlling microbial populations, the growth of spoilage organisms such as Brettanomyces may be inhibited.
Note that inorganic nitrogen (diammonium phosphate) cannot be used by LAB. Check with your supplier for the optimum nutrient product for your particular MLF needs.
Malolactic bacteria are sensitive to excessive amounts of oxygen. The bacteria should not be exposed to large amounts of oxygen after AF is complete. Micro-oxygenation may have a positive impact on the completion of MLF. This impact may be due to the gentle stirring associated with micro-oxygenation that keeps LAB and nutrients in suspension rather than the exposure to oxygen itself.
Some red grape cultivars may have difficulty completing a successful MLF. Some varieties that may experience increased MLF problems include Merlot, Tannat and Zinfandel. This may be related to certain grape tannins negatively impacting the growth and survival of LAB.
Polyphenols can have either stimulatory or inhibitory effects on the growth of wine LAB. This effect depends on the type and concentration of polyphenols as well as on the LAB strain. The tannin fraction of wine tends to complex with other compounds, minimizing their inhibitory effects on MLF. However, in wines that contain a large amount of condensed tannins only, LAB are increasingly inhibited.
MLF nutrients containing polysaccharides have been shown to minimize this effect. This may be due to interactions between the polysaccharides and tannins.
MLF difficulties are usually due to a combination of factors. A stuck or sluggish MLF is usually not the result of one factor alone. It is important, therefore, to both understand and manage the MLF process at each step of the winemaking process. Proper measurement of the process is also vital to be aware when MLF is not proceeding as desired.
Bousbouras, G.E. & Kunkee, R.E., 1971. Effect of pH on malolactic fermentation in wine. Am. J. Enol. Vitic. 22, 121-126.
Britz, T.J. & Tracey, R.P., 1990. The combination effect of pH, SO2, ethanol and temperature on the growth of Leuconostoc oenos. J. Appl. Bacteriol. 68, 23-3 1.
Costello, P.J., Morrison, R.H., Lee, R.H. & Fleet, G.H., 1983. Numbers and species of lactic acid bacteria in wines during vinification. Food Technol. Aust. 35, 14-18.
Davis, C.R., Wibowo, D., Eschenbruch, R., Lee, T.H. & Fleet, G.H., 1985. Practical implications of malolactic fermentation: a review. Am. J. Enol. Vitic. 36, 290-301.
Henick-Kling, T. & Park, Y.H., 1994. Considerations for the use of yeast and bacterial starter cultures: SO2 and timing of inoculation. Am. J. Enol. Vitic. 45, 464-469.
Henick-Kling, T., 1995. Control of malo-lactic fermentation in wine: energetics, flavour modification and methods of starter culture preparation. J. Appl. Bacteriol. Symp. (suppl) 79, 29S-37S.
Henschke, P.A., 1993. An overview of malolactic fermentation research. Wine Ind. J. 2, 69-79.
Ingram, L.O. & Butke, T.M., 1984. Effects of alcohols on micro-organisms. Adv. Microbiol. Physiol. 25, 254-290.
Krieger, 5., 1993. The use of active dry malolactic starter cultures. Austral. New Zealand Wine md. J. 8, 56-62.
Kreiger-Weber, S. and P. Loubser. 2010. Malolactic fermentation in wine. In Winemaking Problems Solved. C.E. Butzke (ed), pp. 88-89.Woodhead Publishing Limited, Cambridge, UK.
Kreiger-Weber, S., A. Silvano and P. Loubser. 2015. Environmental factors affecting malolactic fermentation. In Malolactic Fermentation-Importance of Wine Lactic Acid Bacteria. In Winemaking. R. Morenzoni and K. Specht (eds), pp.131-145. Lallemand Inc., Montreal, Canada.
Kunkee, R.E., 1967. Malo-lactic fermentation. Adv. Appl. Microbiol. 9, 235-279.
Lafon-Lafourcade, S., Carre, E. & Ribereau-Gayon, P., 1983. Occurrence of lactic acid bacteria during the different stages of vinification and conservation of wines. Appl. Environ. Microbiol. 46, 874-880.
Lonvaud-Funel, A. 2001. Interactions between lactic acid bacteria of wine and phenolic compounds. Nutritional aspects II, synergy between yeast and bacteria, Lallemand Technical Meeting, Perugia, Italy.
Loubser, P.A. 2004. Familiarise yourself with malolactic fermentation. Wynboer Technical Yearbook (a Wineland publication). 5:32-33.
Loubser, P., 2005. Bacterial nutrition – essential for successful malolactic fermentation. Wynboer technical yearbook 2005/2006, pp.95-96.
Malherbe, S., F.F. Bauer and M. du Toit. 2007. Understanding problem fermentations-a review. S. Afr. J. Enol. Vitic. 28(2):169-186. Nel, H.A., Moes, C.J. & Dicks, L.M.T., 2001. Sluggish/stuck malolactic fermentation in Chardonnay: possible causes. Wineland Magazine, Wynboer vol. 144, July, pp.1 13-115.
Nielsen, J.C., Pilatte, E. & Prahl, C., 1996. Maitrise de la fermentation malolactique par l’ensemencement direct du yin. Revue Francaise d’Oenologie 160, 12-15.
Nygaard, M. & Prahl, C., 1996. Compatibility between strains of Saccharomyces cerevisiae and Leuconostoc oenos as an important factor for successful malolactic fermentation. Proc. 4 0, Int. Symp. Cool Climate Vitic. Enol., Rochester, NY.
Renouf, V. and M.L. Murat. 2008. L’utilisation de levains malolactiques pour une meilleure maitrise du risqué Brettanomyces. Rev Enol. 126:11-15.
Renouf, V., S. La Guerche, V. Moine and M. Murat. 2009. Techniques for dealing with awkward malolactic fermentations. Wineland Magazine. pp. 82-85.
Vaillant, H., Formisyn, P. & Gerbaux, V., 1995. Malolactic fermentation of wine: study of the influence of some physico-chemical factors by experimental design assays. J. Appl. Bacteriol. 79, 640-650.
Wibowo, D., Eschenbruch, R., Davis, CR., Fleet, G.H. & Lee, T.H., 1985. Occurrence and growth of lactic acid bacteria in wine: a review. Am. J. Enol. Vitic. 36, 301-313.
Zoecklein, B. 2011. Fermentation considerations for the 2011 season. Enology Notes #159. As found on the Wine/Enology Grape Chemistry website