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Sluggish/Stuck malolactic fermentation in Chardonnay: Possible causes

Hannes A Nel¹⁾, Corine J Moes²⁾ & Leon M T Dicks¹⁾
¹⁾ Department of Microbiology, University of Stellenbosch, Stellenbosch 7600, South Africa
²⁾ Technical Research, Distillers Corporation, Stellenbosch 7600, South Africa
Introduction
Malolactic fermentation (MLF) entails the bacterial conversion of L-malic acid to L(+)-lactic acid and carbon dioxide (Wibowo et al, 1985). This secondary fermentation, which may occur during or after alcoholic fermentation, is usually conducted by Oenococcus oeni (previously Leuconostoc oenos, Dicks et al, 1995), but also by other lactic acid bacteria (LAB). O oeni is the preferred starter culture due to its tolerance to low pH and high alcohol levels (Wibowo et al, 1985). Depending on the strain(s) of LAB involved, several byproducts are produced that may impact on the sensory properties of wine. Chemically, the most significant changes observed during the course of MLF are increases in pH and corresponding decreases in titratable acidity. MLF is important for three reasons: (i) deacidification of the wine, (ii) flavour modification and (iii) microbiological stability (Henick-Kling, 1994; Zoecklein et al, 1995).
Although MLF may occur spontaneously, it is considered necessary to inoculate wines with a specific commercial culture to conduct MLF efficiently. The success of induced MLF is, however, not always guaranteed. From practical experience and studies done at ARC Infruitec-Nietvoorbij (Loubser, 1999a, b) and Distillers (internal report), it is clear that slow or incomplete MLF usually occurs when the primary fermentation is performed with yeast strain N96. Wines that suffer from slow or sluggish MLF require more time to convert L-malic acid to L(+)-lactic acid, whereas wines with "stuck" MLF have an abundant amount of unconverted L-malic acid.
Slow or incomplete MLF is especially prominent in Chardonnay wines. However, the problem seems to vary from year to year. This led researchers to believe that stuck or sluggish MLF is influenced by a combination of factors, such as nutritional deficiencies in the must, less optimal pH, high SO2 or alcohol levels, or unfavourable fermentation temperatures (Van der Westhuizen and Loos, 1981). The exact reason(s) for slow or incomplete MLF are not known and may differ from one fermentation to the other. Other possible reasons could be bacteriophage contamination or even antimicrobial compounds (e g fatty acids) produced by yeast. Little is known about the interaction between yeast and lactic acid bacteria. Several papers have been published on the antimicrobial proteins or peptides (bacteriocins) produced by lactic acid bacteria and their effect on malolactic bacteria (Green et al, 1997; Van Reenen et al, 1998).
We have conducted a fermentation experiment with two commercially available yeast cultures to determine if they produce any antibacterial compounds that could lead to stuck or sluggish MLF. We have also tested the interaction among malolactic strains isolated from a commercial starter culture and recorded their influence on MLF.
Experimental Procedure
Sterile Chardonnay grape must (derived from grapes of the Stellenbosch region during the 2000 season) was divided in 25-liter glass containers and fermented at 16øC with yeast strains N96 (Saccharomyces bayanus strain) and VIN 13 (Saccharomyces cerevisiae strain), respectively (Anchor Yeast, SA). The inoculum size was 0.02% (w/v). The presence of lauric acid (C12), myristic acid (C14), pentadecanoic acid (C15), palmitic acid (C16) and stearic acid (C18) were recorded at the end of the alcoholic fermentation. The gas chromatography method described by Marais and Houtman (1979) and M Blom (personal communication, 1997) was used. Bacterial strains isolated from a commercial malolactic starter culture, ViacellTM (Lallemand), were grown to 1.8 ¾ 1011 cfu/ml in acidic grape broth (Dicks et al, 1990) and used to inoculate grape must at 5% (v/v). Autoclaved cells of the malolactic bacteria were used as control. The secondary fermentation was performed in duplicate at 20øC. Viable cell numbers and the conversion of L-malic acid to L(+)-lactic acid were monitored weekly. Standard microbiological methods were used (Sharpe, 1979). The organic acids were determined according to the method described by Schneider and co-workers (1987). In a separate experiment, the malolactic strains isolated from the commercial starter culture were tested for possible antimicrobial activity against each other and against yeast strains VIN13 and N96. The two yeast strains were also tested for antimicrobial activity against the commercial malolactic strains. Antimicrobial activity tests were performed as described by Van Reenen and co-workers (1998).
Results and Discussion
Chardonnay must fermented with the yeast starter cultures yielded very low levels of fatty acids and no significant differences were recorded between fermentations conducted by VIN13 and N96. MLF was completed after 15 weeks. Symptoms of sluggish MLF were observed for N96 and VIN 13 between days 24 to 77 ( Fig 1 ). However, no antimicrobial compounds active against the commercial malolactic starter culture were detected in the wine sampled during this period. This implied that neither the malolactic bacteria, nor the yeast produced any antimicrobial substances that inhibited the growth of the malolactic bacteria. We could also not detect any antibacterial activity in sterile Chardonnay must. Further tests are needed to determine if other LAB, which are usually present in grape must, may produce antibacterial compounds against malolactic starter cultures. The possibility of antibacterial compounds produced by wild yeast is also not ruled out. Numerous other factors can attribute to sluggish MLF. Some possible causes for sluggish or stuck MLF and possible ways to combat this phenomenon are discussed below.
Causes and Recommendations
Bacteriophages
Bacteriophages (bacterial viruses) are probably one of the most neglected reasons for sluggish or stuck MLF. This problem can be overcome if it is effectively assessed and the presence of bacteriophages prevented or restricted to a minimum (Henick-Kling, 1994).
As much as five litres of wine can penetrate the first few millimetres of a standard 300-liter barrel (Berthelot, 2000). The penetration of bacteria and their phages into the wood and their survival in empty barrels is therefore likely to happen. Contamination of fresh wine by these bacteria when aged in re-used barrels is thus very possible. The following precautions may be taken to prevent phage contamination and ensure an active malolactic starter culture:

Prevent the possible build-up of bacteriophages in fermentation tanks from year to year by applying adequate cleaning methods.
Use aseptic inoculation techniques and grow the starter culture in the presence of less than 20 mg/l free SO2 to prevent possible inhibition (Henick-Kling, 1994).
Use a stationary phase culture as inoculum. Cells of O oeni harvested 18 - 24 h after the culture had entered stationary phase, proved to be the most viable and yielded the highest malolactic activity (Krieger and Hammes, 1988). When harvested earlier in the growth phase only 18% of the cells survived after 5 days of MLF. These cells could also not induce MLF.
Stimulate the growth of the starter culture by using an optimal growth medium instead of grape must. The cells should be grown to numbers exceeding 108 cfu/ml before inoculated into must.
Rotate the starter culture. Use different strains of O oeni, or even strains of Lactobacillus spp, which have been preselected and evaluated as suitable malolactic bacteria.
Optimise the growth conditions in the must. Ideal growth conditions for O oeni are 15øC - 20øC, pH 3.4 - 4.2 and in the absence of SO2. Bacteriophages are most active at low temperatures and at the early growth phase of the host (Henick-Kling, 1994).
Use malolactic bacteria harbouring temperate bacteriophages (i e lysogenic cells) as possible starter cultures. These bacteria are more resistant to phage infection. An estimated 50% of O oeni strains isolated from wine contain temperate phages (Patel, 1990).

Managing malolactic fermentation
It is more difficult to accomplish MLF in white and ros‚ wines than in red wines (Pilatte and Nielsen, 2000). The reasons for this are:

White wines normally have a higher acidity and wider pH range (pH 3.0 - 3.5 after alcoholic fermentation).
The low nutrient content in white wines, compared to red wines, is often inadequate to support high cell numbers of malolactic bacteria.
White wines are produced at a lower temperature (18 - 22^oC) compared to red wines (20 - 24^oC).
White wines have less phenolic compounds to which SO2 can bind. Malolactic starter cultures in these wines are thus under more SO2 stress than starter cultures in red wines.

Sulphur dioxide
SO2 is often produced by yeast during alcoholic fermentation. This may inhibit the growth of malolactic bacteria (Eksteen, 2000b; Henick-Kling and Park, 1994; Lonvaud-Funel et al, 1988). The levels of free SO2 produced are dependent on the yeast strain, the availability of nutrients (especially nitrogen) and the presence of compounds in the must to which SO2 binds (Nygaard and Prahl, 1996).
It is of utmost importance to keep the SO2 levels at 50 - 80 ppm for red wines and 20 - 40 ppm for white wines during crushing and separation of the grape must to control the numbers of spoilage bacteria (Eksteen, 2000b). Lactizyme, a product of lysosyme, may be used in combination with SO2, but should only be considered in high-pH wines where the growth of pediococci are favoured. Pediococcus spp can cause volatile acidity or even produce bacteriocins that may inhibit the growth of O oeni (Eksteen, 2000a; Green et al, 1997). It is not recommended to add SO2 to must after alcoholic fermentation (Henick-Kling, 1994).
Parameters influencing the nutrient composition of the must
The use of complex nutrients, such as amino acids and nitrogen sources by yeast during early alcoholic fermentation may retard or even prevent bacterial growth, especially in white wines (Nygaard and Prahl, 1996). This is not surprising, since malolactic bacteria are considered fastidious organisms with limited means of synthesising growth requiring compounds (Fugelsang, 1996; Fourcassier et al, 1992). They survive on low concentrations of hexoses and certain pentoses, organic acids (e g malic- and citric acids) and nitrogen in organic form (amino acids, peptides). Other inorganic elements (Mg++, Mn+ and K+) and vitamins are also essential cofactors in enzymatic reactions. Towards the end of alcoholic fermentation, yeast cell lysis results in the release of nutrients that will favour the growth of malolactic bacteria (Nygaard and Prahl, 1996). A slow alcoholic fermentation will inevitably lead to sluggish or stuck MLF. It is therefore essential that all the nutrients needed by the malolactic bacteria are present in the grapes before crushing. Nutrient supplements for malolactic bacteria are commercially available and are usually used with direct inoculated starter cultures.
The nutrient content of the must and its turbidity is also affected during the clarification methods of white and ros‚ wines (static or dynamic, use of fining agents, temperature conditions, and duration of clarification). When MLF is desired in white wine, the intensity of the clarification should be adjusted in order to prevent any nutrient deficiency, which is likely to interfere with fermentability. Red wines usually have higher nutrient concentrations because of the prolonged maceration on the skins.
Environmental and chemical factors
High levels of herbicides and pesticides left on the grapes, acidic acid accumulation, temperature fluxes and competition between bacteria, are also possible reasons for sluggish or stuck MLF. Must should be free of pesticides, since malolactic bacteria are more sensitive to these residues than yeast. Wild yeast strains and bacteria (e g Lactobacillus brevis) present on grapes or in the cellar often form high levels of acid, which inhibits MLF. Low temperatures and a pH value below 3.4 favours the growth of unwanted yeast strains (Gafner et al, 2000).
The characteristic of the harvest (rainfall, maturity, the condition of the grapes) and wine growing practices (soil type, rootstock type, weeds and nitrogen fertilisation) have a major influence on the levels of acids and nutrients and thus also on the fermentability of the must. The presence of weeds may even cause the levels of malic acid and amino acids in the must to decrease significantly, particularly under dry conditions (Maigre et al, 1995).
Conclusions
No antimicrobial compounds could be detected in sterile Chardonnay must or in must fermented with yeast strains VIN13 and N96, respectively. We could also not detect any antibacterial compounds produced by the strains used as the malolactic starter culture. Other yeast and LAB are currently being screened for the production of antibacterial compounds. When all possible factors are taken into consideration, it is clear that the prevention of sluggish or stuck MLF starts in the vineyard long before the grapes find their way to the cellar. The old clich‚ is still very relevant, viz "no good wine can be made from bad grapes, but bad wine can be made from good grapes".
Acknowledgements
The authors wish to thank Marais Blom and Ludick Arnolds from Distillers for valuable assistance.
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