Using malolactic fermentation to enhance the fruity characters of wine

Utilización de la fermentación maloláctica para potenciar el carácter afrutado del vino

Eveline Bartowsky and Peter Costello
The Australian Wine Research Institute
Glen Osmond, Australia

One of the major goals of red winemaking is to optimize and exemplify varietal fruit and regional character of the grapes through fermentation. In addition to the grape, both yeast and bacteria play a significant role in styling the aroma and flavour of wine. Yeast are well known for their contribution to wine aroma and flavour through alcoholic fermentation (Swiegers et al. 2005), however, bacteria are more often only perceived as agents of the conversion of malic acid to lactic acid through malolactic fermentation (MLF), or, spoilage of wine. While it is widely accepted that the acid conversion during MLF results in a softening of the wine through an increase in pH and decrease in acidity, it is increasingly being recognised that O. oeni can further shape wine aroma through metabolism of a large array of compounds during their growth (Swiegers et al. 2005, Bartowsky and Pretorius 2008). In particular, recent research is indicating that MLF can modulate certain aroma volatiles associated with fruity sensory properties in wine. Oenococcus oeni is the main lactic acid bacteria species associated with MLF, as it can survive and predominate in the often harsh wine environment (low nutrients, high alcohol content and low pH). Although the natural indigenous O. oeni population can potentially grow and conduct MLF, the unpredictable outcomes associated with spontaneous MLF have led to the development of bacteria starter culture preparations with specific O. oeni strains. Such starter cultures are most commonly inoculated after alcoholic fermentation (sequential inoculation), or more recently, inoculation with yeast to have concurrent alcoholic and malolactic fermentations (co-inoculation).

It is well known that malolactic (ML) bacteria are able to influence the buttery aroma of wines due to the production of diacetyl, and that winemaking techniques can be used to manipulate its concentration in wine following MLF (Bartowsky and Henschke 2004). Several recent studies have highlighted the ability of O. oeni to influence various groups of fermentation-derived volatile compounds (fatty acid and acetates esters, acids, higher alcohols), organic acids and amino acids. Many of the volatile compounds contribute to fruity aromas in red and white wines (Francis and Newton 2005); for example, ethyl esters can bestow various fruity characters (berry, pineapple, banana) to wine, however, the longer chained esters (C6-C10) tend to have soapy characters. Several of our studies in Chardonnay and Cabernet Sauvignon wines have shown that MLF can modify the wine ester profile, with the concentrations of ethyl esters and acetate esters tending to increase and decrease, respectively, following MLF (Bartowsky et al. 2008).

Medium- to full-bodied red wines are often described to have intense blackcurrant, dark cherry, raspberry and plum aromas (Iland et al. 2009). An important challenge faced by winemakers is to accentuate such fruity characters, and minimize their loss during the winemaking process. Recent research has suggested that red wine berry fruit aroma is a complex interaction between fruity esters, norisoprenoids, dimethyl sulfide, ethanol and other components. Certain groups of esters have been identified that specifically contributed to red berry and black berry aroma (Escudero et al. 2007, Pineau et al. 2009). Using these analyses it is possible to gain a better understanding how O. oeni metabolism during MLF is able to modify and accentuate fruity characters, especially in red wines. Several case studies are presented which highlight factors that influence the nature and extent of such secondary metabolic activity associated with MLF, including bacterial strain, timing of inoculation of bacterial starter cultures, wine matrix composition and the source of grapes.

Impact of MLF on wine fruity esters and sensory properties

Effects of inoculation regime of MLF with alcoholic fermentation

We have examined the chemical composition of red and white grape juices/wines inoculated either simultaneously or sequentially with alcoholic fermentation using different commercial O. oeni strains. Small scale trials in Chardonnay and Cabernet Sauvignon demonstrated that the concentration of total esters increased following MLF, with a greater increase observed when bacteria were co-inoculated with yeast at the start of alcoholic fermentation. The concentration of total higher alcohols decreased with the yeast/bacteria co-inoculation regime, with minimal changes noted when the bacteria were inoculated sequentially following alcoholic fermentation. Descriptive sensory analysis of the Cabernet Sauvignon wines has enabled us to link compositional analyses with sensorial aroma and flavour changes induced by the bacterial mediated MLF (Bartowsky et al. 2008).

Figura 1. Total black fruit ester concentration in Cabernet Sauvignon wines (Bordertown, South Australia, 2006) following co-inoculation or sequential inoculation for MLF, and sensory ratings of the wines.


Figure 1 shows an example of how co-inoculation of O. oeni with yeast in Cabernet Sauvignon wine can influence both the ester composition and the sensory profile of the wine. This trial was conducted at a South Australian winery on 1000L scale with triplicate fermentations. The wines that were co-inoculated with bacteria and yeast were described as more “fruit driven” with “fresh dark fruit” aromas. Total black fruit ester concentration generally increased with MLF, but higher concentrations were observed with the co-inoculated wines, linking the chemical composition with the sensory aroma and flavour description.

A recent study investigated the effects of inoculating Shiraz grape must with ML bacteria at various stages of alcoholic fermentation (beginning [co-inoculation], mid-alcoholic fermentation, at pressing and post alcoholic fermentation [sequential]) and demonstrated that co-inoculation greatly reduced the overall vinification time (alcoholic + malolactic fermentations) by up to 6 weeks (co-inoculated wines completed within 3 weeks, and sequential inoculation took up to 9 weeks to complete) (Abrahamse and Bartowsky 2011). A principal component analysis (PCA) plot of the respective inoculation treatments and volatile ester (fatty ethyl and acetate esters) and higher alcohol composition (Figure 2) revealed that the co-inoculation treatment produced wine that was well separated and highly distinct from the other MLF inoculation treatments, and from wine which only went through alcoholic fermentation; bacteria inoculation late into or after alcoholic fermentation produced wines which were highly similar to each other. These data clearly demonstrate that the co-inoculation treatment produces wines with a very distinct profile of fruity aroma volatiles.

Figure 2
Figura 2. Fermentation-derived volatile compounds of Shiraz wines (Clare Valley, South Australia, 2008) following different timing regimes of bacteria MLF inoculation. AF- alcoholic fermentation (no MLF). Green vectors indicate acetate esters; orange vectors indicate ethyl esters; pink vectors indicate higher alcohols.


Effects of grape source, ML bacterial strain and wine composition

Studies in Australian Cabernet Sauvignon were undertaken to determine O. oeni strain variation in synthesis of esters that contribute to fruity aromas, as well as the importance of pre-MLF wine composition and viticultural region (source of Cabernet Sauvignon). Certain esters proposed by Escudero et al. (2007) and Pineau et al. (2009) were determined and collectively used as a chemical parameter to gauge the potential berry-fruit sensory characters of the wines.
Metabolism of wine compounds by O. oeni will be determined by the genetic differences between strains and the concentration of metabolites in the wine. Variation in the production of diacetyl which confers buttery characters to wine is well known. Similarly, Figures 3 and 4 highlight that there are O. oeni strain variations in the ability to produce esters.
Wine chemical composition, including alcohol and SO2 concentrations and wine pH have an important impact on the ability of O. oeni strains to grow and complete MLF. Wine pH is also an important factor impacting the metabolic activity of O. oeni. For example, O. oeni will preferentially metabolise sugars at higher pH than organic acids (Krieger et al. 2000). Similarly, our studies have shown that pre-MLF wine pH can influence on the resultant concentrations of fermentation-derived volatile compounds. For two of three bacteria strains tested (ML-A and ML-B), the total fruity ester concentration was much higher for pre MLF wine pH 3.3 compared to the no MLF wine. Conversely, at higher pre MLF wine pH (3.7), these two strains decreased the concentration of total fruity esters decreased relative to the no MLF wines (Figure 3). In contrast, this effect of pre-MLF pH was somewhat reversed with strain ML-C.

Figure 2
Figura 3. Total fruity esters that contribute to red wine fruity aromas in Cabernet Sauvignon wines (South Australia, 2006). Sequential MLF induced with three different commercial Oenococcus oeni strains.


Another factor which will contribute to the potential of fruity esters being produced by O. oeni strains is the initial precursor composition in the wines. We studied this by sourcing Cabernet Sauvignon fruit from four different viticultural regions in South Australia. Prior to MLF, the wines had similar alcohol content (13.9%, 14.7%, 14.4% and 14.0%, I-IV respectively) and were adjusted to pH 3.45. MLF was induced with three O. oeni strains. Strains ML-1 & -2 completely metabolised malic acid within 20-25 days, whereas ML-3 took slightly longer to complete MLF (25-37 days) (it did not complete MLF in the Region I wine).
Figure 4 shows the total fruity esters production by three O. oeni strains the four respective Cabernet Sauvignon wines. Regions III & IV showed a relatively greater increase in fruity ester concentrations and Region II was little affected by MLF. Such variation in the effects of MLF on ester concentration between viticultural regions most likely reflects regional differences in precursor content.

Figure 2
Figura 4. Total fruity esters that contribute to red wine fruity aromas in Cabernet Sauvignon (four different South Australian vineyard regions, 2008). Sequential MLF induced with three different commercial Oenococcus oeni strains.


The authors thank Dr Sibylle Krieger-Weber, Jane McCarthy and Caroline Abrahamse for their research contributions. Part of this research was supported by Lallemand. The AWRI is supported by Australian grapegrowers and winemakers through their investment agency the Grape and Wine Research and Development Corporation, with matching funds from the Australian Government. The AWRI is a member of the Wine Innovation Cluster in Adelaide.


Abrahamse C.E.; Bartowsky, E.J. Timing of malolactic fermentation inoculation in Shiraz grape must and wine: Influence on chemical composition. World Journal of Microbiology & Biotechnology 2011. DOI: 10.1007/s11274-011-0814-3.
Bartowsky, E., Costello, P.; McCarthy, J. (2008) MLF - adding an ‘extra dimension’ to wine flavour and quality. Australian & New Zealand Grapegrower & Winemaker 2011; 533a: 60-5.
Bartowsky, E.J.; Henschke, P.A. The 'buttery' attribute of wine - diacetyl - desirability, spoilage and beyond. International Journal of Food Microbiology 2004; 96: 235-52.
Bartowsky, E.J.; Pretorius, I.S. Microbial formation and modification of flavour and off-flavour compounds in wine. In: Biology of microorganisms on grapes, in must and wine. H. König, G. Unden y J. Fröhlich (eds.) Heidelberg: Springer, 2008: 211-33.
Escudero, A.; Campo, E.; Farina, L.; Cacho, J.; Ferreira, V. Analytical characterization of the aroma of five premium red wines. Insights into the role of odor families and the concept of fruitiness of wines. Journal of Agricultural and Food Chemistry 2007; 55: 4501-10.
Francis, I.L.; Newton, J.L. Determining wine aroma from compositional data. Australian Journal of Grape and Wine Research 2005; 11: 114-26.
Iland, P.; Gago, P.; Caillard, A.; Dry, P. A taste of the world of wine. Campbelltown, South Australia: Patrick Iland Wine Promotions Pty Ltd., 2009.
Krieger, S.A.; Lemperle, E.; Ernst, M. Management of malolactic fermentation with regard to flavor modification in wine. Proceedings of 5th International Symposium on Cool Climate Viticulture and Oenology. Melbourne, Australia: Eds. City, 2000.
Pineau, B.; Barbe, J.C.; Van Leeuwen, C.; Dubourdieu, D. Examples of Perceptive Interactions Involved in Specific "Red-" and "Black-berry" Aromas in Red Wines. Journal of Agricultural and Food Chemistry 2009; 57: 3702-8.
Swiegers, J.H.; Bartowsky, E.J.; Henschke, P.A.; Pretorius, I.S. Yeast and bacterial modulation of wine aroma and flavour. Australian Journal of Grape and Wine Research 2005; 11: 139-73.