Malolactic fermentation (MLF) is a fundamental aspect of winemaking that can be difficult and lengthy. This project characterised the diversity of bacterial fitness that remained undiscovered in the rich resource of the AWRI wine microorganism culture collection (AWMCC). Strains were identified that lend themselves well to challenging environmental conditions. Nutrient additions and other treatment regimens were explored that might facilitate robust and reliable malolactic fermentations. Finally, the characterisation of genetic factors responsible for bacterial fitness and wine quality attributes through gene expression analysis was attempted. Candidate genes that contribute to diacetyl production were identified.
The conversion of malic acid to lactic acid by lactic acid bacteria (known as malolactic fermentation or MLF) is a fundamental aspect of winemaking. MLF is important because it helps provide microbial stability to the finished product through the removal of a potential carbon source for spoilage organisms (malic acid), it reduces acidity and can impart various sensory characteristics, some subtle, others less so.
This project’s primary aim was to improve the reliability of MLF. This goal was approached from multiple angles. The first was to better understand and characterise the bacterial resources available to winemakers. By defining the limits of what was possible using available resources, areas for development were identified. Specifically, the conduct of MLF in low pH or higher ethanol wines was identified as a high priority for achieving tangible improvements in winemaking, given the limited fundamental knowledge and practical resources available to draw upon.
A program of genetic and physiological screening was undertaken to identify strains of O. oeni that could perform better in such conditions, particularly low pH. Pilot and industry trials with a range of bacterial isolates led to information about winemakers’ MLF requirements and enabled the identification of specific bacterial isolates that can conduct efficient MLF at industrial scale.
The second approach to improvement of MLF reliability was through an investigation of process options that had the potential to substantially reduce the overall time taken to get a wine to a point where is can be stabilised and protected through SO2 addition and barrel topping. While MLF is traditionally conducted following completion of alcoholic fermentation, it is also possible for MLF to be conducted concurrently. This is an option increasingly being used by winemakers, but its successful deployment requires a complete understanding of when and where it is most effective such that risks can be managed. Work conducted in this project identified substantial time savings that can be achieved in the production of red wine. Co-inoculation was a reliable and efficient strategy regardless of bacterial strain used. However, the same cannot be said for white and sparkling wine production and some work is still required to better understand how to get co-inoculation to work effectively in these environments.
Other process-driven interventions were also explored, specifically, the use of nutrient additives to support MLF and the impact of oxygen. Nutrient addition has the potential to be stimulatory in nutrient-limiting conditions. Several industry trials over two vintages failed to identify any improvement resulting from nutrient addition. Oxygen or air addition to ferments can be used to stimulate alcoholic fermentation but has the potential to have a negative impact on MLF. Experiments conducted to determine if aeration of alcoholic fermentation could affect subsequent MLF found no inhibitory or stimulatory effects. Work on the potential impact of oxygen use on co-inoculated ferments is ongoing. This area is especially important to winemakers wanting to use oxygen additions in their ferments without adversely affecting MLF, especially when MLF is performed after completion of primary fermentation.
The third approach was to identify genetic markers that might help in more efficiently identifying robust bacterial strains from microbial surveys carried out by others. Both
gene expression studies and genome wide association approaches were used. Candidate genes that could be used as markers for diacetyl production and others that indicated potential variation in strain-specific sugar utilisation in genomically highly related strains were identified. Furthermore, variations in metabolic pathways were found that indicated varied capacity of bacterial strains to utilise citric acid.
The key benefit to industry of this work is the provision of evidence-based advice on how to conduct and manage malolactic fermentation under challenging conditions. In this project, different approaches to the conduct of MLF have been investigated, some substantially outside of standard industry practice, with the aim of pushing the boundaries of what is achievable in MLF. Such risks can be undertaken in a research environment so that commercial risks taken by Australian winemakers can be objectively minimised. This approach contributes to the maintenance of a sustainable and profitable Australian wine industry.
The project benefitted substantially from the involvement of several industry partners including Yalumba, Treasury Wine Estates through their Wolf Blass winery, Hardy’s Wines through their Tintara winery and Pernod Ricard through their Orlando winery at Rowland Flat.