The relationship between grape juice composition and the progress of alcoholic and malolactic fermentation
Difficulties with alcoholic and malolactic fermentation are routinely reported, and can be attributed to a diverse range of causes. Poor fermentation progress can occur even in juices and wines that otherwise satisfy the usual criteria indicative of appropriate fermentation progress (e.g. YAN, Baumé, and SO2). Uncontrolled growth of non-target microorganisms has been reported to be inhibitory to alcoholic fermentation, either through consumption of nutrients, or through the production of secondary metabolites.
Sulfur dioxide additions to bins and crushers are used to control pre-fermentation microbial activity; however, even moderate levels of total SO2 can negatively affect the progress of malolactic fermentation. In addition, some yeasts produce large amounts of SO2 which is inhibitory to malolactic fermentation. This is a particular concern as simultaneous alcoholic and malolactic fermentations are increasingly being used to more efficiently manage scheduling issues associated with conducting malolactic fermentation as a separate process, after alcoholic-fermentation.
Clearly the areas of yeast and bacterial fermentation performance are inter-related, and understanding the risks and capturing opportunities of yeast/bacterial interactions requires an integrated approach as described in this project. Hence this project brings together two previously separate research areas, yeast and bacterial fermentation, in order to realise an integrated approach to the study of alcoholic and malolactic fermentation performance.
The proposed fermentation performance program will study the following:
- yeast/environment interactions, using the barcoded yeast collection to determine strain fitness and implantation efficiency, together with a survey of juice composition across multiple vintages, taking account of transport conditions and other harvest variables to determine their impact on composition (collaboration with Project 3.3.1)
- bacterial/environment interactions, by using model fermentations to identify factors that stimulate or inhibit malolactic fermentation, and through developing a transformation system for Oenococcus oeni to study genetic elements (inter-strain variable regions) and their effects on malic acid utilisation
- pilot and industry trials to evaluate the suitability of uniquely Australian regional isolates of malolactic bacteria, and to determine the robustness of co-inoculated fermentations using a range of winemaking interventions.
Managing interactions between yeast and bacteria
There is no better example of an intimate relationship between yeast and bacteria than when co-inoculation of Saccharomyces cerevisiae and Oenococcus oeni is used to stimulate the simultaneous conduct of alcoholic and malolactic fermentation (MLF). This increasingly common practice can be a boon under ideal conditions as alcoholic and malolactic fermentation can be completed simultaneously. However, the benefits in more trying conditions are not always so obvious. At times, the duration of co-inoculated and sequentially inoculated MLF can be equivalently protracted. The success or failure of co-inoculated MLF rests with the close relationship between S. cerevisiae and O. oeni and this relationship has remained a focus for this project.
The kinetics of SO2 production by wine yeast
Previous work at the AWRI and elsewhere demonstrated the large range of SO2 production potential for different strains of wine yeast. The SO2 produced remains in the wine bound to other metabolites, predominantly acetaldehyde, and is one of the inhibitory factors to consider when conducting sequential MLF. During the conduct of co-inoculated MLF, however, the kinetics of SO2 production may be relevant to the optimal timing for Oenococcus oeni inoculation. For this reason, SO2 accumulation was monitored during alcoholic fermentations conducted by a selection of yeasts with different SO2 production potentials. This work demonstrated that not all yeasts begin SO2 accumulation at the same time or at the same rate and defined a window within 24 hours of yeast inoculation that would give O. oeni the greatest chance of establishing a sustainable population during co-inoculation.
Picking the right time for co-inoculation of O. oeni
In practice, the point of alcoholic fermentation when Oenococcus oeni is inoculated varies considerably from winery to winery. Typical recommendations suggest that inoculation of O. oeni should occur within 48 hours of yeast inoculation. Experimental work was conducted that aimed to provide further guidance about optimal O. oeni inoculation timing, considering the variable kinetics of SO2 production by yeast. An investigation of co-inoculated Chardonnay juice showed a decline in the viability of bacteria at very early inoculation times (two hours post-yeast inoculation) which was ameliorated at 24 hours and exacerbated at 48 hours post-yeast inoculation. These observations align with the kinetics of SO2 production by yeast discussed above. Bacterial survival was also highly dependent on yeast strain, with one high-SO2-producing yeast yielding minimal loss in bacterial viability and MLF completion, while another caused complete decline of the bacterial population and no MLF. This demonstrates that bacterial tolerance to total SO2 is not only dependent on the absolute SO2 concentration and the timing of its production, but that there are other factors contributing to bacterial survival.
Another potential contributing factor to successful MLF is the SO2 tolerance, or lack thereof, of O. oeni itself. Using a co-inoculation model system in Chardonnay juice, the strain-specific tolerance of 20 different O. oeni strains to SO2 was assessed. Surprisingly, this assessment revealed that most strains of O. oeni did not exhibit sensitivity to high concentrations (≤ 60 mg/L) of acetaldehyde-bound SO2. Further exploration of the mechanisms of O. oeni tolerance to acetaldehyde-bound SO2 in co-inoculation may help address delays in MLF induction that can be encountered in white wines with potentially inhibitory SO2 concentrations.
Interspecies microbial interactions – the strange world of Aureobasidium pullulans
Grape juice is a rich environment complete with its own ecology. One of the most abundant organisms present during the early stages of fermentation is a yeast-like fungus belonging to the Aureobasidium genus. While this organism is better known in relation to biotechnological and environmental applications, relatively little is known about its contribution to the progress and outcomes of fermentation. Recent work has sought to better characterise the physiology of strains of Aureobasidium pullulans isolated from spontaneous fermentations. Several compounds of oenological relevance were observed that are novel in the context of wine research. Assessment of the nutrient requirements of A. pullulans suggests that competition with yeast and bacteria is possible under specific conditions. These preliminary investigations will serve as a foundation for future work characterising the impact of this organism on wine production.