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.
Yeast and bacterial interactions during simultaneous alcoholic and malolactic fermentation
Despite significant improvements in malolactic fermentation (MLF) control, stuck or sluggish MLF issues still occur, particularly in white and sparkling base musts and wines. In these cases, knowledge of yeast and malolactic bacteria strain compatibility becomes an essential factor for successful MLF induction. One of the elements of yeast biology that contributes to its compatibility is the amount of SO2 a yeast strain produces. Different yeast strains have an enormous range of SO2 production potential. Laboratory trials have shown that some yeast and bacterial pairs support MLF despite the yeast producing large amounts of SO2. The project team previously correlated the support of MLF in these cases with the transient production of high concentrations of acetaldehyde.
A pilot-scale vintage trial confirmed the previous laboratory findings that early, yeast-derived acetaldehyde can both enhance bacterial survival during co-fermentation in Chardonnay with high SO2-producing yeast and enhance MLF performance. These results show that SO2 production by yeast and the survival of Oenococcus oeni following inoculation into an active fermentation can effectively be decoupled. Such enhancement of O. oeni survival in fermentations with high concentrations of SO2 identifies transient acetaldehyde production as a potential ‘survival factor’ for O. oeni in the difficult conditions of white wine co-fermentation. With the idea that transient acetaldehyde production could become a marker for yeast bacterial compatibility, a survey of the acetaldehyde production potential of a more comprehensive selection of Saccharomyces cerevisiae yeasts was also undertaken.
Quantifying the genetic response of Oenococcus oeni to SO2
It is well known that high total SO2 concentrations are a primary factor in the failure of MLF. Over the previous year, the project team undertook a detailed analysis of the genetic factors activated by O. oeni after exposure to SO2. This analysis revealed that O. oeni does not contain a specific mechanism to counteract the stress produced by SO2 but instead relies on the activation of several general stress-response genes. The identity of the activated genes provides a clue as to how SO2 interacts with the cellular components of O. oeni.
The experiments suggest that SO2 reacts primarily with intracellular proteins, DNA and the cell envelope of O. oeni. The cells respond by trying to repair the damaged proteins or by recycling irreparably damaged ones. DNA damage repair mechanisms are also activated. Addition of a higher but still sub-lethal concentration of SO2 results in arrested growth and an inability to initiate the general stress response. The difference in concentration of free SO2 that elicits either the general stress response or growth arrest is small (5 mg/L). That such a small change in SO2 concentration can produce such different effects highlights the narrow margins between successful and unsuccessful MLF.
Investigating interspecies microbial interactions
With non-Saccharomyces yeast being more commonly directly inoculated into grape juice, often at high cell densities and before inoculation with Saccharomyces cerevisiae, an obvious question to ask is whether some strains are better competitors than others? Are there strain-specific responses of S. cerevisiae to the presence of other organisms in their environment? Competitive experiments performed in the previous year using the collection of 94 barcoded strains of S. cerevisiae showed substantial fitness differences between S. cerevisiae strains in response to the presence of non-Saccharomyces yeast species. These observations identified specific strains of S. cerevisiae that can better perform in a competitive fermentation scenario where a different yeast species is inoculated first.
In the current year, the project team sought to verify the results of the competition experiments with a series of selected S. cerevisiae strains from the barcoded library that had higher or lower fitness when a different yeast species was present. The fermentation performance of the S. cerevisiae strains was evaluated in a medium that had previously been inoculated with specific non-Saccharomyces yeasts. In general, pre-inoculation with a non-Saccharomyces yeast was detrimental to the growth and fermentation performance of S. cerevisiae, irrespective of the non-Saccharomyces species or S. cerevisiae strain pair. However, the pairwise co-inoculation experiments confirmed that some strains of S. cerevisiae were indeed more fit to compete with different yeast species than others. Other strains of S. cerevisiae were unable to complete fermentation if inoculated into a medium in which a non-Saccharomyces yeast had been present for 24 hours.
The general inhibition of S. cerevisiae by non-Saccharomyces yeasts was previously reported by other research groups worldwide. However, the mechanisms of competition between yeast species are still poorly understood. The increasing use of commercial non-Saccharomyces yeast starters and winemaking practices employing spontaneous fermentations are compelling reasons to improve understanding about the interactions between different yeast species.
A set of experiments was conducted to categorise the basis for competition deficiencies evident when readily available commercial non-Saccharomyces species are co-inoculated with S. cerevisiae. Two approaches were used:
- Direct interactions linked to cell-to-cell contact between the two species were evaluated using inter-species co-cultivations.
- Indirect interactions related to depletion of nutrients or production of antimicrobial metabolites were evaluated using the cultivation of S. cerevisiae in non-Saccharomyces culture filtrate.
The results from these experiments demonstrated that not all non-Saccharomyces yeasts affect fermentation by S. cerevisiae in the same way. Inhibition of fermentation by Metschnikowia pulcherrima appears to be related to direct interaction with S. cerevisiae. Removing M. pulcherrima releases growth inhibition. However, species such as Torulaspora delbrueckii change the composition of the grape juice in a way that affects the subsequent growth of S. cerevisiae.
To further resolve how nutritional deficiencies might be inhibiting fermentation by S. cerevisiae, one non-Saccharomyces species was selected and the chemical changes produced in grape-like media after their growth were analysed in detail. In the case of Torulaspora delbrueckii, a depletion of specific amino acids and zinc was observed, which suggested that a deficit of these essential nutritional components might be responsible for the slowed growth and fermentation performance of S. cerevisiae. In subsequent work, correction of nutritional deficiencies by supplementation to pre-non-Saccharomyces concentrations, surprisingly, did not rescue the S. cerevisiae strain from its fermentation performance difficulties. To date, the basis for the inhibition of fermentation by non-Saccharomyces yeasts remains unresolved.