The relationship between grape juice composition and the progress of alcoholic and malolactic fermentation
Abstract
An outstanding problem in oenology is an understanding of the basis for sub-optimal fermentation of fruit whose compositional features are considered sufficient. The interaction between members of the grape microbial community has not been well considered in previous work. This project integrated yeast and bacterial research to explore the impact of oenological ecology on fermentation efficiency. Negative and positive interactions were identified between bacteria, non-Saccharomyces yeast and Saccharomyces cerevisiae. The response of microbial community members to inhibitory compounds was explored, with a particular focus on SO2 tolerance in bacteria and SO2 production by yeast.
Summary
Under the surface of a grape juice fermentation is a complex web of interactions between yeast, bacteria and their environment. Over the course of this project the contribution of individual microorganisms to the modification of grape fermentation composition was considered, starting with Aureobasidium pullulans and progressing to the effects of other non-Saccharomyces yeast found in fermentations. The changes imposed by these yeasts were shown to affect the behaviour of S. cerevisiae yeasts. Likewise, the strain-specific effects of S. cerevisiae on Oenococcus oeni were explored with a particular focus on SO2 production. Finally, a detailed examination of the responses of O. oeni to SO2 were undertaken, including approaches to their acclimatisation, optimal timing of their inoculation and an exploration of yeast-bacterial interactions during co-inoculation.
The physiological characterisation of Aureobasidium pullulans, one the most abundant species in freshly pressed grape juice, revealed its ability to substantially alter the juice matrix, causing depletion of zinc and iron. The results demonstrated that A. pullulans could compete for nutritional resources in juice and affect the fermentation performance of S. cerevisiae. A detailed physiological characterisation of A. pullulans also revealed that this organism contained enzymes capable of decreasing the concentration of galacturonic acid-based polysaccharides and hydrolysable gallotannin. It also produced enzymes capable of stimulating the release of glycosidically linked monoterpenes. These features of A. pullulans are all of oenological relevance.
The metabolic interactions between a wider array of non-Saccharomyces yeast and S. cerevisiae were characterised. This work demonstrated a general inhibition of S. cerevisiae by non-Saccharomyces yeast, with each interaction appearing to operate via different mechanisms. While the characterisation of these varied interactions is not yet complete, initial work suggests that at least some of them are based on a competition for nutrients. Given that many of the non-Saccharomyces yeast investigated in this work are increasingly available as cultures that can be inoculated into juice and must, the work has direct relevance for the management of these more complex fermentation regimes. These organisms are also members of the microbial community naturally present in freshly harvested fruit. Therefore, this research may provide insight into problematic fermentations observed in nutritionally replete fruit.
A survey of strain-specific interactions between an array of different S. cerevisiae yeast and select non-Saccharomyces yeast demonstrated that some S. cerevisiae strains are more competitive in mixed community environments than others. The study points toward selection criteria for yeast performance attributes that are not typically considered.
Unlike S. cerevisiae, bacterial fermentation can benefit from mixed community fermentations. The presence of non-Saccharomyces yeasts was found to stimulate MLF. These disparate observations raise the question of whether non-Saccharomyces yeast can be used to stimulate MLF without detrimental effects on alcoholic fermentation.
In parallel with studies of yeast-yeast interaction, the project team focused on various issues related to the production of and tolerance to SO2. A detailed exploration of two factors known to be inhibitory to yeast, copper and SO2 concentration, was completed. Copper and SO2 arguably have the largest effect on strain-dependent performance. That the mechanisms of resistance to these two inhibitory compounds interact within yeast metabolism was unforeseen and provided a deeper insight into a fundamental metabolic function of yeast, the assimilation of sulfate. The findings have important practical implications for strain development, indicating that a less forcefully driven SSU1 gene may decrease the metabolic burden in commercial yeast strains. It is also informative concerning other fundamental features of fermentation management; that is, the production of hydrogen sulfide and SO2 and, more broadly, nitrogen management.
The findings about the relationship between SO2 tolerance in yeast and SO2 production stimulated a broader analysis of the production potential of a representative set of S. cerevisiae wine strains. SO2 concentrations produced by yeast were found to vary widely, with approximately half of the strains increasing SO2 concentrations and the other half decreasing them. This knowledge provides an immediate initial selection criterion by which wine yeast can be selected as compatible partners for MLF and provides some metrics that can be used by winemakers wishing to control SO2 concentrations in their wines.
A system for assessing the tolerance of O. oeni to SO2 was developed that demonstrated there was little variation in SO2 tolerance among O. oeni strains. A Chardonnay co-inoculation study was useful for understanding the features of SO2 inhibition in a winemaking context. The question about the relative contributions of molecular SO2 and total SO2 to the viability loss experienced by O. oeni, and the inhibition of MLF remains unresolved. The work suggested that a long-held view regarding the potential for consumption of acetaldehyde-bound SO2 by O. oeni may not be correct and led directly to laboratory and pilot-scale experiments investigating interactions between bacteria and SO2-producing yeasts.
The direct effect of SO2 production by yeast on O. oeni survival and MLF progress was assessed through a series of co-inoculation fermentation trials. Although most interactions between bacteria and high-SO2-producing yeast were unsurprisingly negative, one high- SO2-producing yeast was identified that supported maintenance of bacterial viability and efficient MLF progress. This exception to the common observation revealed acetaldehyde as a key metabolite supporting a robust relationship between yeast and bacteria. The yeast strain produced a vast transient excess of this acetaldehyde that appeared to quench the detrimental effects of SO2 for long enough that the bacterium could survive. The work was important for two reasons; firstly, it demonstrated that O. oeni could become acclimatised to high total SO2 concentrations and, secondly, that O. oeni is more than likely exquisitely sensitive to molecular SO2.
An investigation was undertaken of the transcriptional changes associated with SO2 stress in O. oeni. This work showed that major transcriptional changes occurred following exposure to 5 mg/L SO2 and indicated that this compound primarily interacts with intracellular proteins, DNA and the cell envelope of O. oeni. Overall, O. oeni had the ability to counteract mild SO2 stress through direct cellular responses. However, exposure to greater SO2 stress caused irreparable damage. The bacterium was found to lack components that could actively and specifically counteract SO2, such as those found in many yeasts.