A growing number of studies suggest that non-Saccharomyces species may contribute positively to the final taste and flavor of wine during mixed culture fermentation with Saccharomyces cerevisiae. Thus, there has been a growing interest towards creating defined mixed or sequential starter cultures for alcoholic fermentations in which S. cerevisiae at some point grows together with the non-Saccharomyces species. In these kinds of fermentations, the non-Saccharomyces yeasts will grow together with S. cerevisiae during the first 1-2 days after inoculation with S. cerevisiae, but as the fermentation progresses the non-Saccharomyces species will typically die off leaving S. cerevisiae to dominate and complete the fermentation. The present PhD thesis has explored the physiological dynamics of Lachancea thermotolerans (LT) and Saccharomyces cerevisiae (SC) during single and mixed alcoholic fermentations at the metabolome and proteome levels.
In the first study, 1H NMR- and GC-MS-based metabolomics was used to identify metabolites that discriminate single and mixed cultures of LT and SC at two key time-points during mixed culture alcoholic fermentations. Twenty-two metabolites were found when comparing single LT and mixed cultures, including both non-volatiles (carbohydrate, amino acid and acids) and volatiles (higher alcohols, esters, ketones and aldehydes). Fifteen of these compounds (Carbohydrate, acids, higher alcohols, esters, ketones and aldehydes) were discriminatory only at the death phase initiation (T1) and fifteen (amino acid, higher alcohols, esters and aldehydes) were discriminatory only at the death phase termination (T2) of LT in mixed cultures. Eight metabolites (acids, higher alcohols, esters and ketones) were discriminatory at both T1 and T2. These results indicate that specific metabolic changes may be descriptive of different LT growth behaviors. Fifteen discriminatory metabolites (higher alcohols, esters, ketones and aldehydes) were found when comparing single SC and mixed cultures. These metabolites were all volatiles, and twelve metabolites (higher alcohols, esters, ketones and aldehydes) were discriminatory only at T2, indicating that LT-induced changes in volatiles occur during the death phase of LT in mixed cultures and not during their initial growth stage.
In the second study, TMT-based proteomics was used to identify significantly discriminatory proteins (SDPs) that discriminate single and mixed cultures of LT and SC at two key time-points during mixed-culture alcoholic fermentations. These results showed that from the death phase initiation (T1) to termination (T2) of LT in mixed cultures, the protein complexity was significantly increased both in the single and mixed cultures. When comparing single LT and mixed cultures, thirty-three proteins were present only in mixed cultures and forty-three proteins were identified as significantly discriminatory proteins (SDPs) at T1; three proteins were present only in mixed cultures and 155 proteins were identified as SDPs at T2. When comparing single SC and mixed cultures, one protein was present only in mixed cultures and only three proteins were identified as SDPs at T1; four proteins were present only in mixed cultures and ten proteins were identified as SDPs at T2. It indicates that SC grew in the same proteomic environments either in the mixed or single SC cultures; however, LT grew in distinct proteomic environments between the mixed and single LT cultures. Our results also show that, when LT was highly present in mixed cultures at T1, SC up-regulated the protein involved in cell-to-cell contact; compared to LT cells, SC cells have stronger abilities of cell rescue and defense, detoxification and response to nutrient limitation. Taken together, a combination of distinct proteomic environments, cell-to-cell contact and stronger survival abilities of SC may be one of the factors underlying the early death of LT in mixed cultures, not in single LT cultures.
In the third study, the proteomic response of SC to co-cultivation with LT during alcoholic fermentations has been investigated using TMT-based proteomics and flow cytometry sorting. SC cells were sorted from single SC and mixed cultures of SC and LT, respectively, using flow cytometry combined with fluorescence staining. The result showed that the purity of sorted SC cells was above 96% throughout the whole yeast-yeast growth interactions, thereby validating our cell sorting methodology. Twenty-six proteins were identified as significantly regulated proteins (SRPs) when comparing protein expression patterns of SC with and without LT co-cultivation at the death phase initiation (T1) and 32 SRPs were identified at the death phase termination (T2) of LT in mixed cultures. Our results show that, at T1, proteins involved in increasing nutrient sources, cell rescue and resistance to stresses and endocytosis were up-regulated, and proteins involved in proline synthesis and apoptosis were down-regulated. Furthermore, they show that, at T2, proteins involved in protein synthesis in stress responses, were up- and down-regulated, respectively. These data indicate that SC was stressed by the physical presence of LT at T1, using both defensive and fighting strategies to keep itself in a dominant position, and that it at T2 was relieved from the LT-induced stress, increasing its general metabolic machinery to ensure better survival.