# Exploitation of Three Non-Conventional Yeast Species in the Brewing Process
## 4 Discussion  
In winemaking, the controlled use of non-conventional yeast species has been investigated to enhance complexity and give distinctive flavour profiles of wines [22; 23; 24]. Indeed, fermented alcoholic beverages with peculiar and distinctive flavours can be obtained through the yeast inoculation during the fermentation process [11; 21; 25]. In this regard, the potential use of non-conventional yeasts in brewing has been poorly explored. Only recently, some studies focused on flavouring by non-conventional yeasts in brewing [7; 9; 14; 16; 26]. In this work, we investigated three non-conventional yeast species widely found in the wine environment [23; 27; 28]. The three strains chosen within the three species were competitive versus the _S. cerevisiae_ starter strain at the 1:1 inoculation ratio exhibiting similar numbers of viable cells. The competition of the non-conventional species in word under semi-anaerobic conditions was confirmed for the 1:10 and 1:20 ratios (_S. cerevisiae_ brewers' yeast: Non-conventional species). In these last conditions, all three non-conventional yeast species coexisted with brewers' yeast dominating the fermentation process. Similar results were found by [PERSON] et al. [17] where _Candida fibianii_ at a 1:100 ratios (_S. cerevisiae_: _C. fabianii_) dominated the fermentation. However, compared to the results of the present work, a large amount of residual maltose was found. This behaviour is of interest from a practical application point of view. Indeed, the dominance allows displaying the metabolism of the non-conventional yeast in co-culture with the brewing starter, obtaining, at the same time, a distinctive aromatic impact on final product and a correct and complete evolution of the fermentation. In this regard, differences in the analytical composition using the three non-conventional yeasts in pure and in co-culture were found (see Figure 4). All three non-conventional species determined a general decrease of acetaldehyde in all mixed fermentations (1:1, 1:10, 1:20 _S. cerevisiae_/non-conventional yeasts ratios). _L. thermotolerans_, a yeast species widely investigated in wine fermentation, [28; 29] showed a large reduction in pH compared with the _S. cerevisiae_ starter strain. [PERSON] et al. [15] and [PERSON] et al. [8], investigating _L. thermotolerans_ in pure fermentation with the goal of producing sour beer, and found a compared trend in pH reduction. In the word used in this work, _L. thermotolerans_ in pure culture produced a larger amount of lactic acid (1.83 g/L) compared with the quantities shown in the study of [PERSON] et al. [15] (0.24 g/L). Regarding the compounds influencing the biofavor, _L. thermotolerans_ produced beer with a higher ethyl butyrate content and an increase in ethyl acetate (i.e., floral, honey, sweet) in pure and in all of the mixed fermentations compared with _S. cerevisiae_. Moreover, a significant increase in isoamyl acetate content (banana aroma) was exhibited in the trial _S. cerevisiae_/_L. thermotolerans_ at an inoculum ratio of 1:20. These results highlighted that this strain was interesting not only for sour beer production due to its lactic acid production and consequent pH decrease during primary fermentation but also to tailor the aroma profile of the beer. The low ethanol content exhibited by _L. thermotolerans_ pure cultures was also relevant for the production of low alcohol beer. _W. anomalous_ generally studied in apple wine and hard cider production [30; 31], it was poorly investigated in brewing because it is associated with the concept of \"spoilage yeast\" [32]. Only [PERSON] at al. [8] showed that a strain of _W. anomalous_ exhibited a good fermentation performance and led beers with fruit notes such as apple, pear, and apricot. Results of this study showed an increase in ethyl butyrate and ethyl acetate and a reduction in acetaldehyde in all mixed fermentations that positively affect the aromatic profile of the beer.