## 3 Results and Discussion  
### Field Experiment  
The use of mixed fermentation instead a single culture is a practice widely applied in winemaking in order to improve the aroma complexity or mouth-feel of wines [21; 22]. It offers a number of advantages over conventional single-culture fermentations, including higher microbial growth rate and metabolite yield, better utilization of the substrate, and complex formation of aromatic compounds [23]. This work represents the first study on a mixed culture in the coffee beans fermentation process. We experimentally tested the impact of the combination of two selected cultures (_P. fermentans_ YC5.2 and _P. acidilactici_ LPBC161) in terms of the fermentation efficiency and volatile composition of coffee beans. The experiments were performed with individual and combined inoculations in ripe and immature coffee beans, compared to a spontaneous process. The changes in major non-volatiles (sugars, organic acids, and ethanol) and volatiles metabolites were quantified in the course of the fermentation time. The initial pulp sugar concentration of ripe coffee fruits (0.57 and 1.13 g/L glucose and fructose content, respectively) was significantly higher than immature coffee pulp (0.13 and 0.26 g/L glucose and fructose content, respectively; Figure 1). The low levels of sugars in the case of immature coffee are the consequence of incomplete maturation cycle fruits [24]. In all fermentation processes, the sugar concentration showed an increase during the initial 12 h. This phenomenon can be associated with the hydrolysis of pectin, cellulose, sucrose, and other coffee pulp complexes carbohydrates, into monomers of glucose and fructose [25; 26]. After this increase, both glucose and fructose were partially consumed, resulting in a residual concentration of around 0.37 and 1.51 g/L (ripe coffee pulp) and 0.19 and 0.74 g/L (immature coffee pulp) of glucose and fructose, respectively. Residual pulp sugars are generally observed after the coffee fermentation process, mainly associated with the short fermentation cycle [14; 27; 28]. However, fructose consumption was more efficient in the treatments that the yeast was used (i.e., _P. fermentans_-pure culture and combined treatment with _P. fermentans_ and _P. acidilactici_) than _P. acidilactici_ pure culture and spontaneous assay (Figure 1). The yeast's ability to withstand stress tolerance factors and the production of pectinolytic enzymes confer advantages in comparison to lactic acid bacteria [16; 29]. In addition, the low availability of initially willing nutrients in immature coffee pulp [24] may have restricted the development of _P. acidilactici_, showing poor sugar consumption (Figure 1B). Lactic acid bacteria are further distinguished by their limited biosynthetic abilities, being unable to synthesize multiple cofactors, vitamins, purines, pyrimidines, and other nutrients [30]. Lactic acid showed a significant increase through fermentative processes, reaching maximum concentration of \(\geq\)1.45 and \(\geq\)0.53 g/L in the ripe and immature treatments, respectively (Figure 1). Basal concentrations of acetic acid (\(\leq\)0.01 g/L) were detected in all the processes, which can be associated with yeast metabolism or the heterofermentative nature of _P. acidilactici_[31; 32]. Overfermentation acids, such as propionic and butyric acids, were not detected in both ripe and immature treatments. In this sense, the acidification of fermenting coffee pulp can be attributed mainly to lactic acid content. As expected, while the lactic acid concentration increased, the pH decreased progressively during all fermentation processes (Figure 1). Lactic acid is an important end-metabolite associated with coffee fermentation, which assists in the coffee-pulp acidification process without interference in the final product (Figure 1). The pH monitoring is a crucial parameter, since pH values below 4.5 are used as to indicate the end of the coffee fermentation process [33; 34; 35].