# _Forentation_
## 3 Results
### Chemical Properties of Base Ciders  
The physicochemical characteristics of the base ciders are summarized in Table 5. Among the general chemical compounds analyzed, significant differences were observed in glycerol concentrations. Base ciders fermented with pure cultures of _P. kudriazvezii_ and _S. cerevisiae_, as well as those from the mixed Seq S-P strategy, exhibited the highest glycerol levels. Additionally, a significant decrease in sorbitol concentration (approximately 45%) was observed in all ciders compared to the uninoculated apple must. Different letters in the same row indicate significant differences (ANOVA and Tukey test, n = 2). P: _Rudriazvezii_ NPCC1651; S. _cervissie_ NIF8; Sim 1:1: Simultaneous fermentation by S-P ratio 1:10; Seq S-S: Sequential fermentation by _S. cerevisiae_ NIF8 at h after _S. cerevisiae_ NIF8. ND: Not detected. DL: Detection limit. Total cates\({}^{*}\) is the sum of esters  
The effect of the inoculation strategy was slightly evident in the composition of the organic acids in the base ciders. Both lactic and succinic acid concentrations were higher in ciders where _S. cerevisiae_ predominated (S, Sim 1:1, and Seq S-P). Volatile acidity (acetic acid) levels were higher in the ciders dominated by _P. kudriavzevii_ (P and Seq P-S fermentations); however, the concentration was within acceptable limits (2.5 g L\(-\)1). Contrarily, the lowest levels of this acid were found in the ciders produced exclusively by _S. cerevisiae_ (S). On the other hand, malic acid is the predominant acid in the apple must and ciders. The concentration of this acid decreased in all the fermentations analyzed relative to its content in the apple must. The consumption percentages ranged from 27% in the ciders produced by the pure _P. kudriavzevii_ culture to 33-34% in the simultaneous (Sim 1:1 and Sim 1:10) and the sequential Seq S-P fermentations (Table 5). Analysis of volatile compounds of ciders, detected by GC-MS analysis, was carried out to study the effects of the different inoculation strategies on the aroma of the base ciders elaborated from the same apple must (Table 5). Higher alcohols are important aromatic compounds in fermented beverages, produced through the secondary metabolism of yeasts. The most abundant higher alcohols detected in the ciders were isoamyl alcohol (contributing fruity and marzipan notes), followed by isobutyl alcohol (imparting mild alcoholic aromas) and 2-phenylethanol (associated with rose and floral aromas). Base ciders fermented with the pure culture of _P. kudriavzevii_ produced the significantly highest levels of isobutyl alcohol, while the same fermented with the pure culture of _S. cerevisiae_ produced the significantly highest levels of 2-phenylethanol. With regard to the concentrations of isoamyl alcohol, the highest levels were observed in base ciders where _S. cerevisiae_ predominated (S, Sim 1:1, and Seq S-P). These results position Sim 1:10 and Seq P-S fermentations as the trials that produce the base ciders with the lowest levels of total higher alcohols. In addition to higher alcohols, esters also play a crucial role as aroma compounds in cider. The most abundant esters in the base ciders analyzed were, by far, ethyl acetate, followed by isoamyl acetate, benzyl acetate, ethyl hexanoate, and ethyl octanoate. The comparison between the fermentation trials revealed that the base cider produced by the pure culture of _P. kudriavzevii_ exhibited the highest levels of total esters, especially due to the ethyl acetate concentration and, to a lesser extent, benzyl acetate and isobutyl acetate levels. Both isoamyl acetate and ethyl octanoate showed the highest concentrations in base ciders produced by simultaneous inoculations, independently of the _Saccharomyces:Pichia_ ratio. These ciders, along with those produced by sequential fermentation initiated with _S. cerevisiae_ (Seq S-P), showed the highest levels of total esters when ethyl acetate was excluded from the analysis (Table 5).