# _Forentation_
## 4 Discussion
### Physicochemical and Aromatic Composition of Ciders
#### 4.4.2 Considerations of Sequential Fermentations  
As shown in the PCA plot, sequential inoculations exhibited divergent contributions depending on the timing of yeast inoculation. The Seq S-P strategy yielded ciders with lower ethyl acetate and acetic acid levels, likely due to the earlier dominance of _S. cerevisiae_. In contrast, the Seq P-S strategy reflected a stronger influence of _P. kudriavzevii_, resulting in higher levels of these compounds. More specifically, the Seq P-S approach produced ciders with higher levels of acetic acid and ethyl acetate, along with lower levels of lactic acid and succinic acid. This chemical profile closely resembled that of ciders produced by pure _P. kudriavzevii_ inoculation (P), which suggests that the metabolism of this strain is primarily responsible for the production of the acetic acid and ethyl acetate found in P and Seq P-S ciders, compounds that contribute to the perception of sourness and roughness in cider and are undesirable at high concentrations [50; 53]. However, it is important to note that the acetic acid concentrations in P and Seq P-S ciders (0.4 g/L) remained within the acceptable limit of 2.5 g/L set by the Argentine Food Code [54]. Additionally, it has been reported that acetic acid becomes unpleasant at concentrations nearing its sensory threshold of 0.7-1.1 g/L, while values between 0.2 and 0.7 g/L are typically considered optimal [55].  
Ethyl acetate, the main ester in cider and wine, is formed from ethanol and acetic acid. It can impart spoilage characteristics when present at concentrations between 150 and 200 mg/L [53; 55]. In this study, we found that Seq P-S ciders exhibited ethyl acetate levels three times higher than those of Seq S-P ciders, with concentrations surpassing the sensory threshold. However, it is worth noting that when _P. kudriavzevii_ is inoculated in mixed cultures Sim 1:1, Sim 1:10, and Seq S-P, the levels of ethyl acetate decrease drastically in the resulting ciders, falling below the sensory threshold. The greater capacity of species from the genera _Pichia_ to produce ethyl acetate compared to _S. cerevisiae_ wine strains has been well-documented [38]. Subsequently, a study grouping ester production by yeast genera specifically highlighted _Hanseniaspora_ and _Pichia_ as notable producers of ethyl acetate [56]. More recently, ethyl acetate levels similar to those observed in this study were reported in wines produced by mixed inoculation with _P. kudriavzevii_ and a commercial _S. cerevisiae_[17; 57].  
The Seq S-P strategy displayed other favorable attributes compared to Seq P-S, including higher levels of total alcohols, particularly 2-phenylethanol and isoamyl alcohol, which contribute to floral and marzipan/green fruity aromas, respectively. Additionally, Seq S-P ciders exhibited higher levels of 2-phenylethyl acetate (associated with rose, honey, fruity, and flowery notes), isoamyl acetate (linked to banana and pear aromas), and ethyl hexanoate (which imparts apple, banana, and violet-like aromas). These findings support the selection of the Seq S-P strategy over Seq P-S for producing ciders with enhanced aromatic complexity.