# _Forentation_
## 4 Discussion
### Physicochemical and Aromatic Composition of Ciders
#### 4.4.1 Considerations of Simultaneous Fermentations  
Among the two simultaneous inoculation strategies, the Sim 1:1 approach resulted in higher glycerol levels. Glycerol is a desirable compound in cider, as it plays a primary role in enhancing the body and fullness of the final product [50]. Regarding the organic acid profile, the Sim 1:1 cider exhibited higher concentrations of lactic acid and succinic acid than the Sim 1:10 trial. Succinic acid contributes umami notes and a rounded flavor, while lactic acid is a mild-tasting, stable acid compared to malic acid, which is associated with a harsh taste and is more prone to microbial metabolism [25]. It is worth mentioning that among all fermentations, higher levels of lactic acid were detected in ciders where _S. cerevisiae_ dominated the fermentation process (S and Seq S-P), suggesting that its production is characteristic of this strain's metabolism. Lactic acid production by the _S. cerevisiae_ F8 strain has been previously reported [31].  
Higher alcohols are primarily produced by live yeast cells through amino acid deamination during fermentation and positively contribute to the aromatic complexity of fermented beverages when their concentration remains below 300 mg/L [50], as observed for all the ciders obtained in this study. The major fused alcohols identified in the ciders were isoamyl alcohol (contributing fruity and marzipan notes), isobutyl alcohol (imparting mild alcoholic aromas), and 2-phenylethanol (associated with rose and floral aromas). The comparison between the two simultaneous inoculation strategies showed that Sim 1:1 yielded higher levels of higher alcohols compared to Sim 1:10, with notable amounts of 2-phenylethanol and isoamyl alcohol. 2-phenylethanol, known for its rose and honey aromas, is considered one of the most important aromatic alcohols contributing to flavor in fermented beverages [19; 50]. Its levels in the ciders obtained in this study--except for Sim 1:10 and Seq P-S, where it was not detected--were near the detection threshold of 10 mg/L [51] and comparable to those produced by _Saccharomyces_ strains tested in apple must fermentations [5]. However, these levels were not as high as those found in ciders produced by _S. uvarum_ strains [6; 9].  
Esters are a class of volatile compounds known for imparting predominantly pleasant aromas. Produced by yeasts during fermentation as secondary byproducts of sugar metabolism, these compounds are essential contributors to the desirable fruity and floral ester-like character of the beverage. Among them, acetate esters are synthesized through the catalysis of higher alcohols and acetyl-CoA by alcohol acyl-transferases, while ethyl esters are produced via an enzyme-catalyzed condensation reaction between ethanol and an acyl-CoA component [52].  
Regarding the ester profile of the ciders produced through simultaneous inoculation, slightly higher levels of isoamyl acetate (notes of green apple) and slightly lower levels of ethyl hexanoate (pineapple and pear notes) were observed in the Sim 1:1 strategy compared to Sim 1:10. Furthermore, both compounds were present at higher concentrations in Sim 1:1 when compared to all other fermentation trials. Notably, the levels of isoamyl acetate detected in all ciders from this study were substantially higher than those found in ciders produced with _S. uvarum_ strains that exhibited fermentative potential for cidermaking [6].  
Simultaneous inoculations demonstrated a balanced metabolic contribution from both yeasts, resulting in characteristics that could enhance cider aroma and flavor. However, the Sim 1:1 strategy stood out, and, given that the use of 10 times more _P. kudriavzevii_ entails additional production costs, we propose Sim 1:1 as the most promising approach of the two simultaneous inoculation strategies tested for cider production.