## 3 Results and Discussion
### Evaluation of the Effect of Starter Cultures on Bioactive Compounds and Carbohydrates
In addition, in varieties A (MRS: 0.73; YPD: 0.83; PCA: 0.74) and P2 (MRS: 0.86; YPD: 0.78; PCA: 0.79), it was also possible to observe strong and significant correlations between all the microbial groups evaluated and chlorogenic acid. Although the mechanism of production and transformation of bioactive compounds by coffee-related microorganisms has not been fully elucidated, yeasts such as _Hansenispora urarum_, _Pichia kluyveri_, and _Saccharomyces cerevisiae_, as well as the bacteria _Bacillus albus_ and _Lactococcus gariveae_ identified in this study have enzymatic capacity through pectinolytic enzymes and esterases, which contribute to the degradation of mucilage and soluble fibers and the biotransformation of compounds such as chlorogenic acid during the wet fermentation of coffee beans. Microorganisms such as LAB have an enzymatic mechanism rich in transferases and esterases. However, they do not have demethylases. This enzyme has the function of attaching a methyl group to the hydroxyl group they have. Chlorogenic acids, however, are synthesized by esterification, where two compounds, caffeic acid and quinic acid, are linked. For this reason, chlorogenic acid may have a higher content in fermented coffee [10; 34; 37; 38]. In addition, epiphytic lactic acid bacteria and yeasts can protect these compounds by stabilizing the environment through the production of organic acids from the primary metabolism of growing microorganisms, such as lactic acid, which promotes the reduction of chlorogenic acid oxidation [39; 40]. For trigonelline, a strong correlation (0.8) was observed between the yeasts and the P2 variety, as quantified using YPD agar. For caffeine, a correlation was observed only in variety A between lactic acid bacteria (0.83) and mesophilic bacteria (0.75). For variety CP, no correlations were observed between the microbial groups and the bioactive compounds (Supplementary Material--Figure S3). The use of _S. cerevisiae_ CA11 influenced the maximum accumulation of trigonelline and chlorogenic acid in the P2 (trigonelline: 0.07 mg\(\cdot\)L\({}^{-1}\); chlorogenic acid: 0.36 mg\(\cdot\)L\({}^{-1}\)) and CA62 (trigonelline: 0.07 mg\(\cdot\)L\({}^{-1}\); chlorogenic acid: 0.27 mg\(\cdot\)L\({}^{-1}\)) coffee varieties. Compared to using _K. lactis_ B10, the concentration and accumulation of bioactive compounds depended on the coffee variety throughout the fermentation process. The trigonelline concentration was more significant for A (0.06 mg\(\cdot\)L\({}^{-1}\)), P2 (0.07 mg\(\cdot\)L\({}^{-1}\)), and CA62 (0.07 mg\(\cdot\)L\({}^{-1}\)), and the concentration of chlorogenic acid was greater only in P2 (0.36 mg\(\cdot\)L\({}^{-1}\)) and CP (0.33 mg\(\cdot\)L\({}^{-1}\)). However, in the context of spontaneous fermentation, the P2 variety showed a reduction in the concentration of approximately 28% of these compounds throughout fermentation, resulting in coffee beans with lower caffeine concentrations (N: 0.07 mg\(\cdot\)L\({}^{-1}\); CA11: 0.08 mg\(\cdot\)L\({}^{-1}\); B10: 0.08 mg\(\cdot\)L\({}^{-1}\)) and trigonelline (N: 0.05 mg\(\cdot\)L\({}^{-1}\); CA11: 0.07 mg\(\cdot\)L\({}^{-1}\); B10: 0.07 mg\(\cdot\)L\({}^{-1}\)) (Supplementary Material--Table S2). A hierarchical cluster analysis (HCA) was designed to support the multivariate analysis presented. In HCA, the samples are grouped into classes according to their proximity or similarity [41], and the results are shown as a dendrogram (Figure 2B). The samples were separated into three visible groups, in agreement with PCA and with the variability of the data analyzed using the Scott-Knott test. The treatments presented in the blue group represent the biochemical variation in the different coffee varieties during fermentation compared to the treatment without a starter culture, as evidenced by the accumulation of bioactive compounds.