## 3 Results and Discussion
### Evolution of VOC Profiles During Successive Kefir Grain Passages
The second principal component (PC2) showed positive correlations with the glycerol, acetic acid, and yeast count, and negative correlations with the total sugar and lactic acid concentrations. The third principal component (PC3) was primarily characterized by a high LAB count and a greater number of volatile compounds with odor activity values (OAVs) above 1.0. Figure 5B shows the distribution of the 28 beverages based on their scores for the three factors obtained from the PCA. Given the complexity of interpreting the grouping of the 28 kiwifruit-based beverages based on PCA (Figure 5A,B), a third cluster analysis was performed using the three principal factors to prevent overfitting in the formation of clusters. As shown in Figure 5C, two distinct clusters were observed. The first cluster comprised nine beverages from the first kefir grain passage, while the second cluster included the eighteen beverages from the second and third kefir grain passages. As expected, the unfermented kiwi juice was notably distinct, standing out as the most different beverage (Figure 5C). In the first cluster, the number of volatile compounds with an OAV greater than 1.0 ranged from four to twelve: Bev5-24 h (four compounds), Bev2-24 h (six compounds), Bev1-24 h and Bev8-24 h (seven compounds), Bev7-24 h (eight compounds), Bev6-24 h and Bev9-24 h (nine compounds), Bev3-24 h (ten compounds), and Bev4-24 h (twelve compounds). In the second cluster, Bev5-48 h (four compounds) and Bev5-72 h (five compounds) contained the fewest volatile compounds with OAV > 1.0, while Bev6-48 h, Bev3-72 h, and Bev4-72 h, each with fourteen compounds, as well as Bev9-72 h (seventeen compounds), had the highest number of aromatic compounds (Tables 2-4). Interestingly, the nine beverages from the first kefir grain passage exhibited higher concentrations of total sugars (71.10-107.53 g/L), lactic acid (0.41-1.31 g/L), ethanol  
Figure 5: **(A)**: Projection of the independent variables (TS: total sugars, CA: citric acid, QA: quinic acid, LA: lactic acid, AA: acetic acid, EtOH: ethanol, GOH: glycerol, LAB: lactic acid bacteria count, AAB: acetic acid bacteria count), yeasts count, and the number of VOCs with OAVs \(\geq\) 1.0 [nOAVs] from Tables 2–4 on a 3D scatterplot, as a function of PC1, PC2, and PC3. **(B)**: Projection of the 28 kiwifruit-based beverages on the 3D scatterplot, as a function of Factor 1, Factor 2, and Factor 3. Beverages 2–10, 11–19, and 20–28 correspond to those obtained during the first, second, and third kefir grain passages, respectively, following the experimental order outlined in Figure 1B. Beverage 1 is the unfermented kiwi juice (UKJ). **(C)**: Clusters formed by the unfermented kiwi juice and the 27 fermented kiwifruit-based beverages, using the values of Factors 1, 2, and 3 from the PCA as classification variables. (0.36-4.50 g/L), and AAB counts (5.88-6.93 log colony-forming units (CFU)/mL), while they had lower levels of quinic acid (5.49-7.92 g/L), acetic acid (0.07-0.22 g/L), and glycerol (0.18-0.21 g/L) compared to the beverages in the second cluster. All beverages in the second cluster had yeast counts exceeding 10\({}^{6}\) CFU/mL [11], which is the threshold considered to confer probiotic benefits on the host [63]. A detailed analysis of the experimental data revealed that the beverages Bev4-24 h and Bev6-24 h, which contained twelve and nine aromatic compounds with an OAV \(>\) 1.0, respectively, had the highest total sugar concentrations (107.53 and 102.66 g/L, respectively). These values represented 87.02% and 83.09% of the initial total sugar concentration in the unfermented kiwi juice. This suggests that Bev4-24 h and Bev6-24 h are high-calorie beverages, and their consumption could contribute to excessive calorie intake. Notably, while Bev6-24 h contained counts of LAB, AAB, and yeasts exceeding 10\({}^{6}\) CFU/mL, Bev4-24 h had an LAB count higher than 10\({}^{6}\) CFU/mL, with AAB and yeast counts slightly below this threshold.