# Traditional Grain-Based vs. Commercial Milk Refirs, How Different Are They?
## 3 Results and Discussion
### Identification of VOCs and Clustering of Yogurt and Kefir Samples  
VOCs of milk, commercial yogurts (CY1 and CY2), commercial kefirs (CK1, CK2, and CK3), and grain-based traditional kefirs (FN, LS, PN1, PN2, and PN3) were analyzed by HS-GC-IMS. The results of the VOCs profiles are visualized in Figure 4 as a hierarchically clustered heatmap. The clustering algorithm visualizes sample similarities in a cluster dendrogram with the vertical distance corresponding to the similarities between samples. Based on the VOCs, the commercial samples, including milk, yogurts, and commercial kefirs clustered separately from the grain-based traditional kefirs. This unsurprisingly shows that commercial kefirs have a higher similarity to yogurt than to grain-based traditional kefirs. The commercial yogurt-kefir cluster was further divided into two sub-clusters. Here, the commercial yogurts showed higher similarity to milk than to the commercial kefirs, which formed the second sub-cluster. The short distances within the commercial yogurt-kefir sub-clusters suggest a high similarity in the VOCs profiles of the two yogurt samples included in the present study. The grain-based traditional kefir cluster was further divided into two sub-clusters. Sub-cluster 1 contained all PN kefirs, which show high similarity in their VOCs-profiles, while sub-cluster 2 contained FN and LS. By considering the differences in microbial compositions, it seems reasonable that PN1-PN3 are in a different cluster than the other two traditional kefirs LS and FN. PN1-PN3 have more diverse bacterial and yeast communities, e.g., the heterofermatterbacteria _L. mesenteroides_ and the strong aroma producing yeast _Kl. marxianus_. FN and LS kefir showed a larger distance to each other than the three PN kefirs from sub-cluster 1, which can be explained by different bacterial and yeast species in these 2 kefirs. Using a correlation matrix, further sample similarities were investigated. It was noted that not only does CK3 correlate well with CK1 and CK2 with values of 0.898 and 0.833, respectively, but further correlates with the grain-based kefir FN and LS with values of 0.810 and 0.959, respectively. In milk, mainly five VOCs were found: 2-nonanone (#1), 2-heptanone (#4), 2-butanone (#16), acetone (#19), and ethanol (#20) in decreasing order. As shown in Figure 4, a similar abundance of 2-nonanone, 2-heptanone, and acetone, as observed in milk, were detected in all commercial and grain-based kefirs (FN, LS, PN1, PN2, and PN3). Therefore, it was concluded that these three ketones were not originated from kefir fermentation, but from milk. While commercial yogurt (CY1 and CY2) and commercial kefir CK1 and CK2 also contained a similar abundance of acetone, 2-heptanone, and 2-nonanone as milk, CK3 in contrast contained a very low abundance of these three ketones. The abundance of the fourth ketone, 2-butanone (#16), was highest in milk. Especially in the grain-based kefirs FN, LS, and PN3, the abundance of 2-butanone (#16) was clearly lower than in milk, probably due to an assimilation within the microbial fermentation process. Ketones, which were found in yogurt and kefir, but not (or in significantly smaller concentrations) in milk, are acetoin (#9) and 2-pentanone (#10), as well as the diketones pentane-2,3-dione (#11) and butane-2,3-dione (diacetyl, #17). Acetoin was found in CK1 and CK2 and in smaller abundance in CY1, CY2, PN1, PN2, and PN3. 2-pentanone (#10) was found in commercial yogurt as well as in CK1, CK2, LS, PN1, PN2, and PN3, but was absent in CK3 and FN. The  
Figure 4: Hierarchical cluster analysis for VOCs analysis of commercial yogurt (CY1 and CY2), commercial kefirs (CK1, CK2, and CK3) and grain-based kefirs (LS, FN, PN1, PN2, and PN3), showing the peak volume (abundance) of each substance. The grain-based kefir was analyzed after \(47\pm 1\) h of fermentation. diketone pentane-2,3-dion (#11) was only found in CY1 and CY2 and in smaller abundance in CK2 and FN.