# 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
The diketone butane-2,3-dione (#11) was found in all kefir samples, with the highest amount in CK1, CK2, as well as CY2. Three aldehydes were found in this work: 2-methyl propanal (#18), 2-methylbutanal (#12), and 3-methylbutanal (#13). Both methyl butanals (#12 and #13) were previously reported in kefir [19; 20]. All three aldehydes were found in PN1, PN2, and PN3. Both methyl butanals (#12 and #13) were also found in the other kefirs, but in lower abundance. In contrast, 2-methyl propanal (#18) was only found in CK2. A fourth aldehyde, hexanal (#6) was mainly found in the FN-kefir and in smaller abundance in PN3. Aldehydes, such as hexanal (#6), are secondary oxidation products that rapidly develop through hydroperoxides, which are primary oxidation products of unsaturated fatty acids [32]. Due to its low odor threshold (approx. 5 ppb), hexanal is easily detected and it is directly related to oxidative off-flavors [33]. Thus, the presence of hexanal (#6) in the FN kefir may be a consequence of secondary lipid oxidation. Furthermore, various alcohols, which have previously been reported in kefir [19; 34], were also quantified in this work: ethanol (#20), isoamyl alcohol (#8), and 2-methyl-1-propanol (#14) [19]. 2-methyl- and 3-methyl-1-butanol have very similar retention times, differentiation is rather difficult, and both compounds are cumulated here (#8). The ethanol abundance detected in CK1, CK2, and CK3 is similar to the abundance found in milk, while all grain-based kefirs showed an increased content of ethanol. Ethanol production in kefir was previously attributed to yeasts [35], but it was also noted that _Lactococcus_ spp. and _Lactobacillus_ spp. possess a low activity of alcohol dehydrogenase, which converts acetaldehyde to ethanol. Isoamyl alcohols (#8) were absent in the commercial samples and most abundant in PN1, PN2 and PN3 containing a high concentration of _KI. marxianus. Kl. lactis_ was also reported as a producer of both 2- and 3-methylbutanal, which were also detected in higher abundance in PN1-PN3 kefirs [36]. Acids that were detected in the kefir samples are butyric acid (#7) and hexanoic acid (#3). Acetic acid was also tentatively found in all samples, however, the pattern formed by acetic acid in IMS analysis is quite complex. Therefore, a univariate evaluation of acetic acid was not possible. Butyric acid (#7) and hexanoic acid (#3) were found in all yogurt and kefir samples (commercial and grain-based) with a similar abundance. Furthermore, three esters, which were previously reported [19], appear in the grain-based kefirs: ethyl acetate (#15), 3-methylbutyal acetate (#5) and ethyl hexanoate (#2). These esters correspond to alcohols already identified (ethanol and 3-methyl-butanol) with acetic acid and hexanoic acid. Ethyl acetate together with ethyl lactate, benzoic acid, and ethanol were reported as predominant compounds in traditional kefirs [8]. The detection of ethyl hexanoate (#2) in all grain-based kefirs in this test was also reported for _L. kefiranofaciens_ and _L. kefiri_ growth in milk in a recent study [37]. In general, no esters were found in the commercial yogurts and kefirs. Defining the contribution of individual bacteria and yeast species in synthesizing the various metabolites in complex microbial communities is not an easy task, especially when metabolomic characteristics of different species are highly strain-specific [38]. One approach that has been used so far was to analyze the contribution of different microorganisms by elaboration of defined co-cultures. e.g., two or three different microorganisms [36]. This is, however, not truly representative for complex microbial communities, in which an inter-dependency of different isolates or cross-feeding processes with other species are required for the appearance of certain reactions so that the resulting product is accumulated and becomes detectable.