# Traditional Grain-Based vs. Commercial Milk Refirs, How Different Are They?
## 3 Results and Discussion
### Microbial Composition of Kefirs
#### 3.1.2 Identification of Microbial Community Using NGS  
The current study has captured the whole microbial diversity concerning bacteria and yeast communities in milk kefir samples. Regarding the bacterial composition, _Lactobacillus_, specifically _L. kefirnafaciens_ was the most abundant bacterium in all five traditional kefir grains, accompanied by the genus _Lentilactobacillus_ (Figure 2A). In the beverage fraction of traditional kefirs, _L. kefirnafaciens_ also showed the highest relative abundance among bacterial species in kefirs FN and LS, while _L. lactis_ showed the highest relative abundance in kefirs PN1, PN2 and PN3. Unlike the grain-based kefirs, _Lactococcus_ spp. were the dominant bacterial genera in all three commercial kefirs (Figure 2B), besides a lower abundant population of _Lactonacci_ spp. in kefir CK3 and _Leuconostoc_ spp. and _Streptococcus_ (_S._) _thermophilus_ in kefir CK1. Regarding the beverage fraction, a considerable difference in the bacterial population was observed within traditional kefirs. Although the majority of studies have shown the prevalence of _L. lactis_ and _L. mesenteroides_ in kefir beverages [8], the highest abundance of _L. kefirnafaciens_ was surprisingly observed in the beverage fraction of kefirs FN and LS, detected using both targeted qPCR and untargeted NGS. Taxononical analysis using PacBio ITS sequencing revealed that traditional and commercial kefirs were dominated by yeasts _Kz.__turicensis, Kz.__unispora_ and _Kl.__marcianus_ (see Figure 3), which was in agreement to our previous findings [9]. Based on PacBio sequencing results, the commercial kefirs were dominated by one single _Kazachstania_ spp. These findings contradicted with previous studies on commercial kefirs [24], which reported higher community diversity in commercial kefirs [24]. Thus, we sought to validate our PacBio sequencing findings by ITS amplicon sequencing (supplementary method 1). Interestingly, the amplicon sequencing results showed a very high overlap for traditional kefirs, but not for the commercial kefirs. In fact, the amplicon sequencing revealed completely different fungal compositions in commercial kefirs. With amplicon sequencing of ITS2 we detected a completely different fungal composition in commercial kefirs, _Debaryomyces_ (D.) spp., with 99.68% sequence identity to _D. hansenii_ using Blastn [25] dominated the commercial kefirs (Figure S1). _D. hansenii_, which was identified with the highest relative abundance in all commercial kefirs amplicon sequencing (Figure S1) within this study, has also been found by [PERSON] et al. (2021) in high abundance in a Greek commercial kefir [24]. _Cidosporium cladosporoides_ was another species identified by amplicon sequencing in two commercial kefirs Ck1 and Ck3, and although it was not reported to be identified in sequencing of grain-based kefirs, it was detected only in a kefir sample produced by back sloping [8]. Figure 1: Counts of major bacteria and yeast species per unit of beverage (mL) or grain (g) fraction of five traditional kefits after \(4\sigma\pm 1\) h of fermentation and three commercial kefits (CK1, CK2,CK3) using qPCR. * Bacteria in grain, * Bacteria in beverage, * Yeast in grain, and - Yeast in beverage fractions, respectively. Bars represent mean \(\pm\) standard deviation (\(n=3\)). This discrepancy may derive from bias introduced by the sequencing procedures themselves, with DNA extraction, PCR amplification, sequencing, and identifying all contributing to the deviation from the real truth [26]. This bias may become even more pronounced in low biomass communities [27]. Based on our findings, and the results published by others [24], we assume the fungal communities in commercial kefirs have very low abundance.