The Dynamics of the Microbial Community of a Fermented Milk Beverage Produced Using Kefir Grains

Introduction: Despite a significant amount of research devoted to microbial composition of kefir, our understanding of microbial community dynamics during fermentation remains fragmented. Most studies rely on 16S rRNA sequencing or traditional culture methods, which have limited resolution.

Purpose: To evaluate the dynamics of the microbial community composition of a fermented milk drink prepared using kefir grains throughout the entire technological process — from the activation stage of kefir grains to the formation of a product with an expired shelf life, using whole-genome metagenomic sequencing.

Materials and Methods: The study was conducted at the Laboratory of Applied Microbiology and Microbial Genomics, All-Russian Dairy Research Institute (VNIMI). The study objects were lyophilized kefir grains and kefir prepared from them, sampled at different stages of the fermentation process and subsequent storage. The objects of the study were lyophilized kefir grains and kefir prepared from them. Sampling was carried out at the following sequential stages: 0 h — lyophilized grains, 48 ​​h — metabolically active grains, 120 h — kefir starter culture, 122 h — addition of 5% starter culture to milk, 130 h — end of kefir fermentation at +20 °C, 138 h — finished kefir after maturation at +4 °C, and storage at +4 °C for 7, 14, and 21 days (n=10). Total DNA was isolated from each sample, which was used to prepare libraries and perform whole-genome metagenomic sequencing on the GeneMind FASTASeq300 platform in paired-end reading mode (2×150 bp) with a sequencing depth of at least 5 million reads per time point, followed by read filtering and bioinformatics analysis of the taxonomic structure of the microbial community with identification of microorganisms to the species level using Kraken2 and the PlusPF reference database, followed by reclassification and assessment of species representation using Bracken.

Results: Metagenomic analysis of the sequential stages of kefir grain activation, fermentation, and kefir storage revealed that microorganisms from the fungi sequentially colonize the liquid phase, where an ordered succession was observed: ~80% of the kefir grain microbial community was represented by Lactobacillus kefiranofaciens, while the dominant species in the starter culture and kefir were L. cremoris, L. helveticus, and L. lactis. The yeast component was represented by a single species, Kluyveromyces marxianus, whose maximum relative abundance did not exceed ~1.3% at all stages studied.

Conclusion: The dynamics of changes in the microbial composition of a fermented milk drink prepared using kefir grains were analyzed at different stages of fermentation and storage. The data obtained emphasizes the need for further research into the dynamics of the microbiome in kefir production to better understand its impact on the quality and stability of the final product. The results of the study may contribute to the development of more effective methods for managing the microbial community composition in fermented milk drinks, creating standardized starter cultures, and predicting the stability of fermented dairy product quality.

1. Дин Фань, Стоянова Л. Г., & Нетрусов А. И. (2022). Микробиом и метабиотические свойства кефирных зерен и кефиров на их основе. Микробиология, 91(4), 391–409. https://doi.org/10.31857/S0026365622100214

2. Abdalla, A. K., Ayyash, M. M., Olaimat, A. N., et al. (2021). Exopolysaccharides as antimicrobial agents: Mechanism and spectrum of activity. Frontiers in Microbiology, 12, 664395. https://doi.org/10.3389/fmicb.2021.664395

3. Alraddadi, F., Ross, T., & Powell, S. (2022). Evaluation of the microbial communities in kefir grains and kefir over time. International Dairy Journal, 136, 105490. https://doi.org/10.1016/j.idairyj.2022.105490

4. Avila-Reyes, S. V., Márquez-Morales, C. E., Moreno-León, G. R., Jiménez-Aparicio, A. R., Arenas-Ocampo, M. L., Solorza-Feria, J., García-Armenta, E., & Villalobos-Espinosa, J. C. (2022). Comparative analysis of fermentation conditions on the increase of biomass and morphology of milk kefir grains. Applied Sciences, 12(5), 2459. https://doi.org/10.3390/app12052459

5. Baars, T., van Esch, B., van Ooijen, L., Zhang, Z., Dekker, P., Boeren, S., et al. (2023). Raw milk kefir: Microbiota, bioactive peptides, and immune modulation. Food & Function, 14(2), 1000–1017. https://doi.org/10.1039/D2FO03248A

6. Biçer, Y., Telli, A. E., Turkal, G., Telli, N., & Uçar, G. (2025). From raw to fermented: Uncovering the microbial wealth of dairy. Fermentation, 11(10), 552. https://doi.org/10.3390/fermentation11100552

7. Blasche, S., Kim, Y., Mars, R. A. T., Machado, D., Maansson, M., Kafkia, E., et al. (2021). Metabolic cooperation and spatiotemporal niche partitioning in a kefir microbial community. Nature Microbiology, 6(2), 196–208. https://doi.org/10.1038/s41564-020-00816-5

8. Breitwieser, F. P., & Salzberg, S. L. (2019). Pavian: Interactive analysis of metagenomics data for microbiome studies and pathogen identification. Bioinformatics, 35(20), 4180–4182. https://doi.org/10.1093/bioinformatics/btz715

9. Dimitreli, G., Exarhopoulos, S., Apidopoulou, P., Groztidou, O., Georgiou, D., Kalogianni, E. P., & Goulas, A. (2025). Effect of final fermentation pH and pre-drying storage temperature on properties of kefir powder produced by kefir grains. Applied Sciences, 15(5), 2509. https://doi.org/10.3390/app15052509

10. Ding, F., Krasilnikova, A. A., Leontieva, M. R., Stoyanova, L. G., & Netrusov, A. I. (2022). Analysis of kefir grains from different regions of the planet using high-throughput sequencing. Moscow University Biological Sciences Bulletin, 77(4), 286–291. https://doi.org/10.3103/S0096392522040010

11. Dong, X., Shu, G., Kang, J., Zhang, Q., Ma, L., Zhang, M., Chen, H., & Wan, H. (2022). Microbial diversity of six commercially available kefir grains. Acta Universitatis Cibiniensis. Series E: Food Technology, 26, 287–292. https://doi.org/10.2478/aucft-2022-0023

12. Encinas-Vazquez, I. A., Carrillo-Pérez, E., Mártin-García, A. R., Del-Toro-Sánchez, C. L., Márquez-Ríos, E., Bastarrachea, L. J., & Rodríguez-Figueroa, J. C. (2023). Effects of high-intensity ultrasound pretreatment on the exopolysaccharide concentration and biomass increase in cheese whey kefir. Processes, 11(7), 1905. https://doi.org/10.3390/pr11071905

13. Gao, H., Li, X., Chen, X., Hai, D., Wei, C., Zhang, L., & Li, P. (2022). The functional roles of Lactobacillus acidophilus in different physiological and pathological processes. Journal of Microbiology and Biotechnology, 32(10), 1226–1233. https://doi.org/10.4014/jmb.2205.05041

14. González-Orozco, B. D., García-Cano, I., Escobar-Zepeda, A., Jiménez-Flores, R., & Álvarez, V. B. (2023). Metagenomic analysis and antibacterial activity of kefir microorganisms. Journal of Food Science, 88(7), 2933–2949. https://doi.org/10.1111/1750-3841.16614

15. Guron, G. K. P., Chelladhurai, K., et al. (2023). Differential behavior of Lactobacillus helveticus B1929 and ATCC 15009 during milk fermentations. Journal of Dairy Science, 106(7), 4502–4515. https://doi.org/10.3168/jds.2022-22769

16. Kalamaki, M. S., & Angelidis, A. S. (2020). High-throughput, sequence-based analysis of the microbiota of Greek kefir grains from two geographic regions. Food Technology and Biotechnology, 58(2), 138–146. https://doi.org/10.17113/ftb.58.02.20.6581

17. Kotova, I. B., Cherdyntseva, T. A., & Netrusov, A. I. (2016). Russian kefir grains microbial composition and its changes during production process. Advances in Experimental Medicine and Biology, 932, 93–121. https://doi.org/10.1007/5584_2016_2

18. Laureys, D., & De Vuyst, L. (2014). Microbial species diversity, community dynamics, and metabolite kinetics of water kefir fermentation. Applied and Environmental Microbiology, 80(8), 2564–2572. https://doi.org/10.1128/AEM.03978-13

19. Lu, J., Breitwieser, F. P., Thielen, P., & Salzberg, S. L. (2017). Bracken: Estimating species abundance in metagenomics data. PeerJ Computer Science, 3, e104. https://doi.org/10.7717/peerj-cs.104

20. Lu, J., Rincon, N., Wood, D. E., Breitwieser, F. P., Pockrandt, C., Langmead, B., Salzberg, S. L., & Steinegger, M. (2022). Metagenome analysis using the Kraken software suite. Nature Protocols, 18(2), 423–445. https://doi.org/10.1038/s41596-022-00738-y

21. Luo, J., Liu, S., Lu, H., Chen, Q., & Shi, Y. (2023). Microbial community variations and bioconversion improvements during soybean-based fermentation by kefir grains. Foods, 12(8), 1588. https://doi.org/10.3390/foods12081588

22. Marshall, V. M., Cole, W. M., & Brooker, B. E. (1984). Observations on the structure of kefir grains and the distribution of the microflora. Journal of Applied Bacteriology, 57(3), 491–497.

23. Martin, M. (2011). Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal, 17(1), 10–12. https://doi.org/10.14806/ej.17.1.200

24. McGovern, C. J., González-Orozco, B. D., & Jiménez-Flores, R. (2024). Evaluation of kefir grain microbiota, grain viability, and bioactivity from fermenting dairy processing by-products. Journal of Dairy Science, 107(7), 4259–4276. https://doi.org/10.3168/jds.2023-24364

25. Powell, J. E., Witthuhn, R. C., Todorov, S. D., & Dicks, L. M. T. (2007). Characterization of bacteriocin ST8KF produced by a kefir isolate Lactobacillus plantarum ST8KF. International Dairy Journal, 17(3), 190–198. https://doi.org/10.1016/j.idairyj.2006.02.012

26. Plessas, S., Nouska, C., Mantzourani, I., Kourkoutas, Y., Alexopoulos, A., & Bezirtzoglou, E. (2017). Microbiological Exploration of Different Types of Kefir Grains. Fermentation, 3(1), 1. https://doi.org/10.3390/fermentation3010001

27. Prado, M. R., Blandón, L. M., Vandenberghe, L. P., Rodrigues, C., Castro, G. R., Thomaz-Soccol, V., & Soccol, C. R. (2015). Milk kefir: Composition, microbial cultures, biological activities, and related products. Frontiers in Microbiology, 6, 1177. https://doi.org/10.3389/fmicb.2015.01177

28. Saleem, K., Ikram, S., Saeed, F., Afzaal, M., Ateeq, H., Hussain, M., et al. (2023). Nutritional and functional properties of kefir: Review. International Journal of Food Properties, 26, e2280437. https://doi.org/10.1080/10942912.2023.2280437

29. Ströher, J. A., Oliveira, W. d. C., de Freitas, A. S., Salazar, M. M., da Silva, L. d. F. F., Bresciani, L., Flôres, S. H., & Malheiros, P. d. S. (2025). A global review of geographical diversity of kefir microbiome. Fermentation, 11(3), 150. https://doi.org/10.3390/fermentation11030150

30. Tingirikari, J. M. R., Sharma, A., & Lee, H. J. (2024). Kefir: A fermented plethora of symbiotic microbiome and health. Journal of Ethnic Foods, 11, 35. https://doi.org/10.1186/s42779-024-00252-4

31. Walsh, L. H., Coakley, M., Walsh, A. M., Crispie, F., O’Toole, P. W., & Cotter, P. D. (2023). Analysis of the milk kefir pan-metagenome reveals four community types, core species, and associated metabolic pathways. iScience, 26(10), 108004. https://doi.org/10.1016/j.isci.2023.108004

32. Warakaulle, S., Ayyash, M. M., & Kamal-Eldin, A. (2025). Profiling protein hydrolysis and amino acid metabolism in camel and bovine milk fermented by Lactobacillus helveticus, L. bulgaricus, and Streptococcus thermophilus. Scientific Reports, 15(1), 18371. https://doi.org/10.1038/s41598-025-02944-6

33. Wood, D. E., Lu, J., & Langmead, B. (2019). Improved metagenomic analysis with Kraken 2. Genome Biology, 20, 257. https://doi.org/10.1186/s13059-019-1891-0

34. Zhao, J., Zeng, X., Xi, Y., & Li, J. (2024). Recent advances in the applications of Lactobacillus helveticus in the fermentation of plant-based beverages: A review. Trends in Food Science & Technology, 147, 104427. https://doi.org/10.1016/j.tifs.2024.104427