Abstract

Probiotic starters are a biological product based on lactic acid bacteria. Their metabolic characteristics determine the properties of the final products. This study evaluated the bacterial composition of a starter culture at various time intervals during the fermentation of a probiotic dairy product.
The starter consisted of Streptococcus salivarius ssp. thermophiles, Lactobacillus delbrueckii ssp. bulgaricus, Bifidobacterium bifidum, Bifidobactreium animalis ssp. lactis, Bifidobacterium longum, Lactobacillus acidophilus, and Lacticaseibacillus casei. Pasteurized milk served as the object of fermentation. The starter culture was activated in sterilized skimmed milk. Sampling occurred throughout the entire fermentation process (0, 3, 6, 9, 12, 15, and 18 h). To determine the microbiome of the substrates, the authors used the next-generation high-throughput sequencing that targeted V3 of 16S rRNA gene.
The fermentation resulted in a decrease in Bifidobacterium and an increase in Lactobacillus, which peaked (97.5%) after 15 h of fermentation. Each sampling showed that the count of Streptococcus went down. Eventually, Lactobacillus replaced all other genera, including Bifidobacterium, probably, as a result of pH going down during fermentation. The optimal values for the proliferation of Lactobacillus (pH = 4.2–4.4), which were registered after 18 h, turned out to be too low for the productive growth of Bifidobacterium.
The research demonstrated the changes in the bacterial composition of the dairy base during fermentation. The high-throughput sequencing proved to be an efficient tool in controlling probiotic fermentation processes.

Keywords

Bacterial composition, probiotic dairy product, fermentation, starter bacterial culture, Lactobacillus, Bifidobacterium, Streptococcus, 16S rRNA sequencing, Shannon index

FUNDING

The research was supported by the Ministry of Science and Higher Education of the Russian Federation (Minobrnauki) as part of the national project “Science” (FZGW-2020-0001, register no. 075001X39782002) and by the Russian Science Foundation (RSF) (project no. 21-74-00065) as part of studies of biodiversity of bacterial starter culture and fermentation products.

REFERENCES

1. Leeuwendaal NK, Stanton C, O'Toole PW, Beresford TP. Fermented foods, health and the gut microbiome. Nutrients. 2022;14(7). https://doi.org/10.3390/nu14071527
2. Mathur H, Beresford TP, Cotter PD. Health benefits of lactic acid bacteria (LAB) fermentates. Nutrients. 2020;12(6). https://doi.org/10.3390/nu12061679
3. Krasnova IS, Ganina VI, Semenov GV. Fruit and vegetable purees as cryoprotectants for vacuum freeze-dried fermented milk products. Foods and Raw Materials. 2023;11(2):300–308. https://doi.org/10.21603/2308-4057-2023-2-578
4. García-Díez J, Saraiva C. Use of starter cultures in foods from animal origin to improve their safety. International Journal of Environmental Research and Public Health. 2021;18(5). https://doi.org/10.3390/ijerph18052544
5. Yang Q, Rutherfurd-Markwick K, Mutukumira AN. Identification of dominant lactic acid bacteria and yeast in rice sourdough produced in New Zealand. Current Research in Food Science. 2021;4:729–736. https://doi.org/10.1016/j.crfs.2021.10.002
6. Freitas M. The benefits of yogurt, cultures, and fermentation. In: Floch MH, Ringel Y, Walker WA, editors. The microbiota in gastrointestinal pathophysiology. Implication human health, prebiotics, probiotics, dysbiosis. Academic Press; 2017. pp. 209–223. https://doi.org/10.1016/B978-0-12-804024-9.00024-0
7. İspirli H, Dertli E. Isolation and identification of exopolysaccharide producer lactic acid bacteria from Turkish yogurt. Journal of Food Processing and Preservation. 2018;42(8). https://doi.org/10.1111/jfpp.13351
8. Agustinah W, Warjoto RE, Canti M. Yogurt making as a tool to understand the food fermentation process for nonscience participants. Journal of Microbiology and Biology Education. 2019;20(1). https://doi.org/10.1128/jmbe.v20i1.1662
9. Saarela M, Mogensen G, Fondén R, Mättö J, Mattila-Sandholm T. Probiotic bacteria: safety, functional and technological properties. Journal of Biotechnology. 2000;84(3):197–215. https://doi.org/10.1016/s0168-1656(00)00375-8
10. Viability of probiotic Lactobacillus casei in yoghurt: defining the best processing step to its addition [Internet]. [cited 2023 Jan 16]. Available from: https://www.alanrevista.org/ediciones/2013/1/art-8
11. Gao J, Li X, Zhang G, Sadiq FA, Simal-Gandara J, Xiao J, et al. Probiotics in the dairy industry – Advances and opportunities. Comprehensive Reviews in Food Science and Food Safety. 2021;20(4):3937–3982. https://doi.org/10.1111/1541-4337.12755
12. Widyastuti Y, Febrisiantosa A, Tidona F. Health-promoting properties of lactobacilli in fermented dairy products. Frontiers in Microbiology. 2021;12. https://doi.org/10.3389/fmicb.2021.673890
13. Amanat S, Mazloomi SM, Asadimehr H, Sadeghi F, Shekouhi F, Mortazavi SMJ. Lactobacillus acidophilus and Lactobacillus casei exposed to Wi-Fi radiofrequency electromagnetic radiation show enhanced growth and lactic acid production. Journal of Biomedical Physics and Engineering. 2020;10(6):745–750. https://doi.org/10.31661/JBPE.V0I0.1056
14. Fijan S. Microorganisms with claimed probiotic properties: An overview of recent literature. International Journal of Environmental Research Public Health. 2014;11(5):4745–4767. https://doi.org/10.3390/ijerph110504745
15. Cai J, Bai J, Luo B, Nio Y, Tian E, Yan W. In vitro evaluation of probiotic properties and antioxidant activities of Bifidobacterium strains from infant feces in the Uyghur population of northwestern China. Annals of Microbiology. 2022;72. https://doi.org/10.1186/s13213-022-01670-y
16. O’Sullivan A, Farver M, Smilowitz JT. The influence of early infant-feeding practices on the intestinal microbiome and body composition in infants. Nutrition and Metabolic Insights. 2015;8(1). https://doi.org/10.4137/NMI.S29530
17. Cheng J, Laitila A, Ouwehand AC. Bifidobacterium animalis subsp. lactis HN019 effects on gut health: A review. Frontiers in Nutrition. 2021;8. https://doi.org/10.3389/fnut.2021.790561
18. Parhi P, Song KP, Choo WS. Growth and survival of Bifidobacterium breve and Bifidobacterium longum in various sugar systems with fructooligosaccharide supplementation. Journal of Food Science and Technology. 2022;59:3775–3786. https://doi.org/10.1007/s13197-022-05361-z
19. Mills S, Yang B, Smith GJ, Stanton C, Ross RP. Efficacy of Bifidobacterium longum alone or in multi-strain probiotic formulations during early life and beyond. Gut Microbes. 2023;15(1). https://doi.org/10.1080/19490976.2023.2186098
20. Salari A, Hashemi M, Afshari A. Functional properties of kefiran in the medical field and food Industry. Current Pharmaceutical Biotechnology. 2022;23(3):388–395. https://doi.org/10.2174/1389201022666210322121420
21. Landis EA, Oliverio AM, McKenney EA, Nichols LM, Kfoury N, Biango-Daniels, et al. The diversity and function of sourdough starter microbiomes. eLife. 2021;10. https://doi.org/10.7554/ELIFE.61644
22. Savaiano DA, Hutkins RW. Yogurt, cultured fermented milk, and health: A systematic review. Nutrition Reviews. 2021;79(5):599–614. https://doi.org/10.1093/NUTRIT/NUAA013
23. Salazar JK, Gonsalves LJ, Fay M, Ramachandran P, Schill KM, Tortorello ML. Metataxonomic profiling of native and starter microbiota during ripening of gouda cheese made with Listeria monocytogenes-contaminated unpasteurized milk. Frontiers in Microbiology. 2021;12. https://doi.org/10.3389/fmicb.2021.642789
24. Chessa L, Paba A, Dupré I, Daga E, Fozzi MC, Comunian R. Strategy for the recovery of raw ewe's milk microbiodiversity to develop natural starter cultures for traditional foods. Microorganisms. 2023;11(4). https://doi.org/10.3390/microorganisms11040823
25. Wu Y, Li Y, Gesudu Q, Zhang J, Sun Z, Halatu H, et al. Bacterial composition and function during fermentation of Mongolia koumiss. Food Science and Nutrition. 2021;9(8):4146–4155. https://doi.org/10.1002/fsn3.2377
26. Maleke M, Doorsamy W, Abrahams AM, Adefisoye MA, Masenya K, Adebo OA. Influence of fermentation conditions (temperature and time) on the physicochemical properties and bacteria microbiota of amasi. Fermentation. 2022;8(2). https://doi.org/10.3390/fermentation8020057
27. Zhu L, Hou Z, Hu X, Liu X, Dai T, Wang X, et al. Genomic and metabolic features of an unexpectedly predominant, thermophilic, assistant starter microorganism, Thermos thermophilus, in Chinese inner Mongolian cheese. Foods. 2021;10(12). https://doi.org/10.3390/foods10122962
28. Lee HW, Kim IS, Kil BJ, Seo E, Park H, Ham J-S, et al. Investigation of flavor-forming starter Lactococcus lactis subsp. lactis LDTM6802 and Lactococcus lactis subsp. cremoris LDTM6803 in miniature gouda-type cheeses. Journal of Microbiology and Biotechnology. 2020;30(9):1404–1411. https://doi.org/10.4014/jmb.2004.04004
29. Huang F, Sardari RRR, Jasilionis A, Böök O, Öste R, Rascón A, et al. Cultivation of the gut bacterium Prevotella copri DSM 18205T using glucose and xylose as carbon sources. MicrobiologyOpen. 2021;10(3). https://doi.org/10.1002/MBO3.1213
30. Verbrugghe P, Brynjólfsson J, Jing X, Björck I, Hållenius F, Nilsson A. Evaluation of hypoglycemic effect, safety and immunomodulation of Prevotella copri in mice. Scientific Reports. 2021;11. https://doi.org/10.1038/s41598-021-96161-6
31. Shah NP. Bacteria, beneficial | Bifidobacterium spp.: Morphology and physiology. In: Fuquay JW, editor. Encyclopedia of dairy sciences. Academic Press; 2011. pp. 381–387. https://doi.org/10.1016/B978-0-12-374407-4.00043-1
32. Song X, Hou C, Yang Y, Ai L, Xia Y, Wang G, et al. Effects of different carbon sources on metabolic profiles of carbohydrates in Streptococcus thermophilus during fermentation. Journal of the Science of Food and Agriculture. 2022;102(11):4820–4829. https://doi.org/10.1002/jsfa.11845
33. Liu G, Qiao Y, Zhang Y, Leng C, Chen H, Sun J, et al. Metabolic profiles of carbohydrates in Streptococcus thermophilus during pH-controlled batch fermentation. Frontiers in Microbiology. 2020;11. https://doi.org/10.3389/fmicb.2020.01131
34. Pi X, Yang Y, Sun Y, Cui Q, Wan Y, Fu G, et al. Recent advances in alleviating food allergenicity through fermentation. Critical Reviews in Food Science and Nutrition. 2022;62(26):7255–7268. https://doi.org/10.1080/10408398.2021.1913093
35. Ye M, Xu Z, Tan H, Yang F, Yuan J, Wu Y, et al. Allergenicity reduction of cow milk treated by alkaline protease combined with Lactobacillus plantarum and Lactobacillus helveticus based on epitopes. Food Chemistry. 2023;421. https://doi.org/10.1016/j.foodchem.2023.136180
36. Chen JM, Al KF, Craven LJ, Seney S, Coons M, McCormick H, et al. Nutritional, microbial, and allergenic changes during the fermentation of cashew “cheese” product using a quinoa-based rejuvelac starter culture. Nutrients. 2020;12(3). https://doi.org/10.3390/nu12030648
37. Gao J, Li X, Zhang G, Sadiq FA, Simal-Gandara J, Xiao J, et al. Probiotics in the dairy industry – Advances and opportunities. Comprehensive Reviews in Food Science and Food Safety. 2021;20(4):3937–3982. https://doi.org/10.1111/1541-4337.12755
38. Syromyatnikov M, Nesterova E, Gladkikh M, Popov V. Probiotics analysis by high-throughput sequencing revealed multiple mismatches at bacteria genus level with the declared and actual composition. LWT. 2022;156. https://doi.org/10.1016/j.lwt.2021.113055
39. Syromyatnikov MYu, Korneeva OS, Nesterova EYu, Gladkikh MI, Popov ES, Popov VN. High-throughput sequencing of the 16S rRNA gene for evaluation the composition of bacterial starter cultures used for the preparation of fermented milk products. Biotechnology in Russia. 2022;38(1):80–92. (In Russ.). https://doi.org/10.56304/S0234275822010082