Abstract

Microbiological indicators make it possible to reveal potential safety risks in the dairy industry. Bacteriophages affect the lysis of starter cultures because they can disrupt fermentation processes in dairy production. This study featured the seasonal factors that affect the phage status during dairy fermentation, the newly isolated bacteriophages, and the defense systems used by lactococci strains.
The research featured raw milk, cream, and skim milk; whole and skim milk powders; curd and cheese whey; strains of lactococci from different species with different phage resistance (Uglich Biofabrika Ltd; Bioresource Center of All-Russian Collection of Industrial Microorganisms); two new bacteriophages ph. 1622 and ph. 1623. The research relied on a number of standard microbiological, genetic, and mathematical methods. The mesophilic aerobic and anaerobic microbial count was performed by inoculation on a dense nutrient medium (State Standard 32904-2014) while the two-layer inoculation method revealed the bacteriophage titer. The genomic DNA analysis involved a phenol–chloroform extraction followed by precipitation with isopropanol and electrophoretic separation in agarose gel.
The experiments yielded reliable data on the quantitative change of phage particles in the raw material, the seasonal variability of mesophilic lactococci phages, and the genetics of the new industrial bacteriophages. The highest count of phage particles belonged to the samples obtained in the summer whereas the lowest was associated with the winter samples. The count of phage particles correlated with the bacterial contamination of the samples. The phage resistance index in Lactococcus lactis subsp. lactis, L. lactis subsp. cremoris, and L. lactis subsp. lactis biovar. diacetylactis had a seasonal character, the highest variability being recorded in L. lactis subsp. lactis, i.e., an acid former of starters. The DNA and amino acid sequences of phage proteins in phages ph. 1622 and ph. 1623 isolated from industrial samples made it possible to create panels of phage alternative strains.
The seasonal variability in lactic acid bacteria cultures and bacteriophage activity may affect the quality and safety of dairy products. The DNA of ph. 1622 and ph. 1623 differed in restriction patterns, which means they were distinct phages. Comparative genomics revealed their similarity to the well-known L. lactis-infecting c2 phage. The new phages exhibited different lactococcal cell infection mechanisms. The ph. 1623 genome insertion encoded an orphan DNA methyltransferase that could potentially suppress bacterial immune systems. Further research may reveal lactococcal phage sensitivity and defense mechanisms.

Keywords

Bacteriophage, safety, fermented dairy products, cheese, lactococci, starters

FUNDING

The research was supported by the Russian Science Foundation, grant no. 24-14-00181.

REFERENCES

1. Efimochkina NR. Etiology and epidemiological aspects of the most significant foodborne infections. Dairy industry. 2022;(2):30-33. (In Russ.) https://elibrary.ru/ZOQVMG
2. Goroschenko LG. Russian dairy market 2024: Price environment for fermented dairy products and sour cream. Dairy industry. 2024;(5):8-14. (In Russ.) https://elibrary.ru/FUNJVN
3. Ageeva NV, Kochetov VK, Litvinenko EYu. Practical solutions for implementation on enterprises of the food industry of the system food safety management. Izvestiya Vuzov. Food Technology. 2020;(2-3):104-107. (In Russ.) https://doi.org/10.26297/0579-3009.2020.2-3.27
4. Garda-Anaya MC, Sepulveda DR, Saenz-Mendoza AI, Rios-Velasco C, Zamudio-Flores PB, et al. Phages as biocontrol agents in dairy products. Trends in Food Science and Technology. 2020;95:10-20. https://doi.org/10.1016/j.tifs.2019.10.006
5. Sorokina NP, Kucherenko IV, Kuraeva EV, Kushnarenko LV. Up-to-date problems of the bacteria phages. Dairy industry. 2019;(2):32-34. (In Russ.) https://elibrary.ru/YYGBMD
6. Kazak AN, Vasylenko SL, Furik NN. Investigation of bacteriophage prevalence in fermented milk products. Topical Issues of Processing of Meat and Milk Raw Materials. 2013;(8):117-129. (In Russ.)
7. Ganina VI, Mashentseva NG, Ionova II. Bacteriophages of lactic acid bacteria. Food Processing: Techniques and Technology. 2022;52(2):361-374. (In Russ.) https:// doi.org/10.21603/2074-9414-2022-2-2371
8. Sorokina NP. On the problems in the sourdough business of Russia. Dairy industry. 2022;(4):7-10. (In Russ.) https://elibrary.ru/ NLQEOJ
9. Payne LJ, Meaden S, Mestre MR, Palmer C, Toro N, et al. PADLOC: A web server for the identification of antiviral defence systems in microbial genomes. Nucleic acids research. 2022;50(W1):W541-W550.
10. Chen S, Zhou Y, Chen Y, Gu J. fastp: An ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018;34(17):i884-i890. https://doi.org/10.1093/bioinformatics/bty560
11. Seemann T. Prokka: Rapid prokaryotic genome annotation. Bioinformatics. 2014;30(14):2068-2069. https://doi.org/10.1093/bioinformatics/btu153
12. Bouras R, Nepal G, Houtak G, Psaltis AJ, Wormald P-J, et al. Pharokka: A fast scalable bacteriophage annotation tool. Bioinformatics. 2023;39(1):btac776. https://doi.org/10.1093/bioinformatics/btac776
13. Ryabtseva SA, Ganina VI, Panova NM. Microbiology of milk and dairy products: A textbook for universities. Saint Petersburg: Lan’; 2021. 192 p. (In Russ.)
14. Pujato SA, Quiberoni AD, Mercanti J. Bacteriophages on dairy foods. Journal of Applied Microbiology. 2019;126(1): 14-30. https://doi.org/10.1111/jam.14062
15. Ganina VI. The temperature effect on the survival of bacteriophages in the biotechnology of fermented milk products. Dairy industry. 2020;(3):31-32. (In Russ.) https://doi.org/10.31515/1019- 8946-2020-03-32-33
16. Gerasimovich AD, Sidorenko AV. Bacteriophages of lactic acid bacteria Lactococcus lactis and their resistance to physicochemical factors. Microbial biotechnology: Fundamental and applied aspects: Collection of Sci. papers. Minsk, 2020;12:40-58. (In Russ.)
17. Gryaznova MV, Burakova IYu, Smirnova YuD, Nesterova EYu, Rodionova NS, et al. Bacterial composition of dairy base during fermentation. Food Processing: Techniques and Technology. 2023;53(3):554-564. (In Russ.) https://doi.org/10.21603/2074-9414-2023-3-2456
18. Lavelle K, Murphy J, Fitzgerald B, Lugli GA, Zomer A, et al. A decade of Streptococcus thermophilus phage evolution in an Irish dairy plant. Applied and Environmental Microbiology. 2018;84(10):e02855. https://doi.org/10.1128/ AEM.02855-17
19. Lapshevich I. How so? Bacteriophages - The invisible enemy of dairy products. Cheese- and Buttermaking. 2021;(3):16-17. (In Russ.) https://elibrary.ru/CXKKRB
20. Ganina VI. Critically dangerous number of phages in the biotechnology of fermented milk products. Dairy industry. 2022;(3):13-15. (In Russ.) https://doi.org/10.31515/1019-8946-2022-03-13-15
21. Jarvis AW, Lubbers MW, Waterfield NR, Collins LJ, Polzin KM. Sequencing and analysis of the genome of lactococcal phage c2. International Dairy Journal. 1995;5(8):963-976. https://doi.org/10.1016/0958-6946(95)00040-2
22. Cumby N, Edwards AM, Davidson AR, Maxwell KL. The bacteriophage HK97 gp15 moron element encodes a novel superinfection exclusion protein. Journal of Bacteriology. 2012;194(18):5012-5019. https://doi.org/10.1128/jb.00843-12
23. Murphy J, Mahony J, Ainsworth S, Nauta A, van Sinderen D. Bacteriophage orphan DNA methyltransferases: Insights from their bacterial origin, function, and occurrence. Applied and Environmental Microbiology. 2013;79(24):7547-7555. https://doi.org/10.1128/aem.02229-13