koz"Вестник Северо-Казахстанского университета имени Манаша Козыбаева"Bulletin of Manash Kozybayev North Kazakhstan University2958-003X2958-0048М. Қозыбаев атындағы СҚУ10.54596/2958-0048-2026-3-77-89koz-2618Research ArticleБИОЛОГИЧЕСКИЕ НАУКИBIOLOGICAL SCIENCESБИОТЕХНОЛОГИЧЕСКАЯ РАЗРАБОТКА КОРМОВЫХ ПРОБИОТИКОВ НА ОСНОВЕ BACILLUS SUBTILIS С ПРИМЕНЕНИЕМ ЛИОФИЛИЗАЦИИBIOTECHNOLOGICAL DEVELOPMENT OF FEED PROBIOTICS BASED ON BACILLUS SUBTILIS USING FREEZE-DRYINGhttps://orcid.org/0009-0002-1217-7754АбдулжановаМ. А.AbdulzhanovaM.

Алматы

corresponding author, PhD, senior lecturer

Almaty

nurassylbakh@gmail.comhttps://orcid.org/0000-0001-7751-2256БахытжанН. Н.BakhytzhanN.

Алматы

intern, student

Almaty

malika.abdulzhanova@kaznu.edu.kzНАО «Казахский национальный университет имени аль-Фараби»КазахстанNJC “Al-Farabi Kazakh national university”KazakhstanНАО «Казахский национальный университет имени аль-Фараби»; КГУ «Общеобразовательная школа №9» УО города АлматыКазахстанNJC “Al-Farabi Kazakh national university”; MPI “Secondary School No. 9”, Department of Education of AlmatyKazakhstan20263009202603 (71)7789Copyright © Абдулжанова М.А., Бахытжан Н.Н., 20262026Абдулжанова М.А., Бахытжан Н.Н.Abdulzhanova M., Bakhytzhan N.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://vestnik.ku.edu.kz/jour/article/view/2618

Данное исследование было проведено с целью оценки биотехнологического потенциала штамма Bacillus subtilis A-12 для разработки пробиотических кормовых добавок. В качестве основного метода хранения для обеспечения стабильности пробиотической биомассы была использована лиофилизация. В ходе исследования была оценена лиофилизационная толерантность штамма B. subtilis A-12 с использованием различных составов криопротекторных сред. В частности, были протестированы среда на основе желатина, сахарозы и молока, среда на основе сахарозы, среда на основе молока, среда на основе желатина и сахарозы и среда на основе желатина.

Результаты показали, что жизнеспособность клеток B. subtilis A-12 после лиофилизации в значительной степени зависела от состава криопротекторной среды. Среди испытанных составов среда на основе желатина и сахарозы обеспечивала наибольшую жизнеспособность клеток после лиофилизации и показала статистически значимое преимущество по сравнению со средами без углеводов (p < 0,05). Хотя количество жизнеспособных клеток после лиофилизации уменьшилось примерно на 25-35 %, дальнейшие потери составили всего 2-5 % при хранении в течение шести месяцев при температуре 4 °C. Это свидетельствует о долгосрочной стабильности полученной лиофилизированной биомассы.

Кроме того, было установлено, что спорообразующая природа штамма B. subtilis A-12 повышает его устойчивость к дегидратации и тепловому стрессу, что положительно влияет на стабильность лиофилизированного препарата. В результате исследования была разработана экспериментальная пробиотическая кормовая добавка на основе лиофилизированной биомассы B. subtilis A-12. Полученные данные свидетельствуют о перспективности разработанной технологии для практического применения и обосновывают необходимость оценки ее эффективности in vivo на животных моделях в будущем.

This study was conducted to assess the biotechnological potential of the Bacillus subtilis A-12 strain for the development of probiotic feed additives. Lyophilisation was employed as the primary storage method to ensure the stability of the probiotic biomass. During the study, the lyophilisation tolerance of the B. subtilis A-12 strain was assessed using various cryoprotective medium compositions. Specifically, a gelatin-sucrose-milk medium, a sucrose medium, a milk medium, a gelatin-sucrose medium, and a gelatin medium were tested.

The results showed that the post-lyophilisation viability of B. subtilis A-12 cells was significantly dependent on the composition of the protective medium. Among the tested formulations, the gelatin- and sucrose-based medium provided the highest cell viability after lyophilisation and showed a statistically significant advantage compared with carbohydrate-free media (p < 0.05). Although the number of viable cells decreased by approximately 25-35% following lyophilisation, further losses during storage for up to six months at 4°C were only 2-5%. This demonstrates the long-term stability of the obtained lyophilised biomass.

Furthermore, the sporulating nature of the B. subtilis A-12 strain was found to enhance its resistance to dehydration and thermal stress, positively affecting the stability of the lyophilised preparation. As a result of the study, an experimental probiotic feed additive based on lyophilised B. subtilis A-12 biomass was developed. The obtained data indicate that the developed technology is promising for practical application and justify the need to assess its in vivo efficacy in animal models in the future.

Bacillus subtilisпробиотикилиофилизациякриопротекторные средыжизнеспособностькормовые добавкиBacillus subtilisprobioticsfreeze-dryingcryoprotective mediacell viabilityfeed additivesReferencesVieco-Saiz N., Lemâle O., Evans N. P., Quinteiro-Filho W. M., Mellouk A., Consuegra J., Yakout H., Goossens T. (2025). Winning the battle of intestinal peace with Bacillus — a multifaceted approach to animal health, immunity, and future applications in monogastric livestock production [Text] / N. Vieco-Saiz et al. // Frontiers in Microbiology. – Vol. 16. – Art. 1711747. https://doi.org/10.3389/fmicb.2025.1711747.Vieco-Saiz N., Lemâle O., Evans N. P., Quinteiro-Filho W. M., Mellouk A., Consuegra J., Yakout H., Goossens T. (2025). Winning the battle of intestinal peace with Bacillus — a multifaceted approach to animal health, immunity, and future applications in monogastric livestock production [Text] / N. Vieco-Saiz et al. // Frontiers in Microbiology. – Vol. 16. – Art. 1711747. https://doi.org/10.3389/fmicb.2025.1711747.Šimunović K., Rezar V., Štefanič P. et al. (2025). Probiotic effect of Bacillus subtilis PS-216 in broiler chickens: modulation of weight, feed conversion, short-chain fatty acids, microbiota and meat quality [Text] / K. Šimunović et al. // Animal Microbiome. – Vol. 7. – Art. 121. https://doi.org/10.1186/s42523-025-00489-7.Šimunović K., Rezar V., Štefanič P. et al. (2025). Probiotic effect of Bacillus subtilis PS-216 in broiler chickens: modulation of weight, feed conversion, short-chain fatty acids, microbiota and meat quality [Text] / K. Šimunović et al. // Animal Microbiome. – Vol. 7. – Art. 121. https://doi.org/10.1186/s42523-025-00489-7.Cai Y., Xiao C., Tian B., Sandvang D., Meuter A., Song B., Song Z. (2024). Dietary probiotic based on a dual-strain Bacillus subtilis improves immunity, intestinal health, and growth performance of broiler chickens [Text] / Y. Cai et al. // Journal of Animal Science. – Vol. 102. – Art. skae183. https://doi.org/10.1093/jas/skae183.Cai Y., Xiao C., Tian B., Sandvang D., Meuter A., Song B., Song Z. (2024). Dietary probiotic based on a dual-strain Bacillus subtilis improves immunity, intestinal health, and growth performance of broiler chickens [Text] / Y. Cai et al. // Journal of Animal Science. – Vol. 102. – Art. skae183. https://doi.org/10.1093/jas/skae183.Eyre K. E., Pan L., Harper K., Prada e Silva L. F. (2025). The effect of a Bacillus-based probiotic on feed intake and digestibility of a forage and a feedlot diet in Bos indicus steers [Text] / K. E. Eyre et al. // Veterinary and Animal Science. – Vol. 29. – Art. 100463.Eyre K. E., Pan L., Harper K., Prada e Silva L. F. (2025). The effect of a Bacillus-based probiotic on feed intake and digestibility of a forage and a feedlot diet in Bos indicus steers [Text] / K. E. Eyre et al. // Veterinary and Animal Science. – Vol. 29. – Art. 100463.Park J. M., An B. K., Yang T. S., Kim K. E. (2024). Effects of single or combined feeding with Bacillus subtilis and organic acid mixture on egg production, physiological responses, and cholesterol level in chicken eggs [Text] / J. M. Park et al. // Journal of Animal and Feed Sciences. – Vol. 33(4). – P. 486–492. https://doi.org/10.22358/jafs/186959/2024.Park J. M., An B. K., Yang T. S., Kim K. E. (2024). Effects of single or combined feeding with Bacillus subtilis and organic acid mixture on egg production, physiological responses, and cholesterol level in chicken eggs [Text] / J. M. Park et al. // Journal of Animal and Feed Sciences. – Vol. 33(4). – P. 486–492. https://doi.org/10.22358/jafs/186959/2024.Ramlucken U., Lalloo R., Roets Y., Moonsamy G., van Rensburg C. Jansen, Thantsha M. S. (2020). Advantages of Bacillus-based probiotics in poultry production [Text] / U. Ramlucken et al. // Livestock Science. – Vol. 241. – Art. 104215.Ramlucken U., Lalloo R., Roets Y., Moonsamy G., van Rensburg C. Jansen, Thantsha M. S. (2020). Advantages of Bacillus-based probiotics in poultry production [Text] / U. Ramlucken et al. // Livestock Science. – Vol. 241. – Art. 104215.Calaca A. M. M., Figueiredo C. B., Silva M. B., Fernandes J. J. R., Fernandes M. H. M. R., Silva L. F., Couto V. R. M. (2022). Effect of a Bacillus probiotic strain on Nellore cattle finished on pasture during the dry season [Text] / A. M. M. Calaca et al. // Livestock Science. – Vol. 264. – Art. 105068. https://doi.org/10.1016/j.livsci.2022.105068.Calaca A. M. M., Figueiredo C. B., Silva M. B., Fernandes J. J. R., Fernandes M. H. M. R., Silva L. F., Couto V. R. M. (2022). Effect of a Bacillus probiotic strain on Nellore cattle finished on pasture during the dry season [Text] / A. M. M. Calaca et al. // Livestock Science. – Vol. 264. – Art. 105068. https://doi.org/10.1016/j.livsci.2022.105068.Wang B., Middleton A., Gibson R., Khanolkar J., Klymenko O., Wu C.-Y. (2024). Understanding lethal mechanisms and preventive strategies in probiotic tablet production – a review [Text] / B. Wang et al. // Powder Technology. – Vol. 443. – Art. 119905. https://doi.org/10.1016/j.powtec.2024.119905.Wang B., Middleton A., Gibson R., Khanolkar J., Klymenko O., Wu C.-Y. (2024). Understanding lethal mechanisms and preventive strategies in probiotic tablet production – a review [Text] / B. Wang et al. // Powder Technology. – Vol. 443. – Art. 119905. https://doi.org/10.1016/j.powtec.2024.119905.Cutting S. M. (2011). Bacillus probiotics [Text] / S. M. Cutting // Food Microbiology. – Vol. 28(2). – P. 214–220. https://doi.org/10.1016/j.fm.2010.03.007.Cutting S. M. (2011). Bacillus probiotics [Text] / S. M. Cutting // Food Microbiology. – Vol. 28(2). – P. 214–220. https://doi.org/10.1016/j.fm.2010.03.007.Osho S., Bolek K., Saddoris-Clemons K., Humphrey B., Garcia M. (2023). Impact of a direct-fed microbial supplementation on intestinal permeability and immune response in broiler chickens during a coccidia challenge [Text] / S. Osho et al. // Frontiers in Microbiology. – Vol. 14. – Art. 1283393. https://doi.org/10.3389/fmicb.2023.1283393.Osho S., Bolek K., Saddoris-Clemons K., Humphrey B., Garcia M. (2023). Impact of a direct-fed microbial supplementation on intestinal permeability and immune response in broiler chickens during a coccidia challenge [Text] / S. Osho et al. // Frontiers in Microbiology. – Vol. 14. – Art. 1283393. https://doi.org/10.3389/fmicb.2023.1283393.Rahman M. R. T., Fliss I., Biron E. (2022). Insights in the development and uses of alternatives to antibiotic growth promoters in poultry and swine production [Text] / M. R. T. Rahman et al. // Antibiotics (Basel). – Vol. 11(6). – Art. 766. https://doi.org/10.3390/antibiotics11060766.Rahman M. R. T., Fliss I., Biron E. (2022). Insights in the development and uses of alternatives to antibiotic growth promoters in poultry and swine production [Text] / M. R. T. Rahman et al. // Antibiotics (Basel). – Vol. 11(6). – Art. 766. https://doi.org/10.3390/antibiotics11060766.Idowu P. A., Mbambalala L., Idowu A. P. (2025). Probiotics as sustainable alternatives to antibiotic growth promoters: mechanisms, applications, and future perspectives in livestock production [Text] / P. A. Idowu et al. // Letters in Animal Biology. – Vol. 5(1). – P. 129–142. https://doi.org/10.62310/liab.v5i1.258.Idowu P. A., Mbambalala L., Idowu A. P. (2025). Probiotics as sustainable alternatives to antibiotic growth promoters: mechanisms, applications, and future perspectives in livestock production [Text] / P. A. Idowu et al. // Letters in Animal Biology. – Vol. 5(1). – P. 129–142. https://doi.org/10.62310/liab.v5i1.258.Абдулжанова М. А., Бахытжан Н. Н., Мирзагаликова Б. В., Доктыбай Г. (2025). Скрининг и характеристика антагонистически и ферментативно активных штаммов Bacillus для пробиотического применения в птицеводстве [Text] / М. А. Абдулжанова и др. // Ғылым және білім. Наука и образование. Science and Education. – № 3-1 (80). – P. 134–144. https://doi.org/10.52578/2305-9397-2025-3-1-134-144. (In Russian)Абдулжанова М. А., Бахытжан Н. Н., Мирзагаликова Б. В., Доктыбай Г. (2025). Скрининг и характеристика антагонистически и ферментативно активных штаммов Bacillus для пробиотического применения в птицеводстве [Text] / М. А. Абдулжанова и др. // Ғылым және білім. Наука и образование. Science and Education. – № 3-1 (80). – P. 134–144. https://doi.org/10.52578/2305-9397-2025-3-1-134-144. (In Russian)Santivarangkna C., Higl B., Foerst P. (2008). Protection mechanisms of sugars during different stages of preparation process of dried lactic acid starter cultures [Text] / C. Santivarangkna et al. // Food Microbiology. – Vol. 25(3). – P. 429–441. https://doi.org/10.1016/j.fm.2007.12.004.Santivarangkna C., Higl B., Foerst P. (2008). Protection mechanisms of sugars during different stages of preparation process of dried lactic acid starter cultures [Text] / C. Santivarangkna et al. // Food Microbiology. – Vol. 25(3). – P. 429–441. https://doi.org/10.1016/j.fm.2007.12.004.Berner D., Viernstein H. (2006). Effect of protective agents on the viability of Lactococcus lactis subjected to freeze-thawing and freeze-drying [Text] / D. Berner, H. Viernstein // Scientia Pharmaceutica. – Vol. 74. – P. 137–149.Berner D., Viernstein H. (2006). Effect of protective agents on the viability of Lactococcus lactis subjected to freeze-thawing and freeze-drying [Text] / D. Berner, H. Viernstein // Scientia Pharmaceutica. – Vol. 74. – P. 137–149.Abadias M., Benabarre A., Teixidò N., Usall J., Viñas I. (2001). Effect of freeze drying and protectants on viability of the biocontrol yeast Candida sake [Text] / M. Abadias et al. // International Journal of Food Microbiology. – Vol. 65. – P. 173–182.Abadias M., Benabarre A., Teixidò N., Usall J., Viñas I. (2001). Effect of freeze drying and protectants on viability of the biocontrol yeast Candida sake [Text] / M. Abadias et al. // International Journal of Food Microbiology. – Vol. 65. – P. 173–182.Savedboworn W., Teawsomboonkit K., Surichay S., Riansa-Ngawong W., Rittisak S., Charoen R., Phattayakorn K. (2018). Impact of protectants on the storage stability of freeze-dried probiotic Lactobacillus plantarum [Text] / W. Savedboworn et al. // Food Science and Biotechnology. – Vol. 28(3). – P. 795–805. https://doi.org/10.1007/s10068-018-0523-x.Savedboworn W., Teawsomboonkit K., Surichay S., Riansa-Ngawong W., Rittisak S., Charoen R., Phattayakorn K. (2018). Impact of protectants on the storage stability of freeze-dried probiotic Lactobacillus plantarum [Text] / W. Savedboworn et al. // Food Science and Biotechnology. – Vol. 28(3). – P. 795–805. https://doi.org/10.1007/s10068-018-0523-x.Wang J., Wu P., Dhital S., Yu A., Chen X. D. (2025). Impact of freezing and freeze drying on Lactobacillus rhamnosus GG survival: mechanisms of cell damage and the role of pre-freezing conditions and cryoprotectants [Text] / J. Wang et al. // Foods. – Vol. 14. – Art. 1817. https://doi.org/10.3390/foods14101817.Wang J., Wu P., Dhital S., Yu A., Chen X. D. (2025). Impact of freezing and freeze drying on Lactobacillus rhamnosus GG survival: mechanisms of cell damage and the role of pre-freezing conditions and cryoprotectants [Text] / J. Wang et al. // Foods. – Vol. 14. – Art. 1817. https://doi.org/10.3390/foods14101817.Shapiro S. S., Wilk M. B. (1965). An analysis of variance test for normality (complete samples) [Text] / S. S. Shapiro, M. B. Wilk // Biometrika. – Vol. 52(3–4). – P. 591–611. https://doi.org/10.2307/2333709.Shapiro S. S., Wilk M. B. (1965). An analysis of variance test for normality (complete samples) [Text] / S. S. Shapiro, M. B. Wilk // Biometrika. – Vol. 52(3–4). – P. 591–611. https://doi.org/10.2307/2333709.Levene H. (1960). Robust tests for equality of variances [Text] / H. Levene // In: Contributions to Probability and Statistics. Stanford University Press, Palo Alto. – P. 278–292.TUKEY JW. Comparing individual means in the analysis of variance. Biometrics. 1949 Jun;5(2):99-114. PMID: 18151955.Levene H. (1960). Robust tests for equality of variances [Text] / H. Levene // In: Contributions to Probability and Statistics. Stanford University Press, Palo Alto. – P. 278–292.TUKEY JW. Comparing individual means in the analysis of variance. Biometrics. 1949 Jun;5(2):99-114. PMID: 18151955.Kruskal W. H., Wallis W. A. (1952). Use of ranks in one-criterion variance analysis [Text] / W. H. Kruskal, W. A. Wallis // Journal of the American Statistical Association. – Vol. 47(260). – P. 583–621. https://doi.org/10.2307/2280779.Kruskal W. H., Wallis W. A. (1952). Use of ranks in one-criterion variance analysis [Text] / W. H. Kruskal, W. A. Wallis // Journal of the American Statistical Association. – Vol. 47(260). – P. 583–621. https://doi.org/10.2307/2280779.Virtanen P., Gommers R., Oliphant T. E. et al. (2020). SciPy 1.0: fundamental algorithms for scientific computing in Python [Text] / P. Virtanen et al. // Nature Methods. – Vol. 17. – P. 261–272. https://doi.org/10.1038/s41592-019-0686-2.Virtanen P., Gommers R., Oliphant T. E. et al. (2020). SciPy 1.0: fundamental algorithms for scientific computing in Python [Text] / P. Virtanen et al. // Nature Methods. – Vol. 17. – P. 261–272. https://doi.org/10.1038/s41592-019-0686-2.McKinney W. (2010). Data structures for statistical computing in Python [Text] / W. McKinney // Proceedings of the 9th Python in Science Conference. Austin, USA. – P. 56–61. https://doi.org/10.25080/Majora-92bf1922-00a.McKinney W. (2010). Data structures for statistical computing in Python [Text] / W. McKinney // Proceedings of the 9th Python in Science Conference. Austin, USA. – P. 56–61. https://doi.org/10.25080/Majora-92bf1922-00a.Oluwatosin S.O., Tai S.L., Fagan-Endres M.A. (2022). Sucrose, maltodextrin and inulin efficacy as cryoprotectant, preservative and prebiotic – towards a freeze dried Lactobacillus plantarum topical probiotic [Text] / S.O. Oluwatosin, S.L. Tai, M.A. Fagan-Endres // Biotechnology Reports. – Vol. 33. – e00696. https://doi.org/10.1016/j.btre.2021.e00696.Oluwatosin S.O., Tai S.L., Fagan-Endres M.A. (2022). Sucrose, maltodextrin and inulin efficacy as cryoprotectant, preservative and prebiotic – towards a freeze dried Lactobacillus plantarum topical probiotic [Text] / S.O. Oluwatosin, S.L. Tai, M.A. Fagan-Endres // Biotechnology Reports. – Vol. 33. – e00696. https://doi.org/10.1016/j.btre.2021.e00696.Bagad M., Pande R., Dubey V., Ghosh A. R. (2017). Survivability of freeze-dried probiotic Pediococcus pentosaceus strains GS4, GS17 and Lactobacillus gasseri (ATCC 19992) during storage with commonly used pharmaceutical excipients within a period of 120 days [Text] / M. Bagad et al. // Asian Pacific Journal of Tropical Biomedicine. – Vol. 7(10). – P. 921–929. https://doi.org/10.1016/j.apjtb.2017.09.005.Bagad M., Pande R., Dubey V., Ghosh A. R. (2017). Survivability of freeze-dried probiotic Pediococcus pentosaceus strains GS4, GS17 and Lactobacillus gasseri (ATCC 19992) during storage with commonly used pharmaceutical excipients within a period of 120 days [Text] / M. Bagad et al. // Asian Pacific Journal of Tropical Biomedicine. – Vol. 7(10). – P. 921–929. https://doi.org/10.1016/j.apjtb.2017.09.005.Li C., Zhao K., Ma L., Zhao J., Zhao Z. M. (2022). Effects of drying strategies on sporulation and titer of microbial ecological agents with Bacillus subtilis [Text] / C. Li et al. // Frontiers in Nutrition. – Vol. 9. – Art. 1025248. https://doi.org/10.3389/fnut.2022.1025248.Li C., Zhao K., Ma L., Zhao J., Zhao Z. M. (2022). Effects of drying strategies on sporulation and titer of microbial ecological agents with Bacillus subtilis [Text] / C. Li et al. // Frontiers in Nutrition. – Vol. 9. – Art. 1025248. https://doi.org/10.3389/fnut.2022.1025248.Setlow P. (2006). Spores of Bacillus subtilis: their resistance to and killing by radiation, heat and chemicals [Text] / P. Setlow // Journal of Applied Microbiology. – Vol. 101. – P. 514–525. https://doi.org/10.1111/j.1365-2672.2005.02736.x.Setlow P. (2006). Spores of Bacillus subtilis: their resistance to and killing by radiation, heat and chemicals [Text] / P. Setlow // Journal of Applied Microbiology. – Vol. 101. – P. 514–525. https://doi.org/10.1111/j.1365-2672.2005.02736.x.Bodzen A., Jossier A., Dupont S., Mousset P. Y., Beney L., Lafay S., Gervais P. (2021). Increased survival of Lactococcus lactis strains subjected to freeze-drying after cultivation in an acid medium: involvement of membrane fluidity [Text] / A. Bodzen et al. // Food Technology and Biotechnology. – Vol. 59(4). – P. 443–453. https://doi.org/10.17113/ftb.59.04.21.7076.Bodzen A., Jossier A., Dupont S., Mousset P. Y., Beney L., Lafay S., Gervais P. (2021). Increased survival of Lactococcus lactis strains subjected to freeze-drying after cultivation in an acid medium: involvement of membrane fluidity [Text] / A. Bodzen et al. // Food Technology and Biotechnology. – Vol. 59(4). – P. 443–453. https://doi.org/10.17113/ftb.59.04.21.7076.Nguyen H. T., Truong D. H., Kouhoundé S., Ly S., Razafindralambo H., Delvigne F. (2016). Biochemical engineering approaches for increasing viability and functionality of probiotic bacteria [Text] / H. T. Nguyen et al. // International Journal of Molecular Sciences. – Vol. 17(6). – Art. 867. https://doi.org/10.3390/ijms17060867.Nguyen H. T., Truong D. H., Kouhoundé S., Ly S., Razafindralambo H., Delvigne F. (2016). Biochemical engineering approaches for increasing viability and functionality of probiotic bacteria [Text] / H. T. Nguyen et al. // International Journal of Molecular Sciences. – Vol. 17(6). – Art. 867. https://doi.org/10.3390/ijms17060867.Van Engeland C., Haut B., Debaste F. (2025). A closer look at the potential mechanisms of action of protective agents used in the drying of microorganisms: a review [Text] / C. Van Engeland et al. // Microorganisms. – Vol. 13(2). – Art. 435. https://doi.org/10.3390/microorganisms13020435.Van Engeland C., Haut B., Debaste F. (2025). A closer look at the potential mechanisms of action of protective agents used in the drying of microorganisms: a review [Text] / C. Van Engeland et al. // Microorganisms. – Vol. 13(2). – Art. 435. https://doi.org/10.3390/microorganisms13020435.Guyvinska S. O., Paliy A. P., Dunaeva O. V., Paliy A. P., Berezhna N. V. (2018). Biotechnology production of medium for cultivation and lyophilization of lactic acid bacteria [Text] / S. O. Guyvinska et al. // Ukrainian Journal of Ecology. – Vol. 8(2). – P. 5–11. https://doi.org/10.15421/2018_302.Guyvinska S. O., Paliy A. P., Dunaeva O. V., Paliy A. P., Berezhna N. V. (2018). Biotechnology production of medium for cultivation and lyophilization of lactic acid bacteria [Text] / S. O. Guyvinska et al. // Ukrainian Journal of Ecology. – Vol. 8(2). – P. 5–11. https://doi.org/10.15421/2018_302.Грачева И. В., Осин А. В. (2016). Механизмы повреждений бактерий при лиофилизации и протективное действие защитных сред [Text] / И. В. Грачева, А. В. Осин // Проблемы особо опасных инфекций. – № 3. – P. 5–12. https://doi.org/10.21055/0370-1069-2016-3-5-12. (In Russian)Грачева И. В., Осин А. В. (2016). Механизмы повреждений бактерий при лиофилизации и протективное действие защитных сред [Text] / И. В. Грачева, А. В. Осин // Проблемы особо опасных инфекций. – № 3. – P. 5–12. https://doi.org/10.21055/0370-1069-2016-3-5-12. (In Russian)Betoret E., Betoret N., Calabuig-Jiménez L., Barrera C., Dalla Rosa M. (2020). Effect of drying process, encapsulation, and storage on the survival rates and gastrointestinal resistance of Lactobacillus salivarius spp. salivarius included into a fruit matrix [Text] / E. Betoret et al. // Microorganisms. – Vol. 8. – Art. 654. https://doi.org/10.3390/microorganisms8050654.Betoret E., Betoret N., Calabuig-Jiménez L., Barrera C., Dalla Rosa M. (2020). Effect of drying process, encapsulation, and storage on the survival rates and gastrointestinal resistance of Lactobacillus salivarius spp. salivarius included into a fruit matrix [Text] / E. Betoret et al. // Microorganisms. – Vol. 8. – Art. 654. https://doi.org/10.3390/microorganisms8050654.Pyar H. (2016). Investigation of cryoprotection on the viability of freeze-dried probiotics [Text] / H. Pyar // Malaysian Journal of Microbiology. – Art. S02116. https://doi.org/10.21161/MJM.S02116.Pyar H. (2016). Investigation of cryoprotection on the viability of freeze-dried probiotics [Text] / H. Pyar // Malaysian Journal of Microbiology. – Art. S02116. https://doi.org/10.21161/MJM.S02116.García-Robles G., Fajardo-Robledo N. S., Ruvalcaba-Gómez J. M., Solís-Pacheco J. R., Villarruel-López A., Aguilar-Uscanga B. R. (2025). Assessment of the stability and viability of a probiotic formulation with strains of Lactiplantibacillus LH05 and LH06 isolated from breast milk [Text] / G. García-Robles et al. // Journal of Food Science & Nutrition. – Art. 100222. https://doi.org/10.24966/FSN-1076/100222.García-Robles G., Fajardo-Robledo N. S., Ruvalcaba-Gómez J. M., Solís-Pacheco J. R., Villarruel-López A., Aguilar-Uscanga B. R. (2025). Assessment of the stability and viability of a probiotic formulation with strains of Lactiplantibacillus LH05 and LH06 isolated from breast milk [Text] / G. García-Robles et al. // Journal of Food Science & Nutrition. – Art. 100222. https://doi.org/10.24966/FSN-1076/100222.

The authors declare that there are no conflicts of interest present.