Cellular metabolic activity as a marker of cytotoxicity and immunotropicity of probiotics’ derivatives

Keywords: metabolic activity; proliferation of lymphocytes; mouse embryonic fibroblasts; mouse splenocytes


Structural components of cells and metabolites of probiotics with biologically active potential, along with the study of effectiveness, require a series of tests to ensure their safety. The study aims to test the cytotoxicity and potential of structural and metabolic derivatives of Bifidobacterium bifidum and Lactobacillus reuteri to affect the immunocompetent cells using in vitro tests that characterize the metabolic activity of test-cells. Structural components of probiotic bacteria were obtained by the physical method of disintegration – cyclic freezing-thawing. Metabolic derivatives were obtained by cultivation of producers – bifidobacteria and lactobacilli in their own disintegrates. Cultures of mouse embryonic fibroblasts and splenocytes were used as the test cells. MTT and Alamar Blue® were used as redox indicators. According to the MTT test, filtrates that contain structural and metabolic derivates at a concentration of 5% and 10% in the incubation medium did not cause significant changes in the metabolic activity of the embryonic mouse fibroblasts. An increase of up to 20% of content in the incubation medium of filtrates of lactobacilli disintegrates led to a reduction of metabolic activity of test cells by 52.7 ± 6.2%, of filtrates of bifidobacteria disintegrates – by 26.5 ± 6.5%, of filtrates of lactobacterium culture – by 15.7 ± 6.9%, of filtrates of bifidobacterium cultures - by 40.4 ± 6.8%. According to the Alamar Blue® test, filtrates that contained only structural derivatives of lactobacilli and bifidobacteria at concentrations of 5% and 10%, as well as filtrates that contained a complex of structural and metabolic derivatives at a concentration of 5%, did not cause significant changes in the reducing ability of mouse splenocytes. At concentrations of 10%, filtrate containing a complex of structural and metabolic derivatives of lactobacilli, caused the inhibition of metabolic activity of splenocytes by 14.6 ± 3.5%, and bifidobacteria – by 10.0 ± 2.8%. With the contents of the incubation medium at 20% concentration, the filtrate of the disintegrates of lactobacilli decreased the metabolic activity of splenocytes by 12.2 ± 3.0%, and the filtrate of lactobacillus cultures that were grown on their own disintegrates – by 43.2 ± 3.3%. Increasing the content of the disintegrate filtrate and the bifidobacteria culture that were grown on their own disintegrates in the culture medium by up to 20% led to a decrease the metabolic activity of splenocytes by 38.0 ± 2.0%. Thus, the research has shown: the orientation of changes in cellular metabolism under the influence of the studied biologically active derivatives is similar in all model systems, and their intensity depends on the type of test cells, regenerative substrates and concentration of the agent of influence in model systems. The obtained results stimulate further exploration of the immunotropicity of the investigated derivatives of probiotic bacteria and can be used for development of new immunobiological preparations.


Adan, A., Kiraz, Y., & Baran, Y. (2016). Cell proliferation and cytotoxicity as says. Current Pharmaceutical Biotechnology, 17(14), 1213–1221.

Al Kassaa, I. (Ed.). (2017). The antiviral activity of probiotic metabolites. In: New insights on antiviral probiotics. Springer, Cham.

Amrouche, T., Boutin, Y., & Fliss, I. (2006). Effects of bifidobacterial cytoplasm peptide and protein fractions on mouse lymphocyte proliferation and cyto kine production. Food and Agricultural Immunology, 17(1), 29–42.

Ang, L. Y. E., Too, H. K. I., Tan, E. L., Chow, T. K. V., Shek, P. C. L., Tham, E., & Alonso, S. (2016). Antiviral activity of Lactobacillus reuteri protectis against Coxsackievirus A and Enterovirus 71 infection in human skeletal muscle and colon cell lines. Virology Journal, 13(1), 111.

Arora, T., Mehta, A. K., Joshi, V., Mehta, K. D., Rathor, N., Mediratta, P. K., & Sharma, K. K. (2011). Substitute of animals in drug research: An approach towards fulfillment of 4R's. Indian Journal of Pharmaceutical Sciences, 73(1), 1.

Ashraf, R., & Shah, N. P. (2013). Immune system stimulation by probiotic micro organisms. Critical Reviews in Food Science and Nutrition, 54(7), 938–956.

Bauer, J. A., Salvagni, M., Vigroux, J. P. L., Chalvet, L. L. G., & Chiavaroli, C. (2010). Immunomodulatory extracts from Lactobacillus bacteria and me thods of manufacturing and use thereof. U.S. Patent No 20100055082A1. U.S. Patent and Trademark Office, Washington.

Castiblanco, G. A., Yucel-Lindberg, T., Roos, S., & Twetman, S. (2017). Effect of Lactobacillus reuteri on cell viability and PGE2 production in human gingi val fibroblasts. Probiotics and Antimicrobial Proteins, 9(3), 278–283.

Davila, J. C., Rodriguez, R. J., Melchert, R. B., & Acosta, J. D. (1998). Predictive value of in vitro model systems in toxicology. Annual Review of Pharmaco logy and Toxicology, 38(1), 63–96.

Fong, F. L. Y., Shah, N. P., Kirjavainen, P., & El-Nezami, H. (2015). Mechanism of action of probiotic bacteria on intestinal and systemic immunities and antigen-presenting cells. International Reviews of Immunology, 35(3), 179–188.

Griet, M., Zelaya, H., Mateos, M. V., Salva, S., Juarez, G. E., Font de Valdez, G., Villena, J., Salvador, G. A., & Rodriguez, A. V. (2014). Soluble factors from Lactobacillus reuteri CRL1098 have anti-inflammatory effects in acute lung injury induced by lipopolysaccharide in mice. PLoS One, 9(10), e110027.

Habriev, R. U. (2005). Rukovodstvo po eksperimental'nomu (doklinicheskomu) izucheniju novyh farmakologicheskih veshhestv [Manual on experimental (preclinical) study of new pharmacological substances]. Medicina, Moscow (in Russian).

Jensen, G. S., Benson, K. F., Carter, S. G., & Endres, J. R. (2010). GanedenBC 30™ cell wall and metabolites: anti-inflammatory and immune modulating effects in vitro. BMC Immunology, 11(1), 15.

Jozefczuk, J., Drews, K., & Adjaye, J. (2012). Preparation of mouse embryonic fibroblast cells suitable for culturing human embryonic and induced pluri potent stem cells. Journal of Visualized Experiments, 64.

Kang, H. J., & Connolly, E. (2016). Method of improving immune function in mammals using lactobacillus strains with certain lipids. U.S. Patent No 2016/0287702 A1. U.S. Patent and Trademark Office, Washington.

Kim, M. J., Lee, D. K., Park, J. E., Park, I. H., Seo, J. G., & Ha, N. J. (2014). Anti viral activity of Bifidobacterium adolescentis SPM1605 against Coxsackie virus B3. Biotechnology and Biotechnological Equipment, 28(4), 681–688.

Knysh, O. V., Isajenko, O. J., Babych, J. M., Poljans'ka, V. P., Zachepylo, S. V., Kompanijec', A. M., & Gorbach, T. V. (2018). Sposib oderzhannja biolo gichno aktyvnyh deryvativ bakterij probiotychnyh shtamiv [Method for ob taining biologically active derivatives of bacteria of probiotic strains]. Patent of Ukraine for useful model No 122859. Derzhavne Patentne Vidomstvo Ukrainy, Kyiv (in Ukrainian).

Kozlov, I. G., & Andronova, T. M. (2013). Lekarstvennyye vozdeystviya cherez retseptory vrozhdennogo immuniteta [Effect of medicines via innate immu nity receptors]. Allergologiya i Immunologiya, 14(4), 254–259 (in Russian).

Lee, D. K., Jang, S., Kim, M. J., Kim, J. H., Chung, M. J., Kim, K. J., & Ha, N. J. (2008). Anti-proliferative effects of Bifidobacterium adolescentis SPM0212 extract on human colon cancer cell lines. BMC Cancer, 8(1), 310.

Lew, L. C., & Liong, M. T. (2013). Bioactives from probiotics for dermal health: Functions and benefits. Journal of Applied Microbiology, 114(5), 1241–1253.

Motevaseli, E., Shirzad, M., Akrami, S. M., Mousavi, A. S., Mirsalehian, A., & Modarressi, M. H. (2013). Normal and tumour cervical cells respond differ rently to vaginal lactobacilli, independent of pH and lactate. Journal of Medical Microbiology, 62(7), 1065–1072.

O'brien, J., Wilson, I., Orton, T., & Pognan, F. (2000). Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cyto toxicity. The FEBS Journal, 267(17), 5421–5426.

Präbst, K., Engelhardt, H., Ringgeler, S., & Hübner, H. (2017). Basic colorimetric proliferation assays: MTT, WST, and resazurin. In: Gilbert, D., Friedrich, O. (Eds.). Cell Viability Assays. Methods in Molecular Biology, 1601. Humana Press, New York.

Presti, I., D’Orazio, G., Labra, M., La Ferla, B., Mezzasalma, V., Bizzaro, G., Giardina, S., Michelotti, A., Tursi, F., Vassallo, M., & Di Gennaro, P. (2015). Evaluation of the probiotic properties of new Lactobacillus and Bifidobacte rium strains and their in vitro effect. Applied Microbiology and Biotechno logy, 99(13), 5613–5626.

Rampersad, S. N. (2012). Multiple applications of Alamar Blue as an indicator of metabolic function and cellular health in cell viability bioassays. Sensors, 12(9), 12347–12360.

Richards, J. L., Yap, Y. A., McLeod, K. H., Mackay, C. R., & Mariño, E. (2016). Dietary metabolites and the gut microbiota: An alternative approach to control inflammatory and autoimmune diseases. Clinical and Translational Immunology, 5(5), e82.

Riss, T. L., Moravec R. A., Niles, A. L., Benink, H. A., Worzella, T. J., & Minor, L. (2016). Cell viability assays. In: Sittampalam, G. S., Coussens, N. P., Nelson, H., Arkin, M., Auld, D., Austin, C. et al. (Eds.). Assay guidance manual. Eli Lilly & Company, Bethesda.

Rivière, A., Selak, M., Lantin, D., Leroy, F., & De Vuyst, L. (2016). Bifidobacte ria and butyrate-producing colon bacteria: Importance and strategies for their stimulation in the human gut. Frontiers in Microbiology, 7, 979.

Shafran, L. M., Mokienko, A. V., Petrenko, N. F., Gozhenko, A. I., & Nasibullin, B. A. (2010). K obosnovaniyu gormezisa kak fundamentalnoy biomeditsin skoy paradigmy [On the substantiation of hormesis as fundamental biome dical paradigm]. Sovremennye Problemy Toksikologii, 49–50(2–3), 13–23 (in Russian).

Shaikh, A. M., & Sreeja, V. (2017). Metabiotics and their health benefits. Interna tional Journal of Fermented Foods, 6(1), 11–23.

Sharma, C., Singh, B. P., Thakur, N., Gulati, S., Gupta, S., Mishra, S. K., & Pan war, H. (2017). Antibacterial effects of Lactobacillus isolates of curd and human milk origin against food-borne and human pathogens. 3 Biotechnolo gy, 7(1), 31.

Sharma, M., & Shukla, G. (2016). Metabiotics: One step ahead of probiotics; an insight into mechanisms involved in anticancerous effect in colorectal cancer. Frontiers in Microbiology, 7, 1940.

Shenderov, B. A. (2013). Metabiotics: Novel idea or natural development of probiotic conception. Microbial Ecology in Health and Disease, 24(1), 20399.

Shenderov, B. A., & Gabrichevsky, G. N. (2017). Metabiotics: An overview of progress, opportunities and challenges. International Conference on Chronic Diseases & 6th International Conference on Microbial Physiology and Genomics Brussels, Belgium, 2017. Journal of Microbial and Biochemical Technology, 9(4 Suppl), 103.

Stefanov, О. V. (2001). Doklinichni doslidzhennia likars'kyh zasobiv [Preclinical studies of drugs]. Avicena, Kyiv (in Ukrainian).

Sultana, R., McBain, A. J., & O'Neill, C. A. (2013). Strain-dependent augmentati on of tight-junction barrier function in human primary epidermal keratinocy tes by Lactobacillus and Bifidobacterium lysates. Applied and Environmen tal Microbiology, 79(16), 4887–4894.

How to Cite
Knysh, O. V., Isayenko, O. Y., Falko, O. V., Babych, Y. M., Prokopyuk, V. Y., Prokopyuk, O. S., & Pogorila, M. S. (2018). Cellular metabolic activity as a marker of cytotoxicity and immunotropicity of probiotics’ derivatives. Regulatory Mechanisms in Biosystems, 9(2), 223-228. https://doi.org/https://doi.org/10.15421/021833