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

Abstract

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.

Keywords

• metabolic activity; proliferation of lymphocytes; mouse embryonic fibroblasts; mouse splenocytes