Technology for evaluating the ability of probiotic strains to accumulate copper ions in vitro and in vivo using Bacillus bacteria
Authors: Sizentsov Aleksey , Salnikova Elena , Sizentsov Yaroslav , Peshkov Sergey , Barysheva Elena , Naumenko Olga , Romanenko Natalia 
Published: July 2021

Background and Aim: Microorganisms of the genus Bacillus comprising probiotics could have an antitoxic effect that is manifested in the active excretion of toxic substances from the body, as well as heavy metals. This study was conducted to assess the effects of copper ions on probiotic strains based on the members of the genus Bacillus in vitro and in vivo.

Materials and Methods: The following probiotic preparations were selected for this experiment: "Sporobacterin," "Bactisubtil," and "Vetom-2." Sporobacterin liquid, manufactured by Bakoren LLC, is based on Bacillus subtilis 534. Bactisubtil, manufactured by Marion Merrell Dow, Inc., is based on Bacillus cereus 5832. The first part of the experiment (in vitro) was conducted to assess the influence of copper ions on the growth and bioaccumulating ability of probiotic strains. The second part of the experiment (in vivo) was conducted to assess the influence of copper ions on the body of laboratory animals and to determine the ability of probiotic strains to remove copper ions from the body of laboratory animals. Statistical analysis was performed using Statistical Package for the Social Sciences, calculating the average value (M), standard deviation (σ), and standard deviation error (m). p<0.05 was used to denote statistical significance.

Results: In the previous studies, we found the presence of pronounced sorption characteristics of representatives of both the intestinal microbiome and probiotic strains based on them. In this study, we have studied the prospects of using physiological and adaptive mechanisms of resistance of transient probiotic strains in the system of correction of the elemental status of the animal body due to excessive intake of copper ions into the body. The advantage of their use is due to not only sorption but also the high levels of elimination of complexes accumulated on the surface. Analyzing the data, we can state the following. The excessive content of copper ions inhibits the activity of all microorganisms, and the presence of CuSO4 in the nutrient medium reduces the exponential growth phase by 6 h for B. licheniformis. The analysis of data on the bioaccumulating property of the probiotic strains under study shows that B. cereus (part of Bactisubtil) had the most pronounced sorbing effect with the level of accumulation of 23.96%.

Conclusion: We found that probiotic preparations do not affect biochemical indices of blood and biotissues (the muscle and bone tissue, and the cutaneous covering). As a result of determining the ability of Bacillus bacteria comprising the probiotics under this study to accumulate heavy metals by measuring their concentration in the tissues of laboratory animals, the preparations contribute to reducing the toxic effects of copper ions on the body. The cutaneous covering has the greatest accumulation property relative to copper ions. The most effective probiotic under this study in copper ion poisoning was Bactisubtil, and the least effective was Vetom-2.