Comparison of procedures for the extraction of supernatants and cytotoxicity tests in Vero cells, applied to assess the toxigenic potential of Bacillus spp. and Lactobacillus spp., intended for use as probiotic strains

Interest in using Bacillus strains as probiotic components of animal feeds has grown in recent years. However, some of these strains, especially those taxonomically related to the Bacillus cereus group, may have enterotoxigenic activity. Assessment of their toxigenic potential by well-established and robust protocols is required before authorizing their use in animal nutrition. Three methods of extraction and concentration of supernatants of Bacillus and Lactobacillus strains (methanol extraction, ammonium sulphate and ultrafiltration concentration) and three cytotoxic tests in Vero cells (WST-1, LDH and protein synthesis inhibition assays) for the assessment of the cytotoxicity activity of Lactobacillus strains (as probiotic strains in human and animal nutrition) and Bacillus toyonensis BCT-7112T (as animal probiotic strain in animal nutrition-Toyocerin®-) were evaluated in this study. Methanol extraction was not useful under any circumstances. The other two concentration methods (ammonium sulphate and ultrafiltration) were feasible, with slightly greater sensitivity achieved by ultrafiltration. The probiotic strain B. toyonensis BCT-7112T proved to be a non-cytotoxic strain in all the protocols tested. However, some Lactobacillus strains showed cytotoxicity activity, regardless of the protocols applied.