Study On The Cytotoxicity Detection Method And Toxin Types Of Bacillus Metabolites

Bacillus spp.has the prebiotic effects of regulating intestinal flora,stimulating immunity,secreting digestive enzymes and other nutrients.Compared with other probiotics,Bacillus spp.has double advantages in heat resistance and shelf life,and has a wide range of applications in food,feed and medicine.However,Bacillus spp.is not always safe,there are potential safety hazards in the use of Bacillus spp..Recently,it has been found that Bacillus spp.used in food and feed produced toxins,which caused infectious diseases of the hosts.Based on the limitations and shortcomings of the existing in vitro toxicity detection methods of probiotics,we established a rapid detection method of the cytotoxicity of probiotic Bacillus spp.in the paper to provide technical support for the safety evaluation of Bacillus spp.and the breeding of probiotic Bacillus spp..Firstly,using Vero cells as the test cells and MTT colorimetry as cell activity assay method by comparing different cell models and cell activity assay methods to preliminarily establish the cytotoxicity test method.And optimizing the conditions of cytotoxicity test by determining the effects of different Bacillus spp.culture medium,different cytotoxicity test reaction system and reaction time on the cytotoxicity test results of Bacillus spp.metabolites.Secondly,the established cytotoxicity test method was used to detect the cytotoxicity of the crude extracts of 82 strains of Bacillus spp.(including 4 strains of Bacillus cereus,44 strains of Bacillus subtilis,15 strains of Bacillus amyloliquefaciens,10 strains of Bacillus licheniformis,6 strains of Bacillus velezensis,2 strains of Bacillus pumilus and one of Bacillus ginseng)which were selected from different sources by our laboratory.The results showed that among 82 strains of Bacillus spp.,17 strains had cytotoxicity with inhibition rate of Vero cell more than 20%,while 65 strains had no cytotoxicity with inhibition rate of Vero cell less than 20%.Finally,the toxicity of 17 Bacillus spp.strains was further determined by virulence gene PCR detection,hemolysis test and antibody immune response.The results showed that: four strains of Bacillus cereus with strong cytotoxicity carried all the coding genes of enterotoxin and could secrete enterotoxin,and was hemolytic;fve strains of Bacillus amyloliquefaciens,three strains of Bacillus subtilis and one strain of Bacillus velezensis only carried the coding gene hbl D of Hbl,and were not hemolytic;no toxin coding gene and enterotoxin were detected in the three strains of Bacillus velezensis and one strain of Bacillus amyloliquefaciens,and they were not hemolytic either.The thermal stability test of metabolites of Bacillus spp.showed that 13 strains of Bacillus spp.except Bacillus cereus secreted unknown toxins.The rapid detection method for the cytotoxicity of probiotic Bacillus spp.established in this experiment has the advantages of accuracy,sensitivity,short cycle,simple operation and large sample size.Compared with other methods in vitro,it has higher sensitivity in determining the toxicity of Bacillus spp.,which is suitable for different strains of Bacillus species at the cell level.