Aims: Development of an agar-diffusion assay to measure vitamin B2 in biological samples and application of the method to determine the amount of vitamin B2 secreted by bacteria.
Methods and results: A riboflavin-auxotrophic mutant of Bacillus cereus was generated by mini-Tn10 insertion in the ribD gene. ribD mutant sensitivity to exogenous vitamin B2 was investigated by turbidimetric and agar-diffusion assays. In turbidimetric assays, the B. cereus mutant displayed a similar level of sensitivity to vitamin B2 to that of Lactobacillus casei ATCC 7469, the reference organism used for microbiological vitamin B2 quantification. However, only the ribD mutant could be used as an indicator organism in agar-diffusion assays. A total of eight probiotic strains, from five different probiotic formulations, were analysed by the ribD mutant-based assay on agar plates in order to determine their ability to secrete vitamin B2 during growth.
Conclusion: The agar diffusion method with the ribD mutant of B. cereus is highly reproducible, sensitive, rapid, inexpensive, and can be applied to measure the amount of vitamin B2 in different samples.
Significance and impact of the study: The method developed in this study appears to be a good candidate for the screening of vitamin B2 secretion by bacteria growing on solid media.