A riboswitch sensor to determine vitamin B12 in fermented foods

We describe a sensitive and selective method for determination of vitamin B12 content in fermented foods using riboswitch sensor. A riboswitch amplicon from Propionibacterium freudenreichii was cloned in p519NGFP vector in Escherichia coli BL21 (DE3). The expression of green fluorescence protein was revers correlated to the concentrations of adenosylcobalamin. Adenosylcobalamin directly binds to riboswitch region leading to conformational changes in the secondary structure of mRNA, thus inhibiting expression. After various examinations, a standard curve was obtained from 10 to 1000 ng/mL of cyanocobalamin. The limit of determination is 10 ng/mL. The inter-assay coefficients of variation were 7.5% for the range of 10-1000 ng/mL. The recovery of this method was 92.3%. This method has no or less responses to nucleic acid, pseudovitamin B12, vitamin B12 bound to intrinsic factor and haptocorrin. The riboswitch sensor results were similar with HPLC, but they were Ca. 24% lower than the microbiological assay results.