title: Determination of free and bound riboflavin in cow's milk using a novel flavin-binding protein.
abstract: A recently described putative protease from the gut bacterium Bacteroides thetaiotaomicron (termed ppBat) exhibits two tryptophan residues in the interface which enable specific binding of the isoalloxazine heterocycle of riboflavin and its two cofactor forms, FMN and FAD. Recombinant ppBat was used to capture riboflavin from bovine milk directly without any prior preparation steps. The flavin-loaded protein was then re-isolated by means of affinity chromatography to identify and quantify the captured flavins. Free riboflavin concentrations were determined to 197 and 151μg/l for milk with 3.5% and 0.5% fat content, respectively. Total riboflavin concentrations were also determined after acid-treatment of milk and were 4-5 times higher than for free riboflavin. Free FMN and FAD were not detectable and only trace amounts of FMN were found in milk following acid treatment. The method appears to be amenable to develop a direct assay for free riboflavin in milk and other foods.
authors.affiliation: Institute of Biochemistry, Graz University of Technology, A-8010 Graz, Austria.
authors.forename: Julia
authors.initials: J
authors.lastname: Koop
authors.forename: Stefanie
authors.initials: S
authors.lastname: Monschein
authors.forename: E
authors.initials: E
authors.lastname: Pauline Macheroux
authors.forename: Tanja
authors.initials: T
authors.lastname: Knaus
authors.forename: Peter
authors.initials: P
authors.lastname: Macheroux
mesh_terms: D000818:Animals; D002417:Cattle; D008892:Milk / Q000737:chemistry*; D008894:Milk Proteins / Q000737:chemistry*; D011485:Protein Binding; D012256:Riboflavin / Q000737:chemistry*
chemical_list: D008894:Milk Proteins; D012256:Riboflavin
keywords: FAD; FMN; Flavin binding protein; HPLC; Milk; Riboflavin; UV/Vis-absorption spectroscopy; Vitamin detection; flavin adenine dinucleotide; flavin mononucleotide; high performance liquid chromatography; ppBat; putative protease from Bacteroides thetaiotaomicron