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Published on: January 28, 2013
Determination of free and bound riboflavin in cow's milk using a novel flavin-binding protein
Julia Koop1, Stefanie Monschein, E Pauline Macheroux
1Institute of Biochemistry, Graz University of Technology, A-8010 Graz, Austria.
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.
Insights
Researchers developed a novel method using a bacterial protease, ppBat, to directly capture and quantify free riboflavin in milk. This technique offers a new way to measure riboflavin levels in dairy products.
Area of Science:
- Microbiology
- Biochemistry
- Food Science
Background:
- Bacteroides thetaiotaomicron possesses a protease (ppBat) with specific binding capabilities for riboflavin and its cofactors.
- Flavin-binding properties of ppBat are attributed to tryptophan residues at its interface.
- Riboflavin, FMN, and FAD are essential B vitamins with crucial biological roles.
Purpose of the Study:
- To develop a direct assay for quantifying free riboflavin in bovine milk.
- To investigate the feasibility of using recombinant ppBat for flavin capture and analysis.
- To determine free and total riboflavin concentrations in milk with varying fat content.
Main Methods:
- Recombinant ppBat was employed for direct capture of riboflavin from milk.
- Affinity chromatography was utilized for the re-isolation of flavin-loaded ppBat.
- Quantification of captured flavins was performed using established analytical techniques.
- Acid treatment was used to determine total riboflavin content.
Main Results:
- Free riboflavin concentrations were determined to be 197 μg/l (3.5% fat) and 151 μg/l (0.5% fat).
- Total riboflavin concentrations were 4-5 times higher than free riboflavin levels after acid treatment.
- Free FMN and FAD were undetectable, with only trace amounts of FMN found post-acid treatment.
Conclusions:
- The ppBat-based method enables direct measurement of free riboflavin in milk.
- The assay shows potential for application in analyzing riboflavin in various food matrices.
- Milk fat content influences the concentration of free riboflavin.
