A Novel Direct Method for Determination of Riboflavin in Alcoholic Fermented Beverages
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A Novel Direct Method for Determination of Riboflavin in Alcoholic Fermented Beverages Alessandra Bonamore 1 & Maurizio Gargano 1 & Lorenzo Calisti 1 & Antonio Francioso 1 & Luciana Mosca 1 & Alberto Boffi 1 & Rodolfo Federico 2
Received: 27 May 2015 / Accepted: 9 July 2015 / Published online: 23 July 2015 # Springer Science+Business Media New York 2015
Abstract A novel cheap and user friendly method for the determination of riboflavin in wine and beer samples has been developed based on the fluorescence quenching effect given by riboflavin binding to the chicken egg white, riboflavinbinding protein. The method is based on the use of a single diode fluorimeter with emission within riboflavin absorption profile. Linearity was observed over riboflavin concentration from 0 to 500 ng/mL with a limit of detection of 15 ng/mL. Data on real wine and beer samples were obtained and compared with classical high-performance liquid chromatography (HPLC)-based method. The fluorimetric method thus appears much faster and cheaper than the chromatographic one and easily amenable to routine analysis in beverage industry. Keywords Riboflavin . Riboflavin-binding protein . Fluorescence . Wine . Beer Abbreviations RbF Riboflavin RfPB Riboflavin-binding protein
Introduction The deleterious effect of light on wine and beer flavors results in an unpleasant taste described as Bsunlight aroma.^ This
* Alessandra Bonamore [email protected] 1
Department of Biochemical Sciences BA. Rossi Fanelli^, BSapienza^ University of Rome, Piazzale Aldo Moro 5, 00185 Rome, Italy
2
Department of Biology, University of Roma Tre, Viale Guglielmo Marconi 446, 00154 Rome, Italy
effect is due to the presence of riboflavin (vitamin B2) that occurs in fermented beverages as free riboflavin (RbF). Brewers’ yeast, as well as lactic acid bacteria responsible for the malolactic fermentation in wine, produces riboflavin at a rate proportional to growth rate of the microorganisms themselves (Tamer et al. 1988; Castor 1953). In neutral aqueous solution, riboflavin shows UV-visible spectra different for oxidized and reduced states. In oxidized state, it is a yellow pigment with two characteristic absorption bands at 370 and 450 nm while the reduced form shows an absorption spectrum above 300 nm. Moreover, upon excitation at 440–500 nm, oxidized RbF in solution exhibits a strong yellow-green fluorescence emission at 520–530 nm, whereas the reduced form does not fluoresce. Although riboflavin is stable when stored in dry conditions, it is easily degraded in solution as a consequence of photo-activation and subsequent reduction when exposed to light at wavelengths of 370 and 440 nm (Satter and deMand 1977; Duyvis et al. 2002). When wine or beer is exposed to light of wavelength below 450 nm, riboflavin accounts for the photo-generation of thiols (methylmercaptan, H2S) and dimethyldisulfide. The sulfurcontaining amino acids methionine and cysteine, in fact, can be oxidized by light-activated riboflavin to volatile compounds smelling pungently of leek, on
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