Vitamins are important organic compound required for the proper functioning of cells and organisms. Vitamins of special industrial and pharmaceutical interests include riboflavin (vitamin B2) and pyridoxine (vitamin B6). Commercial production of those biological compounds has increasingly relied on microorganisms and requires simple methods for detecting and estimating their level of synthesis during the biotechnological process. In the case of yeast, methods based on autofluorescence, i.e. natural fluorescence emitted by several cellular compounds, including vitamins, may be useful. Considering that the intensity of emitted light is proportional to the intracellular concentration of riboflavin and pyridoxine, autofluorescence may be a convenient method for their quantification. In this report, we demonstrate a simple, rapid, and sufficiently trustworthy spectrofluorimetric method for determining the content of vitamins B2 and B6 in yeast cells which consists of cells growing, harvesting, washing, and resuspending in a buffer, and then measuring the emitted visible light using specific wavelength of excitation (λex=340 nm and λem=385 nm for pyridoxine; λex=460 nm and λem=535 nm for riboflavin). The limits of detection (LOD) and quantification (LOQ) estimated through measurements of vitamin fluorescence were below 0.005 μg/ml for riboflavin and below 0.05 μg/ml for pyridoxine, respectively. In turn, the smallest credible cell density for measuring autofluorescence was set at 1×108 yeast cells/ml. The relative level of the cell's autofluorescence can be expressed in mass units by applying proper calculation formulas. A comparison of the autofluorescence-based method with the reference HPLC-UV method shows that autofluorescence measurement can be used in the screening analysis of vitamin content (especially riboflavin) in microbial cells.
Keywords: autofluorescence; endogenous fluorophores; pyridoxine; riboflavin; yeast.