Numerous beverages, such as multivitamin juice and sports drinks, are enriched with vitamins. These beverages may not be listed on a label as "enriched" or "fortified" with a particular vitamin supplement unless the vitamin is present in "significant amounts", which are defined in Annex XIII to Regulation (EU) No 1169/2011. For beverages, this amount is set at a minimum of 7.5 % of the NRV (nutrient reference values) per 100 ml or 15 % of the NRV per portion for individual portions.
Quantities recommended for the daily dietary intake of vitamins:
Analysis for determining the amount of added vitamin B6 by means of the microbiological microtiter plate test
This analysis is suitable for NAB, juice and other foods.
The VitaFast® microbiological assay for vitamin B6 (pyridoxine) is carried out on microtiter plates and is suitable for the determination of total amount of vitamin B6 in foods and beverages.
Vitamin B6 is added as pyridoxine hydrochloride or as pyridoxine-5-phosphate. Since the phosphorylated vitamin B6 form is not recognized by the microbes in the kit, the beverage is treated with acid phosphatase to determine the amount of supplemental vitamin B6; if necessary, the beverage is diluted with sterile water from the test kit.
The sample must be digested through enzymatic hydrolysis in order to measure the total content of vitamin B6 (naturally occurring and added).
The diluted sample and the vitamin B6 assay medium are added to the wells of the microtiter plate, which are coated with Saccharomyces cerevisiae. Saccharomyces cerevisiae requires vitamin B6 to grow. After an addition of vitamin B6, the microorganism exhibits growth until the vitamin is completely utilized. The plates are incubated at 30 °C for 44–48 h.
The growth of Saccharomyces cerevisiae is dependent upon the concentration of vitamin B6 and is determined through measuring the turbidity. These results are compared with those obtained from a series of standard concentrations. The measurement is performed with a microtiter plate photometer at 610–630 nm (alternatively at 540–550 nm).