Fortification of teabags containing Indian tea (Camellia sinensis) with folate and vitamin B12

Preliminary observations on the dissolution of the vitamins, appearance and organoleptic assessment of vitamins dissolved in brewed tea are described in online supplemental material. The exposure of vitamins to heat following brewing of tea involved teabags being steeped in approximately 95°C hot water (75–100 mL) for 2–5 min before addition of room temperature milk and sugar; this extent of heat exposure is far less than that involved in baking vitamin-fortified bread, where the majority of both vitamins were stable and functionally capable of raising blood vitamin levels among healthy adults.⁴¹ Fortification of teabags was carried out in a darkened room, with windows covered by dark coloured cloth to avoid ultraviolet light induced photodegradation of folic acid⁴² and vitamin B₁₂.⁴³

Each teabag was spiked with pharmacological doses of folate (1 mg folate) plus either of two equally efficacious^{24 37} doses of vitamin B₁₂ (0.1 mg or 0.5 mg, respectively) that were precisely applied onto each teabag for two equal cohorts of women of childbearing-age in Sangli (n=19). As reasoned above (Strategy and Study Design), we also planned to randomly assign a much smaller cohort of women (n=5) to simultaneously receive mock-fortified teabags for 2 months to assess for any changes in serum vitamin levels to unfortified tea; after the 2-month trial, this group would then be given an additional 2 months of full vitamin replacement tablets (ie, with a 2-month delay compared with the other groups).

We used commercially available teabags, each composed of a filter paper bag containing 2 g Brooke Bond Taj Mahal tea—an orange pekoe blend of black tea with characteristic dark-red brew, a strong malty flavour and full body. Both folic acid and vitamin B₁₂ used to fortify tea were of USP-grade and obtained from Sigma-Aldrich (USA); this was in keeping with Food Safety and Standards Authority of India (FSSAI) regulations⁴⁴ on the use of USP-grade vitamins for medical use in humans.

Applying precise amounts of dissolved solutions of vitamin B₁₂ (red colour) and folate (yellow colour) using an Eppendorf pipette left a small coloured stain on each teabag in Group-1 and Group-2. For mock-fortified teabags for controls (Group-0), we used two US Food and Drug Administration-approved food colouring agents, Chefmaster Liqua-Gel Food Golden-Yellow and Super-Red colours (Byrnes & Kiefer Company, Fullerton, California). Diluting 5 mL of stock Golden-Yellow and Super-Red into 50 mL water perfectly matched the colour of stock solutions of folate and vitamin B₁₂, respectively. Thus, women in Group-0 used teabags spiked with 25 µL each of Super-Red plus Golden-Yellow food colouring; women in Group-1 used teabags spiked with 0.1 mg (5 µL) vitamin B₁₂ plus 1 mg (50 µL) folate; and women in Group-2 used teabags spiked with 0.5 mg (25 µL) vitamin B₁₂ plus 1 mg (50 µL) folate. After addition of vitamin B₁₂ and folate (or food colouring) to teabags, they were gently dried using a hair dryer and repacked in original boxes, now specifically coded for Group-0, Group-1 and Group-2.