Purification and Characterization of Corrinoid-Compounds from the Dried Powder of an Edible Cyanobacterium, Nostoc commune (Ishikurage)

Abstract

Summary Vitamin B 12 content (98.8 Ϯ 5.6 g/100 g dry weight) of an edible cyanobacterium, Nostoc commune (Ishikurage) was determined by the Lactobacillus delbrueckii ATCC 7830 microbiological method. Bioautography with vitamin B 12 -dependent Escherichia coli 215 indicated that N. commune contained two (main and minor) corrinoid-compounds. These corrinoid-compounds were purified to homogeneity from the dried algal cells and characterized. The main and minor purified corrinoid-compounds were identified as pseudovitamin B 12 and vitamin B 12 , respectively, on the basis of silica gel 60 TLC, C18 reversed-phase HPLC, 1 H NMR spectroscopy, and UV-visible spectroscopy. These results suggest that the bacterial cells are not suitable for use as a vitamin B 12 source, especially in vegetarians.

Characterization of Corrinoid Compounds in the Edible Cyanobacterium <i>Nostoc flagelliforme</i> the Hair Vegetable

“…12 contents were calculated by subtracting the results for alkali resistant factor from those of total B 12 [8].…”

“…The residual fraction was dissolved in 5.0 mL of distilled water. Concentrated B 12 extracts (1 μL) and authentic and pseudo B 12 (each 50 μg/L) were spotted onto the silica gel 60 TLC sheet and developed in the dark using 2-propanol/ NH 4 OH (28%)/water (7:1:2 v/v) at room temperature (25˚C). After drying the TLC sheet, it was overlaid with agar containing basal medium and precultured E. coli 215, and incubated at 37˚C for 20 h. The gel plate was then sprayed with methanol solution containing 2,3,5-triphenyltetrazolium salt, and B 12 compounds were visualized as red, indicating E. coli growth.…”

“…The eluate was evaporated in a centrifugal concentrator (Integrated Speed VacR System ISS110), and the residual fraction was dissolved in 5.0 mL of distilled water. The purified extract was loaded onto an immunoaffinity column [EASI-EXTRACT ® Vitamin B 12 Immunoaffinity Column (P80) R-Biopharm AG, Darmstadt, Germany], and the corrinoids were purified according to the manufacturer's recommended protocol. Nostoc corrinoids, authentic pseudo B 12 , and B 12 were dissolved in 0.1% (v/v) acetic acid and filtered using a Nanosep MF centrifuge device (0.4 μm, Pall Corp., Tokyo, JAPAN) to separate small particles.…”

“…The purified extract was loaded onto an immunoaffinity column [EASI-EXTRACT ® Vitamin B 12 Immunoaffinity Column (P80) R-Biopharm AG, Darmstadt, Germany], and the corrinoids were purified according to the manufacturer's recommended protocol. Nostoc corrinoids, authentic pseudo B 12 , and B 12 were dissolved in 0.1% (v/v) acetic acid and filtered using a Nanosep MF centrifuge device (0.4 μm, Pall Corp., Tokyo, JAPAN) to separate small particles. We analyzed an aliquot (2 μL) of the filtrate using a LCMS-IT-TOF coupled with an Ultra-Fast LC system (Shimadzu, Kyoto, JAPAN).…”

“…Several studies [5,6] have reported that most of the corrinoids found in certain edible cyanobacteria may not be bioavailable in mammals. Watanabe et al [7] also demonstrated that pseudovitamin B 12 which is inactive in humans, is the predominant corrinoid in the edible cyanobacteria used as a health food by humans. N. flagelliforme, hair vegetable, is already used as health food, but there is no information on about B 12 contents in pure N. flagelliforme and commercially available hair vegetable, or whether the corrinoids are authentic B 12 or inactive corrinoids.…”