J Nutr Sci Vitaminol, 58, 438–441, 2012

Note
Characterization of Vitamin B12 Compounds in the Wild Edible
Mushrooms Black Trumpet (Craterellus cornucopioides) and
Golden Chanterelle (Cantharellus cibarius)
Fumio Watanabe1, Joachim Schwarz2, Shigeo Takenaka3, Emi Miyamoto4,
Noriharu Ohishi1, Esther Nelle2, Rahel Hochstrasser2 and Yukinori Yabuta1
1
School of Agricultural, Biological and Environmental Sciences, Tottori University,
Tottori 680–8553, Japan
2
Medical Practice Dr Med. Joachim Schwarz, Waldstrasse 44, 57520 Dickendorf, Germany
3
Department of Veterinary Science, Graduate School of Life and Environmental Sciences,
Osaka Prefecture University, Osaka 598–8531, Japan
4
Department of Health and Nutrition, Nagasaki International University, Sasebo 859–3298, Japan
(Received July 26, 2012)

Summary This study determined the vitamin B12 content of six wild edible mushrooms
which are consumed by European vegetarians. Zero or trace levels (0.01–0.09 mg/100 g
dry weight) of vitamin B12 were determined in porcini mushrooms (Boletus spp.), parasol
mushrooms (Macrolepiota procera), oyster mushrooms (Pleurotus ostreatus), and black morels
(Morchella conica). By contrast, black trumpet (Craterellus cornucopioides) and golden chante-
relle (Cantharellus cibarius) mushrooms contained considerable levels (1.09–2.65 mg/100 g
dry weight) of vitamin B12. To determine whether C. cornucopioides or C. cibarius contained
vitamin B12 or other corrinoid compounds that are inactive in humans, we purified a cor-
rinoid compound using an immunoaffinity column and identified it as vitamin B12 based on
LC/ESI-MS/MS chromatograms.
Key Words Cantharellus cibarius, cobalamin, Craterellus cornucopioides, edible mushrooms,
vitamin B12

Vitamin B12 (B12) is synthesized only by certain bacte-
ria (1). The B12 synthesized by bacteria is concentrated
mainly in the bodies of higher predatory organisms in
the natural food chain system. Animal foods (i.e., meat,
milk, egg, fish, and shellfish), but not plant foods, are
considered to be the major dietary sources of B12 (2).
Thus, strict vegetarians have a greater risk of develop-
ing B12 deficiency compared with nonvegetarians (3).
The major symptoms of B12 deficiency are neuropathy
and megaloblastic anemia (4). Thus, we need to iden-
tify plant foods that contain high levels of B12 to prevent
vegetarians from developing B12 deficiency.
Wild mushrooms are becoming increasingly impor-
tant in our diet because of their nutritional and medici-
nal characteristics (5, 6). Many species of wild mush-
rooms are consumed widely. Six wild edible mushroom
species are popular with vegetarians in European coun-
tries, i.e., porcini mushrooms (Boletus spp.), parasol
mushrooms (Macrolepiota procera), oyster mushrooms
(Pleurotus ostreatus), black morels (Morchella conica),
black trumpets (Craterellus cornucopioides), and golden
chanterelles (Cantharellus cibarius). However, little infor-
mation is available on the B12 content of these mush-
rooms, particularly whether these mushrooms contain
“true” (authentic) B12 or an inactive corrinoid such as
pseudo B12 (2). If certain edible mushrooms contain
considerable or high levels of B12, they would be good
sources of B12 for vegetarians.
In this study, we analyzed the B12 content of the six
E-mail: watanabe@muses.tottori-u.ac.jp
edible mushrooms consumed by European vegetarians,
and characterized the B12 compounds found in black
trumpet and golden chanterelle mushrooms.
Materials and Methods
Materials. B12 was obtained from Sigma (St. Louis,
Missouri, USA). A B12 assay medium based on Lactoba-
cillus delbrueckii subspecies lactis (formerly L. leichmannii)
ATCC7830 was obtained from Nissui (Tokyo, Japan).
Silica gel 60 thin layer chromatography (TLC) alumi-
num sheets were obtained from Merck (Darmstadt, Ger-
many). The dried mushroom samples were purchased in
Germany and Japan.
Extraction and assay of B12 in edible mushrooms. Each
mushroom sample (approximately 10 g) was homoge-
nized using a mixer (TML160; Tescom & Co., Ltd., Tokyo,
Japan). A portion (5.0 g) of the homogenate was used as
the test sample. Total B12 compounds were extracted by
boiling at pH 4.8 in the presence of 4.031024% KCN
and assayed using a microbiological technique based
on L. delbrueckii ATCC 7830, according to the method
described in the Standard Tables of Food Composition
in Japan (7). L. delbrueckii ATCC 7830 can utilize deoxy-
ribosides, deoxyribonucleotides (known as an alkali-
resistant factor), and B12. Hence, the correct B12 values
were calculated by subtracting the results for the alkali-
resistant factor from those for total B12.
Bioautogram of vitamin B12 compounds using vitamin
B12-dependent Escherichia coli 215. A bioautogram of
B12 compounds was prepared according to a published
method (8). The B12 extract (10 mL) prepared above was

438
 Vitamin B12 in European Wild Mushrooms 439

Table 1. Vitamin B12 content of wild edible mushrooms consumed by European vegetarians.

Vitamin B12 content (mg/100 g dry weight)

Apparent B12 Alkali-resistant factor Corrected B12

Boletus spp.
(Germany) 0.3360.09 0.3160.12 0.0260.03
(Germany) 0.3960.09 0.3260.08 0.0760.03
Macrolepiota procera
(Germany) 1.3260.42 1.2360.39 0.0960.04
Pleurotus ostreatus
(China) 0.2260.09 0.2360.05 0.0160.01
Morchella conica
(Montenegro) 0.1260.04 0.1260.05 0.0060.00
Craterellus cornucopioides
(Bosnia) 2.8860.84 1.2560.45 1.896070
(Bosnia) 2.5560.17 0.7660.14 1.7960.26
(Serbia) 3.4361.29 1.0060.32 2.4361.41
(France) 3.9461.24 1.4960.76 2.6561.46
Cantharellus cibarius
(Germany) 2.0860.60 0.7060.22 1.8260.57
(France) 1.3260.32 0.2360.07 1.0960.27
(Bulgaria) 1.7760.20 0.2960.03 1.4860.55
The countries where the mushrooms were picked are shown in parentheses. The B12 assay is described in the text and it was
performed in triplicate. All values represent the mean6SD.

partially purified and concentrated using a Sep-pak®

Plus C18 cartridge (Waters Corp., Milford, USA) that
had been washed with 5 mL of 75% (v/v) ethanol and
equilibrated with 5 mL of distilled water. The C18 car-
tridge was washed with 5 mL of distilled water and B12
compounds were eluted using 2 mL of 75% (v/v) etha-
nol. The eluate was evaporated in a centrifugal concen-
trator (Integrated SpeedVac® System ISS110; Savant
Instruments Inc., NY, USA). The residual fraction was
dissolved in 1.0 mL of distilled water. Next, 2 mL of
the concentrated B12 extracts as well as authentic and
pseudo B12 (each 10 mg/L) were spotted onto the silica
gel 60 TLC sheet and developed in the dark using 2-pro-
panol/NH4OH (28%)/water (7 : 1 : 2 v/v) at room tem-
perature (25˚C). After the TLC sheet was dried, it was
overlaid with agar containing basal medium and pre-
cultured E. coli 215, and incubated at 37˚C for 20 h.
The gel plate was then sprayed with a methanol solution
containing 2,3,5-triphenyltetrazolium salt and B12 com-
pounds were visualized as red, indicating E. coli growth.
Identification of mushroom B12 compounds by LC/ESI-
MS/MS. The mushrooms samples that contained high
levels of B12 (Craterellus cornucopioides and Cantharellus
cibarius) (each 50 g wet weight, moisture content of
90%) were suspended in 500 mL of distilled water and
homogenized in a mixer (TML160). Each homogenate
was added to 50 mL of 0.57 mol/L acetic buffer, pH 4.5,
with 0.05 g KCN, and boiled for 30 min to extract B12
compounds. The extraction procedures were performed
in a draught chamber (Dalton Co., Tokyo, Japan). The
boiled suspension was centrifuged at 5,000 3g for
10 min. An aliquot (approximately 200 mL) of the
supernatant was placed in Sep-pak®Vac 20 cc (5 g)
C18 cartridges (Waters Corp.) that had been washed
with 75% (v/v) ethanol and equilibrated with distilled
1 2 3 4 5 6 7 8
Fig. 1. E. coli 215 bioautogram analysis of B12 com-
pounds found in various edible mushrooms consumed
by European vegetarians. (1) Authentic B12, (2) pseudo
B12, (3) porcini mushroom (Boletus spp.), (4) parasol
mushroom (Macrolepiota procera), (5) oyster mushroom
(Pleurotus ostreatus), (6) black morel (Morchella con-
ica), (7) black trumpet (Craterellus cornucopioides), and
(8) golden chanterelle (Cantharellus cibarius). The data
show typical bioautograms from three independent
experiments.
water. The C18 cartridges were washed with 30 mL of
distilled water and B12 compounds were eluted using
30 mL of 75% (v/v) ethanol. The remaining superna-
tant was treated in the same way. The combined eluates
were evaporated to dryness under reduced pressure.
The residual fraction was dissolved with 5 mL of dis-
tilled water and centrifuged at 10,000 3g for 10 min
to remove any insoluble material. The supernatant
fraction was loaded onto an immunoaffinity column
[EASI-EXTRACT®B12 Immunoaffinity Column (P80);
R-Biopharm AG, Darmstadt, Germany] and B12 com-
pounds were purified according to the manufacturer’s
 440

TIC TIC
A B C

m/z 678.29 m/z 678.29

Retention time (min) Retention time (min) Retention time (min)

D G E H F I
Watanabe F et al.

Fig. 2. LC/ESI-MS/MS chromatograms of authentic B12, and the B12 compounds purified from black trumpet (C. cornucopioides) and golden chanterelle (C. cibarius). Vitamin B12 was
analyzed using an LCMS-IT-TOF system (Shimadzu) as described in the text. The total ion chromatogram (TIC) of authentic B12 is shown in panel A. Panels B and C show the TICs and
reconstructed chromatograms of the purified B12 compounds (m/z 678.29) from black trumpet (C. cornucopioides) and golden chanterelle (C. cibarius), respectively. The mass spectra of
authentic B12 and the B12 compounds purified from black trumpet (C. cornucopioides) and golden chanterelle (C. cibarius) at 7.35 min are shown in panels D, E, and F, respectively (the
magnified spectrum ranging from m/z 678 to m/z 680 is shown as an insert in each panel). The MS/MS spectra for the m/z 678.292 peak of authentic B12 and the B12 compounds purified
from black trumpet (C. cornucopioides) and golden chanterelle (C. cibarius) are shown in panels G, H, and I, respectively.
 Vitamin B12 in European Wild Mushrooms
recommended protocol. The purified mushroom B12
compounds and authentic B12 were dissolved in 0.1%
(v/v) acetic acid and filtered using a Nanosep MF cen-
trifuge device (0.4 mm; Pall Corp., Tokyo, Japan) to
remove small particles. We analyzed an aliquot (2 mL)
of the filtrate using an LCMS-IT-TOF system coupled to
an Ultra-Fast LC system (Shimadzu, Kyoto, Japan). Each
purified corrinoid was injected into an InertSustain col-
umn (3 mm, 2.03100 mm; GL Science, Tokyo, Japan)
and equilibrated with 85% solvent A [0.1% (v/v) ace-
tic acid] and 15% solvent B (100% methanol) at 40˚C.
B12 compounds were eluted using a linear gradient of
methanol (15% solvent B for 0–5 min, 15–90% solvent
B for 5–11 min, and 90–15% solvent B for 11–15 min).
The flow rate was 0.2 mL/min. The electrospray ion
(ESI) conditions were determined by injecting authentic
B12 into the MS detector to determine optimum param-
eters for detecting parent and daughter ions of the B12
compound. The ESI-MS was operated in the positive
ion mode using argon as the collision gas. The identi-
fication of B12 (m/z5678.292) representing [M12H]21
was confirmed by comparison of the observed molecular
ions and their retention times.
Results and Discussion
The levels of B12 were assayed in six edible mushrooms
that are commonly consumed by European vegetarians
using a microbiological method based on L. delbrueckii
ATCC 7830 (Table 1). Zero or trace levels (0.01–0.09
mg/100 g dry weight) of corrected B12 were found in
Boletus spp., Macrolepiota procera, Pleurotus ostreatus,
and Morchella conica, whereas Craterellus cornucopioides
and Cantharellus cibarius contained considerable lev-
els (1.09–2.65 mg/100 g dry weight) of corrected B12.
High levels (0.12–1.49 mg as B12 equivalent/100 g dry
weight) of the alkali-resistant factor were found in all
mushrooms.
The B12 compounds found in the edible mushrooms
were analyzed using an E. coli 215 bioautogram after
being separated by silica gel 60 TLC (Fig. 1). Each extract
of C. cornucopioides and C. cibarius produced a single
clear spot, the Rf value of which was identical to that of
the authentic B12 form but not to that of the pseudo B12
form that is inactive in humans. No or indistinct spots
were detected in the other mushroom samples. Corri-
noids were purified from the extracts of C. cornucopioi-
des and C. cibarius using an immunoaffinity column and
they were identified by LC/ESI-MS/MS (Fig. 2). Authen-
tic B12 was eluted as a peak with a retention time of
7.35 min. The mass spectrum of authentic B12 mainly
had a divalent ion with an m/z of 678.2932 [M12H]21
(Fig. 2A and D). Its exact mass was calculated from its
formula (C63H88CoN14O14P), i.e., 1354.5674, and the
isotope distribution data showed that B12 mainly existed
as a divalent ion under LC/ESI-MS conditions. The MS/
MS spectra of B12 indicated that a monovalent ion with
an m/z of 359.1008 predominated, mainly because of
the nucleotide moiety of B12 (Fig. 2G). The compounds
purified from C. cornucopioides and C. cibarius were
eluted as several ion peaks, indicating that impurities
441
were still present. The mass spectrum of the main peak
had a retention time of 7.35 min in these purified com-
pounds, containing the B12 divalent ions with m/z val-
ues of 678.2892 and 678.2884 (Fig. 2B, C, E, and F).
The MS/MS spectrum of each compound was identical
to that of authentic B12 (Fig. 2G, H, and I). These results
indicated that black trumpet (C. cornucopioides) and
golden chanterelle (C. cibarius) mushrooms contained
considerable levels of authentic B12, but not pseudo B12
that is inactive in humans. Thus, black trumpet and
golden chanterelle mushrooms could be useful plant B12
sources for vegetarians.
Consumption of approximately 100 g of dried black
trumpet [or approximately 1 kg of fresh mushroom
(moisture content of 90%)] could provide the recom-
mended daily dietary allowance for adults (2.4 mg/d)
(9), although they would not be able to ingest such large
amounts of this mushroom daily. However, a moderate
mushroom intake may contribute slightly to the preven-
tion of severe B12 deficiency in vegetarians.
There is little information available on why C. cor-
nucopioides and C. cibarius contain higher levels of B12
than the other mushrooms tested. Thus, further phylo-
genetic, biochemical, and genetic studies are needed to
determine whether C. cornucopioides and C. cibarius have
the ability to synthesize B12 de novo or if it is derived
from B12 synthesized by bacteria living on the surfaces
of these mushrooms.
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