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

Note Characterization of Vitamin B12 Compounds in the Wild Edible Black Trumpet (Craterellus cornucopioides) and Golden ( 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 ( procera), oyster mushrooms (Pleurotus ostreatus), and black morels ( 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- edible mushrooms consumed by European vegetarians, ria (1). The B12 synthesized by bacteria is concentrated and characterized the B12 compounds found in black mainly in the bodies of higher predatory organisms in trumpet and golden chanterelle mushrooms. the natural food chain system. Animal foods (i.e., meat, milk, egg, fish, and shellfish), but not plant foods, are Materials and Methods considered to be the major dietary sources of B12 (2). Materials. B12 was obtained from Sigma (St. Louis, Thus, strict vegetarians have a greater risk of develop- Missouri, USA). A B12 assay medium based on Lactoba- ing B12 deficiency compared with nonvegetarians (3). cillus delbrueckii subspecies lactis (formerly L. leichmannii) The major symptoms of B12 deficiency are neuropathy ATCC7830 was obtained from Nissui (Tokyo, Japan). and megaloblastic anemia (4). Thus, we need to iden- Silica gel 60 thin layer chromatography (TLC) alumi- tify plant foods that contain high levels of B12 to prevent num sheets were obtained from Merck (Darmstadt, Ger- vegetarians from developing B12 deficiency. many). The dried samples were purchased in Wild mushrooms are becoming increasingly impor- Germany and Japan. tant in our diet because of their nutritional and medici- Extraction and assay of B12 in edible mushrooms. Each nal characteristics (5, 6). Many of wild mush- mushroom sample (approximately 10 g) was homoge- rooms are consumed widely. Six wild edible mushroom nized using a mixer (TML160; Tescom & Co., Ltd., Tokyo, species are popular with vegetarians in European coun- Japan). A portion (5.0 g) of the homogenate was used as tries, i.e., porcini mushrooms (Boletus spp.), parasol the test sample. Total B12 compounds were extracted by mushrooms (Macrolepiota procera), oyster mushrooms boiling at pH 4.8 in the presence of 4.031024% KCN (Pleurotus ostreatus), black morels (Morchella conica), and assayed using a microbiological technique based black trumpets (Craterellus cornucopioides), and golden on L. delbrueckii ATCC 7830, according to the method (Cantharellus cibarius). However, little infor- described in the Standard Tables of Food Composition mation is available on the B12 content of these mush- in Japan (7). L. delbrueckii ATCC 7830 can utilize deoxy- rooms, particularly whether these mushrooms contain ribosides, deoxyribonucleotides (known as an alkali- “true” (authentic) B12 or an inactive corrinoid such as resistant factor), and B12. Hence, the correct B12 values pseudo B12 (2). If certain edible mushrooms contain were calculated by subtracting the results for the alkali- considerable or high levels of B12, they would be good resistant factor from those for total B12. sources of B12 for vegetarians. Bioautogram of vitamin B12 compounds using vitamin In this study, we analyzed the B12 content of the six B12-dependent Escherichia coli 215. A bioautogram of B12 compounds was prepared according to a published E-mail: [email protected] 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 1 2 3 4 5 6 7 8 gel 60 TLC sheet and developed in the dark using 2-pro- Fig. 1. E. coli 215 bioautogram analysis of B12 com- pounds found in various edible mushrooms consumed panol/NH4OH (28%)/water (7 : 1 : 2 v/v) at room tem- perature (25˚C). After the TLC sheet was dried, it was by European vegetarians. (1) Authentic B12, (2) pseudo overlaid with agar containing basal medium and pre- B12, (3) porcini mushroom (Boletus spp.), (4) parasol mushroom (Macrolepiota procera), (5) oyster mushroom cultured E. coli 215, and incubated at 37˚C for 20 h. (Pleurotus ostreatus), (6) black morel (Morchella con- The gel plate was then sprayed with a methanol solution ica), (7) black trumpet (Craterellus cornucopioides), and containing 2,3,5-triphenyltetrazolium salt and B12 com- (8) golden chanterelle (Cantharellus cibarius). The data pounds were visualized as red, indicating E. coli growth. show typical bioautograms from three independent Identification of mushroom12 B compounds by LC/ESI- experiments. MS/MS. The mushrooms samples that contained high levels of B12 (Craterellus cornucopioides and Cantharellus cibarius) (each 50 g wet weight, moisture content of water. The C18 cartridges were washed with 30 mL of 90%) were suspended in 500 mL of distilled water and distilled water and B12 compounds were eluted using homogenized in a mixer (TML160). Each homogenate 30 mL of 75% (v/v) ethanol. The remaining superna- was added to 50 mL of 0.57 mol/L acetic buffer, pH 4.5, tant was treated in the same way. The combined eluates with 0.05 g KCN, and boiled for 30 min to extract B12 were evaporated to dryness under reduced pressure. compounds. The extraction procedures were performed The residual fraction was dissolved with 5 mL of dis- in a draught chamber (Dalton Co., Tokyo, Japan). The tilled water and centrifuged at 10,000 3g for 10 min boiled suspension was centrifuged at 5,000 3g for to remove any insoluble material. The supernatant 10 min. An aliquot (approximately 200 mL) of the fraction was loaded onto an immunoaffinity column ® ® supernatant was placed in Sep-pak Vac 20 cc (5 g) [EASI-EXTRACT B12 Immunoaffinity Column (P80); C18 cartridges (Waters Corp.) that had been washed R-Biopharm AG, Darmstadt, Germany] and B12 com- with 75% (v/v) ethanol and equilibrated with distilled pounds were purified according to the manufacturer’s 440 Watanabe F et al. was was 12 9 compounds purified 12 ) m/z 678.2 C in TI (m

me ti and the B

12 on ti FI eten min are shown in panels D, E, and F, respectively (the respectively E, and F, in panels D, min are shown C is shown in panel A. Panels B and C show the TICs and the B and C show in panel A. Panels is shown 12 ) at 7.35 cibarius ) at C 9 TI )R m/z 678.2 in (m

me ti n io nt EH Rete ). Vitamin B chanterelle ( C. cibarius ). Vitamin compounds purified from black trumpet ( C. cornucopioide s) and golden 12 , and the B 12 ), respectively. The mass spectra ofThe mass spectra chanterelle ( C. cibarius ), respectively. compounds ( m / z 678.29) from black trumpet ( C. cornucopioide s) and golden 12 ) in (m

me ti n s) and golden chanterelle ( C. compounds purified from black trumpet ( C. cornucopioide s) and golden io 12 nt DG Rete and the B 12 LC/ESI-MS/MS chromatograms ofLC/ESI-MS/MS chromatograms authentic B AB 2. reconstructed chromatograms ofreconstructed chromatograms the purified B analyzed using an LCMS-IT-TOF system (Shimadzu) as described in the text. The total ion chromatogram (TIC) ofThe total ion chromatogram system (Shimadzu) as described in the text. authentic B analyzed using an LCMS-IT-TOF ) are shown in panels G, H, and I, respectively. chanterelle ( C. cibarius ) are shown from black trumpet ( C. cornucopioide s) and golden 680 is shown as an insert in each panel). The MS/MS spectra for the m / z 678.292 peak ofThe MS/MS spectra as an insert spectrum from m / z 678 to 680 is shown ranging magnified in each panel). authentic B authentic B Fig. Vitamin B12 in European Wild Mushrooms 441 recommended protocol. The purified mushroom 12B were still present. The mass spectrum of the main peak compounds and authentic B12 were dissolved in 0.1% had a retention time of 7.35 min in these purified com- (v/v) acetic acid and filtered using a Nanosep MF cen- pounds, containing the B12 divalent ions with m/z val- trifuge device (0.4 mm; Pall Corp., Tokyo, Japan) to ues of 678.2892 and 678.2884 (Fig. 2B, C, E, and F). remove small particles. We analyzed an aliquot (2 mL) The MS/MS spectrum of each compound was identical of the filtrate using an LCMS-IT-TOF system coupled to to that of authentic B12 (Fig. 2G, H, and I). These results an Ultra-Fast LC system (Shimadzu, Kyoto, Japan). Each indicated that black trumpet (C. cornucopioides) and purified corrinoid was injected into an InertSustain col- golden chanterelle (C. cibarius) mushrooms contained umn (3 mm, 2.03100 mm; GL Science, Tokyo, Japan) considerable levels of authentic B12, but not pseudo B12 and equilibrated with 85% solvent A [0.1% (v/v) ace- that is inactive in humans. Thus, black trumpet and tic acid] and 15% solvent B (100% methanol) at 40˚C. golden chanterelle mushrooms could be useful plant B12 B12 compounds were eluted using a linear gradient of sources for vegetarians. methanol (15% solvent B for 0–5 min, 15–90% solvent Consumption of approximately 100 g of dried black B for 5–11 min, and 90–15% solvent B for 11–15 min). trumpet [or approximately 1 kg of fresh mushroom The flow rate was 0.2 mL/min. The electrospray ion (moisture content of 90%)] could provide the recom- (ESI) conditions were determined by injecting authentic mended daily dietary allowance for adults (2.4 mg/d) B12 into the MS detector to determine optimum param- (9), although they would not be able to ingest such large eters for detecting parent and daughter ions of the B12 amounts of this mushroom daily. However, a moderate compound. The ESI-MS was operated in the positive mushroom intake may contribute slightly to the preven- ion mode using argon as the collision gas. The identi- tion of severe B12 deficiency in vegetarians. 21 fication of B12 (m/z5678.292) representing [M12H] There is little information available on why C. cor- was confirmed by comparison of the observed molecular nucopioides and C. cibarius contain higher levels of B12 ions and their retention times. than the other mushrooms tested. Thus, further phylo- genetic, biochemical, and genetic studies are needed to Results and Discussion determine whether C. cornucopioides and C. cibarius have The levels of B12 were assayed in six edible mushrooms the ability to synthesize B12 de novo or if it is derived that are commonly consumed by European vegetarians from B12 synthesized by bacteria living on the surfaces using a microbiological method based on L. delbrueckii of these mushrooms. ATCC 7830 (Table 1). Zero or trace levels (0.01–0.09 REFERENCES mg/100 g dry weight) of corrected B12 were found in Boletus spp., Macrolepiota procera, Pleurotus ostreatus, 1) Scheider Z, Stroiñski A. 1987. 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