Food and Nutrition Sciences
Vol.5 No.14(2014), Article ID:48539,7 pages DOI:10.4236/fns.2014.514144
Determination of Vitamin B12 in Chinese Black Tea Leaves
Fei Teng1, Tomohiro Bito1*, Shigeo Takenaka2, Yukinori Yabuta1, Fumio Watanabe1#
1Division of Applied Bioresources Chemistry, The United Graduate School of Agricultural Sciences, Tottori University, Tottori, Japan
2Department of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan
Copyright © 2014 by authors and Scientific Research Publishing Inc.
This work is licensed under the Creative Commons Attribution International License (CC BY).
Received 25 May 2014; revised 28 June 2014; accepted 13 July 2014
We determined vitamin B12 content of Chinese black tea leaves using a microbiological assay based on Lactobacillus delbrueckii ATCC 7830. Trace levels (0.25 - 0.69 μg/100g dry weight) of vitamin B12 were detected in Pu’er, Fu, and Brick tea leaves. However, vitamin B12 content (0.06 - 1.37 μg/100g dry weight) of Ryubao tea leaves significantly varied. To determine whether Chinese black tea leaves contain vitamin B12 or other corrinoid compounds that are inactive in humans, corrinoid compounds were purified from Ryubao tea by an immunoaffinity column chromatography and vitamin B12 was identified by liquid chromatography-electrospray ionization/tandem mass spectrometry. Vitamin B12 content in the tea drink prepared from Ryubao tea leaves was very low (0.8 ng/100mL). Our results indicate that Chinese black tea is usually not a good source of B12, although Ryubao tea leaves with the highest B12 content may be utilized as a source of this vitamin for vegetarians.
Keywords: Chinese Black Tea Leaves, Cobalamin, Ryubao Tea, Vitamin B12
Tea is the second highest consumed nonalcoholic beverage worldwide, and an important dietary source of flavonoid compounds  . Although these tea polyphenols possess therapeutic properties, including anti-cardiovascular and anti-cancer effects in vitro and in vivo, epidemiological and clinical studies suggest an association with moderately reducing the risk of chronic diseases  . Chinese tea is generally divided into at least three categories on the basis of different production methods: non-fermented (green tea), semi-fermented (oolong tea), and fully fermented (black tea)  . Among these, only black tea leaves are withered, rolled, and fermented with bacteria  . Biologically active compounds containing anti-oxidative  , anti-mutagenic  , and anti-hypertriacylglycerolemia  properties have been observed in black tea leaves.
Vitamin B12 (B12) is synthesized by certain bacteria and is mainly concentrated in the bodies of higher predatory organisms in the natural food chain system  . In daily life, we mainly ingest B12 from animal-derived foods (fish, shellfish, meat, and eggs). Thus, strict vegetarians have a greater risk of developing B12 deficiency compared with non-vegetarians  . The major symptoms of B12 deficiency are neuropathy and megaloblastic anemia  . Thus, we need to identify plant foods that contain high levels of B12 to prevent vegetarians from developing B12 deficiency.
Although plant-derived foods generally contain zero or trace B12, Kittaka-Katsura et al.  demonstrated that the Japanese black tea leaf Batabata-cha contains approximately 0.5 μg of B12 per 100 g of dried tea leaves, which is bioavailable in mammals. In addition, they determined that B12 content of two types of Chinese black tea leaves  is similar to that of Batabata-cha. However, B12 content in various types of Chinese black tea leaves and the identification of B12 compounds in Chinese black tea leaves as “true” B12 or inactive corrinoid compounds in humans remain to be established.
Here we describe the characterization of B12 compounds from various Chinese fermented black tea leaves using thin-layer chromatography—Escherichia coli 215 bioautography and liquid chromatography-electrospray ionization/tandem mass spectrometry (LC/ESI-MS/MS).
2. Materials and Methods
Authentic B12 was obtained from Sigma (St. Louis, Missouri, USA). Silica gel 60 thin-layer chromatography (TLC) aluminum sheets were purchased from Merck (Darmstadt, Germany). All other reagents were high-grade and commercially available. Various types of Chinese black tea leaves (Pu’er, Ryubao, Fu, and Brick) were purchased from local markets in Japan (Figure 1).
2.2. Extraction and Assay of B12 from Chinese Black Tea Leaf Samples
Each sample (5 g) of dried Chinese black tea leaves was homogenized in a mixer (TML160; Tescom & Co., Ltd., Tokyo, Japan). A portion (2.0 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 4.0 × 10−4 % KCN and assayed using a microbiological B12 assay based on L. delbrueckii ATCC 7830, according to the method described in the Standard Tables of Food Composition in Japan  . L. delbrueckii ATCC 7830 utilizes deoxyribosides, deoxyribonucleotides (known as alkali-resistant factor), and B12.
The correct B12 values were calculated by subtracting results for the alkali-resistant factor from those for total B12. Tea from 3-g Ryubao leaves (sample H) with the highest B12 content among those tested was extracted for 5 min with 150 mL of boiling water. After cooling down to 40˚C, the extract was used as a tea drink, and B12 was extracted from 50 mL of liquid using the above-mentioned method.
2.3. Bioautography of Corrinoid Compounds Using B12-Dependent E. coli 215
Bioautography of corrinoid compounds was performed as previously described  . B12 extract (50 mL) prepared as described above was partially purified and concentrated using Sep-Pak Plus® C18 cartridge (Waters Corp., Milford, USA) prewashed with 5 mL of 75% (v/v) ethanol and equilibrated with 5 mL of distilled water. The C18 cartridge was washed with 5 mL of distilled water, and B12 compounds were eluted using 2 mL of 75% (v/v) ethanol. The eluate was evaporated in a centrifugal concentrator (Integrated SpeedVac® System ISS110; Savant Instruments Inc., NY, USA) and the residual fraction was dissolved in 2.0 mL of distilled water. Concentrated B12 extracts (1 μL) and authentic B12 and pseudo B12 (50 μg/L each) were spotted onto the silica gel 60 TLC sheet and developed in the dark using 2-propanol/NH4OH (28%)/water (7:1:2 v/v) at room temperature (25˚C). After drying, the TLC sheet was overlaid with agar containing a basal medium and precultured E. coli 215, and incubated at 37˚C for 20 h. The gel plate was sprayed with methanol solution containing 2, 3, 5-triphenyltetrazolium salt and B12 compounds were visualized as red, indicating E. coli growth.
Figure 1. Types of Chinese black tea leaves. Pu’er tea (samples A - G), Ryubao tea (sample H), Fu tea (sample I), and Brick tea (sample J) leaves were used in this study.
2.4. Liquid Chromatography-Electrospray Ionization/Tandem Mass Spectrometry Analysis
Sample H (10 g) containing high levels of B12 was suspended in 500 mL of distilled water and homogenized in a mixer (TML 160). The 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. Extraction procedures were performed in a draught chamber (Dalton Co., Tokyo, Japan). The boiled suspension was centrifuged at 5000× g 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.) prewashed with 75% (v/v) ethanol and equilibrated with distilled 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 supernatant was treated in the same manner. Combined eluates were evaporated to dryness under reduced pressure, and the residual fraction was dissolved in 5.0 mL of distilled water and centrifuged at 10,000 × g for 10 min to remove any insoluble material. The supernatant fraction was loaded onto EASI-EXTRACT Vitamin B12 Immunoaffinity Column (P80) [R-Biopharm AG, Darmstadt, Germany], and corrinoids were purified according to the manufacturer’s recommended protocol. B12 compounds, pseudo B12, and B12 were dissolved in 0.1% (v/v) acetic acid and filtered using a Nanosep MF centrifual device (0.4 μm, Pall Corp., Tokyo, JAPAN) to separate small particles. Aliquots (2 μL) of filtrate were analyzed using LCMS-IT-TOF coupled with an Ultra-Fast LC system (Shimadzu, Kyoto, JAPAN). Each purified corrinoid was injected into an InertSustain column (3 μm, 2.0 × 100 mm, GL Science, Tokyo, JAPAN) and equilibrated with 85% solvent A [0.1% (v/v) acetic acid)] and 15% solvent B (100% methanol) at 40˚C. Corrinoid compounds were eluted using a linear gradient of methanol (15% solvent B for 0 - 5 min, increasing the concentration from 15% to 90% solvent B for 5 - 11 min, followed by decreasing the concentration from 90% to 15% solvent B for 11 - 15 min) at a flow rate of 0.2 mL/min. ESI conditions were determined by injecting pseudo B12 or B12 into the MS detector to ascertain the optimum parameters for detecting the parent B12 compound and daughter ions. ESI-MS was operated in the positive ion mode with argon as collision gas. Pseudo B12 (m/z 672.777) and B12 (m/z 678.292) as [M + 2H]2+ were confirmed by comparing the observed molecular ions and retention times.
3. Results and Discussion
3.1. Vitamin B12 Contents
B12 levels were assayed in 10 Chinese black tea leaves that are commercially available worldwide using the microbiological B12 assay method based on L. delbrueckii ATCC 7830 (Table 1). Traces (0.25 - 0.69 μg/100g dry weight) of the corrected B12 were observed in Pu’er, Fu, and Brick tea leaves. However, Ryubao tea leaves (sample H) contained the highest B12 content (1.37 μg/100g dry weight), which is similar to that previously reported  . To further clarify whether Ryubao tea leaves generally contain high levels of B12, we determined the B12 content of the other Ryubao leaf samples. As shown in Table 2, the corrected B12 content of various Ryubao tea leaves varied (0.06 - 1.37 μg/100g dry weight), and their mean value was calculated as approximately 0.69 μg of B12, which is only slightly higher than that for Pu’er tea leaves (approximately 0.49 μg/100g dry weight). High levels (0.61 - 2.02 μg B12 equivalent/100 g dry weight) of the alkali-resistant factor were detected in all tested Chinese black tea leaves.
3.2. E. coli 215 Bioautography Analysis
B12 compounds identified in Chinese black tea leaf samples A - J were analyzed using the E. coli 215 bioautogram after separation using silica gel 60 TLC (Figure 2). The Ryubao tea leaf extract (sample H) produced a
*Total B12 compounds were extracted from a portion (2.0 g) of each type of black leaf homogenate by boiling at pH 4.5 in the presence of 4.0 × 10−4 % KCN and assayed using the Lactobacillus delbrueckii ATCC 7830 microbiological assay. L. delbrueckii ATCC 7830 utilizes deoxyribosides, deoxyribonucleotides (alkali-resistant factor), and B12. Correct B12 values were calculated by subtracting the results for the alkali-resistant factor from those for total B12 concentration. The B12 assay was performed in triplicate.
Table 2. Vitamin B12 content of various types of Ryubao tea leaves.
*Total B12 compounds were extracted from a portion (2.0 g) of Ryubao tea leaf homogenates by boiling at pH 4.5 in the presence of 4.0 × 10−4 % KCN, and assayed using Lactobacillus delbrueckii ATCC 7830 microbiological assay. Correct B12 values were calculated by subtracting the results for the alkali-resistant factor from those for total B12 concentration. The B12 assay was performed in triplicate.
Figure 2. Escherichia coli 215 bioautogram analysis of B12 compounds detected in various black tea leaf sample. Authentic B12 (1), pseudo B12 (2), and concentrated extracts of various black tea leaf samples A - J. Typical bioautograms from three independent experiments are presented.
Figure 3. LC/ESI-MS/MS chromatograms of authentic B12 and the B12 compounds purified from black tea leaf sample H. B12 compounds were analyzed using the LCMS-IT-TOF system (Shimadzu). Panels (A-1) and (B-1) show total ion chromatograms and those (m/z 678.2914) of authentic B12 and B12 compounds purified from sample H. Mass spectra of authentic B12 and purified B12 compounds at 7.5 min are shown in panels (A-2) and (B-2), respectively (magnified spectrum range from m/z 678 to m/z 680 is shown as an insert in each panel). MS/MS spectra for the peak of authentic B12 at m/z 678.2910 and that of purified B12 compounds at m/z 678.2884 are shown in panels (A-3) and (B-3), respectively.
single, clear spot with an Rf value identical to that of authentic B12. Indistinct spots with the Rf value identical to that of authentic B12 were detected in samples C, D, E, G, and I. The remaining samples showed no spot because of their lower B12 contents.
3.3. LC/ESI-MS/MS Analysis
To more precisely identify the corrinoid compounds present in Chinese black tea leaves, corrinoids were purified from the Ryubao tea leaf extract (sample H) containing high B12 content and identified using LC/ESI-MS/MS (Figure 3). Authentic B12 was eluted as a peak with a retention time of 7.5 min. The mass spectrum of authentic B12 primarily comprised a doubly charged ion with m/z 678.2910 [M + 2H]2+ (Figure 3(A-1) and Figure 3(A-2)). MS/MS spectra revealed a predominant monovalent ion with m/z 359.0994, which was largely attributable to the nucleotide moiety of B12 (Figure 3(A-3)). The corrinoid purified from the Ryubao tea leaf sample H was eluted as several ion peaks, indicating the presence of impurities. The mass spectrum of the main peak with m/z 687.2914 had a retention time of 7.5 min in the purified sample (Figure 3(B-1) and Figure 3(B-2)). The MS/MS spectrum of the purified compound with a monovalent ion with m/z 359.0960 was identical to that of authentic B12 (Figure 3(A-3) and Figure 3(B-3)). These results indicate that the Ryubao tea leaf sample H contained authentic B12 but not pseudo B12 which is inactive in humans.
B12 content in the tea drink prepared from the Ryubao tea leaf sample H was 0.8 ng/100mL of black tea. Therefore, consumption of approximately 300 L of this tea would provide the recommended dietary allowance for adults (2.4 μg/day)   , although ingestion of such large quantities of tea on a daily basis is not recommended. Notably, Kittaka-Katsura et al.  demonstrated that administration of the Japanese black tea drink (B12 content, approximately 2.0 ng/100mL) considerably improves B12 status in B12-deficient rats. Considering these earlier observations and our present findings, we propose that Ryubao tea leaves containing significantly levels of B12 can be utilized as a source of vitamin B12 for vegetarians.
This work was supported by JSPSKAKENHI Grant number 25450168 (FW).
- Serpen, A., Pelvan, E., Alasalvar, C., Mogol, B.A., Yavuz, H.T., Gëkmen, V., Özcan, N. and Özcelik, B. (2012) Nutritional and Functional Characteristics of Seven Grades of Black Tea Produced in Turkey. Journal of Agricultural and Food Chemistry, 60, 7682-7689. http://dx.doi.org/10.1021/jf302058d
- McKay, D.L. and Blumberg, J.B. (2002) The Role of Tea in Human Health: An Update. The Journal of the American College of Nutrition, 21, 1-13. http://dx.doi.org/10.1080/07315724.2002.10719187
- Wang, D., Xiao, R., Hu, X., Xu, K., Hou, Y., Zhong, Y., Meng, J., Fan, B. and Lui, L. (2010) Comparative Safety Evaluation of Chinese Pu-erh Green Tea Extract and Pu-erh Black Tea Extract in Wistar Rats. Journal of Agricultural and Food Chemistry, 58, 1350-1358. http://dx.doi.org/10.1021/jf902171h
- Jhoo, J.-W., Lo, C.-Y., Li, S., Sang, S., Ang, C.Y.W., Heinze, T.M. and Ho, C.-T. (2005) Stability of Black Tea Polyphenol, Theaflavin, and Identification of Theanaphthoquinone as Its Major Radical Reaction Product. Journal of Agricultural and Food Chemistry, 53, 6146-6150. http://dx.doi.org/10.1021/jf050662d
- Feng, Q., Torii, Y., Uchida, K., Nakamura, Y., Hara, Y. and Osawa, T. (2002) Black Tea Polyphenol, Theaflavins, Prevent Cellular DNA Damage by Inhibiting Oxidative Stress and Suppressing Cytochrome P450 1A1 in Cell Cultures. Journal of Agricultural and Food Chemistry, 50, 213-220. http://dx.doi.org/10.1021/jf010875c
- Kobayashi, M., Ichitani, M., Suzuki, Y., Unno, T., Sugawara, T., Yamahira, T., Kato, M., Takihara, T., Sagesaka, Y., Kakuda, T. and Ikeda, I. (2009) Black-Tea Polyphenols Suppress Postprandial Hypertriacylglycer-Olemia by Suppressing Lymphatic Transport of Dietary Fat in Rats. Journal of Agricultural and Food Chemistry, 57, 7131-7136. http://dx.doi.org/10.1021/jf900855v
- Watanabe, F. (2007) Vitamin B12 Sources and Bioavailability. Experimental Biology and Medicine, 232, 1266-1274. http://dx.doi.org/10.3181/0703-MR-67
- Millet, P., Guilland, J.C., Fuchs, F. and Klepping, J. (1989) Nutrient Intake and Vitamin Status of Healthy French Vegetarians and Nonvegetarians. American Journal of Clinical Nutrition, 50, 718-727.
- Scalabrino, G. (2009) The Multi-Faced Basis of Vitamin B12 (Cobalamin) Neurotrophism in Adult Central Nervous System: Lessons Learned from Its Deficiency. Progress in Neurobioogy, 88, 203-220. http://dx.doi.org/10.1016/j.pneurobio.2009.04.004
- Kitta-Katsura, H., Ebara, S., Watanabe, F. and Nakano, Y. (2004) Characterization of Corrinoid Compounds from a Japanese Black Tea (Batabata-cha) Fermented by Bacteria. Journal of Agricultural and Food Chemistry, 52, 909-911. http://dx.doi.org/10.1021/jf030585r
- Kittaka-Katsura, H., Watanebe, F. and Nakano, Y. (2004) Occurrence of Vitamin B12 in Green, Blue, Red, and Black Tea Leaves. Journal of Nutritional Science and Vitaminology, 50, 438-440. http://dx.doi.org/10.3177/jnsv.50.438
- Resources Council, Science and Technology Agency (1995) Standard Tables of Food Composition in Japan—Vitamin K, B6, and B12. Resource Council, Science and Technology Agency, Tokyo, 16-56.
- Tanioka, Y., Yabuta, Y., Miyamoto, E., Inui, H. and Watanabe, F. (2008) Analysis of Vitamin B12 in Food by Silica Gel 60 TLC and Bioautography with Vitamin B12-Dependent Escherichia coli 215. Journal of Liquid Chromatography & Related Technologies, 31, 1977-1985. http://dx.doi.org/10.1080/10826070802197453
- Institute of Medicine (1998) Vitamin B12. In: Dietary Reference Intakes for Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin, and Choline, Institute of Medicine, National Academy Press, Washington DC, 306-356.
- Shibata, K., Fukuwatari, T., Imai, E., Hayakawa, H., Watanabe, F., Takimoto, H., Watanabe, T. and Umegaki, K. (2013) Dietary Reference Intakes for Japanese 2010: Water-Soluble Vitamins. Journal of Nutritional Science and Vitaminology, 59, S67-S82. http://dx.doi.org/10.3177/jnsv.59.S67
*A research fellow of Japan society for the promotion of science.