比较了8种大孔树脂对费氏丙酸杆菌发酵提取液光解转化后形成的羟基钴胺素的吸附性能，考察了上柱料液pH值对吸附量的影响，分析了不同浓度和pH下乙醇溶液作为洗脱剂的解吸情况，研究了上柱速率对吸附量和解吸率的影响。结果表明，静态实验中，LX-50B型大孔树脂对羟基钴胺素的吸附性能最好，当上柱溶液pH为6时，LX-50B树脂饱和吸附量最高，为203.87 mg/g。用pH为2的60%乙醇溶液作洗脱剂时解吸效果最好，羟基钴胺素提取液以2 BV(床柱体积)/h速度上柱吸附饱和，用60%乙醇溶液以0.5 BV/h速度洗脱后，羟基钴胺素浓度提高至1863.32 mg/L，为原提取液浓度的35倍，纯度由0.38%提升至15.73%，提取纯化效果良好。
Propionibacterium freudenreichii is regarded as a GRAS (generally recognized as safe) strain, and has been successfully applied to industrial anaerobic production of deoxyadenosylcobalamin, known as the natural structure of vitamin B12. Generally, due to the unstable of deoxyadenosylcobalamin, it needs to be converted to cyanocobalamin by cyanidation during the subsequent extraction and purification steps. Hydroxycobalamin is another form of vitamin B12 and widely used in the treatment of neurological diseases and cyanide poisoning. For the industrial production of hydroxycobalamin, cyanocobalamin is usually used as the raw material and then deacylated to form hydroxycobalamin. According to this principle, the production of hydroxycobalamin involves multiple chemical reactions, including cyanidation and deacylation, along with the high cost, environmental pollution, and hazardous conditions to the operators. To avoid these disadvantages, the photolysis production of hydroxycobalamin directly from deoxyadenosylcobalamin was proposed. In this work, the adsorption and extraction of hydroxycobalamin in the deoxyadenosylcobalamin photolysis solution were studied. The adsorption properties of 8 kinds of different macroporous resins on hydroxycobalamin were compared, and the effects of pH and ethanol concentration on hydroxycobalamin adsorption and desorption were discussed. The loading and elution velocity were optimized to improve the hydroxycobalamin yield. The results showed that LX-50B macroporous resin had the best adsorption performance for hydroxycobalamin in the static adsorption process. The adsorption capacity of LX-50B macroporous resin reached 203.87 mg/g at pH 6.0. For hydroxycobalamin eluent, 60% ethanol solution with pH 2.0 was the best choice. The optimal loading velocity and elution velocity were 2 and 0.5 BV(bed volume)/h, respectively. Under the above conditions, the concentration of hydroxycobalamin in the elution was 1863.32 mg/L, improved by 35 times compared with the original photolysis solution, while the purity of hydroxycobalamin increased from 0.38% to 15.73%, with an acceptable extraction yield of 92.46%, which achieved to a good extraction and preliminary purification result.
[1]Raux E, Schubert H L, Waren M J.Biosyntheis of cobalamin (Vitamin B12): a bacterial conundrum[J].Cell Mol. Life Sci., 2000, 57(4):1880-1893 [2]吕颖坚，黄俊明.维生素的研究进展[J].中国食品卫生杂志, 2012, 24(4):394-399 [3]Zakharov S, Vaneckova M, Seidl Z, et al.Successful Use of Hydroxocobalamin and Sodium Thiosulfate in Acute Cyanide Poisoning: A Case Report with Follow-up[J].Basic Clin Pharmacol Toxicol., 2015, 117(3):209-212 [4]Martens J H, Barg H, Warren M J, et al.Microbial production of vitamin B12[J].Appl Microbiol Biotechnol., 2002, 58(2):275-285 [5] Salminen S, Wright A, Morelli L, et al.Demonstration of safety of probiotics[J].Int. J. Food Microbiol., 1998, 44(3):93-106 [6] Hodgkin D C, Kamper J, Mackag M, et al.Structure of Vitamin B12 [J].Nature, 1956, 17(3):64-66 [7]Allwyn G C, Laurie M Y, Joseph J S.Time-Resolved spectroscopic studies of B12 coenzymes: A comparison of the primary photolysis mechanism in methyl-,Ethy-,n-propyl,and 5’-deoxyadenosylcobalamin[J].Journal of Chromatogr-aphy., 2002, 124(3):434-441 [8]Eefei C, Mark R C.Nanosecond transient absorption spectroscopy of coenzyme B12[J].The Journal of Biological Chemistry., 1990, 265(22):12987-12994 [9]Feng X H, Li L G.A study on the stability of cobamamide products and tablet dosage[J].Chin J Biochem Pharm., 1997, 18(5):249-250 [10]罗伟，郝常明.维生素的研究进展[J].中国食品添加剂, 2002, 4(3):15-18 [11]张玉明，王雷，王云山，等.一种简便、快速测定发酵液中含量的方法[J].食品与发酵工业, 2005, 31(1):123-125 [12]王雷，张玉明，王云山，等.维生素的光解研究及发酵液中维生素检测新方法的建立[J].中国生物工程杂志, 2006, 26(4):81-85 [13] 资生堂株式会社.维生素B12的纯化方法 [P]. 日本专利，JP1016597A，1989-01-20. [14] Brink N G, Wolf P J.Benzyl alcohol-water countercurrent distribution extraction of vitamin B12 [P]. US2307717A, 1952-08-19. [15] Denkewalter R G, Hughey G B, Kutosh S.Extraction of Vitamin B12-active Compounds from Aqueous Solutions using Solid Trichlorophenol [P]. US2635985A, 1953-04-21. [16]Guan N, Liu L, Shin H D, et al.Systems-level understanding of how Propionibacterium acidipropionici respond to propionic acid stress at the microenvironment levels: Mechanism and application[J].J. Biotechnol., 2013, 167(3):56-63 [17]Wang P, Wang Y S, Liu Y D, et al.Novel in situ Product Removal Technique for Simultaneous Production of Propionic Acid and Vitamin B12 by Expanded Bed Adsorption Bioreactor[J].Bioresour. Technol., 2012, 104(10):652-659 [18]Charles A, Roessner A, Ian S.Fine-tuning our knowledge of the anaerobic route to cobalamin (Vitamin B12)[J].J. Bacteriol., 2006, 188(21):7331-7334 [19]杨洪淼，廖海明，任雪，等.腺苷钴胺有关物质分析方法的改进及杂志研究[J].中国生化药物杂志, 2015, 35(6):157-160
