7 3 Vol. 7 No. 3 2016 3 Journal of Food Safety and Quality Mar., 2016 B 12 * 蒋孟虹, 许苹, 秦峰, 刘浩, 杨美成 (, 201203) 摘要 : 目的 B 12 方法, B 12, (36±1) 19~20 h, 结果 B 12 0.001~0.010 ng/ml, B 12 2.9 μg/100 g 结论 B 12 关键词 : B 12 ; ; Determination of trace vitamin B 12 in enteral nutrition powder by microbiological method JIANG Meng-Hong, XU Ping, QIN Feng, LIU Hao, YANG Mei-Cheng * (Shanghai Institute for Food and Drug Control, Shanghai 201203, China) ABSTRACT: Objective To establish a method for the determination of trace vitamin B 12 in enteral nutrition powder by microbiological method. Methods The test organism was Lactobacillus leichmamii. The medium without vitamin B 12 was used as assay medium. The sample were incubated for 19~20 h at the temperature of (36±1) and measured by microbial turbidimetric method. Results Vitamin B 12 had a good linear relationship in the range of 0.001~0.010 ng/ml, and the content of vitamin B 12 in enteral nutrition powder was 2.9 μg/100 g. Conclusion This method is accurate and reliable, and suitable for the assay of trace vitamin B 12 in enteral nutrition powder. KEY WORDS: vitamin B 12 ; enteral nutrition powder; microbiological method 1 引言 B 12, B,,, B 12, DNA, [1], B 12, B 12,,, [2-4], B 12, B 12 [5-11] [12] [13] * 通讯作者 :,,, E-mail: yangmeicheng@vip.sina.com *Corresponding author: YANG Mei-Cheng, Ph.D., Chief Pharmacist, Shanghai Institute for Food and Drug Control, Shanghai 201203, China. E-mail: yangmeicheng@vip.sina.com
894 7 [14] [15-16],, B 12,, B 12, [16],, B 12 (Lactobacillus leichmannii) B 12, B 12,,, B 12,,, B 12 2 材料与方法 2.1 材料与试剂 (CP225D, Sartorius); ph (Seven easy, Mettler Toledo); (MS 3, IKA); (HVA-85, Hirayama); (KB240, Binder); (UV2450, ) (ATCC 7830, American Type Culture Collection); B 12 ( 96.2%, : 203B, ); ( : 100361526); ( BD ); ( BD ); B 12 ( ); (, ) 2.2 检测方法 2.2.1 测试菌液的制备,, (36±1) 24 h, 2~3, (36±1) 18~24 h, 2000 r/min 2~3 min,, 10 ml,, 2 10 ml, 10 ml,, 550 nm,,, 60%~80% 2.2.2 标准曲线的制作 (1) B 12 (10 μg/ml): 20 mg B 12, ( 25%) B 12 10 μg/ml B 12 (100 ng/ml): 5.0 ml B 12 500 ml B 12 (1 ng/ml): 5.0 ml B 12 500 ml : B 12, 0.02 ng/ml, 0.01 ng/ml (2) 1 4, B 12, 表 1 标准曲线的制备 Table 1 Preparation of standard curve 0.01 ng/ml (ml) 0.02 ng/ml (ml) (ml) (ml) S1 0 0 5 5 S2 0 0 5 5 S3 1 0 4 5 S4 2 0 3 5 S5 3 0 2 5 S6 4 0 1 5 S7 5 0 0 5 S8 0 3 2 5 S9 0 4 1 5 S10 0 5 0 5
3, : B 12 895 2.2.3 试样的处理 1.3 g, 1.0 g, ( 1 )1.2 g, 100 ml ( ), 10 ml, 150 ml,, 121 10 min,, B 12, ph 4.5±0.2, 250 ml, 5 ml 100 ml, 20~30 ml, ph 6.8±0.2, B 12 ph, 2.2.4 待测溶液的制备 2 4, B 12, 表 2 待测溶液的制备 Table 2 Preparation of samples (ml) (ml) (ml) 1 1 4 5 2 2 3 5 3 3 2 5 4 4 1 5 2.2.5 灭菌 接种及培养 121 5 min, 30 50 μl ( S1 ),,, (36±1) 19~20 h 2.2.6 测定, S1,,,, B 12,, B 12, B 12 3 结果与分析 3.1 测试菌液的制备 3.1.1 测试菌液透光率的确定,,,, 60%~80%, 3.1.2 测试菌液加入量的考察,, 25 50 100 200 μl 4,,, 50 μl, 3.2 标准曲线的制作 10 ml, 5 ml,, 10 ml, 0.0005 0.001 0.002 0.003 0.004 0.005 0.006 0.008 0.010 0.015 0.020 0.025 ng/ml,,,, B 12, B 12, B 12,,,,,, 0.001 0.002 0.003 0.004 0.005 0.006 0.008 0.010 ng/ml 3.3 待测液的制备 10 ml, 5 ml, 1 2 3 4 ml, 10 ml, B 12 0.01~0.02 ng/ml ( 1 2 4 8 g), 2 g,
896 7 ( ), 3.4 培养及测定时间的考察, (36±1) 19~20 h,,, 18 h, 1 h 20 h,,,, 30%,, 3.5 标准曲线的绘制 B 12, B 12, ( 1) B 12 0.001~0.010 ng/ml, Y=-5896.983X+92.203, r=0.99 4 结论 B 12,,, B 12, 参考文献 [1],. B 12 [J]., 2012, 24(4): 394 399. Lv YJ, Huang JM. Progress on the studies of vitamin B 12 [J]. Chin Food Hyg, 2012, 24(4): 394 399. [2] Suarez L, Hendricks K, Felkner M, et al. Maternal serum B 12 levels and risk for neural tube defects in a Texas-Mexico border population [J].Ann Epidemiol, 2003, 13(2): 81 88. [3] Molloy AM, Kirke PN, Troendle JF, et al. Maternal vitamin B 12 status and risk of neural tube defects in a population with high neural tube defect prevalence and no folic acid fortification [J]. Pediatrics, 2009, 123(3): 917 923. [4],,. B 12 [J]., 2003, 15(3): 248 252. Lu J, Yang DJ, Wang ZT. Review of chemical methods for determining cyanocobalamin [J]. Chin Food Hyg, 2003, 15(3): 248 252. [5] Guggisberg D, Risse MC, Hadorn R. Determination of vitamin B 12 in meat products by RP-HPLC after enrichment and purification on an immunoaffinity column [J]. Meat Sci, 2012, 90(2): 279 283. [6],,. HPLC B 12 [J]., 2004, (2): 93 96. Hao YP, Jiang JD, Fang YG. Research on the determination 1 B 12 Fig. 1 Standard curve of vitamin B 12 3.6 样品测定,, B 12, ±15%,, B 12, B 12 2.9 μg/100 g method of vitamin B 12 in composite vitamin additives for milk powder and formula by HPLC [J]. China Food Addit, 2004, (2): 93 96. [7],,,. - B 12 [J]. ( ), 2013, 39(2): 185 190. Chu XJ, Tao BH, Lai SY, et al. Quantification of vitamin B 12 in infant formula by dimensional liquid chromatography with solid phase extraction [J]. J Zhejiang Univ (Agric Life Sci), 2013, 39(2): 185 190. [8],,,. / B 12 [J]., 2013, 32(3):
3, : B 12 897 389 392. Wu N, Jiang JD, Tao DL, et al. Determination of vitamin B 12 content in milk powder by immunoaffinity column purification -HPLC method [J]. J Instrumental Anal, 2013, 32(3): 389 392. [9],,,. 8 [J]., 2008, 27(4): 408 411. Liu N, Chen DZ, Tang H, et al. Simultaneous determination of eight water-soluble vitamins in baby milk powder by high performance liquid chromatography [J]. J Instrumental Anal, 2008, 27(4): 408 411. [10],,,. 9 [J]., 2014, 5(3): 899 904. Ren DD, Xie YF, Liu JJ, et al. Simultaneous determination of nine water-soluble vitamins in foods by high performance liquid chromatography [J]. J Food Saf Qual, 2014, 5(3): 899 904. [11] GB/T 5009.217-2008 B 12 [S]. GB/T 5009.217-2008 Determination of vitamin B 12 in health foods [S]. [12] Karmi O, Zayed A, Suheir Baraghethi, et al. Measurement of vitamin B 12 concentration: a review on available methods [J]. IIOAB J, 2011, 2(2): 23 32. [13] Chen JH, Jiang SJ. Determination of cobalamin in nutritive supplements and chlorella foods by capillary electrophoresis-inductively coupled plasma mass spectrometry [J]. J Agric Food Chem, 2008, 56(4): 1210 1215. [14] L Sagaya SK, Thakur MS. Competitive immunoassay for analysis of vitamin B 12 [J]. Anal Biochem, 2011, 418(2): 238 246. [15],,,. B 12 [J]., 2014, 148: 42 44. Wu H, Liu DH, Zhang H, et al. Determination of vitamin B 12 content in milk powder by immunoaffinity column purification HPLC method [J]. China Dairy, 2014, 148: 42 44. [16] GB5413.14-2010 B 12 [S]. GB5413.14-2010 Food safety national standard-determination of vitamin B 12 in food for infants and young children, milk and milk products [S]. 作者简介 ( 责任编辑 : 杨翠娜 ) 蒋孟虹, 硕士, 药师, 主要研究方向为药品 食品检验 E-mail: jmh1122ow@163.com 杨美成, 博士, 主任药师, 主要研究方向为实验室质量管理 药物分析与微生物学检验 E-mail: yangmeicheng@vip.sina.com