Zhu Xuan, Wang Xiaofeng, Zhang Chen, Wang Xiaoqi, Gu Qing
Zhejiang Gongshang University, Key Laboratory for Food Microbial Technology of Zhejiang Province, No. 18 Xuezheng Str., Hangzhou, Zhejiang Province 310018, China.
Zhejiang Gongshang University, Key Laboratory for Food Microbial Technology of Zhejiang Province, No. 18 Xuezheng Str., Hangzhou, Zhejiang Province 310018, China.
Food Chem. 2015 May 15;175:523-8. doi: 10.1016/j.foodchem.2014.11.163. Epub 2014 Dec 11.
We describe a sensitive and selective method for determination of vitamin B12 content in fermented foods using riboswitch sensor. A riboswitch amplicon from Propionibacterium freudenreichii was cloned in p519NGFP vector in Escherichia coli BL21 (DE3). The expression of green fluorescence protein was revers correlated to the concentrations of adenosylcobalamin. Adenosylcobalamin directly binds to riboswitch region leading to conformational changes in the secondary structure of mRNA, thus inhibiting expression. After various examinations, a standard curve was obtained from 10 to 1000 ng/mL of cyanocobalamin. The limit of determination is 10 ng/mL. The inter-assay coefficients of variation were 7.5% for the range of 10-1000 ng/mL. The recovery of this method was 92.3%. This method has no or less responses to nucleic acid, pseudovitamin B12, vitamin B12 bound to intrinsic factor and haptocorrin. The riboswitch sensor results were similar with HPLC, but they were Ca. 24% lower than the microbiological assay results.
我们描述了一种使用核糖开关传感器测定发酵食品中维生素B12含量的灵敏且具选择性的方法。将来自费氏丙酸杆菌的核糖开关扩增子克隆到大肠杆菌BL21(DE3)中的p519NGFP载体中。绿色荧光蛋白的表达与腺苷钴胺素的浓度呈负相关。腺苷钴胺素直接与核糖开关区域结合，导致mRNA二级结构发生构象变化，从而抑制表达。经过各种检测，得到了10至1000 ng/mL氰钴胺素的标准曲线。测定限为10 ng/mL。在10 - 1000 ng/mL范围内，批间变异系数为7.5%。该方法的回收率为92.3%。该方法对核酸、假维生素B12、与内因子结合的维生素B12和运钴胺素蛋白的反应无或较小。核糖开关传感器的结果与高效液相色谱法相似，但比微生物测定结果约低24%。
