微生物法检测叶酸、生物素、维生素B12接种液制备方法的优化

目的解决使用微生物法GB 5009.211-2014、GB 5009.259-2016、GB 5413.14-2010检测食品中叶酸、生物素、维生素B_(12)接种液制备耗时长的问题。方法将标准方法制备的接种液与甘油水按1:1(V:V)比例混匀,分装于小离心管,-80℃冻存。再通过用冻存不同时间的接种液检测样品,分析标准曲线线性、中间精密度和加标回收率来验证制备方法的适用性。结果标准曲线线性关系r~2均0.99,中间精密度叶酸RSD为1.5%～3.2%,生物素RSD为1.7%～4.2%,维生素B_(12) RSD为2.5%～9.3%;回收率方面,叶酸为99.5%～101.0%,生物素98.6%～101.3%,维生素B_(12)90.3%～96.4%。鼠李糖乳杆菌、植物乳杆菌、莱氏曼氏乳杆菌接种液超低温冻存有效时间长分别为6、8、3个月。结论优化后的接种液制备方法缩短了检测周期,操作简便、方法可靠,适合检测使用。     

Synthesis and characterization of thermo‐ and pH‐sensitive block copolymers bearing a biotin group at the poly(ethylene oxide) chain end

A poly(ethylene oxide)‐block‐poly(dimethylamino ethyl methacrylate) block copolymer (PEO‐b‐PDMAEMA) bearing an amino moiety at the PEO chain end was synthesized by a one‐pot sequential oxyanionic polymerization of ethylene oxide (EO) and dimethylamino ethyl methacrylate (DMAEMA), followed by a coupling reaction between its PEO amino and a biotin derivative. The polymers were charac terized with ¹H NMR spectroscopy and gel permeation chromatography. Activated biotin, biotin‐NHS (N‐hydroxysuccinimide), was used to synthesize biotin‐PEO‐PDMAEMA. In aqueous media, the solubility of the copolymer was temperature‐ and pH‐sensitive. The particle size of the micelle formed from functionalized block copolymers was determined by dynamic light scattering. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 102: 3552–3558, 2006     

Fabrication and Characterization of an OTFT‐Based Biosensor Using a Biotinylated F8T2 Polymer

Solution‐processable organic semiconductors have been investigated not only for flexible and large‐area electronics but also in the field of biotechnology. In this paper, we report the design and fabrication of biosensors based on completely organic thin‐film transistors (OTFTs). The active material of the OTFTs is poly(9,9‐dioctylfluorene‐co‐bithiophene) (F8T2) polymer functionalized with biotin hydrazide. The relationship between the chemoresistive change and the binding of avidin‐biotin moieties in the polymer is observed in the output and on/off characteristics of the OTFTs. The exposure of the OTFTs to avidin causes a lowering of ID at V_D = ‐40 V and V_G = ‐40 V of nearly five orders of magnitude.     

Improved ELISA and dot-blot methods for the detection of whey proteins in meat products

Two techniques, ELISA and dot-blot, were applied to the qualitative detection of very low levels of whey proteins in liver pãtés. The use of an avidin-biotin amplification system for both methods led to a useful improvement of the detection limit. The detection level which was 4 g kg^?1 with the classical ELISA method was improved to I g kg^?1 with the amplified ELISA method. Using the dot-blot technique, the results showed that the minimum detectable level was 1–7 g kg^?1 for the classical method with nitrocellulose (NC), 0–7 g kg^?1 for the amplified method with NC, 0–7 g kg^?1 for the classical method with cyanogen bromide-activated NC (activated NC) and 0–3 g kg^?1 for the amplified method with activated NC.     

BIOTIN AND THE METABOLISM OF BAKER'S YEAST

Based on experimental results and the results presented in the literature a model for the metabolism of baker's yeast under biotin deficiency is presented. In these conditions the most sensitive point in the metabolism is the carboxylation of pyruvate to oxaloacetate catalyzed by the biotin-containing pyruvate carboxylase. Because the rate of glycolysis is not affected by biotin-deficiency pyruvate accumulates in the cells and is partially excreted into the medium. The high pyruvate pool in the cells means that the metabolism is mainly fermentative even in vigorously aerated cultures. The oxidation of pyruvate to acetyl-CoA proceeds almost unaffected, as can be seen by the production of elevated amounts of ethyl acetate by yeast grown under sub-optimal biotin concentrations. Acetyl-CoA carboxylase, which also has biotin as the prosthetic group, is not as sensitive to a deficiency of this growth factor as is pyruvate carboxylase. In yeast grown without biotin the amounts of fatty acids and lipids are the same or even higher than in cells grown under optimal conditions. The metabolism from oxaloacetate towards the TCA cycle and glutamate is not affected as strongly as is the metabolism to aspartate, which is present in cells in strictly limited amounts. This causes an over-production of metabolic intermediates, e.g. diazotizable arylamine and hypoxanthine as well as citrulline. Their conversion to normal cell constituents, purine nucleotides and arginine, is dependent on the aspartate available and is thus depressed by lack of biotin.     

生物素和VB1在谷氨酸棒杆菌过量合成缬氨酸中的作用

通过对谷氨酸棒杆菌突变株AATV341进行生物素和VB1添加量的实验，探讨了生物素和VB1添加量对该菌株菌体质量及缬氨酸产量的影响；同时根据对AATV341进行的胞内外某些特定氨基酸和有机酸检测，研究了这两种维生素对碳架代谢流的分布，尤其是对丙酮酸节点处的代谢流量分布的影响。结果表明，生物素添加量在40μg／L时，菌体质量和缬氨酸在胞外的累积明显提高，谷氨酸在胞外累积降低；VB1的适量添加，可提高缬氨酸在胞外的累积，降低发酵液中乙酸和草酰乙酸的含量。     

微生物法测定多种维生素片中的生物素含量

目的利用微生物法测定多种维生素片中生物素的含量。方法在GB 5413.19-2010分析方法的基础上,通过将乳酸杆菌培养基改成MRS肉汤培养基来制备菌悬液,样品经过65℃~70℃水浴超声提取,在630nm波长下用酶标仪测定培养液吸光度,对生物素含量进行定量检测。与国标法进行标准曲线、精密度、准确度对比试验,以及对新建立的方法进行加标回收试验。结果生物素浓度在0.01~0.1 ng/m L范围内与吸光度呈现良好的二次曲线关系,相关系数r20.999,精密度和准确度良好。在80%、100%、120%添加水平下,生物素的回收率分别为99.8%、96.1%、97.2%。结论方法操作简单、灵敏度高、处理量大,适合生产企业的多种维生素片的大批量质量控制和检测。     

婴儿配方食品中生物素测定

植物乳杆菌（ATCC No.8014)对生物素有很高的灵敏性，利用这种特异性，可以定量地测出奶基婴幼儿食品中的生物素的含量。为了达到定量测定的目的，在供繁殖试验菌株所用培养基中供给除生物素以外的所有营养成分，这样植物乳杆菌的生长就会同标准溶液及未知测定溶液的生物素的水平相对应。结果表明，本文法的重现性好，变异系数为3.34%，平均回收率为97%。     

Liquid Hybridization and Solid Phase Detection: A Highly Sensitive and Accurate Strategy for MicroRNA Detection in Plants and Animals

MicroRNAs (miRNAs) play important roles in nearly every aspect of biology, including physiological, biochemical, developmental and pathological processes. Therefore, a highly sensitive and accurate method of detection of miRNAs has great potential in research on theory and application, such as the clinical approach to medicine, animal and plant production, as well as stress response. Here, we report a strategic method to detect miRNAs from multicellular organisms, which mainly includes liquid hybridization and solid phase detection (LHSPD); it has been verified in various species and is much more sensitive than traditional biotin-labeled Northern blots. By using this strategy and chemiluminescent detection with digoxigenin (DIG)-labeled or biotin-labeled oligonucleotide probes, as low as 0.01–0.25 fmol [for DIG-CDP Star (disodium2-chloro-5-(4-methoxyspiro{1,2-dioxetane-3,2′-(5′-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)phenyl phosphate) system], 0.005–0.1 fmol (for biotin-CDP Star system), or 0.05–0.5 fmol (for biotin-luminol system) of miRNA can be detected and one-base difference can be distinguished between miRNA sequences. Moreover, LHSPD performed very well in the quantitative analysis of miRNAs, and the whole process can be completed within about 9 h. The strategy of LHSPD provides an effective solution for rapid, accurate, and sensitive detection and quantitative analysis of miRNAs in plants and animals.