婴幼儿配方乳粉中5种核苷酸的HPLC检测方法

研究了液相色谱法检测婴幼儿配方乳粉中5种核苷酸的液相色谱检测方法。样品经酸沉淀后调节pH值为6.5～7.0,以液相色谱于278 nm,257 nm波长检测。各核苷酸的定量限:胞苷酸CMP为1.6 mg/100 g,尿苷酸UMP为1.7 mg/100 g,鸟苷酸GMP为1.8 mg/100 g,次黄嘌呤核苷酸IMP为1.0 mg/100 g,腺苷酸AMP为2.0 mg/100 g。其样品加标回收率为85%～110%。     

高效阴离子交换色谱法测定婴幼儿配方奶粉中酵母β-葡聚糖

建立一种测定婴幼儿配方奶粉中酵母β-葡聚糖的分析方法。奶粉样品经蛋白酶酶解去除蛋白质,脂肪酶酶解去除脂肪,葡聚糖酶酶解β-葡聚糖产生葡萄糖,经高效阴离子交换色谱-脉冲安培检测,计算β-葡聚糖含量。结果表明,最优酶解条件为：酶解温度50 ℃、酶解时间2.0 h、溶壁酶添加量500 μL。采用PA10阴离子交换柱进行分离葡萄糖,葡萄糖在0.10～50.0 mg/L范围内线性良好（R2＞0.999 5）,添加水平在10.0～50.0 mg/100 g范围的回收率为96.9%～102.1%,精密度试验结果相对标准偏差为2.43%（n=5）,方法检出限为3.0 mg/100 g,定量限为10.0 mg/100 g。该方法灵敏、准确,可用于婴幼儿配方奶粉中酵母β-葡聚糖的测定。     

酶水解-液相色谱法测定婴幼儿食品中的色氨酸

利用酶解水解法,建立了婴幼儿配方乳粉(以下简称乳粉)和特殊医学用途婴儿配方食品(以下简称为医学食品)中游离和总色氨酸的高效液相色谱检测方法。样品经蛋白酶水解后,经C18色谱柱(150 mm×4.6 mm, 3.5μm)分离,以甲醇-0.1%乙酸溶液作为流动相等度洗脱,紫外检测器串联荧光检测器检测,外标法定量。结果表明:在质量浓度为0.2～100μg/mL范围内线性关系良好(r2=0.9999)。添加质量分数在100,500,1000 mg/100 g时,色氨酸的回收率介于99.0%～105.3%之间。色氨酸的检出限为1.5 mg/100g,定量限为5 mg/100 g。本方法操作简便,重现性好,可应用于市售乳粉和医学食品中色氨酸含量的测定,填补了检测标准缺失的空白。     

超高效液相色谱-紫外检测法测定婴幼儿配方乳粉中的核苷酸含量

采用磺酸甜菜碱型两性离子亲水相互作用色谱法,建立婴幼儿配方乳粉中5 种核苷酸（5’-胞嘧啶核苷酸、5’-尿嘧啶核苷酸、5’-腺嘌呤核苷酸、5’-次黄嘌呤核苷酸、5’-鸟嘌呤核苷酸）的测定方法。使用二维固相萃取模式,弱阴离子交换和强阳离子交换,对样品中的核苷酸进行提取,紫外检测器采集数据;用0.001%甲酸-乙腈溶液和pH 5的10 mmol/L磷酸二氢钠溶液作为流动相,等度洗脱。5 种核苷酸在其线性范围内线性关系良好,定量限在0.10～0.15 mg/100 g范围内,方法回收率均在95%以上,精密度在10%以内。该方法快速、灵敏、准确,适合各类婴幼儿配方乳粉中核苷酸的测定。     

酶联免疫吸附法(ELISA)检测婴幼儿配方食品中谷蛋白

介绍了利用ELISA法的醇溶谷蛋白试剂盒测定婴幼儿配方食品中的谷蛋白含量。样品经处理后,用提取液提取样品中谷蛋白,提取液经离心,稀释后,用酶联免疫吸附法直接测定。检测的线性范围是5～80μg/kg,相关系数是0.9988,回收率在115.6%～120.6%之间,证明可以用酶联免疫法测定婴幼儿配方食品中的谷蛋白含量。     

分光光度法检测特殊医学用途婴儿配方乳粉中左旋肉碱

优化前处理方法来提取氨基酸配方和蛋白质水解配方特殊医学用途配方食品中的左旋肉碱,并采用分光光度法进行测定。左旋肉碱与乙酰辅酶A生成游离的辅酶A,游离的辅酶A和2-硝基苯甲酸反应生成黄色物质,利用分光光度法进行外标法定量。考察了酶水解前处理方法的方法学参数,同时在不同基质的样品中对确定的最佳前处理方法进行了实验验证。采用酶水解所得处理液易于过滤且滤液澄清。一种特殊医学用途氨基酸配方食品样品在5 mg/100 g、15 mg/100 g和50 mg/100 g添加水平的加标回收率为96.1%～108.5%,相对标准偏差RSD为3.18%～3.85%。酶水解前处理方法快速、准确、灵敏,适用于批量测定氨基酸配方和蛋白质水解配方特殊医学用途配方食品中左旋肉碱。     

HPLC法测定婴幼儿配方食品中的糠氨酸

建立高效液相色谱法测定婴幼儿配方食品中糠氨酸的分析方法。样品经复溶后,经10.6 mol/L盐酸水解,用迪马铂金ODS色谱柱分离,采用紫外检测器在280 nm下进行检测,外标法定量。方法标准曲线线性相关系数r2=0.999 9,精密度RSD为2.23%,加标回收率为96.56%~106.24%,检出限(S/N=3)为0.38μg/mL,检测限(S/N=10)为1.25μg/mL。分析21个婴幼儿配方食品样品(含11个品牌),结果表明,糠氨酸含量水平在468 mg/100 g~1 467 mg/100 g(以蛋白质计)之间,部分样品的蛋白质质量损伤程度较大。     

钒钼黄分光光度法检测液态特殊食品中磷含量的优化

目的建立钒钼黄分光光度法检测低磷含量液态婴幼儿配方食品、液态特殊医学用途配方食品等特殊食品中磷含量的分析方法。方法试样经消解,磷在酸性条件下与钒钼酸铵生成黄色络合物钒钼黄,于440 nm测定试样溶液中钒钼黄的吸光度值,幵与标准系列比较定量。对3种基质液态特殊食品的磷含量进行测定。结果增大称样量能满足方法准确度和精密度要求;称样量为4 g时,回收率范围为92.4%～107.0%,相对标准偏差为0.8%～3.7%;方法定量限为30 mg/100 g。结论该方法能够准确测定液态配方食品中较低含量的磷,适合液态婴幼儿配方食品、液态特殊医学用途配方食品等特殊食品中磷含量的测定。     

基于超高效液相色谱-三重四级杆串联质谱法的花椒中氨基酸和核苷类成分分析与评价

建立了UPLC-MS/MS法同时测定花椒中17种氨基酸和6种核苷的分析方法，并对不同产地的花椒样品进行主成分分析和聚类分析。样品经水提取后用超高效液相色谱-三重四级杆串联质谱仪测定，采用电喷雾电离源，多反应监测(MRM)模式检测，外标法定量。结果表明，17种氨基酸和6种核苷在各自的线性范围内线性良好，检出限为0.001 3～0.087 8μg/g,定量限为0.004 5～0.292 7μg/g;加标回收率为60.4%～120.4%,相对标准偏差(RSD)为0.6%～13.4%,操作方法简便快速，灵敏度和稳定性较好；14批次花椒样品中TAA在333.5～569.9 mg/kg之间，平均含量为451.3 mg/kg,其中S4氨基酸总量最高；氨基酸中精氨酸含量最高，平均含量为172.7 mg/kg,占TAA的38.3%;核苷总量在14.4～38.5 mg/kg之间，平均含量为27.7 mg/kg,其中S3核苷总量最高；核苷中尿苷含量最高，平均含量为21.6 mg/kg,占核苷总量的77.9%;通过主成分分析和聚类分析发现来自甘肃省陇南市西和县、甘肃省陇南市武都区、甘肃省天水市秦安区的花椒综合...     

液相色谱-串联质谱法测定牛乳基婴幼儿配方奶粉中的乳铁蛋白

目的建立液相色谱-串联质谱法(liquid chromatography-tandem mass spectrometry, LC-MS/MS)测定牛乳基婴幼儿配方奶粉中的乳铁蛋白含量的方法。方法试样中的乳铁蛋白经尿素-碳酸氢铵缓冲液提取,二硫苏糖醇还原,碘乙酰胺烷基化,37℃酶解后,特征肽段用液相色谱-串联质谱正离子模式测定,内标法定量。结果乳铁蛋白在0.00080-0.16 pmol/μL（相当于10~2000 mg/100 g）水平间线性关系良好,相关系数r≥0.9990,检出限为0.0020 pmol/μL,相当于25 mg/100g。检测结果加标回收率为92.00%~101.90%,相对标准偏差2.96%~4.15%。结论该方法准确好、特异性强、灵敏度高,可用于牛乳基婴幼儿配方奶粉中的乳铁蛋白含量的测定。     

高效液相色谱法测定母乳及牛乳中总核苷酸含量

建立高效液相色谱测定母乳和牛乳中总核苷酸含量和组成的分析方法。总核苷酸包括游离核苷酸、游离核苷、核苷聚合物及加合物等不同来源的可利用核苷总量。利用酶解法释放出母乳中的核苷,以硼化聚丙烯酰胺凝胶（Affi-Gel 601）作为固相萃取介质进行样品净化,用反相高效液相色谱仪检测,内标法定量,检测结果以核苷酸计。且针对母乳样品的特性,增加高温灭活步骤进行样品前处理,以检测母乳中核苷酸的天然含量及组成。对所建方法进行全面验证,结果表明其具有良好的线性,相关系数（r2）均在0.999以上;母乳中各核苷酸的检出限和定量限分别在0.18～0.45 mg/L和0.60～1.51 mg/L之间;母乳基质中3 种不同加标水平下总核苷酸的回收率均在99%～108%之间,连续3 d 6 次检测结果的相对标准偏差在0.8%～7.8%之间,显示了良好的准确性及精密度。然后进一步利用所建检测方法,评估母乳及5 种市面常见的牛乳中的核苷酸总量及组成用以验证该方法的适用性。该方法的灵敏度、准确性及精密度均可满足对母乳和牛乳样品中天然的核苷酸含量及组成的测定要求。本研究为今后大样本量调查母乳及牛乳样品中核苷酸的含量和组成提供了更加科学准确的方法。     

食品中核苷类成分的药理作用研究进展

日常摄入的核苷及核苷酸类成分在维持骨髓造血细胞、肠黏膜以及脑组织的正常功能方面具有重要的作用。而膳食是为人体提供该类成分的主要来源之一。本文综述了膳食中核苷及核苷酸对人体免疫、代谢、肝脏、心血管及神经系统等所发挥的生理作用,以及其抗菌和抗病毒等其他生理活性。     

Nucleosides are efficiently absorbed by Na(+)-dependent transport across the intestinal brush border membrane in veal calves

In previous work, a comparatively high capacity for Na(+)-dependent transport of nucleosides across the intestinal brush border membrane (BBM) was observed in dairy cows, which might be related to digestion of the large amount of nucleic acids present in ruminal microorganisms in the ruminant small intestine. If this were the case, the capacity for Na(+)-dependent intestinal nucleoside transport should be much lower in veal calves, in which only small amounts of nucleic acids, nucleotides, and nucleosides reach the small intestine via the milk replacer. To test this hypothesis, we investigated Na(+)-dependent transport of 3H-labeled thymidine and guanosine across the BBM using BBM vesicles (BBMV) isolated from the small intestine of veal calves. In the presence of a transmembrane Na+ gradient both substrates were transported against a concentration gradient. Inhibitory studies showed that thymidine and guanosine are transported by two different transporters with overlapping substrate specificity, one accepting predominantly pyrimidine nucleosides (N2) and one accepting particularly purine nucleosides (N1). Nucleoside transport was inhibited by glucose along the whole small intestine. Maximal transport rates similar to those in dairy cows were obtained for the proximal, mid-, and distal small intestine. These findings suggest that the high absorptive capacity for nucleosides is a genetically fixed property in the bovine small intestine, which is already present in the preruminant state of veal calves. It may contribute to the high digestibility of nucleic acids observed by others in veal calves receiving milk replacer supplemented with RNA. Its main function may be the efficient absorption of nucleosides resulting from the digestion of nucleic acids associated with desquamated enterocytes. Due to the limited de novo synthesis of nucleotides in enterocytes intracellular uptake of nucleosides across the BBM may contribute to nucleic acid synthesis in enterocytes and thus may have a trophic effect on the intestinal epithelium.     

Determination of purine compounds and purine bases in food

The total purine content and the content of RNA, DNA, nucleotides, nucleosides and free purine bases has been determined in commercial raw food. After hydrolysing food samples with acid, the total purine content is enzymatically determined as uric acid. For the determination of the nucleic acid content, a method is chosen that allows for the analysis of the composition of nucleic acids. The amount of purine bound in nucleic acids and of purine bound in nucleotides, nucleosides and free bases is very different. The content of nucleic acids is especially high in the innards of veal, pork and beef. In these samples the quantity of purine bound in nucleotides, nucleosides and bases is very small. In trout and herring, however, more purine is bound in RNA and DNA. The same is true of roe, pork and beef muscle. Peas and beans have the lowest total purine content of all the samples examined.     

Water-Soluble Flavor and Odor Precursors of Meat. 3. Changes in Nucleotides, Total Nusleosides and Bases of Beef, Pork and Lamb During Heating

SUMMARY: Studies were made of the influence of heating on nucleotides and total purine nucleosides and bases of beef, pork and lamb muscle. lnosinic acid was the predominant nucleotide in all three species and it was degraded by heating. Adenylic acid increased during cooking in meat from all three species. Cytidylic, uridylic and guanylic acids were present in relatively low concentrations in meat from all three species and changed little during cooking. A rapid method for estimating total nucleotides resulted in greater variation than a specific method for measuring individual nucleotides.     

Decomposition of the Flavor Enhancers, Monosodium Glutamate, lnosine-5'-Monophosphate and Guanosine-5'-Monophosphate during Canning

Monosodium glutamate (MSG) was found to be very stable to canning conditions. Both flavor nucleotides inosine-5'-monophosphate (IMP) and guanosine-5'-monophosphate (GMP) were lost through hydrolysis, up to 50% or more, depending on the canning conditions. IMP was more stable than GMP with increased hydrolysis for both compounds occurring at lower pH and longer canning time. The loss of IMP and GMP was shown to go via phosphate hydrolysis to the corresponding nucleosides, inosine and guanosine, followed by base hydrolysis of the nucleosides to hypoxanthine and guanine.     

Changes in free nucleotides, nucleosides and bases during thermal processing of goat and sheep meats. Part I. Effect of temperature

Effect of processing temperature on the changes in free nucleotides, nucleosides and bases in goat and sheep meats was investigated. The major changes in nucleotides and related compounds took place during first 30 min of heating and the rates of changes were maximum at 60 degrees C and minimum at 100 degrees C. At 120 degrees C, thermal degradation of inosinic acid proceeds at significant rates but below 100 degrees C major changes are brought about by phosphomonoesterases (5'-mononucleotidases) during initial stages of heating.     

Changes in free nucleotides, nucleosides and bases during preparation of pre-cooked dehdyrated minced meats

Changes in free nucleotides, nucleosides and bases during preparation of pre-cooked dehydrated minced meats from goat and sheep are reported. Major changes took place during cooking stage only; the changes during dehydration were relatively minor. Addition of EDTA at 500 ppm level significantly reduced the rate of dephosphorylation during curing process. Concentration of free nucleotides and related compounds were very low in commercially prepared accelerated freeze dried meat chunks.     

Changes in free nucleotides,nucleosides and bases during Preparation of Pre-Cooked dehydrated minced meats

Changes in free nucleotides, nucleosides and bases during preparation of pre-cooked dehydrated minced meats from goat and sheep are reported. Major changes took place during cooking stage only; the changes during dehydration were relatively minor. Addition of EDTA at 500 ppm level significantly reduced the rate of dephosphorylation during curing process. Concentration of free nucleotides and related compounds were very low in commercially prepared accelerated freeze dried meat chunks.     

Pasteurization of Pacific Oysters by Radiation: Post-Mortem Changes in Nucleotides During Storage at 0-2°C

SUMMARY: The concentration of nucleotides was lower in the adductor muscle of the oyster (1.64 μmoles/g) than in the remaining dark tissues of the oyster (2.75 μmoles/g). The concentration was less in the whole oyster meats (2.87 μmoles/g) than is usually found in fish muscle, or other marine invertebrates.
In addition to the adenine nucleotides and inosine monophosphate, uridine triphosphate, guanosine triphosphate, guanosine diphosphate, guanosine monophosphate and guanosine diphosphate-mannose were found in the fresh oysters. Samples collected in summer had greater concentrations of nucleotides than similar winter samples. lnosine monophosphate formed rapidly from adenosine triphosphate during storage at 0°–2°C, while the turnover rate of inosine monophosphate was slow and reflected low 5'-nucleotidase activity. Hypoxanthine, inosine, guanosine, guanine and uracil were formed during ice storage. The nucleotide breakdown in oysters was not changed by 2 mrads of radiation dose. Total nucleosides and free bases increased during storage of both unirradiated and irradiated samples. During the latter part of the storage period the concentrations of nucleosides and free bases were considerably greater in the irradiated samples. This difference probably is due to the utilization of these compounds by bacteria in the un-irradiated samples. After 15 days of storage bacteria had increased to more than 10'organisms per g, while the counts for irradiated samples were very low (less than 103 per g).