HPLC-MS/MS同时检测大鼠体内三聚氰胺和三聚氰酸含量

目的建立HPLC-MS/MS同时检测大鼠血浆和组织中三聚氰胺和三聚氰酸含量的方法。方法以同位素标记的三聚氰胺和三聚氰酸作为内标物,大鼠血浆和组织经二氯甲烷-乙腈前处理后,经色谱柱分离,串联离子阱质谱采用ESI源按运行时间段切换正、负离子模式进行测定。结果在0.3～75μg/ml范围内,三聚氰酸和三聚氰胺具有良好的线性(r≥0.998)。在0.9、5.0、50μg/ml的给药大鼠的血浆样本中,三聚氰酸和三聚氰胺的回收率分别为97.4%～99.4%、96.8%～98.9%,两者的变异系数为2.43%～6.35%(日内)、1.70%～4.12%(日间)和0.30%～9.17%(日内)、0.75%～3.39%(日间);在0.9、5.0、50μg/ml的给药大鼠肝组织样本中,三聚氰酸和三聚氰胺的回收率分别为90.8%～104.6%、91.7%～106.0%,对应的变异系数为3.70%～5.14%(日内)、2.84%～4.50%(日间)和1.31%～2.21%(日内)、1.12%～3.36%(日间)。结论该方法样品前处理简单,基质干扰小,分析效率高,具有良好的精密度、准确度和特异性,是进行三聚氰酸、三聚氰胺在动物体内的组织代谢和毒性研究的理想分析方法。     

A new approach to measure melamine, cyanuric acid, and melamine cyanurate using surface enhanced Raman spectroscopy coupled with gold nanosubstrates

Vibrational spectroscopic characteristics of melamine, cyanuric acid, and melamine cyanurate were measured using surface-enhanced Raman spectroscopy (SERS) coupled with gold nanosubstrates. Trace amounts of melamine and its analogues (cyanuric acid and melamine cyanurate) were characterized and quantified quickly and accurately by SERS in combination with partial least squares (PLS) analysis. Based on the relationship between the Raman intensity of the most prominent peak at around 676 cm⁻¹ and log values of melamine concentrations, the limit of detection (LOD) of SERS for melamine was estimated to be 2.6 × 10⁻⁷ mol L⁻¹ (∼33 ppb). An approximately 3 × 10⁴ fold of enhancement factor for SERS signals of melamine on gold nanosubstrates was obtained. This result was based upon the comparison of the peak at around 676 cm⁻¹ in the SERS spectra with that of normal Raman spectra of melamine in aqueous solutions. SERS spectra of cyanuric acid acquired from its solid form differ significantly from this compound in an aqueous solution, indicating a possible keto-enol isomerization reaction of cyanuric acid in water. When equal amounts of melamine and cyanuric acid were added together, spoke-like crystals of melamine cyanurate formed instantly, which could be measured and characterized by SERS. This study demonstrates that SERS could provide a fast and ultra-sensitive tool for detection and characterization of melamine and its derivative compounds in aqueous solutions.     

Development of competitive enzyme-linked immunosorbent assays for boscalid determination in fruit juices

Boscalid is a modern, broad-spectrum carboxamide pesticide highly efficient against most fungal diseases affecting valuable crops. In this study, a boscalid-mimicking derivative with a six-carbon spacer arm replacing the chlorine atom at the pyridine ring of the target molecule was synthesized and coupled to carrier proteins. Following rabbit immunization, antibodies against this agrochemical were obtained for the first time, and they were characterised in terms of affinity and specificity, tolerance to solvents, and robustness to changes in buffer pH and ionic strength, using two assay formats. Both of the optimised immunoassays showed limits of detection below 0.1 μg/L. Moreover, matrix effects of grape, peach, apple, and tomato juices were evaluated. Finally, a simple and easy procedure was set up for boscalid determination with spiked samples, affording limits of quantification of 10 μg/L, a value well below the sensitivity levels required for monitoring campaigns of pesticide residue analysis in food.     

Behaviour of tannic acid from various commercial sources towards redox,metal complexing and protein precipitation assays of tannins

Tannic acid is the most commonly used standard for quantitation of tannins. Tannic acid from five commercial sources (Merck, Sigma, Aldrich, Fluka and Apin chemical companies) was tested towards a redox (Folin-Ciocalteu method) and a metal complexing (ferric chloride method) assay, and three protein precipitation assays. Tannic acid from various commercial sources behaved differently towards these tannin assays showing that (i) tannic acid preparation differed from source to source, and (ii) tannic acid from different sources as a standard results in different tannin values for a given sample. It is suggested that the source of tannic acid used as a standard should be stated, and the assay values obtained by different workers should be compared with caution.     

Efficacy of washing meat surfaces with 2% levulinic, acetic, or lactic acid for pathogen decontamination and residual growth inhibition

We compared spray washing at 55.4 °C with 2% levulinic acid to that with lactic or acetic acid for decontamination of pathogenic bacteria inoculated onto meat surfaces, and their residual protection against later growth of pathogenic bacteria. The model systems included Escherichia coli O157:H7 on beef plate, Salmonella on chicken skin and pork belly, and Listeria monocytogenes on turkey roll. In the decontamination studies, acid washes lowered recoverable numbers of pathogens by 0.6 to 1 log/cm(2) as compared to no-wash controls, and only lactic acid lowered the number of pathogens recovered as compared to the water wash. Washing with levulinic acid at 68.3 or 76.7 °C did not result in additional decontamination of E. coli. Acetic acid prevented residual growth of E. coli and L. monocytogenes, and it reduced numbers of Salmonella on chicken skin to below recoverable levels. Overall, levulinic acid did not provide as effective decontamination as lactic acid nor residual protection as acetic acid.     

Reactions of chlorogenic acid and quercetin with a soy protein isolate--influence on the in vivo food protein quality in rats

Plant phenolic compounds are known to interact with proteins producing changes in the food (e. g., biological value (BV), color, taste). Therefore, the in vivo relevance, especially, of covalent phenol-protein reactions on protein quality was studied in a rat bioassay. The rats were fed protein derivatives at a 10% protein level. Soy proteins were derivatized with chlorogenic acid and quercetin (derivatization levels: 0.056 and 0.28 mmol phenolic compound/gram protein). Analysis of nitrogen in diets, urine, and fecal samples as well as the distribution of amino acids were determined. Depending on the degree of derivatization, the rats fed with soy-protein derivatives showed an increased excretion of fecal and urinary nitrogen. As a result, true nitrogen digestibility, BV, and net protein utilization were adversely affected. Protein digestibility corrected amino acid score was decreased for lysine, tryptophan, and sulfur containing amino acids.     

Development of an RP-HPLC method for the simultaneous determination of benzoic acid,sorbic acid,natamycin and lysozyme in hard and pasta filata cheeses

A single method, based on RP-HPLC with UV detection, was developed with the aim of simultaneously quantifying four preservatives in cheeses: benzoic acid, sorbic acid, natamycin and lysozyme.     

用科技创新理念推动吉林省疾病预防控制中心 食品安全风险监测工作持续健康发展

食品安全是关系到人民健康和国际民生的重大问题,食品的安全性越来越引起社会的关注,食品安全风险监测工作正面临着巨大的挑战。本文从注重培养与引进人才,搭建施展技术能力的平台;注重能力提升,在食品安全风险监测工作中发挥技术支撑作用;注重科研和工作的结合,发挥重点实验室作用,为人民健康保驾护航;展望未来,以科技创新为动力,开创食品安全风险监测工作新局面等方面阐述了用科技创新理念推动吉林省疾病预防控制中心食品安全风险监测。目前无论从监测样品的品种、数量和监测的区域,还是监测结果反映出的问题上均有较好发展,为从事食品安全风险监测工作提供参考。     

Glucose,fructose and sucrose increase the solubility of protein–tannin complexes and at high concentration,glucose and sucrose interfere with bisulphite bleaching of wine pigments

Wines were modified with increasing sugar concentrations and decreasing tannin concentrations and analysed by a combination of protein precipitation and bisulphite bleaching. Increasing sugar concentration decreased the precipitation of tannin and protein-precipitable polymeric pigments (PPP). The use of a hydrogen bond disruptor (urea) to reduce protein–tannin and protein–pigment complex formation showed that the effect of sugar concentration occurred by increasing the solubility of the tannin–protein complex, not by interfering with protein–tannin complex formation. By increasing the solubility of pigment–protein complexes, non-protein-precipitable polymeric pigments (nPPP) appeared to increase. There was also an increase in total polymeric pigments at each tannin concentration with increasing glucose and sucrose concentration, indicating that sugar concentration might also affect bisulphite bleaching of wine pigments. While a significant effect of sugar concentration on tannin–protein complex solubility was observed, these effects were greatest at sugar concentrations far in excess of normal wine making conditions. Under normal wine making conditions, sugar concentration will have a negligible effect on protein-precipitable tannin, PPP and nPPP concentrations.     

酶联免疫吸附法与二喹啉甲酸法检测核桃蛋白浓度的比较研究

目的 探究间接酶联免疫吸附法(enzyme-linked immunosorbent assay, ELISA)和二喹啉甲酸 (bicinchoninic acid, BCA)试剂盒2种方法在核桃蛋白浓度检测中的优缺点和适用范围。方法 对本实验室提取的核桃蛋白进行烘烤、高温高压以及微波加热处理后, 用ELISA法和BCA法2种方法分别检测其蛋白质浓度。结果 在不同的热加工处理条件下, ELISA对蛋白浓度测定结果存在显著性差异, 高度依赖热处理的类型。与之相比, 使用BCA法测定蛋白浓度时, 受到热加工处理影响较小。结果提示, 在食物加工中的一些剧烈条件下, 蛋白质的高级结构很容易遭到破坏, 蛋白质结构的改变会干扰其与抗体结合部位的作用, 导致ELISA法测定的结果不准确。BCA法测定蛋白质浓度的准确性较高, 且受加热影响较小。结论 在剧烈的食品加工条件下, 尤其当这种条件引起蛋白结构发生改变后, 对其蛋白质浓度进行测定时, 可选用BCA法进行测定。     

Interference of phenolic compounds in Brassica napus, Brassica rapa and Sinapis alba seed extracts with the Lowry protein assay

The protein content of aqueous extracts of Brassica napus, Brassica rapa and Sinapis alba meal was determined by the Lowry and Kjeldahl nitrogen assays. Phenolic compounds interfered with the Lowry method to different extents based on the lines studied as well as the extraction procedure used. Three ways to correct for this interference were studied; acid precipitation of the protein before analysis, analyzing in the presence and absence of copper and the binding of free phenolics using non-ionic, porous polystyrene (Amberlite XAD-4). Analysis in presence and absence of copper, and using the difference in absorption at 660 nm between these analyses, proved to be the best way to correct for phenolic interference in the Lowry assay. Extractability of Cruciferae seed phenolics may be pH dependant thus the contribution of phenolics to the Lowry protein assay varies with the pH used for extraction.     

Effect of high or low protein ration combined or not with rumen protected conjugated linoleic acid (CLA) on meat CLA content and quality traits of double-muscled Piemontese bulls

A trial was carried out on double-muscled Piemontese bulls to evaluate the effects of two rations differing in crude protein density (HP=14.5% DM and LP=10.8% DM) and top dressed or not with 80 g/d of rumen protected CLA (rpCLA) for a long period (336 d) on meat quality traits and CLA content. Forty-eight bulls were fed one of the four experimental diets based on corn silage and cereals and were slaughtered at an average age and body weight (BW) of 562 ± 18 d and 668 ± 56 kg, respectively. After slaughter the 5th rib cut was dissected into Longissimus thoracis (LT), other muscles (OM), inter-muscular fat (IF), cover fat (CF), and bones. Muscles and fatty tissues were analyzed for proximate composition and fatty acid (FA) profiles. Rib was composed by 81.1, 3.7, 1.6 and 13.6% of muscles, IF, FC and bone, respectively; LT and OM contained only 0.8 and 1.4% of lipid, respectively. The treatments did not influence these values, but rpCLA increased, compared to control, both c9,t11-CLA and t10,c12-CLA concentrations in all the tissues (P<0.01); t10,c12-CLA concentration was increased much more in muscles (+20 times) than in fatty tissues (from +0.2 to +0.9 times). This suggests that in the muscle this isomer is preferentially stored and/or less combusted with respect to other fatty acids. Low protein rations did not exert any influence on carcass and meat quality, as on growth performance, but reduced nitrogen excretion, their use for improving the environmental impact (process quality) of this meat production system is recommended.     

Protocols for the isolation and detection of lactic acid bacteria with bacteriocinogenic potential

The objective of this study was to evaluate culture media and methodologies for isolation and detection of lactic acid bacteria (LAB) capable to produce bacteriocin-like substances. Samples of milk and cheese were pour plated on de Mann-Rogosa-Sharpe agar (MRS) and Kang-Fung-Sol agar (KFS) (both at 35 °C/48 h, under anaerobiosis), from which 389 and 256 LAB cultures were selected. The antagonistic activity of them was evaluated using the spot-on-the-lawn and two culture media: brain-heart infusion agar with catalase (BHI + C) and M17 (both at 35 °C/24 h). The proteinaceous nature of the antagonistic cultures was verified using: spot-on-the-lawn (MRS, 25 °C/24 h, under anaerobiosis) and well-diffusion (cultures amplified on modified MRS broth at 25 °C/24 h, and then neutralized using NaOH). Listeria monocytogenes ATCC 7644 was used as indicator. A larger number of antagonist cultures were isolated from MRS (83 by M17 and 65 by BHI + C) in comparison to KFS (24 by M17 and 15 by BHI + C). The spot-on-the-lawn identified a higher frequency of LAB capable of producing bacteriocin-like substances. MRS was considered to be the best culture media for the isolation of LAB capable to produce bacteriocin-like substances, activity that was better identified using the spot-on-the-lawn methodology.     

Cloning of CDC33: a gene essential for growth and sporulation which does not interfere with cAMP production in Saccharomyces cerevisiae

The CDC33 gene of Saccharomyces cerevisiae belongs to the class II 'START' genes. Its product is required for the initiation of a new cell division cycle (Hartwell, 1974). Many results suggest that the cAMP signalling pathway is one of the major controlling elements of 'START'. Components of this pathway are encoded by class II 'START' genes. The aim of the present study is to determine whether or not the CDC33 gene interferes with the cAMP signalling pathway. We report here the molecular cloning of the CDC33 gene by complementation of the cdc33-1 thermosensitive mutant. The identity of the cloned gene is confirmed by site-specific reintegration and segregation analysis. This gene is transcribed into a 900-nucleotides mRNA and appears to be relatively abundant in the cell. We also show that the CDC33 gene product is essential for sporulation. cdc33-1 mutant cells are able to enter into the resting state. The cAMP intracellular pool is not modified when the cdc33-1 mutant is shifted to the restrictive temperature. The cdc33-1 mutation is not suppressed by other known elements of the cAMP cascade. All these results suggest that the CDC33 'START' gene does not interfere with the cAMP signalling pathway which controls cell division.     

Effects of low protein diets on pigs with a lean genotype. 1. Carcass composition measured by dissection and muscle fatty acid composition

Pigs with a lean genotype were fed diets differing in protein and amino acid contents between 40 and 115 kg live weight. A high protein control regime (C) was compared with one supplying 11% less total protein but the same essential amino acid levels (LP1) and one supplying 16% less protein but lower amino acid levels (LP2). Regime LP2 produced fatter pigs in terms of subcutaneous, intermuscular and intramuscular fat (IMF), the latter measured in longissimus and semimembranosus muscles. The percentage of linoleic acid was lowest and that of oleic acid highest in IMF from LP2 pigs (11.57 and 34.59% respectively in longissimus). Pigs in regime LP1 had more longissimus IMF than C but similar semimembranosus IMF although both muscles had lower percentages of linoleic acid in LP1 than C, suggesting a tendency towards greater fat deposition in LP1. The high IMF content in LP2 produced the most tender, juicy steaks.     

High-performance liquid chromatographic method for the simultaneous detection of the adulteration of cereal flours with melamine and related triazine by-products ammeline, ammelide, and cyanuric acid

Melamine has been used for the adulteration of cereal flours in order to increase their apparent protein content. Crude melamine may contain several by-products, i.e. ammeline, ammelide, and cyanuric acid. The simultaneous analysis of all four chemicals is difficult because of the formation of an insoluble salt between melamine and cyanuric acid. A simple and convenient high-performance liquid chromatography (HPLC) method for the detection of the adulteration of cereal flours with all four chemicals is proposed herein. The precipitate formation between melamine and cyanuric acid was prevented by using alkaline conditions (pH 11-12) for both standards preparation and sample extraction. The method uses matrix-matching, which involves the construction of a calibration curve on a blank (negative control) matrix, which is then used for the quantitation of melamine and by-products in adulterated (positive) samples. Matrix-matching compensates for analyte losses during sample preparation, and for matrix effects. The method was successfully applied to wheat, corn, and rice flours, and is expected to be applicable (with some modifications) to soy flour as well. The method allows for the detection of melamine, ammeline, and ammelide at approximately 5 µg g^-1, and cyanuric acid at approximately 90 µg g^-1 in wheat flour.     

Improvement in food intake and nutritive utilization of protein from Lupinus albus var. multolupa protein isolates supplemented with ascorbic acid

The protein quality of protein isolates from lupin (LPI) (Lupinus albus var. multolupa), prepared by isoelectric precipitation, was assessed by chemical analysis of protein and amino acids and biological analysis of digestive and metabolic utilization of protein by growing rats. The animals were fed isonitrogenous and isocaloric diets adjusted to meet their nutrient requirements, in which lupin protein isolate was the only protein source, complemented with 0.5% methionine. Different LPIs were prepared with addition, or not, of ascorbic acid as antioxidant. Protein isolates had a protein content of 87.8–98.1%. Manganese content of protein isolates was reduced by 72.8–89.5% compared to the raw seed flour. Results from in vivo experiments showed that addition of 0.5% ascorbic acid to LPI incorporated into diets led to a 82.8% increase in daily food intake, when compared to the non-supplemented LPI, reaching similar values to those obtained with a casein–methionine control diet. Digestive and metabolic utilization of protein from LPI, assessed by nitrogen absorption or apparent digestibility coefficient, and by nitrogen balance or percentage of retained to absorbed nitrogen, respectively, was high, when the dietary intake of animals fed the LPI diets was adequate after addition of 0.5% ascorbic acid, although slightly inferior to the values obtained with a casein–methionine control diet. The high nutritive utilization of protein was reflected in excellent growth and nutritional indices assayed. In conclusion, ascorbic acid supplementation led to an improvement in the palatability of the LPI diets and, therefore, in daily food intake, which was reflected in a higher nutritive utilization of protein and improvement in weight gain and the food transformation index.     

Development and validation of an HPLC method with fluorescence detection for the determination of fluorescent whitening agents migrating from plastic beverage cups

An HPLC method with fluorescence detection has been developed and validated for the quantification of six fluorescent whitening agents (FWA) in plastic beverage cups after extraction and in food simulants after migration at 70°C for 2 h. The sensitivity of the method was high with LODs ranging from 0.053 to 0.251 μg kg^?1 and LOQs from 0.107 to 0.504 μg kg^?1. Accuracy and precision were highly acceptable, with recoveries greater than 82% and RSDs (%) below 16%. The expanded combined uncertainty was found to be less than 23% for the measurements of all studied FWAs. In extracting the analytes from food contact materials (FCM), accelerated solvent extraction (ASE) and Soxhlet extraction were applied using ethanol as the extraction solvent. The results obtained for FWA in 10 different food plastic cups, made from different polymers, were compared. The ASE technique proved to be faster, more effective and efficient than Soxhlet extraction. Migration tests with official food simulants from Regulation (EU) No 10/2011 showed that the substances studied could potentially migrate using the selected migration conditions. The most pronounced effect was observed in case of simulant D1 (50% w/v ethanol in water). The analytical method proved to be a simple, fast, sensitive and reliable tool for the simultaneous quantification of six of the most used FWAs in both FCM extracts and food simulants after migration experiments.     

固相萃取柱脱盐-石墨炉原子吸收法测定酱油中铅

目的 建立固相萃取柱脱盐—石墨炉原子吸收法测定酱油中铅的方法.方法 酱油经微波消解后,消解液用乙酸铵调节至pH≈5.5,过经用5 ml 1 mol/L乙酸铵活化后的DigiSEP-Blue柱,将被测元素铅吸附与基体中高盐分离,再分别用8 ml 2 mol/L硝酸、2 ml纯水洗脱,应用石墨炉原子吸收法测定洗脱液中铅含量.结果 用固相萃取柱可将酱油中98％以上的钠盐与被测元素铅分离,消除了石墨炉原子吸收分光光度计测定铅时的基体干扰.低、高两个铅浓度(10和30 ng/ml)的加标平均回收率(n=7)分别为91.3％ ～95.1％,97.9％～98.6％,相对标准偏差为2.1％ ～7.0％,检出限为1.33 ng/g.结论方法准确、灵敏度高,适于高盐样品酱油中铅含量的测定.