酶联免疫法检测乳与乳制品中的氟喹诺酮类药物

研究酶联免疫法检测乳及乳制品中氟喹诺酮类药物的残留,氟喹诺酮EIA微孔板含12个板条,每个板条8孔,用羊抗兔抗体IgG包被.特异性抗体(兔多克隆抗喹诺酮类抗体)、辣根过氧化物酶标记的诺氟沙星(酶结合物)、诺氟沙星标准品或样品被加入到微孔后,经过1小时孵育,特异性抗体被牢固地结合在板条上的羊抗兔抗体IgG上,与此同时,游离的氟喹诺酮类(存在于标准品溶液或样品中)和酶结合物相互竞争与特异性抗体的结合位点结合(即竞争性酶联免疫检测).然后在洗涤过程中除去未结合的酶结合物试剂,加入色原底物(四甲基联苯胺TMB),结合的酶结合物将无色的底物转化为蓝色的产物.滴加终止液终止显色反应,颜色由蓝色变为黄色,在450nm波长下检测吸光度,吸光度值与样品中氟喹诺酮类浓度成反比.本方法单样检测时间为120分钟,具有简便、快速、灵敏等特点,适用于乳与乳制品中氟喹诺酮类残留的检测,该方法为半定量检测检测结果超出相应产品的质量标准,必要时,需用国家标准中规定的仲裁方法或仪器法进行验证.     

Detection of hen’s egg white lysozyme in food: Comparison between a sensitive HPLC and a commercial ELISA method

Recently some cases described allergic reactions to undeclared lysozyme present in foods. In order to protect the allergic consumer, a better control is needed based on reliable and specific detection and quantification methods. The two currently used methods, a commercially available enzyme-linked immunosorbent assay (ELISA) for egg white allergens and a liquid chromatographic method, were compared. Despite the technical specifications of the ELISA kit, ensuring quantitative analysis of lysozyme in absence of other egg white proteins, no reliable quantitative and even qualitative data could be obtained which contrasted with the capabilities of the instrumental technique. A more elaborated evaluation of the methods on spiked samples identified the causes of the observed discrepancies. It was shown that next to a poor sensitivity of the ELISA kit also an insufficient lysozyme extraction and a co-extraction of interfering substances were the causes of the anomalies observed during application of the immunochemical method.     

Validation of a fast real-time PCR method to detect fraud and mislabeling in milk and dairy products

Fast real-time PCR TaqMan assays were developed and validated for species identification in dairy products. Based on the amplification of 12S rRNA and cytB partial genes of mitochondrial DNA, the methods were demonstrated to be sensitive, fast, and species-specific for Bos taurus, Ovis aries, Bubalus bubalis, and Capra hircus. The limit of detection calculated was lower than 1%, and the efficiency was reported to be higher than 96% in every assay. An internal amplification control was used to detect possible false negatives. The method was validated by means of laboratory-prepared samples mixing different species. Moreover, 18 commercial dairy samples were analyzed by both real-time PCR and isoelectric focusing, the official European Union reference method. The 4 TaqMan assays were confirmed to be a useful tool for milk and dairy product authentication.     

HPLC测定肉制品中山梨酸方法的改进

通过分析用HPLC测定肉制品中山梨酸的国家标准GB/T5009.29-2003和行业标准SB/T10389-2004,发现2种方法测定肉制品中山梨酸的前处理方法存在一定的不足,因此,本文建立了一种新型的HPLC测定肉制品中山梨酸的前处理方法——蒸馏法。此法利用山梨酸或其盐在酸性条件下能随水蒸气一同蒸发的特点,在磷酸存在的条件下,用水蒸气蒸馏的方法将样品特别是固体或者半固体样品中的防腐剂山梨酸蒸馏出来,从而达到了固体样品中的山梨酸与干扰物质分离的目的。样品前处理简便快捷,干扰物少,对色谱柱的污染少。在此基础上,以0.02mol/L甲醇和乙酸铵溶液(5∶95)为流动相,流速为1mL/min,紫外检测器在波长230nm条件下测定馏出液中山梨酸含量。结果显示:蒸馏法的平均回收率为90.7%,明显高于使用标准方法所得的平均回收率88.2%。     

高效液相色谱法检测牛奶及其制品中的溶菌酶

目的建立一种测定牛奶及奶酪制品中低含量溶菌酶的高效液相色谱-荧光检测新方法。方法样品经p H 6.0的氯化钠溶液活化,再在低p H值条件下除蛋白后,采用反相色谱柱(PLRP-S 250 mm×4.6 mm,300?,5μm)用A水(0.1%的三氟乙酸(trifluoroacetic acid,TFA)),B乙腈(0.1%TFA)体系作为流动相进行梯度洗脱,使用荧光检测器在激发波长(λ_(ex))276 nm,发射波长(λ_(em))345 nm处检测。结果最优实验条件下,溶菌酶在2.0~30.0 mg/L浓度范围内线性良好,加标实验结果显示回收率和相对标准偏差(relative standard deviation,RSD)分别在92.3%~104.3%和0.81%~3.26%之间,对牛奶和奶酪制品的检出限分别为20和40 mg/kg。结论该方法操作简便,结果准确、可靠,可用于乳制品中溶菌酶含量测定。     

Comparison of extraction conditions for milk and hen's egg allergens

The evaluation of recovery rates by extracting milk powder and egg powder using eleven different extractants gave approximately similar results for both foods. Compared with the other extraction solutions investigated, ‘1% Tween 20® and 0.4% Triton X-100®’ and ‘4% SDS’ are the most suitable extractants to isolate proteins of hen's egg or milk. When comparing calculated protein recovery rates of egg and milk powder extracts, the results clearly indicated that the choice of a suitable extractant is of particular importance. Qualitative investigation of the extracts via LDS-PAGE followed by silver staining as well as immunoblotting confirmed the results of protein quantification. Hence, the immunoblots showed that the extraction agents had no negative influence on the antigenicity of the extracted allergenic proteins. In this study, variation of extraction temperature led neither to any benefit in extraction quality nor to degradation. Changing pH did not reveal any trends, but progressive protein hydrolysis under strong alkaline conditions. Evaluation of recovery rates as well as results of unspecific and specific staining of the extracts showed that an extraction time of 1?h is sufficient for an appropriate sample preparation. For investigations with and without food matrix different results were obtained. In summary, wheat starch did not influence the extraction quality within all examined materials and different extractants. In contrast, using fat powder and dry cake mix, respectively, led to different results in the extraction procedure. When fat powder and dry cake mix were used as food matrices, some protein recovery rates decreased and some increased depending on the allergen material. These results highlight the fact that the suitability of the extractant not only depends on the properties of the allergen but furthermore on the type of matrix containing the allergen.     

Comparison of extraction conditions for milk and hen's egg allergens

The evaluation of recovery rates by extracting milk powder and egg powder using eleven different extractants gave approximately similar results for both foods. Compared with the other extraction solutions investigated, '1% Tween 20? and 0.4% Triton X-100?' and '4% SDS' are the most suitable extractants to isolate proteins of hen's egg or milk. When comparing calculated protein recovery rates of egg and milk powder extracts, the results clearly indicated that the choice of a suitable extractant is of particular importance. Qualitative investigation of the extracts via LDS-PAGE followed by silver staining as well as immunoblotting confirmed the results of protein quantification. Hence, the immunoblots showed that the extraction agents had no negative influence on the antigenicity of the extracted allergenic proteins. In this study, variation of extraction temperature led neither to any benefit in extraction quality nor to degradation. Changing pH did not reveal any trends, but progressive protein hydrolysis under strong alkaline conditions. Evaluation of recovery rates as well as results of unspecific and specific staining of the extracts showed that an extraction time of 1 h is sufficient for an appropriate sample preparation. For investigations with and without food matrix different results were obtained. In summary, wheat starch did not influence the extraction quality within all examined materials and different extractants. In contrast, using fat powder and dry cake mix, respectively, led to different results in the extraction procedure. When fat powder and dry cake mix were used as food matrices, some protein recovery rates decreased and some increased depending on the allergen material. These results highlight the fact that the suitability of the extractant not only depends on the properties of the allergen but furthermore on the type of matrix containing the allergen.     

高效液相色谱法测定奶粉及其他乳制品中的L-羟脯氨酸

建立了柱前衍生反相高效液相色谱法测定奶粉及其他乳制品中L-羟脯氨酸的方法.采用酸水解样品,经6-氨基喹啉基-N-羟基琥珀酰亚氨基甲酸酯(AQC试剂)衍生水解样品中的L-羟脯氨酸,以磷酸盐缓冲溶液、乙腈、水梯度洗脱,流速1.0ml/min,通过symmetry C18柱将L-羟脯氨酸与其他18种氨基酸分离.紫外检测器在248nm波长下检测.测定结果显示,在0～2.05mmol/L范围内,标准曲线呈良好的线性关系(r=0.9991),加样回收率为95.1%～101%,方法精密度RsD(n=7)为1.3%.该方法具有简单、稳定、灵敏度高、准确等优点.     

基因重组蛋清溶菌酶的分离纯化

以毕赤氏基因重组酵母发酵液为研究对象,经过离心分离、超滤、膜分离、阳离子交换树脂吸附后,得到的纯化液,真空干燥得到酶活为20000U·mg~(-1)的蛋清溶菌酶粉末。研究结果表明:发酵液在5000r/min、25℃下离心15min,得到离心液溶菌酶活性为360.1U·mL~(-1),菌体细胞的去除率达99.99%;超滤最佳操作条件:压力为0.15MPa,pH为6.5;一级超滤得到的溶菌酶活性为455.4U·mL~(-1),收率为90.6%,纯化程度提高了23.7%;二级超滤得到的溶菌酶活性为648U·mL~(-1),收率为61.2%,纯化程度提高了42.3%; D152阳离子交换树脂对料液进行离子交换层析,得溶菌酶的酶活性为770.5U·mL~(-1),收率达90%,纯化程度提高了18.9%。     

原料乳中尿素掺假快速检测方法的研究

针对目前市场上出现的原料奶中掺尿素的现状,研究了一种方法可以快速检测掺假乳中的尿素,利用简单的试纸即可检测出乳中尿素。研究了试纸的制作工艺,以及试纸的抗干扰性。结果表明,原料奶中Ca,Zn和Cu到了一定浓度会影响试纸显色,其余金属影响不大。     

Phospholipids in milk and dairy products

A rapid HPLC method has been developed for the analysis of the phospholipids in milk and dairy products. A concentrate of the phospholipids was obtained by chromatography on a cartridge of silica gel. The relative proportions of the different phospholipids were then determined by HPLC, and by means of the addition of an internal standard, dipalmitoyl phosphatidyldimethylethanolamine, the absolute amounts present were also measured. After the reproducibility of the method was demonstrated with fresh whole milk, it was applied to skimmed milk, buttermilk and various products of ultra-high temperature processing. Small differences only were seen in the relative compositions of phospholipids in different milk products, but the absolute amounts varied appreciably. In some samples, it was tentatively concluded that the phospholipids had undergone extensive autoxidation.     

乳及乳制品中残留青霉噻唑酸钾的检测方法

通过分光光度法检测乳及乳制品中青霉素钾的主要酶解产物青霉噻唑酸钾,间接检测了乳及乳制品中是否添加过β-内酰胺酶。通过实验该方法的工作曲线相关系数为0.9997,检测限为2.5 mg/L,回收率在93.2%～106.3%之间,RSD为2.9%～4.5%。在30组样品中应用效果良好,表明该方法简单、快速准确、灵敏度高,适用于乳及乳制品中青霉噻唑酸钾残留量的检测。     

A sensitive HPLC method for measuring bacterial proteolysis and proteinase activity in UHT milk

A sensitive quantitative reversed-phase HPLC method is described for measuring bacterial proteolysis and proteinase activity in UHT milk. The analysis is performed on a TCA filtrate of the milk. The optimum concentration of TCA was found to be 4%; at lower concentrations, non-precipitated protein blocked the HPLC while higher concentrations yielded lower amounts of peptides. The method showed greater sensitivity and reproducibility than a fluorescamine-based method. Quantification of the HPLC method was achieved by use of an external dipeptide standard or a standard proteinase.     

Ultrafiltration-modified chicken egg white lysozyme and its antibacterial action

The aim of the study was to produce a lysozyme preparation with an increased content of enzyme polymeric forms using the ultrafiltration (UF) technique. The effect of selected parameters of the membrane process on the extent of enzyme modification was examined. The study showed that the membrane technique, developed for the production of lysozyme monomer, could also be used in the direct production of a preparation containing enzyme polymers. Under optimal conditions of the modification procedure, the obtained preparation contained 53.3% of lysozyme polymeric forms. The effectiveness of the antibacterial action of UF-modified lysozyme against selected strains of bacteria was determined. Its bacteriostatic activity depended on the applied modification conditions. Among lysozyme preparations modified by UF, the highest bacteriostatic activity against selected strains of bacteria was recorded in the preparation containing 53.3% polymeric forms. The modification procedure facilitates the extension of the antibacterial spectrum of lysozyme against Gram-negative bacteria ( Pseudomonas fluorescens ) .     

A novel antioxidant and antimicrobial peptide from hen egg white lysozyme hydrolysates

Hen egg white lysozyme (HEWL) was hydrolyzed with papain, trypsin and a combination of the two to isolate antioxidant peptides. The prepared hydrolysates were evaluated for antioxidant activity using DPPH and ABTS radical scavenging, metal ion chelation and lipid peroxidation inhibition. The obtained hydrolysate by a combination of the two enzymes exhibited the highest antioxidant activity compared to other hydrolysates and elected for isolation of antioxidant peptides by reverse-phase high-performance liquid chromatography (RP-HPLC). A most potent fraction namely F2 fraction, identified to be NTDGSTDYGILQINSR (MW: 1753.98 ± 0.5 Da) using tandem mass spectrometry. The antimicrobial activity of the F2 peptide was tested using radial diffusion assay (RDA). Our results showed that this peptide has inhibitory effects on both Gram-negative and Gram-positive bacteria. Minimum inhibition concentration (MIC) values of the F2 peptide against Escherichia coli and Leuconostoc mesenteroides bacteria were 355.64 (±2.2) and 442.25 (±2.8) μg/ml, respectively.     

Yersinia enterocolitica in milk and dairy products

Yersinia enterocolitica was first recognized during the 1960's as an important human enteropathogen. The species as later redefined includes both pathogenic and nonpathogenic forms. Pathogenic strains that retain the virulence plasmid can be identified in several animal models and four indirect tests (calcium dependency, autoagglutination, Congo red uptake, serological detection of outer membrane antigen) and by tissue culture assay, serotype, and biotype. Y. enterocolitica and related bacteria have frequently been isolated from raw milk, but none of the isolates, with the possible exception of serotype 05,27, are recognizable as pathogens. Under normal circumstances Y. enterocolitica does not survive pasteurization. If introduced into pasteurized milk, it can grow well at refrigeration temperatures. Two outbreaks of yersiniosis have occurred that involved pasteurized milk. Pigs, which frequently carry pathogenic Y. enterocolitica in their throat, were the probable source in one of these outbreaks. The most rapid enrichment procedure available for isolation of Y. enterocolitica requires 6 d. No isolation method is available for selective isolation of pathogenic Y. enterocolitica in the presence of related bacteria common in milk and other foods.     

Natural antioxidants in milk and dairy products

Milk antioxidants, both lipophilic (conjugated linoleic acid, α‐tocopherol, β‐carotene, vitamins A and D₃, coenzyme Q₁₀, phospholipids) and hydrophilic antioxidants (proteins, peptides, vitamins, minerals and trace elements) play a key role in maintaining pro‐oxidant and antioxidant homeostasis in the human body. Lipophilic antioxidants are characterised by high thermal stability and they are active in all dairy products. Lipophilic and hydrophilic antioxidants interact in the process of deactivating reactive oxygen species and the final products of lipid peroxidation. A negative correlation between milk consumption and the incidence of diet‐dependent diseases confirms that the consumption of milk and dairy products delivers health benefits.     

Oxidised cholesterol in milk and dairy products

Cholesterol is found in animal foods. It can be oxidised in various ways and cholesterol oxidation products (COPs) are formed. Such products are often found in animal foods including dairy products. Recently published results suggest that the contents of COPs in milk and dairy products is very small and does not confirm the results of earlier studies. A higher concentration of COPs can be found only in processed dairy products exposed to harsh storage conditions where the impact of oxygen and light or oxygen and low water activity are concomitant.     

蛋乳发酵酸奶工艺的探讨

鸡蛋与牛奶均具有丰富的营养价值,利用鸡蛋、牛奶等原料通过发酵方法制得的蛋乳发酵酸奶更是具有鸡蛋与酸奶两种物质的营养价值和独特的保健功效。本文综述了国内外蛋乳发酵酸奶的研究进展,并分析了蛋液的添加量、蛋液杀菌条件、发酵菌种比例、接种量和发酵温度和时间等因素对蛋乳发酵酸奶制作的影响。     

Lytic antimicrobial activity of hen egg white lysozyme immobilized to polystyrene beads

ABSTRACT:  Lysozyme [EC 3.2.1.17] was covalently attached to polystyrene resin beads by the sole histidine residue (His-15) through peptide spacers of various lengths. The spacers were amino acid chains composed of 6-aminocaproic acid synthesized with the solid phase peptide synthesis method. Immobilized lysozyme with a spacer length of three 6-aminocaproic acid units (2736 U/g resin with a protein load of 2.21 mg/g resin) displayed the greatest degree of hydrolytic activity against lyophilized Micrococcus lysodeikticus cell wall preparations. Enzymatic activity of immobilized lysozyme was 14.2% of that of the free enzyme. Preparations with longer spacers yielded higher total activity yet the retained activity was constant at about 14% level. A control that consisted of randomly coupled lysozyme to polystyrene beads without an amino acid spacer gave an enzyme activity of 158 U/g with a protein load of 1.24 mg/g resin which equated to 1.4% retained activity. Properties of the immobilized lysozyme system were studied, including stability and activity against soluble compared with insoluble substrates. A kinetics study of the immobilized lysozyme using Eadie–Hofstee plot parameters suggested significant external diffusion effects indicative of deviation from classic Michaelis–Menten kinetic behavior.