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1.
利用奶粉的溶解性,通过水和0.5%次氯酸钠溶液洗涤混有乳源性成分和牛、羊肉骨粉的饲料样品,去除饲料中的乳粉成分后,再使用16S rDNA PCR方法进行动物源性检测。结果表明,实验所建立的方法能够完全区分饲料中的乳粉与肉骨粉。当乳粉含量分别为25%、50%和75%时,混合饲料中牛、羊肉骨粉的检出限分别为2%、0.5%和0.1%。此方法操作简单,容易掌握,可用于鉴别反刍动物饲料中非乳源性成分的牛、羊源性成分。  相似文献   

2.
Polyclonal antibodies raised against the plasmin-released 1-28 phosphopeptide from bovine beta-casein [i.e., beta-CN(f1-28)4P] specifically recognized the tryptic beta-casein 1-25 and 2-25 peptides, whatever the degree of phosphorylation, but were unresponsive to the shortened beta-casein 16-22 phosphopeptide. These antibodies were able to recognize the parent bovine beta-casein as well as the homologous water buffalo protein, but they could not detect the homologous counterparts from ovine and caprine milks. Such antibodies were used in competitive enzyme-linked immunosorbent assays to monitor the plasmin-mediated release of the 1-28 phosphopeptide from beta-casein and to evaluate the residual native beta-casein in bovine cheese sampled during ripening. Applications of these polyclonal antibodies are suggested mainly for estimating the age of hard cheeses and, possibly, for tracing the presence of bovine casein in fresh ovine and caprine cheeses.  相似文献   

3.
应用PCR技术检测饲料中的鱼源性成分   总被引：1，自引：0，他引：1  
PCR方法在动物源性饲料检测中有很好的应用前景。为了有效检测反刍动物饲料中的鱼源性成分，降低疯牛病传播风险，本研究根据鱼线粒体1DNA 6S rRNA序列设计并筛选了鱼源性特异引物，使用Qiagen公司的组织DNA提取试剂盒提取核酸。PCR特异性检测结果表明，引物NC_Fish2480/2501F/ NC_Fish2565/2586R与牛、羊、猪、鸡成分无交叉反应；灵敏性检测结果表明，该引物可以检测到0.1%的鱼源性成分。该方法的建立为饲料中鱼源性成分的PCR检测提供了依据。  相似文献   

4.
The effect of the ripening time on the proteolytic process in cheeses manufactured from mixtures of cow's and ewe's milk during a 167-day ripening period was monitored by capillary electrophoresis of the pH 4.6-insoluble fraction. Totals of 21 and 16 peaks were recognized and matched in the electropherograms obtained with a fused-silica capillary and a neutral capillary (hydrophilically coated), respectively. These peaks corresponded to intact bovine and ovine caseins and their hydrolysis products (e.g., alpha(s1)-casein, gamma-caseins). In 167-day-old cheeses, bovine alpha(s0)-casein (alpha(s1)-casein 9P) had been completely degraded and 6% of the residual bovine alpha(s1)-casein remained intact. Breakdown of the beta-casein fraction was lower than that of the alpha(s)-casein fraction. Finally, partial least-squares regression and principal component regression were used to predict the ripening time in cheeses. The root-mean-square errors in prediction by cross-validation were <7.8 days in all cases.  相似文献   

5.
Mozzarella cheese obtained from buffalo (Bubalus bubalis) milk is a typical Italian product certificated by means of the European Protected Designation of Origin (PDO). Mozzarella cheese can also be obtained from bovine milk or bovine/buffalo milk mixtures, but in this case, it cannot be sold as PDO product, and its label must report the actual ingredients. However, bovine milk in PDO products was frequently detected in the past, suggesting fraudulent addition or accidental contamination. Several methods based on end-point polymerase chain reaction (PCR) have been profitably applied in a large number of tests to detect the presence of undeclared ingredients, also in dairy products. In the present study we report a real-time PCR method able to quantify bovine milk addition to pure buffalo cheese products. We validated a normalized procedure based on two targets: bovine mitochondrial cytochrome b (cyt b) to detect and quantify the bovine DNA and nuclear growth hormone (GH) gene used as a universal reference marker. With the use of this real-time PCR assay, 64 commercial mozzarella di bufala cheese samples purchased at local supermarkets, dairy shops, or directly from cheese manufacturers were analyzed. The results obtained demonstrate that most of the commercial samples were contaminated with bovine milk. Therefore, this assay could be conveniently employed to carry out routine and accurate controls aimed not only to discourage any fraudulent behavior but also to reduce risks for consumer health.  相似文献   

6.
Real-time uniplex and duplex polymerase chain reaction (PCR) assays with a SYBR Green I post-PCR melting curve analysis were evaluated for the identification and quantification of bovine, porcine, horse, and wallaroo DNA in food products. Quantitative values were derived from threshold-cycle (C(t)) data obtained from serial dilutions of purified DNA. The limits of detection in uniplex reactions were 0.04 pg for porcine and wallaroo DNA and 0.4 pg for cattle and horse DNA. Species specificity of the PCR products was tested by the identification of peaks in DNA melting curves, measured as the decrease of SYBR Green I fluorescence at the dissociation temperature. The peaks could be distinguished above the background even at the lowest amount of template DNA detected by the C(t) method. The system was also tested in duplex reactions, by use of either single-species DNA or DNA admixtures containing different shares of two species. The minimum proportions of each DNA species allowing the resolution of T(m) peaks in the duplex reactions were 5% (cattle or wallaroo) in cattle/wallaroo mixtures, 5% porcine and 1% horse in porcine/horse mixtures, 60% porcine and 1% wallaroo in porcine/wallaroo mixtures, and 1% cattle and 5% horse in cattle/horse mixtures. A loss in the sensitivity of the method was observed for some DNA combinations in the duplex assay. In contrast, the results obtained from SYBR Green I uniplex and duplex reactions with single-species DNA were largely comparable to those obtained previously with species-specific TaqMan probes, showing the suitability of that simpler experimental approach for large-scale analytical applications.  相似文献   

7.
The castor seed contains ricin, which is one of the most potent biological toxins and is widely considered to be a threat agent for bioterrorism. In this study, a rapid and sensitive PCR method was applied to the detection of castor contamination in milk and liquid egg samples. The targeting gene sequence of the primer set, Ricin-F4/R4, was not found in either the bovine or chicken genome. Primers against a highly conserved sequence from the 18S ribosomal RNA gene were used as a positive control for DNA extraction and PCR reaction efficiency. The quantity and quality of DNA prepared from castor spiked or nonspiked milk and egg samples obtained from three different DNA extraction methods were compared. The cetyl trimethylammonium bromide (CTAB) method yielded the highest quality of DNA and is most suitable for the sensitive detection of castor DNA by real-time PCR in both milk and liquid egg matrixes. However, taking time and cost into consideration, a commercial kit designed for extraction of DNA from stool samples could be used as an alternative method for the routine extraction of DNA from milk for real-time PCR assays. The egg matrix was found to inhibit PCR amplification and interfere with two of the three methods tested for DNA extraction. Egg yolk had a greater negative effect on PCR amplification than the egg white matrix. Our results affirm the necessity of performing individual validations for each food matrix. Both real-time PCR systems used in this study, TaqMan and SYBR Green I dye, were capable of detecting 100 ng of castor acetone powder, corresponding to 5 ng of ricin, in 1 mL of milk or liquid egg, well below the toxic dose for humans. On the basis of these results, the real-time PCR method for detection of intentional castor contamination is applicable to milk and egg matrixes.  相似文献   

8.
Highly species-specific primers for pork D-loop mtDNA have been designed. Use of these and restrictive PCR amplification conditions has improved a reliable and rapid method for detecting a PCR-amplified 531 bp band from pork. It has been proved useful for detecting both pork meat and fat in meat mixtures, including those dry-cured and heated by cooking. Absence of response in PCR-amplified samples or mixtures from bovine, ovine, chicken, and human was also demonstrated. Furthermore, wild boar and pork samples can be also easily distinguished by a simple AvaII restriction analysis.  相似文献   

9.
基于骨蛋白拉曼光谱特异性的肉骨粉种属鉴别方法   总被引：1，自引：1，他引：0  
为了快速检测肉骨粉的种属来源,该研究开发了一种简便、可靠、科学、高效的肉骨粉种属鉴别方法。以87个肉骨粉(猪,鸡,牛和羊肉骨粉)为研究对象,利用拉曼光谱技术,结合化学计量学方法,建立了基于骨蛋白拉曼光谱特性的肉骨粉种属鉴别分析方法与模型。研究结果表明:根据偏最小二乘判别分析(PartialLeastSquaresDiscriminant Analysis,PLS-DA)模型,发现鸡和哺乳动物(猪,牛和羊)肉骨粉主要在1 659、2 930、2 852、1 246和1 455 cm-1附近的特征谱带具有差异性;猪和反刍动物(牛和羊)肉骨粉主要是在2 852、1 659和1 246 cm-1附近的特征谱带具有差异性;牛和羊肉骨粉主要是在878、853、2 930、2 852、1 246、1 455和1 659 cm-1附近的特征谱带具有差异性,并且PLS-DA模型鉴别肉骨粉的灵敏度和特异度均大于93.8%。研究结果可以丰富肉骨粉种属鉴别方法体系以及为开发便携式拉曼光谱仪提供参考。  相似文献   

10.
一种检测转Bt基因抗虫棉新棉33B和GK-12的PCR方法   总被引：2，自引：1，他引：1  
测定了转基因抗虫棉新棉33B和GK-12的Bt基因表达盒的序列,发现它们在Bt基因和Bt基因与终止子连接区的序列存在差异,而在启动子与Bt基因连接区的序列完全一致。基于这种结构上的差异,设计3条特异性引物MG-P1、MG-P2和MG-P3,建立了检测这两个转基因抗虫棉的双重PCR方法。采用建立的方法,检测了40个转基因棉花样品,其中,只含有新棉33B的Bt基因结构的样品数为32个;只含有GK-12的Bt基因结构的样品数为6个;同时含有两者结构的样品数为2个。  相似文献   

11.
Varieties of market cheese were analyzed for alkaline phosphatase by the modified rapid colorimetric method of the American Public Health Association (APHA) and the official AOAC method, 16.304-16.306. In the APHA method, 5 g cheese (pH less than 7.0) is macerated with 2 mL 1:1 carbonate buffer, or 2 mL water (for cheese with pH greater than 7.0). Addition of 0.1 mL magnesium acetate (1 mg magnesium) to test portions of cheese extracts yielded reproducible and quantitative recovery of added phosphatase. In the AOAC method, macerating 0.5 g cheese with 1 mL borate buffer before adding milk phosphatase improved recovery among cheeses. Addition of magnesium ion increased phosphatase activity in some cheeses. Phosphatases in blue mold-ripened and Swiss cheeses were inactivated by heat faster than was milk phosphatase, yet milk phosphatase added to various soft cheeses was completely inactivated at 60 degrees C for 10 min. The lability of phosphatase was due to the heat-denaturing effect of NaCl present in finished cheeses. Some Mexican style soft cheeses contained both heat-labile and heat-stable phosphatases. These data suggest that the phosphatase test to differentiate milk and microbial phosphatases on the basis of repasteurization and analysis of cheese is no longer valid.  相似文献   

12.
Identification of species-specific DNA in feedstuffs   总被引：2，自引：0，他引：2  
Due to the menace of transmission of spongiform encephalopathies, feed components intended for ruminant nutrition must be checked for the presence of ruminant-derived materials. A sensitive method for the identification of bovine- and ovine- and also swine- and chicken-specific mitochondrial DNA sequences based on Polymerase Chain Reaction (PCR) has been developed. The specificity of the primers for PCR has been tested using samples of DNA of other vertebrate species, which may also be present in rendering plant products intended for feed manufacture. The method allows the detection in concentrate mixtures of 0.01% of the target species derived material. The identity of a sample containing 0.1% of bovine, ovine, swine, and chicken meat-and-bone meal has further been confirmed by sequencing.  相似文献   

13.
Immunoglobulin-rich foods may provide health benefits to consumers. To extend the refrigerated shelf life of functional foods enriched with bovine immunoglobulin G (IgG), nonthermal alternatives such as high-pressure processing (HPP) may offer advantages to thermal processing for microbial reduction. To evaluate the effects of HPP on the immunoactivity of bovine IgG, a soymilk product enriched with milk protein concentrates, derived from dairy cows that were hyperimmunized with 26 human pathogens, was subjected to HPP or heat treatment. To achieve a 5 log reduction in inoculated Escherichia coli 8739, the HPP or heat treatment requirements were 345 MPa for 4 min at 30 degrees C or for 20 s at 70 degrees C, respectively. To achieve a 5 log reduction in natural flora in the enriched soymilk, the HPP or heat treatments needed were 552 MPa for 4 min at 30 degrees C or for 120 s at 78.2 degrees C, respectively. At equivalent levels for a 5 log reduction in E. coli, HPP and heat treatment caused 25% and no detectable loss in bovine IgG activity, respectively. However, at equivalent levels for a 5 log reduction in natural flora, HPP and heat resulted in 65 and 85% loss of bovine IgG activity, respectively. Results of combined pressure-thermal kinetic studies of bovine milk IgG activity were provided to determine the optimal process conditions to preserve product function.  相似文献   

14.
Enumeration of Staphylococcus aureus in foods was collaboratively studied by comparing the present AOAC final action method, 46.062, which uses trypticase soy broth with 10% NaCl to a proposed replacement method which uses the same broth with 1% sodium pyruvate added. Fifteen collaborators analyzed uninoculated samples of milk, tuna salad, and ground turkey, as well as samples inoculated with low (10(2) cells/g), middle (10(4) cells/g), and high (10(6) cells/g) levels of S. aureus. The samples were frozen immediately to maintain the inoculated level of S. aureus in the food. A different strain of S. aureus was used for each food; heat-stressed S. aureus cells were used to inoculate the milk samples. The pyruvate-amended broth significantly (alpha = 0.05) increased enumeration of low, middle, and high levels of S. aureus from milk and ground turkey, and from tuna salad at middle and high levels. The pyruvate-amended media method has been adopted official first action to replace method 46.062.  相似文献   

15.
In this work, a disposable electrochemical immunosensor, based on a competitive assay scheme, was applied to detect polychlorinated biphenyls (PCBs) in food. For this purpose, antibodies against PCBs were directly immobilized onto the carbon surface of a disposable screen-printed electrode. A competition between the PCBs present in the sample and a fixed concentration of an enzyme-labeled PCB was realized and evaluated by electrochemical detection. Alkaline phosphatase was used as the enzyme label, coupled with differential pulse voltammetry (DPV) as the electrochemical technique. The immunosensor was tested on aroclor mixture detection (1242 and 1248) and then on some typologies of food samples to evaluate the possible application for real sample analysis. Samples analyzed were from different matrixes, such as sheep milk, bovine adipose tissue, and bovine muscle. Results obtained were compared with the accredited results according to ISO 17025 methods for PCB detection (HRGC-LRMS) as a confirmatory analysis. Preliminary results show the possibility to use this device as a screening method in food sample analysis. The negligible matrix effect observed may lead to a simplified extraction procedure, and considerable time and consumable savings are the immediate benefits given by the proposed method.  相似文献   

16.
To enforce the labeling regulations of genetically modified organisms (GMOs), the application of reference molecules as calibrators is becoming essential for practical quantification of GMOs. However, the reported reference molecules with tandem marker multiple targets have been proved not suitable for duplex PCR analysis. In this study, we developed one unique plasmid molecule based on one pMD-18T vector with three exogenous target DNA fragments of Roundup Ready soybean GTS 40-3-2 (RRS), that is, CaMV35S, NOS, and RRS event fragments, plus one fragment of soybean endogenous Lectin gene. This Lectin gene fragment was separated from the three exogenous target DNA fragments of RRS by inserting one 2.6 kb DNA fragment with no relatedness to RRS detection targets in this resultant plasmid. Then, we proved that this design allows the quantification of RRS using the three duplex real-time PCR assays targeting CaMV35S, NOS, and RRS events employing this reference molecule as the calibrator. In these duplex PCR assays, the limits of detection (LOD) and quantification (LOQ) were 10 and 50 copies, respectively. For the quantitative analysis of practical RRS samples, the results of accuracy and precision were similar to those of simplex PCR assays, for instance, the quantitative results were at the 1% level, the mean bias of the simplex and duplex PCR were 4.0% and 4.6%, respectively, and the statistic analysis ( t-test) showed that the quantitative data from duplex and simplex PCR had no significant discrepancy for each soybean sample. Obviously, duplex PCR analysis has the advantages of saving the costs of PCR reaction and reducing the experimental errors in simplex PCR testing. The strategy reported in the present study will be helpful for the development of new reference molecules suitable for duplex PCR quantitative assays of GMOs.  相似文献   

17.
An optimized DNA extraction protocol for animal tissues coupled with sensitive PCR methods was used to determine whether trace levels of feed-derived DNA fragments, plant and/or transgenic, are detectable in animal tissue samples including dairy milk and samples of muscle (meat) from chickens, swine, and beef steers. Assays were developed to detect DNA fragments of both the high copy number chloroplast-encoded maize rubisco gene (rbcL) and single copy nuclear-encoded transgenic elements (p35S and a MON 810-specific gene fragment). The specificities of the two rbcL PCR assays and two transgenic DNA PCR assays were established by testing against a range of conventional plant species and genetically modified maize crops. The sensitivities of the two rbcL PCR assays (resulting in 173 and 500 bp amplicons) were similar, detecting as little as 0.08 and 0.02 genomic equivalents, respectively. The sensitivities of the p35S and MON 810 PCR assays were approximately 5 and 10 genomic equivalents for 123 bp and 149 bp amplicons, respectively, which were considerably less than the sensitivity of the rbcL assays in terms of plant cell equivalents, but approximately similar when the higher numbers of copies of the chloroplast genome per cell are taken into account. The 173 bp rbcL assay detected the target plant chloroplast DNA fragment in 5%, 15%, and 53% of the muscle samples from beef steers, broiler chickens, and swine, respectively, and in 86% of the milk samples from dairy cows. Reanalysis of new aliquots of 31 of the pork samples that were positive in the 173 bp rbcL PCR showed that 58% of these samples were reproducibly positive in this same PCR assay. The 500 bp rbcL assay detected DNA fragments in 43% of the swine muscle samples and 79% of the milk samples. By comparison, no statistically significant detections of transgenic DNA fragments by the p35S PCR assay occurred with any of these animal tissue samples.  相似文献   

18.
The nanostructure of Mozzarella cheeses prepared from microfluidized milk was compared with that of control cheeses made from untreated milk. Milk heated to 10 or 54 degrees C and containing 1.0 or 3.2% fat was homogenized by microfluidization at 34 or 172 MPa prior to cheesemaking. The effects on the casein particles and fat globules in the cheese were determined by transmission electron microscopy after 1 day and 6 weeks of storage at 4 degrees C. The micrographs showed that electron-dense regions theorized to be casein submicelles rearranged from a homogeneous configuration to a pattern of clusters during the storage period. The nanostructure of the cheeses made from milk processed under the mildest conditions resembled the controls, but otherwise the fat droplets decreased in size and increased in number as the pressure and temperature were increased. The results indicate that both homogenization temperature and pressure affect the nanostructure of Mozzarella cheese.  相似文献   

19.
In this study, the effects of whey pH at drainage on the physicochemical, sensory, and functional properties of mozzarella cheese made from buffalo milk during storage were investigated. Four cheese samples were manufactured using starter culture at different whey pH values [(A) 6.2, (B) 5.9, (C) 5.6, and (D) 5.3] and analyzed on the 1st, 28th, and 56th day. Ash, calcium, and phosphorus concentrations decreased as the whey pH at drainage was lowered. Cheese yield and calcium recovery were the highest in D cheeses. During storage, expressible serum levels decreased and nonexpressible serum levels increased, indicating an increase in the water holding capacity of the cheeses. Reducing the calcium content of cheeses increased meltability values, but an overly low calcium level (D cheeses) had an adverse effect on the meltability. The melting properties of cheese samples, except D cheeses, were improved with aging. A cheeses were the hardest and D cheeses the softest throughout storage. The 1st day sensory evaluations revealed that C and D cheeses were preferred and that A cheeses were not. All sensory properties of A cheeses were improved with storage. D cheeses were rated inferior to the others at the end of the storage time.  相似文献   

20.
Two mixtures of Propionibacterium freudenreichii commercial strains were tested as adjunct cultures in pasteurized milk Raclette cheese to investigate the ability of propionibacteria (PAB) to enhance flavor development. Cheese flavor was assessed by a trained sensory panel, and levels of free amino acids, free fatty acids, and volatile compounds were determined. The PAB level showed a 1.4 log increase within the ripening period (12 weeks at 11 degrees C). Eye formation, which was not desired, was not observed in PAB cheeses. PAB fermented lactate to acetate and propionate and produced fatty acids by lipolysis, branched chain volatile compounds derived from isoleucine and leucine catabolism and some esters. One of the experimental cheeses received the highest scores for odor and flavor intensity and was characterized by higher frequencies of detection for some minor notes ("propionic"and "whey" odor, "sweet" taste). PAB can therefore be considered as potential adjunct cultures to enhance or modify cheese flavor development.  相似文献   

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