Survey of authenticity of meat species in food products subjected to different technological processes,by means of PCR-RFLP analysis

Polymerase chain reaction (PCR) coupled to restriction-fragment length polymorphism analysis (RFLP) was considered for exploring the incidence of incorrect labelling in food products containing one or more meat species. Universal primers CYT b1/CYT b2, which amplify a variable region of the mitochondrial cytochrome b of vertebrates, and endonucleases PalI, MboI, HinfI and AluI were used for this purpose. Fifty food products, nine of them raw or cured and the other 41 subjected to a variety of technological processes such as pre-cooking and freezing, cooking and smoking, dehydration or sterilisation, were investigated. Twenty of the 50 products declared mixtures of meat species on their labels. Fifteen (30%) of the 50 food samples investigated displayed an incorrect qualitative labelling. While this affected only one (11.1%) of the nine raw/cured products, 14 (34.2%) of the 41 products subjected to some type of heat-processing were not correctly labelled. The undeclared presence of turkey was the most frequent concern, since it was detected in seven food products. The complete absence of a declared species of high commercial value—such as beef or roe-deer—was observed in another four cases. The PCR-RFLP method used here proved to be a rapid and easy-to-perform two-step analytical approach to achieve qualitative meat species identification in raw and cooked food products containing one or more different species.     

Differentiation of cattle species in beef by PCR-RFLP of mitochondrial and satellite DNA

Methods currently used for the identification of the species origin of meat or tissue samples have not been validated for other bovine species than taurine cattle or water buffalo. These methods also do not discriminate between the different bovine species that are used as source of beef. Here, we describe two complementary methods for detection and differentiation of bovine species, which are based on mutations in mitochondrial DNA and centromeric satellite DNA, respectively. The analysis of satellite DNA is especially relevant for the identification of animals that are of hybrid origin.     

PCR-RFLP方法检测和鉴别五种李斯特菌

目的 建立快速检测和鉴别单增李斯特菌、绵羊李斯特菌、英诺克李斯特菌、威尔李斯特菌和格氏李斯特菌等常见李斯特菌的方法。方法 采用PCR-RFLP技术,首先通过引物“Lis1A-Lis1B”对李斯特菌属iap基因进行扩增,扩增产物大小约1.4 kb,然后用限制性内切酶DdeⅠ对PCR产物进行酶切,电泳观察具有种间特异性的酶切谱带进行鉴别。结果 上述5种李斯特菌的PCR扩增产物经内切酶DdeⅠ消化后,得到片段大小不同的,具有种间差异的特异性酶切图谱。结论 本实验建立的PCR-RFLP方法可以用于上述5种常见李斯特菌的快速检测和鉴定。     

Identification of meat species by PCR-RFLP of the mitochondrial COI gene

Meat authenticity verification is pertinent for economical, religious or public health concerns. The present study investigates the use of PCR-RFLP of a part of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene for identification of species origin of raw meat samples of cow, chicken, turkey, sheep, pig, buffalo, camel and donkey. PCR yielded a 710-bp fragment in all species. The amplicons were digested with seven restriction endonucleases (Hind II, Ava II, Rsa I, Taq I, Hpa II, Tru 1I and Xba I) that were selected based on the preliminary in silico analysis. Different levels of polymorphism were detected among samples. The level of COI variation revealed using only Hpa II was sufficient to generate easily analyzable species-specific restriction profiles that could distinguish unambiguously all targeted species. Compared to previously published reports for the determination of meat origin at the molecular level, the approach developed here is much cheaper and faster for routine identification of meats in food control laboratories.     

Quantitative detection of soybean in meat products by a TaqMan real-time PCR assay

In the present work, we propose a normalised real-time quantitative PCR assay to determine the addition of soybean to meat products. The method proved to be a powerful tool for the quantification of soybean protein (dry basis) in the range of 0.01% to 6%, being successfully in-house validated. Its application was effective in the analysis of several meat products, indicating 2% of non-compliance with the food allergen labelling legislation, and some inconsistencies when comparing the declared with estimated amounts of soybean. This work highlights the importance of efficient tools to assess labelling statements of meat products, avoiding fraudulent practices.     

基于多重荧光PCR检测的肉及其制品中鸭DNA成分的鉴别方法

针对现有肉制品中鸭DNA成分检测方法不能检测番鸭DNA成分的问题,通过下载番鸭、家鸭及其相近物种的12SrDNA序列,使用CLUSTAL X2进行序列比对,筛选特异性序列,设计了一对通用引物及两条番鸭、家鸭特异性探针,构建了可同时检测番鸭、家鸭DNA成分的实时荧光PCR方法。结果表明:最终构建的检测方法可在掺伪量为0.1%的情况下,检测出样品中的家鸭或番鸭DNA成分。该方法相比普通PCR或单重荧光PCR检测方法节省了劳动力,提高了检测效率,补充了现有检测方法的不足,为更有效地识别掺伪肉类提供技术支持。     

Conjugated linoleic acid in meat and meat products: A review

Conjugated linoleic acid (CLA) consists of a group of geometric and positional isomers of linoleic acid to which anticancerogenic, antidiabetic, and antiatherogenic effects, as well as effects on immune system, bone metabolism, and body composition are attributed. CLA is found predominantly in milk and meat of ruminants due to the importance of rumen micro-organism in the formation of CLA and its precursors. This review attempts to give an overview of the available data on intramuscular CLA concentrations in meat and meat products originating from different animal species. The factors influencing these concentrations are discussed and the estimated human daily intakes as well as the percentage provided by meat are reported.     

基于PCR多物种鉴定技术及其在肉类 鉴定中的应用

国内外的肉类掺假由来已久,法律上监管部门对此采取了严格监管措施,技术上应用了感官、显微检测和免疫学等相应的检测技术。近几年来,PCR及其衍生技术的快速发展大大推动了肉类鉴定技术的快速发展,尤其在多物种鉴定技术领域建立了如多重PCR、通用单引物多重PCR、通用引物特异性PCR和PCR-RFLP等多项技术。这些技术的发展为肉类及其制品的检测提供了重要手段,代表了检测技术领域发展的方向,但其也各有其优缺点。本文介绍了上述4种多物种鉴定技术及其在肉类鉴定中的应用,并分析了其优缺点,旨在为物种鉴定方法选择及其研究提供参考。     

Chitosan and mint mixture: A new preservative for meat and meat products

Meat is prone to both microbial and oxidative spoilage and therefore it is desirable to use a preservative with both antioxidant and antimicrobial properties. Mint extract alone had good antioxidant activity but poor antimicrobial activity, while chitosan alone showed poor antioxidant activity with excellent antimicrobial properties. Therefore, the potential of chitosan and mint mixture (CM), as a preservative for meat and meat products, was investigated. Addition of chitosan to mint extract did not interfere with the antioxidant activity of mint. In the case of 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, the IC₅₀ value for CM (17.8 μg/ml) was significantly (p ? 0.05) lower than that for mint extract (23.6 μg/ml). CM efficiently scavenged superoxide and hydroxyl radicals. The antimicrobial activities of CM and chitosan were comparable against the common food spoilage and pathogenic bacteria, the minimum inhibitory concentration being 0.05%. CM was more effective against Gram-positive bacteria. The shelf life of pork cocktail salami, as determined by total bacterial count and oxidative rancidity, was enhanced in CM-treated samples stored at 0–3 °C.     

A survey of the use of soy in processed Turkish meat products and detection of genetic modification

To screen for possible illegal use of soybeans in meat products, the performance characteristics of a commercial polymer chain reaction (PCR) kit for detection of soybean DNA in raw and cooked meat products were established. Minced chicken and beef products containing soybean at levels from 0.1% to 10.0% were analysed by real-time PCR to amplify the soybean lectin gene. The PCR method could reliably detect the addition of soybean at a level of 0.1%. A survey of 38 Turkish processed meat products found only six samples to be negative for the presence of soybean. In 32 (84%) positive samples, 13 (34%) contained levels of soy above 0.1%. Of soybean positive samples, further DNA analysis was conducted by real-time PCR to detect whether genetically modified (GM) soybean had been used. Of 32 meat samples containing soybean, two samples were positive for GM modification.     

Advances in ingredient and processing systems for meat and meat products

Changes in consumer demand of meat products as well as increased global competition are causing an unprecedented spur in processing and ingredient system developments within the meat manufacturing sector. Consumers demand healthier meat products that are low in salt, fat, cholesterol, nitrites and calories in general and contain in addition health-promoting bioactive components such as for example carotenoids, unsaturated fatty acids, sterols, and fibers. On the other hand, consumers expect these novel meat products with altered formulations to taste, look and smell the same way as their traditionally formulated and processed counterparts. At the same time, competition is forcing the meat processing industry to use the increasingly expensive raw material “meat” more efficiently and produce products at lower costs. With these changes in mind, this article presents a review of novel ingredient systems and processing approaches that are emerging to create high quality, affordable meat products not only in batch mode but also in large-scale continuous processes. Fat replacers, fat profile modification and cholesterol reduction techniques, new texture modifiers and alternative antioxidant and antimicrobial systems are being discussed. Modern processing equipment to establish continuously operating product manufacturing lines and that allow new meat product structures to be created and novel ingredients to be effectively utilized including vacuum fillers, grinders and fine dispersers, and slicers is reviewed in the context of structure creation in meat products. Finally, trends in future developments of ingredient and processing systems for meat products are highlighted.     

Meat species identification and Halal authentication analysis using mitochondrial DNA

A method utilizing PCR-restriction fragment length polymorphism (RFLP) in the mitochondrial genes was developed for beef (Bos taurus), pork (Sus scrofa), buffalo (Bubalus bubali), quail (Coturnix coturnix), chicken (Gallus gallus), goat (Capra hircus), rabbit (Oryctolagus cuniculus) species identification and Halal authentication. PCR products of 359-bp were successfully obtained from the cyt b gene of these six meats. AluI, BsaJI, RsaI, MseI, and BstUI enzymes were identified as potential restriction endonucleases to differentiate the meats. The genetic differences within the cyt b gene among the meat were successfully confirmed by PCR-RFLP. A reliable typing scheme of species which revealed the genetic differences among the species was developed.     

Survey of the authenticity of prawn and shrimp species in commercial food products by PCR-RFLP analysis of a 16S rRNA/tRNA mitochondrial region

A novel PCR-RFLP method was evaluated as a tool to assess the incidence of incorrect labelling of prawns and shrimps in commercial food products. The whole method can be performed in less than 8 h in only one day of work. PCR amplification with primers 16Scru4/16Scru3, targeted to the amplification of a ca. 530 bp region of 16S rRNA and tRNA^Val mitochondrial genes, was coupled to restriction analysis with AluI, TaqI or HinfI. Forty-one commercial food products were considered. The molecular method considered allowed the identification of up to 17 different prawn and shrimp species in all the processed products considered. Seven (28%) of the 25 food products declaring one or more species in their labels were incorrectly labelled. Authentication was successfully assessed in commercial peeled products subjected to industrial processing, in which none of the products displayed labelling at species level. Overall, incorrect labelling was detected in 10 (24.4%) of the 41 commercial products tested, while another 16 samples (39%) exhibited incomplete labelling. The molecular method evaluated in this study proved to be a rapid and easy-to-perform two-step analytical approach to achieve species identification of commercial whole specimens of frozen prawns and shrimps and in peeled processed products where such raw materials are included as added-value ingredients.     

速测盒法快速测定肉及肉制品中亚硝酸盐

目的 了解亚硝酸盐速测盒的可靠性,为现场监督执法及基层快速检测提供有力的技术支撑。方法采用速测盒方法检测亚硝酸盐标准溶液、肉及肉制品中亚硝酸盐添加情况,并与《GB 5009.33-2016食品中亚硝酸盐与硝酸盐的测定》第二法(盐酸萘乙二胺法)进行对比。结果速测盒对亚硝酸盐最低检出限可达到0.1 mg/L;检测样品时,速测盒与盐酸萘乙二胺法阴性符合率为97.8%,阳性符合率为100.0%。不同环境温度下,只需将反应时间控制在5 min以上,则不会对检测结果产生影响。样品经简单处理后,显色剂滴加到样品提取液中,混匀后反应3～5 min,即可观察结果。检测单个样品20 min内即可出结果。结论速测盒法具有快速、准确、方便、灵敏等特点,适用于肉及肉制品中亚硝酸盐现场定性分析。     

Comparison of different methods for total lipid quantification in meat and meat products

This study was aimed to evaluate the efficiency of six extraction methods for the quantification of total lipid content in meat and meat products: standard Soxhlet method (with and without previous acid hydrolysis), continuous Soxhlet method (with and without previous acid hydrolysis), and those methods based in the use of a mixture of chloroform and methanol, and described by Folch, Less, and Sloane (1957) and Bligh and Dyer (1959). Lipid content was determined in nine different meat products with different fat contents and physico-chemical features: cooked turkey breast, fresh pork loin, cooked ham, dry-cured ham, mortadella, beef burger, fresh sausage, dry-cured sausage and salami. The most effective methods for determining fat content in the studied meat products were the method described by Folch et al. (1957) and the Soxhlet with previous acid hydrolysis method. The Soxhlet method without previous acid hydrolysis adequately extracted lipids only in those meat products with very high fat content. The use of the method described by Bligh and Dyer (1959) gave rise to the lowest lipid contents in all the studied meat products.     

Immunological detection of keratan sulfate in meat products with and without mechanically separated chicken meat

Keratan sulfate is a glycosaminoglycan found in the structure of cartilage proteoglycans, aggrecan and fibromodulin. This study was undertaken to detect this glycosaminoglycan in meat products containing mechanically separated chicken meat (MSCM) having cartilage particles. Dry-defatted samples of MSCM and meat products with or without MSCM were digested with papain, and a non-dialyzable fraction from each papain digest was examined by immunodiffusion analysis using anti-keratan sulfate monoclonal antibody (IgM). No precipitine line was formed with the antibody for all samples of meat products without MSCM, while a sample of MSCM and all samples of meat products with MSCM gave clear precipitine lines with the antibody. The immunodiffusion test described here appears to be a simple sensitive specific method for qualitative analysis of keratan sulfate, which in combination with other methods may be useful for detection of MSCM in meat products.     

Development of PCR assays for detection of Escherichia coli O157:H7 in meat products

A multiplex polymerase chain reaction (PCR) procedure based on fliC_h7 and rfbE genes was developed for the detection of Escherichia coli O157:H7 in raw pork meat and ready-to-eat (RTE) meat products. Two different DNA extraction procedures were evaluated for application on meat products. MasterPure™ DNA Purification kit in combination with immunomagnetic separation was found to be the best method in a meat system. The optimized PCR included an enrichment step in brilliant green bile 2% broth at 37 °C. This method was applied to artificially inoculated meat and RTE meat products with different concentrations of E. coli O157:H7. The results indicate that the PCR assay developed could sensitively and specifically detect E. coli O157:H7 in raw pork meat and RTE meat products in approximately 10 h, including a 6 h enrichment step. Thus, this method could be proposed for screening E. coli O157:H7 in raw pork and RTE meat products.     

A fast and accurate method for controlling the correct labeling of products containing buffalo meat using High Resolution Melting (HRM) analysis

The substitution of high priced meat with low cost ones and the fraudulent labeling of meat products make the identification and traceability of meat species and their processed products in the food chain important. A polymerase chain reaction followed by a High Resolution Melting (HRM) analysis was developed for species specific detection of buffalo; it was applied in six commercial meat products. A pair of specific 12S and universal 18S rRNA primers were employed and yielded DNA fragments of 220 bp and 77 bp, respectively. All tested products were found to contain buffalo meat and presented melting curves with at least two visible inflection points derived from the amplicons of the 12S specific and 18S universal primers. The presence of buffalo meat in meat products and the adulteration of buffalo products with unknown species were established down to a level of 0.1%. HRM was proven to be a fast and accurate technique for authentication testing of meat products.     

膜芯片用于肉制品中动物源性成分检测的研究

目的研究膜芯片技术检测肉类及其制品中动物源性成分,验证该技术的准确性及可行性。方法利用膜芯片检测肉制品中的动物源性成分,对样品中猪、牦牛、驴及羊源性成分的灵敏度、检出限进行相关实验,并就实际样品的检测与国家标准进行比较。结果该技术用于动物源性成分检测特异性良好,4种动物源性检测的灵敏度为0.1 ng,检出限为0.1%(w:w),适用的样品种类较广,实际样品的检测结果与国家标准荧光PCR法一致。结论该技术可作为快速筛选的方法,为肉制品的动物源性成分鉴定和掺假检测提供理论依据。     

Semiquantitative detection of male pork tissue in meat and meat products by PCR

Consumer awareness has increased concerning castration of piglets without analgesia or anaesthesia. On the other hand the occurrence of boar taint is not tolerated by consumers. Currently no reliable methods exist for the on-line detection of boar taint in the slaughterhouse or for genetic sexing of pigs. Therefore, as an alternative the detection of male pork meat was sought. Based on detection of a length polymorphism of the sex chromosomal amelogenin gene a reliable, specific and highly sensitive PCR method for qualitative and semi-quantitative determination of male pork tissue in meat and meat products was determined. A set of 25 male and 25 female meat samples could be correctly identified and mixtures with as little as 0.1% male meat content could be detected. Therefore the method can be used for production and control of specific meat products containing low amounts of male pork meat and thus avoiding boar taint.