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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Identification of the species of origin of fresh,cooked and canned meat and meat products using antisera to thermostable muscle antigens by ouchterlony's double diffusion test

Antisera to thermostable muscle antigens (TMA) from 14 species of bovidae were raised in goats and/or sheep. To achieve species specificity the antisera were absorbed with serum from the other species. While the absorbed antisera to TMA of buffalo, impala, eland, waterbuck, wildebeest and oryx were rendered specific, the antiserum to cattle TMA cross-reacted with buffalo fresh meat antigens (FMA) and cooked meat antigens (CMA) but not with buffalo thermostable muscle antigens. Fresh and cooked muscle antigens from these two species could be differentiated by the antiserum to buffalo TMA. A similar approach was used to differentiate the FMA, CMA, and TMA of kongoni, topi and wildebeest. Antiserum to cattle TMA proved useful in detecting the presence of beef meat in meat products that had undergone commercial sterilisation.     

肉及肉制品碘含量检测新方法的验证

对即将实施的碘含量检测新方法进行验证分析,通过选取具有代表性的样本进行实验,研究了新方法对肉及肉制品检测结果准确性和精密度的影响.结果表明,与现有检测方法相比,新检测方法的准确性更好、精密度更高,更加适用于肉及肉制品中碘含量的测定.     

Determination of Vitamin B12 in meat products by RP-HPLC after enrichment and purification on an immunoaffinity column

A quantitative method for the determination of Vitamin B12 in meat products by RP-HPLC and UV detection was developed and compared to the reference method (microbiological assay, MBA). Vitamin B12 was extracted with 50 mM sodium acetate buffer in the presence of sodium cyanide. For the quantification of total Vitamin B12, it was necessary to release protein-bound Vitamin B12 by pepsin treatment. Cyanocobalamin was detected as total Vitamin B12 after purification and enrichment on an immunoaffinity column. The calibration with five concentrations of Vitamin B12 was linear with a regression coefficient r2 > 0.99. The method was validated at three different concentration levels (5-15 ng/g) with salami showing good recovery rates between 80 and 108% and low relative standard deviations between 1.50 and 7.26% (n = 6). The detection limit was found to be 2 ng/g. The Vitamin B12 levels of 50 meat products measured by the developed procedure were similar or significantly lower than those determined by the MBA.     

Polymerase chain reaction assay for identification of chicken in meat and meat products

The aim of this study was to develop polymerase chain reaction (PCR) assay for specific detection of chicken meat using designed primer pair based on mitochondrial D-loop gene for amplification of 442 bp DNA fragments from fresh, processed and autoclaved meat and meat products. The PCR result was further verified by restriction digestion with HaeIII and Sau3AI enzymes for specific cutting site in amplified DNA fragments. The specificity of assay was cross tested with DNA of cattle, buffalo, sheep, goat, pig, duck, guinea fowl, turkey and quail, where amplification was observed only in chicken without cross reactivity with red meat species. However positive reaction was also observed in quail and turkey. In this study, no adverse effects of cooking and autoclaving were found on amplification of chicken DNA fragments. Thus, the detection limits was found to be less than 1% in admixed meat and meat products. The developed assay was found specific and sensitive for rapid identification of admixed chicken meat and meat products processed under different manufacturing conditions.     

Biologically active polyamines in beef, pork and meat products: A review

Dietary polyamines (PAs) putrescine (PUT), spermidine (SPD) and spermine (SPM) participate in an array of roles in human metabolism. Nevertheless, under some physiological conditions they can be undesirable. Meat and meat products are among important sources of PAs in human nutrition, mainly of SPM. The usual contents of PUT, SPD and SPM in fresh beef and pork are <2, <5 and 20-40mgkg(-1), respectively. Current information on changes of PAs during meat storage corresponds with PUT formation by bacterial activity mainly of pseudomonads and Enterobacteriaceae. However, data on SPD and SPM changes during meat chill-storage have been inconsistent. Culinary processing of meat probably does not change SPD and SPM levels. PUT can be formed in different meat products in relation to the microbial population of the raw materials used and the hygienic level of manufacturing process. SPD and SPM contents seem to remain stable during processing of non-fermented meat products or decrease during dry-cured ham ripening. PUT contents increase commonly to 60-140mgkg(-1) in dry spontaneously fermented sausages, however, contents up to several hundreds mgkg(-1) are not extraordinary. Starter cultures are usually able to decrease PUT formation considerably. SPD and SPM contents in dry fermented sausages are comparable with levels typical for fresh meat. Data on SPD and SPM changes during ripening and storage are inconsistent. A decrease of the both polyamines during a storage period has been usually reported.