Thermostable muscle antigens suitable for use in enzyme immunoassays for identification of meat from various species

A method for the extraction of thermostable muscle antigens for use in enzyme immunoassays is described. The method yields antigens devoid of contaminating proteins which reduce the adsorption of the antigens on to the plate. The effect of such proteins is stimulated by the addition of gelatin. Gelatin (5 mg ml^?1) results in 100% inhibition of the antigen adsorption on to the plate.     

Studies on characterisation of thermostable antigens of adrenals and muscle tissues of meat animals

The characteristics of heat-stable, ethanol-precipitable antigens (BE) of adrenals and muscle tissues of cattle, buffalo, sheep, goat and pig were studied. The antigens were subjected to protein analysis. The distribution patterns of the antigens employing sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) as well as polyacrylarnide gel isoelectric focusing revealed that the heat-stable antigens of a dreds contained only one component corresponding to troponin T, whereas muscle BE showed associated low molecular weight components along with troponin T as major component. Negative results of thymol-H₂SO₄ staining of SDS-PAGE gels indicated that the thermostable antigens did not belong to the mucoprotein class.     

Identification of meat species by TaqMan-based real-time PCR assay

In this study, a convenient, sensitive and specific real-time PCR assay was described for the species identification and their quantification in raw and cooked meat products. Specific primers and TaqMan probes were designed on the mitochondrial ND2, ND5 and ATP 6-8 genes for donkey, pork and horse, respectively, and the performance of the method was tested. In the results, no cross-reaction was observed between the donkey and pork species specific primer-probe systems and non-target species (bovine, ovine, chicken and turkey). Only one cross reaction was observed between the horse species specific primer-probe set and 100 ng pork DNA at the ct 33.01 level (corresponding to 0.01 ng horse DNA). The real-time quantitative assay used in this study allowed the detection of as little as 0.0001 ng template DNA from pure meat for each species investigated and experimental meat mixtures. In conclusion, it can be suggested that the TaqMan probe assay used in this research might be a rapid and sensitive method for the routine meat species identifications studies in raw or cooked meat products.     

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.     

A strategy for molecular species detection in meat and meat products by PCR-RFLP and DNA sequencing using mitochondrial and chromosomal genetic sequences

Molecular species detection in food has become common in the last 10 years. The methods are sensitive enough to detect small, but relevant, amounts of one species in composed food. We have developed a strategy for detecting different animal species in food by molecular means. This strategy uses a combination of published PCR systems and new developed PCR primer systems for the detection of porcine, bovine, ovine, avian, cervine and equine DNA by PCR followed by restriction analysis (PCR-RFLP). In some cases, analysis is completed by DNA sequencing. The species detection system includes an amplification control and so is in accordance with the relevant food standards.     

Duplex PCR approach for the detection and quantification of donkey,horse and mule in raw and heat‐processed meat products

Donkey‐related products have been paid more attention for their high nutritional value to human beings. Due to donkey resource scarcity, coupled with gradually increasing market demand, adulterated donkey meat products with other low‐cost animal meat, especially with the similar species horse and mule, are often found in market. Therefore, detection of species fraud in donkey meat products is important for consumer protection and food industries. In this study, a simple and highly specific duplex PCR method, based on the simultaneous amplification of fragments of the mitochondrial ATP synthase subunit 8/6 and ND2, was developed and optimised for the identification of horse, donkey and mule species in raw and heat‐processed meat products. To the best of our knowledge, it was the first time for this strategy applied to these three genetically related animal meat products differentiation to date. The duplex PCR generated a 153‐bp and 83‐bp amplification products for horse and donkey, respectively. While for mule, both of the two length amplification products are appeared on the agarose gel. Target meat species could be detected at a level of 1%, and the results indicated that the duplex PCR assay could be used in the authentification of donkey‐related products with high specificity, cost‐effectiveness and simplicity.     

Species identification by oligonucleotide hybridisation: the influence of processing of meat products

We have evaluated the influence of meat processing on the results obtained with a species identification test by DNA oligonucleotide hybridisation. Freezing and thawing of meat did not cause a substantial reduction in the hybridisation signal. Heating of meat at 100°C or 120°C, however, led to signal reduction caused by DNA degradation, but identification was still possible. The signal is highly influenced by the kind of tissues processed. Four extensively processed products with 50 g kg⁻¹ species admixtures were tested. Admixtures could be detected in three products but no hybridisation signal was observed with corned beef. We conclude that the quantification of admixtures by hybridisation is not better than with most alternative methods of species identification, as the strength of the signal depends on factors such as tissue origin and sample processing. © 1999 Society of Chemical Industry     

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.     

Modeling and predicting spoilage of cooked, cured meat products by multivariate analysis

A cooked, cured meat product is a perishable product spoiled mainly by lactic acid bacteria (LAB). LAB cause discoloration, slime formation, off-odors and off-flavors as the result of their metabolic activity producing various products. These microbial products in conjunction with the microbial population could be used to assess the degree of spoilage of this type of product. The spoilage evaluation was achieved by following a multivariate approach. Cluster analysis, principal component analysis and partial least square regression were employed to associate spoilage with microbiological and physicochemical parameters. The developed model was capable of giving accurate predictions of spoilage describing the spoilage associations. The study might contribute to the improvement of quality assurance systems of meat enterprises.     

Lead and cadmium in meat and meat products consumed by the population in Tenerife Island, Spain

The aim of this study was to determine the levels of lead and cadmium in chicken, pork, beef, lamb and turkey samples (both meat and meat products), collected in the island of Tenerife (Spain). Lead and cadmium were measured by graphite furnace atomic absorption spectrometry (GFAAS). Mean concentrations of lead and cadmium were 6.94 and 1.68 µg kg^-1 in chicken meat, 5.00 and 5.49 µg kg^-1 in pork meat, 1.91 and 1.90 µg kg^-1 in beef meat and 1.35 and 1.22 µg kg^-1 in lamb meat samples, respectively. Lead was below the detection limit in turkey samples and mean cadmium concentration was 5.49 µg kg^-1. Mean concentrations of lead and cadmium in chicken meat product samples were 3.16 and 4.15 µg kg^-1, 4.89 and 6.50 µg kg^-1 in pork meat product, 6.72 and 4.76 µg kg^-1 in beef meat product and 9.12 and 5.98 µg kg^-1 in turkey meat product samples, respectively. The percentage contribution of the two considered metals to provisional tolerable weekly intake (PTWI) was calculated for meat and meat products. Statistically significant differences were found for lead content in meats between the chicken and pork groups and the turkey and beef groups, whereas for cadmium concentrations in meats, significant differences were observed between the turkey and chicken, beef and lamb groups. In meat products, no clear differences were observed for lead and cadmium between the various groups.     

Identification of cattle, llama and horse meat by near infrared reflectance or transflectance spectroscopy

Visible and near infrared reflectance spectroscopy (VIS-NIRS) was used to discriminate meat and meat juices from three livestock species. In a first trial, samples of Longissimus lumborum muscle, corresponding to beef (31) llamas (21) and horses (27), were homogenised and their spectra collected in reflectance (NIRSystems 6500 scanning monochromator, in the range of 400-2500 nm). In the second trial, samples of meat juice (same muscle) from the same species (20 beef, 19 llama and 19 horse) were scanned in folded transmission (transflectance). Discriminating models (PLS regression) were developed against “dummy” variables, testing different mathematical treatments of the spectra. Best models indentified the species of almost all samples by their meat (reflectance) or meat juice (transflectance) spectra. A few (three of beef and one of llama, for meat samples; one of beef and one of horse, for juice samples) were classified as uncertain. It is concluded that NIRS is an effective tool to recognise meat and meat juice from beef, llama and horses.     

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.     

Potential applications of plant based derivatives as fat replacers,antioxidants and antimicrobials in fresh and processed meat products

Growing concern about diet and health has led to development of healthier food products. In general consumer perception towards the intake of meat and meat products is unhealthy because it may increase the risk of diseases like cardiovascular diseases, obesity and cancer, because of its high fat content (especially saturated fat) and added synthetic antioxidants and antimicrobials. Addition of plant derivatives having antioxidant components including vitamins A, C and E, minerals, polyphenols, flavanoids and terpenoids in meat products may decrease the risk of several degenerative diseases. To change consumer attitudes towards meat consumption, the meat industry is undergoing major transformations by addition of nonmeat ingredients as animal fat replacers, natural antioxidants and antimicrobials, preferably derived from plant sources.     

Water distribution and mobility in meat during the conversion of muscle to meat and ageing and the impacts on fresh meat quality attributes--a review

This paper reviews current knowledge on the distribution and mobility of water in muscle (myowater) ante- and post mortem and factors affecting these in relation to fresh meat quality parameters; water-holding capacity (WHC), tenderness and juiciness. NMR transverse relaxometry (T(2)) using bench-top Low-Field Nuclear Magnetic Resonance (LF-NMR) has characterised myowater distribution and mobility as well as structural features in meat which directly affect WHC. The current literature demonstrates that WHC is correlated to the water located outside the myofibrillar network (extra-myofibrillar). This review identifies the critical stages which affect the translocation of water into the extra-myofibrillar space and thus the potential for decreased WHC during proteolysis (the conversion of muscle to meat). This review discusses how the intrinsic properties of the water held within the meat could contribute to juiciness and tenderness. Tenderness has been shown to correlate to T(2), however breed and species differences made it difficult to draw firm conclusions. Further understanding of the inherent water properties of fresh meat and the factors affecting water distribution and mobility using NMR technologies will increase the understanding of WHC and tenderisation of fresh meat.     

Control of food spoiling bacteria in cooked meat products with nisin,lacticin 3147, and a lacticin 3147-producing starter culture

Nisin, in the form of the commercial product Nisaplin, and lacticin 3147 in whey powdered form were added to minced pork-meat in amounts of 0.15% (w/w) and 1.5% (w/w), respectively. The meat was cooked and inoculated with a Staphylococcus aureus strain of meat origin and a Listeria innocua strain at a level of 10⁷ or 10⁵ CFU g^–1. The batches were stored vacuum-packaged for 21 days at 8 °C. Nisin and lacticin 3147 immediately reduced the L. innocua population at the time of inoculation. Nisin showed higher inhibitory activity than lacticin 3147. During the storage period, a slight L. innocua growth was observed in the batches inoculated with the larger inoculum, and a bacteriostatic effect was observed against Listeria in the batches inoculated with 10⁵ CFU g^–1. Nisin maintained a constant S. aureus population in the cooked batch inoculated with 10⁷ CFU g^–1, although the bacteriocin was capable of reducing the amount of S. aureus by 90% in the batch inoculated with 10⁵ CFU g^–1. On the other hand, lacticin 3147 did not show an inhibitory effect against S. aureus in the cooked meat. The starter culture Lactococcus lactis DPC 303-T4 (containing the conjugative plasmid encoding production of lacticin 3147) was inoculated in a portion of a Longissimus dorsi pork muscle with brine. L. lactis DPC 303-T4 performed a good fermentation, but lacticin 3147 production was not found after 7 days at 12 °C of storage.     

Use of species-specific antisera to adrenal heat-stable antigens for the identification of raw and cooked meats by agar gel diffusion and counter immunoelectrophoretic techniques

Rabbit antisera to adrenal heat-stable and ethanol-precipitable antigens of buffalo, cattle, sheep, goat and pig were used to develop an agar gel precipitation test and a counter immunoelectrophoretic method for the identification of homologous species in raw, partially heated and boiled meat extracts. Immunoabsorption was necessary to make the primary antisera species specific. The specific antisera can be recommended for identification of the species of origin of meats and their mixtures (5-10% adulteration) in raw, partially heated and cooked states even in the case of closely related species, viz cattle and buffalo, sheep and goat.     

An evaluation of a method to differentiate the species of origin of meats on the basis of the contents of anserine,balenine and carnosine in skeletal muscle

The high performance liquid chromatographic method proposed by Carnegie for determining the species of origin of meats in cooked products has been evaluated. The dipeptides anserine, balenine (ophidine) and carnosine have been extracted from the skeletal muscle of animals in New Zealand and compared with results obtained in Australia. The presence of significant quantities of balenine in red deer meat is reported for the first time. Limitations of the method are discussed.     

Identification of the fish species of raw or cold-smoked salmon and salmon caviar by single-strand conformation polymorphism (SSCP) analysis

Differentiation between ten salmonid fish species belonging to the genera Salmo, Oncorhynchus and Salvelinus was achieved by single strand conformation polymorphism analysis (SSCP) of PCR products of mitochondrial and nuclear genes. Amplicons (300–460 bp in size) of the genes for cytochrome b, parvalbumin and growth hormone gave species-specific patterns of single-stranded DNA in native polyacrylamide gel electrophoresis (PAGE). The method was successfully used to identify products from raw or cold-smoked salmon, as well as from salmon roe.     

Effect of lyophilized water extracts of Melissa officinalis on the stability of algae and linseed oil-in-water emulsion to be used as a functional ingredient in meat products

Previous work pointed out the possibility to enhance the nutritional value of meat products using long chain ω − 3 PUFA enriched emulsions. Oil-in-water emulsions elaborated with a mixture of algae and linseed oils (15:10) in order to be used as functional ingredient were stabilized with BHA (butylhydroxyanisol) or with a lyophilized water extract of Melissa officinalis L. (Lemon balm). The lipid profile of the oil mixture showed a high amount of DHA (31.7%), oleic (25.4%) and α-linolenic acid (12.7%) resulting in a very low ω − 6/ω − 3 ratio (0.12). The lyophilized extract of M. officinalis showed a high antioxidant activity (being 62 ppm of the lyophilized water extract of Melissa equivalent to 200 ppm of BHA, using the DPPH assay as reference), and high total phenolic content. Studying the oxidation process in the emulsions during 15 days at room temperature, it could be concluded that this extract was as efficient as BHA in order to control the thiobarbituric acid reactive substances (TBARS) formation.     

A databank able to be used for identifying and authenticating commercial flatfish (Pleuronectiformes) products at the species level using isoelectric focusing of native muscle proteins

Summary A computerized databank of IEF protein patterns for use in identifying flatfish species (in total 17 species, including 15 commercial ones) is presented. The databank includes all species regulated by the Belgian Law (22 May 1996) on the use of official names for fishes and seafood products. It was found that interspecimen similarity of the IEF patterns, as processed by digitization, was always larger than interspecies similarity, which allows for unequivocal authentication of unknown samples, as long as the authentic pattern is available in the databank. The databank was used to authenticate 17 commercial fish fillets.