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.     

Methodology for meat species identification: A review

This review covers methods available for the recognition of the species of animal whose meat is present in raw materials or meat products. Isolation of species-specific proteins is cumbersome; however, isoelettric focusing followed by recognition of the pattern of protein bands is particularly effective for speciation. Immunoassay has been established as a powerful technique for the determination of food protein analytes in suitable extracts of unresolved mixtures. Simple immunodiffusion using antisera to serum proteins is sufficient to provide a check on the identity of suspect raw samples; routine control would involve extensive sampling programmes. Enzyme-linked immunosorbent assay procedures are also appropriate, but attempts to quantify individual meat species in mixtures have not been successful due to the variability of residual blood levels. Species identification in heat-processed meat products is hindered by progressive denaturation of the protein markers, leading to loss of solubility and antigenicity; the effects of heating can be minimised by a judicious choice of analyte and its solubilisation by renaturation.     

Capillary electrophoresis for bovine and ostrich meat characterisation

The objective of this study was to characterise, compare and quantify the water soluble protein (WSP) and salt soluble protein (SSP) fractions from bovine and ostrich muscle by using sodium dodecyl sulphate polymer-filled capillary gel electrophoresis (CE-SDS). Samples were raw ostrich leg and eye round beef collected 24 and 48 h, respectively, after sacrifice from local slaughter houses. WSP were extracted with cold double distilled deionized water and SSP with 0.6 M NaCl/0.01 M phosphate buffer pH 6 with 0.5% polyphosphates. Separation of WSP and SSP extracts was achieved by CE-SDS. Quantitative data for individual proteins was generated by constructing a calibration curve using bovine serum albumin (BSA) as a standard. The WSP profiles showed differences for bovine and ostrich meat, both qualitatively and quantitatively and could be employed for species differentiation. Quantitative data derived for WSP and SSP from bovine and ostrich muscle showed significant differences among individual proteins. A comparison of protein profiles form ostrich and bovine meat should be useful for meat species differentiation and muscle characterisation for establishing relations to meat quality.     

The determination of isolated soybean protein in raw and pasteurized meat products

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to determine the isolated soy protein content in raw and pasteurized meat products. This method determined soy protein (±0.5%) by using an internal standard protein (haemocyanin) to compensate for variations in the meat. The detection limit for meat products was 0.5%. Several possible meat and non-meat interferences were examined and none were found to interfere. The assay cannot be used on retorted products.     

Easy determination of the addition of soybean proteins to heat-processed meat products prepared with turkey meat or pork-turkey meat blends that could also contain milk proteins

The addition of non-meat proteins to processed meat products is limited by regulations. Therefore, this work has investigated the determination of added soybean proteins in commercial heat-processed meat products prepared with turkey meat or pork-turkey meat blends that could also contain milk proteins. The method consisted of extracting proteins from the meat products in a Tris-HCl buffer (pH 8) and analysing the extract by high-performance liquid chromatography with a linear gradient water-acetonitrile containing 0.05% (v/v) TFA. This method enabled the detection and quantitation of up to 0.08 and 0.28% (w/w), respectively, of soybean proteins (related to 6 g initial product) in these products. Satisfactory precision and recovery data were established. Accuracy was evaluated by a comparison of soybean protein contents determined by the proposed method and the existing AOAC official method based on an enzyme-linked immunosorbent assay (ELISA) from which no statistically significant differences were observed.     

Protein and lipid oxidation in meat: A review with emphasis on high-pressure treatments

BackgroundHigh-pressure (HP) treatment is considered a food safety process that can stabilize meat by inactivating microorganisms. However, HP treatment can induce modifications of components, such as lipids and proteins, and favor their oxidation by promoting the formation of radicals. Oxidation is one of the most important factors in the non-microbial degradation of meat. Lipid oxidation has been extensively investigated in meat because the products of the reaction can readily react with proteins, leading to organoleptic modifications and the loss of nutritional value. In contrast, the interest in protein oxidation is more recent.Scope and approachThe objective of this work is to review the literature on the effect of HP treatment on lipid and protein oxidation in comparison with that of classical meat processing to identify a possible link between protein and lipid oxidation and the underlying mechanisms.Key findings and conclusionsMicroorganisms were efficiently inactivated after treatment at pressures above 400 MPa. This pressure seems to be critical for the initiation of lipid oxidation, which was probably related to the no hemic iron in the meat, rather than the hemic compounds. Lipid and protein oxidation are closely related, as shown by the observed pattern of each reaction, which depends on the type of meat, the treatment used, and the methods used to assess the reactions. As it was observed to be for cooked meat products, the impact of HP could be greater for meat products that underwent cold storage. Better control of the quality of meat products subjected to HP treatments requires that the entire process, extending from the raw product to its conservation and consumption, be considered.     

9274份肉及肉制品食源性致病菌监测结果分析

目的 了解吉林省9274份肉及肉制品食源性致病菌污染情况,为防控食源性疾病提供科学依据。方法从吉林省9个地市级行政区采集市售6类肉及肉制品样品共9274份,包括生畜肉、生禽肉、熟肉制品、调理肉制品、冷冻肉糜制品和动物血液及制品。按照国家标准方法检测10种食源性致病菌。结果 全部9274份样本食源性致病菌总阳性检出率为3.9%(366/9274)。检出率最高为调理肉制品 (13.0%,63/483),其次是生禽肉(5.6%,107/1900)和生畜肉(5.0%,71/1428)。检出的主要致病菌为单核细胞增生李斯特菌、金黄色葡萄球菌和沙门菌。生禽肉中弯曲菌检出率(7.5%,31/411)和产气荚膜梭菌检出率(3.9%,7/180)均高于沙门菌检出率(3.5%,8/231)。生禽肉、生畜肉中未检出小肠结肠炎耶尔森菌。动物血液及制品未检出单细胞增生李斯特菌、弯曲菌和小肠结肠炎耶尔森菌。冷冻肉糜制品未检出沙门菌。熟肉制品未检出大肠埃希氏菌O157、志贺菌和蜡样芽胞杆菌。熟肉制品各年度检出率范围为1.3%-4.4%。结论 吉林省市售的肉及肉制品较长时间受到不同程度的食源性致病菌污染,存在食源性疾病发生的风险。     

Detection of soya proteins in heated meat products by "blotting" and "dot blot"

Soy proteins (isolates, concentrates and texturates) as well as meat products containing soya isolate were analysed by SDS-electrophoresis. The separated proteins were blotted on nitrocellulose and stained with a selective immunoperoxidase system with the following sequence: primary (anti-soya) serum, goat anti-rabbit IgG serum and peroxidase-antiperoxidase complex (rabbit allotype). By developing the blot with a peroxidase substrate the antigenic soya fractions were visualised while the meat proteins did not stain. All major (reduced) soya fractions alpha, alpha', beta conglycinin, the acid and basic subunits of glycinin as well as some minor fractions became visible with a commercially available anti-soya serum as primary antiserum. The pattern thus obtained provides a high evidence for the presence of soya protein in meat products. Detection level is about 0.02% of soya protein. During a 24-h incubation at room temp. (before heat processing) of a meat product containing soya product and raw liver a remarkable loss of antigenic material was observed.     

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.     

Proteomic approach for the detection of chicken mechanically recovered meat

Mechanically recovered meat is cheaper than raw meat and thus has been incorporated into many meat-derived products. EU regulations exclude mechanically recovered meat from the definition of meat; as a consequence analytical procedures are needed to differentiate it from hand-deboned meat. The present pilot study has utilized a proteomic approach to find potential markers for the detection of chicken mechanically recovered meat. Intact proteins were extracted from raw meat and then analyzed with OFF-GEL electrophoresis followed by SDS-PAGE and identification of potential markers by nano-LC-MS/MS. It was shown that it is possible to extract, separate and identify key proteins from processed meat material. Potential chicken mechanically recovered meat markers--hemoglobin subunits and those similar to myosin-binding protein C were also identified.     

Detection of chicken and turkey meat in meat mixtures by using real-time PCR assays

In this study, TaqMan-based real-time Polymerase Chain Reaction (PCR) techniques were developed for the detection of chicken and turkey meat in raw and heat-treated meat mixtures. Primers and TaqMan probe sets were designed to amplify 86 bp and 136 bp fragments for the chicken and turkey species, respectively, on the mitochondrial NADH dehydrogenase subunit 2 gene. In the results, it was possible to detect each species at the level of 0.1 pg template DNA with the TaqMan probe technique without any cross-reactivity with nontarget species (bovine, ovine, donkey, pork, and horse) while the detection level was 1 pg template DNA using conventional PCR. The TaqMan probe assays used in this study allowed the detection of as little as 0.001% level of both species in the experimental meat mixtures, prepared by mixing chicken and turkey meat with beef at different levels (0.001% to 10%). In conclusion, TaqMan probe assays developed in this research are promising tools in the specific identification and sensitive quantification of meat species even in the case of heat-treated meat products, and suitable for a rapid, automated, and routine analysis.     

肉及肉制品中磷酸盐含量分析

目的探究生鲜肉中磷酸盐本底含量。方法以不同品种肉及肉制品为研究对象,采用国家标准食品中磷酸盐的测定斱法对其迚行磷酸盐含量测定,比较分析肉及肉制品不同品种间磷酸盐含量差别。结果肉制品中磷酸盐含量在0.46～10.32g/kg,超限量比率为26.67%,生鲜肉中磷酸盐含量在1.37～6.65g/kg,参考GB 2760-2014肉制品中磷酸盐限量5.0 g/kg计算,超限量比率为34.00%。结论生鲜肉中磷酸盐本身含量较高,对熟肉制品的磷酸盐含量会存在一定影响,继而导致熟肉制品的超限量比率较高。     

Analysis of commercial soya additives in meat products

The quantitative aspects of the analysis of commercial soya additives in meat products have been investigated using a polyacrylamide gel electrophoresis technique. Extraction of the proteins from the food products was carried out either in 8 M -urea and 1%, 2-mercaptoethanol at 18 to 20 °C for 16 h (Method 1), or 10 M -urea and 4%, 2-mercaptoethanol at 100 °C for 30 min (Method 2). The proteins in the extracts were separated by electrophoresis on gels containing 6% polyacrylamide and either 6 M -urea (Method 1) or 8 M -urea (Method 2) using a 0.06 M -Tris-glycine buffer, pH 8.6. Densitograms of the stained protein bands were used for the quantitative estimation of the soya proteins. Both methods were found to give quantitative results for the analysis of soya protein additives in beefburgers and sausages. Method 1 was also found to give quantitative results for meat pie fillings and canned meat loaf products, which had been autoclaved at 115 °C for 40 min.     

Proximate composition and mineral contents of the blue crab (Callinectes sapidus) breast meat, claw meat and hepatopancreas

The objective of this study was to determine the nutritional composition of the breast, claw meat and hepatopancreas of the blue crab (Callinectes sapidus). Samples were subjected to proximate (protein, fat, ash and moisture) and calcium, magnesium, phosphorus, potassium and sodium (Ca, Mg, P, K and Na) analyses. Protein, fat, ash and moisture of the breast, claw meat and hepatopancreas of the blue crab averaged 19.05, 0.59, 2.10 and 76.85 g/100 g, respectively. The results have revealed that this species is a rich source of protein, Ca, Mg, P and Na. Claw meat had higher protein concentrations (19.55 g/100 g) than both breast meat and hepatopancreas (18.81 g/100 g). Na was predominant element among minerals analysed. There were significant differences between Ca, Mg, P, K and Na contents of claw, breast meat and hepatopancreas of the blue crab. Information on the nutrient composition is needed to facilitate the processing, utilisation and marketing of blue crab products. Blue crab could balance human nutrition and could be used as an alternative dietary supplement of proteins and mineral matter.     

Analysis of soyabean proteins in meat products: a review

The use of soyabean proteins as meat extenders has spread significantly due to the interesting nutritional and functional properties that are present in soyabean proteins. Together with these, health and economical reasons are the major causes for the addition of soyabean proteins to meat products. Nevertheless, despite the good properties associated to soyabean proteins, there are many countries in which the addition of these proteins is forbidden or in which the addition of soyabean proteins is allowed up to a certain extent. Thus, the need of analytical methods enabling the detection of added soyabean proteins in meat products is obvious. Microscopic, electrophoretic, immunologic, and chromatographic methods are the most widely used for this purpose. However, the detection of soyabean proteins in meat products presents difficulties related to the composition (meat species, meat quality, soyabean protein source, presence of other non-meat proteins, etc.) and the processing of the meat products, and, although these analytical methods have tried to overcome all these difficulties, there is still not a method enabling quantitative assessment of soyabean proteins in all kinds of meat products.     

2015~2018年玉溪市售肉及肉制品细菌性污染状况调查

目的 调查2015~2018年玉溪市售肉及肉制品的细菌性污染状况。方法 采用国家标准方法对161份2015~2018年玉溪市售肉及肉制品进行细菌总数、大肠埃希氏菌、致泻大肠埃希氏菌、金黄色葡萄球菌、沙门氏菌、单核细胞增生李斯特氏菌、空肠弯曲菌、小肠结肠炎耶尔森氏菌及产气荚膜梭菌检测分析。结果 本次调查的161件肉及肉制品中, 单核细胞增生李斯特氏菌、沙门氏菌、金黄色葡萄球菌和空肠弯曲菌的总检出率依次为2.91%、9.68%、7.14%和5.22%; 冷却肉类生肉制品的致病菌检出率最高(37.04%), 与其他肉类制品的检出率间有显著性差异(P<0.05); 同一细菌性污染物指标, 在不同季度的检出率差异均无统计学意义(P>0.05); 食源性致病菌中金黄色葡萄球菌超标率最高(28.57%); 城市肉类样品检出率略高于农村; 致病菌在流通环节的总检出率高于餐饮服务环节, 其差异均无统计学意义(P>0.05); 研究中的6种动物性肉类样品的细菌性污染状况不一, 其检出率间存在显著性差异(P＜0.05)。结论 2015~2018年玉溪市售肉类制品中细菌性污染较为严重, 应进一步加强相关产品的全过程监管。     

Investigation of methods to detect mechanically recovered meat in meat products - IV: Immunology

The possibility of using immunological techniques as a method for the detection of mechanically recovered chicken meat in meat products has been investigated in this preliminary study. Antibodies were raised against a low molecular weight fraction (≤ 30 kDa) of chicken bone marrow proteins and an enzyme-linked immunosorbent assay (ELISA) developed. The system was used to test for the presence of mechanically recovered meat (MRM) in a range of product types, from raw chicken meat through to heat processed samples. The results show that it is possible to raise antibodies to chicken bone marrow proteins which show a strong reactivity with chicken and turkey MRM but show little reaction with extracts of MRM and hand deboned meat of other common meat species. However, blood, skin and soya all affected the accuracy of the ELISA.

This study has demonstrated the potential for the use of an immunological procedure as a rapid test for MRM. The selectivity of the antiserum would, however, have to be increased before this procedure could be considered as a suitable technique for the detection of MRM in meat products.     

Sensory acceptability of raw and extruded bovine rumen protein in processed meat products

The use of bovine rumen protein (raw and extruded) as a replacement for extruded soy protein concentrate in three meat products (pork sausage, chicken hamburger, and kibbe) was investigated. Similarity between rumen and soy protein meat products was assessed using triangle tests and sensory acceptability evaluated by consumer panelists using a nine-point hedonic scale. The addition of raw rumen protein was detected in all meat product types tested, while extruded rumen protein was only detected in kibbe. The addition of raw rumen protein decreased the acceptability of pork sausage aroma and flavor, but improved kibbe appearance, texture and overall acceptability. The addition of extruded rumen protein reduced the acceptability of chicken hamburger texture, but improved pork sausage flavor. Replacement of soy protein by bovine rumen protein is feasible based upon sensory results, but depended upon its form and the type of meat product to which it was added.     

Stability of haem pigments in model systems and cooked meat

The stability of sheep haemoglobin and myoglobin in aqueous solution at 80, 100 and 121°C for 1 h was measured by subjecting portions of the heated solutions to electrospray mass spectrometry (ESMS). ESMS dissociates haem proteins into the globin chains and the haem moiety and, with haemoglobin, degradation of the α-(15047·5 Da) and β-(16073·3 Da) chains was observed at all heating temperatures. Under the same conditions, sheep myoglobin dissociated into the globin (16923·2 Da) and haem parts but the globin was stable and few degradation products were observed in the ESMS trace (mass range 4-20 kDa) even after 1 h at 121°C. There did seem to be limited breakdown of the globin due to loss of 170 Da. From the amino acid sequence, it is postulated that this is due to loss of GLY-LEU from the N-terminus. Methods for extracting myoglobin from raw and cooked meat were then investigated. Water was adequate for myoglobin extraction from raw meat but urea solution was required for adequate extraction of cooked meat samples. Sheep meat was heated at 80, 100 and 121°C in sealed cans, extracted and the mass profile in the range 4-20 kDa measured. Myoglobin was the major peak when samples were heated for 10, 20, 30 and 40 min. After that time, other peaks appeared although the myoglobin globin chain was still apparent. The results are discussed in relation to using myoglobin as a marker for meat speciation.     

Detection of roe deer,red deer,and hare meat in raw materials and processed products available in Poland

To allow detection of meat from the most popular game species in Poland, we developed a PCR-based method for identification of roe deer (Capreolus capreolus), red deer (Cervus elaphus), and hare (Lepus europaeus). The designed primers were based on the noncoding, mitochondrial D-loop region. Amplicon sizes ranged from 116 to 255 bp. The primers exhibited no cross-reactivity with the DNA from common slaughter and other game species. The detection limit of the assay was established to be below 0.001 % in raw red deer (C. elaphus) and hare (L. europaeus) meat, and below 0.01 % in raw roe deer (C. capreolus) meat, whereas <0.5 % of hare and red deer meat in processed samples could be detected. The PCR-based assay was used for authentication of 17 samples of raw game meat and 32 samples of game meat-containing products available in Polish markets. Analysis of all tested raw meat and processed products revealed the presence of DNA of investigated species in concordance with producers’ declarations.