Authentication Of Meat Species Research Articles

Volatile Compounds for Discrimination between Beef, Pork, and Their Admixture Using Solid-Phase-Microextraction-Gas Chromatography-Mass Spectrometry (SPME-GC-MS) and Chemometrics Analysis.

This study addresses the prevalent issue of meat species authentication and adulteration through a chemometrics-based approach, crucial for upholding public health and ensuring a fair marketplace. Volatile compounds were extracted and analyzed using headspace-solid-phase-microextraction-gas chromatography-mass spectrometry. Adulterated meat samples were effectively identified through principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA). Through variable importance in projection scores and a Random Forest test, 11 key compounds, including nonanal, octanal, hexadecanal, benzaldehyde, 1-octanol, hexanoic acid, heptanoic acid, octanoic acid, and 2-acetylpyrrole for beef, and hexanal and 1-octen-3-ol for pork, were robustly identified as biomarkers. These compounds exhibited a discernible trend in adulterated samples based on adulteration ratios, evident in a heatmap. Notably, lipid degradation compounds strongly influenced meat discrimination. PCA and PLS-DA yielded significant sample separation, with the first two components capturing 80% and 72.1% of total variance, respectively. This technique could be a reliable method for detecting meat adulteration in cooked meat.

Halal Concerns on Lard in Food Products and its Detection Methods

Lard refers to a creamy, white, soft substance made from pig fat and used in cooking and baking. Lard not only has the risk of biological complications and health problems associated with regular consumption, but also has other issues such as the restriction on use in the food industry from the perspective of the Muslim religion. There are few literature studies that show several issues related to the adulteration of food products, especially halal meat. This issue must be resolved to avoid confusion for Muslims. Therefore, laboratory analysis on meat species authentication is highly necessary. There are different available methods to detect different species in food samples, including Polymerase Chain Reaction (PCR), Electronic Nose (e-nose), Duplex PCR-Enzyme-linked Oligonucleotide Assay (ELONA), Fourier Transform Infrared (FTIR) Spectroscopy, Differential Scanning Calorimeter (DSC), and Rapid PCR. This article aims to review the application of lard in food, cases related to halal and methods to detect the presence of lard.

Interlaboratory Validation of a DNA Metabarcoding Assay for Mammalian and Poultry Species to Detect Food Adulteration.

Meat species authentication in food is most commonly based on the detection of genetic variations. Official food control laboratories frequently apply single and multiplex real-time polymerase chain reaction (PCR) assays and/or DNA arrays. However, in the near future, DNA metabarcoding, the generation of PCR products for DNA barcodes, followed by massively parallel sequencing by next generation sequencing (NGS) technologies, could be an attractive alternative. DNA metabarcoding is superior to well-established methodologies since it allows simultaneous identification of a wide variety of species not only in individual foodstuffs but even in complex mixtures. We have recently published a DNA metabarcoding assay for the identification and differentiation of 15 mammalian species and six poultry species. With the aim to harmonize analytical methods for food authentication across EU Member States, the DNA metabarcoding assay has been tested in an interlaboratory ring trial including 15 laboratories. Each laboratory analyzed 16 anonymously labelled samples (eight samples, two subsamples each), comprising six DNA extract mixtures, one DNA extract from a model sausage, and one DNA extract from maize (negative control). Evaluation of data on repeatability, reproducibility, robustness, and measurement uncertainty indicated that the DNA metabarcoding method is applicable for meat species authentication in routine analysis.

Design of Mismatch Primers to Identify and Differentiate Closely Related (Sub)Species: Application to the Authentication of Meat Products.

Single-nucleotide polymorphisms (SNPs) are powerful molecular markers for the identification and differentiation of closely related organisms. A variety of methods can be used to determine the allele that is present at a specific locus in the genome, including real-time PCR by using an allele-specific primer. In order to increase the selectivity for the target allele, deliberate mismatch bases at the 3' end of the allele-specific primer may be introduced. This strategy has already been used for the identification and differentiation of microorganisms and plants. We have recently developed real-time PCR assays involving mismatch primers for the identification and differentiation of closely related deer species (red deer, fallow deer, sika deer) or the discrimination of wild boar and domestic pig in game meat products. These methods are applicable to detect meat species adulteration in food products.In this chapter, we offer a protocol for the design of PCR primer/probe systems suitable for meat species authentication in food. We address the retrieval and alignment of sequences, primer design by using a commercial software and the introduction of deliberate mismatch bases. In addition, we describe how the suitability of primer/probe systems can be tested in silico and in practice. We use the design of PCR primer/probe systems for wild boar and domestic pig as example.

Analytical approaches of meat authentication in food

SummaryMeat industry sector is likely exposed to illegal practices which involve replacement of meat ingredients with other animal species. Meat products are commonly modified during processing, and the cases have been rampant in certain part of the world. Meanwhile, individual consumption patterns are affected by religious belief and health reason. For instance, Muslims and Jews are prohibited from consuming pork and allergic reaction to certain meat species affects choices of meat products. Thus, food authentication is vital as it offers protection to consumers from fraudulent act. Numerous authentication techniques have been developed to tackle food fraud issues. Authentication methods are categorised mainly as targeted analysis which provides quantitative outcome and non‐targeted analysis which offers qualitative result. In the present review, both targeted and non‐targeted analyses for meat species authentication are discussed based on previous research conducted using these methods and how they serve the purpose for authentication.

Authentication of meat species and net muscle proteins: updating of an old concept

The aim of the study was to develop an efficient method for assessment of meat origin and determination of net muscle protein (NMP; based on amino acids and β-alanylhistidine dipeptides) in meat products using the ultra-high performance liquid chromatography coupled with mass spectrometry. As an important result, a database of ratios of particular amino acids to 3-metylhistidine, applicable for muscle protein origin confirmation, was established. Based on the specific ratios of 1-methylhisitidine/3-methylhistidine, revealing of undeclared addition of 2% of chicken meat to the pork was enabled. Similar outcome was achieved by considering the ratios of β-alanylhistidine dipeptides. In the case of chicken-pork and pork-beef admixtures, as low additions as 0.5 and 2% of chicken or pork adulterant could be recognized. The ratio of 4-hydroxyproline/3-methylhisitidine was shown to be diagnostic for detection undeclared addition of 0.5 and 1.5% of connective tissues into the pork and chicken meat, respectively. On the basis of 3-methylhisitidine concentration, the conversion factor F = 292 ± 4 was calculated for quantification of NMP (%) content.

Differentiation between wild boar and domestic pig in food by targeting two gene loci by real-time PCR

Studies indicate that many meat products are not authentic, most frequently because the meat species differ from those given on the food labels. At present, DNA based methods play the most important role in meat species authentication. Discrimination of wild boar and domestic pig meat in food is challenging because it is differentiation on the subspecies level. We developed and validated two singleplex real-time PCR assays targeting SNP rs81416363 on chromosome 9 and a duplex real-time PCR assay targeting SNP g.299084751 C > T in the NR6A1 gene located on chromosome 1. The singleplex real-time PCR assays led to some ambiguous results for Mangalica and Krškopolje pig breeds and wild boar individuals from Germany, the duplex real-time PCR assay particularly for the Turopolje pig breed. We demonstrate that the probability of misclassification can be substantially reduced if the results of both the singleplex real-time PCR assays and the duplex real-time PCR assay are taken into consideration. 86 (91.5%) of a total of 94 individuals, comprising 64 domestic pigs (14 different breeds and 6 cross-breeds) and 30 wild boars (from Austria, Germany, Romania, USA and Estonia), were classified correctly.

Corrigendum to “Comparison of Two Multiplex PCR Systems for Meat Species Authentication” [Journal of Food Quality and Hazards Control 6 (2019) 8-15

The article's abstract is no available.

Comparison of Two Multiplex PCR Systems for Meat Species Authentication

Background: Meat species adulteration has become a problem of concern. This study aimed to compare two previously published multiplex Polymerase Chain Reaction (PCR) methods for meat species authentication. Methods: The primers used in the first multiplex PCR involved species-specific reverse primer for sheep, goat, cattle, pig, and donkey with universal forward primer. In the second multiplex PCR, the primers included species-specific forward and reverse primer for pork, lamb, ostrich, horse, and cow. The extracted DNA was then amplified with species-specific primers and with mix primers separately in the respective multiplex PCR. Results: The first multiplex PCR was accompanied with cross reactivity, whereas the second multiplex PCR was specific as expected for pork, lamb, ostrich, horse, and cow. The first set of multiplex PCR showed not always amplification of all species-specific DNAs with a mixture of DNA from mentioned animals. Regarding the second set of primers, the extracted DNA of different meat species was amplified with corresponding species primers as simplex PCR resulting in specific amplicons for species DNA prepared from sheep, ostrich, horse, pig, and cattle with the specific PCR products of 119, 155, 253, 100, and 311 bp, respectively. Conclusion: Based on the present investigation, we recommend the multiplex PCR with the second set of primers included species-specific forward and reverse primers for species authentication of five meat types, including pork, lamb, ostrich, horse, as well as cow.

MRM–MS of marker peptides and their abundance as a tool for authentication of meat species and meat cuts in single-cut meat products

The abundance of protein markers in different types of meat cuts was explored in the context of authentication of raw meat (pork, beef and chicken) and processed meat products. Peptides originating from myoglobin (Mb) and myosin (My) were analyzed using multiple reaction monitoring mass spectrometry (MRM–MS). Analytical protocol was optimized for good repeatability (CV < 10%) and high sensitivity. The MS signal intensity of Mb marker peptides in raw pork depended significantly on the cut type (e.g. ham vs knuckle). Importantly, a similar pattern in the abundance of the marker peptides was found for processed meat products made of different types of pork cuts, despite the food processing applied. This suggests the protocol can be used for authentication of raw pork cuts and processed products made of different cuts of pork. More uniform contents of Mb markers were found in raw beef cuts, and for My markers in raw chicken cuts.

Cross priming amplification with nucleic acid test strip analysis of mutton in meat mixtures

A simple, sensitive, accurate and affordable rapid detection of meat species authentication is urgently needed in food industry. In this study, a cross priming amplification (CPA) combining nucleic acid test strip (CPA-Strip) assay for rapid detection of mutton from meat mixture were developed and its feasibility was investigated. In an isothermal CPA system, cytochrome b (cytb) gene as target was amplified at 63°C for 60min. The nucleic acid strip was able to show the corresponding test line in the presence of target gens in 5min. Non-targeting gene interference was not evident. The CPA-Strip has been applied for the detection of 0.1–100% mutton in a thermal treated meat mixtures with a detection limit of a detect limit of 1%. CPA-Strip assay would be a promising simple, rapid and sensitive method for identification of target species in raw and processed meat mixtures.

OFFGEL electrophoresis and tandem mass spectrometry approach compared with DNA-based PCR method for authentication of meat species from raw and cooked ground meat mixtures containing cattle meat, water buffalo meat and sheep meat

The present study compared the accuracy of an OFFGEL electrophoresis and tandem mass spectrometry-based proteomic approach with a DNA-based method for meat species identification from raw and cooked ground meat mixes containing cattle, water buffalo and sheep meat. The proteomic approach involved the separation of myofibrillar proteins using OFFGEL electrophoresis, SDS-PAGE and protein identification by MALDI-TOF MS. Species-specific peptides derived from myosin light chain-1 and 2 were identified for authenticating buffalo meat spiked at a minimum 0.5% level in sheep meat with high confidence. Relative quantification of buffalo meat mixed with sheep meat was done by quantitative label-free mass spectrometry using UPLC-QTOF and PLGS search engine to substantiate the confidence level of the data. In the DNA-based method, PCR amplification of mitochondrial D loop gene using species specific primers found 226bp and 126bp product amplicons for buffalo and cattle meat, respectively. The method was efficient in detecting a minimum of 0.5% and 1.0% when buffalo meat was spiked with cattle meat in raw and cooked meat mixes.

Multiplex PCR in Species Authentication: Probability and Prospects—A Review

Food forgery is one of the most articulated socio-economic concerns which contributed to increase people’s awareness on what they eat and how and where it is produced. Consumers are anxious about the consequences of food falsification on their choices, religious rituals, health, and hard-earned fortunes. The recent scandals of horse and rat meats in Europe and China have given us a brainstorming apprehension on the detection, differentiation, and identification of meat products. To restore consumers’ trust and protect wildlife in natural habitats, researchers and policy-making and policy-implementing authorities have massively monitored all steps in the production of foods and food materials. Analytical approaches based on lipids, proteins, and DNA have been proposed for the authentication of meat species under pure and complex matrices. However, protein and lipid-based methods are less effective since the target biomarkers could be modified throughout the processing treatments. On the other hand, DNA-based species identification schemes have gained wider acceptance and reliability because of the superior stability and universality of DNA in all tissues and cells. We systematically presented here major species detection schemes with special emphasis on multiplex polymerase chain reaction (PCR) of both end-point and real-time platforms. We believe this short but comprehensive review would serve as a reference guide for the developers and users of multiplex PCR and others DNA-based techniques.

Authentication of species in meat products by genetic techniques

In the present work, a method for the authentication of meat products was developed, using Polymerase Chain Reaction (PCR) followed by Forensically Informative Nucleotide Sequencing (FINS). This study describes the use of sequencing of the cytochrome b gene of the mtDNA in a wide variety of species to diagnose adulteration of meat through the substitution from one species to another that have less commercial value. The main importance of this work is to deal with a wide variety of species that have not previously been analyzed. This methodology strategy allows the authentication of meat species in all kind of products, fresh, or precooked products, the more usual format for marketing in that species. This method shows a specificity of 100%. The developed methodology was validated and finally applied to 20 commercial samples including some that had been subjected to intensive thermal treatment. In 15% of the products analyzed, the name of the species displayed in the label was not in agreement with the identified species. The main novelty of this work lies in the fact that it allows the identification of a large number of meat species not analyzed so far in previous works. In this work are included meat species, which can be easily found in our markets, and a wide variety of others whose consumption is common in other parts of the world. Therefore, this technique can be used as a routine method to avoid the mislabeling in the marketing of these products and to assess their correct traceability.

Application of Species-Specific Polymerase Chain Reaction and Cytocrome b Gene for Different Meat Species Authentication

Application of Species-Specific Polymerase Chain Reaction and Cytocrome b Gene for Different Meat Species Authentication