PROCEDURES FOR THE IDENTIFICATION AND DETECTION OF ADULTERATION OF FISH AND MEAT PRODUCTS

The addition or exchange of cheaper fish species instead of more expensive fish species is a known form of fraud in the food industry. This can take place accidentally due to the lack of expertise or act as a fraud. The interest in detecting animal species in meat products is based on religious demands (halal and kosher) as well as on product adulterations. Authentication of fish and meat products is critical in the food industry. Meat and fish adulteration, mainly for economic pursuit, is widespread and leads to serious public health risks, religious violations, and moral loss. Economically motivated adulteration of food is estimated to create damage of around € 8 to 12 billion per year. Rapid, effective, accurate, and reliable detection technologies are keys to effectively supervising meat and fish adulteration. Various analytical methods often based on protein or DNA measurements are utilized to identify fish and meat species. Although many strategies have been adopted to assure the authenticity of fish and meat and meat a fish products, such as the protected designation of origin, protected geographical indication, certificate of specific characteristics, and so on, the coverage is too small, and it is unrealistic to certify all meat products for protection from adulteration. Therefore, effective supervision is very important for ensuring the suitable development of the meat industry, and rapid, effective, accurate, and reliable detection technologies are fundamental technical support for this goal. Recently, several methods, including DNA analysis, protein analysis, and fat-based analysis, have been effectively employed for the identification of meat and fish species.


INTRODUCTION
At present, there is no harmonized definition of food fraud in the European Union (EU) 2017. However, it is commonly accepted that the term 'food fraud' covers any violation of food law that is an intentional and deceptive misrepresentation of food for financial gain (van Ruth et al., 2017; EC, 2019. Food fraud is about "any suspected intentional action by businesses or individuals to deceive purchasers and gain undue advantage therefrom. Spink and Moyer (2011) have elaborated on this definition and describe seven types of food fraud: adulteration, tampering, over-run, theft, diversion, simulation, and counterfeit. These intentional infringements to the EU agri-food chain legislation may hinder the proper functioning of the internal market and may also constitute a risk to humans. However, existing databases that monitor food fraud Such as the Rapid Alert System for Food and Feed (RASFF) and HorizonScan have their categorizations (Bouzembrak et al., 2018). RASFF has six categorizes for fraud (Improper, fraudulent, missing or absent health certificates; illegal importation; tampering; improper, expired, fraudulent or missing common entry documents or import declarations; expiration date; mislabeling) as does HorizonScan (adulteration/substitution, fraudulent health certificate/documentation, produced without an inspection, unapproved premises, expiry date changes).
Four key operative criteria are referred to for distinguishing whether a case should be considered as fraud or as non-compliance: if a case matches all four criteria, then it could be considered a suspicion of fraud: violation of EU rules, deception of customers, undue advantage and intention. Meat and fish are food categories that are highly vulnerable to adulteration. Although there are various national and international laws for supervising the quality and safety of fish, meat, and meat and fish products, meat adulteration is still widespread. Most meat adulteration is economically motivated, such as the lowcost addition of duck meat and fish to mutton (Wang et al., 2019a), which causes consumers to suffer economic losses. Meat and fish adulteration may lead to serious public health risks, such as exposure to toxins, pathogens, or allergens in these products (

MEAT AND FISH ADULTERATIONS
The demand for meat and fish products is high and as a result, meat is one of the most highly-priced food commodities; therefore, a prime target for food fraud (Cawthorn et al., 2013). The examples of adulteration are presented in Table 1.  Ram and Baidoun, 1996) Uncovering of adulterated meat products is important for several reasons. Allergic individuals and those who hold religious beliefs that specify allowable intake of certain species have a special interest in proper labeling. Proper labeling is also important to help fair-trade. The need for analytical species-specific methods is clearly illustrated by the following examples: Hsieh, Chai and Hwang (2007) found, with the use of immunoassays, meat from undeclared animal species in 15.9% of cases in raw products and 22.9% of cases in cooked products analyzing a total of 902 meat products. In a more recent investigation performed on 100 meat products, also with the use of immunoassays, meat from undeclared species was found in 22.0% of cases, primarily with poultry substituting beef (Ayaz et al., 2006). The provenance of food, especially meat products, is a sensitive topic but there are tools available to support producers in demonstrating compliance with legislators and other authorities. Since the level of awareness about food quality and safety has recently increased, food fraud has become a major global issue. Hence, the identification of meat and fish products adulteration with unfavorable and inappropriate animal species is important from health, economic, and religious points of view (Mousavi et al., 2015). Currently, the protein-based techniques (e.g. electrophoresis, isoelectric focusing, ELISA, and chromatography) have been utilized for meat and fish adulteration. These methods are laborious, expensive, and sophisticated instrumentation

DNA TECHNOLOGIES
As a prerequisite for accurate species quantification, DNA has to comply with minimum requirements about yield, purity, and integrity. Yield is an important parameter since food DNA has to be in a sufficient amount to allow the reliable and repeatable downstream analysis of meat species Although often underestimated, DNA isolation is a crucial step for molecular analysis of food due to its heterogeneity in terms of composition and processing. The presence of chemical inhibitors, proteins, and/or damaged DNA are common situations in meat food analyses. Moreover, the extraction methods themselves can further influence the yield, purity, and integrity of DNA depending on the type of food matrix ( Of the different DNA markers used for fish species identification, mitochondrial DNA (mtDNA) possesses several advantages over nuclear DNA for studies of speciation in fish products. It is relatively more abundant in total nucleic acid preparations than nuclear DNA, with the copy number of the mitochondrial genome exceeding that of the nuclear genome several folds (Alberts et al., 1994). Research on fish mitochondrial DNA (mtDNA, mitogenome) has led to substantial advances in the fields of species authentication and population biology (Miya et al., 2001). Mitochondrial DNA tends to be maternally inherited so that individuals normally possess only one allele and thus sequence ambiguities from heterozygous genotypes are generally avoided. The relatively high mutation rate compared to nuclear genes has tended to result in the accumulation of enough point mutations to allow the discrimination of even closely related species. It should however be noted that mitochondrial DNA also exhibits a degree of intraspecific variability and so care has to be taken when studying differences between organisms based on single base polymorphisms (Chow and Inogue, 1993). However, the use of nuclear markers may be useful for fish species discrimination because of the existence of introns of different sizes which allow sometimes the amplification of species-specific DNA fragments (Ferguson et al., 1995).
The comparative analysis of the commonly applied meat adulteration DNA techniques is present in Table 2.

Polymerase chain reaction-restriction fragment length polymorphism
Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is a technique for variation analysis by using restriction endonuclease digestion to identify specific sequences of conserved regions of DNA amplified by using PCR. PCR-RFLP is a sensitive, accurate, and versatile method for meat authenticity verification LAMP is simple and easy to perform once the appropriate primers are prepared, requiring only four primers, a DNA polymerase, and a regular laboratory water bath or heat block for reaction (Notomi et al., 2000).

PCR
The direct PCR method has the characteristics of high sensitivity, high resolution, and specificity, so it is commonly used in meat authenticity and origin traceability developed species-specific PCR methods of the mitochondrial D-loop to detect pork adulteration in commercial beef and/or chicken products, and the methods were able to detect as little as 1% pork in heat-treated pork-beef-chicken mixtures. However, the conventional single-species PCR method could only detect one specific species of adulterant in products (Kumar et al., 2015), which is of low commercial value because there might be many other adulterants in the products. This method provides very accurate and reproducible quantitation of gene copies. Unlike other quantitative PCR methods, realtime PCR does not require post-PCR sample handling, preventing potential PCR product carry-over contamination and resulting in much faster and higher throughput assays (Heid et al., 1996). Multiplex PCR assays with multiple species-specific primers have been greatly developed since they offer multiple target detection in a single reaction (

PCR-RFLP
In PCR-RFLP, a conserved region of the DNA sequence is amplified using PCR, followed by digestion with restriction enzymes, which can reveal genetic variation between species (Partis et al., 2000). In a search for fast and simple genetic techniques, PCR-RFLP has gained acceptance among fish species identification methods, since it is much easier to perform and less costly than conventional DNA sequencing and nucleotide sequence analysis (Meyer et al., 1995). This method has been used for the discrimination of mackerel species

Real-time PCR
Real-time PCR is performed by monitoring the fluorescence signal, which allows for deducing the initial quantity of the target genes without additional steps (Xu et al., 2018). The real-time PCR method has a very large dynamic range of starting target molecule determination (at least five orders of magnitude). Real-time quantitative PCR is extremely accurate and less labor-intensive than current quantitative PCR methods (Heid et al., 1996). SYBR Green and TaqMan technology are commonly used in quantitative methods (the working principle is outlined in the review of Kumar et al., 2015). SYBR Green technology can only detect a single species, but the detection cost was lower than that of TaqMan technology. Li et al. (2019) developed a novel reference primer-based mitochondrial 12S rRNA for the quantitative determination of goat meat adulterated with pork by using real-time PCR. The method showed high specificity and sensitivity for goat meat mixed with pork within the 10% to 100% mixture-level range. TaqMan technology has higher specificity and sensitivity than those of SYBR Green technology. More importantly, it can be used for multispecies detection (Xu et al., 2018).

Droplet digital PCR
Droplet digital PCR (ddPCR) is a new method for nucleic acid detection and quantification. The principle of this method is to perform independent PCR on a large number of small reactors in the form of droplets that contain or do not contain one copy of the target molecule template in each reactor, to achieve "single-molecule template PCR amplification" (

RAPD
The RAPD technique involves PCR amplification with a single primer to generate a collection of DNA fragments or fingerprint, which is expected to be consistent for the same primer, DNA, and conditions used ( Partis and Wells, 1996). The main advantages of RAPD are (i) it does not require previous knowledge of DNA sequences of the species under study and (ii) it targets many sequences in the DNA of the sample, producing DNA patterns that allow comparison of many loci simultaneously. However, RAPD analysis presents some disadvantages: (i) it may not be practical to identify the species of origin in products containing mixtures of species (Martínez and Malmheden Yman, 1998) and (ii) it does not seem to be adequate for analysis of severely degraded material, as in autoclaved samples (Martínez and Malmheden Yman, 1998).

DNA barcoding and next-generation sequencing
The above reviewed DNA-based technologies are mainly targeted detection methods, but in meat adulteration detections, many unknown meat species should be identified (Cottenet et al., 2020). Following this need, an untargeted detection technology named DNA barcoding had been developed (Cavin et al., Hebert et al., 2003). DNA barcoding is particularly successful when applied to seafood because of several reasons: i) in comparison to other animal sources (e.g. cattle, sheep, goat, horse) the number of species is higher, so the effectiveness of the technique is enhanced; ii) classical identification approaches are not useful in many cases (following industrial processing, morphological characteristics are often lost and classical identification processes are no longer effective) and iii) identification can often proceed beyond species level, allowing the identification of local varieties and hence the origin of the product. Through PCR amplification and sequencing of specific gene fragments, and then search it in the Barcode of Life Data (BOLD) system and the U.S. National Center for Biotechnology Information database, the adulterated meat species could be identified (Fiorino  et al., 2018). The early DNA barcoding technology mainly relied on Sanger DNA sequencing for an approximately 650 bp region of COI and the CtyB gene of the animal species (Böhme et al., 2019). DNA Barcoding application can be applied to authenticate labeling and certification labels. This technique has aided several researchers in discovering mislabeled/substitution incidences, for example, Filonzi, et al., (2010) found halibut were substituted with pangasius However, when there are multiple adulterated ingredients in meat products, the traditional Sanger sequencing will generate multiple or overlaying sequencing peaks, resulting in false sequence information. Therefore, a DNA metabarcoding method had been constructed to implement multispecies identification in complex samples using next-generation sequencing (NGS) technology. Furthermore, for processed meat products, DNA can be degraded to small fragments (<200 bp) depending on the treatment (Cavin et al., 2018). Thus, a mini-barcoding method, which focuses on shorter DNA fragments ( successfully applied a commercial NGS Food Authenticity Workflow to identify untargeted meat species, 46 pure and mixture meat species were successfully tested, including some close-related species, such as bison versus beef and red deer versus reindeer. Furthermore, the method was also suitable for processed (grounded, cooked, and canned) samples identification. However, DNA barcoding technology also has some disadvantages, such as expensive sequencing costs, time-, and sample-consuming (Fiorino et al., 2018).

PROTEIN TECHNOLOGIES
Meat adulteration detection by using PCR methods is usually affected by many factors, such as poor trace quantitative analysis, sampling pollution, and DNA degradation in meat processing  , 2017). Moreover, DNA extraction is time-consuming and must be optimized for each particular case to ensure that enough DNA was obtained for the analysis (Song et al., 2017). Protein is the main component of meat. The specific protein composition and three-dimensional structure of specific proteins have certain conservation and specificity between species, which is suitable for meat adulteration detection. Moreover, some protein molecules are tissue-specific and can be used for the identification of less valuable additives, such as connective tissue, blood plasma, or milk preparations (Jiang et al., 2018; Montowska and Spychaj, 2018; Ofori and Hsieh, 2015). The comparative analysis of the commonly applied meat adulteration protein techniques is present in Table 3.

Enzyme-linked immunosorbent assay
EIA/ELISA uses the basic immunology concept of an antigen-binding to its specific antibody, which allows detection of very small quantities of antigens such as proteins, peptides, hormones, or antibodies in a fluid sample. There are two kinds of immunoassay techniques used in meat adulteration detection: enzyme-linked immunosorbent assay (ELISA) and immunosensors. ELISA is the most widely applied immunoassay method of meat adulteration detection (Thienes et al., 2018). However, immune techniques are characterized by their simplicity of sample preparation, absence of the need for complex equipment and qualified personnel, and high productivity of serial testing. As well, for food authentication, electrochemical immunosensors are an alternative detection tool and are highly feasible for on-site usage; therefore, there is only one previously reported immunosensor for meat authentication (Lim and Ahmed, 2016). The principle of immunosensor methods is similar to that of ELISA methods, but the former uses a biosensor to transmit and amplify the optical, electrical, or other signals of the immune response to a detectable signal, so the sensitivity of the method is better than that of ELISA. The immunosensor technique has been widely used in food allergy, pesticide residue, and milk adulteration analyses, among others. However, only a few reports have utilized immunosensing for meat adulteration detection

Protein mass spectrometry analysis
Modern mass spectrometers can accurately measure thousands of compounds in complex mixtures over a given liquid chromatography method, depending on the desired outcome and method duration. This stream of analytical chemistry has wide-ranging applications across food, pharma, environmental, forensics, clinical, and research (Broadbent et al., 2020). Recently, mass spectrometry technologies based on protein and peptide analysis have rapidly evolved and have been increasingly applied for meat species identification.   Since the amino acid sequence of peptides is more stable than DNA during meat processing, they have an incomparable advantage in meat adulteration identification, especially for highly processed meat products and similar meat species (Prandi et al., 2017).