الفهرس 
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Abstract
Meat fraud of costlier meat with cheaper meat is one of the most common practices of adulteration prevalent in Egyptian meat industry without any consideration of economic, religious, fair trade, legal or public health concerns. Therefore, meat authentication needs specialized attention in the management of food safety and quality systems. Generally, the wide spreading of meat adulteration in Egypt may be due to the absence of oblivious routine strategy performed by Governmental veterinary directorates and food safety organization for authentication of meat species in adulterated meat
Therefore, The current study was conducted through experimental adulteration of fresh beef with mutton, pork donkey and dog meats. The fresh meat and fat samples of five animal species were collected randomly, beef, mutton and pork meats from Central Cairo abattoir, donkey meats from The Giza Zoo, while dog meats from Pet’s veterinary clinic in Cairo in a period of 2021. All samples were collected in a clean disposable polyethylene bag and stored at -20°C for analysis.
In the presnt study there are three experimental parts:
Objective 1:
Multiplex PCR assay for simultaneous detection of beef meat fraud with different species.
This work was designed to investigate different concentrations of beef adulteration with other meat types (donkey, mutton, dog, and pork) using multiplex PCR. Fresh meat samples were minced experimentally to make meat mixture for mimicking adulteration. Three meat mixtures (beef – donkey, beef – dog and beef – pork). The first mixture containing beef adulterated with different concentration (0.5 % 5%, 10%, 30%, and 50%) of donkey meat. The second mixture containing beef meat adulterated with different concentration (0.5% 5%, 10%, 30%, and 50%) of dog meat. The third mixture containing beef meat adulterated with different concentration (0.5,% 5%, 10%, 30%, and 50%) of pork meat. Each mixture was prepared to final weight of 50gm. For authentication of meat species using Multiplex PCR technique.
The obtained results cleared that, the multiplex PCR assay for five meat species was run effectively, clarifying five unique PCR fragments. These PCR sections compared the particular sizes expected for the five designated species. The results showed successful amplification of the target cyt b gene sequences with the expected amplicon sizes (271pb) for beef, (274pb) for mutton, and (808pb) for dog meat. Amplification of the target mt DNA, and 12S rRNA-tRNA Val gene sequences with the expected amplicon sizes (359pb) for donkey, and (290 bp) for pork. The developed multiplex PCR assay was sensitive enough to detect all adulterated meat under mixed matrices even in very low concenteration 0.5% (w/w). It was concluded that the multiplex PCR could greatly minimize the cost for detection of meat adulteration.
Objective 2:
Detection of Mutton Meat Fraud with Beef Meat as a genetically related species using Multiplex PCR and GC/MS/MS.
Mutton meat is delicious and high-priced meat. Its tallow is a flavoring precursor to being authenticated by using different meat species. Mutton authenticity by beef that is genetically related to each other makes it difficult to detect fraud of products, mainly if the substitution is applied with fat, not only meat. So, accurate methods for detection must be applied to evaluate meat fraud based not only on protein but also on fat substitution.
This work planned to evaluate the mutton substitution by meat and fat of beef using multiplex PCR and Gas chromatography-Tandem Mass Spectrometry (GC/MS/MS).
Fresh meat and fat specimens (mutton and beef) were collected from Al-Basateen Central Abattoir of Cairo, Egypt (50 gm. each). Meat samples were minced and prepared as pure beef (100%), pure mutton meat (100%), mutton authenticated with beef 0.5%, mutton authenticated with beef 5%, mutton authenticated with beef 30%, and mutton authenticated with beef 50% to be examined by multiplex PCR, while for GC/MS/MS, fatty acid profile evaluation fat samples were prepared as pure mutton fat (100%), pure beef fat (100%), and a mixture of beef and mutton fats (50%).
The achieved results revealed that: Regarding The Multiplex PCR detects cytochrome b in mutton meat adulterated with different concentrations of beef, even with very low concenteration (0.5%). PCR products amplified at 274 bp for mutton and 271 bp for beef.
Concerning the Fatty acid profile of pure mutton (tallow) samples and mutton authenticated with beef by GC/MS/MS. It was found that it contained 39.48 mg/100 gm. of total saturated fatty acids (TSFA), 60.48 mg/100 gm. of total unsaturated fatty acids (TUSFA), and 3.4 mg/100 gm. of trans-fatty acids (TFA), while after being authenticated with beef, these results changed to 49.58, 49.57, and 6.37 mg/100gm for TSFA, TUSFA, and TFA, respectively. The current study concluded that, The Multiplex PCR and GC/MS/MS evidenced to be accurate and applicable for the recognition of mutton meat authentication.
Objective 3:
Multiplex PCR for detection of cattle, pork and sheep meat authenticity.
Aim of this study was to verify the multiplex PCR assay to identify meat authenticity with three different types of meat species (two of them are genetically related) at different concentrations.
Fresh meat samples of different animal species beef (B), pork (P), and mutton (Sh) were collected (50 gm. Each) from Al-Basateen Abattoir, Cairo, Egypt. All samples were collected in a clean disposable polyethylene bag and stored at -20 °C for analysis.
Meat samples were prepared and minced separately, then mixed experimentally by moulinex mixer with different concentrations using digital scale as the following (five) groups: group (1) B 99%: P 0.5%: Sh 0.5%; group (2) B 90%: P 5%: Sh 5%; group (3) B 80%: P 10%: Sh 10%; group (4) B 50%: P 25%: Sh 25%. group (5) pure samples with 100% concentration for each species as a positive control and also negative control were examined. All mixtures were kept at −20 °C until used.
Multiplex PCR based on species specific genes was used. Results revealed that all three species under study (cattle, pig, and sheep) were positive by multiplex PCR. Moreover, different concentrations of experimentally authenticated samples were detected even at low concentration (0.5% for adulterated species). Multiplex PCR using mt.DNA target gene had differentiated between different meat species even they are genetically related as beef and mutton. So, multiplex PCR is a reliable, accurate and sensitive technique for meat authenticity evaluation even for closely related species.
It was concluded that:
The developed Multiplex PCR assay was sensitive enough to detect all experimentally adulterated fresh meat under mixed matrices even in very low concenteration 0.5% (w/w).
The Multiplex PCR could greatly minimize the cost, time and labours for detection of meat adulteration. The Multiplex PCR using mt.DNA gene is a reliable, accurate and sensitive technique for meat authenticity evaluation even for closely related species. The Fatty acid profile using Gas chromatography-Tandem Mass Spectrometry (GC/MS/MS) evidenced to be accurate and applicable methods for the recognition of mutton substitution by meat and fat of beef.