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\title{Studying the Presence of Adultery, Fraudulent Imitation and Food Pathogens within Processed Meat Products (Such as Salami, Sausage, Braised Meat) using DNA Typing and PCR Procedures*}
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\begin{document}

             \author[1]{Harun  Cerit}

             \affil[1]{  Istanbul University}

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\date{\small \em Received: 8 December 2014 Accepted: 31 December 2014 Published: 15 January 2015}

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\begin{abstract}
        


Adulteration of meat with cheaper ambiguous meats of different origin during preparation of meat products is a common practice in many countries. Because meat adulteration and mislabeling are illegal and raise many health, religious, cultural and economic issues. In this study, 500 ready to eat raw meat samples (minced meat, lahmacun ingredients, kebap, stew and meatball samples / 100 samples for each type) were collected from different types of plants that were located in Istanbul. The samples were explored if they had different animal originated DNA residues (pork, chicken, cattle, sheep, horse, donkey, cat, dog, mouse, cockroach and house fly) by PCR procedures. According to the results, total of 52 samples were determined as adulterated and different originated animal DNA samples were found (chicken, horse and sheep DNA residues). It was concluded that to apply total quality management and food security systems are very important to decrease the risk factors for both products and the public health.

\end{abstract}


\keywords{PCR, species identification, ready to eat meat products.}

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\let\tabcellsep& 	 	 		 \par
Özet-Et ve ürünlerinde taklit ve ta??i? uygulamalar? gerek kar amac?n? yükseltmek amac? ile illegal bir biçimde yap?lmakta, gerekse birden fazla et ürünü i?leyen i?letmelerde kaza / yetersiz hijyen ve san istasyon uygulamalar? sonucu meydana gelebilmektedir. Et ve ürünlerinde taklit ve ta??i?ler ekonomik, dini inançlar, sa?l?k, kültürel, tüketiciyi aldatma yönünden önemli sorunlara yol açabilmektedir. Bu çal??mada 500 adet tüketime haz?r halde sat??a sunulmu? olan çi? et örne?i (k?yma, lahmacun iç malzemesi, kebap, köfte ve sulu yemeklerde kullan?lmak üzere haz?rlanm?? etler olmak üzere) ?stanbul'da bulunan farkl? sat?? noktalar?ndan toplan?lm?? ve söz konusu örneklerde 9 adet farkl? hayvana ait (domuz, tavuk, s???r, koyun, at, e?ek, kedi, köpek, fare, hamamböce?i ve ev sine?i olmak üzere) DNA örnekleri PCR prosedürleri kullan?larak ara?t?r?lm??t?r. Elde edilen sonuçlara göre 52 adet örnekte farkl? hayvan türlerine ait (tavuk ,at ve koyun olmak üzere) DNA kal?nt?lar? saptanm??t?r. Sonuç olarak özellikle et ve ürünlerini üreten i?letmelerde toplam kalite yönetimi ve optimal hijyen uygulamalar?n?n kontrollü bir biçimde uygulanmas?n?n taklit ve ta??i? uygulamalar?n?n minimize edilebilece?i sonucuna var?lm??t?r.\par
Author ? ?: This article is an excerpt of the study supported by the Istanbul University Scientific Researches Project UnitwithIssue Number of 33896/2013. e-mail: hcerit@istanbul.edu.tr Anahtar sözcükler: PCR, tür tayini, tüketime haz?r et ürünleri. 
\section[{I. Introduct?on}]{I. Introduct?on}\par
he composition of food is a major concern of consumers today. In the case of adulterated meat product consumption, several factors including economic, food safety (allergy) and moral reasons (religious belief), trigger such apprehensions. Among these concerns, consumers are most sensitive because of religious factors and do not tolerate even trace amounts of adulteration of meat products with forbidden meats like pork. Hygiene and right labeling notified on the label of any food stuff are very important criteria especially for public health.\par
Although food safety practices is one of the top priority policies of European Union, the information on the labels of meat and meat products does not provide food safety guarantee for the period "from the stable to table" (1,2).\par
According to the latest "Meat and Meat Products Manifest announced in our country in February 2013 (3), production of meat products containing meat from different animal species has been banned.\par
Meat and meat products are species-wise safe if they are acquired from healthy animals and processed under hygienic conditions. However, in the frauds and adulterations which are used in order to cut down the costs and increase the profits, meat from inappropriate animals (horse, donkey, and hog) might be mixed in the aforementioned meat and meat products. Besides, in facilities which process several animal products (like facilities processing both cattle and poultry), foreign animal meat might be indeliberately adulterated in the meat products. Besides, due to poor hygienic standards, there may be a possibility of meat and meat products to be adulterated by the wastes and/or tissues of mice and / or insects.\par
Before the introduction of DNA (Deoxyribonucleic Acid) typing method, methods such as Ouchterlony method, SDS-PAGE, ELISA, isoelectric to specify the animal type in meat and meat products. Some of these are based on protein analysis and immunological tests \hyperref[b3]{(4,} {\ref 5)}. However, in case of cooked and processed meat products, heat and continuity of temperature causes the denaturation of type-specific proteins and this decreases the reliability of these methods. PCR (Polymerize Chain Reaction) procedures based on DNA isolation are relatively more stable and are considered to be the most reliable method to specify the animal species of meat and meat products, especially for the short primary strands consisting of specific locus in heat treated products \hyperref[b5]{(6)}. This study aimed to examine various meat and meat products (kebaps, lahmacun ingredients, minced meat, stews, various meat balls etc.) which are presented in various sales points (restaurants, butcher shops, groceries etc.) in Istanbul region, to determine their ingredients through DNA typing method and to specify the different animal tissues / residuals in these products. 
\section[{II. Mater?als and Methods}]{II. Mater?als and Methods} 
\section[{a) Specimen Handling}]{a) Specimen Handling}\par
Random sampling method has been used in this study. From 500 different sales points in the Istanbul region (250 sales points from Asian side and 250 sales points from European side), 500 meat and meat product samples have been collected. As required by the asepsis and antisepsis norms, samples have been placed in sterile containers and transferred to the laboratory in these containers which have +4°C internal heat. b) DNA Extraction DNA of all the isolates are extracted using commercial DNA extraction kits and in accordance with kit protocol. Extracts have been kept at -20ºC, to be used as target DNA in PCR process. 
\section[{c) PCR}]{c) PCR}\par
50-100 mg tissue from the meat samples have been put into a microcentrifuge tube as small pieces. 400 ?L solutions SH has been added and blended with vortex. 8 ?L Proteinase K and 40 ?L solution SLS have been added to the mixture. After blending properly, the mixture has been kept waiting for two hours at 60°?, in order for the cells to stretch. After the incubation at 60°?, 300 ?L Solution SP has been added and blended with vortex for 30 seconds. The mixture has been centrifuged at 12.000 rpm for 30 minutes. The supernatant has been transferred to a clean tube and 500 ?L isopropanol has been added. 
\section[{Table 2 :}]{Table 2 :}\par
Type-specific primer sets used in PCR procedure \hyperref[b14]{(15,}\hyperref[b16]{16,}\hyperref[b17]{17,}\hyperref[b18]{18,}\hyperref[b19]{19)}.\par
Tablo 2 : PCR prosedüründe kullan?lan türe spesifik primer setleri \hyperref[b14]{(15,}\hyperref[b16]{16,}\hyperref[b17]{17,}\hyperref[b18]{18,}\hyperref[b19]{19)}  
\section[{Type}]{Type}\par
Primer Direction Sequence After blending with vortex, the mixture has been incubated for an hour at -20 °?. Then, it has been centrifuged at 12.000 rpm for 20 minutes. Supernatant has been removed. The remaining pellet has been gently vortexed by 1 ml 70\% ethanol and has been distributed, then centrifuged at 13.000 rpm for 5 minutes. Ethanol has been removed and the subsided DNA has been left to dry. After ethanol completely vaporized, 150 ?L Solution SE has been added to the pellet and kept waiting for one night at room temperature, in order for the DNA to dissolve. The dissolved DNA has been measured with UV Spectrometers and diluted to the point of 50 ng/?L concentration. After that, heat treatment protocol has been applied for 10 seconds at 95°C and 15 seconds at 60°C. The second and third steps are repeated for 5 times as 3 cycles (7,8,9,10,11). 
\section[{III. Results}]{III. Results}\par
18 (3.6\%) of the samples showed chicken DNA, 33 (6.6\%) of them showed sheep DNA and 1 (0.2\%) of them showed horse DNA. None of them showed pork, donkey, cat, dog, mice, cockroach and fly DNA. The detailed refraction of the results can be seen in Table \hyperref[tab_2]{3}. The positive results have been determined through Realtime PCR procedures.   The nutritious choices are determined by life styles, religious beliefs, cultures, diets and health conditions. Pursuant to community health, customs, traditions and beliefs, to determine the source of animals of the consumed meat and meat products has been one of the main research subjects for food scientists \hyperref[b11]{(12)}. In many countries, food fraud and adulteration in food products, especially in meat and meat products are done either deliberately in order to increase the profit margin or involuntarily as a result of not following the food safety standards, especially in facilities which process more than one animal species.\par
A study conducted in USA (United States of America) has analyzed raw minced meat and determined 15.9\% of the samples to be containing extraneous animal DNAs . Hsieh et al. (  {\ref 13}) has conducted another study in USA in 1996 and reported that 90\% of the minced meat samples has been adulterated with poultry, either deliberately or unintentionally. Turky?lmaz et al. \hyperref[b13]{(14)}, studied 121 meat and meat product samples using the AGID method and determined horse meat in 3 (2.5\%) of them and pork meat in 2 (1.7\%). Turk et al. \hyperref[b14]{(15)}, studied 223 samples and determined pork meat in 16 (7.1\%), horse meat in 12 (5.3\%) and mixture of pork and horse meat in 6 (2.6\%). The results of our study in general examination are lower than the results of Hsieh et al. \hyperref[b16]{(16)}, similar to those of Turky?lmaz et al. (  {\ref 14}) and Turk et al. \hyperref[b14]{(15)} The different results which have been reported in world and Turkey literature may originate from many reasons, such as the physical conditions of the sales points, whether the food safety products have been applied or not, the differences in the supervision processes, the deficiencies of the facilities which process more than one animal species and/or usage of the same equipment, the deliberateness of adulterations and the staff's lack of information about the procedures.\par
In this study, the highest extraneous DNA in the bovine meat samples was sheep DNA (6.2\%). 96\% (30 of the 31 mutton positive samples) of these positive samples have been collected from kebap shops. Since mutton meat is used commonly in kebap shops, mixture of bovine and mutton meat can be a microbiological threat to consumers.\par
Out of the 500 samples collected, 68 (13.6\%) were determined to be risky for human consumption according to the plate count parameter. 39 (57.4\%) of these "risky" samples contain meat from different animal species. On the other hand, 29 (42.6\%) of these samples contained only one type of meat. Plate count is an indicator of not only food hygiene but also of the tools used in production, food contact surfaces and hands of the staff who contact food. If the plate count is high, it may mean that food, contact surfaces, tools and hands may be carrying potential pathogens and saprophytes.\par
In a study conducted to determine the food intolerance reactions, 22\% of the subjects showed food intolerance and if the foods causing the intolerance are consumed again, the reactions repeated themselves in 15\% of the subjects \hyperref[b17]{(17,}\hyperref[b18]{18)}. Food intolerance may cause chronic inflammatory diseases such as chronic headache, abnormal weight gain, abnormal weight loss, dermatological problems, autoimmune diseases, fibromyalgia, migraine, stomach diseases, bowel diseases such as inflammatory bowel disease (IBD), malabsorptions, rheumatic diseases, shortness of breath, asthma, depression, anxiety, Type 2 diabetes, hypertension, metabolic syndrome, hypothyroidism, chronic rhinitis, eczema, acne, edematous eyelids, urinary diseases, Crohn's disease, cardiovascular diseases \hyperref[b19]{(19,}\hyperref[b20]{20)}. Literature shows intolerance against food of animal origin. The intolerance, which is determined to be more common in males can cause the abovementioned clinical symptoms and some of them can be life threatening. According to WHO (World Health Organization), half of the world population has food intolerance and 1 billion people have been diagnosed with it. WHO predicts that by the year 2015, the count would reach 2.5 billion \hyperref[b21]{(21)}.\par
Whether done deliberately in order to increase the profit margin or accidentally by the facilities which process meat from more than one animal species, adulteration is an illegal practice which deceives the consumer in the sense of health, religion, culture and economy. Another point to be kept in mind is that adulterated meat and meat products pose a greater microbiological risk for consumer health as well. DNA typing also used in our study is a very efficient way of detecting foreign meat species in meat and meat products.\par
Whatever the reason of the adulteration maybe, it results in deficient hygiene conditions and this is a serious threat for the facility, staff and product and consumer health. Besides, microorganisms which reproduce in meat and meat products because of hygiene deficiency can quickly develop single or multi resistance to antibiotics through complex genetic interactions. Our study shows that adulterated products pose a statistically meaningful higher risk for consumer health than unadulterated products. Total quality management systems and food safety practices should be applied together with the official inspection of the state authorities; programs to raise consumer awareness and continuous training programs for the staff responsible for food production should also be carried into effect. All these would be beneficial to reduce the incidence of the adulteration practices. 
\section[{V. ACKNOWLEDGEMENT}]{V. ACKNOWLEDGEMENT}\par
Thanks to Seda Cingay for her help in translation -proof reading.\begin{figure}[htbp]
\noindent\textbf{5}\includegraphics[]{image-2.png}
\caption{\label{fig_0}Pork Forward / Reverse 5 '}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{1}\includegraphics[]{image-3.png}
\caption{\label{fig_1}Figure 1 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{233}\includegraphics[]{image-4.png}
\caption{\label{fig_2}Figure 2 :Figure 3 : 3 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{1} \par 
\begin{longtable}{P{0.26683991683991687\textwidth}P{0.19615384615384615\textwidth}P{0.3057172557172557\textwidth}P{0.0812889812889813\textwidth}}
Region\tabcellsep Sample name\tabcellsep Sales point\tabcellsep Total number of samples\\
?stanbul Europe\tabcellsep Lahmacun ingredients\tabcellsep Kebap shop/restaurant\tabcellsep 50\\
?stanbul Europe\tabcellsep Minced Meat\tabcellsep Butcher shop\tabcellsep 50\\
Istanbul Europe\tabcellsep Kebap\tabcellsep Kebap shop/pedlar wrap point\tabcellsep 50\\
?stanbul Europe\tabcellsep Meat balls\tabcellsep Restaurant\tabcellsep 50\\
?stanbul Europe\tabcellsep Stews\tabcellsep Restaurant\tabcellsep 50\\
Istanbul Asia\tabcellsep Lahmacun ingredients\tabcellsep Kebap shop/restaurant\tabcellsep 50\\
?stanbul Asia\tabcellsep Minced meat\tabcellsep Butcher shop\tabcellsep 50\\
Istanbul Asia\tabcellsep Kebap\tabcellsep Kebap shop/pedlar wrap point\tabcellsep 50\\
?stanbul Asia\tabcellsep Meat ball\tabcellsep Restaurant\tabcellsep 50\\
?stanbul Asia\tabcellsep Stew\tabcellsep Restaurant\tabcellsep 50\\
TOTAL\tabcellsep \tabcellsep \tabcellsep 500\end{longtable} \par
 
\caption{\label{tab_1}Table 1 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{3} \par 
\begin{longtable}{P{0.325531914893617\textwidth}P{0.16954787234042554\textwidth}P{0.1740691489361702\textwidth}P{0.11303191489361701\textwidth}P{0.06781914893617022\textwidth}}
Region\tabcellsep Sample (RAW)\tabcellsep Sales point\tabcellsep Extraneous DNA\tabcellsep DNA positive samples\\
Istanbul Europe -?stanbul Asia\tabcellsep Lahmacun ingredients\tabcellsep Kebap shop\tabcellsep Chicken\tabcellsep 11\\
?stanbul Europe -?stanbul Asia\tabcellsep Minced meat\tabcellsep Butcher shop\tabcellsep Chicken\tabcellsep 5\\
?stanbul Europe\tabcellsep Kebap\tabcellsep Kebap shop\tabcellsep Chicken\tabcellsep 2\\
?stanbul Europe -?stanbul Asia\tabcellsep Kebap\tabcellsep Kebap shop\tabcellsep Sheep\tabcellsep 30\\
?stanbul Europe\tabcellsep Minced meat\tabcellsep Butcher shop\tabcellsep Sheep\tabcellsep 3\\
Istanbul Asia\tabcellsep Minced meat\tabcellsep Butcher shop\tabcellsep Horse\tabcellsep 1\\
TOTAL\tabcellsep \tabcellsep \tabcellsep \tabcellsep 52\end{longtable} \par
 
\caption{\label{tab_2}Table 3 :}\end{figure}
 			\footnote{© 2015 Global Journals Inc. (US) Global Journal of Medical Research ( ) G Studying the Presence of Adultery, Fraudulent ?mitation and Food Pathogens within Processed Meat Products (Such as Salami, Sausage, Braised Meat) using DNA Typing and PCR Procedures*} 			\footnote{Studying the Presence of Adultery, Fraudulent ?mitation and Food Pathogens within Processed Meat Products (Such as Salami, Sausage, Braised Meat) using DNA Typing and PCR Procedures*} 		 		\backmatter  			  				\begin{bibitemlist}{1}
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