Polycyclic Aromatic Hydrocarbons in Grilled Meats from Restaurants

Polycyclic aromatic hydrocarbons are a group of lipophilic compounds that can be generated during the preparation of food items at elevated temperatures. They are regarded as potentially genotoxic and carcinogenic to human beings, related to increased incidence of breast and colorectal cancers, oxidative DNA injury, and bad effects on children neuro-differentiation. Thus, they are considered a public health concerns. A total of thirty samples of grilled beef steak, beef kofta and chicken (ten each) were collected from different restaurants. The samples were extracted by magnesium sulfate and sodium acetate in acetonitrile then purified in magnesium sulfate, primary, secondary amine and silica gel, and finally measured by gas chromatography-mass spectrometry (GC-MS). Benzo[a]pyrene was recorded with the highest average level (3.63µg/kg) in grilled kofta samples, but it was not detected in chicken samples. On the other hand, PAH4, PAH8 and ƩPAHs content were more abundant in grilled beef steak (5.32, 9.97 and 56.91µg/kg). Meanwhile, they recorded the least concentrations of grilled chicken from different restaurants. Furthermore, benzo[a]pyrene exceeded the permissible limits of the European Commission and Egyptian National Food Safety Authority in grilled kofta samples; further studies are needed to investigate the limits of exposure to these harmful compounds from meats and other food items. ــــــــــــــــــــــ ـــــــــــــــــــ


INTRODUCTION
Polycyclic aromatic hydrocarbons (PAHs) are very stable pollutants of foodstuffs and included various sources such as contaminated soils, polluted air and water, cooking procedures, food processing, pseudo-curing with smoking flavor agents or wood generation (Reinik et al., 2007). Foodstuff contamination with PAHs may occur during extreme heat processing, drying, toasting, roasting or frying by direct pyrolysis of food nutrients and due to direct deposition of PAHs from smoke produced via incomplete combustion of different thermal agents (Rey-Salgueiro et al., 2008). Also, these harmful compounds in grilled meats result from partial charcoal pyrolysis that leads to the formation of PAHs in grilled food; the heat on the surface of muscle foods forms these contaminants by direct pyrolysis of food nutrients. Additionally, meat drips that are formed during the grilling and fall on hot charcoal lead to the formation of PAHs, which are carried by the smoke, and adsorbed on the surface of the meat (Costa et al.,

2009).
Human exposure to PAHs is related to a 30-50% increase in the incidence of breast cancer (White et al., 2016), oxidative DNA injury (Yu et al., 2021), and bad effects on neuro-differentiation which occur over a long period in late gestation or early childhood (Slotkin et al., 2017). Additionally, they can cause cardiopulmonary and cardiovascular diseases by producing reactive oxygen species, resulting in oxidative stress and inflammatory responses (Ramesh et al., 2004).
This study aimed to investigate the content and profile of polycyclic aromatic hydrocarbons (PAHs) in different grilled meat types, including beef steak, beef kofta and broiler chicken from different restaurants in Fayoum Governorate, Egypt. Beef steak is made by slicing beef into thin chips. Salt and black pepper are added, while kofta is made by adding chopped vegetables and ground spices to minced beef, then molded into bars and grilled or fried.

Chemicals
Mixed 18 PAHs standards containing 10mg/L of each PAHs congener (Dissolved in cyclohexane) manufactured by Supelco ® (Bellefonte, PA, USA) was used, deionized water was obtained by pobel ® water distiller (Desa 0040 model, Spain), the salts kits were got from Agilent ® Technologies (Palo Alto, CA, USA) and finally, acetonitrile obtained from Merck ® (Darmstadt, Germany).

Sampling and sample preparation
Grilled meats samples (Beef steak, Beef kofta and chicken) were purchased from various restaurants in Fayoum Governorate, Egypt during the period from January to July 2021. Ten samples were taken from each product(Sample weight was 1kg of grilled beef kofta and beef steak and one whole grilled broiler chicken, grilled chicken were deboned, then all samples were homogenized and kept in a deep freezer in foil-wrapped bottles till examination at Faculty of Agriculture Laboratories, Fayoum University.

Extraction and clean-up procedures
The technique formerly designed by Chen et al. (2013) was used, five grams of homogenized sample were mixed with 10 mL of deionized water in a polypropylene tube for 1 minute, then 10 mL acetonitrile solvent was poured and the mixture was exposed to strong shaking for another minute. The content was replaced in a QuEChERS column having 6 grams of MgSO4 and 1.5 grams of sodium acetate, subsequently vortexed for 1 minute and centrifuged at 1431 g for 5 minutes. Then 6 mL of the uppermost layer was transferred into the clean-up column with 0.4g of PSA, 1.2g of magnesium sulfate and 0.4g of silica gel particles for purification. The content was then put in a centrifuge at 1431 g for 5 minutes and 1µl aliquot from the uppermost layer was injected into a gas chromatograph coupled with a mass spectrometer for PAHs analysis.

Gas chromatographic analysis
The PAHs standard curve was obtained by integrating the compound concentration alongside the peak area. The content of each congener in the sample was estimated according to its particular calibration curve, 1µl of the sample extract was introduced into an Agilent HP-5MS gas chromatograph, and eighteen PAHs congeners were identified before elapsing for forty minutes period. Helium gas was used as the carrier gas with a flowing speed of one milliliter per minute; the temperature of the injector was adjusted at 290°C. The splitless mode was selected, and the temperature programming was adjusted on the following: 70°C at first, then elevated to 195°C with the level of fifteen centigrade per minute then continued for another two and half minutes. After then increased to 240°C at the level of fifteen centigrade per minute, then lasted for 17 minutes, upraised to 270°C at the level of five centigrade per minute, and elevated to 310°C at the level of fifteen centigrade per minute then was fixed for ten minutes, PAHs concentrations in tested samples were quantified by matching times of retention and mass spectrum of masses for unidentified peaks to that of previously injected reference materials.

Statistical analysis
The analysis of variance (ANOVA) was made and the means were calculated via SPSS 13.0 statistical package (SPSS, 2008); for descriptive statistics, PAHs concentrations that were lower than the apparatus limit of detection were given a zero value.

RESULTS
Regarding beef steak samples, and from data shown in    , 12.6, 7.15, 0.73, 32.31, 19.22, nd, and 4.04µg/kg wet weight, respectively. Significant variances (P≤0.05) were present in the mean values of PAHs congeners between grilled samples of beef steak, chicken and kofta from different restaurants, which can be attributed to the usage of different coal kinds, as the production of parent PAHs from the coal burning includes both the parent PAHs released from the coal itself and the parent PAHs formed during the combustion process, because the PAHs level and composition within coal itself fluctuates depending on coal rank, and fuel source (Stout and Emsbo-Mattingly, 2008). The presence of these harmful compounds in grilled meats results from partial charcoal pyrolysis that leads to the formation of PAHs in grilled food; the heat on the surface of muscle foods forms these contaminants by direct pyrolysis of food nutrients. Additionally, meat drips that are formed during the grilling and fall on hot charcoal lead to the formation of PAHs, which are carried by the smoke, and adsorbed on the surface of the meat (Costa et al.,  2009).
Also, this variation in total PAHs levels in different grilled meat types from different restaurants, which ranged from 29.95 to 56.91µg/kg, is probably due to variation in fat amount, as fatty meat when grilled, more fat exudes on the hot coal and, by the high temperature, PAHs are generated then carried by smoke to the surface of the grilled meats (Reinik et al.,  2007). Additionally, this difference may be attributed to the fact that some restaurants marinate meats with different edible oils before grilling, which creates several levels of PAHs depending on the oil type (Farhadian et al., 2012).
The abundance of light non-carcinogenic PAHs in grilled meats that ranged from 83-99% in this study could be explained, by the fact that smoke composition itself affects their content, as these low molecular weight compounds are usually found in higher levels in the smoke (Gomes et al., 2009). Moreover, the high levels of PAHs, especially benzo[a]pyrene in kofta may result from the high lipid amounts in kofta because beef fat is added to kofta components to facilitate binding and molding; this fat is pyrolyzed to produce more PAHs (EC-SCF, 2002).

CONCLUSION
It could be concluded that PAHs concentrations between the three groups of grilled meats dishes were found to be significantly different at P≤ 0.05, also, benzo[a]pyrene recorded the highest level in grilled kofta samples, but it was not detected in chicken samples. On the other hand, PAH4, PAH8 and ƩPAHs were more abundant in grilled steak and recorded; meanwhile, they recorded the lowest concentrations in grilled chicken from different restaurants. Furthermore, benzo[a]pyrene exceeded the permissible limits in grilled kofta but was below permissible limits in other investigated food items.