Polycyclic aromatic hydrocarbons are a group of compounds formed when organic compounds such as wood, coal, garbage as well as foods are incompletely combusted. These compounds have been found to be potential carcinogenic compounds even in low concentrations as well as one of the environmental pollutants and are also present in some food categories. Their presence in foods is due mainly to the different processing and cooking methods such as smoking, roasting, baking and frying (Tfouni et al, 2009).
Fifteen of these compounds; benzo(a)anthracene, cyclopenta(c,d)pyrene, chrysene, benzo(b)flouranthene, benzo(j)flouranthene, benzo(k)flouranthene, benzo(a)pyrene, indenol(1,2,3-cd) pyrene, dibenzo(a,e)pyrene, dibenzo(a,i)pyrene, dibenzo(a,h)pyrene, dibenzo(a,l)pyrene,5-methyl chrysene, benzo(g,h,i)perylene and dibenz(a,h)anthracene have been identified as doubtless mutagenic and carcinogenic by the scientific committee on food (Commission Regulation,2006).
According to this committee, benzo(a)pyrene B(a)P, can be used as a marker for the occurrence and effect of these carcinogenic PAHs in food. Humans mainly come into contact with this carcinogenic compound through consumption of foods contaminated with this compound.
B(a)P is defined by Irwin et al (1997) as a high molecular weight of five ring Polycyclic aromatic hydrocarbon, and an Environmental Protection Agency (EPA) priority pollutant and classified carcinogen.
Most human health concerns for B(a)P relate to cancer as well as immunosuppressant, genetic damage and various reproductive problems. B(a)P is known to produce tumors in test animals from single exposures to very small quantities.
B(a)P has been shown to cause genetoxic effects in a broad range of prokaryotic and mammalian cell assay systems. In prokaryotes, B(a)P tested positive in DNA damage assays and in both reverse and forward mutation assays. In mammal cell culture assays, B(a)P tested positive in DNA damage assays, chromosomal effects assays and cell transformation assays (Irwin et al, 1997).
Various concentrations of B(a)P have been detected in various smoked foods smoked with different fuel, either wood or charcoal. Fredrick M (1990) detected 200µg/kg of B(a)P in sausages smoked with wood and 50µg/kg in ground meat smoked with charcoal. Rivera et al (1996) also detected 16µg/kg of B(a)P in meat smoked with charcoal. Gomaa et al (1993) also extracted 3.9µg/kg concentrations of B(a)P from pork and chicken smoked with charcoal.
Gray areas still exist because even though smoked foods are a major characteristic of the Ghanaian dish, Ghanaians still remain in the dark so far as this carcinogenic compound is concerned. Literature is devoid of B(a)P concentration levels of smoked foods on the Ghanaian market.
Although this compound is present in small amount, it is one of the most carcinogenic compounds ever found and it produces tumors in mice, rats and monkeys. Such knowledge combined with the fact that these compounds are present in many foods characteristic of the Ghanaian diet gives this project an extra sense of urgency.
This project therefore seeks;
To determine the B(a)P concentrations in smoked herring, tilapia, salmon, sausages and beef.
To compare the B(a)P concentrations in these smoked foods and compare with the standard allowable intake of B(a)P as prescribed by FAO and WHO.
To find out which of the fuels used for smoking gives the highest concentration of B(a)P.
The methods employed in this work will be extraction of the B(a)P using an organic solvent since PAHs are hydrophobic. This will be followed by clean-up procedures because no matter whatever solvent used for the extraction, meat and fishes are complicated matrices and are therefore bound to contain other hydrophobic and non polar compounds as well as other PAHs. Detection and quantification will be done using High Performance Liquid Chromatography (HPLC) with Fluorescence detection.
Food samples high in fats are expected to have higher B(a)P concentrations as well as foods smoked with woods.
LITERATURE REVIEW
Sausages, beef, tilapia, salmon and herring are foods that are enjoyed by all groups of people in Ghana. These foods are mostly eaten smoked. Smoked sausages, beef and tilapia are enormously appreciated by consumers and are a good success on the market. These foods are common at the fast food joints and due to the busy lives of people living in the urban areas these smoked foods have become a very common diet on their menu. Restaurants are bustling with smoked tilapia and sausages. Smoked beef, popularly known as "khebab" joints are common sights along the road sides in the urban areas of Ghana.
Smoked salmon and herring are also enjoying high patronage on the Ghanaian market. Ghanaians' light soup, palm-nut soup or even groundnut soups will never be devoid of either of these fish. Not to talk about the various sauces of Ghanaians (garden eggs stew, vegetables stew etc ).
Smoking involves exposing the meat or fish sometimes pre-treated to the smoke. The meat or fish is laid on mesh trays and the smoke is produced by the combustion or smoldering of wood or charcoal.
Different kinds of fuels are used for smoking foods by people from different places. The Chinese can use uncooked rice, sugar and tea as their source of fuel for smoking. In New Zealand, sawdust from the native manuka (tea tree) is commonly used for hot smoking fish. In Iceland dried sheep dung is used for cold smoking (http/en. wikipedia org/wiki/smoking-cooking). In Ghana wood and charcoal are normally used as the source of fuel for smoking.
Charcoal is derived from pyrolysis of wood. The smoke from both fuels contain apart from the PAHs other compounds like chlorinated dioxins, cadmium, arsenic, toluene, nitrogen oxides carbon monoxides among others. Others are aldehydes, ketones, esters, alcohols, phenols etc. Smoking foods has gained popularity because of the presence of the phenol compounds present in the smoke that are able to impart desirable sensory attributes to the food. These compounds are formed due the thermal degradation of wood followed by the oxidation of some of the products of combustion and incomplete combustion in limited oxygen supply. The chemicals mentioned are just a few out of the lot because combustion can lead to thousands of chemical combinations amongst which are sweet smelling vanilla and others also being the most toxic compounds that one can ever think of (Nestrick et al,1994).
Smoking is an aged old method of food preservation and even in modern days it is one of the commonest method of processing meat and fish. Smoking is capable of preserving foods because smoke contains compounds that are capable of inhibiting the growth of microorganism. Due to the advent of refrigeration especially in the urban areas, smoking is not used for its preservation capability currently but rather to impart some sensory attributes to the food like flavor, aroma and other organoleptic appeal. However people in the remote areas where electricity is still a fantasy find smoking of fish and meat as a necessary and indispensable preservation tool. In such areas, especially fish and game are smoked and kept to reduce waste at times of bumper harvest and also as a means of increasing protein availability to the local folks.
Smoke also contains carbonyl compounds hence its ability to impart flavor and aroma. Other important compounds in smoke are the phenolic compounds that prevent oxidation and subsequent rancidity of fatty foods. There are basically two main types of smoking foods based on the temperature for smoking: hot smoking which is done at 120-180oF and cold smoking which is done at a temperature of 90-120oF (http://www.answers.com/topic/smoked-meat).
Despite the fact that this method of cooking makes the food more appealing, tastier and attractive, it comes with its own associated problems.
First and foremost the health risks of these smokers cannot be overemphasized especially the women who use the traditional ovens where more firewood and constant turning are required. Smoke entered their eyes and lungs, they burn their fingers and are exposed to the heat directly. However, according to one woman smoker, "the Chorkor smoker has come to our aid". Depletion of the forest of trees used as fuel for smoking is one area of worry to the environmentalist. The Chorkor smoker is an improved oven for smoking where some of the above mentioned problems have been addressed.
Epidemiologic studies have shown that people living in areas where eating of smoked foods are common are at greater risks of getting various range of cancers like stomach and colon cancers.
Charbroiling of the meat and fish convert the proteins and fats near the surface to oxidized products like peroxides and oxides which are equally probable human carcinogens (http://www.helium.com/items/66683-dangers of-eating-smoked food).
There are also the PAHs present in the smoke that can get adsorbed on to the food. These compounds are known to be ubiquitous, present in the atmosphere, water, sediments, air and tobacco smoke (Falcon et al , 2004). PAHs can undergo photo degradation and oxidation in the presence of light. Right after smoking, as reported by (Simko) 1991, the B(a)P concentration in smoked fish was 10.6µg/kg and after seven days the concentration level reduced to 1.3µg/kg. This presupposes that after extraction of the PAH awaiting HPLC analysis, the samples need to be prevented from exposure to sunlight in order not to get inaccurate results. The samples need to be kept in the refrigerator at a temperature of 4oC.
Amongst the various compounds making the PAHs is the carcinogen marker Benzo(a)pyrene. Below is the structure of benzo(a)pyrene.
C:\Users\Ezy\Desktop\research tools\benzo1_files\200px-Benzo-a-pyrene.png
Fig1
Retrieved from http://en .wikipedia.org/wiki/Benzo(a)pyrene.
The main way through which humans come into contact with B(a)P is through eating of food contaminated with the compound, inhaling and also through skin contact. The distribution of PAHs and for that matter B(a)P in smoked foods is very alarming. The B(a)P can penetrate 6mm deep into the smoked food product (Stolywho et al, 2004) .
Strictly speaking, B(a)P is a pro-carcinogenic compound; it is the intermediates that are carcinogenic. When consumed, B(a)P is first metabolized by the system of enzymes known as the michrosomal cytochrome P450 system to several arene oxides. These arene oxides are further hydrated to dihydrodiols by the epoxide hydrase enzymes and are further epoxidized to diol epoxides or react covalently with glutathione. Some of these intermediate metabolites have high potential of being carcinogenic. The bay region diol epoxide intermediates are considered as the ultimate carcinogenic and once these regions are formed, nothing stops them from binding covalently to DNA to disrupt the integrity of the DNA structure. This subsequently leads to the initiation of mutagenesis and carcinogenesis (ASTDR, 1995).
B(a)P has a molecular weight of 252.3g/mol and it is pale yellow in colour (IARC,1983). Its melting point is 179-179.3oC (Weast, 1987), with boiling point of 495oC at 760 mm/Hg (Aldrich, 1996). It has a faint aromatic odor (ASTDR, 1995).
Different concentration levels of B(a)P has been found in various food categories as well as in foods smoked with either woods or charcoal. There are no fast straight jacket procedures for determining concentration levels of B(a)P since different procedures have been used by different researchers. However basically, determination of B(a)P in involves extraction of the B(a)P using an organic solvent, followed by clean-up procedures to get rid of unwanted compounds that may be present in the food matrices as well as other compounds making up the PAHs and then detection and quantification of the B(a)P using HPLC.
Ilze et al (2008) determined the concentration levels of B(a)P in various meat smoked with different types of wood. Their analytical sample preparation method employed extraction of PAH with cyclohexane, liquid-liquid extraction with N, N dimethylformamide/water, back extraction with cyclohexane, followed up by clean-up on silica phase extraction (SPE) column. Quantification was done using gas chromatography-mass spectrometry. They found that samples smoked with apple tree and alder contained the least B(a)P concentration with 6.04µg/kg and 9.43µg/kg respectively whilst meat smoked with Aspen had the highest concentration of 35.07µg/kg and therefore concluded the type of wood smoked has an influence on the amount of B(a)P produced. The meat smoked with charcoal had 10µg/kg concentration of B(a)P.
Nieva Cano et al (2001), determined the concentration levels of PAH in non-fatty, non-smoked mashed potatoes and toasted bread using ethyl ether-methylene chloride (1:1) sonication as their extraction method and subsequent separation by HPLC with flourimetric detection. A Hypersil Green PAH column was used with a gradient acetonitrile-water as the mobile phase together with ten excitation and emission wavelength pairs. Total PAH found in mashed potato were in the range 9.35-17.1µg/kg and 7.38-18.0µg/kg in toasted bread. However they detected an unquantifiable level of B(a)P concentration in non-fatty, non-smoked potato and mashed potato. It can be deduced from this work that the method used for cooking has an adverse effect on the concentration of B(a)P that may be present.
Falcon et al (2004), decided to develop a fast, simple and economic method to determine the concentration of the potential PAHs that re developed during the roasting of the coffee bean that forms integral stage in the coffee making process for the coffee bean to be able to develop a characteristic flavor. The method they employed was extraction with hexane, clean-up with silica cartridges, concentration to dryness and injection of the acetonitrile solution of the residue for HPLC analysis with fluorescence detection. Concentration of B(a)P were found at levels between the detection and quantification limits of the method used of 0.03-0.1µg/kg in instant coffees (Falcon et al, 2004).
Farhadian et al (2010), studied the effects on PAH specifically on Benzo(a)Pyrene, Benzo(b)Flouranthene and Flouranthene by using two different types of treatments, preheating (steam and microwave) and wrapping (aluminum and banana leaf) of the meat samples prior to charcoal grilling. PAHs were extracted using tandem solid-phase extraction. The results showed that applied pre-heating and wrapping treatments affect the PAHs levels in the charcoal grilled meat. No carcinogenic PAHs Benzo(a)Pyrene and Benzo(b)Flouranthene were detected in the samples, at least not within quantifiable range after steam and microwave pre heating and wrapping in the aluminum and banana leaf. Significant reduction Flouranthene content up to 46%and 81% in beef and chicken samples, respectively, were observed in these studies.
(Stolywho et al, 2004), detected 50µg/kg in the outer parts of heavily smoked fish from traditional kilns, while 0.1µg/kg was detected in mild hot-smoked fish from smoke houses supplied with conditional wood smoke from external generators. The method they employed was the contemporary analytical procedure based on the extraction of the PAHs from the matrix of the fish followed by the clean-up procedure, separation by HPLC and quantification and detection by fluorescence detection.
This result could be attributed to the f act that smoking under mild conditions produces less contamination. Much the same way using conditioned wood of which much of the toxins, undesirable resins as well as gums have been removed produces less contaminant.
Lorenzo et al (2010), determined the PAH content in two traditional smoked sausages in Spain. Determination and quantification were performed by HPLC with fluorescence detection. The results showed that Androlla sausage had PAH levels of 36.45µg/kg whiles Bortillo had 29.39µg/kg. Phenanthrene compound of PAH detected whilst none of the samples exceeded the maximum limit of 5µg/kg set for B(a)P.
The mean level of B(a)P in different types of sausages was found to be 0.22µg/kg (Garcia Falcon et al, 1996). Rivera et al (1996) also extracted 3.9µg/kg concentrations of B(a)P from chicken smoked with charcoal.
Gomaa et al (1993) also extracted 3.9µg/kg concentrations of B(a)P from pork and chicken smoked with charcoal.
The fat content of the food being smoked seems to have a positive correlation to the concentration of B(a)P. This is because as the food is being smoked the fat gets to drip onto the hot fire. This produces more smoke and hence more PAHs are produced. The more the PAH, the higher the concentration of the B(a)P.
FAO and WHO have set maximum permissible concentration of B(a)P in food to be 10µg/kg (Joint FAO/WHO Expert Committee on Food Additives, 1987). Turkish Food Codex (2002) has been set as 1µg/kg, while a level of 5µg/kg has been set by the European Commission (2005).
Pathetically, nothing can be said about the B(a)P levels in smoked food products found on the Ghanaian market even though smoked food products happen to form a major category of the Ghanaian delicacies.
A survey conducted showed that most Ghanaians know practically nothing about the existence of this silent killer compound, B(a)P not even most of the elite in the society. Is it therefore surprising that, cancer cases and deaths due to cancer are on the ascendancy in Ghana? Hardly..
witt
MATERIALS AND METHODS
Smoked herring and salmon were obtained from local fish smokers at Asafo in Kumasi. Smoked sausages, tilapia and beef were obtained from the smokers at Ayigya and the campus of Kwame Nkrumah University of Science and Technology.
Sample preparation
The sample preparation was done according to the method of (Hong Z et al, 2009), with slight modification.
2.0g of homogenized sample was weighed into a centrifugal tube. 10mL of 0.4mol/KOH-ethanol: water (9:1 v/v) was added and the mixture heated in water bath at 60oC whilst stirring continuously. The solution was transferred into microwave extraction tanks and the centrifugal tube cleaned twice with 5mL cyclohexane to ensure effective cleaning. The solution was heated for 10 minutes at 100oC with microwave power at 230kW and then cooled until 30oC. The solution was transferred into 30mL glass tubes. The mixture was centrifuged at300 rpm for 15 minutes. The supernatant fluid was transferred into 10mL glass tubes. The supernatant fluid was evaporated until dryness with N2/vacuum evaporator at 45oC. The mixture was redissolved with 3mL of water/methanol (90:10 v/v).
Clean up
The clean-up procedure was adopted from Ledicia S et al's (2009) method. This was done by using 30mL of the extract directly with Sep-Pack silica plus cartridges, adding 10mL n-hexane to avoid losses. The eluate was evaporated till dryness under vacuum evaporation (20oC). The eluate was redissolved to a final volume of 0.5mL acetonitrile and filter through 0.45µm PVDF syringe filter prior to HPLC with fluorescence detection analysis.
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY WITH FLOURESENCE DETECTION
The HPLC analysis was done using the specification as prescribed by Tfouni et al (2009) with slight modification.
The analysis was carried out using Adept Cecil HPLC apparatus equipped with Rinary pump, and RF-10Axl fluorescence detector (excitation wavelength 290nm and emission wavelength 430nm). A C18 column (250Ã-46nm, 5µm particle size) at 30oC was used and the mobile phase consisted of 75% acetonitrile and 25% water at a flow rate of 1ml/min. The injection volume was 20µl.
STATISTICAL ANALYSIS
Data were processed using the one analysis of variance ANOVA using the Tukey for unequal N test at 95% confidence interval.
