Molecular Products and Particulate Characterization of Emissions from High Temperature Cooking of Goat Meat

Efforts to understand the formation characteristics of molecular toxins and particulate matter from various combustion systems, has gained intense attention in the recent past. Accordingly, this work investigates the evolution of organic toxins and particulate matter from a goat meat sample at various pyrolysis temperatures. To simulate cooking conditions, 10 mg of meat sample was heated under atmospheric conditions in an air depleted environment in a thermal degradation reactor and the smoke effluent passed through a transfer column and collected over 10.0 mL dichloromethane for GC-MS analysis. The major selected toxins reported in this study include indole, 2-(1-methyl) quinoline, phenol, 2ethylthiophenol, 2,3-dimethylhydroquinone, and 1,1’-biphenyl. At the highest pyrolysis temperature (700  ̊C), the mean particle size of particulate emissions was estimated to be 7.72 ± 0.61 μm while at 500  ̊C, the particle size of emissions was found to be 3.52 ± 0.31 μm. The decomposition profile of meat was monitored between 300 and 525  ̊C, and the highest mass loss was recorded between 300 and 450 ̊ C (~ 36%). Most of the organic toxins from high temperature cooking of goat meat were mainly phenolics whereas the particulate emissions at 500  ̊C and 700  ̊Cwere approximately PM2.5 and PM10 respectively.


INTRODUCTION
Although many studies have been conducted on the thermal degradation of various biomass materials, few studies have been investigated on the combustion of goat meat at high cooking temperatures.In this regard, the concept of potentially toxic by-products from various combustion sources has attracted interest because of the health and environmental impacts induced by organic toxins [1,2].For instance, many extremely mutagenic heterocyclic amines have been proven to be multi-site tumor initiators in long-term animal studies such as monkeys and rodents [3].Combustion pollution therefore has adverse effects on respiratory and cardiovascular systems coming up as acute reduction in lung function, aggravation of asthma, increased risk of pneumonia among humans of all ages including neonates [4].On the other hand, the concentration of respirable particulate matter (PM) in ambient air has become a topic of considerable importance in the recent past [4].Presently there is still a fundamental lack of understanding underlying the mechanisms of their toxicity but one of the widely accepted hypotheses is that toxicity of particulates depends on their size and also on their composition [5].Molecular products such as phenols and heterocylic amines for instance are well-established carcinogensas well as mutagens [1,6] while phenols are capable of disrupting sexual hormone functions, which ultimately may result in sterility of many animals including higher order animals including man [7].
Most of these organic toxins are released into the environment as a result of direct emissions from incomplete combustion processes and high temperature cooking procedures [8,9].In the search for possible correlation between diet and cancer, the highly mutagenic heterocyclic amines and phenols present in cooked foods have attracted a great deal of concern [3,10].It has been established in previous studies that rodents fed on a diet laced with heterocyclic amines developed tumors of the breast, liver, lung, and prostate cancer [3].Moreover, exposure to airborne fine particles, of ≤ 2.5 microns (PM2.5) or ≤ 10 microns (PM10), is associated with deaths in excess of 50 000 yearly in the USA as well as various chronic respiratory ailments [11].HCAs become capable of damaging DNA when they are metabolized by specific enzymes in the body, following a process called bio-activation which in turn initiates the development of cancer risks and other cellular damaging consequences [12].
This study therefore investigates some of the bio-hazardous by-products from high temperature cooking such as nitro-polycyclic aromatic hydrocarbons (nitro-PAHs), phenolic compounds such as phenol, 2-ethylthiophenol, 2,3-dimethylhydroquinone, and polycyclic aromatic hydrocarbons (PAHs) such as 1,1'-biphenyl.These compounds are considered hazardous to human health.Particulate emissions and their surface morphology are investigated using a scanning electron microscope with a view to classifying the particulate emissions as PM2.5 or PM10.Moreover, the Density functional framework (DFT) has been used to explore the energetics of free radical formation from selected bio-hazardous compounds reported in this study.

Materials
The heater (muffle furnace) was purchased from Thermo Scientific Inc., USA while the reactor was locally fabricated in our laboratory by a glass-blower.The meat sample (lean goat meat with general composition of proteins, fats, water, minerals and carbohydrates [13]) investigated in this work was purchased from a common meat joint (Kikopey meat joint in Nakuru County, Kenya) and used without further treatment.Kikopey meat complex center is a tourist area located along the busy Nairobi-Nakuru High way specialized in the preparation of barbecue from goat meat.

Sample preparation and treatment
The meat sample 10 mg was weight and packed in a quartz reactor (volume 1.6 cm 3 ) and then placed in an electrical furnace whose maximum heating temperature is 1000 ˚C.The heating rate of the muffle furnace was ~ 5 ˚C s -1 at 1 atmosphere.The meat sample was heated in an air depleted atmosphere in a quartz reactor and the smoke effluent was allowed to pass through a transfer column and collected over 10.0 mL dichloromethane and kept in crimp top amber vials for a total pyrolysis time of 15 minutes to simulate average cooking time.This combustion experiment was conducted under conventional pyrolysis described elsewhere [14,15] and the evolution of molecular products was monitored between 300 and 525 ˚C.To understand the surface characterization of soot from high temperature cooking of goat meat, two cooking temperatures were examined (500 and 700 ˚C).

GC-MS characterization of selected combustion by-products
GC-MS analysis was carried out using an Agilent Technologies 7890A GC system coupled with an Agilent Technologies 5975C inert XL Electron Ionization/Chemical Ionization (EI/CI) with a triple axis mass selective detector, using HP-5MS 5% phenyl methyl siloxane column (30 m x 250 µm x 0.25 µm).The oven temperature was set at 50 ºC for 3 min then ramped to 10 ºC/min to 250 ºC for 2 min, then held constant at 250 ºC for 20 min.Ultra High Purity (UHP, 99.999%) helium was used as the carrier gas at the flow rate of 1 mL/min [15]. 2 µL of smoke effluent extract was injected in the split ratio 50:1 at an injection temperature of 250 ºC.To ensure that the right compound was detected, standards were run through the GC-MS system and the peak shapes and retention times compared with the compounds of interest [16].Standards were used in identification of compounds in conjunction with NIST data base, enhanced data software package developed by Agilent technologies, and thorough literature searches.The two libraries gave consistent structures of the compounds of interest therefore resulting to sufficiently high confidence in the pyrolysis products reported in this work.Accordingly, critical emphasis has been given to those products which can easily be correlated with the structure of the starting material (meat) especially for those compounds where standards were not available [16,17].All the results presented in this study are averaged replicates of two data points.

Calibration of pyrolysis products from goat meat
A stock solution was prepared by weighing 0.1 g of each individual standard and dissolved in a10 mL volumetric flask containing analytical grade dichloromethane (purity ≥ 99.9%).The details of the calibration procedure are described elsewhere [15,17]. 2 µL of each standard (for pyrolysis products whose standards were available) solution was injected into the GC-MS and the peak area of each standard compound was then plotted against the mass of the compound in µg/mL solution and then linearly regressed to obtain the calibration curve of the compound [15].The R 2 value was ≥ 0.95 for all standards.The weight percent yield of each compound was determined using equation 1 according to references [15,17]: where Y represents yield of the pyrolysis product in Wt %.

SEM characterization of meat char
Particulate emission of meat at 500 ˚C and 700 ˚C conducted at 15 minutes residence time was dissolved in dichloromethane through a porous tube diluter and transferred into amber vials.About 5 mg of sample was added to 1 mL methanol and gold grids were dipped into the prepared sample.Twisters were used to pick the gold grids from the sample.The grids were allowed to dry in the open before putting them into the analysis chamber of the SEM (JEOL JMS 7100F) [18,19].The sample was analyzed under high vacuum to ensure no interference of air molecules during analysis.The SEM machine was then switched on and imaging of the sample conducted.The lens was varied at various resolutions until a clear focus of the sample was observed.The details of this procedure are reported elsewhere [19,20].Image J computer program was used to determine the size of the soot particles and a distribution curve of soot size was then determined using Igor computer software [19].The mean sizes of the soot particles at 500 and 700 ˚C were reported and presented as Gaussian distribution curveswhere the peak of the curve shows the average of the particle size.

Computational methodology
The use of computational models plays a critical role in the environmental regulatory processes; this is because complex relationship between environmental emissions, the quality of the environment, and human and ecological impacts can be clearly elucidated by modeling procedures [21].Density functional theory (DFT) optimizations at B3LYP/6-31G quantum level has been performed on all the molecular compounds and their corresponding free radicals investigated in this work.All calculations have been carried out using the Gaussian '09 Computational program [22,23].Nonetheless, when using DFT, the choice of basis set is considered to be inconsequential because the convergence of DFT to the basis-set limit with increasing size of basis set is relatively quick, and as such, small basis sets are used [24].More often, diffuse functions on basis sets are not used for DFT calculations, as these lead to linear dependencies and a bad convergence of the self-consistent-field (SCF) Kohn-Sham equations for larger molecules [24].

RESULTS AND DISCUSSION
It is evident from this study that various classes of organic toxins were released from the combustion of goat meat.Clearly, nearly all organic toxins reached a maximum yield at about 450 ˚C as presented in Figure 1.The major toxic group of compounds in the heterocyclic amine category was 2-(1-methyl) quinoline and indole with respective maximum %wt yields of 0.84 and 1.80 (Figs.1A and B).In the phenolic category, 2-ethylthiophenol gave the highest yield at 450 ˚C of 1.27 %wt, Figure 1a.On the other hand, 1,1'-biphenyl in the class of PAHs was the major product giving a %w yield of 1.34 at 450 ˚C (cf. Figure 1b).Other major products included 2,3-dimethylhydroquinone, phenol and 2-ethenylnaphthalene with 0.65, 0.24 and 0.93 %wt respectively at 450 ˚C.Interestingly below 400 ˚C, most molecular toxics are released at low levels.Therefore, these results suggest a safe cooking temperature region of about 300 ˚C or even lower.Although, it would appear that at high temperatures > 450 ˚C, the yields of reaction products decrease, the resultant residue is largely char which is basically aromatic and therefore non edible.The yields of virtually all pyrolysis products investigated in this work, increase sharply at 400 ˚C and decrease suddenly above 450 ˚C (Figure 1).
The quantitative release of bio-hazardous compounds were monitored in a GC-MS and presented in Figure 2. The structures as well as their retention times and molecular masses of the pyrolysis products are presented in Table 1, vide infra.Long chain molecular compounds were also detected and presented in Figure 3.The overlay chromatograms at 400 ˚C and 500 ˚C show clearly the intensity of each reaction product at various retention times.
Remarkably, whereas the concentration of most species as observed in Figure 2 decreased significantly at 500 ˚C, the concentration of indole appears to be comparably high.The oxygenated molecular products (phenols in particular) are expected to decompose at high temperatures to form mainly CO, water and other small molecules including hydrogen [14,25].The decrease in the concentration of 1,1'-biphenyl is anticipated because of the scission of the biphenyl bond leading to the formation of other by-products of combustion especially indole and other indole derivatives in presence of nitrile radicals in the combustion system.This is because; meat is largely a protein and is bound to contain amino acids.Therefore, the dominance of nitro-PAHs and indole derivatives during high temperature cooking of meat is

B
not unusual.At high temperatures, many complex reactions occur including intermediates, radical formation, and new products of aromatic nature [26].Although long chain molecular products reported in this work (Figure 3) have significantly high concentrations, they will not be discussed in detail.Nevertheless, they are believed to be toxic due to their high molecular masses and the possibility of covalently bonding to biological structures such as DNA and lipids to cause severe health problems.Furthermore, these long chain compounds can transform to free radicals at high temperatures and their potency cannot be ignored.
The compounds presented in Figure 3 were not quantified, however; they are very important components of meat and were tentatively identified from the NIST and enhanced data libraries.These compounds can possibly be correlated with the compounds found in a meat matrix.
Because of the toxic nature of phenolics and the carcinogenic nature of PAHs, quantum chemical calculations of these compounds (2,3-dimethyl hydroquinone and 1,1'-biphenyl) will be investigated further to determine their energetics in relation to their biological action in causing ill health.The results may be generalized as to the toxicology behavior of other related compounds including phenol and 2-ethenylnaphthalene.The toxicological effects of heterocyclic amines including indole and 2-(1-methyl) quinoline will be discussed in general.Nevertheless, the chemistry of 2-ethylthiophenol believed to be a reaction product of methionine amino acid (or any other S-based amino acid) is of scientific interest although this will not be investigated further.Nonetheless, consensus of opinion in literature indicates that thiophenols exhibit both environmental and human toxicity [27,28].It is evident from the compounds detected from this study that emissions from high temperature cooking of red meat are poisonous and subsequently injurious to human health.For instance, phenols (phenol and 2,3-dimethyl hydroquinone) provoke mutagenesis and carcinogenesis towards humans and other ecosystems through generation of organic radicals and reactive oxygen species (ROS) [29].More importantly, phenol at high temperatures is capable of forming phenoxy radicals that are transient reactive species capable of reacting with biological tissues causing extensive cellular damage and eventually cancer [14,30].

Decomposition profile of red meat
The decomposition characteristics of red meat over the temperature range of 300 -525 ˚C gave interesting results (Figure 4).The initial sharp decrease in % char to ~ 60% at 300 ˚C is very remarkable and can be attributed to high mass loss of water in the meat sample.The greatest mass loss was registered between 300 ˚C and 450 ˚C (approximately 36% mass loss).This is consistent with other results of biomass pyrolysis which indicate the highest mass loss of biomass pyrolysis occurs in this temperature region [31].
The lowest mass loss was recorded at 525 ˚C (  17% ).Interestingly, between500 ˚C and 525 ˚C, the mass loss was fairly constant.Above this temperature zone, the char is largely aromatic and few oxygenated compounds are often formed [14,31].

Mechanistic Description of Radical formation
To investigate the energetics of selected molecular compounds as well as their corresponding free radicals, quantum chemical calculations were conducted using the density functional theory (DFT/B3LYP) in conjunction with 6-31G basis set [32].It can be noted that 2,3-dimethylhydroxyphenoxy radical (1) is at resonance with benzoquinone type radicals (2,3dimethyl hydroxy-2-benzoquinone (2) and 2,3-dimethylhydroxy-6-benzoquinone (3) radicals), Scheme 1. Accordingly, radicals 1, 2, and 3 are resonance hybrids.It was found that radicals 1 and 2 were formed via an enthalpic barrier of 70.79 kcalmol -1 .Formation of radical 3 nonetheless was predicted to be highly endothermic (128.89kcalmol -1 ).This enthalpy is ~ 1.8 times the energy observed for the formation of radicals 1 and 2. Clearly, the conversion of radical 1 to radical 2 is barrierless i.e. the change in enthalpy for the conversion of radical 1 to radical 2 is ~ 0 kcalmol -1 .The characteristic behavior of phenoxy radical and its associated health effects is well-documented in literature and will not be described further in this study [15,30,33] Remarkably, 2,3-dimethylhydroquinone can undergo hydrogen abstraction to form a benzoquinone type radical, usually considered extremely reactive and a potential cause for oxidative stress and possibly cardiac arrest [26,30].Following H abstraction, the resultant benzoquinone and phenoxy radicals exhibit electron deficient characteristics.The benzoquinone type radical is stabilized by electron donating groups (methyl groups) and thus it is believed to be an environmentally persistent free radical [30,34].Such environmentally persistent free radicals can cause irreparable damage to the biological genetic make-up and consequently carcinogenesis and other cancer related ailments.Scheme 1 shows the mechanistic formation of some of the possible radicals from 2,3-dimethylhydroquinone at high temperatures.Radicals produced during high temperature cooking of food materials are therefore very reactive and capable of causing oxidative stress, cancers, and coronary health problems including cardiac arrest [6,26].

Scheme 1. Mechanistic formation of radicals from 2,3-dimethyl hydroquinone
The carcinogenicity of indole and its derivatives are well-known in literature [14,35].Nitrogen-containing compounds are present in high temperature cooking (≥120 ˚C) processes such as frying, baking and grilling [36].High temperature pyrolysis ≥ 500 ˚C for various food materials has been shown to result in indole formation and other nitrogen containing compounds [35][36][37].On the other hand, 1,1'-biphenyl can undergo rapture of the biphenyl bond (C-C bond) at high temperatures, resulting in the formation of toxic benzyl radicals which can immediately convert to benzene in the presence of hydrogen radical 'pool'.Scheme 2 shows how benzyl radical can be formed from 1,1'-biphenyl.It is reported in literature that benzene can cause hematopoietic disorders, leukemia as well as cancer [37] and hence may pose potential risks to both environmental and human health.
The scission of the biphenyl bond is accompanied by an endothermicity of 110.48 kcalmol -1 whereas the formation of benzene from benzyl radical proceeds with an enthalpic change of -111.61 kcalmol -1 .This suggests that at high temperatures, benzyl radical is formed in the gas phase but in the presence of hydrogen radicals, it can be converted to the wellestablished carcinogen, benzene [38,39] which was detected in low amounts in this study.Whereas the health consequences of organic by-products of combustion are well documented in literature, the toxicology action of soot particles is yet to be understood [4].Stabilized structures of the reacting radicals of hydrocarbons with conjugated structures and their derivatives are critical intermediates to soot nucleation [40].For instance, in the pyrolysis of benzene, the aromatic growth is initiated by the formation of biphenyl as presented in Scheme 2 [41].

Particulate characterization of emissions from high temperature cooking of goat meat
At an associated magnification of x 400, the size of particulate matter from cooked meat at temperatures of 500 ˚C and 700 ˚C were significantly different.Clearly, at 500 ˚C, the particle size of emissions was larger ~ 7.72 ± 0.61µm (Figure 5) as compared to the particles at 700 ˚C Scheme 2. Mechanistic formation of benzyl radical and benzene from 1,1'-biphenyl Eurasian J Anal Chem 55 ~ 3.52 ± 0.31 µm (Figure 6).This implies that the higher the cooking temperature, the smaller the soot particles.Nevertheless, the particulate size from high temperature cooking of red meat falls well under PM10 classification of particulate matter at 500 ˚C, whereas at 700 ˚C the particulate size is ~ PM2.5.Therefore, our results corroborate studies reported elsewhere in literature regarding particulate emissions from high temperature cooking of red (goat) meat [42].Particles of this size are known to cause respiratory ailments including lung cancer, bronchitis and composed of mainly organics such as phenols, PAHs, and possibly HCAs since they originate from the thermal degradation of red meat [43].In many countries, about 80-90% of people spend most of their times indoors thus, exposure to harmful emissions as a consequence of high temperature cooking and other combustion sources is very high [11,37].

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Statistically, as reported in Table 2, there is a significant difference in the mean sizes of particulate emissions released at 500 ˚C and 700 ˚C at 95% confidence interval since the calculated F-value = 37.53 is greater than the F-critical = 4.04, then the difference between the two sets of data is significant.P-value = 0.000 and thus less than 0.05 level of confidence.
Epidemiological studies have revealed that exposure to particles of aerodynamic diameter of PM2.5 and PM10 enhances lung cancer, morbidity, and cardiopulmonary death [37].The aerodynamics of airborne particulate matter and the deposition characteristics in the human lung has shown that airborne particulate matter with an aerodynamic average diameter less than 2.5 µm (PM2.5) is the fraction of the particles with the largest impact in human health [44].Despite intense interest in the toxicity of PM2.5 adPM10the mechanism by which they causes ill health is poorly understood [11].Combustion particulate matter is known to contain free radicals and thus airborne fine particles from combustion especially PM2.5 may contain radicals which are injurious to human health [11,45].The fine particles provide a carrier for deposition of the radicals deep in the human respiratory tract.These radicals consequently initiate immune system responses that can activate the production of more radicals as well as other species that can cause damage to DNA and/or induce damage to the respiratory airway [45].Nonetheless, the health effects of airborne fine particles are still a matter of critical scientific debate [44].

CONCLUSION
The organic toxins reported in this investigation (phenols, PAHs, and nitro-PAHs) are harmful by-products of combustion implicated in numerous diet problems such as gouts, aging, and cancer related illnesses.On the other hand, the particulate nature of soot from combustion of red meat suggests that at 500 ˚C and 700 ˚C the particulate emissions are approximately PM10and PM2.5respectively and are responsible for various health respiratory problems such as whizzing of the lungs and lung damage.The application of quantum chemical calculations (DFT/B3LYP) has revealed interesting results on the stability of benzoquinone type radicals.It is evident 2,3-dimethylhydroxyphenoxy radical and benzoquinone type radical (2,3-dimethylhydroxy-2-benzoquinone radical) interconvert to one another with ~ 0 kcalmol -1 .Accordingly, the results presented in this study are perhaps the first on the pyrolysis of goat meat representative of actual cooking environments.We believe these results form a basis to the investigation of other forms of meats such as chicken, pork, and beef.

Figure 1 .
Figure 1.Yield (GC -Area counts) distribution of molecular toxins from high temperature cooking of goat meat

Figure 2 .
Figure 2. GC-MS chromatogram for the pyrolysis of red meat at 400 ˚C (red line) and 500 ˚C (blue line)

Figure 3 .
Figure 3.Long chain molecular compounds detected in this work (cf.Figure 1)

Figure 1 )
Figure 3.Long chain molecular compounds detected in this work (cf.Figure 1)

Figure 4 .
Figure 4. % yield of char from pyrolysis of red meat

Figure 5 .Figure 6 .
Figure 5. SEM image and particle size distribution (Gaussian green) of soot particles of meat at 500 ˚C

Table 1 .
Major molecular toxins determined in this study

Table 2 .
Mean sizes of particulate emissions formed at 500 and 700 ˚C