Toxicological assessment of crude palm oil (Elaeis guineensis Jacq.) used in deep frying of akara (cowpea paste finger food)

I. Felzenszwalba, J.L. da Costa Mazzeib, S. Feitosac, C.A. Fortes Aiubd and D.T. de Almeidac,* aUniversidade do Estado do Rio de Janeiro, Instituto de Biologia Roberto Alcântara Gomes. Av 28 de Setembro, 87 fds. 4° andar, Vila Isabel 20551-030 Rio de Janeiro, RJ–Brasil bFundação Oswaldo Cruz, Far-Manguinhos Instituto de Tecnologia em Fármacos. Rua Sizenando Nabuco, 100, Manguinhos, 21041-250 Rio de Janeiro, RJ–Brasil cEscola de Nutrição, Universidade Federal da Bahia, Av. Araújo Pinho, 32, Canela, Salvador-Bahia, CEP: 40110-150, Brazil dUniversidade Federal do Estado do Rio de Janeiro, Departamento de Genética e Biologia Molecular, Rua Frei caneca 94, Centro, 20211040 Rio de Janeiro, RJ–Brasil *Corresponding author: deliata@uol.com.br; delia@ufba.br


INTRODUCTION TOP
Palm oil is derived from the fruit of the palm tree Elaeis Guineensis Jacq. and is consumed as crude palm oil (CPO) in Africa, Southeast Asia and Brazil. Unre ned palm oil is orange-red in color and contains triglycerides with approximately 50% saturated fatty acids, 40% monounsaturated and 10% polyunsaturated fatty acids, along with carotenoids and vitamin E (Norhaizan et al., 2013;Sampaio et al., 2013). By fractionation, a liquid fraction (palm olein, rich in unsaturated compounds) and a solid fraction (palm stearin, rich in saturated compounds) can be obtained and used for different purposes in the food industry (Lin, 2011).
In Brazil, crude palm oil is known as azeite de dendê and it is an ingredient in most dishes from Bahia, such as moqueca, vatapá, xinxin de galinha, caruru, and acarajé (akara) (Almeida et al., 2013;Oliveira, 2009). Akara is a paste of cowpea (Vigna unguiculata L. Walp) sold as a street nger food and it has been described as the most common food product containing cowpeas in Africa (Reber et al., 1983) and Brazil. It's also a cultural and touristic Brazilian icon made and sold by typically clothed women called baianas de acarajé (IPHAN, 2005). In the process of making akara, cowpea is split, decorticated and macerated into a paste. After seasoning with grated onions and salt, akara paste is whipped, shaped into balls with a wooden spoon and deep-fried in CPO.
During the frying process, the oil undergoes hydrolysis, oxidation and polymerization reactions leading to changes in avor stability and quality, color, and texture of the fried food and its nutritional quality (Dobarganes, 2000a;Saguy and Dana, 2003;Velasco et al., 2008). The frying temperature and time, type of heating, frying oil composition, oil turnover rate, oxygen content, antioxidants and type of fryer affect the deterioration of oil during deep-fat frying (Soriano et al., 2002).
These chemical reactions in frying oil originate volatile products, which are partially eliminated during frying and new non-volatile compounds are also formed (Saguy and Dana, 2003). These new compounds have higher polarity and constitute the so-called total polar compounds (TPC), which include triglyceride polymers (TGP), triglyceride dimers (TGD), oxidized triglyceride monomers (oxTGM), diglycerides (DG) and free fatty acids (FFA) (Dobarganes et al., 2000b). In general, fresh re ned oils contain total polar compounds ranging from 3.2% to 3.8%. It is recommended that frying oils which contain more than 24-27% TPC should be discarded (Berger, 2005;Saguy and Dana, 2003).
Several studies were performed to evaluate the mutagenic potential in vegetable oils used in deep frying (Fong et al., 1980;Scheutwinkel-Reich et al., 1981;Taylor et al., 1982;Van Gastel et al., 1984), the effects of repeated ingestion of heated palm oil by rats (Adam et al., 2009;Farag et al., 2010;Isong et al., 1997), and they showed that the fraction of polar compounds isolated from the oxidized oils is toxic to laboratory animals (Pantzaris, 1998). Besides that, the Ames test (Salmonella/microsome assay) is a rapid, safe and predictive test for mutagenicity in mammals (Claxton et al., 1987;Maron and Ames, 1983;Carpes et al., 2013), in which Hageman et al. (1988 have detected mutagenic activity in polar material obtained from deep-frying fats. The Ames test is a widely used methodology to detect mutagenic chemicals (Claxton et al., 1987;Maron and Ames, 1983;Carpes et al., 2013), however, bacteria is less able to repair DNA damage than a mammals ability. A macrophage lineage is used due to its ability to promote responses involving oxidative stress. In this way, RAW 264.7 is one of the most commonly used for giving the clearest results and being extremely sensitive to lipopolysaccharide endotoxin from gram-negative bacteria such as Salmonella (Claxton et al., 1987;Maron and Ames, 1983).
Changes in oil quality during deep frying are still a major issue from the health perspective. Nevertheless, there are few studies in the literature regarding the consequences of consuming CPO deep fried foods. It has been observed that the baianas de acarajé sell akara daily for 5h hours and continuously reuse the fried palm oil, hence this frying oil may have toxic compounds present. Therefore, the aim of this work was to evaluate CPO mutagenic and genotoxic activities before and after 25 hours of frying akara, and to quantify its TPC (%) contents. inerted with nitrogen gas and stored in an amber bottle at -20 °C (Jorge and Gonçalves, 1998) and thawed for analysis (initial sample, CPO).

MATERIALS AND METHODS
The preparation of akara was performed by a baiana de acarajé according to her traditional practice. 5 kg of crude mass of akara were bought daily from her traditional source at the same fair. Frying was always performed outdoors. First the raw mass was placed in an aluminum pan and seasoned with grated onions and salt, then whipped and shaped into balls (60-80 g each) with a wooden spoon. Then, 5 L of CPO in an enameled pan were heated for 12 minutes in the presence of an onion (baiana's traditional practice -an intention of making the oil quality lasts longer the oil quality longer during frying). Subsequently, 5 of those balls were successively added to the oil and deep-fried during approximately 07/12/2019 Felzenszwalb grasasyaceites.revistas.csic.es/index.php/grasasyaceites/rt/printerFriendly/1485/1538 3/8 6 minutes, with some intervals in which the oil remained with only the onion, while the baiana whipped the mass again, and a total of 110 akaras/day was obtained -simulating akara's average quantity at the baiana's point of sale (Rogerio, 2010).
The total frying time per day was 5 intermittent hours and the frying process was performed on 5 consecutive days -totaling 25 hours. The temperature ranged from 143 to 188 °C in the beginning (day 1) and 159-178 °C in the end (day 5).
Every frying day began with 5 L in the enameled pan. On day 1, only fresh oil was used, and on subsequent days a mixture of the previous day's oil (used oil) was made with fresh oil. The oil turnover was performed daily with 2 L at the most. On days 1 and 2 only fresh CPO was added, and on the other days, the replacement was made with a mixture of fresh plus used oil, according to the baiana's techniques.
Then, at the end of each frying day, the oil was left undisturbed in a pan with a lid, at room temperature, until the food waste settled, and was ltered afterwards, stored until the next frying day and the same procedure was repeated over four subsequent days, for a total time of 25 hours.
At the end of each day and the frying process (25 hours), a new aliquot of 20 mL was ltered in a glass wool lter, obtaining the last sample (FPO of frying) and the others for TPC (%) content analysis.

Total Polar compounds TOP
Total polar compound (TPC) content was determined gravimetrically according to a mini column method described previously, with slight modi cations (Dobarganes et al., 2000b). In brief, approximately 0.5 g of crude palm oil were dissolved in the elution solvent and introduced into a glass column lled with a silica gel slurry and elution solvent. This elution solvent was a mixture of light petroleum (b.p. 40-60 °C) and diethyl ether 94:6 (v:v). A chromatographic glass column with an internal diameter of 1 mm and a length of 15 mm, containing 5 g of silica gel (with a particle size 0.063-0.200 mm and 70-230 mesh) adjusted to a water content of 5% was used. Non-polar compounds were eluted with 60 mL of elution solvent and the polar compound fraction was eluted with 50 mL diethyl ether. A dropping funnel was used and the ow rate was adjusted to approximately 1.5 mL·min −1 . Solvent was removed by rotary evaporation and the ask was ushed with a stream of nitrogen to ensure dryness. The completeness of fractionation was evaluated by analytical thin-layer chromatography (TLC) with an elution system of petroleum ether:diethyl ether:acetic acid (70:40:1; v:v:v).
The reported data of TPC were obtained from duplicate measurements of each sample and expressed in terms of the mean and standard error (SE). A regression analysis was also applied to these data.

Salmonella/microsome assay TOP
The mutagenic evaluation of CPO and FPO was performed by the pre-incubation method of Salmonella/microssome assay as described by Maron and Ames (1983), with slight modi cations (Mortelmans and Zeiger, 2000). Brie y, 100 μL of different concentrations of CPO or FPO (all dilutions were in DMSO) and 100 μL of stationary growth cultures of Salmonella Typhimurium strains TA97, TA98, TA100, and TA102 were added to 500 μL of 0.2 M of a sodium phosphate buffer (pH 7.4) or to 500 μL of an S9 mix. After pre-incubation of the mixture at 37 °C for 30 min, 2 mL of top agar were added to it and the contents poured onto a minimal glucose agar plate. The plates were incubated at 37 °C and the numbers of His + were scored after 72 h. Each assay (in triplicate) was repeated twice to con rm the results.
Aroclor 1254-induced rat liver S9 fraction was purchased from Molecular Toxicology Inc. (Moltox™, USA). The metabolic activation mixture (S9 mix) was prepared according to Maron and Ames (1983) and used as described earlier (Aiub et al., 2003). The criterion for a positive mutagenic response was the number of reverting colonies in the test assay amounting to at least twice the number of spontaneous reverting (Mutagenic Index, MI≥2) (Maron and Ames, 1983).
The mutagenic response was considered positive when the number of revertant colonies in the test was at least twice the number of spontaneous revertants and no cytotoxicity was detected (survival rates <70%). Signi cant statistical differences between negative and tested concentrations under the same experimental conditions were veri ed by statistical analysis (Student t-test, p≤0.05) (Stankevicins et al., 2008).

Survival experiments TOP
To determine the citotoxic effect of CPO and FPO, samples from the pre-incubation assay mixture in the Salmonella/microssome assay were diluted in 0.9 % NaCl (w/v) to obtain a suspension containing 2×10 2 cells·mL −1 . An aliquot of 100 μL of this suspension was poured onto a nutrient agar plate. The plates were incubated at 37 °C for 24 h and colonies were counted. Citotoxicity was considered positive when the cell survival rate was <70% of the rate observed for the negative control (Aiub et al., 2003).

RAW 264.7 cell culture TOP
A murine macrophage cell line was used from a culture at con uence and was incubated in an atmosphere of 5% CO 2 , at a temperature of 37°C . Cell detachment was carried out mechanically by scraping. After that the suspension was centrifuged at 5000 rpm for 5 min and cells were re-suspended in Modi ed Eagle Medium (MEM) Ca 2+ , 1.8 mM (Gibco®) supplemented with 10% fetal bovine serum, 1.76 g·L −1 of NaHCO 3 , 0.88 g.L −1 of pyruvate, 22 mg·L −1 of aspartic acid and 17 mg·L −1 of L -serine. Cell viability was determined after trypan blue staining and the cells were plated on 24-well plates at a density of 2×10 5 cells/well and then incubated for 24 h.

Micronucleus assay TOP
The micronucleus assay was performed as previously described (Aiub et al., 2011), with modi cations. Cells were treated with 100 μL of various dilutions of CPO or FPO in DMSO, corresponding to 10% of the total volume of each well and then the plates were incubated for 24 h. After this period, the medium was removed from the wells, cells were washed with 1 mL of MEM Ca 2+ , 1.8 mM and reincubated, and after that with 1 mL of MEM Ca 2+ , 1.8 mM, supplemented with fetal bovine serum for 24 h in an atmosphere of 5% CO 2 . N-methyl-N-nitro-N-nitrosoguanidine  To determine the mitotic index and number of micronucleated cells, as well as percentages of necrotic and apoptotic cells, 1000 cells per concentration were analyzed in a uorescence microscope (Reichert Univar) at an excitation wavelength of 350nm. This experiment was conducted in quintuplicate. The data were analyzed using a one-way ANOVA and the Tukey-Kramer multiple comparison test. Results were considered to be statistically signi cant at p<0.05 (Aiub et al., 2011, Carpes et al., 2013. GraphPad Instat® software, version 3.01 (GraphPad Software, Inc., USA) was used for all analyses.

Mutagenic activity TOP
There was no signi cant (p>0.05) increase in the number of reverting colonies at any of the tested concentrations of CPO and FPO over the respective negative control plates in all four tested strains, both with or without the S9 mix (Tables 1 and 2).  and TA102. Cytotoxicity was detected when survival rates <70%.

Micronucleus evaluation TOP
The results from the assay with CPO and FPO are presented in Table 3. Both samples did not induce micronuclei formation, apoptosis or necrosis on macrophage cells. Moreover, they did not induce an increase in mitotic division. There was no signi cant difference between the results obtained with the oils sampled before and after frying (p<0.05).

Total Polar compounds TOP
TPC contents increased linearly with frying time with high correlation coe cients (R 2 =0.96; TPC=15.24+0.6 time) (Table 4). After 25 hours of heating, the oil showed around 29% polar compounds, above the established limit in most of the current regulation regarding the discharging of frying oils and fats for human consumption (Berger, 2005;Saguy and Dana, 2003). It was observed that the values were higher from 15 h of frying (Table 4), due to the greater heating time and replenishment with reused oil. Crude palm oil is largely used in Brazilian cuisine and it is essential in akara deep frying. The latter is now regarded as one of Brazil's immaterial national treasures (IPHAN, 2005) and a cultural and touristic icon in the city of Salvador (Bahia, Brazil). During deep-frying, the high temperatures used in the presence of oxygen and water induced important chemical changes in the palm oil, inevitably reducing its shelf life and directly affecting its nutritional characteristics (Matthäus, 2007). Therefore, it is necessary to carry out toxicological studies to determine the security of the CPO used in this process.
No mutagenic or citotoxic activity were detected in the CPO used in the traditional akara frying process in all tested strains (Tables 1 and 2).
Furthermore, it was observed that CPO did not induce genotoxic potential in the micronucleus assay in macrophages (Table 3). These results were contrary to the expected ones since the TPC content in the analyzed frying oil sample (25 h) exceeded the considered safe limit for health (Table 4) (Berger, 2005). Nevertheless, some researches had found no mutagenic activity in deep-frying oils (Scheutwinkel-Reich et al., 1981;Taylor et al., 1982;Van Gestel et al., 1984), and only Fong et al. (1980) had observed mutagenicity in peanut oil, however it had been correlated to an a atoxin B1 contamination in the oil.
As already mentioned, TPC include TGP, TGD, oxTGM, DG, and FFA (Dobarganes et al., 2000b). As DG is measured as a fraction of TPC, it may be misleading to use TPC in judging oil quality. The higher amounts of total polar compounds in palm oils are mainly due to a higher level of DG contents (4.0-7.5%) compared to other vegetable oils (2-3%) (Berger, 2005;DeMarco et al., 2007). Otherwise, the CPO processing in Bahia displays high FFA %, a consequence of the poor condition of the oil extraction (Almeida et al., 2013). Thus TPC elevation in samples (CPO and FPO) may be due to the predominance of such fractions which are not associated with the toxicity of heated oils (González-Munõz et al., 1998;Morita et al., 2008).
One unique characteristic of crude palm oil is its high content of carotenoids and tocopherols. Carotenoids, which impart to CPO its distinctive orange-red color, together with tocopherols and tocotrienols contribute to stability. A research made by our group demonstrated that the mean of carotenes in the industrial CPO processed in Bahia were 526.77 μg·g −1 (Almeida et al., 2013) and this carotenoid value is extremely high when compared to other oils: sun ower (8.5±0.7 mg·L −1 ) and extra virgin olive oil (11.0±0.3 mg·L −1 ) (Andreu-Sevilla et al., 2008). On the other hand, CPO contains unusually high levels of tocotrienols (Norhaizan et al., 2013;Berger, 2005). Most other common vegetable oils have negligible contents of tocotrienols (Lin, 2011). In CPO, the presence of these antioxidants seems to offer some oxidative protection to the oil through a mechanism in which they are oxidized prior to triglycerides (Lin, 2011). Together with CPO constitution factors, the baiana de acarajé practice of heating oil in the presence of a whole onion considerably preserves the oil quality with a signi cant reduction in the levels of lipid peroxidation indicators as well as a reduction in tocoferol loss (Oyewole and Olayinka, 2007).

CONCLUSIONS TOP
In despite of its high TPC levels, no cytotoxic, mutagenic or genotoxic activities were detected in the crude palm oil used in the traditional akara frying process. This may be due to the CPO natural antioxidant content and akara frying practices such as the addition of an onion to the frying oil, and fresh oil replacement at the beginning. Therefore, the CPO used in akara deep-frying do not offer toxicological risks to consumers.