Akara is cowpea paste which is deep-fried in crude palm oil (CPO;
Palm oil is derived from the fruit of the palm tree
In Brazil, crude palm oil is known as
During the frying process, the oil undergoes hydrolysis, oxidation and polymerization reactions leading to changes in flavor stability and quality, color, and texture of the fried food and its nutritional quality (Dobarganes,
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,
Several studies were performed to evaluate the mutagenic potential in vegetable oils used in deep frying (Fong
The Ames test is a widely used methodology to detect mutagenic chemicals (Claxton
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
30 L of integral CPO (a mixture of liquid and solid phases) industrialized at Nazaré city (state of Bahia, Brazil) and conditioned in two tinplate cans with a capacity of 15 L each were purchased at São Joaquim Fair, in Salvador (Bahia). This oil was placed in a stainless steel recipient and heated up to 45 °C, allowing oil fusion and homogenization to be used in frying. At first, 20 mL (time 0) of it were filtered in a glass wool filter, inerted with nitrogen gas and stored in an amber bottle at –20 °C (Jorge and Gonçalves,
The preparation of akara was performed by a
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
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 filtered 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 filtered in a glass wool filter, obtaining the last sample (
Total polar compound (TPC) content was determined gravimetrically according to a mini column method described previously, with slight modifications (Dobarganes
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.
The mutagenic evaluation of CPO and FPO was performed by the pre-incubation method of
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 (
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%). Significant statistical differences between negative and tested concentrations under the same experimental conditions were verified by statistical analysis (Student t-test, p ≤ 0.05) (Stankevicins
To determine the citotoxic effect of CPO and FPO, samples from the pre-incubation assay mixture in the
A murine macrophage cell line was used from a culture at confluence and was incubated in an atmosphere of 5% CO2, 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 Modified Eagle Medium (MEM) Ca2+, 1.8 mM (Gibco®) supplemented with 10% fetal bovine serum, 1.76 g·L−1 of NaHCO3, 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 2×105 cells/well and then incubated for 24 h.
The micronucleus assay was performed as previously described (Aiub
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 fluorescence 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 significant at p < 0.05 (Aiub
There was no significant (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 (
Mutagenic evaluation of crude palm oil before akara frying (CPO) in strains TA97, TA98, TA100 and TA102 of
Strain | CPO | −S9 | +S9 | ||||
---|---|---|---|---|---|---|---|
Mean±SD |
M.I |
%Survival |
Mean±SD |
M.I |
% Survival |
||
TA97 | 0 | 132±22 | 1.0 | 100 | 107±14 | 1.0 | 100 |
1:128 | 138±38 | 1.0 | 97 | 130±19 | 1.2 | 100 | |
1:64 | 134±11 | 1.0 | 90 | 114±9 | 1.1 | 100 | |
1:32 | 115±14 | 0.9 | 91 | 119±15 | 1.1 | 100 | |
1:16 | 123±28 | 0.9 | 90 | 134±11 | 1.2 | 100 | |
1:8 | 158±66 | 1.2 | 89 | 118±6 | 1.1 | 100 | |
PC | 483±62 | 3.7 | 100 | 1016±418 | 9.5 | 100 | |
TA98 | 0 | 27±4 | 1.0 | 100 | 42±3 | 1.0 | 100 |
1:128 | 22±2 | 0.8 | 100 | 42±4 | 1.0 | 92 | |
1:64 | 21±3 | 0.8 | 100 | 45±8 | 1.1 | 100 | |
1:32 | 20±3 | 0.8 | 100 | 37±4 | 0.9 | 90 | |
1:16 | 22±12 | 0.8 | 100 | 56±5 | 1.3 | 100 | |
1:8 | 31±11 | 1.1 | 100 | 64±6 | 1.5 | 100 | |
PC | 145±5 | 5.4 | 90 | 224±16 | 5.3 | 86 | |
TA100 | 0 | 70±8 | 1.0 | 100 | 186±25 | 1.0 | 100 |
1:128 | 68±40 | 1.0 | 100 | 189±17 | 1.0 | 100 | |
1:64 | 68±47 | 1.0 | 100 | 190±12 | 1.0 | 100 | |
1:32 | 86±4 | 1.2 | 100 | 216±5 | 1.2 | 99 | |
1:16 | 99±13 | 1.4 | 100 | 221±1 | 1.2 | 100 | |
1:8 | 65±9 | 0.9 | 99 | 231±11 | 1.2 | 100 | |
PC | 444±6 | 6.0 | 100 | 2304±153 | 12.4 | 100 | |
TA102 | 0 | 345±64 | 1.0 | 100 | 315±31 | 1.0 | 100 |
1:128 | 356±45 | 1.0 | 83 | 431±88 | 1.4 | 100 | |
1:64 | 301±15 | 0.9 | 84 | 374±23 | 1.2 | 100 | |
1:32 | 337±18 | 1.0 | 79 | 332±74 | 1.1 | 100 | |
1:16 | 312±10 | 0.9 | 88 | 297±4 | 0.9 | 92 | |
1:8 | 295±39 | 0.9 | 87 | 416±23 | 1.3 | 87 | |
PC | 2207±286 | 6.4 | 89 | 1016±103 | 3.2 | 100 |
Number or revertant colonies per plate: mean values and standard deviation (SD) of at least three experiments
MI: Mutagenic index: number of
Survival calculated in relation to the negative control. Concentration 0 refers to the negative control (solvent): 100 µL of DMSO. The doses of the positive controls (PC) per plate in the absence of S9 mix were 0.5 µg of 4-nitroquinoline-1-oxide to TA97 and TA98; 1.0 µg of Sodic Azide to TA100 and 0.5 µg of Mitomicin C to TA102. The doses per plate of the positive controls in the presence of S9 mix were 1µg of 2-Aminoantracen to TA97 and TA100 and 20 µg of Benzo[α]piren to TA98 and TA102. Cytotoxicity was detected when survival rates <70%
Mutagenic evaluation of crude palm oil after akara frying (FPO) in strains TA97, TA98, TA100 and TA102 of
Strain | FPO | −S9 | +S9 | ||||
---|---|---|---|---|---|---|---|
|
|
||||||
Mean±SD |
M.I |
%Survival |
Mean±SD |
M.I |
% Survival |
||
TA97 | 0 | 341±41 | 1.0 | 100 | 202±8 | 1.0 | 100 |
1:128 | 321±91 | 0.9 | 100 | 238±7 | 1.2 | 100 | |
1:64 | 319±42 | 0.9 | 100 | 116±22 | 0.6 | 100 | |
1:32 | 335±15 | 1.0 | 100 | 265±37 | 1.3 | 100 | |
1:16 | 238±47 | 0.7 | 100 | 287±22 | 1.4 | 100 | |
1:8 | 233±59 | 0.7 | 95 | 240±6 | 1.2 | 100 | |
PC | 2162±499 | 6.3 | 70 | 2388±399 | 11.8 | 78 | |
TA98 | 0 | 19±3 | 1.0 | 100 | 51±4 | 1.0 | 100 |
1:128 | 25±4 | 1.3 | 100 | 59±5 | 1.1 | 100 | |
1:64 | 35±5 | 1.9 | 100 | 52±0 | 1.0 | 100 | |
1:32 | 29±2 | 1.5 | 97 | 65±3 | 1.3 | 100 | |
1:16 | 31±8 | 1.6 | 76 | 53±6 | 1.0 | 100 | |
1:8 | 13±3 | 0.7 | 100 | 69±1 | 1.3 | 100 | |
PC | 322±146 | 17.3 | 94 | 239±12 | 4.7 | 82 | |
TA100 | 0 | 115±15 | 1.0 | 100 | 221±9 | 1.0 | 100 |
1:128 | 109±8 | 1.0 | 100 | 228±14 | 1.0 | 100 | |
1:64 | 137±13 | 1.2 | 100 | 242±31 | 1.1 | 100 | |
1:32 | 118±15 | 1.0 | 100 | 266±20 | 1.2 | 100 | |
1:16 | 108±1 | 0.9 | 100 | 281±30 | 1.3 | 100 | |
1:8 | 111±12 | 1.0 | 100 | 260±0 | 1.2 | 100 | |
PC | 593±214 | 5.2 | 86 | 986±61 | 4.5 | 85 | |
TA102 | 0 | 341±41 | 1.0 | 100 | 483±87 | 1.0 | 100 |
1:128 | 374±7 | 1.1 | 88 | 599±63 | 1.2 | 100 | |
1:64 | 336±30 | 1.0 | 90 | 639±26 | 1.3 | 100 | |
1:32 | 287±18 | 0.8 | 90 | 383±49 | 0.8 | 100 | |
1:16 | 264±9 | 0.8 | 100 | 911±64 | 1.9 | 100 | |
1:8 | 267±4 | 0.8 | 100 | 859±33 | 1.8 | 100 | |
PC | 2163±499 | 6.3 | 75 | 1747±260 | 3.6 | 81 |
Number or revertant colonies per plate: mean values and standard deviation (SD) of at least three experiments
MI: Mutagenic index: number of
Survival calculated in relation to the negative control. Concentration 0 refers to the negative control (solvent): 100 µL of DMSO. The doses of the positive controls (PC) per plate in the absence of S9 mix were 0.5 µg of 4-nitroquinoline-1-oxide to TA97 and TA98; 1.0 µg of Sodic Azide to TA100 and 0.5 µg of Mitomicin C to TA102. The doses per plate of the positive controls in the presence of S9 mix were 1 µg of 2-Aminoantracen to TA97 and TA100 and 20 µg of Benzo[α]piren to TA98 and TA102. Cytotoxicity was detected when survival rates <70%.
The results from the assay with CPO and FPO are presented in
Micronucleus induction assay in macrophages using CPO and FPO
Samples | Concentration | ‰ Mi.I.±SD |
% Apoptosis | % Necrosis | % Micronucleus±SD |
---|---|---|---|---|---|
CPO | NC | 7.9±2.8 | 3.6±1.2 | 1.0±0.3 | 3.8±1.2 |
1:128 | 4.4±1.4 | 1.7±0.9 | 1.0±0.2 | 3.2±0.3 | |
1:64 | 4.5±0.8 | 1.4±0.5 | 0.8±0.3 | 3.0±1.5 | |
1:32 | 11.6±0.4 | 2.7±2.8 | 0.6±0.5 | 2.9±1.3 | |
1:16 | 8.4±0.8 | 3.5±0.2 | 1.2±0.3 | 4.4±0.6 | |
1:8 | 8.9±1.5 | 2.8±0.5 | 0.8±0.2 | 4.1±1.3 | |
PC | 6.7±1.9 | 4.7±0.8 | 1.2±0.1 | 7.9±1.1 | |
FPO | NC | 7.9±2.8 | 3.6±1.2 | 1.0±0.3 | 3.8±1.2 |
1:128 | 5.0±0.9 | 1.9±0.3 | 0.9±0.4 | 3.0±0.6 | |
1:64 | 6.4±0.1 | 1.9±1.8 | 0.6±0.4 | 2.8±1.2 | |
1:32 | 6.0±3.2 | 1.7±0.9 | 0.7±0.2 | 2.6±0.3 | |
1:16 | 5.4±3.4 | 1.5±0.5 | 1.0±0.2 | 1.9±0.6 | |
1:8 | 6.4±3.1 | 1.5±0.2 | 0.9±0.4 | 2.8±1.4 | |
PC | 6.7±1.9 | 4.7±0.8 | 1.2±0.1 | 7.9±1.1 |
Mitotic index per thousand. The doses of the negative and positive controls were 10% of DMSO and 0.5 µM of MNNG, respectively. The experiment was performed in quintuplicate.
TPC contents increased linearly with frying time with high correlation coefficients (R2=0.96; TPC = 15.24 + 0.6 time) (
Total polar compound (TPC) content (%w/w) of crude palm oil during 25 h of akara frying.5 days, 5h/day
Time (h) | Mean | Std. Error | % |
---|---|---|---|
14.08 | 0.04 | — | |
5 | 20.25 | 1.09 | 44.12 |
10 | 20.09 | 0.40 | 42.68 |
15 | 24.73 | 0.17 | 75.63 |
20 | 27.51 | 0.22 | 95.38 |
29.81 | 0.48 | 111.71 |
The percentage of increase in TPC after 25 h of frying
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,
No mutagenic or citotoxic activity were detected in the CPO used in the traditional akara frying process in all tested strains (
As already mentioned, TPC include TGP, TGD, oxTGM, DG, and FFA (Dobarganes
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
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.
The authors were supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Process n° 482790/2010-5, Brazil), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and SR2/UERJ, FAPESB (Termo N°: BOL1784/2010, Brazil), Associação das Baianas de Acarajés e Mingau da Cidade de Salvador-Bahia-Brazil and Alessandra Quirino for technical assistance.