Mutagenesis, Vol. 17, No. 1, 63-66,
January 2002
© 2002 UK Environmental Mutagen Society/Oxford University Press
Modulation of mutagenic activity in meat samples after deep-frying in vegetable oils
Department of Food Science and Toxicology, Faculty of Pharmacy, University of Navarra, E-31080 Pamplona, Spain
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Abstract |
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Previous studies have been carried out on the influence of frying fats on the formation of food mutagens, but most of them have been performed on model systems or under cooking conditions that are more frequent in northern countries. The objective of this work was to study the overall mutagenic activity generated in hamburgers and frankfurters deep-fried under cooking conditions that are normal practice in Spain and other Mediterranean countries, in order to determine if there was any modulation of the mutagenic activity with respect to other cooking conditions previously studied. Hamburgers were prepared from beef purchased in a butcher's shop. Frankfurters as well as the oils [olive, marc olive (`orujo'), sunflower and soya bean oil] and butter were purchased in a local supermarket. The samples were fried in a teflon-coated frying pan at 170–180°C for 10, 20 or 30 min. The mutagens were extracted and the mutagenic activity evaluated using the Salmonella mammalian microsome assay with strain TA98. Two independent assays were carried out for each experimental condition. All the hamburgers showed a mutagenic activity that was more than four times higher than that of the controls. Frankfurters showed a lower mutagenic activity than hamburgers (fried under the same conditions) because they have a lower protein content and a higher fat content. Hamburgers fried in olive oil for 10 min showed a significant increase in the number of revertants with respect to the other oils, probably due to the fact that the temperature reached was
10°C higher. Longer frying times significantly increased the number of revertants in samples fried in oils, except in olive oil, probably due to its lower content of polyunsaturated fatty acids. |
Introduction |
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The presence of high mutagenic activities in the charred surface of beef and fish grilled over a naked flame or charcoal was first demonstrated by Sugimura and co-workers (Sugimura et al., 1977
). Soon after, it was observed that mutagenic activity was also produced during the cooking of meat under normal domestic conditions (Commoner et al., 1978) and several studies confirmed that moderate temperature cooking at 190–200°C, such as frying and broiling, induced high levels of mutagenic activity (Pariza et al., 1979; Spingarn and Weisburger, 1979; Felton et al., 1981). Compounds responsible for the said mutagenic activity were identified as heterocyclic amines (HAs) and more than 15 have been isolated from different sources (Chen et al., 1990).
Several mechanisms have been implicated in the formation of HAs. The Maillard reaction has been suggested to play an important role, with the precursors being creatine/creatinine, amino acids and sugars (for a review see Skog, 1993). In long-term rodent bioassays some HAs have shown carcinogenic effects (for a review see Ohkagi et al., 1991). Various epidemiological studies have been carried out to demonstrate an association between the consumption of well-done red meat and the occurrence of colon cancer, although inconsistent results have been obtained (Knekt et al., 1994; Muscat and Wynder, 1994). There are also several reports that try to evaluate human exposure to said types of compounds by different procedures (Sinha and Rothman, 1997; Keating et al., 1999; Keating and Bogen, 2001). Therefore, we think it important to better understand which conditions of normal cooking practice in different countries or cultures are safer from the point of view of mutagenicity risk.
Some authors have investigated whether or not fatty acids and oils, added to a model system with the components of the Maillard reaction (creatinine, glucose and glycine) in adequate proportions, increased the yields and species of food mutagens formed. In these studies it was observed that the fat content significantly affected mutagen formation (Johansson and Jägerstad, 1993) and that the addition of corn or olive oil to a model system heated for 30 min at 180°C almost doubled the yield of MeIQx (2-Amino-3, 8-dimethylinidazo [4,5-f] quinoxaline) formed (Johansson et al., 1993).
Nilsson et al. (1986) studied the effect of frying lean pork meat at 200 and 250°C in different fats and found small differences at 200°C and a significant increase in mutagenic activity at 250°C. Johansson et al. (1995) studied the effect of different fats (butter, margarine, rape seed oil and sunflower seed oil) on the formation of HAs during frying of beefburgers and observed that the amount of several HAs was always lower in the meat than in the pan residue and that the total amount was lower after frying in sunflower seed oil or margarine than after frying with the other fats.
The objective of the present work was to study the overall mutagenic activity generated in ground beef patties and frankfurters when deep-fried with several classes of oils under cooking conditions that are normal practice in Spain and other Mediterranean countries, in order to determine if there was any modulation of the mutagenic activity with respect to other cooking conditions (i.e. those more common in northern countries) previously studied. The presence of mutagens has been evaluated using the Salmonella mammalian microsome assay with strain TA98, which is accepted as a reliable method for the detection of mutagenicity in food.
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Materials and methods |
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Samples
Beef was purchased in a butcher's shop and chopped up in order to homogenize the samples. Hamburgers weighing 100 g were prepared, measuring 1 cm in thickness and 10 cm in diameter. They were conserved at –20°C until use.
Frankfurters of two different brands, as well as the several oils [olive, marc olive (`orujo'), sunflower and soya bean oil] and butter used for cooking, were purchased in a local supermarket.
Analytical methods
The general composition of the hamburgers and frankfurters was determined before cooking by standard analytical techniques: total protein content (AOAC, 1990a), total fat content (AOAC, 1990b), moisture content (ISO, 1973) and ash (AOAC, 1923).
Cooking methods
The hamburgers and frankfurters were fried in a teflon-coated frying pan 18 cm diameter which was placed over heat with 200 ml of oil, in butter or without fat. The starting frying temperature was the maximum recommended for all the oils (170°C) except olive oil (recommended 180°C). When the food was placed directly in the pan (without oil) the temperature was 180°C. When the predetermined temperature was reached, the samples, which were at 4°C, were introduced into the pan. External and internal temperature were controlled during each process. Hamburgers were fried for 10, 20 or 30 min and frankfurters for 10 min. Each experiment used two ground beef patties or frankfurters.
Extraction
The method for extraction of mutagens was based on that previously developed by Bjeldanes et al. (1982). It consisted of a liquid–liquid extraction procedure at different pH values, followed by a solid–liquid extraction phase. First, the external crust of the hamburgers or hot dogs was separated (50 g), because it is known that mutagens are formed in the outer part. Subsequently, these 50 g of meat were suspended in a double volume of water, acidified with 0.1 N HCl to pH 2 and centrifuged at 8000 r.p.m. for 15 min. The supernatant was collected and the sediment suspended in a double volume of water, acidified and centrifuged. This operation was repeated twice. The final sediment was discarded. The supernatants collected were combined, brought to pH 12 with 0.1 N NaOH and filtered through a cellulose filter (Whatman no. 1). It was then passed through a polymer resin (Amberlite XAD-2; Supelco) in a 2.5x50 cm column (Sigma) and eluted with acetone. Finally, the solvent was evaporated and the extract dissolved in 3 ml of dimethylsulfoxide (DMSO). Four serial 1:2 dilutions of the concentrated extracts in DMSO were performed in order to test five different concentrations of the extracts in the mutagenicity assays.
Mutagenicity assays
The Salmonella typhimurium His- reversion test (Ames test) was carried out with strain TA98, kindly provided by Prof. Bruce Ames. The preincubation procedure was performed both with and without S9 mix, prepared with the S9 fraction of Aroclor-induced Wistar rat liver homogenate (10%) following the method of Maron and Ames (1983). Fifty microliters of the different extract concentrations or the controls were tested in duplicate plates in each assay. A solvent control treated with DMSO and two positive controls [10 µg/plate 2-aminofluorene (2-AF) with metabolic activation and 20 µg/plate 4-nitro-o-phenylendiamine (NPD) without metabolic activation] were always included. After 48 h incubation at 37°C the revertant colonies were counted. Two independent assays were carried out for each experimental condition.
A sample was considered mutagenic if the number of revertants was at least 2-fold above the control level and if it produced a dose-related increase in the number of revertants with respect to the negative control.
Statistical analysis
The dose–response relationship for each mutagenicity assay was evaluated with a regression test. The comparisons between the number of revertants obtained under the different cooking conditions were performed by one-way and two-way ANOVA and using the Tukey test a posteriori. The computer program SPSS 7.0 was used for the calculations.
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Results |
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The general composition of the meat samples was determined before treatment and the weight loss was calculated after cooking (Table I
). During frying the oil temperature oscillated between +8 and –28°C from the initial frying temperature, both over short and long frying times (Table I). Positive results were obtained for all of the samples under the conditions assayed, always in the presence of S9. The linear regression test results were significant in each case and the correlation coefficients obtained were close to 1.
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Since the same quantity (50 g) of sample was used when extracting the mutagens, the comparisons between the different cooking conditions were carried out with the His+ revertant values obtained in the extract without dilution, i.e. the maximum value in each case. It was observed that as the cooking time was increased the number of revertants found also increased (Figure 1
). With a 10 min cooking time the average number of revertants was approximately four or five times the basal value (18 ± 8), except in the case of olive oil, for which it was eight times higher; with 20 and 30 min cooking time the values reached were approximately 10 times higher (Figure 1). In order to test the statistical differences between cooking times, a one-way ANOVA was applied and the differences between them were found to be very significant (0.006 > P > 0.0001). The Tukey test carried out a posteriori showed very significant differences between 10 and 30 min for all the cooking conditions, between 10 and 20 min after cooking in sunflower, marc olive oil, butter and without any fat, and between 20 and 30 min after frying in olive oil.
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In order to compare the results obtained with the different fats, the only cooking time studied was 10 min. A one-way ANOVA was performed and very significant differences were found among the different fats used (F = 7.200, P = 0.001). The Tukey test carried out a posteriori showed differences between olive oil and the rest of the fats used.
With respect to the frankfurters, the number of revertants obtained after the same heating treatment was much lower in comparison with hamburgers (Table II). A two-way ANOVA (brands and fats) was carried out. The interaction was non-significant (F = 0.760, P = 0.584), but very significant differences appeared between brands (F = 12.605, P = 0.0001) and between fats (F = 50.449, P = 0.0001).
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Discussion |
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Deep-frying in a large volume of olive oil, marc olive oil (`orujo') or sunflower oil is a common cooking practice in Spain and other Mediterranean countries. To our knowledge, the influence of such conditions on the mutagenic activity generated in meat samples has not been explored until now. In this work we have compared deep-frying with cooking without any fat or with some butter, both of which can be considered a more common cooking practice in northern Europe.
We have found that hamburgers fried in olive oil for 10 min show a significantly greater number of revertants than hamburgers under any other condition. This could be explained by the higher temperature reached by the olive oil, 10°C above the mean temperature maintained with the other oils and butter. In samples fried without any fat, although the range of temperature maintained during the thermal treatment was similar to that of olive oil, the number of revertants was lower, probably due to less efficient heat transmission in the absence of fat (Holtz et al., 1985; Nilsson et al., 1986; Övervik et al., 1987). The number of revertants obtained with the samples fried in sunflower oil were lowest, although the differences were not statistically significant except with olive oil. These results are in agreement with those of Johansson et al. (1995).
From the point of view of mutagenicity risk, frying in a large volume of vegetable oil could be considered safer than cooking directly in the pan or on a grill, because in the first case the temperature can be controlled by the cook, who will not surpass the oil smoke temperature (170–180°C). Also, for the same reason, sunflower oil could be considered better than olive oil, as the smoke temperature is 10°C lower. In contrast, a pan or grill can reach higher temperatures which are not controlled under normal home cooking conditions.
Longer heat treatments of 20 or 30 min produced an additional number of revertants in hamburgers fried in butter or in any oil, except olive oil. This difference could be explained by the higher stability of olive oil due to its lower content of polyunsaturated fatty acids (PUFAs) (11.2% in olive oil versus 50% in sunflower oil and 56.8% in soya bean oil). This is consistent with the fact that samples fried in sunflower oil and soya bean oil, those with a higher content of PUFAs, show a progressive and escalating increase in mutagenic activity with longer cooking times.
In this work, for hamburgers fried without any fat, the maximum number of revertants was obtained at 20 min. These results differ from those of Berg et al. (1990), where maximum mutagenic activity was found after 10 min frying of pork at 200°C. The difference may be attributed to the different temperature used, 10°C lower in our case.
We have found a much lower mutagenic activity in the frankfurter samples, which could possibly be attributed to their different compositions. These results coincide with those of Augustsson et al. (1999). Frankfurters have approximately half the level of protein and double the content of fat of hamburgers. The lower level of protein is directly related to the components of the Maillard reaction. With respect to the fat present in the meat samples, two opposite effects are possible. The fat may contribute to better transmission of heat and, therefore, would facilitate the formation of mutagens, but when it is present in high proportions it can also dilute the precursors of the Maillard reaction, thus inhibiting mutagenic activity.
The fats used to fry the frankfurters seem to modulate the mutagenic activity but the differences are not important nor consistent between brands. Nevertheless, the statistical test gave significant differences, probably because the data were very homogeneous, as can be deduced from the coefficients of variation.
In conclusion, several factors modulate the mutagenic activity generated in meat samples under thermal treatment. It is confirmed that the composition of the sample, together with the temperature of cooking, is one of the main factors to be taken into account. Deep-frying in a large volume of vegetable oil in the way it is done in Spain and other Mediterranean countries for normally used cooking times (
10 min) induces a considerable number of revertants: between 2- and 8-fold of the control, and similar to the number of revertants obtained when the same samples are fried without any fat or with a small amount of butter (a more common practice in northern countries). Frying in olive oil for normally used cooking times (10 min) can induce a higher number of revertants than cooking in sunflower oil, because the smoke temperature is higher and therefore the temperature reached in the meat is probably also higher.
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Acknowledgments |
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We thank Laura Stokes for her helpful assistance with production of the manuscript.
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Notes |
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1 To whom correspondence should be addressed. Tel: +34 948 425653; Fax: +34 948 425652; Email: acerain{at}unav.es
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References |
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Received on June 4, 2001; accepted on August 31, 2001.
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