Food Chemistry 68 (2000) 175178

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Inuence of the addition of antioxidants in vivo on the fatty acid composition of sh llets
L.S. Sant'Ana a,*, J. Mancini-Filho b
a

rio de Tecnologia de Produtos Agropecua rios, Faculdade de Ciencias Agrono micas, Universidade Estadual Paulista CAUNESP Laborato (UNESP), Botucatu, SP, Brazil b FCF/USP/Sa o Paulo, Brazil Received 2 March 1999; received in revised form 11 May 1999; accepted 1 June 1999

Abstract In this study, the inuence of the addition of antioxidants in vivo on the fatty acid composition of the esh of a freshwater sh known as ``pacu'' (Piaractus mesopotamicus) is veried. Four groups (one being the control group) of juvenile ``pacu'' were cultured on isocaloric and isoproteic diets. The lipid source was soybean oil and diets were added with either 100 ppm of a-tocopheryl acetate, or 100 ppm of BHT or 1.4 g of rosemary extract (Herbalox1)/kg diet. The fatty acid composition of the lipids of the dierent groups was determined before and after irradiation at 2 and 3 kGy, respectively, for the evaluation of the protective eects of the dierent antioxidants. Similarly, thiobarbituric acid reactive substances (TBARS) were determined from irradiated and nonirradiated samples. The results showed that the use of antioxidants altered the fatty acid composition of the llets. TBARS and irradiation conrmed their important role in protecting against lipid oxidation. Among all the antioxidants used, tocopherol was the most ecient, as shown by the highest percentage of polyunsaturated fatty acids (PUFA), by the lowest values of TBARS and by the analyses of the individual fatty acid levels at dierent irradiation doses. Signicant statistical dierences were observed only in 17% of the fatty acids in the llets of the groups. # 1999 Elsevier Science Ltd. All rights reserved.
Keywords: Antioxidants; Fatty acid; Freshwater sh; Lipid oxidation

1. Introduction Some of the major changes that occur during processing, distribution, and nal preparation of food are due to oxidation. Oxidation of lipids initiates other changes in the food system which aect nutritional quality, wholesomeness, safety, colour, avour, and texture (Shahidi & Wanansudara, 1992). Antioxidants are used by the food industry to delay the oxidation process (Brand-Williams, Cuvelier & Berset, 1995). One way to improve product stability prior to processing is by dietary manipulation. An example is the increase in the concentration of natural antioxidant vitamin E in the tissues through an elevated dietary supplementation (Gatlin, Bai & Erikson, 1992). The activity of an antioxidant can be estimated by quantitatively determining primary or secondary products
* Corresponding author. E-mail address: lssantana@laser.com.br (L.S. Sant'Ana).

from the autoxidation of lipids or by monitoring other variables, such as the studies on accelerated oxidation conditions or model systems tests (Shahidi & Wanasundara, 1992). Careful consideration concerning the applicability of the method to biological systems and data interpretation are essential, as the complexities of food and/or biological systems can often contradict the validity of the existing analytical systems (Namiki, 1990). Irradiation is a physical method of food processing consisting of exposing food to radiation during a limited period of time (Olszyna-Marzys, 1992). The autoxidative process induced by irradiation in fat is much the same as that which occurs without irradiation. However, with irradiation, it is quite accelerated (IGGFI, 1992). Irradiation continues to oer a well-known and very useful method of producing radicals and of studying their important reactions for biology, both in vitro and in vivo (Pryor, 1978).

0308-8146/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S0308-8146(99)00172-7

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L.S. Sant'Ana, J. Mancini-Filho / Food Chemistry 68 (2000) 175178

In the present study, we have veried the eciency of in vivo use of three antioxidants: a-tocopheryl acetate, 3,5-di-tert-butyl-4-hydroxytoluene (BHT) and rosemary extract Herbalox1. Irradiation was used to evaluate protective eects of all those dierent antioxidants. 2. Material and methods During 63 days of the most appropriate period JulySeptember (Carneiro, Chain & Dias, 1995), the experiments were carried out in the Aquaculture Centre in UNESP, Brazil. It was demonstrated that, from April to September, when the temperature was more agreeable, sh fed on pelleted diet gained more weight. With an average weight of 198.8939.6 g, 80 juvenile ``pacu'' (Piaractus mesopotamicus) were randomly divided into four groups of 20 sh, one group being control. The sh were cultured for 9 weeks (63 days) in 50 m2 tanks with brick walls and a ground earth bottom supplied with continuous water. The juvenile ``pacu'' were fed on isocaloric and isoproteic diets. The composition of the experimental diets was identical for all groups with the presence of added antioxidants in the dierent groups, except in the control. The lipid source was soybean oil and the diet for each group was added with either 100 ppm of a-tocopheryl acetate, or 100 ppm of 3,5-di-tert-butyl-4-hydroxytoluene (BHT) or 1.4 g of rosemary extract Herbalox1/Kg diet. Each sample was individually packed in a commercial polyethylene bag with a sealing clamp and maintained under iced conditions. They were transported to the irradiation facility Embrarad S. A., Sa o Paulo, cooled with packs of ice and irradiated to average doses of 2 and 3 kGy using a 60Co irradiator (Dose rate: 3 kGy/h). The TBARS were analysed from nonirradiated and irradiated samples to determine the extent of lipid oxidation on an acid extract, according to Wyncke (1970). The lipids were extracted by the method of Folch, Lee and Sloane Stanley (1957). The fatty acid composition was determined on the lipid extracts after methylation with sulfuric acid and ammonium chloride (Hartman & Lago, 1973). The fatty acid methyl esters (FAME) were analysed using a CG-500 chromatograph, equipped with fused silica capillary column Carbowax-20 M (30 m, 0.25 mm ID) and ame ionization detector (FID). Nitrogen was used as the carrier gas. Thermal gradient from 150 to 230 C at 6 C/min. Injector and FID temperature were 250 C. Heptadecanoic acid (C17, Sigma) was added to all samples as an internal standard before preparation of FAME. Statistical analyses were developed using a GraphPad Instat, 2.01 version, GraphPad Software. Dierences between mean numbers were determined by analysis of variance, followed by the Tukey test.

3. Results and discussion The fatty acid composition after dierent irradiation doses for each treatment is present in Tables 1, 2, 3 and 4. The results of the Tukey tests applied to individual fatty acids at dierent irradiation doses demonstrated signicant dierences in 75% of the fatty acids in the control group. The Tukey test applied to the groups with BHT or rosemary extract Herbalox1 in the diet showed signicant dierence in 42% of the fatty acids. However, for the group with tocopherol, only 17% of fatty acids had a signicant dierence. Similar work on antioxidant activities of sage and rosemary extracts and tocopherol has been done in a model meat system showing that all three antioxidants were eective as antioxidants, although vitamin E was more eective than the plant extracts (Wong, Hashimoto & Shibamoto, 1995). Saturated fatty acid (SFA), monounsaturated fatty acid (MUFA) and polyunsaturated fatty acid (PUFA) revealed similar results for the dierent antioxidants. In the control group dierences were observed between the irradiation doses for SFA, MUFA and PUFA, whereas no signicant dierences were observed for the tocopherol group in any class of fatty acid. Similarly, n-3, n6 and n-9 fatty acid showed the same results.

Table 1 Fatty acid composition of ``pacu'' esh fed (control diet) before and after irradiationa Fatty acid 14:0 16:0 16:1 18:0 18:1 n-9 18:2 n-6 18:3 n-3 20:3 n-6 20:4 n-6 20:5 n-3 22:5 n-6 22:6 n-3 SFAc MUFAd PUFAe n9 n6 n3
a b

0 kGyf 2.060.07a 18.000.06a 4.440.22a 7.840.06 39.860.19 21.540.07a 1.400.06 1.030.01a 2.060.02a 0.220.01a 0.750.03a 0.820.02a 27.900.10a 44.301.18a 27.810.09a 39.860.19 25.380.10a 2.430.05a

2 kGyf 2.520.23b 20.771.58b 5.880.22b 9.480.96 37.792.18 17.850.12b 1.450.15 0.860.07b 1.730.19b 0.290.04b 0.640.09a 0.710.18a 32.772.77b 43.672.40b 23.550.36b 37.792.18 21.080.47b 2.470.12a

3 kGyf 2.280.15a,b 19.770.39a,b 6.180.33b 8.220.60 39.701.28 20.870.51c 1.460.02 0.660.03c 1.050.07c 0.170.02a 0.300.01b 0.290.02b 30.281.12a,b 45.881.57a 24.200.47b 39.701.28 22.290.48c 1.920.05b

AVb * * *** NS NS *** NS *** *** ** *** ** * *** *** NS *** ***

Meanstandard deviation (n=31). AV, analysis of variance, NS=not signicant *p<0.05 **p<0.01 ***p<0.001. c SFA, Saturated fatty acid. d MUFA, Monounsaturated fatty acid. e PUFA, Polyunsaturated fatty acid. f Within each row, means followed by dierent letters are signicantly dierent. TukeyKramer test (p<0.05).

L.S. Sant'Ana, J. Mancini-Filho / Food Chemistry 68 (2000) 175178 Table 2 Fatty acid composition of ``pacu'' esh fed (BHT diet) before and after irradiationa Fatty acid 14:0 16:0 16:1 18:0 18:1 n-9 18:2 n-6 18:3 n-3 20:3 n-6 20:4 n-6 20:5 n-3 22:5 n-6 22:6 n-3 SFAd MUFAe PUFAf n9 n6 n3
a b

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Table 4 Fatty acid composition of ``pacu'' esh fed (tocopherol) diet before and after irradiationa Fatty acid 14:0 16:0 16:1 18:0 18:1 n-9 18:2 n-6 18:3 n-3 20:3 n-6 20:4 n-6 20:5 n-3 22:5 n-6 22:6 n-3 SFAc MUFAd PUFAe n9 n6 n3
a b

0 kGyg 2.660.29 21.270.55 2.950.42 9.260.24 35.471.19 18.890.22 1.600.06 1.020.09a 2.590.16a 0.410.03a 0.920.14a 1.000.02a 33.190.85 40.421.08 26.412.23a 35.471.68 23.400.25a 3.010.02a

2 kGyg 2.400.24 20.250.53 4.830.31 8.900.64 36.820.89 19.380.31 1.630.21 0.970.08a,b 2.530.13a 0.380.04a 0.870.17a 1.060.10a 31.551.32 41.651.10 26.800.50a 36.820.89a 23.740.47a 3.060.17a

3 kGyg 2.640.10 21.030.01 5.410.27 9.660.13 36.970.29 19.200.08 1.470.04 0.800.05b 1.540.22b 0.240.01b 0.490.08b 0.570.14b 33.330.09 42.380.54 24.310.54b 36.970.29b 22.030.42b 2.280.12b

AVb NSc NS NS NS NS NS NS * *** * ** ** NS NS *** NS ** ***

0 kGyf 2.140.06 20.180.99 4.910.25 8.540.89 36.151.54 19.270.73 1.350.07 1.210.12 3.400.79a 0.340.05 1.300.19a 1.300.11 30.861.91 41.061.78 28.101.39 36.151.54 25.121.42 2.990.10

2 kGyf 2.340.09 21.100.40 4.880.21 8.990.98 36.470.39 19.230.27 1.280.07 1.090.14 2.510.33a,b 0.300.03 0.850.19b 0.970.15 32.421.44 41.360.49 26.230.97 26.470.39 23.680.92 2.550.06

3 kGyf 2.200.16 20.870.56 5.130.26 7.970.41 37.830.27 19.421.07 1.470.10 1.010.06 2.090.12b 0.340.08 0.680.03b 1.000.016 31.050.81 42.950.08 26.020.24 37.8300.27 23.200.98 2.820.29

A.V.b NS NS NS NS NS NS NS NS * NS ** NS NS NS NS NS NS NS

Meanstandard deviation (n=31). AV, analysis of variance. c NS, not signicant *p<0.05 **p<0.01 ***p<0.001. d SFA, Saturated fatty acid. e MUFA, monounsaturated fatty acid. f polyunsaturated fatty acid. g Within each row, means followed by dierent letters are signicantly dierent. Tukey-Kramer test (p<0.05).

Meanstandard deviation (n=31). AV, analysis of variance, NS, not signicant. c SFA, saturated fatty acid. d MUFA, monounsaturated fatty acid. e PUFA, polyunsaturated fatty acid. f Within each row, means followed by dierent letters are signicantly dierent. TukeyKramer test (p<0.05).

Table 3 Fatty acid composition of ``pacu'' esh fed (rosemary extract Herbalox1 diet) before and after irradiationa Fatty acid 14:0 16:0 16:1 18:0 18:1 n-9 18:2 n-6 18:3 n-3 20:3 n-6 20:4 n-6 20:5 n-3 22:5 n-6 22:6 n-3 SFAc MUFAd PUFAe n9 n6 n3
a b

0 kGyf 2.520.23 20.771.58 4.880.22a 9.480.96 37.792.18 18.850.13 1.450.15 0.860.07a 1.730.19a 0.290.04 0.640.09a 0.740.09a 32.772.77 43.672.40 23.550.37a 37.792.18 21.080.47a 2.470.11a

2 kGyf 2.360.12 20.730.11 5.360.57a,b 8.891.09 37.940.32 19.160.35 1.590.20 0.830.01a,b 1.700.07a 0.280.07 0.590.02a 0.380.10b 31.990.95 43.300.25 24.740.70b 37.940.32a 22.270.42b 2.460.28a

3 kGyf 2.310.11 20.080.31 4.740.12b 9.710.26 39.550.47 19.250.40 1.390.10 0.740.03b 1.340.10c 0.160.01 0.360.02b 0.590.02a 32.110.43 44.290.59 23.600.20a,b 39.550.47b 21.680.26a,b 1.920.20b

AVb NS NS * NS NS NS NS * * NS ** ** NS NS * NS * *

meanstandard deviation (n=3.1). AV, Analysis of variance, NS, not signicant *p<0.05 **p<0.01 ***p<0.001. c SFA, saturated fatty acid. d MUFA, monounsaturated fatty acid. e PUFA, polyunsaturated fatty acid. f Within each row, means followed by dierent letters are signicantly dierent. TukeyKramer test (p<0.05).

In the tocopherol group, a signicant dierence was observed only in the highly unsaturated fatty acid. Wolters, Tilbury and Konings (1987) suggested that fatty acyl chains containing more double bonds were more radiosensitive. Oxidative changes in sh esh are quantied by the measurement of TBARS, in nonirradiated and irradiated samples. The results are shown in Table 5. The values of TBARS evaluated in mg malonaldehyde/g sh llet, and the standard curve were y=0.168(r=0.9968). The data show that TBARS in the sh esh of control, BHT and rosemary extract had statistically signicant dierences between nonirradiated and irradiated samples. However, for the tocopherol group a signicant dierence was observed only at the 3 kGy dose. These results suggest that the tocopherol had the best protection against oxidation when compared with the eects of other antioxidants. Buckley and Morrissey (1992) suggest that because a-tocopherol is an integral part of the membrane, it consequently stabilises membrane lipids. Galvin, Morrissey and Buckley (1998) suggests that in chicken, supplementation at levels of at least 200 mg of a-tocopheryl acetate/kg diet is required to stabilise irradiated breast and thigh meat (dose 2.5 and 4 kGy) following cooking and during storage.

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L.S. Sant'Ana, J. Mancini-Filho / Food Chemistry 68 (2000) 175178 dutivo de pacu, Piaractus mesopotamicus (Holmberg, 1887). Publ. ACIESP, 84, 1320. Folch, J., Lee, M., & Sloane Stanley, G. H. (1957). A simple method for isolation and purication of total lipids from animal tissue. J. Biol. Chem., 226, 497509. Frigg, M., Prabuck, A. L., & Ruhdel, E. U. (1990). Eect of dietary vitamin E levels on oxidative stability of trout llets. Aquaculture, 84, 145158. Galvin, K., Morrissey, P. A., & Buckley, D. J. (1998). Eect of dietary a-tocopherol supplementation and gamma-irradiation on a-tocopherol retention and lipid oxidation in cooked minced chicken. Food Chemistry, 62, 185190. Gatlin III, D. M., Bai, S. C., & Erickson, M. C. (1992). Eects of dietary vitamin E and synthetic antioxidants on composition and storage quality of channel catsh, Ictalarus punctatus. Aquaculture, 106, 323332. Hartman, L., & Lago, B. C. A. (1973). Rapid preparation of fatty, methyl esters from lipids. Lab. Pract., 22, 457477. Hampson, J. W., Fox, J. B., Lakritz, L., & Thayer, D. W. (1996). Eect of low dose gamma radiation on lipids in ve dierent meats. Meat Science, 42, 271276. ICGFI DOCUMENT 14. (1992). Training manual on operation of food irradiation facilities (4th. ed., pp. 132). FAO/IAEA/WHO. Namiki, M. (1990). Antioxidants/antimutagens in food. Crit. Rev. Food Sci. Nutr., 29, 273300. O'Keee, T. M., & Noble, R. L. (1978). Storage stability of channel catsh (Ictalurus punctatus) in relation to dietary level of a-tocopherol. J. Fish. Res. Board Can., 35, 457460. Olszyna-Marzys, A. E. (1992). Radioactivity and food preservation. Nutr. Rev., 50, 162165. Pryor, W. A. (1978). The formation of free radicals and the consequences of their reaction in vivo. Photochem. Photobiol., 28, 787801. Shahidi, F., & Wanasundara, P. D. (1992). Phenolic antioxidants. Crit. Rev. Food Sci. Nutr., 32, 67103. Wolters, H., Tilburg, C. A. M., & Konings, W. T. (1987). Radiation-induced lipid peroxidation: inuence of oxygen concentration and membrane lipid composition. Int. J. Radiat. Biol., 51, 619 628. Wong, J. W., Hashimoto, K., & Shibamoto, T. (1995). Antioxidant activities of rosemary and sage extracts and vitamin E in a model meat system. J. Agric. Food Chem., 43, 27072712. Wyncke, W. (1970). Direct determination of the thiobarbituric acid value in trichloroacetic acid extracts of sh as a measure of oxidative rancidity. Fette-Scifen Anstrichmittel, 72, 10841087 .

Table 5 TBARS levels (mg malonaldehyde/g sh llet) in irradiated and nonirradiated sha,c
Treatment Control Tocopherol BHT Rosemary extract
a b

0 kGy 0.4010.048a 0.1760.055a 0.2000.037a 0.1350.050a

2 kGy 0.6810.092b 0.3640.098a,b 0.3490.014b 0.3880.111b

3 kGy 0.6340.091b 0.4600.103b 0.3930.058b 0.4620.076b

AVb * * ** **

Meansstandard deviation (n=3). AV, analysis of variance. c Within each row, means followed by dierent letters are signicantly dierent (p<0.05).

Various researchers have also found that feed which includes tocopherol supplementation decreases lipid oxidation in the esh of various sh species, such as trout (Frigg, Prabuck & Ruhdel, 1990) and channel catsh (Gatlin III et al., 1992; O'Keefe & Noble, 1987). Hampson, Fox, Lakritz and Thayer (1996) showed a dose dependence of malonaldehyde production due to irradiation in pork, lamb, beef and turkey muscle. However, no signicant statistical dierence was found in the range 010 kGy. According to the results, the use of antioxidants was important in protecting against lipid oxidation and of all the three antioxidants used in this trial, tocopherol was the most ecient. References
Brand-Williams, W., Cuvelier, M. E., & Berset, C. (1995). Use of a free radical method to evaluate antioxidant activity. Food Science and Technology, 28, 2530. Buckley, D. J., & Morrissey, P. A. (1992). Vitamin E and meat quality. La Roche: F. Homann. Carneiro, D. J., Chaim, S. H. S., & Dias, T. C. R. (1995). Efeito do processamento de dietas comerciais so bre o desenvolvimento pro-