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Radiation Physics and Chemistry 75 (2006) 297300 www.elsevier.com/locate/radphyschem

Effect of irradiation and storage on the antioxidative activity of cashew nuts

M.G. Sajilata, R.S. Singhal
Food and Fermentation Technology Department, Institute of Chemical Technology, University of Mumbai, Matunga, Mumbai 400019, India Received 7 February 2005; accepted 24 July 2005

Abstract Food irradiation, a cold process employed for preservation of food has been studied extensively for its benecial and undesirable effects on food constituents. Since nuts have been shown to contain several antioxidants, and ionizing irradiation is known to result in the formation of free radicals, investigation on the antioxidative potential of cashew nuts after irradiation and subsequent storage was undertaken by assessing their ability to inhibit lipid peroxidation using the 1,3-diethyl-2-thiobarbituric acid (DETBA) assay. Irradiation at 0.251.00 kGy and subsequent storage was found to considerably reduce antioxidative activity in the cashew nuts. r 2005 Elsevier Ltd. All rights reserved.
Keywords: Irradiation; Cashew nuts; Antioxidative activity; Storage

1. Introduction Food irradiation is a physical process involving treatment of food with ionizing radiation that can effectively delay a number of problems such as ripening of fruits, bacterial growth, insect infestation and reduced shelf life. The forms of ionizing energy, which may be used in food processing, include g-rays (from 60Co or 137 Cs), X-rays and accelerated electrons (electron beams) (Kader, 1986). An undesirable effect of irradiation is the formation of lipid oxides by the reaction of membrane lipids and other lipids in foods with oxygen radicals produced by g-rays (Ahn et al., 1998). Reducing oxygen and temperature levels can decrease formation of these oxides during irradiation. g-rays interact directly with lipid molecules to form cation radicals or excited lipid
Corresponding author. Fax: +91 22 24145614.

E-mail address: rekha@udct.org (R.S. Singhal).

molecules. These products may generate lipid oxides and small amounts of fatty acids, aldehydes, esters, ketones and other compounds. If the radiolytic compounds from saturated and unsaturated compounds are compared, it can be seen that the major hydrocarbons, fatty acids and symmetric ketones are produced in lesser quantities from the unsaturated compounds (Vajdi and Nawar, 1978). More recently, interest in the adverse biological effects of free radicals has encouraged research on antioxidant vitamins and other constituents of plants that act as antioxidants in the body. There is overwhelming evidence to indicate that free radicals cause oxidative damage to lipids, proteins and nucleic acids. Nuts contain many different antioxidative components. Besides vitamin A, vitamin C and b-carotene, nuts are also known to contain antioxidant phytochemicals such as avonoids, phenolic compounds, luteolin, tocotrienols, isoavones, ellagic acid as well as other components like plant sterols (Rainey and Nyquist, 1997). Some

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avonoids such as catechin are reported to demonstrate strong antioxidative activities (Bors et al., 1990). Previous work from our institute has shown irradiation at 0.251.00 kGy to arrest insect infestation in cashew nuts (Bhattacharjee et al., 2003a, b). Since ionizing radiation is known to produce free radicals and antioxidants have been reported to protect foods from oxidative damage, it was of interest to study the effect of irradiation and subsequent storage on the antioxidative potential of cashew nuts. Any decrease in antioxidative activity could be correlated with the scavenging of free radicals, produced on irradiation, by the naturally occurring antioxidants in cashew nuts. A simple uorometric method, which could quickly estimate the antioxidative activity of ethanol extracts of samples, was used to evaluate the antioxidative activity of nuts (Furuta et al., 1997).

2. Materials and methods 2.1. Raw materials Cashew nuts were procured from the local market of Mumbai city. 2.2. Chemicals and reagents 1,3-diethyl-2-thiobarbituric acid (DETBA) was obtained from Aldrich Chemical Company Inc, Milwaukee, WI, USA. Linoleic acid was obtained from Sisco Research Laboratories, Mumbai. SDS and vitamin C were procured from S.D. Fine Chemicals Ltd., Mumbai. All other chemicals were of analytical grade. 2.3. Irradiation of cashew nuts Twenty-ve gram cashew nuts packed in heat-sealed polyethylene pouches were placed in Al containers and passed by a 60Co source for irradiation at dosages of 0.25, 0.5, 0.75 and 1 kGy using the Food Package Irradiator at BARC, Mumbai. The dose was controlled by the exposure time of each container to the source. The temperature and dose rate for all samples were 30 1C and 28 Gy/min, respectively. The dose range within the samples was 720% of the actual dose. The control and irradiated samples were stored in plastic containers at room temperature (2830 1C) under identical conditions. 2.4. Assessment of antioxidative activity A uorometric assay for evaluating antioxidative activities was principally based on inhibition of lipid peroxidation accompanied by autoxidation of linoleic

acid (Furuta et al., 1997). The standard procedure used with few modications was as follows: An extract of the powdered sample was prepared in 80% ethanol using a cyclomixer, followed by centrifugation at low temperature (8 1C) and re-extraction and centrifugation, and then diluting the supernatant to 25 ml with 80% ethanol. To 50200 mL of the extract in a test tube, 200 mL of linoleic acid (20 mg/100 mL absolute alcohol) and required quantity of diluent was added so as to make the nal volume to 400 mL. This was incubated at 80 1C for 1 h. To stop the autoxidation of linoleic acid, incubated samples were cooled in an ice bath and to them were sequentially added 200 mL BHA (36 mg/10 mL absolute ethanol), 400 mL ascorbic acid (80 mg/25 mL water) and 200 mL 8% SDS. BHA and ascorbic acid were used to minimize oxidation during the DETBA test. SDS was used to increase the afnity between linoleic acid and other reagents. The degree of autoxidation was measured by the DETBA test (Suda et al., 1994). Briey, 3.2 mL of 12.5 mM DETBA in a sodium phosphate buffer was added to the autoxidized samples. The solution was mixed and heated at 9095 1C for 10 min and then cooled in an ice bath. To extract the DETBA-reactive substance, 8 mL of ethyl acetate was added and the mixture was shaken on a cyclomixer. The supernatant was pipetted out and passed through a bed of sodium sulfate and the uorescence intensity of the ethyl acetate layer was measured at an excitation wavelength of 515 nm and an emission wavelength of 555 nm using a Perkin Elmer uorometer (Model L 30). A control containing no additives represented 100% lipid peroxidation and was used as a blank. Lipid peroxidation was calculated using the following formula: % lipid peroxidation Sample reading 100. Blank reading

A low lipid peroxidation level indicated a high antioxidative activity. All data are expressed as mean7SD of values from three independent replications of the experiment.

3. Results and discussion Fig. 1 shows the extent of lipid peroxidation brought about by different concentrations of extracts from unirradiated cashew nuts and that after immediate irradiation at 0.251.00 kGy. Fig. 2 shows similar data after 2 months of storage at a room temperature of 2830 1C. Data were also collected after 4 and 6 months of storage (gures not shown). In order to understand the effect of irradiation on the antioxidant activity, regression equations correlating percentage of lipid peroxidation with the concentration of the nut extract was developed, from which the concentration required to

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M.G. Sajilata, R.S. Singhal / Radiation Physics and Chemistry 75 (2006) 297300
Control 70 0.25 kGy 0.5 kGy 0.75 kGy 1 kGy

299

60

50

40

30

20 0 500 1000 1500 2000 2500 3000 3500 4000 4500

Cashew nut extract (ppm)

Fig. 1. Percent lipid peroxidation brought about by different concentrations of cashew nut extracts from unirradiated and irradiated cashew nuts at 0 months of storage. Results are expressed as mean7SD%.

90 80 70

control

0.25 kGy

0.5 kGy

0.75 kGy

1 kGy

60 50 40 30 20 10 0 0 5000 10000 15000 20000 25000 30000

inhibit 50% lipid peroxidation was calculated. This was done for control cashew nuts and those irradiated at 0.251.00 kGy for the entire storage period of 6 months. The results are compiled in Table 1. As can be seen, the antioxidative activity of cashew nuts decreased immediately on irradiation and further decreased on subsequent storage. The decrease in antioxidant activity in irradiated cashew nuts could be potentiated by the combined effects of irradiation and storage at room temperature in air. Irradiation is known to produce free radicals, and antioxidants from foods have been shown to scavenge free radicals. In the present work, immediately after irradiation, there was a decline in antioxidative activity, which further decreased during storage. Since cashew nuts are rich in fats (47%) and proteins (20%), the stable radicals could be contained chiey in the lipid and protein fractions. Fats are particularly susceptible to changes in odor and avor. The food processing industry has been able to resolve some of the issues relating to changes in the quality of foods by lowering processing temperatures to refrigeration or freezing levels, and/or by excluding oxygen (Diehl, 1979) from foods to be processed with the use of vacuum packaging or packaging in nitrogen atmosphere. Cashew nuts are reported to contain antioxidants such as vitamin E. Tocopherols have been shown to be very sensitive to irradiation in the presence of oxygen, which could be contributing to the decrease in antioxidant activity (Urbain, 1986). Diehl (1981) reported the loss of atocopherol in stored irradiated (1 kGy) rolled oats to be greater than that in non-irradiated oats under the same conditions. The substantial loss of antioxidative activity shown by the nuts after irradiation and subsequent storage could be ascribed to free radical scavenging by the naturally occurring antioxidants, and also loss of vitamin E due to storage in the presence of oxygen.

% lipid peroxidation

% lipid peroxidation

Cashew nut extract (ppm)

Fig. 2. Percent lipid peroxidation brought about by different concentrations of cashew nut extracts from unirradiated and irradiated cashew nuts after 2 months of storage. Results are expressed as mean7SD%.

4. Conclusion Irradiation and subsequent storage of cashew nuts showed a decrease in antioxidative activity indicating free radical damage.

Table 1 Concentration of control and irradiated cashew nuts required to effect 50% lipid peroxidation Storage time (months) Irradiation dose (kGy) Control 0 2 4 6 1045 3144 4128 5198 0.25 1311 6820 8367 8419 0.5 1765 10 156 11 235 14 285 0.75 2417 13 157 11 740 14 513 1 2833 14 500 13 005 14 125

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300 M.G. Sajilata, R.S. Singhal / Radiation Physics and Chemistry 75 (2006) 297300 exclusion of atmospheric oxygen. Z. Lebensm. Unters. Forsch. 169, 276280. Diehl, J.F., 1981. Effects of combination processes on the nutritive value of food. In: Combination Processes in the Food Irradiation. International Atomic Agency, Vienna, Austria, pp. 349366. Furuta, S., Nishiba, Y., Suda, I., 1997. Fluorometric assay for screening antioxidative activity of vegetables. J. Food Sci. 62 (3), 526528. Kader, A.A., 1986. Potential applications of ionizing radiation in postharvest handling of fresh fruits and vegetables. Food Technol. 40 (6), 117121. Rainey, C., Nyquist, L., 1997. Nutsnutrition and health benets of daily use. Nutr. Today 32, 157163. Suda, I., Furuta, S., Nishiba, Y., 1994. Fluorometric determination of 1,3-diethyl-2-thiobarbituric acid-malondialdehyde adduct as an index of lipid peroxidation in plant materials. Biosci. Biotechnol. Biochem. 58 (1), 16061608. Urbain, W.M., 1986. Radiation chemistry of food components and of foods. In: Food Irradiation. Academic Press Inc., Harcourt Brace Jovanonich Publishers, New York, pp. 3782. Vajdi, M., Nawar, W.W., 1978. Comparison of radiolytic compounds from saturated and unsaturated triglycerides and fatty acids. J. Am. Oil Chem. Soc. 55, 849850.

Acknowledgements The authors gratefully acknowledge Dr. Arun Behere, Food Technology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400085, for arranging for irradiation of cashew nuts.

References
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