DNA comet assay: A simple screening technique for identification of some irradiated foods

DNA Comet Assay method was carried out to detect irradiation treatment of some foods like meat, spices, beans and lentils. The fresh meat of cow and duck were irradiated up to radiation doses of 3 kGy, the spices (cardamoms and cumin black) were irradiated to radiation doses of 5, 10, 15 and 20 kGy while the beans (black beans and white beans) and lentils (red and green lentils) were irradiated to 0.5 and 1 kGy. All the foods were then analyzed for radiation treatment using simple microgel electrophoresis of single cells or nuclei (DNA Comet Assay). Sedimentation, lysis and staining times were adjusted to get optimized conditions for correct and easy analysis of each food. Using these optimized conditions, it was found out that radiation damaged DNA showed comets in case of irradiated food samples, whereas in non-treated food samples, round or conical spots of stained DNA were visible. Shape, length and intensity of these comets were also radiation dose dependent. Screening of unirradiated and irradiated samples by Comet Assay was successful in the case of all the foods under consideration under the optimized conditions of assay. Therefore, for different kinds of irradiated foods studied in the present study, the DNA Comet Assay can be used as a rapid, simple and inexpensive screening test.     

Detection of irradiated food: DNA fragmentation in grapefruits

Employing the simple microgel electrophoresis of single cells - ‘comet assay’ - on grapefruit seeds enabled a rapid identification of irradiated fruits. Fruits were exposed to radiation doses of 0, 0.1, 0.2, 0.3, 0.4 and 0.5 kGy covering the range of potential commercial irradiation for insect disinfestation and quarantine purposes. Seeds were isolated, crushed, and the cells embedded in an agarose layer. After lysis of the cells, they were subjected to microgel electrophoresis for 2.5 minutes, and then stained. Fruits irradiated with 0.2 kGy and higher doses showed typical DNA fragmentation, the DNA fragments stretching or migrating out of the cells forming a tail towards the anode, giving the damaged cells an appearance of a comet. With increasing dose a longer extension of the DNA from the nucleus towards the anode is observed. Undamaged cells will appear as intact nuclei without tails. The DNA comet assay is thus a rapid and inexpensive screening technique to detect irradiated grapefruits. Suspected samples may subsequently be analysed by officially validated methods for detection of irradiated foods.     

Detection of radiation treatment of beans using DNA comet assay

A simple technique of microgel electrophoresis of single cells (DNA Comet Assay) enabled a quick detection of radiation treatment of several kinds of leguminous beans (azuki, black, black eye, mung, pinto, red kidney and white beans). Each variety was exposed to radiation doses of 0.5, 1 and 5 kGy covering the permissible limits for insect disinfestation. The cells or nuclei from beans were extracted in cold PBS, embedded in agarose on microscope slides, lysed between 15 and 60 min in 2.5% SDS and electrophoresis was carried out at a voltage of 2 V/cm for 2–2.5 min. After silver staining, the slides were evaluated through an ordinary transmission microscope. In irradiated samples, fragmented DNA stretched towards the anode and the damaged cells appeared as a comet. The density of DNA in the tails increased with increasing radiation dose. However, in non-irradiated samples, the large molecules of DNA remained relatively intact and there was only minor or no migration of DNA; the cells were round or had very short tails only. Hence, the DNA comet assay provides an inexpensive, rapid and relatively simple screening method for the detection of irradiated beans.     

Detection of irradiated quail meat by using DNA comet assay and evaluation of comets by image analysis

A simple technique of microgel electrophoresis of single cells (DNA comet assay) was used to detect DNA comets in irradiated quail meat samples. Obtained DNA comets were evaluated by both photomicrographic and image analysis. Quail meat samples were exposed to radiation doses of 0.52, 1.05, 1.45, 2.00, 2.92 and 4.00 kGy in gamma cell (gammacell ⁶⁰Co, dose rate 1.31 kGy/h) covering the permissible limits for enzymatic decay and stored at 2 °C. The cells isolated from muscle (chest, thorax) in cold PBS were analyzed using the DNA comet assay on 1, 2, 3, 4, 7, 8 and 11 day post irradiation. The cells were lysed between 2, 5 and 9 min in 2.5% SDS and electrophorosis was carried out at a voltage of 2 V/cm for 2 min. After propidium iodide staining, the slides were evaluated through a fluorescent microscope. In all irradiated samples, fragmented DNA stretched towards the anode and damaged cells appeared as a comet. All measurement data were analyzed using BS 200 ProP with software image analysis (BS 200 ProP, BAB Imaging System, Ankara, Turkey). The density of DNA in the tails increased with increasing radiation dose. However, in non-irradiated samples, the large molecules of DNA remained relatively intact and there was only minor or no migration of DNA; the cells were round or had very short tails only. The values of tail DNA%, tail length and tail moment were significantly different and identical between 0.9 and 4.0 kGy dose exposure, and also among storage times on day 1, 4 and 8. In conclusion, the DNA Comet Assay EN 13784 standard method may be used not only for screening method for detection of irradiated quail meat depending on storage time and condition but also for the quantification of applied dose if it is combined with image analysis. Image analysis may provide a powerful tool for the evaluation of head and tail of comet intensity related with applied doses.     

Identification of irradiation treatment of spices by thermoluminescence of contaminating minerals

Five spices, cumin, coriander, clove, cinnamon and black pepper were irradiated by gamma-ray doses of 1.0 and 5.0 kGy and thermoluminescence (TL) method was used for identification of the irradiation treatment. The TL response of the minerals isolated from irradiated samples was much higher as compared to the mineral particles from unirradiated control samples. For the normalisation of results the separated minerals were reirradiated to a normalisation dose of 1.0 kGy and the TL glow curve was recorded a second time. By comparing the glow curves of irradiated and unirradiated samples, finding the ratio of the areas of first and second glow curves (TL₁/TL₂) and comparing the shapes of the glow curves, all the irradiated and unirradiated samples were identified correctly.     

Application of different techniques to identify the effects of irradiation on Brazilian beans after six months storage

Four different techniques to detect the effect of irradiation in beans were investigated. Two types of Brazilian beans, Phaseolus vulgaris L., var. carioca and Vigna unguiculata (L.) Walp, var. macaçar, were irradiated using a ⁶⁰Co source with doses ranging from 10.0 kGy. After 6 months storage at ambient temperature the detection tests were carried out. Firstly, germination tests showed markedly reduced root growth and almost totally retarded shoot elongation of irradiated beans as compared to non-irradiated beans. Secondly, DNA fragmentation was studied using a microgel electrophoresis. Irradiated cells produced typical comets with DNA fragments migrating towards the anode. DNA of non-irradiated cells exhibited a limited migration. Thirdly, electron spin resonance for detection of cellulose radicals was tested, since it was expected that these free radicals are quite stable in solid and dry foods. However, only in beans irradiated with 10 kGy a small signal could be detected. Fourtly, thermoluminescence, a method to analyze mineral debris adhering to food, turned out to be a good choice to detect irradiation effects in beans, even after 6 months of storage. The results indicate that three of these four techniques proposed, can be used to detect the effect of irradiation in these two varieties of Brazilian beans at a dose level useful for insect disinfestation (1 kGy).     

Identification of irradiated pulses by thermoluminescence of the contaminating minerals

Thermoluminescence (TL) response of contaminating minerals from six samples of pulses commonly consumed in Pakistan has been studied for identification of irradiation treatment. The samples were irradiated by Co-60 gamma rays at 0.3, 0.5 and 1.0 kGy, or by 10 MeV electrons using an accelerator at 0.75 and 2.2. kGy. Generally, the TL intensity for minerals separated from irradiated samples was higher than for unirradiated samples. To normalize the results, separated minerals deposited on stainless steel discs were re-irradiated by a normalizing dose and TL response was redetermined. The ratio of the area of the first glow curve to the second glow curve was more than 0.8 for all irradiated samples and less than 0.33 for most of the unirradiated samples. For those unirradiated samples where the ratio of the glow curves was more then 0.03, the shapes of the glow curves were compared. Taking this criterion into consideration, all 21 unirradiated and irradiated samples of pulses were identified correctly. Therefore, a normalization procedure by re-irradiation of minerals and analysis of TL glow curve shapes lead to unequivocal identification of radiation treatment of pulses.     

Thermoluminescence of contaminating minerals for the detection of radiation treatment of dried fruits

Several types of dry fruits (pistachio nut, dried apricot, almond and raisins) have been investigated for detection of their radiation treatment by gamma rays or electron beam using thermoluminescence (TL) measurements. These samples were irradiated to 1.0–3.0 kGy (gamma rays) or 0.75–3.9 kGy (10 MeV electron beam). Thermoluminescence glow curves for the contaminating minerals separated from the dry fruits were recorded between the temperature range of 50°C and 500°C. In all the cases, the intensity of TL signal for the irradiated dry fruits was 1–3 orders of magnitudes higher than the TL intensity of the corresponding unirradiated control samples allowing clear distinction between the irradiated and unirradiated samples. These results were normalized by re-irradiating the mineral grains with a gamma-ray dose of 1.0 kGy, and a second glow curve was recorded. The ratio of intensity of the first glow curve (TL₁) to that after the normalization dose (TL₂), i.e. (TL₁/TL₂) was determined and compared with the recommended threshold values. These parameters, together with comparison of the shape of the first glow curve, gave unequivocal results about the radiation treatment of the dry fruit samples.     

Thermal analysis applied to irradiated propolis

Propolis is a resinous hive product, collected by bees. Raw propolis requires a decontamination procedure and irradiation appears as a promising technique for this purpose. The valuable properties of propolis for food and pharmaceutical industries have led to increasing interest in its technological behavior. Thermal analysis is a chemical analysis that gives information about changes on heating of great importance for technological applications. Ground propolis samples were ⁶⁰Co gamma irradiated with 0 and 10 kGy. Thermogravimetry curves shown a similar multi-stage decomposition pattern for both irradiated and unirradiated samples up to 600°C. Similarly, through differential scanning calorimetry , a coincidence of melting point of irradiated and unirradiated samples was found. The results suggest that the irradiation process do not interfere on the thermal properties of propolis when irradiated up to 10 kGy.     

Antimicrobial properties of natural substances in irradiated fresh poultry

This study was undertaken to determine if a combined treatment (marinating in natural plant extracts or vacuum) with irradiation could have a synergetic effect, in order to reduce the dose required for complete elimination of Salmonella on fresh poultry. The effect of these combined treatments on the shelf-life extension was also evaluated. The fresh chicken legs were irradiated at 0, 3 and 5 kGy. The poultry underwent microbial analysis(mesophilic and Salmonella detection). For each treatment, the total microbial count decreased with increase of irradiation dose. The marinating treatment have a synergistic effect with irradiation treatment to reduce the total microbial count and controlling the proliferation during storage at 4°C. Irradiation of fresh chicken pieces with a dose of 3 kGy appears to be able to extend the microbial shelf-life by a factor of 2. When the chicken is marinating and irradiated at 3 kGy or when irradiated at 5 kGy without marinating, the microbial shelf-life is extended by a factor of 7 to 8. No Salmonella was found during all the experiment in the chicken in air and marinated. However, a presence of Salmonella was found in samples irradiated at 5 kGy under vacuum, in unirradiated samples and samples irradiated at 3kGy in air and under vacuum.     

Investigation of the irradiation history of the Iranian dates and pistachio nuts using thermoluminescence technique

Three different varieties of Iranian fresh dates and five types of raw and salted pistachio nuts have been tested for identification of irradiation histories. Doses of 2.5, 5, 7.5 and 10 kGy from a gamma cell Gc-220 have been administrated to the samples under investigation.TL response versus dose for date and for pistachio nuts have been obtained. The effect of added ingredients such as salt in pistachio nuts, and moisture in date samples on the TL response have been studied. The fading of TL intensity of the irradiated dates and pistachio nuts have also been measured. Based on the latter results, it appears possible to identify the irradiated dates (10 kGy), within (1–2) months post-irradiation.In the salted pistachio nuts, the salt itself gives a very significant and distinguishable response. In the unsalted case, however, the difference between the irradiated and unirradiated samples seem difficult to detect due to partial overlapping of the respective responses.     

Polymerization of calcium caseinates solutions induced by gamma irradiation

Solutions of calcium caseinate (5%) combined with propylene glycol (PG) or triethylene glycol(TEG) (0, 2.5% and 5%) and used for the development of edible films and coatings, were irradiated at doses between 0 to 128 kGy. Solutions were chromatographed through toyopearl HW 55F resin to observe the effect of irradiation on cross-link reactions. In unirradiated calcium caseinate solutions, two peaks could be observed (fractions 30 and 37) while samples irradiated at 64 kGy and 128 kGy showed one shifted peak at fraction 32 and 29 respectively. No effect of the plasticizers was observed. According to proteins standards of knowed molecular weights, the molecular weight of calcium caseinate increased approximately 10 times when irradiated at 128 kGy and 5 times when irradiated at 64 kGy. The physico-chemical properties of bio-films prepared with the irradiated solutions, demonstrated that tensile strength at break increased with increase of irradiation dose. A maximum dose was obtained at 16 kGy.     

Effect of gamma irradiation on the microbiological quality and on the functional properties of proteins in dry red kidney beans (Phaseolus vulgaris)

Gamma-irradiation was found to affect the physicochemical properties of dry red kidney beans. The highest dose used (8 kGy) significantly (P0.05) modified the extent of deamidation, the number of sulfhydryl groups, as well as the solubility and the hydrophobicity of the protein. Deamidation, protein solubility and hydrophobicity all increased with the irradiation dose while the number of sulfhydryl groups was reduced by the treatment. Furthermore, irradiation also affected the outgrowth of natural filamentous fungi contaminants present on the dry beans. A dose of 1.5 kGy reduced the number of filamentous fungi by 2 log cycles immediately after treatment. However, the highest dose used (3 kGy) did not eliminate the filamentous fungi completely. Moreover, the filamentous fungi population was a lot less diversified on the irradiated samples. Species of Aspergillus sp. and Penicillium sp. were more abundant on the unirradiated beans while the beans irradiated at 3 kGy contained were predominantly infected by species of Rhizopus sp. , Cladosporium sp. and Alternaria sp.     

A study of the physiological changes and the nutritional qualities of irradiated apples and the effect of irradiation on apples stored at room temperature

The effects of γ-irradiation on the metabolism and nutritional qualities of cold Delicious apples and on the lethality of verticillate pathogenic fungi have been studied. The storage effect of irradiation on apples at room temperature has been observed. The results showed that the respiratory intensity of irradiated apple at 0.3–0.5 kGy was near or lower than that of unirradiated apple after 15 days irradiation. The amount of ethylene release was obviously inhibited when fruits were irradiated with 0.3–0.7 kGy. The flesh firmness of apple irradiated with 0.3–0.9 kGy was higher than that of unirradiated apple with the increasing of storage time. The negative correlations between the flesh firmness and the activities of pectinesterase (PE), polygalacturonase (PG) were observed when the dosage was lower than 1.5 kGy. The 2.0 kGy irradiation damaged the ultrastructure of cells, induced the softening of apple. When apples were irradiated with 0.7–2.0 kGy, the contents of 4 important volatile components of apple would be decreased. However, the dosage mentioned above had no effects on the pure chemicals. The studies showed that there was no significant effect of irradiation with 0.3–2.0 kGy on the nutritional qualities of apples and this dosage range could effectively control the verticillate pathogenic fungi. The result of storage experiments showed that the rotting fruits were obviously decreased by 0.3–0.9 kGy irradiation.     

Effect of γ-irradiation on cotton-cellulose

The effect of high-energy irradiation on cotton-cellulose was studied in inert atmosphere and in the presence of oxygen. A decrease in the degree of polymerization from 1200 to 330 was measured after irradiation with a dose of 15 kGy (dose rate 100 Gy/h) and further gradual decrease was measured when the dose was increased up to 100 kGy. At lower doses a slight increase in crease recovery angle was found as compared to the unirradiated sample. In the FTIR spectra of the same sample the appearance of the absorbance of carbonyl groups (band at 1730–1750 cm⁻¹) was found as a consequence of the oxidative degradation. Cracks on the surface of the fibers were observed by scanning electron microscope on samples irradiated with doses higher than 100 kGy.     

Effect of gamma irradiation on microbiological,chemical and sensory characteristics of licorice root product

Licorice root products were irradiated at doses of 0, 5, 10, 15 and 20 kGy in a ⁶⁰Co package irradiator. Irradiated and unirradiated samples were stored at room temperatures. Microbial population on product, chemical changes and sensory properties of produced solution of licorice root products were evaluated after 0 and 12 months of storage. The results indicated that gamma irradiation reduced the counts of microorganisms on licorice root products. D₁₀ of total count and klebsiella spp. were about 1.4 and 0.7 kGy, respectively. The mineral ions (Na, Ca and K) concentration in solution produced from irradiated products were lower than non-irradiated ones. Glycyrrhezinic acid and maltose concentration in solution produced from irradiated products were higher than non-irradiated ones. Sensory evaluation indicated that no significant differences (P<0.05) were found between solution produced from irradiated and unirradiated products in color, flavor, texture, or taste.     

Effect of radiation processing on nutritional and sensory quality of minimally processed green gram and garden pea sprouts

In the present study, radiation processing of minimally processed green gram and garden pea sprouts was carried out at doses 1 and 2 kGy. The effect of this treatment on different quality parameters like vitamin C content, total carotenoids content, sensory quality, texture, and color was determined over a storage period of 12 days at two different temperatures, a 4 and 8 °C. It was observed that treatment of irradiation (1 and 2 kGy) and storage period did not have any significant effect on vitamin C content of control as well as irradiated sprout samples stored at 4 and 8 °C. Total carotenoids content of sprouts stored at 4, as well as at 8 °C, for 12 days remained almost unchanged after irradiation as well as during storage. Sensory evaluation studies showed that irradiation had no significant effect (p>0.05) on the ratings of any of the sensory attributes in green gram as well as garden pea sprouts and, thus, did not alter the overall acceptability of the irradiated sprouts. Textural studies revealed that there was no significant change (p>0.05) in the firmness of irradiated sprouts (1 and 2 kGy) as compared to control samples at both the temperatures. Storage period of 12 days also did not affect the firmness of sprouts significantly. Color measurement results indicated no drastic change in the color coordinates of the green gram samples except greenness of controls stored at both the temperatures, which showed insignificant decrease in the a^* values. Thus, the nutritional as well as sensory quality of minimally processed green gram and garden pea sprouts did not alter significantly after gamma irradiation with a dose of 1 and 2 kGy.     

Effect of gamma-irradiation and refrigerated storage on the improvement of quality and shelf life of pear (Pyrus communis L., Cv. Bartlett/William)

Gamma-irradiation alone and in combination with refrigeration was tested consecutively for 3 years for extending the shelf life of pear. Matured green pears were irradiated in the dose range of 0.8–2.0 kGy and stored under ambient (temperature 25±2 °C, RH 70%) and refrigerated (temperature 3±1 °C, RH 80%) conditions. Dose range of 1.5–1.7 kGy extended the storage life of pear by 14 days under ambient conditions. Control unirradiated pears were almost fully ripe within 8 days, while as the pears irradiated in the dose range of 1.5–1.7 kGy were fully ripe within 22 days of ambient storage. Irradiation dose of 1.5–1.7 kGy significantly inhibited the decaying of pears upto 16 days of ambient storage. Irradiation in combination with refrigeration prevented the decaying of pears upto 45 days as against the 35% decay in unirradiated samples. Irradiation dose of 1.5–1.7 kGy also gave an extension of 8 and 4 days during additional ambient storage of the pears following 30 and 45 days of refrigeration, respectively.     

An electron spin resonance study of gamma-irradiated grapes

The ESR spectra of the seeds, skins and stalks of unirradiated and γ-irradiated Cape black grapes have been obtained. In the spectra of all parts of the grape a single line (g ca. 2.004) is observed both before and after irradiation. New spectral features are observed after irradiation with doses of between 2 and 10 kGy. Some of these features decline in intensity over a period of several days. However, in the case of stalks, new spectral features are readily observed over the shelf-life of the fruit and in samples irradiated to a dose of only 2kGy.     

Effects of radiation and storage time on conductive NBR/LDPE blends

The electrical conductivity, σ, of conductive acrylonitrile-butadiene rubber (NBR) mixed with different concentrations (1, 3 and 5 phr) of low-density polyethylene (LDPE) is presented. The effect of gamma radiation doses in the range 5 to 50 kGy on the electrical conductivity was determined for all samples. Storage of the unirradiated and irradiated samples for 7 days at 60 or 100 °C has a great influence on the electrical conductivity of these blends. The variation in the conduction mechanisms through these blends on both irradiation and storage is also discussed.