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.     

Application of a rapid screening method to detect irradiated meat in Brazil

Based on the enormous potential for food irradiation in Brazil, and to ensure free consumer choice, there is a need to find a convenient and rapid method for detection of irradiated food. Since treatment with ionising radiation causes DNA fragmentation, the analysis of DNA damage might be promising. In this paper, the DNA Comet Assay was used to identify exotic meat (boar, jacaré and capybara), irradiated with ⁶⁰Co gamma rays. The applied radiation doses were 0, 1.5, 3.0 and 4.5 kGy. Analysis of the DNA migration enabled a rapid identification of the radiation treatment.     

Detection of irradiated fresh fruits treated by e-beam or gamma rays

Since about 1990, the amount of commercially irradiated food products available worldwide has increased. Commercial irradiation of foods has been allowed in Brazil since 1973 and now more than 20 different food products are approved. Among these products are a number of fresh fruits which may be irradiated for insect disinfestation, to delay ripening and to extend shelf-life. Today, there is a growing interest to apply radiation for the treatment of fruits instead of using fumigation or e.g. vapour-heat treatments, and an increased international trade in irradiated fruits is expected. To ensure free consumer choice, methods to identify irradiated foods are highly desirable. In this work, three detection methods for irradiated fruits have been employed: DNA Comet Assay, the half-embryo test and ESR. Both electron-beam (e-beam) and gamma rays were applied in order to compare the response with these two different kinds of radiation. Fresh fruits such as oranges, lemons, apples, watermelons and tomatoes were irradiated with doses in the range 0, 0.50, 0.75, 1.0, 2.0 and 4.0 kGy. For analysis, the seeds of the fruits were utilized. Both DNA Comet Assay and the half-embryo test enabled an easy identification of the radiation treatment. However, under our conditions, ESR measurements were not satisfactory.     

Detection of irradiation treatment of foods using DNA ‘comet assay’

Microgel electrophoresis of single cells (DNA comet assay) has been investigated to detect irradiation treatment of some food samples. These samples of fresh and frozen rainbow trout, red lentil, gram and sliced almonds were irradiated to 1 or 2 kGy using 10 MeV electron beam from a linear accelerator. Rainbow trout samples yielded good results with samples irradiated to 1 or 2 kGy showing fragmentation of DNA and, therefore, longer comets with no intact cells. Unirradiated samples showed shorter comets with a significant number of intact cells. For rainbow trout stored in a freezer for 11 days the irradiated samples can still be discerned by electrophoresis from unirradiated samples, however, the unirradiated trouts also showed some longer comets besides some intact cells. Radiation treatment of red lentils can also be detected by this method, i.e. no intact cells in 1 or 2 kGy irradiated samples and shorter comets and some intact cells in unirradiated samples. However, the results for gram and sliced almond samples were not satisfactory since some intact DNA cells were observed in irradiated samples as well. Probably, incomplete lysis has led to these deviating results.     

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.     

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).     

Improvement of the ESR detection of irradiated food containing cellulose employing a simple extraction method

Fruit may be irradiated at rather low doses, below 1 kGy in combination treatments or for quarantine purposes. To improve the ESR detection sensitivity of irradiated fruit de Jesus et al. (Int. J. Food Sci. Technol. 34 (1999) 173.) proposed extracting the fruit pulp with 80% ethanol and measuring the residue with ESR using low power (0.25 mW) for detection of ‘cellulosic’ radicals. An improvement in ESR sensitivity using the extraction procedure could be confirmed in this paper for strawberries and papayas. In most cases, a radiation dose of 0.5 kGy could be detected in both fruits even after 2–3 weeks storage. In addition, some herbs and spices were also tested, but only for a few of them the ESR detection of the ‘cellulosic’ signal was improved by previous alcoholic extraction.

As an alternative to ESR measurements, other detection methods like DNA Comet Assay and thermoluminescence were also tested.     

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.     

Identification of irradiated wheat by germination test, DNA comet assay and electron spin resonance

In several countries, there has been an increase in the use of radiation for food processing thus improving the quality and sanitary conditions, inhibiting pathogenic microorganisms, delaying the natural aging process and so extending product lifetime. The need to develop analytical methods to detect these irradiated products is also increasing. The goal of this research was to identify wheat irradiated using different radiation doses. Seeds were irradiated with a gamma ⁶⁰Co source (Gammacell 220 GC) in the Centro de Energia Nuclear na Agricultura and the Instituto de Pesquisas Energéticas e Nucleares. Dose rate used were 1.6 and 5.8 kGy/h. Applied doses were 0.0, 0.10, 0.25, 0.50, 0.75, 1.0, and 2.0 kGy. After irradiation, seeds were analysed over a 6 month period. Three different detection methods were employed to determine how irradiation had modified the samples. Screening methods consisted of a germination test measuring the inhibition of shooting and rooting and analysis of DNA fragmentation. The method of electron spin resonance spectroscopy allowed a better dosimetric evaluation. These techniques make the identification of irradiated wheat with different doses possible.     

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.     

Effect of dose rate on inactivation of microorganisms in spices by electron-beams and gamma-rays irradiation

Total aerobic bacteria in spices used in this study were determined to be 1 × 10⁶ to 6 × 10⁷ per gram. A study on the inactivation of microorganisms in spices showed that doses of 6–9kGy of EB (electron-beams) or γ-irradiation were required to reduce the total aerobic bacteria in many However, a little increase of resistance was observed on the inactivation of total aerobic bacteria in many spices in case of EB irradiation. These difference of radiation sensitivities between EB and γ-rays was explained by dose rate effect on oxidation damage to microorganisms from the results of radiation sensitivities of Bacillus pumilus and B. megaterium spores at dry conditions. On the other hand, these high dose rate of EB irradiation suppressed the increase of peroxide values in spices at high dose irradiation up to 80 kGy. However, components of essential oils in spices were not changed even irradiated up to 50 kGy with EB and γ-rays.     

Identification of irradiated foods by monitoring radiolytically produced hydrocarbons

Many countries allow the treatment of foods with low doses of ionizing radiation to reduce microbial and insect infestations, inhibit maturation, and extend shelf life. Therefore, a reliable method is needed to identify irradiated foods and to determine their compliance with respect to allowable absorbed radiation dose. Several approaches for the identification of irradiated foods have been developed such as measurement of radiolytic products, chemiluminescence, and thermoluminescence, and the use of electron spin resonance spectroscopy to measure free radicals trapped in bone. A method for the determination of radiolytically produced hydrocarbons was developed in our laboratory to evaluate the utility of monitoring these compounds as indicators of food irradiation. The method involves the extraction of the radiolytic hydrocarbons from foods and their quantitation by gas chromatography. Concentrations of the radiolytically produced hydrocarbons increased linearly with radiation doses ranging from 0 to 6 kGy. The limit of detection appears to be approximately 1 kGy. The method was found to be useful for the identification of gamma-irradiated foods such as shrimp, frog legs, pork, beef, and poultry. Results of the method evaluation studies of these food matrices as well as factors affecting hydrocarbon production and determination will be presented.     

Effect of gamma irradiation on the antimicrobial and free radical scavenging activities of Glycyrrhiza glabra root

The efficacy of gamma irradiation as a method of decontamination for food and herbal materials is well established. In the present study, Glycyrrhiza glabra roots were irradiated at doses 5, 10, 15, 20 and 25 kGy in a cobalt-60 irradiator. The irradiated and un-irradiated control samples were evaluated for phenolic contents, antimicrobial activities and DPPH scavenging properties. The result of the present study showed that radiation treatment up to 20 kGy does not affect the antifungal and antibacterial activity of the plant. While sample irradiated at 25 kGy does showed changes in the antibacterial activity against some selected pathogens. No significant differences in the phenolic contents were observed for control and samples irradiated at 5, 10 and 15 kGy radiation doses. However, phenolic contents increased in samples treated with 20 and 25 kGy doses. The DPPH scavenging activity significantly (p<0.05) increased in all irradiated samples of the plant.     

Preliminary study on detection of irradiated foodstuffs from the Romanian market

In order to fulfil the European task for market survey in food irradiation the first Romanian laboratory for detection of irradiated foodstuffs was established at IRASM Irradiation Centre. In this preliminary study, a wide range of Romanian food samples (spices, vegetables and meat) gamma irradiated at IRASM have been studied using different detection methods: (1) DNA comet assay, (2) thermoluminescence (TL) and (3) electron spin resonance (ESR) for foodstuffs containing bone or cellulose. The results suggest that there is no general available detection method and there is no perfect detection method. In conclusion, in order to carry out a correct identification of radiation treatment of a food sample it is recommended to use at least two standardised detection methods.     

Mediate gamma radiation effects on some packaged food items

For most of prepackaged foods a 10 kGy radiation dose is considered the maximum dose needed; however, the commercially available and practically accepted packaging materials must be suitable for such application. This work describes the application of ionizing radiation on several packaged food items, using 5 dehydrated food items, 5 ready-to-eat meals and 5 ready-to-eat food items irradiated in a ⁶⁰Co gamma source with a 3 kGy dose. The quality evaluation of the irradiated samples was performed 2 and 8 months after irradiation. Microbiological analysis (bacteria, fungus and yeast load) was performed. The sensory characteristics were established for appearance, aroma, texture and flavor attributes were also established. From these data, the acceptability of all irradiated items was obtained. All ready-to-eat food items assayed like manioc flour, some pâtés and blocks of raw brown sugar and most of ready-to-eat meals like sausages and chicken with legumes were considered acceptable for microbial and sensory characteristics. On the other hand, the dehydrated food items chosen for this study, such as dehydrated bacon potatoes or pea soups were not accepted by the sensory analysis. A careful dose choice and special irradiation conditions must be used in order to achieve sensory acceptability needed for the commercialization of specific irradiated food items.     

Effect of gamma irradiation on trichromatic values of spices

The effect of γ-irradiation on trichromatic color values L*, a*, and b* was determined in black pepper, oregano, and allspice samples irradiated at average doses from 5 kGy to 30 kGy. Trichromatic values a* measured in methanol extracts of treated spices immediately after the irradiation process were significantly changed, but the subsequent storage of allspice and oregano caused much more distinctive alteration of these color values than the irradiation itself. Additionally, the differences in redness between the reference (non-irradiated) allspice and oregano samples and samples treated by γ-radiation gradually disappeared during the storage period. On the contrary, the post-irradiation storage of black pepper samples did not reveal any changes of a* values. Presented at the XVIIIth Slovak Spectroscopic Conference, Spišská Nová Ves, 15–18 October 2006.     

ESR spectroscopy for detecting gamma-irradiated dried vegetables and estimating absorbed doses

In view of an increasing demand for food irradiation technology, the development of a reliable means of detection for the control of irradiated foods has become necessary. Various vegetable food materials (dried cabbage, carrot, chunggyungchae, garlic, onion, and green onion), which can be legally irradiated in Korea, were subjected to a detection study using ESR spectroscopy. Correlation coefficients (R²) between absorbed doses (2.5–15 kGy) and their corresponding ESR signals were identified from ESR signals. Pre-established threshold values were successfully applied to the detection of 54 coded unknown samples of dried clean vegetables (chunggyungchae, Brassica camestris var. chinensis), both non-irradiated and irradiated. The ESR signals of irradiated chunggyungchae decreased over a longer storage time, however, even after 6 months of ambient storage, these signals were still distinguishable from those of non-irradiated samples. The most successful estimates of absorbed dose (5 and 8 kGy) were obtained immediately after irradiation using a quadratic fit with average values of 4.85 and 8.65 kGy being calculated.     

Addition of Transition Metals to Improve Physico-Mechanical Properties of Radiation-Vulcanized Natural Rubber Latex Films

The effect of transition metals as a promoter of physico-mechanical properties of radiation-vulcanized natural rubber latex (RVNRL) films was investigated. RVNRL films were prepared by the addition of transition metals (Fe, Mn, etc.) of different concentrations (0–30 ppm) in natural rubber (NR) latex and irradiated with various radiation doses (0–20 kGy). The concentrations of metal ions and radiation doses were optimized and found to be 20 ppm and 12 kGy, respectively. Tensile strength, tear strength, and cross-linking density of the irradiated rubber films were increased with increasing concentration of metal ions as well as radiation doses. The mechanical properties of the films were enhanced by approximately 20% at the optimum conditions. In contrast, elongation at break, permanent set, and swelling ratio of the films were decreased under the same conditions. The comparative effect of metal ions can be explained by Fajan's rules, reported in this article.     

Effect of gamma irradiation on the thiamine, riboflavin and vitamin B6 content in two varieties of Brazilian beans

The effect of ⁶⁰Co gamma rays on the content of several B-vitamins in two varieties of Brazilian beans has been studied. Carioca (Phaseolus vulgaris L. var. Carioca) and Macaçar beans (Vigna unguiculata L. Walp, var. Macaçar) were irradiated at doses of 0, 0.5, 1.0, 2.5, 5.0 and 10 kGy, and subsequently stored at ambient temperature for 6 months. The content of vitamin B₁, B₂ and B₆ was analysed by HPLC. In addition, the optimum cooking time was established for each dose and bean variety. A taste panel evaluated sensory properties. Only slight changes were measured for thiamine and riboflavin, whereas a dose-dependent decrease was noted for pyridoxine, which, however, was significant only at the highest doses of 5 and 10 kGy. Cooking time was considerably reduced with increasing radiation dose, but accompanied by a loss of the sensory quality. However, at the disinfestation dose up to 1 kGy, acceptable ratings were obtained for the sensory evaluation. In conclusion, for insect disinfestation of Brazilian beans radiation processing is a promising technology.     

Effects of ionizing radiation on mechanically deboned chicken meat during frozen storage

Summary Ionizing radiation was used for the purpose of reducing bacterial contamination in mechanically deboned chicken meat (MDCM). Irradiated and non-irradiated MDCM stored at -18±1 °C, and samples were taken at zero time and at 30-day intervals up to 90 days for 2-thiobarbituric acid reactive substances, sensory and microbiological analyses. The results obtained for the microbiological, sensory and lipid oxidation evaluations, showed that the MDCM samples irradiated with doses of 3.0 and 4.0 kGy were all considered acceptable during 90 days of frozen storage.