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.     

Consumer attitudes to radiation and irradiated potatoes at “radiation fair” in Osaka, Japan

“Radiation Fair” has been held in summer vacation season in August for more than 10 years in Osaka, the largest city of western Japan, for the purpose of public education and information transfer of radiation and radiation-related technology. We distributed questionnaires to the visitors for recent 3 years to inquire their status toward radiation and irradiated products including irradiated potatoes as well as impression toward the displays. According to the survey results, more than 60% of the kids visitors were satisfied with this exhibition as informative, more than half of the older visitors (16 years old and upward) indicated that they recognized the word of “radiation” when they were at elementary school and the most significant sources of this information were school lessons and the mass media. Consumer's image toward radiation seems to shift to more “positive” when correct knowledge about radiation is given. More than half of consumers did not know “irradiated potatoes” but the percentage indicating that irradiated potatoes was definitely hazardous was less than 10%.     

Market trials of irradiated chicken

The potential market for irradiated chicken breasts was investigated using a mail survey and a retail trial. Results from the mail survey suggested a significantly higher level of acceptability of irradiated chicken than did the retail trial. A subsequent market experiment involving actual purchases showed levels of acceptability similar to that of the mail survey when similar information about food irradiation was provided.     

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.     

Genotoxic properties of 2-dodecylcyclobutanone, a compound formed on irradiation of food containing fat

When food containing fat is treated by ionizing radiation, a group of 2-alkylcyclobutanones is formed. These components contain the same number of carbon atoms as their precursor fatty acids and the alkyl group is located in ring position 2. Thus, from palmitic acid 2-dodecylcyclobutanone is derived. To date, there is no evidence that the cyclobutanones occur in unirradiated food. Therefore, these components cannot be considered inherent to food, and for questions pertaining to risk assessment of irradiated food it would be advisable to determine the genotoxic and toxic potentials of cyclobutanones. Measurements of DNA damage in cells exposed to 2-dodecylcyclobutanone, employing the single cell microgel electrophoresis technique, have been carried out. In vitro experiments using rat and human colon cells indicate that 2-docylcyclobutanone in the concentration range of about 0.30 – 1.25 mg/ml induces DNA strand breaks in the cells. Simultaneously, a concentration related cytotoxic effect is observed as was determined by trypan blue exclusion. To which extent these in vitro findings are of relevancy for the in vivo human exposure situation needs to be investigated in further studies. In vivo tests in rats are in progress.     

Rheological changes in irradiated chicken eggs

Pathogenic bacteria may cause foodborne illnesses. Humans may introduce pathogens into foods during production, processing, distribution and or preparation. Some of these microorganisms are able to survive conventional preservation treatments. Heat pasteurization, which is a well established and satisfactory means of decontamination/disinfection of liquid foods, cannot efficiently achieve a similar objective for solid foods. Extensive work carried out worldwide has shown that irradiation is efficient in eradicating foodborne pathogens like Salmonella spp. that can contaminate poultry products. In this work Co-60 gamma irradiation was applied to samples of industrial powder white, yolk and whole egg at doses between 0 and 25 kGy. Samples were rehydrated and the viscosity measured in a Brookfield viscosimeter, model DV III at 5, 15 and 25°C. The rheological behaviour among the various kinds of samples were markedly different. Irradiation with doses up to 5 kGy, known to reduced bacterial contamination to non-detectable levels, showed almost no variation of viscosity of irradiated egg white samples. On the other hand, whole or yolk egg samples showed some changes in rheological properties depending on the dose level, showing the predominance of whether polimerization or degradation as a result of the irradiation. Additionally, irradiation of yolk egg powder reduced yolk color as a function of the irradiation exposure implemented. The importance of these results are discussed in terms of possible industrial applications.     

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.     

Antioxidant 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 synergistic effect, in order to prevent the lipid oxidation resulting in the development of undesirable flavours. The fresh chicken legs were irradiated at 0,3 and 5 kGy. The fatty acids composition of lipids was identified using gas liquid chromatography. The effect of irradiation treatment combined with a pre-treatment on the fatty acids composition was followed. The day after irradiation, ten panallists were asked to evaluate, using the instruction scaling, the overall appearance, the odor, the flavor and the overall acceptability of the samples. The major fatty acids identified in lipids were oleic acid, palmitic acid, palmitoleic acid and stearic acid. Pre-treatments have a significant effect on linoleic acid (C18:2) and higher fatty acids. The unsaturated fatty acids derived from phospholipids appeared to be more affected by the irradiation dose: however, marinating have better protection on C18:2 derived from phospholipids. The results of sensory evaluation have shown a significant better odor and flavor for the irradiated chicken at 5 kGy than the control. No significant difference have been found between the marinated chicken, the chicken irradiated under vacuum and the control.     

Protein quality of irradiated Brazilian beans

Beans are a major source of dietary protein in Brazil. However, high losses due to insect infestation occur after each harvest. To combat these losses, radiation processing of beans offers promise as an alternative to chemical treatment, provided the nutritional quality of beans is not impaired by the radiation treatment. Conflicting results have been published about the effect of radiation on the biological value of legume proteins. Therefore, two varieties of Brazilian beans were studied: 1) Phaseolus vulgaris L., var. carioca and 2) Vigna unguiculata (L.) Walp, var. macaçar. The beans were irradiated with doses of 0, 0.5, 1.0, 2.5, 5.0 and 10 kGy. Since irradiated beans will be consumed after appropriate storage, the beans under study were stored for 6 months at ambient temperature. Protein quality was measured by a biological assay employing the nitrogen balance approach in weanling rats. The animals were fed with optimally cooked beans, which were the only source of protein (10%). Nitrogen contents of legumes, diets, animal urine and faeces were determined by Kjeldahl analysis. The indices for apparent protein quality: net protein utilisation, digestibility and biological value were not influenced by irradiation. Thus, radiation treatment of Brazilian beans offers considerable promise as an effective insect disinfection process, without impairing the biological quality of the valuable bean protein.     

A study on chemical composition of spices irradiated by electron beam

Quantitative changes in common organic acids and inorganic acids from spices irradiated by electron beam were studied by Dionex-4000i ion chromograph. The results showed that the acids content of either chilli or the five-spice powder⁽³⁾ irradiated with a dose of 9.94 kGy did not undergo significant changes in comparison with the control samples. The flavour composition in the five-spice powder irradiated by electron beam was also determined by Finnigan MAT-8230B gas chromatograph-mass spectrometer, and compared to the results by heating treatment (120°C, 30min). The comparison indicated that the effect of electron beam treatment on flavour composition was less than that of heating.     

The influence of gamma irradiation on flavonoïds content during storage of irradiated clementina

The influence of Gamma irradiation on content of some important flavonoïds (flavonones glycosides and polymethoxylated flavones) was evaluated during storage of Moroccan clementina treated at a mean dose of 0.3 kGy and stored three months at 3′C. Results shows that at day one, gamma irradiation induced degradation of small quantities of these flavonoïds, however after 14 days of storage, the content of these compounds was significantly higher (p≤0.05) in irradiated samples. Irradiation stimulated biosynthesis of flavonoïds after 14 days of storage. Hesperidin was the major flavanones compounds in clementines. Nobiletin and Heptamethoxyflavone were the major polymethoxylated flavones in clementines. Our study demonstrated that the content of these compounds was significantly higher (p≤0.05) in irradiated samples.     

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.     

A new irradiation plant in Italy: Technical features and activities performed

Since 1995, a new Hitesys Co. pilot/industrial irradiation plant has been operating in Aprilia, 50 km south of Rome. The plant, which was jointly designed by ENEA and Hitesys Co., is equipped with a highly flexible automatic materials transport system which is also suitable for continuous process repetition or, when upturned, for double-side material treatment. The main features of the irradiation plant are:

• - the radiation source: EB-machine LINAC type (s band) with maximum electron beam energy of 10 MeV and beam power of 1000 W;

• - the bunker: external type, shielded by ordinary concrete, equipped with a suitable entrance/exit maze in order to allow easy handling and managing of materials as well as to facilitate equipment and machinery maintenance and inspection activities.

An intense program of R&D activities and technological services has already been performed in relation to industrial processes and environmental applications, as well as many other radiation technology applications, including agriculture related processes.     

Analysis of irradiated food

Foods, e.g. chicken, shrimps, frog legs, spices, different dried vegetables, potatoes and fruits are legally irradiated in many countries and are probably also exported into countries, which do not permit irradiation of any food. Therefore all countries need analytical methods to determine whether food has been irradiated or not. Up to now, two physical (ESR-spectroscopy and thermoluminescence) and two chemical methods (o-tyrosine and volatile compounds) are available for routine analysis. Several results of the application of these four mentioned methods on different foods are presented and a short outlook on other methods (chemiluminescence, DNA-changes, biological assays, viscometric method and photostimulated luminescence) will be given.     

The effect of ionizing radiation on microbiological decontamination of medical herbs and biologically active compounds

Several thousand tons of medical herbs are produced annually by pharmaceutical industry in Poland. This product should be of highest quality and microbial purity. Recently, chemical methods of decontamination are recognized as less safe, thus irradiation technique was chosen to replace them in use. In the Institute of Nuclear Chemistry and Technology the national program on the application of irradiation to the decontamination of medical herbs is in progress now. The purpose of the program is to elaborate, on the basis of research work, the facility standards and technological instructions indispensable for the practice of radiation technology.     

Pork fat peroxidation by gamma-irradiation

In this paper, pork fat peroxidation by γ-irradiation and the possible effects of oxygen, UV-irradiation and storage after the γ-irradiation have been investigated. It has been found that the level of peroxides in irradiated pork increases linearly with the increasing absorbed dose. The chemical yield of peroxides formed in the irradiated fat is about 4.2 and independent on the sample temperature or absorbed dose rate, but dependent on storage time of sample before γ-irradiation. The irradiated pork exhibits some unusual features as following: 1) the peroxide content in irradiated pork is higher than that in unirradiated one; 2) the peroxide content in irradiated pork increases gradually on storage and is essentially constant in unirradiated one, which is very useful for the detection of irradiated pork; 3) the further peroxidation in irradiated pork is much more susceptible to UV radiation than that in unirradiated pork.     

Crosslinked polyethylene foams, via EB radiation

Polyethylene foams, produced by radio-induced crosslinking, show a smooth and homogeneous surface, when compared to chemical crosslinking method using peroxide as crosslinking agent. This process fosters excellent adhesive and printability properties. Besides that, closed cells, intrinsic to theses foams, imparts opitmum mechanical, shocks and insulation resistance, indicating these foams to some markets segments as: automotive and transport; buoyancy, flotation and marine: building and insulation: packaging: domestic sports and leisure goods. We were in search of an ideal foam, by adding 5 to 15% of blowing agent in LDPE. A series of preliminary trials defined 203° C as the right blowing agent decomposition temperature. At a 22.7 kGy/dose ratio, the lowest dose for providing an efficient foam was 30 kGy, for a formulation comprising 10% of azodicarbonamide in LDPE, within a 10 minutes foaming time.     

Calculation of the electronic and optical properties of indium tin oxide by density functional theory

Density functional theory (DFT) was used to calculate the bulk electronic and optical properties of indium tin oxide (ITO). The ITO model was constructed replacing indium atoms with tin atoms in the cubic unit cell of indium oxide. To allow more possibilities for tin atom substitution than afforded by the forty-atom primitive cell of indium oxide all eighty atoms of the unit cell were included in the stoichiometry (In_32−xSn_xO₄₈) using periodic boundary conditions. A number of properties of ITO were calculated including the optical band gap, charge carrier density and plasma frequency. The dependence of the electronic and optical properties of ITO on a variety of parameters such as the tin content, cubic lattice parameter and the distance between adjacent tin atoms was investigated. The electronic and optical properties agreed well with experimental data and allowed insight into the origin of the electronic and optical properties of ITO.     

EPR detection of foods preserved with ionizing radiation

The applicability of the epr technique for the detection of dried vegetables, mushrooms, some spices, flavour additives and some condiments preserved with ionizing radiation is discussed. The epr signals recorded after exposure to gamma rays and to beams of 10 MeV electrons from linac are stable, intense and specific enough as compared with those observed with nonirradiated samples and could be used for the detection of irradiation. However, stability of radiation induced epr signals produced in these foods depends on storage condition.

No differences in shapes (spectral parameters) and intensities of the epr spectra recorded with samples exposed to the same doses of gamma rays (⁶⁰Co) and 10 MeV electrons were observed     

Rapid detection of irradiated frozen hamburgers

DNA comet assay can be employed as a rapid and inexpensive screening test to check whether frozen ground beef patties (hamburgers) have been irradiated as a means to increase their safety by eliminating pathogenic bacteria, e.g. E. coli O157:H7. Such a detection procedure will provide an additional check on compliance with existing regulations, e.g. enforcement of labelling and rules in international trade. Frozen ready prepared hamburgers from the market place were ‘electron irradiated’ with doses of 0, 1.3, 2.7, 4.5 and 7.2 kGy covering the range of potential commercial irradiation. DNA fragmentation in the hamburgers was made visible within a few hours using the comet assay, and non-irradiated hamburgers could be easily discerned from the irradiated ones. Even after 9 months of frozen storage, irradiated hamburgers could be identified. Since DNA fragmentation may also occur with other food processes (e.g. temperature abuse), positive screening tests shall be confirmed using a validated method to specifically prove an irradiation treatment, e.g. EN 1784 or EN 1785.