Detection of Phakopsora pachyrhizi by polymerase chain reaction (PCR) and use of germination test and DNA comet assay after e-beam processing in soybean

Soybean harvest is the main agribusiness in Brazil, which is the second largest exporter in the world and has a revenue of billions of dollars. Asian dust is caused by the fungus Phakopsora pachyrhizi and its dissemination is difficult to control, since it occurs through wind dispersion. Actually P. pachyrhizi is found in different parts of the world. Electron beam treatment could be an alternative process to minimize these losses, especially for the grains exportation industry. Besides the possibility of being disconnected when not in use, this source does not need to be reloaded, is easily available and, streamlines the process and reduces logistics costs. The present work aims to identify, by the polymerase chain reaction technique (PCR), the P. pachyrhizi fungus presence in the irradiated soybeans and the possibility to use radiation treatment as a sanitary alternative. Doses 0, 1.0, 2.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 kGy (IPEN-CNEN/SP Electron Accelerator) were applied and two fast-screening methods were performed: DNA comet assay (for the detection of DNA damage) and germination test (for the measurement of roots inhibition). These tests are very easy to carry out and measure damage response depending on radiation dose.     

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.     

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.     

Determination of total <Emphasis Type="Italic">trans</Emphasis> fats and oils by infrared spectroscopy for regulatory compliance

The mandatory requirement in many countries to declare the amount of trans fat present in food products and dietary supplements has led to a need for sensitive and accurate methodologies for the rapid quantitation of total trans fats and oils. Capillary gas chromatography (GC) and infrared spectroscopy (IR) are the two methods most commonly used to identify and quantify trans fatty acids for food labeling purposes (see the article by Delmonte and Rader in this ABC issue for a detailed presentation of GC methodology). The present article provides a comprehensive review of the IR technique and the current attenuated total reflection (ATR) Fourier-transform (FT) IR methodologies for the rapid determination of total trans fats and oils. This review also addresses potential sources of interferences and inaccuracies in FTIR determinations, particularly those done at low trans levels. Recent observations have shown that the presence of saturated fats caused interferences in the FTIR spectra observed for trans triacylglycerols. The recognition and resolution of previously unresolved quantitative issues improved the accuracy and sensitivity of the FTIR methodology. Once validated, it is anticipated that the new negative second-derivative ATR-FTIR procedure will make IR spectroscopy more suitable than ever, and a rapid alternative and/or complementary method to GC, for the rapid determination of total trans fats for regulatory compliance. Figure Infrared light bouncing inside an internal reflection crystal     

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.     

Reduction of the IgE-binding ability and maintenance of immunogenicity of gamma-irradiated Dermatophagoides farinae

House dust mites, Dermatophagoides farinae (DF) and Dermatophagoides pteronyssinus, are major allergens in the most common indoor allergen and are important risk factor for asthma. The modified antigen has been studied to treat allergic disorder. This study was carried out to measure possibility of modified allergen using gamma irradiation to treat allergy such as asthma. DF solutions (2 mg/ml) as target allergen were irradiated with Co-60 at 50 and 100 kGy. Conformational alternation of irradiated DF was observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Levels of anti-irradiated DF mouse IgGs (sub-isotypes) against intact DF were measured similar to that of anti-intact DF IgGs. The binding abilities of house dust mite-allergic patients’ IgE were reduced depending on radiation dose, and irradiation could inhibit the binding ability of patients’ IgE more than 40%. This study has shown that the binding ability of IgE was reduced by conformational alteration by irradiation and the irradiated DF had epitopes capable to induce immunogeniciy.     

Analytical methods to identify irradiated food—a review

The ability to analytically identify irradiated food—complementary to certification—helps to enhance consumers confidence. It makes it possible to check compliance with existing regulations (e.g. enforcement of labelling, control of prohibition) and to facilitate international food trade. An enormous effort—both at the world-wide international level (ADMIT) and at European (BCR)/national level—has led to several validated and even standardized (CEN) detection methods for a large number of different food products. In this short review, the present methods are summarized, limitations and new developments drafted.     

Radiation grafting of pH and thermosensitive N-isopropylacrylamide and acrylic acid onto PTFE films by two-steps process

Polytetrafluoroethylene (PTFE) was grafted (g) with acrylic acid (AAc) by γ-ray pre-irradiation method to get PTFE-g-AAc films, then N-isopropylacrylamide (NIPAAm) was grafted onto PTFE-g-AAc films with γ-ray to get (PTFE-g-AAc)-g-NIPAAm. PTFE films were irradiated in air at a dose rate of 3.0 kGy h^–1 and different radiation dose. The irradiated films were placed in glass ampoules, which contained aqueous solutions with different monomer concentration (AAc), and then they were heated at different temperatures and reaction time. NIPAAm onto PTFE-g-AAc was carried out with the same procedure with monomer concentration of 1 mol L⁻¹. The thermosensitivity of the samples was defined and calculated as the ratio of the grafted samples swelling at 28 and 35 °C, and pH sensitivity defined as the ratio of the grafted samples swelling at pH 2 and 8.     

Irradiated bivalve mollusks: Use of EPR spectroscopy for identification and dosimetry

High energy radiation treatment of foodstuff for microbial control and shelf-life extension is being used in many countries. However, for consumer protection and information, the European Union has adopted the Directives 1999/2/EC and 1999/3/EC to harmonize the rules concerning the treatment and trade of irradiated foods in EU countries. Among the validated methods to detect irradiated foods the EU directives also include Electron Paramagnetic Resonance (EPR/ESR) spectroscopy.We describe herein the use of EPR for identification of four species of bivalve mollusks, i.e. brown Venus shells (Callista chione), clams (Tapes semidecussatus), mussels (Mytilus galloprovincialis) and oysters (Ostrea edulis) irradiated with ⁶⁰Co γ-rays. EPR could definitely identify irradiated seashells due to the presence of long-lived free radicals, primarily CO₂⁻, CO₃³⁻, SO₂⁻ and SO₃⁻ radical anions. The presence of other organic free radicals, believed to originate from conchiolin, a scleroprotein present in the shells, was also ascertained. The use of one of these radicals as a marker for irradiation of brown Venus shells and clams can be envisaged. We also propose a dosimetric protocol for the reconstruction of the administered dose in irradiated oysters.     

Applications of Thermal Analysis on the Marine Phytoplankton,Tetraselmis Suecica

Marine microalgae represent an essential link in the planktonic food web and some of them are commonly utilised in aquaculture systems as food for larval and juvenile stages of fishes, crustacea and mollusca. However, the caloric content and the biochemical composition of these microrganisms vary in relation to ageing and to several environmental conditions; so if the parameters under which phytoplankton grow are not suitable, marine microalgae can supply small quantities of energy and essential nutrients. The aim of this work was to study the effects of temperature on the marine planktonic alga Tetraselmis suecica using thermogravimetry (TG) and differential thermal analysis(DTA). Marked differences have been observed between exponentially, stationary and senescence phases probably due to both the presence of different biomolecules produced during algal growth and to the differences in the thermal properties of these intracellular molecules. This revised version was published online in July 2006 with corrections to the Cover Date.     

Influence of different methods of controlling microwave sintering

The ceramic industry uses enormous amounts of energy to produce products at high temperatures. Energy-saving measures based on sintering process improvements are examined. Oxide ceramics were irradiated by two different methods during microwave sintering: intermittent use of the same high power levels (time-control method) and continuous use of lower, increasing levels of power (power-control method). We found that: 1) the power consumption and efficiency of the time-control method were lower than those of the power-control method; 2) the power-control method gave more precise control of temperature under our experimental conditions; 3) sample densification did not differ greatly between the two methods; and 4) grain growth did not differ greatly between the two methods.     

Identification of radiolytic marker compounds in the irradiated beef extract powder by volatile analysis

In order to develop a new detection method for marker compounds from irradiated powdery foods, two different extraction methods such as solid phase microextraction (SPME) and purge and trap (P&T) methods, were used to detect radiolytic volatile compounds as marker compounds by testing beef extract powder. Beef extract powder was irradiated using a ⁶⁰Co source (γ-irradiation) at 1, 3, 5 and 10 kGy, and then divided into 0 and 30 days of storage (30±1 °C). In 0 day of storage, each concentration of 1,3-bis(1,1-dimethylethyl)benzene as a marker compound detected simultaneously by the extraction methods, increased linearly with irradiation dose (p<0.05) and had the same trend after 30 days of storage. Meanwhile, four other compounds such as tridecane, hexadecane, 2-octene and 2-decanone were excluded from possible marker compounds because these compounds were detected initially but soon disappeared. Another reason is that these compounds were detectable in non-irradiated samples. In this study, therefore, 1,3-bis(1,1-dimethylethyl) benzene was selected as a maker compound in irradiated beef extract powder.     

Stress Response Kinetics of Two Nisin Producer Strains of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> spp. <Emphasis Type="Italic">lactis</Emphasis>

The purpose of this study is to determine the survival and nisin production behaviors of two strains of Lactococcus lactis under different stress conditions that represent the food ecosystem. In this respect, the survival ratios of two nisin producers were determined under different pH, temperature, NaCl, and bile salt concentrations. Then, nisin production levels of the strains were determined at each stress conditions. Both strains had similar growth or inactivation patterns under the same stress conditions. NaCl and bile salt stresses on the survival ratio of the strains could be successfully described by the exponential decay function, whereas Gaussian function produced good fits for temperature and pH stresses. The nisin activity of two nisin producers (in their mid-exponential and/or early stationary phase) decreased dramatically under all stress conditions, except osmotic (NaCl) and low temperature applications. The results of this study showed that two nisin producers had similar adaptive responses under severe stress conditions, which could be described by appropriate mathematical equations. Moreover, the effect of harsh environment on the nisin activity of L. lactis strains depends on the stress factors applied.     

Effects of gamma irradiation on the yields of volatile extracts of Angelica gigas Nakai

The study was carried out to determine the effects of gamma irradiation on the volatile flavor components including essential oils, of Angelica gigas Nakai. The volatile organic compounds from non- and irradiated A. gigas Nakai at doses of 1, 3, 5, 10 and 20 kGy were extracted by a simultaneous steam distillation and extraction (SDE) method and identified by GC/MS analysis. A total of 116 compounds were identified and quantified from non- and irradiated A. gigas Nakai. The major volatile compounds were identified 2,4,6-trimethyl heptane, α-pinene, camphene, α-limonene, β-eudesmol, α-murrolene and sphatulenol. Among these compounds, the amount of essential oils in non-irradiated sample were 77.13%, and the irradiated samples at doses of 1, 3, 5, 10 and 20 kGy were 84.98%, 83.70%, 83.94%, 82.84% and 82.58%, respectively. Oxygenated terpenes such as β-eudesmol, α-eudesmol, and verbenone were increased after irradiation but did not correlate with the irradiation dose. The yields of active substances such as essential oil were increased after irradiation; however, the yields of essential oils and the irradiation dose were not correlated. Thus, the profile of composition volatiles of A. gigas Nakai did not change with irradiation.     

Comparative identification of irradiated herbs by the methods of electron paramagnetic resonance and thermoluminescence

Non irradiated and γ-irradiated dry herbs savoury (Savoury), wild thyme (Thymus serpollorium) and marjoram (Origanum) with absorbed dose of 8 kGy have been investigated by the methods of elecrtron paramagnetic resonance (EPR) and thermoluminescence (TL). Non-irradiated herbs exhibit only one weak siglet EPR signal whereas in irradiated samples its intensity increase and in addition two satelite lines are recorded. This triplet EPR spectrum is attributed to cellulose free radical generated by irradiation. It has been found that upon keeping the samples under the normal stock conditions the life-time of the cellulose free radical in the examined samples is ∼60–80 days. Thus the conclusion has been made that the presence of the EPR signal of cellulose free radical is unambiguous indication that the sample under study has been irradiated but its absence can not be considered as the opposite evidence. In the case when EPR signal was absent the method of TL has been used to give the final decision about the previous radiation treatment of the sample.     

Light-induced fading of the PSL signal from irradiated herbs and spices

Reliability of the photo-stimulated luminescence (PSL) technique, as screening method for irradiated food identification, has been tested with three kinds of herbs and spices (oregano, red pepper and fennel), prepared in two different ways (granular: i.e. seeds and flakes, or powdered), over a long period of storage with different light exposures. The irradiated samples kept in the dark gave always a positive response (the sample is correctly classified as “irradiated”) for the overall examination period. The samples kept under ambient light conditions, in typical commercial glass containers, exhibited a reduction of the PSL signal, more or less pronounced depending on the type of food and packaging. The different PSL response of the irradiated samples is to be related to the quantity and quality of the mineral debris present in the individual food. It was also found that, for the same type of food, the light-induced fading was much stronger for the flaked and seed samples than for the corresponding powder samples, the penetrating capability of light being much more inhibited in powdered than in whole seeds or flaked form samples. The observed light bleaching of the PSL signal in irradiated herbs and spices is of practical relevance since it may lead to false negative classifications.     

Gas chromatographic/mass spectrometric and microbiological analyses on irradiated chicken

Ionizing radiation is widely used as treatment technique for food preservation. It involves among others reduction of microbial contamination, disinfestations, sprout inhibition and extension of shelf life of food. However, the commercialization of irradiated food requires the availability of reliable methods to identify irradiated foodstuffs. In this paper, we present results on the application to irradiated chicken of this method, based on the detection, in muscle and skin samples, of the peaks of ions 98 Da and 112 Da, in a ratio approximately 4:1, typical of radiation induced 2-dodecylcyclobutanones (2-DCB). Aim of the work was also to study the time stability of the measured parameters in samples irradiated at 3 and 5 kGy, and to verify the efficacy of the treatment from a microbiological point of view. Our results show that, one month after irradiation at 3 kGy, the method is suitable using the skin but not the muscle, while the measured parameters are detectable in both samples irradiated at 5 kGy. The microbial population was substantially reduced even at 3 kGy.