Inactivation of Escherichia coli O157 and Salmonella Enteritidis in raw beef liver by gamma irradiation.

Irradiation of ground beef and beef liver inoculated with Escherichia coli O157 466 and DT66 and Salmonella Enteritidis 3313 were performed with gamma rays from cobalt-60 at refrigerated and frozen temperatures under air- and vacuum-packaged conditions. Results showed that D10 values for all pathogens in frozen beef liver were higher than those in frozen ground beef samples, with significant differences observed between the D10 values of E. coli O157 466 and S. Enteritidis 3313 under air-packaged conditions, as well as in E. coli O157 DT66 and S. Enteritidis 3313 under vacuum-packaged conditions. To verify effective bacterial inactivation under high bacterial-contamination levels (105-107 CFU/g), survival/death interfaces of E. coli O157 DT66 and S. Enteritidis 3313 inoculated in beef liver under vacuum-packaged and frozen conditions were constructed, with results suggesting that doses from 5.3 kGy to 5.5 kGy and 8.2 kGy-8.5 kGy would be sufficient to kill 105 CFU/g of E. coli O157 and S. Enteritidis 3313, respectively, at a 95%-99% predicted confidence interval. These results suggested that food matrixes containing high amounts of antioxidants (such as beef liver) and treated under frozen and vacuum-packaged conditions require additional consideration and evaluation for applications of irradiation treatment.

Modifications of azoxymethane-induced carcinogenesis and 90-day oral toxicities of 2-tetradecylcyclobutanone as a radiolytic product of stearic acid in F344 rats.

A 90-day oral toxicity test in rats was performed to evaluate the toxicity of 2-tetradecylcyclobutanone (2-tDCB), a unique radiolytic product of stearic acid. Six-week-old male and female F344 rats (n=15/group) were given 2-tDCB at concentrations of 0, 12, 60 and 300 ppm in a powder diet for 13 weeks. Slight dose-dependent increases in serum total protein and albumin in male rats were found, but these changes were not considered to be a toxic effect. The fasting, but not non-fasting, blood glucose levels of the male rats in the 300 ppm group and female rats in the 60 and 300 ppm groups were lower than those of the controls. Gas chromatography-mass spectrometry analysis showed dose-dependent accumulation of 2-tDCB in adipose tissue, notably in males. Next, we performed an azoxymethane (AOM)-induced two-stage carcinogenesis study. After injection of 6-week-old male F344 rats (n=30/group) once a week for 3 weeks, the animals received 2-tDCB at concentrations of 0, 10, 50 and 250 ppm in a powder diet for 25 weeks. The incidences of colon tumors for the 2-tDCB dosages were 34%, 45%, 40% and 37%, respectively, and were not statistically significant. These data suggest that 2-tDCB shows no toxic or tumor-modifying effects under the present conditions, and that the no-observed-adverse-effect level for 2-tDCB is 300 ppm in both sexes, equivalent to 15.5 mg/kg b.w./day in males and 16.5 mg/kg b.w./day in females.

Distribution of radioactive cesium ((134)Cs plus (137)Cs) in rice fractions during polishing and cooking.

We investigated the distribution of cesium-134 ((134)Cs) and cesium-137 ((137)Cs) during polishing and cooking of rice to obtain their processing factors (Pf) and food processing retention factors (Fr) to make the information available for an adequate understanding of radioactive Cs dynamics. Polishing brown rice resulted in a decreased radioactive Cs concentration of the polished rice, but the bran and germ (outer layers) exhibited higher concentrations than brown rice. The Pf values for 100% polished rice and outer layers ranged from 0.47 to 0.48 and 6.5 to 7.8, respectively. The Fr values for 100% polished rice and outer layers were 0.43 and 0.58 to 0.60, respectively. The distribution of radioactive Cs in polished rice and outer layers was estimated at approximately 40 and 60%, respectively. On the other hand, cooked rice showed significantly lower levels of radioactive Cs than polished rice, and transfer of radioactive Cs into wash water was observed. The Pf and Fr values for cooked rice were 0.28 and 0.65 to 0.66, respectively. From these results, we can calculate that if the radioactive Cs concentration in brown rice is 100 Bq/kg, the concentrations of Cs in polished rice and cooked rice will be 47 to 48 Bq/kg and 13 Bq/kg, respectively.

Genotoxic potential and in vitro tumour-promoting potential of 2-dodecylcyclobutanone and 2-tetradecylcyclobutanone, two radiolytic products of fatty acids.

The DNA-damaging and tumour-promoting effects of two 2-alkylcyclobutanones (2-ACBs), which are found in irradiated fat-containing foods, were investigated by use of the comet assay and in an azoxymethane (AOM)-induced colon-carcinogenesis study in rats, respectively. We conducted genotoxicity tests of 2-dodecylcyclobutanone (2-dDCB) and 2-tetradecylcyclobutanone (2-tDCB) according to the test guidelines for chemicals or drugs. In addition, a cell-transformation assay with Bhas 42 cells was performed to investigate their promoting potential in vitro. The Salmonella typhimurium mutagenicity assay (Ames test), conducted with five tester strains, revealed that neither 2-dDCB nor 2-tDCB possessed mutagenic activity. Moreover, both in the in vitro chromosomal aberration test on CHL/IU cells and the in vivo bone-marrow micronucleus test where mice were given 2-dDCB and 2-tDCB (orally, up to 2000 mg/kg bw/day), we did not detect any clastogenic effects. Furthermore, DNA strand-breaks were not detected in the in vitro comet assay with CHL/IU cells, and DNA adducts derived from 2-dDCB and 2-tDCB were not detected in the colon tissues of the mice used for the micronucleus tests, in rats from a repeated dose 90-day oral toxicity test (0.03% 2-tDCB in the diet), or in rats from the AOM-induced carcinogenesis study (0.025% 2-tDCB in the diet). An in vitro tumour-promotion assay with Bhas 42 cells revealed that the number of transformed foci increased significantly following treatment of cells in the stationary phase with 2-dDCB or 2-tDCB for 10 days. Our results indicate that neither 2-dDCB nor 2-tDCB were genotoxic chemicals. However, they exhibited promoting activity, at least in vitro, when Bhas 42 cells were continuously exposed to these chemicals at toxic doses.

Characterization of brown rice as a certified reference material for Fukushima accident-related radioactivity measurements.

We developed a certified reference material of brown rice to measure radioactivity from the Fukushima Daiichi Nuclear Power Plant accident. The rice was planted in the spring of 2011, just after the Fukushima accident occurred, and it was harvested in the autumn of 2011. The certified value of radioactivity concentration in the rice was 33.6 Bq kg(-1) of Cs-134 and 51.8 Bq kg(-1) of Cs-137 on August 1, 2012. The reference material is being widely distributed by the National Metrology Institute of Japan. To determine the radioactivity and its uncertainties in the brown rice, we employed gamma-ray spectrometry with a high-purity germanium detector and Monte Carlo simulation.

Comparison of scavenging capacities of vegetables by ORAC and EPR.

Reactive oxygen species (ROS) are considered to be causative agents of many health problems. In spite of this, the radical-specific scavenging capacities of food samples have not been well studied. In the present work, we have developed an electron paramagnetic resonance (EPR) spin trapping method for analysis of the scavenging capacities of food samples for multiple ROS, utilising the same photolysis procedure for generating each type of radical. The optimal conditions for effective evaluation of hydroxyl, superoxide, and alkoxyl radical scavenging capacity were determined. Quantification of radical adducts was found to be highly reproducible, with variations of less than 4%. The optimised EPR spin trapping method was used to analyse the scavenging capacities of 54 different vegetable extracts for multiple radicals, and the results were compared with oxygen radical absorption capacity values. Good correlations between the two methods were observed for superoxide and alkoxyl radicals, but not for hydroxyl.

[Application of electron spin resonance spectroscopy for the detection of irradiated crustaceans (prawn, shrimp, and crabs)].

Prawn, shrimp and crabs sold in Japan are mostly imported from overseas. Detection of irradiated crustaceans is very important for quality assurance. In this study, we used ESR to detect radiation-induced radicals after irradiation of prawn, shrimp and crabs of major species. No radiation-induced radicals were detected in prawn (black tiger prawn) or shrimp (white leg shrimp). Radiation-induced radicals due to hydroxyapatite were detected in the claws of snow crab, red king crab, and swimming crab. Our results indicate that ESR measurement on the claw parts of these three species of crab can be used to determine their irradiation history.

Effect of gamma irradiation on soybean allergen levels.

The levels of food allergens in gamma-irradiated soybean (0, 2.5, 5, 7.5, 10, 20, and 30 kGy) were investigated by immunoblotting and ELISA, using allergen-specific antibodies and patient serum. After 3 months of storage, Coomassie brilliant blue (CBB) staining indicated similar total protein profiles among the treatments, but that some proteins were degraded by irradiation at high doses. Immunoblotting with specific antibodies for major soybean allergens (ß-conglycinin, Gly m Bd 30 K, soybean trypsin inhibitor, and Gly m 4) resulted in apparent band profiles and intensities that were not significantly changed by irradiation. Competitive inhibition ELISA analyses suggested that there were no significant changes in the allergen contents, except for a decrease in the soybean trypsin inhibitor. The patient IgE binding allergenic protein patterns were not changed by irradiation up to 30 kGy. ELISA using patient serum also revealed that the IgE reactivity to the irradiated soybean extract did not increase from the level of the control, but that the reactivity to some patient serum IgE was significantly decreased by irradiation.

Catalases CAT1 and CAT3 are not key enzymes in alleviating gamma irradiation-induced DNA damage, H2O2 accumulation, or lipid peroxidation in Arabidopsis thaliana.

Gamma irradiation increased catalase activities at 0.1 kGy and decreased them at 10 kGy in Arabidopsis wild type and catalase-deficient mutants, cat3-1 and cat1 cat3. Irradiation induced DNA damage, H2O2 accumulation, and lipid peroxidation in both mutants as well as the wild type. Thus catalases might not be key enzymes protecting gamma irradiation-induced damage.

Distribution of radioactive cesium ((134)Cs Plus(137)Cs) in a contaminated Japanese soybean cultivar during the preparation of tofu, natto, and nimame (Boiled Soybean).

We investigated the fate of radioactive cesium ((134)Cs plus (137)Cs) during the production of tofu, natto, and nimame (boiled soybean) from a contaminated Japanese soybean cultivar harvested in FY2011. Tofu, natto, and nimame were made from soybean grains containing radioactive cesium (240 to 340 Bq/kg [dry weight]), and the radioactive cesium in the processed soybean foods and in by-product fractions such as okara, broth, and waste water was measured with a germanium semiconductor detector. The processing factor is the ratio of radioactive cesium concentration of a product before and after processing. For tofu, natto, nimame, and for the by-product okara, processing factors were 0.12, 0.40, 0.20, and 0.18, respectively; this suggested that these three soybean foods and okara, used mainly as an animal feed, can be considered safe for human and animal consumption according to the standard limit for radioactive cesium of soybean grains. Furthermore, the ratio of radioactive cesium concentrations in the cotyledon, hypocotyl, and seed coat portions of the soybean grain was found to be approximately 1:1:0.4.

Dynamics of radioactive cesium (134Cs and 137Cs) during the milling of contaminated Japanese wheat cultivars and during the cooking of udon noodles made from wheat flour.

The fate of radioactive cesium ((134)Cs plus (137)Cs) during the milling of contaminated Japanese wheat cultivars harvested in FY2011, and during the cooking of Japanese udon noodles made from the wheat flour, was investigated. Grain samples containing various radioactive cesium concentrations (36.6 to 772 Bq/kg [dry weight]) were milled using a laboratory-scale test mill to produce eight fractions: three break flours (1B, 2B, and 3B), three reduction flours (1M, 2M, and 3M), bran, and shorts. The concentrations of radioactive cesium were found to be highest in the bran fractions of all the samples tested, with 2.3- to 2.5-fold higher values than that of the whole grain. Shorts contained radioactive cesium levels similar to that of the whole grain. In contrast, radioactive cesium concentrations in other fractions were found to be less than half the concentration in whole grain. The average processing factor (PF) value calculated for patent flour (0.401 ± 0.048), made from the mixture of 1B, 2B, 1M, and 2M for human consumption, or for low-grade flour (0.467 ± 0.045), made from the mixture of 3B and 3M, was found to be less than 0.5; whereas the average PF value (2.07 ± 0.232) for feed bran (mixture of bran and shorts), which has been used mainly as livestock feed in Japan, was over 2.0. Boiling udon noodles (made from patent flour) resulted in a substantial reduction (>70 % of initial amount) of radioactive cesium. Moreover, radioactive cesium was reduced further (<10 % of the initial amount) in the subsequent rinsing process, and the PF value of boiled noodles was recorded as 0.194. These results demonstrated that patent flour containing radioactive cesium can be made safe for human consumption by adopting the standard limit for radioactive cesium in wheat grain and that radioactive cesium in udon noodles is substantially reduced by cooking.

Effect of γ irradiation on the fatty acid composition of soybean and soybean oil.

Food irradiation is a form of food processing to extend the shelf life and reduce spoilage of food. We examined the effects of γ radiation on the fatty acid composition, lipid peroxidation level, and antioxidative activity of soybean and soybean oil which both contain a large amount of unsaturated fatty acids. Irradiation at 10 to 80 kGy under aerobic conditions did not markedly change the fatty acid composition of soybean. While 10-kGy irradiation did not markedly affect the fatty acid composition of soybean oil under either aerobic or anaerobic conditions, 40-kGy irradiation considerably altered the fatty acid composition of soybean oil under aerobic conditions, but not under anaerobic conditions. Moreover, 40-kGy irradiation produced a significant amount of trans fatty acids under aerobic conditions, but not under anaerobic conditions. Irradiating soybean oil induced lipid peroxidation and reduced the radical scavenging activity under aerobic conditions, but had no effect under anaerobic conditions. These results indicate that the fatty acid composition of soybean was not markedly affected by radiation at 10 kGy, and that anaerobic conditions reduced the degradation of soybean oil that occurred with high doses of γ radiation.

Molecular mechanisms of apoptosis induction by 2-dodecylcyclobutanone, a radiolytic product of palmitic acid, in human lymphoma U937 cells.

The irradiation of fat-containing food forms 2-dodecylcyclobutanone (2-DCB) from palmitic acid (PA). In this study, we investigated whether 2-DCB and PA induce apoptosis in human lymphoma U937 cells. We found that cell viability decreased by 2-DCB and apoptosis was induced by 2-DCB and PA. 2-DCB and PA significantly enhanced the formation of intracellular reactive oxygen species (ROS). Apoptosis induced by 2-DCB and PA was strongly prevented by an antioxidant, N-acetyl-L: -cysteine. The treatment with 2-DCB and PA resulted in the loss of mitochondrial membrane potential, and Fas, caspase-8 and caspase-3 activation. Pretreatment with a pan-caspase inhibitor (z-VAD) significantly inhibited apoptosis induced by 2-DCB and PA. Moreover, 2-DCB and PA also induced Bax up-regulation, the reduction in Bcl-2 expression level, Bid cleavage and the release of cytochrome c from the mitochondria to the cytosol. In addition, an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) was observed after the treatment with 2-DCB and PA. Our results indicated that intracellular ROS generation, the modulation of the Fas-mitochondrion-caspase-dependent pathway and the increase in [Ca(2+)](i) involved in apoptosis are induced by 2-DCB and PA in U937 cells.

Age- and time interval-specific gamma radiation-induced DNA damage in adult maize weevils, Sitophilus zeamais Motschulsky, assessed using comet assays.

The gamma radiation-induced DNA damage in adult maize weevils, Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae), was assessed using single-cell electrophoresis (comet assay). Analysis of DNA damage following 0.5 and 1.0 kGy of gamma radiation was performed using cells from 1- and 15-day-old adults. Gamma-irradiated adults from both age groups showed typical DNA fragmentation, whereas cells from non-irradiated adults showed more intact DNA than young S. zeamais. Investigations using the comet assay showed that tail length, % tail DNA and % DNA damage all increased in adults of both age groups when compared to the control insects. A maximum comet length of 227.33 µm was recorded for 15-day-old adults at 24h after irradiation with 1.0 kGy and a minimum of 50.12 µm for 1-day-old adults at 0 h after irradiation with 0.5 kGy. The percentage of DNA damage increased up to 57.31% and 68.15% for 1- and 15-day-old adults, respectively, at 24h after irradiation with 1.0 kGy, whereas only 8.58% and 12.22% DNA damage were observed in the control batches. The results also showed that percentage of DNA damage increased at 24h after irradiation compared to that at 0 h. However, further studies are needed to confirm these results.

Detection of irradiated food using 2-alkylcyclobutanones as markers: verification of the european committee standardization method EN1785 for the detection of irradiated food containing lipids.

2-Alkylcyclobutanones (ACBs) are specific radiolytic products in irradiated lipid-containing food and can be used to detect irradiation of foodstuffs. EN1785, a European Committee Standardization Method, can detect 2-dodecylcyclobutanone (DCB) and 2-tetradecylcyclobutanone (TCB), which are ACBs, using GC/MS, thereby allowing judgement as to whether foodstuffs have been irradiated. In this study, the performance of EN1785 as a qualitative test in a single laboratory was evaluated and its applicability to beef, pork, chicken and salmon was verified. In the performance evaluation test, lipids extracted from unirradiated food using the Soxhlet extraction method were used as negative samples. Further, negative samples, to which DCB and TCB were added at 0.05 µg/g lipid (equivalent to the amount generated in food when irradiated at 0.5 kGy or more), were used as positive samples. For each food type examined, 4 negative and 16 positive samples were analyzed by EN1785 to verify the method's ability to detect irradiation. All of the negative samples were judged negative and all of the positive samples were judged positive. Thus, the method should be able to detect irradiation in beef, pork chicken and salmon irradiated at 0.5 kGy or higher. Next, to confirm that this is the case, the same types of food examined above, both unirradiated and irradiated at doses of 0.5-4 kGy, were analyzed by the method. All of the unirradiated samples were judged negative and all of the irradiated samples were judged positive. In a laboratory different from the one where the aforementioned evaluation was conducted, a performance evaluation test was carried out. Blind coded samples, including unirradiated and irradiated samples, were then analyzed in the laboratory according to EN178S. Ten samples (2 unirradiated and 8 irradiated samples) were analyzed for each type of food and the verified method was found to be 100% accurate. Even after the irradiated foodstuffs had been frozen for 6-9 months, it was still possible to judge whether the foodstuffs had been irradiated or not using the EN1785 method.

[Development and single laboratory validation for thermoluminescence detection of irradiated foods using X-ray irradiation].

The thermoluminescence (TL) method using X-rays was investigated for the purpose of detection of irradiated food, and the method was validated at a single laboratory level. A small X-ray irradiator was developed as an alternative radiation source for normalization, and X-ray irradiation conditions equivalent to gamma-ray irradiation from (60)Co were established. Gamma-ray irradiated spices were used for the method validation. The detection limits (MDL) and lower limit of integrated TL intensities (MDL×10) for the spices were checked and the separation of silicate minerals from the spices was confirmed to be sufficient for TL analysis. There was no significant difference in TL glow ratio obtained using two sets of X-ray irradiation equipment including the newly developed equipment. Repeatability and intermediate precision showed no influence of analysts, X-ray irradiation equipments, or measurement days on the TL ratios. From these results, this detection method was validated in a single laboratory.

Identification of irradiated prawn (Penaeus monodon ) using thermoluminescence and 2-alkylcyclobutanone analyses.

Thermoluminescence (TL) and 2-alkylcyclobutanone (2-ACB) analyses were performed to identify irradiated prawns ( Penaeus monodon ). With the TL method, minerals were extracted from prawns using acid hydrolysis. The experimental results satisfied the evaluation criteria of European Norm (EN) 1788, even after low-dose irradiation (0.5 kGy) and a 60 day storage at -20 °C. With the 2-ACB method, 2-dodecylcyclobutanone (2-DCB) and 2-tetradecylcyclobutanone (2-TCB) were successfully extracted from prawns by direct solvent extraction with purification using a conventional silica column and a sulfoxide column, which was used for 2-ACB for the first time. Both 2-ACB derivatives were absent from the non-irradiated samples but were identified in all irradiated samples by gas chromatography-mass spectrometry. Moreover, 2-DCB and 2-TCB production correlated with the applied dose (2.5-10 kGy), and the correlation did not diminish after 60 days of storage at -20 °C for any dose. Therefore, these two techniques provide rapid, simple, and promising methods for routine investigation of frozen prawns.

Combined effect of low-dose irradiation and acidified sodium chlorite washing on Escherichia coli O157:H7 inoculated on mung bean seeds.

The effect of low-dose irradiation (0.75 and 1.5 kGy) in combination with acidified sodium chlorite (ASC) on the reduction of Escherichia coli O157:H7 on mung bean seeds was examined. Washing with ASC (0.2, 0.5, 0.8, and 1.2 g/L sodium chlorite and 1.0 g/L citric acid) for 2 h reduced the E. coli O157:H7 population from 5.2 to 2.3-3.3 log CFU/g, depending on the concentrations of sodium chlorite. Gamma ray irradiation at 0.75 and 1.5 kGy resulted in reductions of about 1.8 and 2.8 log CFU/g, respectively. Therefore, a single treatment with ASC washing or gamma ray irradiation at 0.75 or 1.5 kGy could not achieve the complete elimination of E. coli O157:H7 on mung bean seeds. Conversely, low-dose irradiation (0.75 and 1.5 kGy) followed by washing with ASC (0.5-1.2 g/L) reduced the population of E. coli O157:H7 to below the detection limit (<1 log CFU/g). However, E. coli O157:H7 was detected in most samples in the enrichment and germination studies. When the treatment order was reversed (ASC washing followed by low-dose irradiation), the E. coli O157:H7 population was also observed to be below the detection limit. Under this treatment, fewer samples (16.7%) were shown to be positive in the enrichment and germination studies, and complete elimination was not achieved. The germination rates of mung bean seeds were not affected by ASC washing and gamma irradiation; however, the yield and length of sprouts were decreased by gamma irradiation.

Detection of gamma radiation-induced DNA damage in maize weevil, Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae) assessed using the comet assay.

PURPOSE: The Deoxyribonucleic Acid (DNA) Comet assay, being a quick, simple, sensitive, reliable and fairly inexpensive method for measuring DNA strand breaks, has been used to assess DNA damage caused by gamma radiation in developmental stages of maize weevil Sitophilus zeamais Motschulsky. MATERIALS AND METHODS: An analysis of DNA damage following gamma radiation ((60)Cobalt) treatments at a dose of 0.5 and 1.0 kGy (Kilo Gray) has been carried out with cells obtained from the larvae, pupae and adults of S. zeamais. RESULTS: Gamma radiation induced significant damage at DNA level in the experimental larvae, pupae and adults as manifested by increased strand breaks compared to intact cells from non-irradiated ones. Comet assay revealed that tail length and % tail DNA varied significantly for all developmental stages of S. zeamais. CONCLUSIONS: This study suggests that the comet assay would be a potentially useful tool for detecting DNA damage in insect pest control strategies due to its advantages in the requirement of small numbers of cells per sample and sensitivity in detecting low levels of DNA damage.

Physicochemical and microbiological qualities of steamed and irradiated ground black pepper (Piper nigrum L.).

The effects of steam and irradiation treatments on the physicochemical properties (moisture content, pH, extractable yield, reducing sugar, soluble pigment, antioxidant activity, piperine, Hunter's color, and sensory attributes) and microbiological quality (total aerobic bacteria, coliforms, and yeasts and molds) of ground black pepper stored at refrigerated and room temperatures for 6 months were compared and evaluated. Irradiation resulted in a higher microbial reduction in pepper, with minimal effects on the proximate composition, functional components, color, and sensory attributes of the spice. Steamed peppers appeared darker, and a considerable decrease in the piperine content was observed after treatment and storage. This study illustrates that irradiation is a better decontamination method than steam treatment in eliminating microorganisms without apparently affecting the quality of the powdered spice. Storage at 4 degrees C enhanced the microbial quality and minimized the loss of piperine content in ground black peppers.