Genetically modified (GM) papaya has not yet been approved for importation into, or cultivation in the European Union (EU) and Japan. A DNA extraction method using the Qiagen DNeasy Plant Mini Kit (PM method) and a method using a buffer containing cetyltrimethyl ammonium bromide (CTAB method) have been adopted as the official Japanese methods for detecting GM foods. However, the amounts of DNA extracted from papaya by these methods are very low. Therefore, we investigated an extraction method to obtain a high yield of DNA from raw or freeze-dried fresh papaya using the Promega Wizard DNA Clean-Up Resin System (WCR). The incubation for the extraction was carried out at 58°C without proteinase K for 15 min. The extract was applied to a mini-column, then the column was washed with 80% isopropyl alcohol, and genomic DNA adsorbed on the column was eluted with TE buffer. The WCR method gave a higher yield of genomic DNA, and was simpler and faster than the PM method or CTAB method. In addition, it could be used to extract genomic DNA from fresh papaya at various stages of ripeness. Based on these results, we propose that the present method using WCR is the most practical and useful way to extract genomic DNA for the purpose of detecting GM papaya.
Contamination of spices with pathogens has been reported worldwide, and Salmonella might result in foodborne infections. In this study, we investigated the survival of Salmonella in black pepper and red pepper, and the growth of the surviving Salmonella in cooked food. Salmonella Enteritidis, Salmonella Weltevreden and Salmonella Senftenberg were inoculated into spices, and their survival during storage was examined. In black pepper, S. Enteritidis was no longer viable after storage for 28 days, but S. Weltevreden and S. Senftenberg remained viable. In red pepper, S. Weltevreden and S. Senftenberg survived for 28 days although S. Enteritidis was not viable after 7 days. Salmonella Weltevreden and Salmonella Senftenberg were inoculated into cooked food, and their survival during storage was determined. In potato salad, egg salad, namul and kimchi as cooked foods, both pathogens grew at 30°C, but not at 10°C. Our results indicate that cooked food should be stored at low temperature after addition of spices, such as black pepper and red pepper, following the cooking.
A simple method for the simultaneous determination of four aconitine analogues (AC; aconitine, HA; hypaconitine, MA; mesaconitine, JA; jesaconitine) in Aconitum plants (Aconitum subcuneatum NAKAI) and a food that caused food poisoning was developed, using liquid chromatography tandem mass spectrometry (LC/MS/MS). Aconitine analogues were extracted with 1 mmol/L HCl and then cleaned up with an Oasis HLB cartridge. The LC separation was performed with an octadecylated silica column (Develosil ODS-HG-5, 2.0 mm i.d.×50 mm) at a flow rate of 0.2 mL/min, using A solution (5 mmol/L ammonium acetate dissolved in 0.1% acetic acid) and B solution (acetonitrile-THF=1 : 3), 90%A (0 min)→60%A (15 min)→const. (2 min). Mass spectral acquisition was performed in the positive mode and the analogues were targeted using multiple reaction monitoring (MRM) with electrospray ionization (ESI). The recoveries of aconitine analogues were 93-99% from Aconitum plants. The detection limits of AC, HA, MA and JA were 0.4, 0.4, 0.3 and 0.5 ng/g, respectively. The aconitine analogues, except JA, were detected in food that caused food poisoning at the level of 2.6-29.7 μg/g. These results indicate that the developed method is suitable for the determination of aconitine analogues in Aconitum plants and foods that cause food poisoning.
Comparative sequence analysis was performed on the 18S rRNA gene (1,676 bp), 26/28S rRNA gene D2 region (321 bp) and lys2 (997 bp) to evaluate the gene index for rapid, accurate and convenient identification of Byssochlamys spp. and related species. The results showed that 26 strains (11 species) of the clade could be identified or grouped by means of each gene sequence. The highest resolution to discriminate these species was observed with lys2, but 26/28S rRNA gene D2 region was considered to be the best index for convenient identification. In addition, phylogenetic analysis based on these genes indicated that genus Byssochlamys is not monophyletic, although species in the clade are closely related to each other. Re-classification will be necessary, based on detailed morphological observations.
Because there is a great difference between the toxicity of inorganic arsenic (As) and organic As in food, the JECFA has set a PTWI value for inorganic As (iAs) rather than for total As. The difference in As toxicity makes it necessary to extract iAs completely from food samples for toxicological analysis, but complete extraction of As from most foods including seaweed has not been achieved to date. We developed a partial-digestion method that uses nitric acid as a solvent in order to extract almost all arsenicals from the solid matrix of hijiki (Hizikia fusiforme, a brown alga) samples. In this method, organic As species were not converted into iAs. HPLC/ICP-MS was then used to determine the concentration of iAs. Total As was measured by hydride generation-atomic absorption spectrometry. The adopted conditions for 0.1 g of ground fine powder sample were: 2 mL of 0.3 mol/L nitric acid; heating, 80°C for 1 hr. Intra-laboratory validation of the method showed good precision and accuracy. The repeatability and intermediate precision for iAs were 1.5% and 1.5%, respectively. The LOD and LOQ for iAs were 0.14 and 0.46 mg/kg dry weight, respectively. Recovery studies performed by spiking 0.5 mg/kg dry weight as the LOQ level and by spiking 3 mg/kg dry weight as the iAs concentration of an un-spiked hijiki sample showed good accuracy. The method was applied to hijiki samples after a water soaking process and a water soaking and simmering process. The results suggested that the As concentration in hijiki after both processes was lower than that before the treatments and that the water soaking and simmering process reduced the iAs concentration much more effectively than the water soaking process.
Arsenic (As) uptake in human occurs via the food chain mainly. The Joint FAO/WHO Expert Committee on Food Additives has established the provisional tolerable weekly intake level for As as an inorganic As (iAs) value, because iAs in food is much more toxic than organic As. In this study, we studied an acid based partial-digestion method for the complete extraction of arsenicals from rice. HPLC/ICP-MS was used to determine the concentration of iAs selectively. The conditions adopted to extract arsenicals from a 0.5 g of finely ground rice sample were addition of 2 mL of 0.15 mol/L nitric acid and heating at 80°C for 2 hr. The LOD and LOQ for iAs were 0.0024 and 0.0079 mg/kg dry weight, respectively. Recovery studies showed good accuracy. When the method was applied to ten short-grain brown rice samples, the iAs concentrations were 0.108-0.227 mg/kg dry weight and the total As concentrations were 0.118-0.260 mg/kg dry weight. Although dimethylarsinic acid was also detected in most samples, the percentage of iAs content in total As content was 62.2-96.3%. Thus, iAs was the principal As species in the short-grain brown rice samples tested.
Methods using high-performance liquid chromatography with fluorescence detection (HPLC-FL) and using liquid chromatography with tandem mass spectrometry (LC/MS/MS) were developed for simultaneous determination of ochratoxin A (OTA), ochratoxin B (OTB) and citrinin (CIT) in cereal, fruit, and coffee products. The samples were extracted with ethyl acetate under an acidic condition, and then cleaned up with liquid-liquid separation. The test solutions were analyzed by reverse-phase HPLC-FL and LC/MS/MS. Mass spectral acquisition was performed in positive ion mode by applying multiple reaction monitoring. The performances of both detectors were almost equivalent. The recoveries of OTA and OTB were 87-111%, and that of CIT were 70-88%. The limits of quantification (S/N≥10) of OTA, OTB and CIT was 0.1 μg/kg or less. These methods were considered to be useful for the determination of the three mycotoxins at low levels (0.1 μg/kg).
Mold species that grew on the surface of retailed strawberries (10 packs, 211 strawberries) and cherries (18 packs, 441 cherries) during storage at 25°C were isolated and identified to evaluate the state of mold growth. Mold growth was observed on 208 (98.6%) of the 211 strawberries and 193 (43.8%) of the 441 cherries. The mold species most frequently isolated from strawberries was Botrytis cinerea, being observed in 81.0% of the strawberries examined, followed by Cladosporium and Alternaria alternata. The mold most frequently isolated from cherries was Alternaria (28.7%), followed by Penicillium, Botrytis, and Cladosporium. The frequency of cherries on which mold growth was observed varied among packs. Mold tended to grow more often in the areas of the fruits in contact with adjacent fruits.
Ochratoxin A (OTA), ochratoxin B (OTB) and citrinin (CIT) in commercial foods were simultaneously determined and confirmed with high-performance liquid chromatography (HPLC) and liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS). The samples examined were made up of cereal, fruit, coffee, and cacao products. The limits of quantification (S/N≥10) of OTA, OTB and CIT were 0.1 μg/kg or less. Aflatoxins (AF), deoxynivalenol (DON) and fumonisins were also surveyed. Of 157 samples examined, 44 were contaminated with OTA at levels of 0.11 to 4.0 μg/kg. At least 2 positive samples were labeled as domestics. In most positive samples, the OTA level was low, less than 1 μg/kg. The highest incidence of OTA was observed in cacao powder (10/12), followed by instant coffee (5/7), cocoa (5/8) and raisin (6/13). OTB was found in fruit and cacao products containing relatively high levels of OTA. Co-occurrence of OTA, CIT and DON was found in cereal products, and co-occurrence of OTA and AF was found in cacao products. Approximately 30% of naturally contaminated OTA in roasted coffee bean moved into the extract solution when brewed with paper filter.
Inoculation tests of Staphylococcus aureus were performed to evaluate the risk of toxic hazard in cheese manufacturing processes. S. aureus was inoculated into pasteurized milk or cheese curd, and the survival and growth were examined. S. aureus grew only slightly or decreased in cell number under the manufacturing condition of semi-hard type cheese or soft-type cheese. Under the conditions of the fresh cheese making process, S. aureus slightly increased in cell number, though no enterotoxin was detected. In processed cheese, S. aureus did not grow at all. Growth inhibition of S. aureus by lactic acid produced from starter culture was suggested to be the cause of growth inhibition in the natural cheese.