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Aflatoxins (AFs) are known to be oncogenic mycotoxins. This study investigated the mitigation effects of lactic acid bacteria (LAB) isolated from four types of vegetable, cucumber, Chinese cabbage, Japanese radish and eggplant, which are used to make Japanese traditional fermented pickles, on AFs. Using aflatoxin M1 (AFM1) binding assay for screening, four representative strains were selected (one from each vegetable) from total 94 LAB strains, based on the highest binding ratio. The ranges of the binding ratio of these representative strains to aflatoxin B1 (AFB1), aflatoxin B2, aflatoxin G1, aflatoxin G2 and AFM1 were 57.5%-87.9% for the LAB strain derived from cucumber, 18.9%-43.9% for the LAB strain derived from Chinese cabbage, 26.4%-41.7% for the LAB strain derived from Japanese radish, and 15.0%-42.6% for the LAB strain derived from eggplant. The strains isolated from cucumber, Chinese cabbage, Japanese radish and eggplant were identified as Lactococcus lactis subsp. lactis, Weissella cibaria, Leuconostoc mesenteroides and Leu. mesenteroides, respectively. An in vitro binding assay of the four strains under acidic conditions showed that the number of living bacteria decreased, while the binding ratio increased in some strains, suggesting that the LAB maintained their capacity to bind aflatoxins even in an environment that imitated the stomach. An in vivo experiment using L. lactis subsp. lactis derived from cucumber revealed that the bacteria significantly inhibited the absorption of AFB1 into blood. These results showed that the LAB used for Japanese vegetable pickles was an effective binding agent of AFs and suggested that they might play a role in mitigating AF absorption.

Ciguatera poisoning (CP) is one of the most abundant seafood poisonings in the world. CP frequently occurred in the tropical and subtropical Indo-Pacific Ocean and the Caribbean Sea. In Japan, CP cases have been reported annually, from the subtropical regions, including Okinawa Prefecture and Amami Islands, Kagoshima Prefecture. The principal toxins, named ciguatoxins (CTXs), are bio-synthesized by benthic dinoflagellate of genera Gambierdiscus and Fukuyoa. They are bio-transferred herbivorous animals to carnivorous fishes via the food chain.The Ogasawara Islands comprise more than 30 islands, Mukojima Islands, Chichijima (Bonin) Islands, Hahajima Islands, Iwo Islands, Nishinoshima, Minamitorishima, and Okinotorishima, which locate in the tropical to subtropical regions. The Mukojima Islands, Chichijima Islands, and Hahajima Islands locate approximately the same latitude as Okinawa. The distance from Tokyo is approximately 1,000 km for Chichijima, 1,700 km for Okinotorishima (the southernmost tip of Japan), and 1,900 km for Minamitorishima (the easternmost tip of Japan). These islands exist in a wide range of waters, latitudes from 20°25' to 27°44' North and longitudes from 136°04' to 153° 59' East. We collected 65 specimens of a grouper, Variola louti, the most frequent species implicated in CP in Japan, from the waters around the Chichijima, Mukojima, and Hahajima islands. The fish flesh specimens were analyzed CTXs using the liquid chromatography-tandem mass spectrometer (LC-MS/MS). While the peak whose retention time is almost identical to that of CTX1B was detected in all specimens on our routine protocol, no 52-epi-54-deoxyCTX1B nor 54-deoxyCTX1B was detected. The peak retention time was quite different from that of CTX1B when re-analyzing by changing the analytical column. Thus, the CTXs in the specimens in the waters of these islands seemed to be undetectable levels.

Boiled noodles are considered to be one of the most perishable foods due to their high moisture content and high water activity. Thus, the hygiene control measures based on HACCP manuals has been recommended in the noodle manufacturing industry. However, there were several cases in which post-packaged products manufactured at the Boiled noodles factory of small-to-medium size company detected a viable cell count higher than their voluntary standards. To identify the source of microbial contamination, an investigation based on the Plan-Do-Check-Act (PDCA) cycle was conducted. The results showed that the bacteria causing the contamination were environmental bacteria. Secondary contamination occurred during the cooling process after sterilization. Airborne environmental bacteria and oxygen may have been introduced into the rinsing and cooling water tank by the strong water flow during the rinsing and cooling process, inducing growth of microorganisms in the cooling water and contaminating the final product. This is a new finding, as such occurrence was not listed in the HACCP manual and should be contributed to plan HACCP system.

Staphylococcal food poisoning (SFP) is caused by Staphylococcus aureus, and its typical symptom of vomiting is evoked by staphylococcal enterotoxins (SEs). SEs are classified as classical and new types. SEQ is a new-type enterotoxin predicted to have a high potential risk for SFP. To elucidate the correlation between the number of S. aureus cells and the production of SEs as well as classical and new-type enterotoxins in the food environment, the numbers of S. aureus strain cells carrying sea and seq genes and the production of SEA and SEQ protein were examined under 3 pHs values (pH 6.0, 7.0 and 8.0) and 2 NaCl concentrations (0.5 and 1.0%) conditions. The experiments were performed at 25℃, resembling the setting of scrambled eggs at room temperature after cooking. By 24 hr after incubation, the cell number in the scrambled egg was ≥107/10 g, reaching 109/10 g by 48 hr under all conditions. The productions of both SEA and SEQ were detected in the scrambled egg under all conditions by 48 h. SEQ was detected from 24 hr at all 3 pH values in the egg containing 1.0% NaCl, whereas in the egg containing 0.5% NaCl, it was detected from 24 hr at pH 6.0 and from 48 hr at other pHs. The SEQ production was consistently 100-1,000 times less than that of SEA. These results suggest that the new-type enterotoxin SEQ has the potential to evoke symptoms related to SFP following the consumption of egg products cooked under relative lower pH and water activity.

It has been suggested that a myxosporean parasite, Unicapsula seriolae, is responsible for food-borne disease associated with the ingestion of raw greater amberjack. In this study, we quantified U. seriolae in greater amberjack meats involved in food-poisoning episodes. U. seriolae DNA was detected in 26 samples out of 29 samples by means of quantitative real-time PCR(qRT-PCR). The major symptoms were diarrhea and vomiting within 12 hours after consumption. No seasonal trend in the outbreaks was apparent. The number of spores in samples with qRT-PCR-detected U. seriolae DNA ranged from 1.9×105 to 1.7×107 spores/g. However, no spores were detected in greater amberjack purchased from markets. These results indicate that U. seriolae was responsible for the outbreaks. The copy number of DNA in the positive samples was more than 107 copies/g. The ingestion amount was known in 11 of the incidents, and the minimum quantity of spores that caused symptoms was estimated to be 3.8×106 spores/g.

To validate an LC-MS/MS method using a strong anion exchange cartridge for simultaneous determination of fumonisin B1, B2 and B3 in corn, an inter-laboratory study was performed in 9 laboratories using one fumonisin-negative corn sample, three spiked corn samples (FB1: 100-1,000 μg/kg, FB2 and FB3: 10-100 μg/kg) and two naturally contaminated corn samples. The recoveries were in the ranges of 79.7-87.2% for FB1, 78.6-103.2% for FB2 and 80.1-92.8% for FB3. The relative standard deviations for repeatability (RSDr) ranged from 3.7 to 8.0% for FB1, from 2.6 to 15.3% for FB2 and from 4.3 to 9.7% for FB3. The relative standard deviations for reproducibility (RSDR) for FB1, FB2 and FB3 were in the ranges of 6.3-10.1%, 5.9-18.7% and 9.3-16.0%, respectively. The HorRat values for all analytes ranged from 0.2 to 0.9. The difference of the trueness between the two kinds of commercially available anion exchange cartridges used in this study was not significant (p>0.05). Surveillance for fumonisins in corn grits was performed using the validated method. All of the samples were contaminated with fumonisins and the mean concentrations for FB1, FB2 and FB3 were 118.1, 37.3 and 17.9 μg/kg, respectively. These results indicated that the method for simultaneous determination of FB1, FB2 and FB3 in corn was successfully developed and validated.

To validate an LC-MS/MS method for simultaneous determination of deoxynivalenol (DON) and its acetylated derivatives, 3-acetyl-deoxynivalenol (3ADON) and 15-acetyl-deoxynivalenol (15ADON), in wheat using a multifunctional column, an inter-laboratory study was performed in 9 laboratories using one blank wheat sample, three spiked wheat samples (10, 50, 150 µg/kg) and one naturally contaminated wheat sample. The recoveries ranged from 98.8 to 102.6% for DON, 89.3 to 98.7% for 3ADON, and from 84.9 to 90.0% for 15ADON. The relative standard deviations for repeatability (RSDR) and reproducibility (RSDR) of DON were in the ranges of 7.2-11.3% and 9.5-22.6%, respectively. For 3ADON, the RSDR ranged from 5.3 to 9.5% and the RSDR ranged from 16.1 to 18.0%, while for 15ADON, the RSDR ranged from 6.2 to 11.2% and the RSDR ranged from 17.0 to 27.2%. The HorRat values for the three analytes ranged from 0.4 to 1.2. These results validate this method for the simultaneous determination of DON and its acetylated derivatives, 3ADON and 15ADON.

We evaluated the sensitivity and specificity of an immunochromatography kit, Single-path Emetic Tox Mrk (Merck), which targets a marker protein for the detection of Bacillus cereus that produces emetic toxin. Strains were isolated after outbreaks of food poisoning, and from retail prepared foods and food products. The strains were examined for the presence of the emetic toxin-synthetase gene by PCR. All 58 emetic strains isolated from the food poisoning cases showed a positive reaction in the immunochromatography kit. No emetic strains gave false negative result. Among 47 non-emetic strains, only two strains isolated from the food poisonings and one strain isolated from food products showed a false positive reaction in the test. We concluded that this method has high sensitivity and specificity. The test can be used for detection of emetic toxin-producing B. cereus not only from food poisoning cases, but also in food products.

A rapid, sensitive convenient method for determination of aflatoxin M₁ (AFM₁) in cheese and butter by HPLC was developed and validated. The method employs a safe extraction solution (mixture of acetonitrile, methanol and water) and an immunoaffinity column (IAC) for clean-up. Compared with the widely used method employing chloroform and a Florisil column, the IAC method has a short analytical time and there are no interference peaks. The limits of quantification (LOQ) of the IAC method were 0.12 and 0.14 µg/kg, while those of the Florisil column method were 0.47 and 0.23 µg/kg in cheese and buffer, respectively. The recovery and relative standard deviation (RSD) for cheese (spiked at 0.5 µg/kg) in the IAC method were 92% and 7%, respectively, while for the Florisil column method the corresponding values were 76% and 10%. The recovery and RSD for butter (spiked at 0.5 µg/kg) in the IAC method were 97% and 9%, and those in the Florisil method were 74% and 9%, respectively. In the IAC method, the values of in-house precision (n=2, day=5) of cheese and butter (spiked at 0.5 µg/kg) were 9% and 13%, respectively. The IAC method is superior to the Florisil column method in terms of safety, ease of handling, sensitivity and reliability. A survey of AFM₁ contamination in imported cheese and butter in Japan was conducted by the IAC method. AFM₁ was not detected in 60 samples of cheese and 30 samples of butter.

Aflatoxin M1 (AFM1) is a hydroxylated metabolite of aflatoxin B1 (AFB1), which has been found in the milk of dairy cattle fed AFB1-contaminated feeds. Since AFM1 has been evaluated as a possible human carcinogen, the cancer risk arising from AFM1 contamination in milk is a serious problem in food safety. To evaluate the risk of AFM1 contamination in milk, it is necessary to analyze the risk factors of AFB1 contamination in corn provided for concentrated feed in Japan. The AFM1 level in domestic raw bulk milk was measured at three sampling times, January, February and June in 2004. The AFB1 contamination in corn supplied to cows was determined at the same time as the sampling of raw milk. The AFM1 contamination levels in milk in January, February and June 2004 were 0.011, 0.007 and 0.005 ng/g, respectively. The AFB1 contamination level in the corn of the concentrated feed was higher from October of 2003 to February of 2004 than from April to June in 2004. This study provides evidence that AFM1 contamination level in milk is parallel to that of AFB1 in corn of concentrated feed, so monitoring of the AFB1 level in corn is important to prevent the risk of AFM1 contamination in milk in Japan.

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 degrees C, but not at 10 degrees 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.

Activation and inactivation of aflatoxin B1 (B1) by microsomes and cytosol prepared from the liver of various mammalian and avian species were studied in vitro by determining the microsomal activity to bind aflatoxin to calf thymus DNA and the cytosol activity to inhibit the hamster microsome-mediated aflatoxin-DNA binding. The microsomal activity to bind aflatoxin to DNA was higher in day-old duckling and female chicken than in the other species, being similar in the male hamster, male chicken, both sexes of Japanese quail and laying duck, and lower in the male rat and male mastomys than in the other species. The hamster cytosol inhibited the aflatoxin-DNA binding markedly in the presence of glutathione (GSH) but not at all in its absence. In contrast, the avian cytosol showed a similar level of aflatoxin-DNA binding regardless of the presence or absence of GSH, suggesting that the contribution of cytosol glutathione S-transferase (GST) to B1 detoxification is negligible in the avian species. Nevertheless the cytosol of the avian species such as the Japanese quail and chicken showed apparent inhibitory activity toward aflatoxin-DNA binding. Relative susceptibility of the mammalian and avian species to the toxic and carcinogenic effects of B1 could be explained by the combined activities of microsomes and cytosol, indicating the importance of the opposite activities of microsomes and cytosol toward aflatoxin-DNA binding in the species difference in susceptibility.