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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.

Although tumor necrosis factor-α (TNF-α) is a known major inflammatory mediator in inflammatory bowel disease (IBD) and has various effects on intestinal epithelial cell (IEC) homeostasis, the changes in IECs in the early inflammatory state induced during short-time treatment (24 h) with TNF-α remain unclear. In this study, we investigated TNF-α-induced alterations in IECs in the early inflammatory state using mouse jejunal organoids (enteroids). Of the inflammatory cytokines, i.e., TNF-α, IL-1β, IL-6, and IL-17, only TNF-α markedly increased the mRNA level of macrophage inflammatory protein 2 (MIP-2; the mouse homologue of interleukin-8), which is induced in the early stages of inflammation. TNF-α stimulation (3 h and 6 h) decreased the mRNA level of the stem cell markers leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) and polycomb group ring finger 4 and the progenitor cell marker prominin-1, which is also known as CD133. In addition, TNF-α treatment (24 h) decreased the number of Lgr5-positive cells and enteroid proliferation. TNF-α stimulation at 3 h and 6 h also decreased the mRNA level of chromogranin A and mucin 2, which are respective markers of enteroendocrine and goblet cells. Moreover, enteroids treated with TNF-α (24 h) not only decreased the integrity of tight junctions and cytoskeletal components but also increased intercellular permeability in an influx test with fluorescent dextran, indicating disrupted intestinal barrier function. Taken together, our findings indicate that short-time treatment with TNF-α promotes the inflammatory response and decreases intestinal stem cell activity and barrier function. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10616-021-00487-y.

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.

The first study reporting the morphological characterization of Sarcocystis sybillensis was performed in 1983; however, without any molecular analysis. Sarcocystis nipponi has been recently described as a species synonymic to S. sybillensis. We reconfirmed the presence of S. sybillensis in Japanese sika deer (Cervus nippon) captured in its native territory; and performed its molecular and phylogenetic characterization. The morphological characteristics of the sarcocysts were consistent with those of S. nipponi and S. sybillensis described in the first report. However, the nucleotide sequence of 18S rRNA gene of S. sybillensis showed only 91.9% identity to that of S. nipponi, suggesting low homology among the concerned Sarcocystis spp. Accordingly, S. sybillensis was found to occupy a clade distinct from that of S. nipponi in a phylogenetic tree of Sarcocystis. Therefore, the present study provides essential information on 18S rRNA-based molecular characterization of S. sybillensis and disproves the existing notion of morphology-based species synonymity of S. sibillensis and S. nipponi. These results also suggest that S. sybillensis belongs to type 2 Sarcocystis.

Aspergillus section Versicolores species, except Aspergillus sydowii, produce a carcinogenic mycotoxin sterigmatocystin (STC). Since these fungi are found in varied environmental milieu including indoor dust and food products, our aim was to develop a sensitive and convenient assay to detect STC producing fungal strains. We made use of a high discrimination DNA polymerase (HiDi DNA polymerase), for single nucleotide polymorphism (SNP)-based PCR amplification. Using specific primer pairs based on the SNPs between A. sydowii and other strains of Aspergillus section Versicolores, we succeeded in amplifying the genomic DNA all target strains except A. sydowii. These results confirm that the SNP-based PCR amplification technique, using a high discrimination DNA polymerase, was a reliable and robust screening method for target fungal strains.

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 M

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.

Baker's yeast is an attractive host with established safety and stability characteristics. Many yeast-based biosensors have been developed, but transmembrane signal transduction has not been used to detect membrane-impermeable substances using antigen-antibody interactions. Therefore, we created Patrol Yeast, a novel yeast-based immunosensor of various targets, particularly toxic substances in food. A membrane-based yeast two-hybrid system using split-ubiquitin was successfully used to detect practically important concentration ranges of caffeine and aflatoxins using separated variable regions of an antibody. Moreover, enterohemorrhagic Escherichia coli O157 was detected using a specific single-chain antibody, in which Zymolyase was added to partially destroy the cell wall. The incorporation of secreted Cypridina luciferase reporter further simplified the signal detection procedures without cell lysis. The methodology is more cost-effective and faster than using mammalian cells. The ability to detect various targets renders Patrol Yeast a valuable tool for ensuring food and beverage safety and addressing other environmental and technological issues.

A survey of the contamination of foods by sterigmatocystin (STC) was performed by an analytical method based on LC-MS/MS. STC was extracted from samples with acetonitrile/water (85/15, v/v) and then purified with immunoaffinity columns. The method was validated by a small-scale inter-laboratory study using spiked wheat samples. Mean recoveries of STC were 100.3% and 92.5% from two samples spiked at 0.5 and 5.0 µg/kg, respectively. A total of 583 samples were analysed between 2016 and 2018, and STC was detected in 19.9% of all samples at0.05 μg/kg (limit of quantification). The foods that were contaminated by STC were wheat flour, Job's tears products, rye flour, rice, buckwheat flour, white sorghum, barley products, azuki bean and corn flour. STC was not found in beer or wine. The occurrence of STC in domestic wheat flour (44.4%), Job's tears products (41.7%) and rye flour (29.9%) accounted for the three highest values. The highest mean concentrations were obtained for Job's tears products (0.3 μg/kg) and rye flour (0.3 μg/kg). The maximum contamination level was present in a sample of rye flour (7.1 μg/kg). Although the contamination levels were low, these results indicate that STC frequently contaminates Japanese retail foods. A continuous survey is required to assess exposure to STC in Japan.

Contamination of agricultural crops by mycotoxins has increased because of the expansion of mycotoxin-producing fungi along with global warming. In this study, the fungal microflora of brown rice grains cultivated in Kyushu region in the southern part of Japan was investigated. A total of 75% of rice samples examined in this study showed less than 30% of fungal contamination rates with a median rate of 12.5%. Some isolates of Aspergillus flavus showed the ability to produce aflatoxins (AFs) (AFB1 production was 62.5-70.4 ng/mL) . Furthermore, AF-producing Aspergillus flavus survived during storage and Aspergillus creber, which produced sterigmatocystin, was detected in a stored rice sample. Although AFs or sterigmatocystin-contamination was not detected in any rice samples, these mycotoxin-producing fungi are distributed and can survive during storage under the natural conditions in Japan. Employing suitable storage conditions is important for preventing mycotoxin contamination of brown rice grains.

Game meat potentially harbors a number of parasitic and bacterial pathogens that cause foodborne disease. It is thus important to monitor the prevalence of such pathogens in game meats before retail and consumption to ensure consumer safety. In particular, Sarcocystis spp. and Shiga toxin-producing Escherichia coli (STEC) have been reported to be causative agents of food poisoning associated with deer meat consumption. To examine the prevalence of these microbiological agents on-site at a slaughterhouse, the rapid, simple and sensitive detection method known as the "DNA strip" has been developed, a novel tool combining loop-mediated isothermal amplification and a lateral flow strip. This assay has achieved higher sensitivity and faster than conventional PCR and is suitable for on-site inspection.

Exposure to sterigmatocystin (STC) raises concerns on developmental neurological disorders. The present study investigated the effects of maternal oral STC exposure on postnatal hippocampal neurogenesis of offspring in rats. Dams were exposed to STC (1.7, 5.0, and 15.0 ppm in diet) from gestational day 6 until day 21 post-delivery (weaning), and offspring were maintained without STC exposure until adulthood on postnatal day (PND) 77, in accordance with OECD chemical testing guideline Test No. 426. On PND 21, 15.0-ppm STC decreased type-3 neural progenitor cell numbers in the subgranular zone (SGZ) due to suppressed proliferation. Increased γ-H2AX-immunoreactive (+) cell numbers in the SGZ and Ercc1 upregulation and Brip1 downregulation in the dentate gyrus suggested induction of DNA double-strand breaks in SGZ cells. Upregulation of Apex1 and Ogg1 and downregulation of antioxidant genes downstream of NRF2-Keap1 signaling suggested induction of oxidative DNA damage. Increased p21WAF1/CIP1+ SGZ cell numbers and suppressed cholinergic signaling through CHRNB2-containing receptors in GABAergic interneurons suggested potential neurogenesis suppression mechanisms. Multiple mechanisms involving N-methyl-d-aspartate (NMDA) receptor-mediated glutamatergic signaling and various GABAergic interneuron subpopulations, including CHRNA7-expressing somatostatin+ interneurons activated by BDNF-TrkB signaling, may be involved in ameliorating the neurogenesis. Upregulation of Arc, Ptgs2, and genes encoding NMDA receptors and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors suggested synaptic plasticity facilitation. On PND 77, ARC+ granule cells decreased, and Nos2 was upregulated following 15.0 ppm STC exposure, suggesting oxidative stress-mediated synaptic plasticity suppression. Inverse pattern in gene expression changes in vesicular glutamate transporter isoforms, Slc17a7 and Slc17a6, from weaning might also be responsible for the synaptic plasticity suppression. The no-observed-adverse-effect level of maternal oral STC exposure for offspring neurogenesis was determined to be 5.0 ppm, translating to 0.34-0.85 mg/kg body weight/day.

Ciguatera fish poisoning (CFP) is one of the most frequently encountered seafood poisoning syndromes, it is caused by the consumption of marine finfish contaminated with ciguatoxins (CTXs). The majority of CFP cases result from eating fish flesh, but a traditional belief exists among people that the head and viscera are more toxic and should be avoided. Unlike the viscera, scientific data to support the legendary high toxicity of the head is scarce. We prepared tissue samples from the fillet, head, and eyes taken from five yellow-edged lyretail (Variola louti) individuals sourced from Okinawa, Japan, and analyzed the CTXs by LC-MS/MS. Three CTXs, namely, CTX1B, 52-epi-54-deoxyCTX1B, and 54-deoxyCTX1B, were confirmed in similar proportions. The toxins were distributed nearly evenly in the flesh, prepared separately from the fillet and head. Within the same individual specimen, the flesh in the fillet and the flesh from the head, tested separately, had the same level and composition of toxins. We, therefore, conclude that flesh samples for LC-MS/MS analysis can be taken from any part of the body. However, the tissue surrounding the eyeball displayed CTX levels two to four times higher than those of the flesh. The present study is the first to provide scientific data demonstrating the high toxicity of the eyes.

To understand fungal contamination in the indoor environment of the disaster region, a field survey was performed to measure the number of fungal counts and identify isolates in the indoor air of prefabricated temporary housing, privately independent-housing, and rented apartments flooded by the East Japan Great Earthquake disaster tsunami. As a result, the period with the highest detected fungal count was from the rainy season to summer in independent-housing and rented apartments. Moreover, in the temporary housing, the fungal number increased further in winter as indicated by the maximum fungal-number throughout the measurement period. The detection frequency of Aspergillus species was relatively higher in the indoor air of temporary housing than in typical housing in the non-disaster area. Since Aspergillus is known as an allergenic genus, it requires careful attention to the health risk for residents. The extremely high level of fungal condensation in indoor air possibly occurred due to high relative humidity and loss of heat insulation in the building attics. It is suggested that this problem commonly happened in the cold region including the entire disaster region of the East Japan Great Earthquake.

The circumstances in which organisms live induce polymorphism in their genes, including fungal allergen genes, leading to altered structures and functions of proteins, related to their pathogenicity. Major allergen genes of Aspergillus fumigatus, Asp f 1, Asp f 2, and Asp f 3, were examined in 59 strains [environment and animal/human-body origin] to determine their nucleotide sequences, and then categorized. The location and number of IgE epitopes on the allergen molecules were predicted using a computer software. The Asp f 1 gene was classified into two groups (f1-1 and f1-2). One of the groups possessed one-nucleotide mutation point with one amino-acid substitution. The mutated Asp f 2 gene accompanying 6-amino acid substitution was classified into 7 groups (f2-1 to f2-7). Six of the groups possessed a newborn IgE epitope. The Asp f 3 gene contained two mutations, resulted in three groups (f3-1 to f3-3) without any amino-acid substitutions. Category E, consisting of groups f1-1, f2-5, and f3-2, was specific to an environmental origin. Our findings suggest that nucleotide mutation of the fungal allergen genes, associated with the origin of the fungus, modifies the structure of proteins, and affects their pathogenic properties, such as the localization of IgE epitopes.

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 ≥10

Entoloma sarcopum is widely known as an edible mushroom but appears morphologically similar to the poisonous mushrooms E. rhodopolium sensu lato (s. l.) and E. sinuatum s. l. Many cases of food poisoning caused by eating these poisonous mushrooms occur each year in Japan. Therefore, they were recently reclassified based on both morphological and molecular characteristics as sensu stricto species. In this study, we analyzed the nucleotide sequences of the rRNA gene (rDNA) cluster region, mainly including the internal transcribed spacer regions and mitochondrial cytochrome oxidase 1 (CO1) gene, in E. sarcopum and its related species, to evaluate performances of these genes as genetic markers for identification and molecular phylogenetic analysis. We found that the CO1 gene contained lineage-specific insertion/deletion sequences, and our CO1 tree yielded phylogenetic information that was not supported by analysis of the rDNA cluster region sequence. Our results suggested that the CO1 gene is a better genetic marker than the rDNA cluster region, which is the most widely used marker for fungal identification and classification, for discrimination between edible and poisonous mushrooms among Japanese E. sarcopum and related species. Our study thus reports a new genetic marker that is useful for detection of Japanese poisonous mushrooms, Entoloma.

Citreoviridin (CTV) is a mycotoxin that is produced by Aspergillus terreus, Eupenicillium ochrosalmoneum and Penicillium citreonigrum, and CTV has been detected in a wide range of cereal grains throughout the world. Furthermore, it is especially a serious problem in regions where rice is consumed as a staple food. Moreover, CTV is a well-known yellow rice toxin, and outbreaks of beriberi have occurred due to consumption of rice that is contaminated by CTV even in the recent years. Although CTV biosynthetic genes of A. terreus have been described, those of P. citreonigrum remain unclear, which is concerning since P. citreonigrum is the main cause of CTV contamination in rice. In the present study, we determined the draft genome of the P. citreonigrum strain IMI92228 and revealed the presence of all four genes that form a gene cluster and that are homologous to the CTV biosynthesis genes of A. terreus. The expression of these four homologous genes were highly correlated with CTV production, suggesting that they may play an important role in CTV biosynthesis in P. citreonigrum. We concluded that the gene cluster is a CTV biosynthesis cluster of P. citreonigrum. The findings will contribute to the understanding of the biosynthetic pathway of CTV and will ultimately lead to improvements in the CTV management of agricultural products.

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.

Sterigmatocystin is a genotoxic and hepatocarcinogenic mycotoxin that contaminates foods and environments worldwide. Sterigmatocystin is produced as a precursor to aflatoxin B1 or as an end product by certain Aspergilli. Aspergillus section Versicolores is one of the major sections including sterigmatocystin-producing species and is thus a potential health and environmental hazard. Recently, the taxonomy of this section was revised and classified into 14 species on the basis of molecular phylogenetic analysis. However, investigation of the distribution and sterigmatocystin production of each species has been limited; in particular, its distribution in foods has been scarcely reported. In this study, we collected isolates of Aspergillus section Versicolores from various foods and environments in Japan and investigated their distribution and sterigmatocystin production. The isolates were classified into nine species or species groups, which revealed that A. creber, A. puulaauensis/tennesseensis and A. sydowii are the main species/species groups in Japan. In addition, A. versicolor sensu stricto was detected with some frequency, specifically in foods. Furthermore, the two species A. creber and A. versicolor sensu stricto frequently produced sterigmatocystin. It is therefore important for food safety to intensively monitor these two species and distinguish them from other species, especially A. sydowii, which is not considered to produce sterigmatocystin.

Fumonisins are well known as mycotoxins produced by various Fusarium species. Recently Aspergillus niger has been reported to be a fumonisin B2 (FB2) producer. Aspergillus niger is a member of Aspergillus section Nigri. Members of this section are common food contaminants and are also distributed widely in the environment. This study aimed to determine 1) optimum culture conditions of A. niger for fumonisin production including growth medium, temperature and incubation period and 2) fumonisin production among isolates of Aspergillus section Nigri and closely related species isolated from Japanese food and environmental samples. Growth on Czapek yeast extract broth +5% NaCl (CYBS) at 28°C for 7 days resulted in the highest levels of FB2 production as determined by quantitative LC-MS/MS of culture extracts. Sixty-two isolates were collected from various foods in domestic markets as well as from soil and air. The isolates principally separated into two groups; A. niger and A. luchuensis/A. piperis/A. tubingensis, following molecular phylogenetic analysis. ELISA using the tip culture method was shown to be suitable for screening of the fumonisin-producing strains. Phylogenic analysis of Aspergillus section Nigri isolates from food and environmental samples indicated that fumonisin producing strains could be grouped into the A. niger clade. Nineteen of 35 (54%) isolates classified as A. niger were FB2 producers. The current study suggests that FB2-producing A. niger are distributed throughout several regions of Japan.

Recently, the Sarcocystis parasite in horse and deer meat has been reported to be a causative agent of acute food poisoning, inducing nausea, vomiting and diarrhea. Compared with other causative agents, such as bacteria, viruses and other parasites, in deer meat, the Sarcocystis species parasite, including its stability under various conditions, is poorly understood. In this study, we assessed the viability of Sarcocystis spp. and the activity of their diarrhea toxin (a 15-kDa protein) in deer meat under conditions of freezing, cold storage, pH change and curing. In addition, the heat tolerance was assayed using purified bradyzoites. The results showed that the species lost viability by freezing at -20, -30 and -80°C for

The objective of this study was to determine the contamination of aflatoxin B1 (AF-B1) when keeping various dairy feeds in a farm environment. The study was carried out from March to May 2011 and involved 63 small holder dairy farms belonging to a single dairy cooperative in Chiang Mai province, Thailand. All feed samples used for milking cows including 4 commercial concentrates (CC1 to CC4), by-products from local corn processing factories fermented in plastic bags (SIL), and corn and cob meal or corn dust (CCD). Feed samples were collected 2 times at before and after storage. Farmers were requested to store CC1 to CC4 and CCD for a month and SIL for a week using their routine on-farm storage arrangements. All samples were measured for their AF-B1 concentrations by ELISA. Results showed that AF-B1 concentrations of CC1 to CC4, SIL and CCD before storage were 5.1, 4.1, 4.0, 4.2, 5.5 and 5.5 μg/kg, respectively, and after storage the concentration of AF-B1 were 9.7, 6.5, 9.8, 12.3, 11.4 and 20.0 μg/kg, respectively. CCD at after storage was the only feed that had mean level more than 20 μg/kg. Concentrations of AF- B1 at before storage in all feeds were significantly lower than after storage (P0.01), and the increased ratio of AF-B1 levels was approximately 2 to 3 times. The study concluded that increased AF-B1 levels are related to feed types and farm conditions.

Citreoviridin (CTV) in an inhibitor of mitochondrial ATPase that has been isolated from molded yellow rice and linked to the human disease Shoshin-kakke (acute cardiac beriberi). The disease results from a deficiency of thiamine, however, purified CTV can reproduce the symptoms in experimental animals. The link between CTV and Shoshin-kakke has been difficult to resolve, in part because cases of the disease are rare. In addition to rice, CTV has been found in maize, pecan nuts, and wheat products. A method to screen for CTV and its geometric isomer, iso-CTV, in commodities was developed, based upon the isolation of two novel monoclonal antibodies (mAb). In an antigen-immobilized competitive enzyme-linked immunosorbent assay format (CI-ELISA), the observed IC50s for CTV were 11 ng/mL and 18 ng/mL (mAbs 2-2 and 2-4, respectively). The assays were relatively tolerant to methanol and acetonitrile, which allowed their application to the detection of CTV in spiked polished white rice. For quantification, a standard mixture of CTV and iso-CTV was used, along with matrix matched calibration. The dynamic range of the ELISA using mAb 2-4 was equivalent to 0.23 to 2.22 mg/kg in rice. Recoveries over the range of 0.36 to 7.23 mg/kg averaged 97 &plusmn, 10%. The results suggest that the mAb 2-4-based immunoassay can be applied to the screening of white rice for CTV. Both mAbs were also observed to significantly enhance the fluorescence of the toxin.

This study was conducted to give a preliminary estimation of deoxynivalenol (DON) dietary exposure in Japanese university students (n = 30, aged 22–25 years) using a biomarker approach and to examine the correlation between wheat food intake and DON exposure levels. Spot urine samples were collected from 30 students of Azabu University, Tokyo. Urine samples were treated with enzyme digestion (for total DON measurement) and without (for unconjugated DON analysis) before clean-up using an immuno-affinity column and analysis using an LC-MS method, with a 13C15- DON internal standard used for accurate quantification. The limit of detection for this method is 0.5 ng/mL urine. The geometric mean (95% CI) of DON concentration was 2.03 (1.64 – 6.87) ng per mL urine. Ninety of the urine samples had detectable levels of urinary DON. The DON dietary intake exposure estimation suggested that one out of the 30 subjects had an intake of DON that exceeded Joint FAO/WHO Expert Committee on Food Additives (JECFA) provisional maximum tolerable daily intake (PMTDI) level. Mean ratio of free DON to total DON was determined to be 19%. Wheat intake assessed using a basic food frequent questionnaire method did not show a significant correlation with the urinary DON level.

Citreoviridin (CTVD), a mycotoxin called yellow rice toxin, is reported to be related to acute cardiac beriberi, however, its toxicokinetics remain unclear. The present study elucidated the toxicokinetics through in vivo experiments in swine and predicted the human toxicokinetics by comparing the findings to those from in vitro experiments. In vivo experiments revealed the high bioavailability of CTVD (116.4%) in swine. An intestinal permeability study using Caco-2 cells to estimate the toxicokinetics in humans showed that CTVD has a high permeability coefficient. When CTVD was incubated with hepatic S9 fraction from swine and humans, hydroxylation and methylation, desaturation, and dihydroxylation derivatives were produced as the predominant metabolites. The levels of these products produced using human S9 were higher than those obtained swine S9, while CTVD glucuronide was produced slowly in human S9 in comparison to swine S9. Furthermore, the elimination of CTVD by human S9 was significantly more rapid in comparison to that by swine S9. These results suggest that CTVD is easily absorbed in swine and that it remains in the body where it is slowly metabolized. In contrast, the absorption of CTVD in humans would be the same as that in swine, although its elimination would be faster.

To elucidate whether Kudoa septempunctata was able to live in the human intestine, we assessed viability of K. septempunctata sporoplasms under conditions that mimicked human and ragworm digestive tracts. To study the effect of osmotic pressure on viability, sporoplasms were incubated in 0.9 or 3.4 % sodium chloride solutions, which roughly corresponded to the osmotic pressure in human or ragworm tissues, respectively. While viability in 3.4 % sodium chloride did not change after 72 h, it dropped to 21 % in 0.9 % sodium chloride. To study the effect of temperature on viability, sporoplasms were incubated at 37, 15, or 25 °C, which were representative of human, winter ragworm, or summer ragworm temperatures, respectively. Viability decreased sharply to 8.4 % after 48 h at 37 °C, but remained essentially unchanged at 15 and 25 °C. In addition, sporoplasms showed strong susceptibility to bile. These results indicate that K. septempunctata could not live in the human intestine for a long time.

Kudoa septempunctata is the causative agent of a food-borne disease associated with the ingestion of raw olive flounder. As the current qRT-PCR method for its detection is time-consuming, a rapid and simple method is required. Recently, a new real-time loop-mediated isothermal amplification (LAMP) method and an immunochromatography method, whose sensitivities are intended to be compatible with that designated in the official analytical method (10(5) spores/g olive flounder), have been developed. To validate these new methods, we performed an inter-laboratory study across seven laboratories. Both methods could not detect less than 10(4) spores/g; however, these methods were able to detect more than 10(5) spores/g in olive flounder samples. These results demonstrated that the sensitivities of these methods were compatible with the designated level in the official analytical method. We concluded that these new methods were acceptable as the screening methods for K. septempunctata.

Psocids inhabit stored foods and cereals and cause serious problems with regard to food hygiene. They are a major target for pest control around the world, including Japan. Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelidae) is a representative species of household psocid that prefers to feed on fungi while also spreading fungi in the site it inhabits; it is therefore recognized as an indicator of fungal generation. The present study investigated the characteristics of fungi that influence the feeding behavior of this fungivorous insect using four fungus species (Aspergillus tubingensis, Aspergillus flavus, Penicillium chrysogenum, Cladosporium cladosporioides) that are representative fungi found in Japanese food factories and food storage facilities. The results of preference and feeding tests performed using an arena device (diameter: 9 cm) and a handmade olfactometer revealed that P. chrysogenum was the most attractive fungus for feeding. Based on the comparison of multiple characteristics of P. chrysogenum and the three other fungus species, the factors that were considered to evoke the feeding behavior of L. bostrychophila were a velvety appearance with hyphae that were shorter than the height of the insect as well as specific odors. The present study clarified the common characteristics of the fungi preferred by L. bostrychophila, which may be used to predict pest occurrence and develop agents for trapping and monitoring, thus leading to effective integrated pest management.