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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.

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.

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.

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.

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.

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.

To investigate the developmental exposure effect of diacetoxyscirpenol (DAS) on postnatal hippocampal neurogenesis, pregnant ICR mice were provided a diet containing DAS at 0, 0.6, 2.0, or 6.0 ppm from gestational day 6 to day 21 on weaning after delivery. Offspring were maintained through postnatal day (PND) 77 without DAS exposure. On PND 21, neural stem cells (NSCs) and all subpopulations of proliferating progenitor cells were suggested to decrease in number in the subgranular zone (SGZ) at ≥ 2.0 ppm. At 6.0 ppm, increases of SGZ cells showing TUNEL+, metallothionein-I/II+, γ-H2AX+ or malondialdehyde+, and transcript downregulation of Ogg1, Parp1 and Kit without changing the level of double-stranded DNA break-related genes were observed in the dentate gyrus. This suggested induction of oxidative DNA damage of NSCs and early-stage progenitor cells, which led to their apoptosis. Cdkn2a, Rb1 and Trp53 downregulated transcripts, which suggested an increased vulnerability to DNA damage. Hilar PVALB+ GABAergic interneurons decreased and Grin2a and Chrna7 were downregulated, which suggested suppression of type-2-progenitor cell differentiation. On PND 77, hilar RELN+ interneurons increased at ≥ 2.0 ppm; at 6.0 ppm, RELN-related Itsn1 transcripts were upregulated and ARC+ granule cells decreased. Increased RELN signals may ameliorate the response to the decreases of NSCs and ARC-mediated synaptic plasticity. These results suggest that DAS reversibly disrupts hippocampal neurogenesis by inducing oxidative cellular injury and suppressed differentiation of granule cell lineages. The no-observed-adverse-effect level of DAS for offspring neurogenesis was determined to be 0.6 ppm (0.09–0.29 mg/kg body weight/day).

To investigate the developmental exposure effect of citreoviridin (CIT) on postnatal hippocampal neurogenesis, pregnant ICR mice were dietary exposed to CIT at 0, 1, 3 and 10 ppm from gestation day 6 to postnatal day (PND) 21 on weaning. Offspring were maintained through PND 77 without CIT exposure. Male offspring were analyzed. At 10 ppm on PND 21, weak changes suggestive of neural stem cell reduction and progenitor cell proliferation were observed. Number of hilar CALB1+ interneurons reduced, suggesting an influence on neurogenesis. In contrast, number of hilar SST+ interneurons increased and Bdnf and Ntrk2 transcripts upregulated in the dentate gyrus, suggesting a facilitation of BDNF-TRKB signaling for progenitor cell proliferation. Transcript expression changes of an outside regulatory system suggested suppressed function of GABAergic interneurons, especially of PVALB+ interneurons for compensation on neural stem cell reduction. At ≥ 3 ppm, number of ARC+ mature granule cells increased, and at 10 ppm, number of hilar GRIA1+ cells increased and Gria2 and Gria3 upregulated, suggesting an operation of AMPA receptor membrane trafficking on the increase of ARC-mediated synaptic plasticity. On PND 77, all the transcript expression changes of the neurogenesis regulatory system except for Grin2d were inverted, suggesting an operation of a homeostatic mechanism on CIT-induced disruptive neurogenesis. Simultaneous downregulation of Grin2a and Grin2d suggests suppression of GABAergic interneuron function to adjust neurogenesis at the normal level. The no-observed-adverse-effect level of CIT for offspring neurogenesis was determined to be 1 ppm, translating to 0.13–0.51 mg/kg body weight/day of maternal oral exposure.

4,15-Diacetoxyscirpenol (4,15-DAS) is a type A trichothecene mycotoxin produced by Fusarium species. Four modified forms of 4,15-DAS including 7-hydroxydiacetoxyscirpenol, 7,8-dihydroxydiacetoxyscirpenol, 4β,8α,15-triacetoxy-3α,7α-dihydroxy-12,13-epoxytrichothec-9-ene and 4,15-diacetylnivalenol were purified from cultures of F. equiseti. An analytical method using a multifunctional column has been developed for the simultaneous determination of 4,15-DAS, its four modified forms, T-2 toxin, HT-2 toxin and neosolaniol in cereals. The performance of the current method was evaluated, and a total of 248 samples of five different commodities were analyzed for over two years by this method. 4,15-DAS was detected in Job’s tears products, corn flour and azuki bean, but it was not found in wheat flour or rye flour. The four modified forms of 4,15-DAS were detected in samples of Job’s tears products, contaminated by 4,15-DAS. This is the first report on quantification of the modified forms of 4,15-DAS in cereals.

Vibrio parahaemolyticus carrying the tdh gene, encoding the thermostable direct hemolysin (TDH), or the trh gene, encoding the TDH-related hemolysin (TRH), are both considered virulent strains. There are, however, disproportionally fewer reports of infections caused by seafood contaminated with trh-positive strains than by seafood contaminated with tdh-positive strains. Bivalves such as clams and oysters are the major seafood varieties associated with the infections. In this study, the prevalence of strains possessing the tdh and trh genes was investigated in Japan in 74 samples collected in 2007-2008 and in 177 samples collected in 2010 of domestic bivalves, bloody clams, hen clams, short-neck clams, and rock oysters. The tdh-positive and trh-negative, tdh-negative and trh-positive, and tdh-positive and trh-positive samples represented 5.4%, 12.2%, and 4.1% of all samples collected in 2007-2008, and 5.1%, 18.6%, and 5.6% of all samples collected in 2010, respectively. As determined by polymerase chain reaction, the prevalence of tdh negative and trh positive in all samples was two to four times higher than that of tdh positive and trh negative. In the samples collected in 2010, the tdh-negative and trh-positive V. parahaemolyticus (20 samples) was more often isolated than tdh-positive and trh-negative V. parahaemolyticus (7 samples). The most common serotype of tdh-positive isolates (22 of 24 strains) was pandemic O3:K6. The trh-positive isolates (61 strains) were various serotypes including OUT:KUT. In 330 V. parahaemolyticus outbreaks and sporadic infections in Japan, most outbreaks and sporadic infections were caused by tdh-positive and trh-negative strains (89.4%). The frequencies of infections caused by tdh-negative and trh-positive, and both tdh- and trh-positive strains were 1.2% and 3.0%, respectively. This finding suggests that the virulence of trh might be less than that of tdh, although trh-positive V. parahaemolyticus frequently contaminated bivalves.

Commercially available rice grains in Thailand were examined to isolate the monoverticillate Penicillium species responsible for toxic yellowed rice. Penicillium species were obtained from seven out of 10 rice samples tested. Among them, one Penicillium citreonigrum isolate and six Penicillium brocae isolates were morphologically identified. The P. citreonigrum isolate produced the mycotoxin citreoviridin on a yeast extract sucrose broth medium. Mycotoxin surveys showed that citreoviridin was not detected in any samples, but one out of 10 rice samples tested was positive for aflatoxin B1 at a level of 5.9 μg/kg. An Ames test revealed that methanol extracts from rice grains inoculated with selected P. brocae isolates were positive for strains TA100 and YG7108 of Salmonella typhimurium, suggesting the presence of base-pair substitution and DNA alkylation mutagens. Our data obtained here demonstrated that aflatoxin B1 and toxic P. citreonigrum were present on domestic rice grains in Thailand, although limited samples were tested. Penicillium brocae, which may produce mutagenic metabolites, was isolated for the first time from the surface of Thai rice grains.

A survey of the contamination of wheat, barley, and Japanese retail food by four Fusarium mycotoxins, deoxynivalenol (DON), zearalenone (ZEN), T-2 toxin (T-2), and HT-2 toxin (HT-2), was performed between 2010 and 2012. A method for the simultaneous determination of the four mycotoxins by liquid chromatography-tandem mass spectrometry was validated by a small-scale interlaboratory study using two spiked wheat samples (DON was spiked at 20 and 100 μg/kg and ZEN, T-2, and HT-2 at 6 and 20 μg/kg in the respective samples). The recovery of the four mycotoxins ranged from 77.3 to 107.2%. A total of 557 samples of 10 different commodities were analyzed over 3 years by this validated method. Both T-2 and HT-2 were detected in wheat, wheat flour, barley, Job's tears products, beer, corn grits, azuki beans, soybeans, and rice with mixed grains. Only T-2 toxin was detected in sesame seeds. The highest concentrations of T-2 toxin (48.4 μg/kg) and HT-2 toxin (85.0 μg/kg) were present in azuki beans and wheat, respectively. DON was frequently detected in wheat, wheat flour, beer, and corn grits. The contamination level of wheat was below the provisional standard in Japan (1,100 μg/kg). The maximum contamination level of DON was present in a sample of a Job's tears product (1,093 μg/kg). ZEN was frequently detected in Job's tears products, corn grits, azuki beans, rice with mixed grains, and sesame seeds. A sample of a Job's tears product presented the highest ZEN contamination (153 μg/kg). These results indicate that continuous monitoring by multiple laboratories is effective and necessary due to the percentage of positive samples detected.

Kudoa septempunctata is a newly identified causative agent of foodborne diseases associated with consuming raw olive flounder. Qualitative PCR and quantitative real-time PCR have been used as notification methods to identify K. septempunctata in Japan. However, these methods require expensive equipment and are time-consuming (2-3 h for screening). To address these problems, in this study, we developed new rapid and simple methods using real-time loop-mediated isothermal amplification (LAMP) and nucleic acid sequence based amplification-nucleic acid chromatography (NASBA-NAC). Using these methods, the total procedure required approximately 45 min and did not require any expensive equipment. With regard to validating these new methods in comparison with the notification methods used in Japan, we performed an inter-laboratory study of 5 laboratories using samples that included olive flounders infected with 4 different amounts of K. septempunctata. These results demonstrated that the sensitivity of NASBA-NAC was equivalent to that of qualitative PCR, and that the sensitivity of real-time LAMP was equivalent to that of quantitative real-time PCR, which indicated that these new methods were acceptable screening methods for identifying K. septempunctata.

The National Food Surveillance System in Japan was formed in 1998 to monitor the contamination of retail foods with bacterial pathogens. Approximately 2000-3000 samples were tested annually, and the data from food categories that had more than 400 samples collected during 1998-2008 were analysed. With regard to meat, the frequency of positive samples for Salmonella in chicken for raw consumption and ground chicken was 12.7% and 33.5%, respectively. Moreover, Shiga toxin-producing Escherichia coli (STEC) O157 was found in ground meat, organ meat and processed meat, although at a low frequency (0.1%). The prevalence of Campylobacter jejuni/coli was 13.3% and 20.9% in chicken for raw consumption and ground chicken, respectively. In vegetables and fruit, Salmonella was detected in cucumber, lettuce, sprout and tomato samples at a frequency of around 0.1-0.2%. With regard to seafood, Salmonella was found in 0.5% of oysters for raw consumption. Seafood was not contaminated with STEC O157 or Shigella. Serotype Infantis was the most frequently detected serotype of Salmonella in seafood, followed by the serotypes Typhimurium, Schwarzengrund and Manhattan. In ground chicken, 72.2% of the strains were identified as the serotype Infantis. E. coli, as an indicator of food hygiene, was detected in all food categories. The results show the prevalence of the above-mentioned pathogens in the retail food supplied in Japan; further, they indicate that consumption of raw food carries the risk of contracting food-borne infections.

To investigate the diversity of stress tolerance levels in Vibrio parahaemolyticus, 200 V. parahaemolyticus strains isolated from various coastal environments, seafood, and human clinical cases were exposed to acid, low-osmolality, freezing-thawing, and heat stresses. Tolerance against acid stress was higher in the virulent (tdh- and/or trh-positive) strains than in the avirulent (tdh- and trh-negative) strains. Tolerance against low-osmolality, freezing-thawing, and heat stresses was higher in the clinical strains of tdh- and/or trh-positive V. parahaemolyticus than in the coastal environment- and seafood-originated strains of tdh and/or trh-positive V. parahaemolyticus. Tolerance against acid stress was higher in the strains isolated from coastal seawater at ≤15°C than in the strains isolated at ≥20°C. Tolerance against heat stress was higher in the avirulent strains than the virulent strains, and in the strains isolated from coastal seawater at ≥20°C than the strains isolated from coastal seawater at ≤15°C. Therefore, this study demonstrated that the diversity of stress tolerance levels in V. parahaemolyticus strains depended on their source and whether they harbored virulence genes. In particular, there was significantly greater tolerance against acid in the virulence gene-harboring strains and strains isolated from low-temperature seawater. Because the stress tolerances of V. parahaemolyticus have direct influences for the survival in environment and food, it is important for the prevention of foodborne infection to control the stress-tolerant strains.

We aimed to develop a new food-processing approach using pectin to reduce gastrointestinal absorption of mycotoxins. When Ca2+ is added to low-methoxyl pectin, a gel resembling an egg box-like structure forms that is able to trap certain molecules. We examined whether or not low-methoxyl amidated pectin (LMA) and low-methoxyl non-amidated pectin (LMNA) trapped the mycotoxin deoxynivalenol (DON) after being ingested. We first determined the trapping effects of LMA and LMNA on DON in vitro under conditions similar to those in the human stomach, with results showing that LMA gel trapped DON to a greater extent than the LMNA gel. We then performed in vivo experiments and demonstrated that the LMA gel containing DON reduced DON's absorption from the gastrointestinal tract. This new food-processing technique holds great promise for reducing the bioavailability of DON in contaminated food and may be useful in mitigating the effects of other mycotoxins.

Kudoa septempunctata is a myxosporean parasite of Paralichthys olivaceus (olive flounder) and causes a foodborne illness that affects more than 100 cases in Japan each year. We previously reported that the consumption of raw olive flounder meat containing a high concentration of K. septempunctata spores induces transient but severe diarrhea and emesis through an unknown mechanism. Here, we demonstrate that K. septempunctata sporoplasm plays an important role in mediating the toxicity of K. septempunctata. When K. septempunctata spores were inoculated in Caco-2 human intestinal cells, K. septempunctata sporoplasms were released from spores, and they invaded the cells. Electron microscopic observations revealed that the sporoplasm invasion severely damaged the Caco-2 cells. The inoculation of K. septempunctata spores eliminated the transepithelial electrical resistance (TER) across the cell monolayer. Inhibiting the invasion of the sporoplasms prevented the observed loss in cell layer integrity, as illustrated by the rapid elimination of the TER. These results suggest that the invasion by sporoplasms severely damaged individual intestinal cells, resulting in a loss of cell monolayer integrity.

The contamination levels of beauvericin and four enniatins, A, A1, B and B1, in 207 samples of wheat flour and corn grits on the Japanese market were determined by an analytical method based on LC-MS/MS. The toxins were extracted from samples with acetonitrile–water (85:15, v/v) and then purified with C18 cartridges. The method was validated in a single laboratory using spiked samples at two levels; the recovery of the five toxins ranged from 91.1% to 113.8%. Enniatin B was frequently detected in imported wheat flour (81.8%) and domestic wheat flour (85.6%), and the highest concentration of enniatin B was present in a domestic wheat sample (633 μg kg−1). In corn grits, beauvericin was found in 34% of the samples, but enniatins were not detected at all. The maximum concentration of beauvericin in corn grits was 26.1 μg kg−1. Deoxynivalenol and nivalenol in the same samples were determined by a method using an immunoaffinity column. Co-contamination of deoxynivalenol and enniatins was observed in 61% of the imported wheat samples and in 58% of the domestic wheat samples. These results suggest the need for a risk assessment for cyclic depsipeptide mycotoxins in Japan and a study on the synergistic effect of deoxynivalenol and enniatins.

Vibrio parahaemolyticus has been one of the most important foodborne pathogens in Japan since the 1960s, and a large epidemic was caused by the pandemic serotype O3:K6 from 1997 to 2001. V. parahaemolyticus infections, however, have sharply declined since that time. Data on serotypes isolated from 977 outbreaks were collected and analysed. Total and pathogenic, thermostable direct hemolysin (TDH) gene-positive V. parahaemolyticus were qualitatively and quantitatively detected in 842 seafood samples from wholesale markets in 2007–2009. Strains isolated from patients and seafood were analysed by serotyping, tdh -PCR, group-specific PCR for pandemic strains, and pulsed-field gel electrophoresis (PFGE). The sharp decrease in the infections from 1999 onwards was noted not only for O3:K6 infections but also for other serotypes. The change in the seafood contamination situation from 2001 to 2007–2009 was characterised by a decrease to three-fourths in the frequency of tdh -positive samples, although that decrease was small compared to the 18-fold decrease in the cases of V. parahaemolyticus outbreaks. PFGE detected the pandemic O3:K6 serotype in the same profile in seafood and patients from 1998 to the present. Because of no large decrease in seafood contamination by V. parahaemolyticus from the production to distribution stages and the presence of pandemic O3:K6 serotype in seafood to the present, it was suggested that the change of seafood contamination was unrelated to the sharp decrease in V. parahaemolyticus infections. V. parahaemolyticus infections might be prevented at the stages after the distribution stage.

The effects of secondary starter molds of common mold-ripened cheeses on the Shiga toxin-producing Escherichia coli (STEC) O157 were assessed in 3 model systems. In the 1st model, 8 STEC O157 strains were incubated in the spent culture of Penicillium camemberti or Penicillium roqueforti under mild acidic conditions at 25 °C. In the spent cultures of the mold at pH 4.8 to 5.0, the lag times of STEC O157 growth were significantly shorter than those observed in fresh medium. Analyses of the spent culture of P. camemberti showed that the causative agents of the growth enhancement were produced by the mold in response to an acidic environment and were not fully inactivated in heat treatment. In the 2nd model, P. camemberti and STEC O157 were cocultured in acidified milk at 25 °C. The population of STEC O157 reached 108 CFU/mL in the presence of the mold, whereas the population steadily declined in the absence of the mold. Although this growth enhancement was partially attributable to alkalization by the mold, it was observed even when the pH of this model was stabilized. In the 3rd model, 2 STEC O157 strains were incubated in the spent cultures of molds at pH 4.5 at 10 °C. In the spent culture, proportions of injured cells were significantly lower and D values were significantly higher than those in control, except one STEC O157 strain in the spent culture of P. camemberti. These results showed that the molds could enhance the growth and survival of STEC O157 by changing the environment. Practical Application: This study demonstrated that molds in foods can improve the growth and survival of the Shiga toxin-producing Escherichia coli O157. Because microbial interactions are ubiquitous in food, our results provide an important insight for understanding the behavior of microorganisms in food.

Summary To determine the amounts of staphylococcal enterotoxin A (SEA), a novel and sensitive enzyme-linked immunosorbent assay (ELISA) was developed. Protein A, which is produced by Staphylococcus aureus, interferes with the reaction between SEA and anti-SEA immunoglobulin G (IgG), resulting in a false-positive reaction. Chicken IgY was introduced as a capture antibody in the sandwich ELISA system, as IgY binds less efficiently to protein A. When the anti-SEA IgG antibody was used as the capture and detection antibodies (IgG-IgG ELISA), the background levels of protein A increased, thus resulting in a false-positive reaction. A 0.01 ng mL−1 concentration of protein A significantly increased the absorbance value of the blank wells. When the anti-SEA IgY antibody was used as the capture antibody, 1000 ng mL−1 of protein A did not affect the absorbance value. The ELISA system using anti-SEA IgY as a capture antibody and anti-SEA IgG as a detection antibody (IgY-IgG ELISA) showed a detection limit of

A comprehensive and quantitative analysis of the mycoflora on the surface of commercial fruit was performed. Nine kinds of fruits grown in Japan were tested. Overall fungal counts on the fruits ranged from 3.1 to 6.5 log CFU/g. The mean percentages of the total yeast counts were higher than those of molds in samples of apples, Japanese pears, and strawberries, ranging from 58.5 to 67.0%, and were lower than those of molds in samples of the other six fruits, ranging from 9.8 to 48.3%. Cladosporium was the most frequent fungus and was found in samples of all nine types of fruits, followed by Penicillium found in eight types of fruits. The fungi with the highest total counts in samples of the various fruits were Acremonium in cantaloupe melons (47.6% of the total fungal count), Aspergillus in grapes (32.2%), Aureobasidium in apples (21.3%), blueberries (63.6%), and peaches (33.6%), Cladosporium in strawberries (38.4%), Cryptococcus in Japanese pears (37.6%), Penicillium in mandarins (22.3%), and Sporobolomyces in lemons (26.9%). These results demonstrated that the mycoflora on the surfaces of these fruits mainly consists of common pre- and postharvest inhabitants of the plants or in the environment; fungi that produce mycotoxins or cause market diseases were not prominent in the mycoflora of healthy fruits. These findings suggest fruits should be handled carefully with consideration given to fungal contaminants, including nonpathogenic fungi, to control the quality of fruits and processed fruit 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.

Penicillium islandicum produces luteoskyrin (LUT), a yellow rice toxin that has been found frequently in rice. However, conventional analytical methods for determining LUT are limited, are complicated, and exhibit low sensitivity. In this study, an analytical method more sensitive and simple based on high-performance liquid chromatography combined with electrospray ionization mass spectrometry was developed. The cleanup procedure of the method was one step, using a solid-phase extraction cartridge. An isocratic mobile-phase system, consisting of acetonitrile-water-acetic acid (50:49:1 [vol/vol/vol]) at a flow rate of 0.2 ml/min, was utilized to obtain the best resolution. Our method showed good linearity (r = 0.9993, 0.5 to 50 ng/g) and high repeatability (relative standard deviation = 8.9 and 5.1% at levels of 0.5 and 10 ng/g, respectively) in the fortification test. The detection and quantification limits for the method in multiple-reaction monitoring mode were 0.1 and 0.3 ng/g, respectively. The average recovery of LUT in spiked rice at 0.5 and 10 ng/g was 80.7 and 85.2%, respectively. The method developed in this study should be applicable to survey LUT in rice, with high sensitivity, selectivity, and rapidity.

An interfering substance that is not 5-hydroxymethylfurfural appears in some apple juices during high-performance liquid chromatography (HPLC) analysis of patulin based on the AOAC 995.10 method. Because this interfering substance could cause the overestimation of patulin in the apple juices, we tried to identify the substance and to develop an improved method of analyzing patulin free from the influence of this substance. We isolated the substance from the apple juice and identified it as adenosine based on its mass spectrometry, proton nuclear magnetic resonance, and photo diode array spectra. Because of the chemical properties of adenosine, changes in the extraction method under acidic conditions and the HPLC conditions (wavelength and analytical column) were effective for avoiding the influence of adenosine and more specifically for analyzing the patulin. The most effective and simple improvement of the official method was the use of column in-point carbon contents greater than 15.5%.

Deoxynivalenol (DON) and nivalenol (NIV), which are produced by Fusarium fungi as secondary metabolite, are among the mycotoxins known as trichothecenes and they naturally occur in cereal grains of bread making wheat. In this study, contamination levels of these mycotoxins in various wheat flour samples containing domestic flour used for the mass production of bread and related products were collected and analyzed in Japan. Samples of flour and bread were collected from nine prefectures, and their trichothecene levels were measured by a validated High Performance Liquid Chromatography-Mass Spectrometry system. The average concentrations of DON and NIV in flour samples collected were 31.3 ± 28.9 and 8.5 ± 3.7 μg/kg, whereas those in bread samples were 8.6 ± 5.1 μg/kg and 3.4 ± 2.0 μg/kg, respectively. These results suggest that the percentage of DON and NIV remaining after converting flour into bread using industrial equipment and baking yeast were estimated as approximately 74.4 and 65.8 %, respectively.

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.

Immunobiological effects of nivalenol (NIV), a trichothecene mycotoxin produced by Fusarium nivale, were examined in male F344 rats after 90-day dietary exposure at doses of 0, 0.4, 1.5, and 6.9 mg/kg body weight/day (0, 6.25, 25 and 100 ppm, respectively) in a subchronic toxicity study. With regards to the serum immunoglobulin levels, a slight increase of IgM was observed only at 6.9 mg/kg (26% increase), while levels of IgG and IgA did not fluctuate at any dose. Flow cytometric analysis of splenic cells revealed a dose-dependent decrease of T lymphocyte/B lymphocyte (CD3(+)/B220(+)) ratio from 1.5mg/kg and an elevated CD4(+)helper/CD8(+)cytotoxic T lymphocyte ratio at 6.9 mg/kg. Furthermore, increases of natural killer (NK) activity of splenic lymphocytes against YAC-1 target cells were observed at all doses, while the magnitude of changes was similar between 1.5 and 6.9 mg/kg. At 6.9 mg/kg, the reduction of the ratio of NKR-P1A(+) splenic cells, which is an indicator of NK cells in the spleen, was apparent. As with other previous studies of NIV, decreased body weight was observed from 1.5 mg/kg during the experiment in the present study. In summary, NIV affected immune function in rats after 90-day dietary exposure, the effects being apparent from 0.4 mg/kg judging from the increase of NK activity, although nutritional suppression might have influenced the immunological changes appeared from 1.5mg/kg.

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.

The retention of deoxynivalenol in noodles and bread made from naturally-contaminated flour was examined by a chemical analysis (HPLC) and bioassays. The retention level of deoxynivalenol obtained from both assays was reduced by boiling process, although only the bioassays showed it to have been reduced by baking. This study is the first to estimate the exposure to deoxynivalenol from the consumption of the final products of wheat flour in Japan.

The levels of formaldehyde (FA) and acetaldehyde (AA) in polyethylene terephthalate (PET) bottles and in commercial mineral water are reported. All the water samples bottled in Japan contained detectable levels of FA (10.1-27.9 microg l(-1)) and AA (44.3-107.8 microg l(-1)). Of 11 European bottled water samples, eight did not contain either FA or AA, while the remaining three had detectable levels of FA (7.4-13.7 microg l(-1)) and AA (35.9-46.9 microg l(-1)). In three North American bottled water samples, two contained FA (13.6 and 19.5 microg l(-1)) and AA (41.4 and 44.8 microg l(-1)), and one did not. Regardless of the region of origin, all the sterilized water samples contained FA and AA, whilst in contrast, none of the unsterilized water without carbonate contained FA or AA. Of the carbonated water samples, three contained FA and AA, and one did not. When fortified with FA and AA, the commercial water sample without otherwise detectable FA and AA was able to reduce levels, although the commercial water sample containing FA and AA could not. The presence of bacteria in the commercial water samples was investigated using an ATP-based bioluminescent assay and heterotrophic plate count method. The commercial water without FA and AA contained heterotrophic bacteria, whilst the commercial water with FA and AA did not contain detectable bacteria. It is suggested that in this case both FA and AA migrated from PET materials, but were subsequently decomposed by the heterotrophic bacteria in the unsterilized water.

Antibacterial effects of catechins, the major green tea polyphenols, were studied using Clostridium and Bacillus spores. Incubation with crude catechins decreased the number of C botulinum and C butyricum spores but not B cereus spores. Furthermore, the effects of six catechin derivatives on spores were investigated. (−)-Epicatechin gallate (ECg), (−)-epigallocatechin (EGC), (−)-epigallocatechin gallate (EGCg) and (+)-gallocatechin gallate (GCg) were more effective in decreasing C botulinum and C butyricum spore numbers than (+)-catechin (C) and (−)-epicatechin (EC). The vegetative growth of C botulinum and B cereus was inhibited by crude extracts of the catechins. Specifically, purified GCg and EGCg inhibited the vegetative growth of C botulinum and B cereus. The inhibitory effect of ECg on B cereus was similar to that of GCg. However, toxin-production by B cereus was not inhibited by catechin. Damage to the membrane of C butyricum spores by catechin derivatives was shown using fluorescent microscopy. This study shows that low concentrations of catechins, although requiring a long exposure time, inhibited the growth of bacterial spores. However, the effects of the purified derivatives of the catechins were not the same and GCg and EGCg were found to be the most potent. Spores that are generally resistant to many disinfectants were sensitive to catechins. Copyright © 2005 Society of Chemical Industry

Kudoa septempunctata in olive flounder meat was inactivated using 3 distinct freezing methods：liquid freezing for 5 min, air blast freezing at －30℃ for 5 h, and －80℃ for 1 h. The fracture curve of olive flounder meat subjected to liquid freezing resembled that of meat stored at 4℃, indicating that the structure of olive flounder muscle was well preserved. In contrast, air blast freezing induced the disappearance of the fracture point in the fracture curve, indicating that there was deterioration in the meat quality. Liquid freezing preserved the transparency of olive flounder meat to the same degree as that of meat stored at 4°C. However, air blast freezing induced meat cloudiness. These results indicate that liquid freezing can be used for K. septempunctata inactivation without affecting the meat quality.

Food poisoning has been reported after the consumption of raw horsemeat in Japan. Diarrhea with a short incubation period is a common symptom in such cases of food poisoning. Cysts found in horsemeat ingested by patients have been identified as Sarcocystis fayeri based on morphological and genetic evaluation and findings from experimental feeding of cysts to dogs, which resulted in the excretion of sporocysts. The extracts of the horsemeat containing the cysts produced a positive enterotoxic response in the rabbit ileal loop test. Intravenous injection of a 15-kDa protein isolated from the cysts induced diarrhea and lethal toxicity in rabbits, and the protein produced enterotoxicity in the ileal loop test as did the extracts of the horsemeat containing the cysts. The partial amino acid sequence of the 15-kDa protein was homologous to the actin-depolymerizing factor of Toxoplasma gondii and Eimeria tenella. These findings indicate that the 15-kDa protein of S. fayeri is a toxin that causes food poisoning after consumption of parasitized horsemeat.

More than 27 outbreaks per year of food poisoning caused by consuming horse meat were reported in Kumamoto Prefecture (including Kumamoto City) from January 2009 to September 2011. It was found that the causative agent of the outbreaks was a protein with a molecular weight of 15 kDa that had originated from bradyzoites of Sarcocystis fayeri parasitizing the horse meat. Rabit ileal loop tests showed that pepsin treatment of homogenates of frozen horse meat containing the cysts of S. fayeri induced loss of toxicity, presumably by digestion of the proteinous causative agent(s). Slices of horse meat containing the cysts were frozen at below -20°C for various periods. The cysts were collected after thawing the slices, then treated in an artificial stomach juice containing pepsin. The bradyzoites of the cysts kept at -20°C for 48 hr or more completely disappeared. Simultaneously, the 15 kDa protein also disappeared in the frozen cysts. After notifying the public and recommending freezing treatment of horse meat, no subsequent cases of food poisoning were reported. This indicates that freezing of horse meat is effective to prevent the occurrence of food poisoning caused by consuming raw horse meat containing S. fayeri.

Species of the genus Fusarium are well-known plant pathogens and mycotoxigenic fusaria are associated with health hazards to humans and animals. There is a need to understand the mechanisms of mycotoxin production by Fusarium species and to predict which produce mycotoxins. In this study, the Fusarium phylogenetic tree was first inferred among trichothecene producers and related species. We reconstructed the maximum likelihood (ML) tree based on the combined data from nucleotide sequences of rDNA cluster regions, the β-tubulin gene (β-tub) and the elongation factor 1α gene (EF-1α). Second, based on this tree topology, the ancestral states of the producing potential of type A and B trichothecenes (TriA and TriB), zearalenone (ZEN), moniliformin (MON), beauvericin (BEA) and enniatins (ENN) were reconstructed using the maximum parsimony (MP) method based on the observed production by extant species as reported in the literature. Finally, the species having the potential to produce each of these six mycotoxins was predicted on the basis of the parsimonious analysis. The ML tree indicated that the Fusarium species analysed in this study could be divided into two major clades. Clade I was divided into four distinct subclades: I-a, I-b, I-c and I-d. Furthermore, the parsimony reconstruction suggested that the potential for producing MON and ZEN was gained or lost only once, and that the producing potential for TriA and TriB, BEA and ENN was repeatedly gained and lost during the evolutionary history of the Fusarium species analysed in this study. Interestingly, the results showed the possibility that several species, about which reports were scarce with regard to mycotoxin production, have the potential to produce one or more of the six evaluated in this study. The phylogenetic information therefore helps one to predict the mycotoxin-producing potential by Fusarium species, and these "phylotoxigenic relationships" may be useful for predicting the pathogenicity of fungi.

Kudoa septempunctata is a myxosporean parasite that infects Paralichthys olivaceus (olive flounder). Previously, we reported that the consumption of raw P. olivaceus meat containing a high concentration of K. septempunctata spores induces transient but severe diarrhoea and emesis. In this study, we investigated the cytokine production of mouse macrophage-like RAW 264 cells stimulated with K. septempunctata. When the RAW 264 cells were incubated with the spores of K. septempunctata for 24 h, they secreted tumour necrosis factor α (TNF-α) and several chemokines, such as IP-10, MIP-1β, and MIP-2. The secretion of TNF-α was induced in a dose-dependent manner in a bioassay using L929 cells and mouse TNF-α-specific enzyme-linked immunosorbent assay (ELISA). To identify the macrophage receptor of K. septempunctata, activation of HEK 293 cells expressing one of the Toll-like receptors (TLR) was measured using an NF-κB-dependent reporter assay. TLR2-expressing HEK 293 cells were strongly activated following stimulation with the spores. These results suggested that K. septempunctata was recognised by TLR2 on the macrophages, which were then activated and produced TNF-α.

The authors performed exposure and risk assessments based on surveillance studies of retail foods in Japan that were undertaken during the past six years (2004–2010). The exposure to ochratoxin A (OTA) and fumonisins (FBs) in different age groups, including toddlers and young children (1–6 years old), older children (7–14 years old), adolescents (15–19 years old) and adults (over 20 years old) was simulated, and the risk of these mycotoxins was evaluated by comparing the provisional maximum tolerated daily intake (PMTDI) for FBs and the provisional maximum tolerated weekly intake (PMTWI) for OTA established by the FAO/WHO Joint Export Committee on Food Additives. The exposure assessment for both mycotoxins in each age group in Japan indicated that the highest exposure occurred in toddlers and children, but in all cases the percentage of the PMTWI and PMTDI at the 99th percentile of exposure was less than 35% for OTA and 10% for FBs.

To evaluate the relationship between bacterial genotypes and stress resistance patterns, we exposed 57 strains of Shiga toxin-producing Escherichia coli (STEC) O157 to acid, freeze-thaw, heat, osmotic, oxidative, and starvation stresses. Inactivation rates were calculated in each assay and subjected to univariate and multivariate analyses, including principal component analysis (PCA) and cluster analysis. The stx genotype was determined for each strain as was the lineage-specific polymorphism assay (LSPA6) genotype. In univariate analyses, strains of the stx 1 stx 2 genotype showed greater resistance to heat than strains of the stx 1 stx 2c genotype; moreover, strains of the stx 1 stx 2 genotype showed greater resistance to starvation than strains of the stx 2 or stx 2c genotypes. LSPA6 lineage I (LI) strains showed greater resistance to heat and starvation than LSPA6 lineage II (LII) strains. PCA revealed a general trend that a strain with greater resistance to one type of stress tended to have greater resistance to other types of stresses. In cluster analysis, STEC O157 strains were grouped into stress-resistant, stress-sensitive, and intermediate clusters. In stx genotypes, all strains of the stx 1 stx 2 genotype were grouped with the stress-resistant cluster, whereas 72.7% (8/11) of strains of the stx 1 stx 2c genotype grouped with the stress-sensitive cluster. In LI strains, 77.8% (14/18) of the strains were grouped with the stress-resistant cluster, whereas 64.7% (11/17) of LII strains were grouped with the stress-sensitive cluster. These results indicate that the genotypes of STEC O157 that are frequently associated with human illness, i.e., LI or the stx 1 stx 2 genotype, have greater multiple stress resistance than do strains of other genotypes.

In this study, enumeration methods for fungi in foods were evaluated using fruits that are often contaminated by fungi in the field and rot because of fungal contaminants. As the test methods, we used the standard most probable number (MPN) method with liquid medium in test tubes, which is traditionally used as the enumeration method for bacteria, and the plate-MPN method with agar plate media, in addition to the surface plating method as the traditional enumeration method for fungi. We tested 27 samples of 9 commercial domestic fruits using their surface skin. The results indicated that the standard MPN method showed slow recovery of fungi in test tubes and lower counts than the surface plating method and the plate-MPN method in almost all samples. The fungal count on the 4th d of incubation was approximately the same as on the 10th d by the surface plating method or the plate-MPN method, indicating no significant differences between the fungal counts by these 2 methods. This result indicated that the plate-MPN method had a number agreement with the traditional enumeration method. Moreover, the plate-MPN method has a little laborious without counting colonies, because fungal counts are estimated based on the number of plates with growing colonies. These advantages demonstrated that the plate-MPN method is a comparatively superior and rapid method for enumeration of fungi.

BACKGROUND: Members of the genus Fusarium are well known as one of the most important plant pathogens causing food spoilage and loss worldwide. Moreover, they are associated with human and animal diseases through contaminated foods because they produce mycotoxins. To control fungal hazards of plants, animals and humans, there is a need for a rapid, easy and accurate identification system of Fusarium isolates with molecular methods. RESULTS: To specify genes appropriate for identifying isolates of various Fusarium species, we sequenced the 18S rRNA gene (rDNA), internal transcribed spacer region 1, 5.8S rDNA, 28S rDNA, β-tubulin gene (β-tub), and aminoadipate reductase gene (lys2), and subsequently calculated the nucleotide sequence homology with pair-wise comparison of all tested strains and inferred the ratio of the nucleotide substitution rates of each gene. Inter-species nucleotide sequence homology of β-tub and lys2 ranged from 83.5 to 99.4% and 56.5 to 99.0%, respectively. The result indicated that sequence homologies of these genes against reference sequences in a database have a high possibility of identifying unknown Fusarium isolates when it is more than 99.0%, because these genes had no inter-species pair-wise combinations that had 100% homologies. Other markers often showed 100% homology in inter-species pair-wise combinations. The nucleotide substitution rate of lys2 was the highest among the six genes. CONCLUSION: The lys2 is the most appropriate genetic marker with high resolution for identifying isolates of the genus Fusarium among the six genes we examined in this study. Copyright © 2011 Society of Chemical Industry

The antimicrobial action of dietary polysaccharides on eight food-borne pathogenic bacteria were examined. Among the polysaccharides, the carrageenans showed the most pronounced inhibitory effect, the growth of all the bacterial strains except Listeria monocytogenes being significantly inhibited by them, particularly by ι-carrageenan. A growth-inhibition experiment using Salmonella enteritidis showed that the inhibitory effect of the carrageenans was not bactericidal but bacteriostatic. The removal of sulfate residues eliminated the bacteriostatic effect of ι-carrageenan, suggesting that the sulfate residue(s) in carrageenan played an essential role in this effect. The results of the present study suggest that dietary polysaccharides, and particularly carrageenans, may act as effective preservatives in various types of processed food.

To identify a rapid method for extracting a large amount of DNA from fungi associated with food hygiene, extraction methods were compared using fungal pellets formed rapidly in liquid media. Combinations of physical and chemical methods or commercial kits were evaluated with 3 species of yeast, 10 species of ascomycetous molds, and 4 species of zygomycetous molds. Bead grinding was the physical method, followed by chemical methods involving sodium dodecyl sulfate (SDS), cetyl trimethyl ammonium bromide (CTAB), and benzyl chloride and two commercial kits. Quantity was calculated by UV absorbance at 260 nm, quality was determined by the ratio of UV absorbance at 260 and 280 nm, and gene amplifications and electrophoresis profiles of whole genomes were analyzed. Bead grinding with the SDS method was the most effective for DNA extraction for yeasts and ascomycetous molds, and bead grinding with the CTAB method was most effective with zygomycetous molds. For both groups of molds, bead grinding with the CTAB method was the best approach for DNA extraction. Because this combination also is relatively effective for yeasts, it can be used to extract a large amount of DNA from a wide range of fungi. The DNA extraction methods are useful for developing gene indexes to identify fungi with molecular techniques, such as DNA fingerprinting.

The intake of total aflatoxins (AFT) and aflatoxin B(1) (AFB(1)) from food in Japan was estimated from AFT and AFB(1) concentration and frequency data in 24 foods (884 samples) from a 3-year retail market survey from the summer of 2004 to the winter of 2006, and by food consumption data from the National Health and Nutrition Survey performed in 2005. The AFT and AFB(1) survey revealed that peanut, peanut products, cocoa, chocolate, pistachio, white pepper, red pepper, almond, job's tears, buckwheat and corn grits are considered to be contributors of AFT (or AFB(1)) intake in Japan (maximum AFB(1) (AFT) levels ranged from 0.21 to 28.0 microg kg(-1) (from 0.21 to 9.0 microg kg(-1))) in AFT-contaminated food. A probabilistic approach using the Monte Carlo method was carried out to simulate an estimate of the AFT (or AFB(1)) intake distributions in each age group in Japan. In this study, AFB(1) intake ranged from 0.003 to 0.004 ng kg(-1) body weight day(-1) (from lower to upper limits), and the potential risk for cancer using a formula devised by the Joint Food and Agricultural Organization/World Health Organization (FAO/WHO) Expert Committee on Food Additives (JECFA) was estimated at 0.00004-0.00005 person/year/100,000 persons, even though this was in the higher levels (95.0th percentile) of the consumer population. The results suggest that the current dietary intake of AFB(1) in Japan has no appreciable effect on health.

Between 2004 and 2007 we examined foods from Japanese retail shops for contamination with ochratoxin A (OTA) and fumonisins B(1), B(2), and B(3). A total of 1,358 samples of 27 different products were examined for OTA, and 831 samples of 16 different products were examined for fumonisins. The limits of quantification ranged from 0.01 to 0.5 microg/kg for OTA and 2 to 10 microg/kg for the fumonisins. OTA was detected in amounts higher than limits of quantification in wheat flour, pasta, oatmeal, rye, buckwheat flour and dried buckwheat noodles, raisins, wine, beer, coffee beans and coffee products, chocolate, cocoa, and coriander. OTA was found in more than 90% of the samples of instant coffee and cocoa, and the highest concentration of OTA, 12.5 microg/kg, was detected in raisins. The concentration of OTA in oatmeal, rye, raisins, wine, and roasted coffee beans varied remarkably from year to year. Fumonisins were detected in frozen and canned corn, popcorn grain, corn grits, cornflakes, corn soups, corn snacks, beer, soybeans, millet, and asparagus. The highest concentrations of fumonisins B(1), B(2), and B(3) were detected in corn grits (1,670, 597, and 281 microg/kg, respectively). All of the samples of corn grits were contaminated with fumonisins, and more than 80% of the samples of popcorn grain and corn snacks contained fumonisins. OTA and fumonisins were detected in several food products in Japan; however, although Japan has not set regulatory levels for these mycotoxins, their concentrations were relatively low.