Publications by authors named "Tomoya Yoshinari"

51 Publications

Disruption of postnatal neurogenesis and adult-stage suppression of synaptic plasticity in the hippocampal dentate gyrus after developmental exposure to sterigmatocystin in rats.

Toxicol Lett 2021 Oct 11;349:69-83. Epub 2021 Jun 11.

Laboratory of Veterinary Pathology, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo, 183-8509, Japan; Cooperative Division of Veterinary Sciences, Graduate School of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo, 183-8509, Japan; Institute of Global Innovation Research, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo, 183-8509, Japan. Electronic address:

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 p21 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.
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http://dx.doi.org/10.1016/j.toxlet.2021.06.006DOI Listing
October 2021

A 28-day repeated oral dose toxicity study of enniatin complex in mice.

J Toxicol Sci 2021 ;46(4):157-165

Laboratory of Veterinary Pathology, Tokyo University of Agriculture and Technology.

Enniatins are so-called "emerging mycotoxins" that commonly occur in milligrams per kilogram levels in grains and their derived products, as well as in fish, dried fruits, nuts, spices, cocoa, and coffee. The present study investigated the 28-day repeated oral dose toxicity of enniatin complex in CD1(ICR) mice. Enniatin B, enniatin B1, and enniatin A1 at a ratio of 4:4:1 were administered to male and female mice at doses of 0 (vehicle controls), 0.8, 4, and 20 mg/kg body weight/day. In life parameters did not change during the study period, with the exception of slight reductions in food consumption in male mice administered 4 and 20 mg/kg and in female mice administered 20 mg/kg. Body and organ weights did not change, and no alterations in hematology, blood biochemistry, or histopathology parameters were observed at the end of the administration period. Thus, we determined that the no-observed-adverse-effect level of enniatin complex was 20 mg/kg/day for both sexes under the present experimental conditions.
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http://dx.doi.org/10.2131/jts.46.157DOI Listing
January 2021

Transformation of Selected Trichothecenes during the Wheat Malting Production.

Toxins (Basel) 2021 02 11;13(2). Epub 2021 Feb 11.

Research Centre for Registration of Agrochemicals, Institute of Plant Protection-National Research Institute, 60-318 Poznań, Poland.

The transformation of deoxynivalenol (DON), nivalenol (NIV), and their glucosides (DON3G and NIV3G) during the malting of grains of two wheat varieties was studied. The concentration of DON3G and NIV3G started to increase significantly before the concentration of DON and NIV increased. This may reflect the transformation of the parent mycotoxin forms into their glucosides due to xenobiotic detoxification reactions. After a sharp rise during the last 2 days of the process (day 6 and 7), the DON concentration reached 3010 ± 338 µg/kg in the Legenda wheat-based malt and 4678 ± 963 µg/kg in the Pokusa wheat-based malt. The NIV concentration, at 691 ± 65 µg/kg, remained the same as that in the dry grain. The concentration of DON3G in the Legenda and Pokusa wheat-based malt was five and three times higher, respectively, than that in the steeped grain. The concentration of NIV3G in the Legenda wheat-based malt was more than twice as high as that in the steeped grain. The sharp increase in the concentration of DON at the end of the malting process reflected the high pathogen activity. We set aside some samples to study a batch that was left undisturbed without turning and aeration, for the entire period of malting. The concentration of DON in the malt produced from the latter batch was 135% and 337% higher, for Legenda and Pokusa, respectively, than that in the malt produced from the batch that was turned and aerated. The NIV concentration was 22% higher in the latter batch.
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http://dx.doi.org/10.3390/toxins13020135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7918639PMC
February 2021

[Discovery of Putative Anti-Kudoa septempunctata Chemical Agents by Comprehensive Assay].

Shokuhin Eiseigaku Zasshi 2020 ;61(5):183-185

National Institute of Health Sciences.

We screened 360 chemicals and discovered that 71 chemicals had anti-Kudoa septempunctata effect. Especially 19 and seven of 71 chemicals were antibiotics and antibacterial agents/disinfectants, respectively. The other 45 chemicals were pesticides, natural toxins, industrial chemicals and medicines for non-infectious diseases. Nineteen antibiotics that possessed anti-Kudoa effect contained four tetracyclines, one steroid, two macrolides, one aminoglycoside, three β-lactams, one quinolone, two rifamycines, one polyene, one novobiocine, one sulfonamide and two nitroimidazoles. To use these drugs for prevention of Kudoa infection, the further study is need for the determination of effective dose.
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http://dx.doi.org/10.3358/shokueishi.61.183DOI Listing
January 2021

Prevalent Human Gut Bacteria Hydrolyse and Metabolise Important Food-Derived Mycotoxins and Masked Mycotoxins.

Toxins (Basel) 2020 10 13;12(10). Epub 2020 Oct 13.

Rowett Institute, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK.

Mycotoxins are important food contaminants that commonly co-occur with modified mycotoxins such as mycotoxin-glucosides in contaminated cereal grains. These masked mycotoxins are less toxic, but their breakdown and release of unconjugated mycotoxins has been shown by mixed gut microbiota of humans and animals. The role of different bacteria in hydrolysing mycotoxin-glucosides is unknown, and this study therefore investigated fourteen strains of human gut bacteria for their ability to break down masked mycotoxins. Individual bacterial strains were incubated anaerobically with masked mycotoxins (deoxynivalenol-3-β-glucoside, DON-Glc; nivalenol-3-β-glucoside, NIV-Glc; HT-2-β-glucoside, HT-2-Glc; diacetoxyscirpenol-α-glucoside, DAS-Glc), or unconjugated mycotoxins (DON, NIV, HT-2, T-2, and DAS) for up to 48 h. Bacterial growth, hydrolysis of mycotoxin-glucosides and further metabolism of mycotoxins were assessed. We found no impact of any mycotoxin on bacterial growth. We have demonstrated that , and hydrolyse DON-Glc, HT-2 Glc, and NIV-Glc efficiently and have confirmed this activity in and (DON-Glc only). and efficiently de-acetylated T-2 and DAS, but none of the bacteria were capable of de-epoxydation or hydrolysis of α-glucosides. In summary we have identified key bacteria involved in hydrolysing mycotoxin-glucosides and de-acetylating type A trichothecenes in the human gut.
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http://dx.doi.org/10.3390/toxins12100654DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7601956PMC
October 2020

[Assessment of Modified Forms of Fumonisins in Corn-Based Products Retailed in Japan by an Alkaline Hydrolysis Method].

Shokuhin Eiseigaku Zasshi 2020 ;61(4):119-125

National Institute of Health Sciences.

Fumonisins, which are secondary metabolites produced by some Fusarium species, are detected mainly in corn and corn-based products. Recently, the presence of modified forms of fumonisins in fumonisin-contaminated food products has been reported. In order to evaluate the health risk of modified forms of fumonisins to the Japanese population, we analyzed modified forms of fumonisins in corn-based products retailed in Japan. The modified and free forms of fumonisins in food samples were hydrolyzed by alkaline treatment. The resulting hydrolyzed fumonisins were quantified by LC-MS/MS, and total fumonisins (sum of modified and free forms) was calculated. A total of 166 samples of corn-based products were analyzed over two years. The relative ratios of mean total fumonisins to mean free fumonisins in the cornflakes, corn snacks, corn flour and powdered corn soup samples were 4.7, 2.8, 2.1 and 1.2, respectively. Total fumonisins in the residual solid of five cornflake and three corn snack samples obtained after extraction with methanol-water (3 : 1) were quantified. In the cornflakes and corn snacks samples, 56-72 and 83-98% of the modified forms of fumonisins were present in the residual solid, respectively. The average daily intake of fumonisins from cornflakes and corn snacks by the Japanese population was estimated at 1.1 to 3.9 ng/kg body weight/day when the results of free fumonisins were used for the estimate, but when the results of total fumonisins were used, average daily intake increased about three times and was estimated at 3.3 to 12.5 ng/kg body weigh/day. These results indicate that a risk assessment of fumonisins, including the modified forms of fumonisins, is necessary in order to evaluate the true risk of fumonisins to Japanese people.
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http://dx.doi.org/10.3358/shokueishi.61.119DOI Listing
November 2020

Transformations of Selected Toxins and Their Modified Forms During Malt Loaf Production.

Toxins (Basel) 2020 06 11;12(6). Epub 2020 Jun 11.

Department of Pesticide Investigation, Institute of Plant Protection-National Research Institute, Wladysława Wegorka 20, 60-318 Poznan, Poland.

An increasing number of studies have found that modified mycotoxins, such as free mycotoxins, naturally occur in food, and severely impact food safety. The present study investigated concentrations of trichothecenes nivalenol (NIV), deoxynivalenol (DON), and zearalenone (ZEN), together with their modified forms, nivalenol-3-glucoside (NIV-3G), deoxynivalenol-3-glucoside (DON-3G), and zearalenone-14-glucoside (ZEN-14G) and zearalenone-14-sulfate (ZEN-14S), respectively, at successive stages of malt loaf production (flour, dough kneading/fermentation, loaf baking). Toxins in bakery products originate in flour produced from wheat grain that is naturally contaminated with . Mycotoxin concentrations were determined using high-performance liquid chromatography-high resolution mass spectrometry, and did not significantly change during the successive stages of bread production. After the dough kneading/fermentation stage, concentrations of NIV-3G and DON-3G were slightly increased, whereas those of ZEN and ZEN-14S were slightly decreased. The largest average decrease (21%) was found in ZEN-14G. After the baking stage, the average concentrations of NIV-3G, DON-3G, ZEN-14S, and ZEN-14G in the loaf crumb and crust decreased by 23%, 28%, 27%, and 20%, respectively, compared with those in the dough. During this technical process, the concentration of ZEN-14G in loaf crumb significantly decreased by an average of 48%, and those of ZEN, ZEN-14S, and ZEN-14G in loaf crust decreased by an average of 29%, 42%, and 48%, respectively. Considering the possibility of modified mycotoxins degradation to free forms, as well as the ability to synthesize them from free forms during technological processes, it would be prudent to consider them together during analysis.
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http://dx.doi.org/10.3390/toxins12060385DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7354580PMC
June 2020

A New Protocol for the Detection of Sterigmatocystin-producing Aspergillus Section Versicolores Using a High Discrimination Polymerase.

Biocontrol Sci 2020 ;25(2):113-118

Department of Life and Environmental Sciences, Azabu University.

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.
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http://dx.doi.org/10.4265/bio.25.113DOI Listing
April 2021

Whole Genome Analysis Revealed the Genes Responsible for Citreoviridin Biosynthesis in .

Toxins (Basel) 2020 02 15;12(2). Epub 2020 Feb 15.

Graduate School of Life and Environmental Sciences, Azabu University, Kanagawa 252-5201, Japan.

Citreoviridin (CTV) is a mycotoxin that is produced by , and , 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 have been described, those of remain unclear, which is concerning since is the main cause of CTV contamination in rice. In the present study, we determined the draft genome of the 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 . 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 . We concluded that the gene cluster is a CTV biosynthesis cluster of . 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.
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http://dx.doi.org/10.3390/toxins12020125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7077241PMC
February 2020

Detection of Kudoa hexapunctata and Kudoa neothunni from retail raw tuna in Japan using a novel duplex polymerase chain reaction.

Parasitol Int 2020 Apr 28;75:102048. Epub 2019 Dec 28.

Division of Microbiology, National Institute of Health Sciences, 3-25-26 Tonomachi, Kawasaki-ku, Kawasaki, Kanagawa 210-9501, Japan. Electronic address:

Kudoa hexapunctata was taxonomically separated from Kudoa neothunni, but their main host is tuna. K. hexapunctata has been identified as causative agent of foodborne diseases associated with the ingestion of raw Pacific bluefin tuna (PBT) in Japan, but K. neothunni has not. Therefore, it is clinically and epidemiologically important to detect and distinguish these two species. In the present study, we developed a novel duplex polymerase chain reaction (dPCR) targeting the 28S rRNA gene sequences of K. hexapunctata and K. neothunni. The dPCR amplified the desired genetic regions of each species, and the detection limit was 10 copies/reaction. A total of 36 retail tuna samples from different fishing ports were purchased and tested by dPCR. Thirty-one tested positive for K. hexapunctata and four tested positive for K. neothunni. Several retail PBT samples were examined in some of the fishing ports, and among these samples, the detection rates of K. hexapunctata was higher than 85%, and the rates were similar between wild and farmed PBT. The detection rates of K. hexapunctata in wild and farmed retail PBT were 75% and 71%, respectively, in May. However, the rates in June and July were 100% for both. K. hexapunctata and K. neothunni myxospores were not observed in the dPCR-positive samples, except in juvenile PBT, suggesting that the number of parasites was insufficient to cause foodborne disease. Thus, dPCR is a useful method for detecting and distinguishing K. hexapunctata and K. neothunni, and can be used in epidemiological studies of these parasites.
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http://dx.doi.org/10.1016/j.parint.2019.102048DOI Listing
April 2020

Developmental exposure to diacetoxyscirpenol reversibly disrupts hippocampal neurogenesis by inducing oxidative cellular injury and suppressed differentiation of granule cell lineages in mice.

Food Chem Toxicol 2020 Feb 10;136:111046. Epub 2019 Dec 10.

Laboratory of Veterinary Pathology, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo, 183-8509, Japan; Cooperative Division of Veterinary Sciences, Graduate School of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo, 183-8509, Japan; Institute of Global Innovation Research, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo, 183-8509, Japan. Electronic address:

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).
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http://dx.doi.org/10.1016/j.fct.2019.111046DOI Listing
February 2020

Selected Trichothecenes in Barley Malt and Beer from Poland and an Assessment of Dietary Risks Associated with their Consumption.

Toxins (Basel) 2019 12 9;11(12). Epub 2019 Dec 9.

Department of Food Analysis, Prof. Waclaw Dabrowski Institute of Agricultural and Food Biotechnology, Rakowiecka 36, 02-532 Warsaw, Poland.

Eighty-seven samples of malt from several Polish malting plants and 157 beer samples from the beer available on the Polish market (in 2018) were tested for mycotoxins (deoxynivalenol (DON), nivalenol (NIV)), and their modified forms ((deoxynivalenol-3-glucoside (DON-3G), nivalenol-3-glucoside (NIV-3G), 3-acetyldeoxynivalenol (3-AcDON)). DON and its metabolite, DON-3G, were found the most, among the samples analyzed; DON and DON-3G were present in 90% and 91% of malt samples, and in 97% and 99% of beer samples, respectively. NIV was found in 24% of malt samples and in 64% of beer samples, and NIV-3G was found in 48% of malt samples and 39% of beer samples. In the malt samples, the mean concentration of DON was 52.9 µg/kg (range: 5.3-347.6 µg/kg) and that of DON-3G was 74.1 µg/kg (range: 4.4-410.3 µg/kg). In the beer samples, the mean concentration of DON was 12.3 µg/L (range: 1.2-156.5 µg/L) and that of DON-3G was 7.1 µg/L (range: 0.6-58.4 µg/L). The concentrations of other tested mycotoxins in the samples of malt and beer were several times lower. The risk of exposure to the tested mycotoxins, following the consumption of beer in Poland, was assessed. The corresponding probable daily intakes (PDIs) remained a small fraction of the tolerable daily intake (TDI). However, in the improbable worst-case scenario, in which every beer bottle consumed would be contaminated with mycotoxins present at the highest level observed among the analyzed beer samples, the PDI would exceed the TDI for DON and its metabolite after the consumption of a single bottle (0.5 L) of beer.
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http://dx.doi.org/10.3390/toxins11120715DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6949925PMC
December 2019

Determination of sterigmatocystin in foods in Japan: method validation and occurrence data.

Food Addit Contam Part A Chem Anal Control Expo Risk Assess 2019 Sep 26;36(9):1404-1410. Epub 2019 Jun 26.

Department of Food and Life Sciences, Azabu University , Kanagawa , Japan.

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 at >0.05 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.
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http://dx.doi.org/10.1080/19440049.2019.1628359DOI Listing
September 2019

[Study of Tea and Coffee on Inactivation of Kudoa septempunctata].

Shokuhin Eiseigaku Zasshi 2019 ;60(2):22-25

National Institute of Health Sciences.

The inhibition of Kudoa septempunctata by green tea extract, black tea extract, and coffee extract were studied. Incubation of about 10 Kudoa spores with green tea extract, black tea extract, or coffee extract at 25℃ for 4 hr reduced the survival ratio of Kudoa to 0%. While coffee extract and green tea extract contain approximately 2 and 1 mM of caffeine, respectively, the incubation of Kudoa spores with 2 and 1 mM of caffeine reduced its survival ratio to 68.2 and 93.3%, respectively. Although green tea extract and black tea extract contain over 1 mM of catechin, incubation with 0.01 mM of catechin was enough to reduce the survival ratio of Kudoa to 20%. These results suggested that green tea extract, black tea extract, and coffee extract have strong inhibitory effects on Kudoa and the effects of green tea extract and black tea extract are mainly manifested through catechin.
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http://dx.doi.org/10.3358/shokueishi.60.22DOI Listing
May 2019

Contamination of Wheat Cultivated in Various Regions of Poland during 2017 and 2018 Agricultural Seasons with Selected Trichothecenes and Their Modified Forms.

Toxins (Basel) 2019 02 1;11(2). Epub 2019 Feb 1.

Department of Food Analysis, Prof. Waclaw Dabrowski Institute of Agricultural and Food Biotechnology, Rakowiecka 36, 02-532 Warsaw, Poland.

Cross-interaction of antibodies within the immunoaffinity columns used in this study facilitated the simultaneous determination of nivalenol (NIV), deoxynivalenol (DON), their glucoside derivatives (NIV-3G, DON-3G), and 3-acetyl-deoxynivalenol (3-AcDON) in wheat grain harvested in various regions of Poland. In Poland, 2018 was a warm, dry agricultural season, and hence, was relatively less favourable for cereal cultivation than 2017. Data on the natural occurrence of NIV-3G in wheat grain are among the first published in the literature. DON was the most frequently found mycotoxin in the tested samples; the percentage occurrence of DON-positive samples was 92% in 2017 and 61% in 2018. Moreover, DON concentrations were generally higher in 2017 samples (5.2⁻1670.7 µg/kg) than those in 2018 samples (range 5.0⁻461.7 µg/kg). A similar pattern was found for DON-3G. However, no statistically significant differences between the samples from the two agricultural seasons were observed for the other three mycotoxins that were analysed, and their concentrations were generally considerably lower. DON was strongly correlated with DON-3G (correlation coefficient = 0.9558), while NIV was strongly correlated with NIV-3G ( = 0.9442). The percentage occurrence of NIV-3G- and DON-3G-positive samples was 14% in 2017 and 49% in 2018. The NIV-3G/NIV ratio was 5.9⁻35.7%, while the DON-3G/DON ratio range was 3.2⁻53.6%. In 2018, wheat samples from Southern Poland exhibited statistically significantly higher levels of DON than those from Northern Poland. The dry and hot summer of 2018 not only reduced wheat yields, but also limited development of spp. Therefore, grain harvested that year was generally contaminated with relatively low levels of mycotoxins. Lower levels of DON were also accompanied by lesser amounts of DON-derivatives.
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http://dx.doi.org/10.3390/toxins11020088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6409988PMC
February 2019

Fumonisin-production by section isolates from Japanese Foods and Environments.

Food Saf (Tokyo) 2018 Jun 29;6(2):74-82. Epub 2018 Jun 29.

National Institute of Health Sciences, Tonomachi 3-25-26, Kawasaki-ku, Kawasaki-shi, Kanagawa 220-9501, Japan.

Fumonisins are well known as mycotoxins produced by various species. Recently has been reported to be a fumonisin B (FB) producer. is a member of section . 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 for fumonisin production including growth medium, temperature and incubation period and 2) fumonisin production among isolates of section 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 FB 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; and , 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 section isolates from food and environmental samples indicated that fumonisin producing strains could be grouped into the clade. Nineteen of 35 (54%) isolates classified as were FB producers. The current study suggests that FB-producing are distributed throughout several regions of Japan.
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http://dx.doi.org/10.14252/foodsafetyfscj.2018005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6989198PMC
June 2018

[Quantitative Analysis of Unicapsula seriolae in Greater Amberjack Associated with Unidentified Food-Borne Disease].

Shokuhin Eiseigaku Zasshi 2018 ;59(1):24-29

Azabu University.

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 1.9×10 to 1.7×10 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 10 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 3.8×10 spores/g.
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http://dx.doi.org/10.3358/shokueishi.59.24DOI Listing
July 2018

Development of an Analytical Method for Simultaneous Determination of the Modified Forms of 4,15-Diacetoxyscirpenol and their Occurrence in Japanese Retail Food.

Toxins (Basel) 2018 04 26;10(5). Epub 2018 Apr 26.

Department of Food and Life Sciences, Azabu University, 1-17-71 Fuchinobe, Chuo-ku, Sagamihara, Kanagawa 252-5201, Japan.

4,15-Diacetoxyscirpenol (4,15-DAS) is a type A trichothecene mycotoxin produced by 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 . 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.
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http://dx.doi.org/10.3390/toxins10050178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5983234PMC
April 2018

A Kudoa septempunctata antigen induces production of IgE in BALB/c mice.

Parasitol Res 2018 Jan 3;117(1):303-306. Epub 2017 Nov 3.

Division of Microbiology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya, Tokyo, 158-8501, Japan.

Kudoa septempunctata, a myxosporean parasite, is the causative agent of a foodborne illness associated with consumption of raw Paralichthys olivaceus (olive flounder). Because the lag phase of this illness is short (from 1 to 12 h), it is possible that an allergic response is relevant to this illness. To test whether a K. septempunctata antigen is the possible allergen, we injected a myxospore extract into BALB/c mice and measured IgE levels in serum. When the mice were injected with the myxospore extract, the total serum IgE concentration increased significantly after the second immunization as compared to the negative control. After the third immunization, total IgE concentration in the immunized mice reached 26.5 ng/ml and was almost equivalent to that of egg albumin-injected mice. Western blot analysis revealed that IgE antibodies-in serum samples that were collected from myxospore extract-injected mice-bound to at least two Kudoa proteins with molecular weight between 28 and 36 kDa. These results suggested that a K. septempunctata antigen is the allergen. Further studies are needed to clarify the contribution of allergy to the foodborne illness caused by K. septempunctata.
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http://dx.doi.org/10.1007/s00436-017-5665-xDOI Listing
January 2018

Intracellular superoxide level controlled by manganese superoxide dismutases affects trichothecene production in Fusarium graminearum.

FEMS Microbiol Lett 2017 Nov;364(21)

Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.

The intracellular superoxide level is a clue to clarification of the regulatory mechanism for mycotoxin production in Fusarium graminearum. In this study, we focused on two manganese superoxide dismutases (SODs) of the fungus, FgSOD2 and FgSOD3, to investigate the relationship of the superoxide level to trichothecene production. Recombinant FgSOD2 and FgSOD3 showed SOD activity, and they were localized mainly in the mitochondria and cytoplasm, respectively. Trichothecene production and mRNA levels of Tri5 and Tri6, which encode a trichothecene biosynthetic enzyme and a key regulator of trichothecene production, respectively, were greatly reduced in gene-deletion mutants of FgSod2 and FgSod3 (ΔFgSod2 and ΔFgSod3). Significant increases in the cytosolic and mitochondrial superoxide levels were observed in ΔFgSod2 and ΔFgSod3, respectively. These results suggested that the cellular superoxide level affects trichothecene production in F. graminearum.
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http://dx.doi.org/10.1093/femsle/fnx213DOI Listing
November 2017

Inhibitory Activities of Blasticidin S Derivatives on Aflatoxin Production by Aspergillus Flavus.

Toxins (Basel) 2017 05 26;9(6). Epub 2017 May 26.

Division of Microbiology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.

Blasticidin S (BcS) is a protein synthesis inhibitor which shows strong growth inhibitory activity against a number of microorganisms. However, BcS inhibited aflatoxin production by without affecting its growth. In order to obtain information about the structure-activity relationship of BcS as an aflatoxin production inhibitor, BcS derivatives were prepared and their aflatoxin production inhibitory activities were evaluated. Among five derivatives, blasticidin S carboxymethyl ester, deaminohydroxyblasticidin S, and pyrimidinoblasticidin S showed inhibitory activity, while the others did not. The IC value for aflatoxin production of the carboxymethyl ester derivative was one-fifth of that of BcS although their antimicrobial activities were almost the same. These results indicate that the inhibitory activity of BcS against aflatoxin production was enhanced by esterification of its carboxyl group and that the carboxymethyl ester derivative might be more suitable for practical use than BcS because of the specificity of the carboxymethyl ester derivative, which inhibited aflatoxin production more than BcS.
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http://dx.doi.org/10.3390/toxins9060176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5488026PMC
May 2017

Masked trichothecene and zearalenone mycotoxins withstand digestion and absorption in the upper GI tract but are efficiently hydrolyzed by human gut microbiota in vitro.

Mol Nutr Food Res 2017 04 6;61(4). Epub 2017 Feb 6.

Fera Science Ltd, Sand Hutton, York, UK.

Scope: Cereal grains are commonly contaminated with Fusarium mycotoxins and their plant-derived masked metabolites. The fate of masked mycotoxins in the human gut is poorly understood. Here we assess the metabolism and transport of glucoside metabolites of common trichothecenes (deoxynivalenol, nivalenol, T-2 toxin) and zearalenone compounds (zearalenone, α- and β-zearalenol) in the human gut in vitro.

Methods And Results: Masked mycotoxins were incubated with artificial digestive juices and absorption was assessed in differentiated Caco-2/TC7 cells. Colonic metabolism was studied using fecal batch cultures from five donors and mycotoxins were detected using LC-MS/MS. All masked mycotoxins were stable under upper GI tract conditions and no absorption was observed. Free trichothecenes were absorbed intact whereas free zearalenone compounds were absorbed and metabolized to undetected compounds by Caco-2/TC7 cells. Human gut microbiota efficiently hydrolyzed all masked mycotoxins. Trichothecenes were fully recovered as parent mycotoxins whereas 40-70% of zearalenone compounds were further metabolized to unknown metabolites.

Conclusion: Our results demonstrate that masked trichothecenes will reach the colon intact to be released as parent mycotoxins by gut microbiota, hence contributing to mycotoxin exposure. Masked zearalenone compounds are metabolized by gut microbiota and epithelial cells and the identity and toxicity of metabolites remain to be determined.
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http://dx.doi.org/10.1002/mnfr.201600680DOI Listing
April 2017

Cryopreservation of Kudoa septempunctata sporoplasm using commercial freezing media.

Parasitol Res 2017 Jan 23;116(1):425-427. Epub 2016 Sep 23.

The Graduate School of Life and Environmental Sciences, Azabu University, 1-17-71 Fuchinobe Chuo-ku, Sagamihara, Kanagawa, 252-5201, Japan.

Cryopreservation methods for Kudoa septempunctata have not been established. This prevents an effective study of K. septempunctata, which cannot be artificially cultivated in the laboratory. In this study, we attempted to establish a cryopreservation method for K. septempunctata sporoplasm using Cellbanker® 1, a commercial preservation medium for mammalian cells. Spores were purified from the meat of Paralichthys olivaceus (olive flounder). These purified spores were suspended in Cellbanker® 1 and were stored at -80 °C for up to 16 months. Although the spores stored at -80 °C for 16 months were damaged, the sporoplasms maintained its amoeba-like indeterminate morphology, and their motility was well preserved. The viability of sporoplasms was variable among vials but was not below 70 %. In addition, the sporoplasms stored at -80 °C for 16 months could decrease the transepithelial electrical resistance of Caco-2 cells. These results indicate that this cryopreservation method using Cellbanker® 1 could preserve the viability and pathogenesis of K. septempunctata sporoplasm.
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http://dx.doi.org/10.1007/s00436-016-5262-4DOI Listing
January 2017

Occurrence of beauvericin and enniatins in wheat flour and corn grits on the Japanese market, and their co-contamination with type B trichothecene mycotoxins.

Food Addit Contam Part A Chem Anal Control Expo Risk Assess 2016 Oct 14;33(10):1620-1626. Epub 2016 Sep 14.

a National Institute of Health Sciences , Tokyo , Japan.

The contamination levels of beauvericin and four enniatins, A, A, B and B, 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). 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. 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.
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http://dx.doi.org/10.1080/19440049.2016.1228126DOI Listing
October 2016

Evaluation of Four Commercial Kits Based on Immunochromatography for Screening Aflatoxin M1 in Milk.

Shokuhin Eiseigaku Zasshi 2016 ;57(3):76-9

National Institute of Health Sciences.

In Japan, a regulatory limit of 0.5 μg/kg was set in 2015 for aflatoxin M1 (AFM1) in milk. A method using an immunochromatographic kit has been adopted as the official screening method, and criteria for the kit have been set. In order to confirm whether commercial immunochromatographic kits for detecting AFM1 satisfy these criteria, the performance of four kits was evaluated by performing spike-and-recovery experiments using AFM1-free milk samples and milk samples spiked at seven levels (100-700 ng/kg). With the two qualitative kits, determinations of blank samples were all negative and those of the samples spiked at 500 ng/kg were all positive. With the two quantitative kits, the measured values of the blank samples were all less than 100 ng/kg and the recoveries of the samples spiked at 500 ng/kg were all more than 70%. The detection limits of the two quantitative kits were both less than 100 ng/kg. These results indicate that all four immunochromatographic kits meet the criteria and can be used to screen AFM1 in milk.
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http://dx.doi.org/10.3358/shokueishi.57.76DOI Listing
February 2017

Survivability of Kudoa septempunctata in human intestinal conditions.

Parasitol Res 2016 Jun 2;115(6):2519-22. Epub 2016 Apr 2.

The Graduate School of Life and Environmental Sciences, Azabu University, 1-17-71 Fuchinobe Chuo-ku, Sagamihara, Kanagawa, 252-5201, Japan.

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.
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http://dx.doi.org/10.1007/s00436-016-5036-zDOI Listing
June 2016

Identification and Characterization of a New Enterotoxin Produced by Clostridium perfringens Isolated from Food Poisoning Outbreaks.

PLoS One 2015 19;10(11):e0138183. Epub 2015 Nov 19.

Division of Microbiology, National Institute of Health Sciences, Tokyo, Japan.

There is a strain of Clostridium perfringens, W5052, which does not produce a known enterotoxin. We herein report that the strain W5052 expressed a homologue of the iota-like toxin components sa and sb of C. spiroforme, named Clostridium perfringens iota-like enterotoxin, CPILE-a and CPILE-b, respectively, based on the results of a genome sequencing analysis and a systematic protein screening. In the nicotinamide glyco-hydrolase (NADase) assay the hydrolysis activity was dose-dependently increased by the concentration of rCPILE-a, as judged by the mass spectrometry analysis. In addition, the actin monomer of the lysates of Vero and L929 cells were radiolabeled in the presence of [32P]NAD and rCPILE-a. These findings indicated that CPILE-a possesses ADP-ribosylation activity. The culture supernatant of W5052 facilitated the rounding and killing of Vero and L929 cells, but the rCPILE-a or a non-proteolyzed rCPILE-b did not. However, a trypsin-treated rCPILE-b did. Moreover, a mixture of rCPILE-a and the trypsin-treated rCPILE-b enhanced the cell rounding and killing activities, compared with that induced by the trypsin-treated rCPILE-b alone. The injection of the mixture of rCPILE-a and the trypsin-treated rCPILE-b into an ileum loop of rabbits evoked the swelling of the loop and accumulation of the fluid dose-dependently, suggesting that CPILE possesses enterotoxic activity. The evidence presented in this communication will facilitate the epidemiological, etiological, and toxicological studies of C. perfringens food poisoning, and also stimulate studies on the transfer of the toxins' gene(s) among the Genus Clostridium.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0138183PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4652906PMC
June 2016

Search for aflatoxin and trichothecene production inhibitors and analysis of their modes of action.

Biosci Biotechnol Biochem 2016 17;80(1):43-54. Epub 2015 Sep 17.

a Department of Applied Biological Chemistry , The University of Tokyo , Tokyo , Japan.

Mycotoxin contamination of crops is a serious problem throughout the world because of its impact on human and animal health as well as economy. Inhibitors of mycotoxin production are useful not only for developing effective methods to prevent mycotoxin contamination, but also for investigating the molecular mechanisms of secondary metabolite production by fungi. We have been searching for mycotoxin production inhibitors among natural products and investigating their modes of action. In this article, we review aflatoxin and trichothecene production inhibitors, including our works on blasticidin S, methyl syringate, cyclo(L-Ala-L-Pro), respiration inhibitors, and precocene II.
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http://dx.doi.org/10.1080/09168451.2015.1086261DOI Listing
September 2016

Development of a rapid method for the quantitative determination of deoxynivalenol using Quenchbody.

Anal Chim Acta 2015 Aug 8;888:126-30. Epub 2015 Aug 8.

Department of Food and Life Science, Azabu University, 1-17-71 Fuchinobe, Chuo-ku, Sagamihara, Kanagawa 252-5201, Japan. Electronic address:

Quenchbody (Q-body) is a novel fluorescent biosensor based on the antigen-dependent removal of a quenching effect on a fluorophore attached to antibody domains. In order to develop a method using Q-body for the quantitative determination of deoxynivalenol (DON), a trichothecene mycotoxin produced by some Fusarium species, anti-DON Q-body was synthesized from the sequence information of a monoclonal antibody specific to DON. When the purified anti-DON Q-body was mixed with DON, a dose-dependent increase in the fluorescence intensity was observed and the detection range was between 0.0003 and 3 mg L(-1). The coefficients of variation were 7.9% at 0.003 mg L(-1), 5.0% at 0.03 mg L(-1) and 13.7% at 0.3 mg L(-1), respectively. The limit of detection was 0.006 mg L(-1) for DON in wheat. The Q-body showed an antigen-dependent fluorescence enhancement even in the presence of wheat extracts. To validate the analytical method using Q-body, a spike-and-recovery experiment was performed using four spiked wheat samples. The recoveries were in the range of 94.9-100.2%. The concentrations of DON in twenty-one naturally contaminated wheat samples were quantitated by the Q-body method, LC-MS/MS and an immunochromatographic assay kit. The LC-MS/MS analysis showed that the levels of DON contamination in the samples were between 0.001 and 2.68 mg kg(-1). The concentrations of DON quantitated by LC-MS/MS were more strongly correlated with those using the Q-body method (R(2) = 0.9760) than the immunochromatographic assay kit (R(2) = 0.8824). These data indicate that the Q-body system for the determination of DON in wheat samples was successfully developed and Q-body is expected to have a range of applications in the field of food safety.
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http://dx.doi.org/10.1016/j.aca.2015.07.020DOI Listing
August 2015

Multi-mycotoxin screening reveals separate occurrence of aflatoxins and ochratoxin a in Asian rice.

J Agric Food Chem 2015 Apr 13;63(12):3104-13. Epub 2015 Mar 13.

†Food Safety Laboratory, Applied Sciences Group, Health Sciences Authority, 11 Outram Road, Singapore 169078.

The determination of important regulated mycotoxins in rice has been reported previously but not in the individual matrix of white, brown, red, and basmati rice with respect to the matrix effect, recovery, and stability. A total of 190 Asian rices were examined for regulated mycotoxin contamination by the LC-ESI-MS/MS method. Significant variation (p < 0.05) in the matrix effect was observed for fumonisins. Methanol improved the limits of detection (LOD) for HT-2 from 50 μg/kg to 2.3 μg/kg by promoting ionization efficiency of the ammonium-adduct. LOD and limits of quantitation ranged from 0.1 to 18 μg/kg and 0.2-31 μg/kg, respectively. All analytes degraded by more than 50% on storage, except fumonisins. Acetic acid (1%) provided significant improvement (p < 0.05) in recovery for all analytes in selected white rice from Thailand and China. Mean recovery ranged from 70 to 120%. RSD values were lower than 15% for all analytes. Five AFB1 and single OTA positive samples were detected. No correlation between mycotoxin contamination and rice species (r = 0) exists.
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http://dx.doi.org/10.1021/acs.jafc.5b00471DOI Listing
April 2015
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