Takumi Yoshizawa

Metabolism of three trichothecene ycotoxins, T-2 toxin, diacetoxyscirpenol and deoxynivalenol, by bovne rumen microorganisms

The three trichothecene mycotoxins T-2 toxin, diacetoxyscirpenol (DAS) and deoxynivalenol (DON) were incubated in vitro for 12, 24 and 48 h with rumen microorganisms obtained from a fistulated dairy cow. Gas chromatographic and gas chromatographic-mass spectrometric analyses of extracts indicated all three toxins were biotransformed to a variety of deepoxy and deacylated products. DON was partially converted to a product identified as deepoxy DON. DAS was rapidly converted to four products including 15-monoacetoxyscirpenol (MAS), scirpentriol and two new compounds identified as 15-acetoxy-3 alpha,4 beta-dihydroxytrichothec-9,12-diene (deepoxy MAS) and 3 alpha,4 beta,15-trihydroxytrichothec-9,12-diene (deepoxy scirpentriol). T-2 toxin was also completely biotransformed to the products HT-2, T-2 triol and two new metabolites identified as 15-acetoxy-3 alpha,4 beta-dihydroxy-8 alpha-(3-methylbutyryloxy) trichothec-9,12-diene (deepoxy HT-2) and 3 alpha,4 beta,15-trihydroxy-8 alpha-(3-methylbutyryloxy)trichothec-9,12-diene (deepoxy T-2 triol).

Natural co-occurrence of aflatoxins and Fusarium mycotoxins (fumonisins, deoxynivalenol, nivalenol and zearalenone) in corn from Indonesia.

Sixteen corn samples collected from Indonesia were analysed for aflatoxins (AF), fumonisins (FM), trichothecenes and zearalenone (ZEA) using high-performance liquid chromatography and gas chromatography-mass spectrometry. AF were detected in 11 (69%) samples at a mean level of 119 ng/g (maximum 487 ng/g) and FM in all of the samples at a mean level of 895 ng/g (maximum 2970 ng/g). Deoxynivalenol (DON), nivalenol (NIV) and ZEA were each detected in two (12%) samples; 21 and 32 ng/g, 49 and 169 ng/g, and 11 and 12 ng/g, respectively. All of the AF-contaminated samples were co-contaminated with FM. Mycological study showed all of the AF-contaminated samples were infected with A. flavus/A. parasiticus, and the FM-contaminated samples were either infected with F. moniliforme (50%), F. proliferatum (12%), F. nygamai (6%) or F. decemcellulare (38%). Supportive mycological studies showed that Fusarium species isolated from Indonesian corn were capable of producing a mean level of 10,000 micrograms/g FM. Based on these results, the correlation between FM-producers and AF-producers on kernel infection and contamination of these mycotoxins in corn was discussed. This is the first report on the natural co-occurrence of AF and various Fusarium mycotoxins, including DON and NIV in corn from Indonesia, and also the first report on the natural occurrence of DON in corn from hot areas of Southeast Asia.

Mycotoxins (trichothecenes, zearalenone and fumonisins) in cereals associated with human red-mold intoxications stored since 1989 and 1991 in China.

Two corn powder samples implicated in the human food poisoning that occurred in Guangxi province in 1989, and eight wheat and two barley samples linked to an episode that involved about 130,000 people in gastrointestinal disorders in Anhui province in 1991 were analyzed for trichothecenes including deoxynivalenol (DON), nivalenol (NIV) and their esters, zearalenone (ZEA) and fumonisins (FMs) by gas chromatography/mass spectroscopy and high performance liquid chromatography, and T-2 toxin by enzyme-linked immunosorbent assays. DON was detected in all samples as a major trichothecene (16-51,450 microg kg(-1)), and NIV was in one corn, one barley and all wheat at relatively low levels (10-6935 microg kg(-1)). ZEA was found in all corn and barley, and six wheat samples (46-3079 microg kg(-1)). In addition, 3-acetyl-DON (2544 microg kg(-1)) and 15-acetyl-DON (2537 microg kg(-1)) were detected separately in one corn and one wheat sample. The highest levels of these mycotoxins were found in one wheat sample associated with the human intoxication in Anhui province. FMs in corn were below 1000 microg kg(-1). Risks of DON and ZEA on the people who consumed the causative cereals were assessed.

A Practical Method for Measuring Deoxynivalenol, Nivalenol, and T-2 + HT-2 Toxin in Foods by an Enzyme-linked Immunosorbent Assay Using Monoclonal Antibodies

We have developed and tested an enzyme-linked immunosorbent assay system for individual measurement of deoxynivalenol, nivalenol, and T-2 + HT-2 toxin using monoclonal antibodies for 3,4,15-triacetyl-nivalenol, for both 3,4,15-triacetyl-nivalenol and 3,15-diacetyl-deoxynivalenol, and for acetyl-T-2 toxin. The assay system comprised three kits (desinated the DON + NIV kit, the NIV kit, and the T-2 + HT-2 kit). The practical performance of the enzyme-linked immunosorbent assay system was assessed by assaying trichothecene mycotoxins in wheat kernels. The enzyme-linked immunosorbent assay system meets all the requirements for use in a routine assay in terms of sensitivity (detection limit: deoxynivalenol 80 ng/g, nivalenol 80 ng/g, T-2 toxin 30 ng/g), reproducibility (total coefficient of variation: 1.9–6.2%), accuracy (recovery: 93.8–112.0%), simplicity and rapidity (time required: <2 h), mass handling (>42 samples/assay), and a good correlation with gas chromatography–mass spectrometry (r=0.9146–0.9991). Components derived from the wheat extract did not interfere with the assay kits. The enzyme-linked immunosorbent assay system is a useful alternative method to gas chromatography–mass spectrometry, liquid chromatography–mass spectrometry, or liquid chromatography–ultraviolet absorption for screening cereals and foods for trichothecene mycotoxin contamination.

Occurrence of fumonisins and aflatoxins in corn from Thailand

Eighteen corn samples collected from Thailand were analysed for fumonisins and aflatoxins by high performance liquid chromatography. Fumonisins B1 and B2 were detected in 16 (89%) and 12 (67%) samples at levels ranging from 63 to 18800 ng/g (mean, 1790 ng/g) and from 50 to 1400 ng/g (mean, 251 ng/g), respectively. Fusarium moniliforme and F. proliferatum isolated from Thai corn showed the production of fumonisins in corn grit cultures. Aflatoxins B1 and B2 were found in 15 and 10 samples at mean levels of 72 and 15 ng/g, respectively. It was noted that as many as 72.2% (13/18) of samples were coincidentally contaminated with fumonisins and aflatoxins. Based on these results, the interaction of fumonisin-producers and aflatoxin-producers on kernel infection and contamination of these mycotoxins in corn is discussed. This is the first report on the natural co-occurrence of fumonisins and aflatoxins in Thailand.

Fusarium toxins in wheat from an area in Henan Province, PR China, with a previous human red mould intoxication episode

Wheat samples of the 1998 and 1999 crops from Puyang, an area in Henan Province, PR China with a previous human red mould intoxication episode, were analysed for trichothecenes and zearalenone (ZEA). For the 1998 Puyang crop, deoxynivalenol (DON) was the predominant toxin detected abundantly and frequently at a level of up to 14000 μgkg-1 (mean 2850 μgkg-1) in 30 of 31 (97%) wheat samples. Among these were 21 (70%) with a DON level that exceeded the Chinese regulation of 1000 μgkg-1. Nivalenol (NIV) and 15-acetyl-DON (15-ADON) were also found at 578 μgkg-1 (one sample) and 59–1800 μgkg-1 (mean 365 μgkg-1, 20 samples), respectively. ZEA co-occurred in 21 samples at 9–1400 μgkg-1 (mean 209 μgkg-1). Twenty-five (89%) wheat samples from Zhumadian, a region without a history of human red mould intoxication in the same province, contained low levels of DON (53–1240, mean 223 μgkg-1) with seven (25%) co-contaminated with ZEA (10–217, mean 108 μgkg-1). All were free from 15-ADON and NIV. Significant differences in DON, 15-ADON and ZEA concentrations between both areas were found. DON (<1000 μgkg-1) and ZEA (5–113 μgkg-1) were also detected in the 1999 Puyang wheat. Proper environmental conditions for Fusarium species surviving winter combined with unusual high precipitation during wheat flowering were responsible for a high concentration of Fusarium mycotoxins in the 1998 Puyang wheat.

Updated profile of aflatoxin and Aspergillus section Flavi contamination in rice and its byproducts from the Philippines.

Our goal was to develop an updated profile of aflatoxin (AF) and AF-producing fungi contamination in rice and its byproducts from the Philippines. The total AF levels in 78 samples of polished and brown rice, determined by an immunoaffinity column clean-up method coupled with HPLC (detection limit: 25 ng/kg), ranged from <0.025–2.7 µg/kg (mean of positive samples: 0.37 µg/kg) and 0.03–8.7 µg/kg (mean of positive samples: 2.7 µg/kg), respectively. The incidence (% of positive samples) of AF in polished and brown rice were 94% and 100%, respectively. The AF levels in polished rice imported from Thailand and Vietnam were approximately 20% of the levels found in locally produced polished rice. AF levels decreased as the rice progressed through the various stages in milling. Fungi recovered include toxigenic Aspergillus flavus and A. parasiticus with an incidence ranging from 14% in rice bran to 78% in rough rice and producing <0.025−6200 µg/kg total AF in in vitro cultures on rice. All samples of rice bran and rice hull contained AF at levels ranging from 0.27–11 µg/kg. The estimated potential daily intake of AFB1 from rice is between 0.1 and 7.5 ng/kg of body weight/day, the mean of which is 1.0 ng representing 9.1–5.3 times the estimated tolerable daily intake for AFB1 reported to date for Asia. Thus, Filipinos have a potentially high risk of exposure to AF that can be easily controlled through proper post-harvest handling and storage of rice and its byproducts.

Occurrence of aflatoxin M1 in domestic milk in Japan during the winter season.

A total of 208 samples of commercial pasteurized milk gathered from retail outlets across Japan during the winter season were analysed for aflatoxin M1 (AFM1). Japan was divided into 11 regions from north to south, and nine to 45 milk samples from each region were randomly purchased between December 2001 and February 2002. Each milk sample was cleaned up by an immunoaffinity column, and AFM1 was quantified by liquid chromatography with fluorescence detection in four independent laboratories. The limit of detection of the method was 0.001 μg kg−1. The identity of the putative AFM1 in milk sample was confirmed by the formation of AFM1 hemi-acetal with trifluoroacetic acid. Based on the results obtained with spiked samples (0.05 μg AFM1 kg−1), the mean recovery was 91.4%, the relative standard deviation for repeatability was 4.6%, and the relative standard deviation for reproducibility was 8.0% among four independent laboratories. AFM1 was detected in 207 (99.5%) of 208 milk samples at 0.001–0.029 μg kg−1, with a mean of 0.009 μg kg−1 and a 90th percentile of 0.014 μg kg−1. No significant difference of the level of AFM1 contamination was observed among the regions.

Effects of heating procedures on deoxynivalenol, nivalenol and zearalenone levels in naturally contaminated barley and wheat

The influence of heating temperature and time on deoxynivalenol (DON), nivalenol (NIV) and zearalenone (ZEA) contents in naturally co-contaminated barley and wheat was investigated intending to establish the basis for a decontamination model of Fusarium mycotoxins in cereals. The standard toxins and whole barley powder samples were heated in a convection oven at 140, 160, 180, 200, or 220°C, and kernel subsamples (200 g each) were roasted in an experimental rotary gas-fired roaster at 150, 180 or 220°C. All treatments resulted in a time–temperature-dependent decomposition of the toxins; the logarithm of the toxin remaining % presented a linear relationship with heating time. The lines equations were used to estimate the half (H) and decimal (D) decomposition times (time required to destroy 50 or 90% of the toxin, respectively). DON and NIV H and D decomposition times were similar and 50% shorter for heated standards than for whole barley powder. ZEA standard values were considerably longer, while whole barley powder values were comparable with those of DON and NIV. At 220°C, D decomposition times of DON, NIV and ZEA heated standards were 11, 10 and 85 min, respectively, while the values obtained in whole barley powder were the same for the three toxins (25 min). The determination of H and D decomposition values constitutes a basis to understand the heating stability nature of each toxin.

Evaluation and application of a simple and rapid method for the analysis of aflatoxins in commercial foods from Malaysia and the Philippines.

For application to the analysis of aflatoxins (AF) in commercial peanut and corn products, the ISOLUTE multimode column (IMC, solid phase multifunctional column) method was validated by comparing with the modified Florisil column (MFC) method. Twenty-two peanut and eight corn products from Malaysia and the Philippines were analysed for AFB1, AFB2, AFG1 and AFG2 firstly by the MFC method and then by the IMC method. For peanut products, 14 out of 22 samples were positive by the two methods in the range of 1-378 micrograms/kg of AF, and correlation coefficients (r) for AFB1 and AFB2 were 0.987 and 0.997, respectively. For corn and corn products, all the samples were positive in the range of 1-130 micrograms/kg, and r values were 0.992 and 0.805 for AFB1 and AFB2 respectively. Thus, the results were significantly (p < 0.01) in close agreement, particularly for lower range of 1-50 micrograms/kg of AF concentrations in all the samples. For the occurrence of AF, 11 (65%) of peanut products from Malaysia were contaminated with AF at a mean level of 50 micrograms/kg (maximum 180 micrograms/kg) and two (40% products from the Philippines were contaminated with as high as 375 micrograms/kg and 177 micrograms/kg of AF, respectively. All the corn products from the Philippines were contaminated with AF at a mean level of 44 micrograms/kg (maximum 130 micrograms/kg). Contamination of commercial foods with high levels of AF is a very important issue to both the countries since these foods are very popular among children.