In conjunction with the international current situation about the regulation for mycotoxins in food, the natural occurrence of trichothecenes (DON and NIV) and aflatoxins in Asia is overviewed. Updated information is very limited, however, being difficult to assess the exposure of Asian populations to the mycotoxins. Comprehensive surveillances are recommended to be conducted nationwide in individual countries using adequate sampling plans and validated methods. Recent advances in molecular genetic approach for identifying Fusarium graminearum and Aspergillus flavus are also reviewed briefly.

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Recently, PCR-based methods have been developed to identify important fungi isolated from various sources. In this paper, we describe the outline of these methods for detecting mycotoxigenic fungi, except aflatoxin-producing fungi, and briefly describe our real-time PCR work for toxigenic Fusarium species responsible for natural occurrence of trichothecene mycotoxins in wheat and barley. In addition, the problems to be solved in the application of PCR-based methods are discussed.

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Heteroduplex panel analysis (HPA) was previously developed for genetic identification of Aspergillus section Flavi isolates, utilizing PCR-amplified fragments of the internal transcribed spacer (ITS) regions of the rRNA gene. Application of HPA to a field study demonstrated that a new type of FP-1 belonging to section Flavi has been predominantly distributed throughout sugarcane field soil in the southernmost islands of Japan, and such trend may be also the case in Vietnam. All of the 71 type FP-1 isolates tested were able to produce aflatoxins B and G. The morphological observations of the type FP-1 isolates showed that a major part of them had broad interfaces with Aspergillus parasiticus and the remainder did with Aspergillus flavus. The phylogenetic analysis based on the ITS sequences indicated that type FP-1 formed an independent Glade positioned between A. parasiticus and A. flavus, and was more closely related to the former species. We also found that A. nomius has been distributed in sugarcane field soil and/or sugarcane stems in Japan and Vietnam. Until now, 19 HPA types were identified among a total of 351 section Flavi isolates: F-1 to F-6 of A. flavus/A. oryzae, P-1 and P-2 of A. parasiticus/A. sojae, T-1 of A, tamarii, T-2 of A, pseudotamarii, T-3 of A. caelatus, N-1 to N-5 of A. nomius, TN-1 and TN-2 of A. bombysis, and FP-1 of a new genotype that is potentially aflatoxigenic.

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This report gives a retrospective of studies done on aflatoxins and aflatoxigenic fungi in rice and its by-products. Previous studies on rice employed analytical methods with detection limits ranging from 0 .1 to 5μg/kg, which could mean that lower levels were overlooked, thus, underestimating the actual incidence of aflatoxins in the commodity. Aflatoxin production in rice is relatively minimal. However, high aflatoxin levels have been found in samples from some countries in Asia and Africa, which can be attributable to inadequate postharvest system. Incidence of aflatoxins in more than 2800 samples of rice and its products surveyed all over the world appears to have increased from 7% in the 70s to about 31% from 2000 onwards. Levels of AFB₁ and total AF in samples analyzed were mostly beyond European Union regulatory limits; some even have levels above 20μg/kg, the amount of aflatoxins allowed in foods by Codex Alimentarius. Incidence of aflatoxigenic species, particularly A. flavus and A. parasiticus, in rice and its by-products appears to be concentrated in the tropical and subtropical regions, where prevailing environmental conditions are conducive to their growth. Due to increasing stringency of regulatory limits, there is a need to develop more sensitive methods that can detect the lower levels of aflatoxins found in rice and its by-products and ascertain the extent of contamination in these commodities. In order to regulate these commodities, continued monitoring is necessary to provide data that can serve as basis for exposure assessment studies. Other urgent areas for research are those of improved and integrated approaches to control aflatoxins and aflatoxigenic fungi at all stages of production and marketing.

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Various fungi such as Aspergillus sp. and Fusarium sp. were isolated from traditional foodstuffs and raw materials, and have been screened their mycotoxigenicity including aflatoxin, ochratoxin, deoxynivalenol (DON) fumonisin and zearalenone by HPLC, and new advanced enzyme linked immunosorbent assay (ELISA) methods. Penicillium sp. were also isolated and confirmed the Penicillium mycotoxins including patulin and citrinin from apple, pear, citrus and grape. An assay based on multiplex PCR was also applied for screening of potential mycotoxin producing fungi in fermented foods and grains. As the published results, various mycotoxigenic fungi were contaminated in Korean foodstuffs, but systematic screening was not performed, so more continuing studies were necessary for understanding the distribution of mycotoxigenic fungi.

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A liquid chromatography/mass spectrometry (LC/MS) method based on time of flight MS (TOF-MS) with a real time reference mass correction technique using dual spray-ESI source was developed for the determination of Fusarium mycotoxins [fumonisinB1(FMB1), fumonisinB2(FMB2), fumonisinB3(FMB3), zearalenone (ZEN), α-zearalanol (α-ZAL), β-zearalanol(β-ZAL), α-zearalenol(α-ZOL), β-zearalenol (β-ZOL) ]and Aspergillus mycotoxins [ochratoxin A(OTA)] in corn, rice, and feed. Samples were cleaned-up with solid phase extraction(SPE) mini column. Detection of the mycotoxins was carried out in exact mass chromatograms with a mass window of 0.03 Da. Calibration curves were linear from 1 to 100ng/mL. The limits of detection ranged from 0.1 to 6.1 μg/kg in foodstuffs analyzed in this study. The LCTOF-MS method was found to be suitable for the screening of multiple mycotoxins in foodstuffs.

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Patulin is a highly toxic and carcinogenic mycotoxin that produced by some species of Penicillium, Aspergillus and Byssochlamys. This toxin is heat stable and usually contaminates apple- and hawthorn-based products. In this paper, natural occurrence, producer fungi invasion, analytical methods and control measures for patulin in Chinese agricultural commodities are reviewed.

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The two kind of way were proposed to improving analysis productivity for the mycotoxins analysis. The select ion of suitable ion probe depend on the target compounds to get higher sensitivity. It was shown that the atomospheric pressure photo ionization (APPI) probe is best for the nivalenol and deoxynivalenol analysis. The use of the high resolution mass spectrometer was used to increase probability of identification. It was able to recognize the small mass number differentiates.

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Nivalenol (NIV) is a trichothecene mycotoxin produced by Fusarium species and it frequently co-contaminates wheat and barley with deoxynivalenol in Japan. Whereas deoxynivalenol has been evaluated with regards to its toxicity profile, nivalenol has not been done so because of poor information about its toxicity. In the present study, we examined the subchronic toxicity of NIV in male and female F344 rats by exposure through diets at doses of 0, 6.25, 25, and 100 mg/kg for 90 days. Most changes in the general, histopathological and biochemical aspects were found at 100 mg/kg in both sexes. Hematologically, white blood cell counts decreased at 100 mg/kg in males and from 6.25 mg/kg in females. The results of the immune function in males revealed that the ratio of helper/cytotoxic T lymphocytes and the B/T cell ratio decreased from 25 mg/kg but serum immunoglobulin levels did not change at any dose. Taken together, NIV targets the female reproductive system as well as hematopoietic and immune systems in rats. Based on the hematology data, the no-observed-effect level of NIV was determined to be less than 6.25 mg/kg.

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Five-week-old female ICR:CD-1 mice were inoculated orally with 10 mg/kg b.w. of T-2 toxin. Hematological and blood biochemical examinations and histopathological examination of the liver were done up to 48 hours after treatment (HAT). In addition, microarray analysis was done on the gene expression profile of the liver at 0.5, 3 and 24 HAT. In the T-2 toxin-treated group, the levels of AST and ALT increased while those of total cholesterol, total protein, blood glucose and fibrinogen decreased at and after 3 HAT. The coagulation test revealed the prolongation of both prothrombin time (PT) and activated partial thromboplastin time (APTT). Histopathologically, dead hepatocytes were sporadically observed at and after 3 HAT. Dead hepatocytes at the early stage were characterized by cellular swelling and those at the late stage by pyknosis with condensed eosinophilic cytoplasm, respectively. Microarray analysis on the liver revealed the up-regulated expression of oxidative stress-, cell cycle- and apoptosis-related genes and the down-regulated expression of lipid metabolism-, glycogen metabolism-, drug metabolism- and blood coagulation-related genes. Especially, c-fos and c-jun mRNAs expression was significantly elevated immediately after T-2 toxin-treatment and kept high level until 24 HAT. The present study clarified that T-2 toxin affects liver function including blood coagulation system and that morphological characteristic of dead hepatocytes shifts from cellular swelling (necrosis) to pyknosis with condensed eosinophilic cytoplasm (apoptosis). From the results of microarray analysis, T-2 toxin was considered to damage hepatocytes mainly through oxidative stress and apoptosis.

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Fumonisins are produced by Fusarium verticillioides and occur in maize and maize-based foods. Their effects on human health are unclear, however, epidemiological and experimental evidence suggest that they increase the risk of neural tube defects (NTDs) in populations routinely consuming foods prepared from contaminated maize. When given orally to CD1 mice and other laboratory species, fumonisin B1 (FB1) was fetotoxic, but not teratogenic. However, intraperitoneal (ip) injection of FB1 to inbred LM/Bc mice at the critical time for neural tube closure elicited NTDs in a dose-dependent manner. To determine if CD1 mice are susceptible to induction of NTDs by fumonisins, 10 to 100 mg/kg body weight (BWt) FB1 was given ip to females on gestation days 7 and 8. FB1 was fetotoxic at maternal doses≥45 mg/kg BW. The number of litters with one or more NTD-affected fetuses increased in a dose-dependent manner, reaching a maximum of 55 % (six of 11 litters) at 100 mg/kg BW. A no observed adverse affect level was not established as 10 % of litters at the lowest dose, 10 mg/kg BW FB₁, were NTD positive. These results demonstrated that NTD induction by FB1 in mice is not unique to the LM/Bc strain. They further showed that CD1 mice are less sensitive to NTD induction by FB 1 than LMBc mice. Comparative investigations using these and other strains will be useful for determining the mechanisms of NTD induction in fumonisin-treated mice and the relevance of these animal models to humans.

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Acute aflatoxicosis outbreaks sporadically occur among subsistence farmers in Makueni and Kitui districts, Eastern Province, Kenya. Over the past 25 years, four documented outbreaks of acute aflatoxicosis affecting 467 individuals and resulting in 178 deaths have occurred in the same region. The outbreaks occurred in the southeastern part of Eastern Province between the months of April and July. The outbreaks resulted from the consumption of aflatoxin contaminated homegrown maize. Analysis of homestead and market maize samples collected from the affected region showed widespread contamination with aflatoxin at concentrations above 20 μg/kg. Approximately 18% of the samples were contaminated with aflatoxin at concentrations above 1, 000 μg/kg. Consumption of the contaminated maize (once or multiple times) could have resulted in estimated mean aflatoxin ingestion of 50 to 2, 500 ng/kg bw/day, with a few individuals probably ingesting as high as 18, 000 ng aflatoxin/kg bw/day. The exact cause of the aflatoxin contamination of the maize crop in this specific region has not been fully established. Circumstantial evidence suggests that weather and agricultural practices may have created favorable conditions for the proliferation of a prevalent and highly aflatoxigenic Aspergillus strain in the maize crop resulting in the production of high levels of aflatoxin in the harvested maize.

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Aflatoxins are a welknown group of mycotoxins. Among mycotoxins, aflatoxin B₁ is the most potential mutagen, hepatotoxin, hepatocarcinogen and teratogen. It is commonly found in agricultural, food and feed products including corn, peanut, dried medicinal herbs milk and dairy products. The risk of aflatoxin exposure and liver carcinogens are clearly high and frequently reported in Southeast Asian warm and humid countries, such as Thailand and southern China. The incidence of liver cancer and cholelangiocarcinoma in Thailand is the highest one among other types of cancer. Other co-carcinogens and co-tumor promoters of hepatocarcinogenesis, such as liver fluke and nitrosamines, could be involved and are yet to be further studied. The cause of liver hepatocarcinoma in animals and human has been suggested to be due to the contamination of aflatoxins in feed and food products. The risk of liver cancer is even much higher among the carriers of the hepatitis B virus which is considered to be a liver-tumor promoter. Aflatoxin contamination causes health damage to human and farm animals and nationally and internationally economic loss to food supplies and food markets as well as to agricultural farmers and business. Prevention and control measure of mycotoxins are therefore necessary and encouraged to reduce the damage of agricultural products and the decrease the risk and incidence of liver diseases and hepatoma. Researchers are actively looking for methods to control aflatoxin in susceptible crops and food products. Fungal inhibitors, chemicals to inactivate aflatoxins have been found and practically used. Some chemopreventive agents in hepatocarcinogen-risk consumers are also urgently needed. Classical methods for plant disease prevention and routine technologies for controlling plant pathogens have generally been unsuccessful. Legal limitation and safety limits of the aflatoxin contamination in food, milk and their products are still not fully effective in Thailand and other developing countries. Therefore, the surveillance, monitoring and control for the fungal growth and aflatoxin contamination in crops, commodities, feeds and foods have been emphasized to practice, however, more efforts and research projects have to be done. The newer strategies and guidelines for more effective control of aflatoxins are essentially needed in each country. Some of those problematic decontamination and controlling methods were generally reviewed and discussed.

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Method validation is one of the key activities to ensure that analytical data and the method used to produce it have been verified before use. Harmonized collaborative validation (HCV) is the most prestigious form of method validation but it is not necessary to validate all methods by HCV. As each method has it's fit for purpose, the various forms of method validation have their fit for purpose. In the area of method validation, AOAC International leads the world, but there are currently many issues regarding the process of method validation itself. However, the use of validated methods is beginning and it is one of the keys for laboratory quality assurance.

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The occurrence of mycotoxin in a large number of foods is a major health concern for Malaysia, as some mycotoxins such as aflatoxin are genotoxic carcinogenic substances where there is no threshold level below which no harmful effect is observed. For most mycotoxin, current scientific and technical knowledge and improvements in production and storage techniques do not prevent the development of moulds that produce these toxins. As such, setting limits that are practical and achievable and monitoring mycotoxins levels become necessary, which Malaysia has carried out. Monitoring of aflatoxin in raw peanuts imported into Malaysia in 2004 indicated a detection rate of 50% and 6% were detected with total aflatoxin exceeding 15μg/kg [Maximum level in Malaysia's Food Regulation]. In 2004 and 2005, samples of raw peanut taken from retailed market showed 81% were detected with aflatoxin and 38% were detected with more than 15μg/kg of total aflatoxin. The current practice of storage and the natural high temperatures and humidity levels contributed to the high occurrence at the retail market. In comparison roasted peanut in-shell was found to be much less contaminated. However, the rice was found to be relatively safe. A total of 96 rice sample taken in 2005 showed no trace of aflatoxin contamination. Only 18 out of 200 rice samples analyzed (9%) contaminated with traces of zearalenone ranged between 6 to 11μg/kg and none contaminated with ochratoxin. Determination of aflatoxin was performed by HPLC with photochemical reactor after cleaned up using immuno affinity column. Zearalenone and ochratoxin were determined by HPLC fluorescence detection after cleaned up using Immuno Affinity Column. In this paper, the mycotoxin monitoring results in 2004 and 2005 is reviewed and health hazard using risk assessment approached related to aflatoxin is assessed.

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Taiwan is located in the subtropical zone, and the typical weather is hot and humid which benefit the growth of fungi. Mycotoxins are the toxic metabolites produced by some species of fungi genera such as Aspergillus, Penicillium and Fusarium. Surveys of mycotoxins in market foods have been implemented by Bureau of Food and Drug Analysis (BFDA) in Taiwan for several years. The results were used as database to establish related regulations by Department of Health (DOH) and provided for local health authorities to prevent unsafe food products from being consumed. Aflatoxin, ochratoxin A, fumonisin, deoxynivalenol (DON), patulin and T-2 toxin were tested and surveyed in various kinds of foods during the past ten years. From 1997 to 2006, a total of 1, 056 peanut product samples were tested and 339 samples (32.1%) contained aflatoxins. Among them, 65 samples which contained aflatoxins above 15μg/kg exceeded action levels of Taiwan. According to the results of the surveys in 2005 and 2006, most of violative products were imported from a foreign country. In 2002, aflatoxin M₁ was tested in 113 dairy products including fresh milk, milk powder, infant formula and drinking yogurt. Most of them contained a trace of aflatoxin M₁, but none exceeded action levels of Taiwan. In 2000, 2003, 2005 and 2006, 441 samples were collected and examined for ochratoxin A, and 68 samples (15.4%) contained less than 5μg/kg, including grain products, wine, coffee products and so on. In 2002, 76 corn products were tested and 11 samples (14.5%) contained fumonisins (B₁+B₂) ranging from 0.05 to 0.16mg/kg. The survey of DON in rice, flour, noodle, corn, oat and other products was implemented in 2004. One hundred and fifty samples were tested and the results showed that 3 samples contained DON ranging from 0.11 to 0.45mg/kg. In 1999 and 2005, 122 commodities including baby juice, juice drink, pure apple juice, mixed juice and apple paste were collected and tested for patulin. The results showed that 13 apple juices contained patulin ranging from 8.6 to 39.9μg/kg. In the survey of T-2 toxin in 2005, 31 food products were tested and 4 samples (12.9%) contained T-2 toxin ranging from 0.7 to 8.9μg/kg. The results showed that the violative rate of aflatoxins in imported peanut products grew in recent years. Other mycotoxins including ochratoxin A, fumonisins, DON, patulin and T-2 toxin were detected in some food commodities, but the contamination levels were pretty low.

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Various kinds of fruit juice such as apple, orange, guava, passion fruit have been collected from supermarkets and drinking trolleys in Bangkok and suburb since 2003 for determinating of patulin contamination. Patulin was extracted from fruit juice with ethyl acetate by liquid-liquid partition extraction, cleaned up with silica gel column and determined by HPLC-UV detector. It was found that 14 samples out of 103 samples (14%) were contaminated with patulin in the range 4.55-35.97μg/L. The result showed that there was not serious contamination of patulin in fruit juice especially apple juice since the maximum regulation level of patulin in apple juice is set at 50μg/L (Regulation (EC) No 1425/2003).

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Aflatoxin B₁, B₂, G₁, G₂ and M₁ were examined in commercial food and foodstuffs including nuts, cereals, beans spices and dairy products in Tokyo, Japan. Aflatoxins were determined and confirmed by HPTLC or HPLC after purification with Florisil column chromatography. The limit of quantification was 0.1μg/kg for each aflatoxin. Aflatoxins were found in nuts (pistachio nut, peanut), cereals (coix seed, corn, buckwheat, etc.), crude sugar, beans (beans for bean paste), spices (nutmeg, paprika, red pepper, etc.), and dairy product. The highest incidence of aflatoxin contamination was found in nutmeg, and highest level of aflatoxin B₁ was found in pistachio nut. More than 10μg/kg of aflatoxin B₁ were sometimes found in imported nuts, cereals, beans and spices. These samples were eliminated on the order of the government.

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Aflatoxins are highly toxic and carcinogenic substances mainly produced by Aspergillus f lavus and Aspergillus parasiticus. Contamination of agricultural commodities with aflatoxins is a very serious problem all over the world. Aflatoxin biosynthesis has been extensively studied, and now it is known that at least 18 enzyme steps are involved in the pathway from acetyl CoA to aflatoxins. Many genes encoding the enzymes and the transcription factors have been isolated, and those functions have been studied. In the genomes of A. parasiticus and A. flavus, at least 25 genes related to aflatoxin biosynthesis make a 70-kb gene cluster. We have investigated many enzyme reactions involved in aflatoxin biosynthesis by using cell-free systems. Our biochemical studies showed the aflatoxin biosynthetic pathway has many unique characters as a secondary metabolism.

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Specific inhibitors for mycotoxin production are useful not only to prevent mycotoxin contamination in foods and feeds without incurring rapid spread of resistant strains, but also to know the regulatory mechanism of mycotoxin production by fungi at the molecular level. We have been studying some aflatoxin production inhibitors, aflastatins A and B, and blasticidin A, this past decade. All these compounds are metabolites of Streptomyces and have similar unique structures. In this proceedings, we describe our recent work on the stereochemistry of these compounds and a fluorescence probe of blasticidin A to investigate its localization in the fungal hypha. Furthermore, we present detailed biological activity of dioctatin A as a strong inhibitor of aflatoxin production by Aspergillus parasiticus. Our results indicate that dioctatin A has pleiotropic effects on regulatory mechanisms of fungal secondary metabolite production and differentiation, which lead to inhibition of aflatoxin production.

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NIV chemotypes of F graminearum were widely distributed in western part of Japan and the isolation frequency was higher than that of DON chemotypes, and moreover, their virulence would be considerably high. We tested hypothesis that prevalence of NIV chemotype in Japan is related to rice cropping. Artificial inoculation of F graminearum caused disease on rice and salmon-pink sporodochia were observed on the diseased rice glumes. Our preliminary tests using isolates differing in trichothecene chemotype suggested that virulence of F graminearum isolates against rice may be different from that against wheat and barley. Use of appropriate fungicides is most important and effective practice to control FHB. Therefore, re-evaluation of registered fungicides and screening new candidates for control of mycotoxin contamination are considered mandatory. We found that six chemicals; metoconazole, tebuconazole, captan, thiofanate-methyl, oxine-copper, and phosphorous acid would control DON and NIV. On the contrary, treatment of azoxystrobin significantly increased the level of DON and NIV compared to the control plots, even though it reduced disease severity of FHB. Lodging also seems to be an important risk factor in mycotoxin production. A case-control study and invention trials were done to clarify effect of lodging on the accumulation of DON and NIV. The concentration of DON+NIV in lodged crop was higher than that of non-lodged one and the mycotoxins production was increased 30% even after 5-7 days lodging. These results suggest that appropriate management practices e.g. cultivar or fertilization to avoid lodging should be required for reduction of mycotoxin contamination.

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Fusarium head blight (FHB) is a devastating disease of important cereal crops resulting in significant yield loss and mycotoxin contamination. Although reducing fungal infections is the most desirable method to solve the problems, genetic approach and cultural practices have achieved limited success in terms of effectiveness and costs. In this paper, we briefly summarize progress of our studies on wheat and Fusarium genes, which may be useful in biotechnology for Fusarium resistance and mycotoxin decontamination in the future.

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Mycotoxins have the ability to impact health of humans and animals. Especially livestock animals are very often affected by receiving mycotoxins via contaminated feed. While adsorption to clay-based minerals seems to work very well for deactivating aflatoxins in animal feed this approach has not been scientifically proven for other mycotoxins. Microbial degradation or biotransformation poses an alternative strategy to alleviate and ameliorate mycotoxins in animal feedstuffs. Many mixed and pure cultures of microorganisms have been described to degrade various mycotoxins. But for a practical application those microorganisms have to meet many requirements (metabolites must be nontoxic, rapid degradation of mycotoxin, safety of microorganisms applied, ability to work in gastrointestinal environment etc.). During the last decade our research group was able to isolate and characterize microorganisms (Eubacterium BBSH 797, T, mycotoxinivorans, bacterium #144) which are capable of detoxifying deoxynivalenol, ochratoxin A, zearalenone and fumonisins. These microbes also meet the above mentioned requirements.

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Mycotoxins are secondary metabolites produced by the certain strain of fungi when environmental factors are favorable. Exposure to mycotoxins can cause lowered performance, sickness or death of human and animal including birds. Mycotoxins attract worldwide attention because of the significant economic losses associated with their impact on human and animal productivity and both domestic and international trade. No single method can be successfully eliminated mycotoxins contamination from food and feed. The cooperation and management systems should be emphasized for the prevention and control of mycotoxins. The system might be different from one to another depending on each commodities nature. Hence, to understand the crop and farmer practices from planting, harvesting, on farm-storage, off farm storage, transportation, processing and selling are very important for team leader to solve the problem. As it is known that the growth of fungi in crop and agricultural product is the main cause of toxin formation and related to the concentration produced. However, three main factors which involve to mycotoxins production are the ability of toxigenic fungi, substrate (commodities) and environment such as temperature moisture content and relative humidity. If one of these three factors is lacked, mycotoxins could not be produced. After knowing crop processing steps and factors involving mycotoxins production, appropriate prevention and control measures at each steps could be managed. Mycotoxins prevention and control in food and feed would not be success if there was no cooperation among farmers, local dealers, manufactures, retailers, exporters, governments and private sectors involved. To build up awareness and strengthen cooperation with all of them by set up the meeting, brainstorming for solution and training on nature and important of mycotoxins including routine monitoring should be set up in the management action plan. Moreover, educate and encourage the consumers to buy or consume only safety food must be announced. Consequently, the mycotoxins management in food and feed will be automatically operated by producers.

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We attempted the identification of Fusarium species by a DNA sequence analysis to practically validate the utility of a molecular approach for fungal identification and reveal its limitations. We sequenced three regions, the 5' end of the large-subunit (D2 region) and the internal transcribed spacer 1 and 2 (ITS1 and ITS2) regions, in the rRNA genes. The DNA sequences of 38 Fusarium strains isolated from unpolished rice harvested in Japan were analyzed and compared for similarity to entries in the GenBank. Based on this comparison, it was estimated that all these three regions, as a minimum, must be compared with the database to identify Fusarium fungi at the species level. According to the sequence differences in the three regions, the 38 isolates were classified into 13 groups. Out of the 13 groups, 6 groups (20 isolates in total) were able to be identified as the definite species based only on the sequence data. For the other 6 groups (17 isolates in total), the candidate species were restricted on the basis of the sequence similarity. The restriction of candidate species rendered it easy to identify the Fusarium isolates at the species level with the aid of their morphologies. Only one isolate could not be identified. It was verified in this study that a DNA sequence comparison with the GenBank database was useful and reliable for the identification of the Fusarium species.

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Rapid strip test kits (AgraStrip) were validated to test total aflatoxins in corn using ethanol extraction method at cut-off levels of 4 μg/kg, 10 μg/kg and 20 μg/kg, respectively. The test is a one-step lateral flow immunochromatographic test based on a competitive immunoassay format. The strip membrane contains a test zone and a control zone. A positive sample with total aflatoxins greater than the cut-off level will result in no visual line in the test zone. Alternatively, a negative sample with total aflatoxins less than the cut-off level will form a visible line in the test zone. One line will always be visible in the control zone regardless of the presence of aflatoxins. The test is a rapid semi-quantitative method with assay results to be obtained within 5 minutes. Validation studies assessed the accuracy of test kits by using ethanol as sample extraction solvent. Results indicated that the tests are accurate and effective for semi-quantitive measuring total aflatoxins greater or less than the cutoff levels in corn.

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Aflatoxin M₁(AFM₁) is well-known as a metabolite of AFB₁ and possible to exist in milk or dairy products. As cows have polluted feed by AFB₁, AFM₁ also will be secreted in milk. We could not detect low content AFM₁ in foods about ten years ago, but recently according to the progress of technology in analytical chemistry, very small amount of AFM₁(μg/kg) can be detected with various instrument of analysis. We would like to introduce the new methods of AFM₁ analysis by LCMS equipped with APPI (atmospheric pressure photo ionization) system. As APPI system is a selected ionization method in comparison with ESI (electro spray ionization) for AFs, 0.002 μg/kg ofAFM₁ in milk can be detected.

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DOA-Aflatoxin ELISA Test Kit is an in-house (ELISA test) kit produced by Department of Agriculture (DOA) Ministry of Agriculture and Cooperatives. The test kit had been validated for their precision and accuracy for aflatoxin B₁ detection in agricultural commodities. However, proficiency testing of analysis using DOA-Aflatoxin ELISA test kit is an essential element of test kit quality assurance. The analysis performance could be compared the analytical results with those from other laboratories with different methods. The test materials of animal feed, pistachio paste, peanut butter slurry, peanut and chilli powder were prepared and sent to our laboratory by FAPAS (Food Analysis Performance Assessment Scheme). The analysis was performed for aflatoxin B₁ using DOA-Aflatoxin ELISA test kit. The satisfactory results of Zscore were obtained from the tests. Performance in a FAPAS proficiency testing, therefore is considered satisfactory if a participant's Z-score lies within the range of-2 and +2.

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Polyclonal antibodies against fumonisin B 1 (FB1) were produced in the rabbits after immunizing the animal with FmBI-brovine serum albumin. A direct competitive enzyme-link immunosorbent assay (ELISA) was used to characterize the antibodies and for analysis of the toxin in corn samples. The serum against FB1 was obtained from the rabbit after 5 weeks of immunization. The highest antibody titer was found in the sera of rabbit 10 weeks after immunization with the titer of 1:25, 000 The conjugation of FB 1-HRP was achieved by a water soluble carbodiimide (EDPC). The appropriate time of the reaction between FB 1 and FB 1-HRP in competition to bind the antibody at the bottom of the well was 60 minutes. A good correlation was found between FB 1 level in naturally contaminated corn samples analyzed by direct ELISA and the high pressure liquid chromatography (HPLC) method. The correlation coefficient of a linear regression between ELISA and HPLC data was found to be 0.9732.

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Ochratoxin A (OTA) is one of the important mycotoxins found in foods and is expected for determination in many exported foods. The AOAC method was reviewed and modified to determine OTA in roasted coffee and corn surveyed in Bangkok and nearby provinces during the year 2004-2005. The extraction method was cooperated with Immunoaffinity Column (IAC), analyzed by high performance thin layer chromatography(HPTLC) then followed by densitometer for quantitative analysis. This method was employed to analyze OTA in corn and resulted good recovery (58.4-86.0 %) and LOD and LOQ showed value of 0.28 and 0.5 μg/kg, respectively. Of 24 corn samples were found OTA positive 58% (14/24), but none of these should the level higher than 5 μg/kg (as the EU standard allowed). In case of roasted coffee, the result revealed only 3 samples positive among 44 surveyed samples (<1-4.6 μg/kg). Due to the interferences during extraction which may affect OTA in roasted coffee analysis, the recovery at concentration of 5 μg/kg was lower compared to corn analysis. Recovery was 76.8% (ranged 1-20 μg/kg), the LOQ and the LOD were 1 and 0.33 μg/kg, respectively. This study concluded that the situation of OTA contamination in both corn and roasted coffee marketed in Bangkok and nearby provinces is low and these foods are safe and wholesome consume as food or feed.

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Zearalenone (ZON) and its four metabolites, α-zearalenol (α-ZOL), .β-zearalenol (β-ZOL), α-zearalanol (α-ZAL) and β-zearalanol (β-ZAL), have been shown to competitively bind to estrogen receptors (ERs) in a number of in-vitro tests. As the binding affinities of these substances to ERs are different, a simultaneous quantitative determination is necessary for the accurate risk assessment. Therefore liquid chromatography/tandem mass spectrometry using electrospray ionization (LC-ESI-MS/MS) was evaluated for the applications to beer and related raw materials. In this method, Zearalanone (ZAN), which doesn't occur in nature, was used as an internal standard for quantification. Sample preparation for malt was performed by the extraction of the commodities with a mixture of acetonitrile and water, followed by solid phase extraction (SPE) with C-18 cartridge. As for beer products, samples were degassed and applied directly to the same cartridge. After separation on a phenyl column, quantification limits of 1.1-1.6 μg/kg in malt and 0.2-2.0 μg/kg in beer were achieved with the negative ionization mode.
To further characterize the ZON metabolisms in brewing, ZON was added to wort at the level of 100 μg/kg and fermentation tests were conducted for up to 10 days using two commercial strains of Saccharomyces pastrianus. After fermentation, ZON levels were decreased to 1.0 μg/kg in both experiments. Instead of ZON, β-ZOL, which shows lower estrogenic activity than ZON, was detected mainly at levels of 78.3 ng/g and 88.2 μg/kg. α-ZAL and β-ZAL which show higher estrogenic activity than ZON were not detected in both cases.

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This research was undertaken to study the toxic effects of low level aflatoxin B₁ (AFB₁) on hepatic enzyme activities and white blood cell parameters in fresh water fish Nile tilapia (Oreochromis niloticus). Two hundred and seventy fresh water fish Nile tilapia, 2 months old, were acclimated for 7 days and then entered into an experiment with a completely randomized design. They were divided by weight into three groups of 30 fish per group and tested in triplicate. They were fed commercial feed, which was confirmed to be free from AFBI through analysis by ELISA. One of the three groups was orally intubated and given a single dose of olive oil, as a control. The other groups were orally intubated and given a single dose of 20 or 40 μg/kg body weight (bw) AFB₁.On the 1^st, 2^nd, 4²th, 8^th, 10^th, 16^th, 20^th, 24^th, 30^th and 48^th hour of the experiment, nine fish of each group (three fish of each replication) were randomly selected. Blood was collected from the caudal vessel and monocyte, lymphocyte and neutrophil counts were determined. Glutamic oxaloacetic transaminase (GOT), gutamic pyruvic transaminase (GPT), alkaline phosphatase (ALP) and gamma glutamyltransferase (GGT) were measured in serum samples. On the 1^st and 2^nd hour of the experiment, average cell counts of monocytes and lymphocytes were significantly higher in the AFB₁-treated groups than in the control group, whereas neutrophil counts were significantly lower in the AFB₁-treated groups than in the control group. After the 4^th hour of the experiment, the results showed the same pattern and there were significant differences between the 20 μg/kg bw AFB₁ group and the 40 μg/kg bw AFB₁ group. GOT, GPT, ALP and GGT levels were significantly increased in the AFB₁-treated groups, in comparison to the control group. Results of this study show that the hepatic enzyme activity and the white blood cell counts are a potential indicator for low level AFB₁ intoxication.

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Mycotoxins, which induce mitochondrial swelling, were compared with regard to the swelling patterns. Mycotoxins were tentatively classified into three groups type I, II, and III. The type I mycotoxins induced the swelling which went on to the final stage, the complete disruption of mitochondria, and the swelling velocity proportionally increased corresponding to the increase of mycotoxin concentrations. The type II mycotoxins showed a very swift swelling which turned very soon into a slow swelling. The swelling magnitude in stead of swelling velocity increased corresponding to the increase of mycotoxin concentrations. The type III mycotoxins showed a brief lag time before the initiation of swelling. A few mycotoxins such as emodin, luteoskynn, rubroskyrin, secalonic acid and versicolorins, which are uncouplers of oxidative phosphorylation, did not induce mitochondrial swelling.

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The situation of aflatoxins contamination in risky foods such as peanut and peanut products, dried chili and products during the year 2004 in Thailand was investigated. The survey of peanut-process chains from crops to retail and whole sale markets revealed that malpractices during drying process in the field, dehulling, grading and unsanitary warehouse caused increasing risk of aflatoxins contamination. In this research, contamination of total aflatoxins >20 μg/kg in peanut and products were found positive 55 in 183 samples (30 %), in which the roasted ground peanuts were the most risky food (27/35 or 77.14 %). Sorting houses and 12 small enterprises were observed based on GMP regulation. Some small enterprises had poor hygienic practices but aflatoxin level in food was low compared to the sorting house. Sorting of raw peanuts was the key to reduce aflatoxins contamination. Hand sorting was done in one factory as the model practice and was successful to reduce aflatoxins contamination that were found only 0.02-3.05μg/kg in sorted raw peanuts. The survey of dried chili proceses in 7 small enterprises were found that poor practices in raw material storage could cause the possibility of fungi growth. Aflatoxins in dried chili and products found positive in 47 of 75 samples (62.67 %), however, aflatoxins contamination in those foods was lower than that Thai-standard allowed (20μg/kg). Isolated fungi in dried chili (40 samples) indicated the potential of aflatoxins production when tested in rice-media. Using of chlorine dioxide 10 mg/L and 20 mg/L for 2 min reduced fungi population as much as 9.37-12.70 %.

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Aflatoxin is a toxic secondary metabolite produced by certain strains of Aspergillus flavus. The contamination of aflatoxin occurred in a wide variety of food and feed and has been implicated in a range of human and animal diseases. Because it is mainly concerning with human consumption, hence the method used for controlling aflatoxin should be safe to human. The aim of this study was to select the varieties of edible herbs which commonly grow in Thailand and can inhibit aflatoxin production or fungal growth in order to use as aflatoxin control agent in food and feed. Generally, most herbs were known to inhibit fungal growth. In this study we tried to select herbs which directly do inhibit aflatoxin production aside from growth inhibition. Sixteen varieties of edible herbs which commonly grow in Thailand were selected for the tests. Tip culture method was employed to characterize the efficiency of herb extracts to inhibit aflatoxin production or mycerial growth. Amount of aflatoxin production and mycerial weight of each treatment were determined as percent inhibition from control. Six varieties of herbs were found to cause high percentage of aflatoxin production inhibition (64-99 %) whereas percent inhibition of mycerials weight was low or no inhibition. They were Pluyllanthus amarus, Zingiber afcinale, Thuybergia taurifolia, Occimum basilicum, Boesenbergia pandurata and Tumaric sp. The other 5 varieties Syzyglum aromaticum, Occimum tenuif forum, Cymbopogon citrates, Alpinia nigra and Allium sativum gave high percentage of mycerial weight inhibition(50-70 %) which enhanced to have high percentage of aflatoxin inhibition(80-99 %). The effectiveness of these common herbs can be utilized as biological control of aflatoxin production or fungal growth in food and feed.

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Efficacy of yeast cell wall on detoxification of aflatoxin in diet of tilapian fish was evaluated. Three levels of aflatoxin preparation (1, 10 and 50 mg/kg) and 0.2 % yeast cell wall (YCW) were mixed with fish feed. After 8 weeks of feeding experiment, fish fed aflatoxin without adding YCW showed the reduction of body weight gain. Inclusion of YCW in the diets prevented the growth inhibitory effect of aflatoxin. In addition, fish fed YCW showed protection against the aflatoxin induced liver damage indicated by the reduction of hepatocellular enzyme. These results indicate that inclusion of YCW in aflatoxin contaminated feeds can reduce aflatoxicosis in the tilapian fish.

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Practical and effective methods for the detoxification of aflatoxins in contaminated foods and feedstuffs are highly needed to reduce deleterious effects of these mycotoxins exposed to human and animal health. Previously, hectorite clay was shown to effectively bind aflatoxin B₁ (AFB₁) in vitro. In this study, AFB₁ was isothermally mixed with hectorite at 25, 37, and 45 °C to study the adsorption process. The isotherm studies estimated the maximal capacity and affinity constant for AFB₁ binding. The linearlized constant isotherm curves of hectorite indicated that numbers of binding sites remained constant throughout the whole ranges of AFB₁ concentration and the equilibrium has not reached under this experimental condition. These data may suggest that hectorite clay could be effective for AFB₁ binding.

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The objective of this research is to evaluate the ability of clay minerals to adsorb aflatoxin B₁ (AFB₁) in vitro. Fourteen clay minerals from different sources in Thailand and commercial adsorbents including bentonites and toxin binders were tested for AFB₁ adsorption capacities. Our results indicated that the clay minerals were capable of sequestering AFB₁ from aqueous solution differently. The experimental data were fitted to modified Freundlich model better than Langmuir model to investigate adsorption capacities and affinity constant. The S-shaped isotherms were observed for these clay minerals and commercial adsorbents. The clay minerals had maximum capacity (Q_max) ranged from 7.10 × 10^-4 mol/kg to 4.76 × 10^-3 mol/kg whereas the commercial adsorbents had Q_max ranged from 8.21 × 10^-4 mol/kg to 4.52 × 10^-3 mol/kg. The distribution constant (K_d) for the clay mineral samples and commercial adsorbents were in the ranges of 2.15 × 10⁵-6.70 × 10⁷ and 2.52 × 10⁵-4.71 × 10⁵, respectively. This in vitro method may be useful for accurately predicting the efficacy of adsorbents before testing in vivo.

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