Occurrence of ochratoxin A (OTA) and ochratoxin B (OTB) in 32 Vietnamese roasted coffees, 30 Thai roasted coffees and 38 Thai instant coffees was performed using an immunoaffinity column-HPLC method. Twenty-six of Vietnamese roasted coffees were contaminated with overall average 0.75 μg/kg of OTA and 11 samples were contaminated with overall average 0.20 μg/kg of OTB. Four of Thai roasted coffees were contaminated with overall average 0.17 μg/kg of OTA and one sample was contaminated with 0.56 μg/ kg of OTB. Twenty-eight of Thai instant coffees were contaminated with overall average 2.19 μg/kg of OTA, and OTB from these samples was not detected. There was no sample more than OTA regulatory limits of European Union. Therefore, it seems that the risk of ochratoxins in retail coffees in Vietnam and Thailand was acceptably low. This report is the first on occurrence of OTB in Vietnamese roasted coffee and ochratoxins in Thai instant coffee.
The reverse-phase HPLC-fluorescence (FL) analysis of pre-column trifluoroacetic acid (TFA) derivatized extract of cultured medium of an Aspergillus flavus strain gave an unexpected peak nearly identical to aflatoxin G1 (AFG1). This peak was proven to be aflatoxin M1 (AFM1), not AFG1, by LC-MS/MS analysis. Therefore, we examined some chromatographic conditions to separate AFM1 and AFG1 clearly on reverse-phase HPLC analysis.
We examined the localization of the full-length protein and zinc finger domain of the trichothecene regulator, TRI6. When fused to an enhanced green fluorescent protein (EGFP) gene, nuclear accumulation of the zinc finger domain, but not the full-length protein, was observed.
We examined the effect of using xylose as a carbon source in the medium on trichothecene production by Fusarium graminearum. By frequently adjusting the pH of the submerged culture in response to the pH changes of the known trichothecene- inducing sucrose-agmatine culture, trichothecene became detectable from the xylose liquid culture initiated at neutral pH. In contrast to the low pH requirement of the xylose medium for trichothecene production, the fungus produced the mycotoxin in sucrose liquid culture at neutral pH.
For the detection of new glucoside derivatives of mycotooxins (masked mycotoxins), screening based on the accurate mass was carried out through the high resolution mass spectrometry measurement with LC-MS (LC-Orbitrap MS). Consequently, mono-glucoside derivatives of type B trichothecenes (nivalenol and fusarenon-X) in wheat grains infected with Fusarium strains and those of the type A trichothecenes (T-2 toxin, HT-2 toxin, neosolaniol, diacetoxyscirpenol, and monoacetoxyscirpenol) in corn powder reference material were detected, respectively. Di-glucoside derivatives were also found for T-2 toxin and HT-2 toxin. These findings indicate that the presence of masked mycotoxins is not limited to some specific mycotoxins such as deoxynivalenol and zearalenone, but likely with the other Fusarium mycotoxins.
Ochratoxin A (OTA) is a secondary metabolite of fungi such as A. ochraceus and P. verrucosum, which occur mainly in stored foods. OTA contamination has been found in various food commodities including cereals, coffee, cocoa, beer and wine. Food Safety Commission of Japan (FSCJ) conducted a risk assessment of OTA as a Self-tasking risk assessment, based on the studies on toxicokinetics, acute toxicity, subacute toxicity, genotoxicity, chronic toxicity and carcinogenicity, as well as the contamination survey in foods and estimated exposure to OTA in Japan. This paper summarizes the risk assessment of OTA reported in 2014.
Proceeding of the 77th meeting
Symposium "Production of new biomaterials by microorganisms in various environments"
The fungal cell-wall is a complex structure composed of cross-linked polysaccharides and glycoproteins. Chitin and β-1,3-glucan are the main skeletal polysaccharides of most fungi. On the other hands, α-1,3-glucan has been found in various fungal species and has an important role in some fungi. For example, α-1,3-glucan acts as a virulence factor in some pathogenic fungi. Considering this background, α-1,3-glucanase (EC220.127.116.11) has been studied as a biological control agent of pathogenic fungi. α-1,3-Glucanases are classified into two families, 71 and 87, of glycoside hydrolases (GH) based on their amino acid sequences. Type 71 α-1,3-glucanase have been found in fungi, and type 87 enzymes have been found in bacteria. We isolated type 87 α-1,3-glucanase (Agl-KA) from culture filtrate of Bacillus circulans KA-304. Agl-KA hydrolyzed the fungal cell-wall α-1,3-glucan and released the protoplasts from Schizophyllum commune mycelia in cooperation with GH family type 19 chitinase. Here, we report that domain structure and function of Agl-KA. Furthermore, we briefly discuss potential future applications of bacterial α-1,3-glucanases.
Toxicity evaluation research on type B trichothecene mycotoxins has accumulated various data about their toxicity mechanisms. However, except for end products such as DON or NIV, individual data for derivatives are insufficient because those data were yielded as part of the information about end products. To address this matter, we introduce some speculation about the toxicity characteristics of these trichothecene derivatives using gene expression data derived from DNA microarray analysis. Yeast cell studies suggest that high-sensitivity mycotoxins induce widespread gene expression changes, and that sensitivity has a relationship with plasma membrane transporters. Additionally, these data and previous studies suggest that the toxicity of type B trichothcenes influences mitochondrial ribosomes.
In Aspergillus section Flavi fungi, there are aflatoxigenic and non-aflatoxingenic species. However, those relations between aflatoxigenic and non-aflatoxigenic species as well as strains which lost aflatoxin-producing ability are not clearly understood yet. Also a basic question, why fungi produce aflatoxin, has not yet been answered.
Trichothecene mycotoxins are secondary metabolites of Fusarium species, such as F. graminearum, and other molds. They are major mycotoxins that can cause diarrhea, vomiting, and alimentary toxic aleukia when consumed via contaminated cereal grains. Since trichothecenes have emerged as a significant threat to food and feed safety, determination of these mycotoxins in cereals is important for securing the health of humans and animals. In this review, we summarize the current understanding of the trichothecenes determination using LC-MSn, immunoassay, TLC and bioassay.
Actinomycetes produce an array of natural compounds in the form of secondary metabolites including antibiotics, and are currently the most useful microorganisms for this purpose. Within actinomycetes, members of the genus Streptomyces have been extensively studied due to their ability to synthesize a vast array of important secondary metabolites. Understanding of Streptomyces secondary metabolism could lead not only to achieve a high-yield production of antibiotics but also to awaken silent gene clusters for the discovery of novel natural compounds. Streptomyces hormones, exemplified by γ-butyrolactone-type autoregulators and butenolide-type autoregulators, and their cognate receptors could provide a clue to decipher the complicated regulatory mechanism for such secondary metabolism. In this mini-review, we describe our recent studies on the regulatory mechanism for actinomycetes secondary metabolism controlled by Streptomyces hormones and the application of our knowledge of the signaling cascades in the discovery of novel natural compounds.
Mangrove forests area are distributed in most tropical and subtropical coast regions of the world. Some of the potency of mangrove plants may be due to mutualistic endophytes fungal associated with host plants. In fact, some chemical materials are isolated from the fermentation broth of endophytes from mangrove plants. Several fungi such as producing chemical materials are adapted to sodium chloride (NaCl). It could be shown that NaCl has a profound influence on the regulation of secondary metabolites biosynthesis in fungal species. In this project, we started a research program to discover the endophytic fungi from mangrove plants that show the growth increase of secondary metabolites under the NaCl including fermentation condition. Over 100 fungal strains were collected from the twigs and petiols of mangrove plants, on NaCl supplemented agar plate. The AcOEt extracts obtained from these cultures have been done by TLC analysis. Then, a fungal strain, Eurotium rubrum IM-26 was selected because of large productivity or characteristic of secondary materials profile in the NaCl including fermentation condition (exposed salt stress). TLC-guided fractionation of the extract resulted in the isolations of eurotinone (1), its analogues compounds (3, 4, 5), and torosachrysone (2) form IM-26 strain. These structures were elucidated by spectroscopic methods. Although these compounds do not contain a chloride atom, compounds 1, 2 and 5 increased under the 3 % NaCl concentration in fermentation condition. The biosynthetic pathways of them could be activated by NaCl in this species.
In filamentous fungi, transcription of lignocellulolytic enzyme genes is triggered by recognition or uptake of a low molecular sugar derived from polysaccharides at the cell surface, followed by signal transduction. However, if easily metabolizable carbon sources like glucose co-exist, the transcription is inhibited by carbon catabolite repression. Understanding of these regulatory mechanisms is extremely important for efficient industrial production of plant biomass degrading enzymes. In recent years, genome-wide analyses have been performed in mainly Aspergilli, Trichoderma reesei, and Neurospora crassa, and a number of specific transcriptional factors were identified by using comprehensive gene disruption library or functional analysis of function-unknown genes that are co-regulated with lignocellulolytic enzyme genes. Furthermore, recent research showed that responses to cellulosic signal are often modulated by light-sensing machinery and by crosstalk with other alreadyknown signaling pathways. In this review, we summarizes researches on regulatory mechanisms of lignocellulolytic enzyme genes with reference to functions and activation mechanisms of transcription factors as well as recognition of the inducer to signal transduction.