Penicillium expansum was cultured in apple juice media prepared from six different commercial apple juices. The patulin production was profoundly affected by the differences in apple juices, whereas fungal growth was generally not. The maximum concentration of patulin was 7.3-fold of that in the media containing minimum concentration of patulin. The six apple juices were concentrated by evaporation and reconstructed to the original volumes by adding Milli-Q water. P. expansum was cultured in the media prepared from reconstructed apple juices, and the patulin concentration and fungal growth were determined. Evaporation of apple juice tended to decrease patulin production and to increase fungal growth, suggesting that the volatile compounds promote patulin production and inhibit fungal growth. The volatile compounds in the apple juice were then analyzed by GC-MS. The 13 compounds of which concentrations were largely decreased by evaporation were selected for evaluation of their stimulation of patulin production. Seven out of the 13 compounds, 2-methylpropyl acetate, ethyl butyrate, ethyl 2-methylbutanoate, 3-methyl-1-butanol, hexyl acetate, 1-hexanol, and 2-methylbutanoic acid, increased the patulin production of P.expansum concentration-dependently; 2-methylbutanoic acid and its ethyl ester were highly effective. Our results indicated that the composition of volatile compounds in apple juice media largely affects the patulin production and the growth of P. expansum.
A method to determine aflatoxin M1 (AFM1) levels by using an immunoaffinity column-based clean-up procedure and HPLC with fluorescence detection was validated by an inter-laboratory study among ten laboratories in Japan. Using the validated method, we surveyed AFM1 contamination in powdered formula. Samples for validation included a blank, three levels (blind pairs) of AFM1 spiked into liquid milk, naturally contaminated liquid milk, and naturally contaminated powdered formula. All samples were frozen and sent to the ten participating laboratories. For the liquid milk spiked at 1.0, 0.5, and 0.05 μg/kg levels, recoveries were 89.9, 91.6, and 88.2%, respectively. The repeatability relative standard deviation (RSDr) and reproducibility relative standard deviation (RSDR) were less than 7.4 and 8.1%, respectively. The recovery, RSDr, and RSDR of the powdered formula were 94.5, 8.9, and 11.9%, respectively. The RSDr and RSDR of the naturally contaminated milk were 13.3 and 20.9%, respectively. The Horwitz ratio (HorRat) values of all six samples were less than 1.0. For surveillance, 108 commercial powdered formulae were obtained in Japan. The average value of AFM1 in the powdered formulae was 0.002 μg/L, as ready-for-infant liquid milk (14 g powdered formula in 100 mL water). The highest contamination was 0.025 μg/L.
A simple, fast, accurate analytical method of Fusarium mycotoxin zearalenone (ZEA) from edible oils was developed by analyte-extension of the method for aflatoxins in oils. Oil samples were extracted with a mixture of acetonitrile and water (9 + 1, v/v) followed by the clean-up with a multifunctional column. ZEA content was determined by high-performance liquid chromatography with fluorescence detector (HPLC-FLD). The method was in-house validated for two matrices, blended oil and olive oil. The recovery rates were between 100.4% and 105.2 %, while relative standard deviations (RSDs) were between 0.9% and 2.3%.
Plants have immunity which is activated against broad range of microbes upon recognition of ‘non-self’ molecules called PAMPs （pathogen-associate molecular patterns） such as flagellin on bacterial flagella and chitin oligomer derived from fungal cell walls. However, pathogens have developed mechanisms to overcome this host immunity for successful infection. We have studied immune evasion mechanisms in fungal plant pathogens using an ascomycete rice pathogens Magnaporthe oryzae as a model. We have revealed that M.oryzae masked cell wall PAMPs with α-1,3-glucan, a non-degradable polysaccharide in plants. Indeed, M.oryzae mutant lacking α-1,3-glucan rapidly activated host immunity. Interestingly, ascomycete Cochliobolusmiyabeanus and basidiomycete Rhizoctinia solani concealed cell wall PAMPs with α-1,3-glucan during rice infection. In supporting of this finding, transgenic rice plants secreting a bacterial α-1,3-glucanase became resistant to these fungal pathogens. Our finding suggested that a wide range of fungal pathogens use α-1,3-glucan for evading host immunity. Thus, α-1,3-glucan may be a efficient target to controlling various fungal diseases. Use of α-1,3-glucanase-producing bacteria and α-1,3-glucanse will have potential applications in sustainable crop protection techniques.
To get a deeper insight into cellulase gene regulation mechanisms in Aspergillus aculeatus, we screened cellulose induction deficient mutants from the T-DNA insertion mutant library and identified a novel Zn（II）2Cys6 transcription factor, which was designated as the cellobiose response regulator （ClbR）. Functional analysis of clbR by gene disruption and overexpression revealed that ClbR regulates the cellulose and cellobiose-responsive induction of the cellulase and xylanase genes in A. aculeatus. Furthermore, we demonstrated the genetic interaction between ClbR and its paralog on the regulation of manR gene which is recently identified as a pathway specific transcriptional activator.
A simultaneous and multiple determination method for the major five Fusarium toxins (deoxynivalenol, nivalenol, T-2 toxin, HT-2 toxin, and zearalenone) contaminating wheat and barley was developed with liquid chromatography tandem-mass spectrometry (LC-MS/MS), and a harmonized collaborative validation of the method was conducted by the participants of 12 laboratories. Although there are many reports on the simultaneous and multiple determination of Fusarium toxins by instrumental analysis, only a few of them are developed to the harmonized collaborative validation. The variance in the extraction efficiency and the matrix effects on the ionization were occasionally innegligible with the mycotoxin analysis by LC-MS/MS, however, the employment of internal standards with the present method apparently contributed to compensate for these effects. This is the first report of a simultaneous and multiple determination method for both type A and B trichothecenes along with ZEA by LC-MS/MS that was validated through the harmonized collaborative validation. On the other hand, the accurate mass and high-resolution measurement was carried out with LC-MS (LC-Orbitrap MS) for the screening of new masked mycotoxins. Consequently, monoglucoside derivatives of type B trichothecenes (nivalenol and fusarenon-X) in wheat grains and those of type A trichothecenes (T-2 toxin, HT-2 toxin, neosolaniol, diacetoxyscirpenol, and monoacetoxyscirpenol) in corn powder sample were detected, respectively. Di-glucoside derivatives were also found for T-2 toxin and HT-2 toxin. Our 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 toxins.
Fusarium toxins are a group of mycotoxins produced by many kinds of Fusarium species, and frequently detected in field crops such as wheat, barley, maize and corn. They cause mycotoxicosis, a serious health hazard to humans and domestic animals. Therefore, collecting the information about fusarium toxin contamination in daily foods is crucial. Occurrence data of deoxynivalenol, a major fusarium toxin, have been collected in worldwide, but that of other toxins is limited. In this study, we examined foods from Japanese retail shops for contamination with four kinds of fusarium toxins, deoxynivalenol, T-2 toxin, HT-2 toxin and zearalenone.
Novel bioactive substances that regulate trichothecene production are in great demand both as tools for basic research and as control agents for reduction of mycotoxin contamination. Using compounds from the RIKEN Natural Product Depository (NPDepo) library, we are now screening for activators and inhibitors of trichothecene production. In this symposium, we describe the outlines of our recent progress on the regulation mechanisms of trichothecene production.
Indole-diterpenes are a group of compounds that contain mycotoxins including paxilline and lolitrem B. Recently, similarity of indole-diterpene biosynthetic pathways has been revealed by isolating and analyzing indole-diterpene biosynthetic gene clusters. We found an indole-diterpene compound terpendole E as the first natural product inhibitor of kinesin Eg5 by screening of cell cycle inhibitors. We found that terpendole E is a key biosynthetic intermediate of indole-diterpens by isolating and analyzing the terpendole E biosynthetic gene cluster.
It has been suggested that somatic homologous recombination is one of the important mechanisms of genetic variations in asexual life cycle of filamentous fungi. Recently, a simple detection/selection system of somatic homologous recombination was constructed by using nonfunctional fluorescence antibiotic markers in the rice blast fungus Pyricularia oryzae. Here we have summarized the detection systems of somatic homologous recombination and the current advancements of genome responses for unsuitable conditions in eukaryotes. We focused on somatic homologous recombination events induced by chemical stress treatment, and discussed the application of this mechanism to a biological marker of several mycotoxins.
Hepatocellular carcinoma (HCC), the most common liver cancer, occurs mainly in men. Even though the causative factors for HCC are well known to be hepatitis B/C viruses, aflatoxin B1 (AFB1) and abuse of alcohol, our knowledge about the processes of HCC development is very little due to their complexity. By this reason, molecular -targeted drugs for HCC is currently very limited. In this review, we described that based on the recent reportssex-determining factors including Sry (sex determining region Y) and sex hormones play a crucial role in onset of male HCC and its conversion into malignant tumor via cancer stem cell (CSC). These findings may confer the useful information for development of promising chemoprevention and anti-cancer drugs against male HCC.