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タイトル: MALDI-TOF/MS及びARISAを用いたキノコ子実体迅速同定手法に関する研究
著者: 菅原, 涼太
発行日: 9-Jun-2016
抄録: Mushrooms provide contradict properties. One aspect is as a favorite food widely accepted in the world, and another is pathogenesis of food poisoning sometimes in lethal. The identification of mushroom was traditionally used morphological analysis. However identification of fungi, including mushroom, has been approached by genomic analysis in recent decade. This study focus on characterization and identification of wild mushrooms by functional metabolites, rDNA sequences and protein compositions. Especially, the probability of matrix-assisted laser desorption-ionization mass spectrometry (MALDI-TOF/MS) based protein analysis is spotlighted for rapid and reliable identification of mushrooms. Two hundreds and five samples of fruiting bodies, grown wild in Hokkaido island including a campus of our university, were collected from the period from June '12 to November '14. The samples, hot-air blowed and powdered, were extracted with alkaline hot water for beta-glucan, with 50% ethanol for total polyphenol (TPP) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity. The ISOIL beads beating kit (Nippon gene) was used for DNA analyses of sequencing or automated rRNA intergenic spacer analysis (ARISA). Ribosomal proteins, being mainly present, were extracted from the cap of raw fruiting bodies with formic acid-acetonitrile mixture (1+1) for MALDI-TOF/MS analysis. The functional features were evaluated by colorimetric assays of Congo red reagent for beta-glucan, of Folin-Denis reagent for TPP and of DPPH radical scavenging activity for antioxidant ability. The rDNA inter spacer (ITS) regions were sequenced for the species identification and also submitted to a polymorphic evaluation by ARISA. The protein mass spectra were obtained by MALDI-Biotyper (Bruker daltnics). Beta-glucan contents were shown clear differences from each species to species beyond genus and family, although TPP and DPPH radical scavenging activity were also recognized similar tendency but accompanied with a little ambiguity. The DNA sequencing identified 175 samples as independent species or genus, however failed to obtain sequencing data mainly due to mixed fluorescence signals for residual 21 samples. The protein profiles obtained with MALDI-TOF/MS well distinguished not only species but also each part of fruiting bodies such as stem, cap or spore within the same sample. The best spectrum was obtained from the extracts of cap-portion of fruiting bodies. Mushroom database of mass profiling spectrum (MPS) was finally constructed through the measurements of 113 species cap samples which provided data qualified. MALDI-TOF/MS analyses of these samples were achieved correct identification of between species as much as 109 samples (96%). The results of ARISA identification, discriminating 1bp size differences in DNA fragment within ±0.1% accuracy, were well consistent with the result of sequencing identification, however 10 of 31 species were not differentiated from other species because of the same fragment sizes. However, ARISA was effectively applied for the evaluation of a mushroom suspected an origin of as the food poisoning. The mushroom was stored at room temperature for 1 week then the fruiting body was deteriorated from original shape ever accompanied with some fungi like microbe. DNA sequencing could not be applied for such mixed samples since they provide complicated signals. The ARISA fragments of the mushroom clearly differentiated from the size of toxic mushrooms suspected as a candidate. This finding leads to the estimation of no relation of the mushroom to the cause of food poisoning. For rapid and reliable identification of mushroom species, more importantly removal of toxic mushrooms, would be possible by verification with indicative-protein databases of fruiting bodies obtained by MALDI-TOF/MS. Further, ARISA would be useful to confirm the authenticity of mushroom-processed foods with expected or unexpected contamination of toxic mushrooms.
URI: http://hdl.handle.net/10659/4803
学位授与年月日: 2014
学位名: 酪農学
学位授与機関: 酪農学園大学
出現コレクション:学位論文(修士)

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