The effects of treatment methods and conditions on the quality of the main spectrum profile (MSP) of yeast were investigated. The MSP is the standard spectrum used for database registration while constructing an in-house database for identifying food-derived yeast through matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). MSPs were generated for 75 strains of 22 food-derived yeast species using MALDI-TOF MS Autoflex Speed and MALDI Biotyper OC Ver. 3.0. After culturing yeast on potato dextrose agar at 28℃ for 5d, the colonies were treated with ethanol‐formic acid mixture and subjected to MALDI-TOF MS analysis. Using these conditions, MSPs were generated for 72 strains, except for 2 strains of Brettanomyces custersianus and 1 strain of Sterigmatomyces elviae. For these three strains, the MSP was generated by disrupting the cells with beads using a yeast disruption kit followed by ethanol‐formic acid treatment. In some cases, it was difficult to extract proteins from yeast cells, and the extraction efficiency of proteins decreased because of the production of viscous substances. It was necessary to select the optimal treatment method depending on the species and strain. The prepared MSPs of yeast cultured on various media were verified, and the highest identification match rate of 72.2％ was achieved when the strains were cultured on potato dextrose agar. Yeast strains were accurately identified through MALDI-TOF MS analysis when the colony was treated with ethanol‐formic acid, even though the strains could not be identified when samples were prepared using the recommended preparation method. Notably, it was difficult to classify Zygosaccharomyces bailii, Zygosaccharomyces parabailii, Zygosaccharomyces pseudobailii, Zygosaccharomyces mellis, and Zygosaccharomyces siamensis as independent species using MALDI-TOF MS Autoflex Speed. To accurately identify these closely related species, a dendrogram is necessary. This study showed that it is important to prepare and measure samples under the same culture and protein extraction conditions used for MSP preparation to accurately identify unknown yeast species.

　MALDI-TOF MS is attracting attention as a rapid microbial identification method. Among microbial identification methods using MALDI-TOF MS, the fingerprint method has become mainstream owing to its simplicity. In this method, the spectrum of a sample is compared with the spectra of known bacterial species registered in a database, and the microbial species in the database with the highest match rates are the identification results. Therefore, obtaining identification results unless there is a large amount of data stored in the database is difficult. Furthermore, the databases created by the manufacturers generally contain only a small amount of data on food-related microorganisms. Accurate identification of food-related microorganisms therefore requires the establishment of an in-house database of food-related microorganisms. For that purpose, ensuring the quality of the registered data is crucial to obtain correct identification results. In this study, Main spectra (MSP), standard spectrum for database registration, were obtained by preparing changing samples with different culture periods and different extraction methods for bacteria and yeast, which are typical food-related microorganisms. The MSP was then used to investigate the effects of culture period and sample extraction methods on microbial identification. An MSP with a high Score Value was obtained when all the following conditions were satisfied : 1) many peaks on the high molecular side with a mass-to-charge ratio (m/z) of 6,000 or higher were obtained, 2) high peak intensity was obtained, 3) spectrum showed stable baseline on the low molecular mass side without rising, and 4) spectrum showed no peaks on the low-molecular mass side larger than those on the high-molecular mass ide. The results of this study emphasize the importance of increasing the efficiency of extracting microbial components in sample preparation to ensure data quality. Furthermore, to improve the extraction efficiency of cellular components from microorganisms producing extracellular polymeric substances etc., it is important to (1) shorten the culture period, and (2) perform physical disruption using beads in addition to extraction with formic acid. Finally, regarding non-spore-forming bacteria and asexual yeast, it was clarified that changes in metabolism because of extended culture periods do not significantly affect the quality of spectra as long as the microbial components are extracted well.

　近年，魚醤油は，日本の各地で独自のものがつくられているが，長い熟成期間と高い塩分濃度が問題となる。食塩は，腐敗細菌の生育抑制のため，添加されるが，同時にプロテアーゼの阻害剤となるため，魚醤油の熟成期間が長くなる。今までに食塩を添加せずに魚醤油に圧力処理することで微生物の生育を抑制し，大幅に熟成期間を短縮できることが報告されている。しかし，圧力処理によるプロテアーゼ活性変化について明らかにされていない。そこで本研究では，熟成に関わるプロテアーゼ活性に及ぼす圧力処理の影響について検討した。また，魚醤油の熟成における圧力処理の有用性を求めるため，食塩添加魚醤油の熟成に及ぼす圧力処理の影響についても検討した。プロテアーゼ活性は，アゾカゼインを基質として，圧力処理による活性変化を求めた。ニジマス魚醤油は，ニジマス重量の20％に相当する食塩を添加し，45℃で調製し，圧力処理は，100 MPaの圧力レベルで調製した。魚醤油の熟成は，pH，可溶性窒素量，アミノ態窒素量や遊離アミノ酸量で検討した。その結果，内臓プロテアーゼは，圧力処理により食塩存在下でも未加圧より活性が上昇し，ニジマス魚醤油は，食塩濃度15％以下であれば，圧力処理は，未加圧より熟成期間を短縮できた。ニジマス魚醤油への圧力処理は，食塩濃度を調整し，魚醤油の熟成期間を短縮させ，従来の方法より高い品質で熟成することができる加工技術と考える。