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.

　We extracted protease from rainbow trout internal organs and investigated the proteases activities under high-pressure condition. The proteases activities were increased by about 1.5-fold under the high-pressure condition compared to the control condition. Furthermore, the protease activity under 100 MPa (15%-NaCl) was approx. 1.7 times higher than the activity of control (0.1 MPa, 15% NaCl). Also, in order to investigate the usefulness of high-pressure treatment (100 MPa for 50 days, 45℃) in the processing of rainbow trout fish sauce, the effect of high-pressure treatment on the fermentation of rainbow trout fish sauce with the addition of NaCl was evaluated. Pressurized rainbow trout fish sauce without the addition of NaCl showed the characteristic of adjusting the NaCl concentration and the shortest fermentation period. Also, pressurized rainbow trout fish sauce with the addition of NaCl showed a shorter fermentation period than the non-pressurized fish sauce, although the NaCl concentration was 15%. In this study, high-pressure treatment for rainbow trout fish sauce is useful technology to adjust the NaCl concentration and shorten the fermentation period at 0－15% NaCl concentration compared to the conventional process.