The expansion of the in-house database is indispensable for improving the accuracy of microbial identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). To this end, it is important to ensure the high quality of the data to be registered in order to obtain accurate identification results. In this study, the effects of the formation of bacterial spores and yeast ascospores on mass spectra were investigated. A main spectrum (MSP, a spectrum that becomes a standard for registration) was prepared using spore-forming bacteria and ascospore-forming yeast cells cultured by different methods and for different incubation times. The score values indicating the similarities of the spectra were compared, and the culture conditions and sample preparation methods that can obtain accurate species identification results, even in the case of spore and ascospore formation, were determined. First, Bacillus subtilis and Bacillus cereus were investigated as spore-forming bacteria, and the spore formation was greatly affected by the oxygen supply amount and medium composition. When the rates of spore formation differed greatly, the mass spectra obtained also showed considerable differences. When cultured on a standard agar medium, by using the MSP obtained from bacterial cells on the third day of culture for both B. cereus and B. subtilis, a high score value was obtained, indicating accurate bacterial species identification results. This high score value was achieved even for the mass spectral data obtained from the bacterial cells on the first day without spore formation and on the fifth day when spore formation was observed to some extent. However, when ascospore-forming yeast Pichia manshurica was cultured on a potato dextrose agar (PDA) plate medium, the ascospore formation rate differed with the strains. In the ethanol-formic acid extraction method, which is the bacterial cell extraction method recommended by the manufacturer, the bacterial cell extraction rate decreased because of ascospore formation. Through cell crushing using beads conducted to increase the cell protein extraction efficiency, it was shown that species identification is possible even upon ascospore formation.

　Edible canna (Canna edulis, C. discolor) is a perennial herb of the ginger family Cannaceae and is a useful plant resource cultivated on a small scale in tropical and subtropical regions. However, no studies of the components and functionality have reported on edible canna stems and leaves. Therefore, in this study, we analyzed the nutritional characteristics of edible canna leaves before and after lactic acid fermentation and the functionality of their 80% methanol extracts (N-MEECL and F-MEECL) in vitro. Edible canna leaves contained large amounts of polyphenols and rich in proteins. Lactic acid fermentation of the leaves reduced the total polyphenol content and antioxidant capacity. An 80% methanol extract of nonfermented edible canna leaves (N-MEECL) contained rutin, chlorogenic acid, quercetin, piceatannol, and neochlorogenic acid. On the contrary, in the 80% methanol extract of fermented edible canna leaves (F-MEECL), in which the leaves were fermented by lactic acid, the four polyphenols, except piceatannol, were significantly reduced. N-MEECL and F-MEECL contained free amino acids. After fermentation, the amounts of amino acids slightly increased. On the contrary, hydrolysis of N-MEECL and F-MEECL with hydrochloric acid increased the amounts of amino acids. N-MEECL and F-MEECL inhibited the degranulation of Rat basophilic leukemia (RBL-2H3) cells. The effects of F-MEECL on degranulation inhibition were significantly larger compared with those of N-MEECL, indicating that the degranulation inhibitory effect was enhanced by fermentation. The five polyphenols in N-MEECL showed stronger degranulation inhibitory effects on RBL-2H3 cells than on N-MEECL. However, none of the four polyphenols other than piceatannol were present after fermentation. The compounds in F-MEECL that cause degranulation were considered to be distinct from the four polyphenols in N-MEECL, aside from piceatannol. Moreover, β-hexosaminidase release inhibitory activity test of RBL-2H3 cells degranulation by the 21 amino acids and imino acid samples was determined. However, none of the effects on degranulation inhibition were larger compared with those of the five respective polyphenols. Thus, it is unlikely that the enhanced degranulation inhibitory effects of F-MEECL are caused by the amino acids. Our findings suggest that lactic acid fermentation of edible canna leaves induces the formation of compounds that inhibit RBL-2H3 cells degranulation.

　The total polyphenol content and H-ORAC values of 15 traditional Okinawan vegetables were determined and compared with those of spinach. Nishiyomogi, Hosobawadan, Botanboufu, and Suizenjina exhibited higher total polyphenol contents and H-ORAC values than those of spinach. The total polyphenol content and H-ORAC values exhibited a high correlation (r = 0.986, p < 0.01), suggesting that polyphenols are the major contributors to the antioxidant activity of Okinawan traditional vegetables in water systems. The effects of storage temperature on the H-ORAC values and total polyphenol content of six traditional Okinawan vegetables were also investigated. The behavior of the total polyphenol content and H-ORAC values during storage differed among the vegetables. In Nishiyomogi and Suizenjina, the total polyphenol content decreased on the fifth day of storage at 25 ℃. In contrast, for Botanboufu, significant increases in the total polyphenol content and H-ORAC values were observed after the second day of storage at 25 ℃ and on the fifth day of storage at 15 and 25 ℃ compared to the first day of storage. No increase or decrease in the total polyphenol content or H-ORAC values was observed in the other vegetables during storage.