Metabolite profiling of the fermentation process of "yamahai-ginjo-shikomi" Japanese sake.

Sake is a traditional Japanese alcoholic beverage prepared by multiple parallel fermentation of rice. The fermentation process of "yamahai-ginjo-shikomi" sake is mainly performed by three microbes, Aspergillus oryzae, Saccharomyces cerevisiae, and Lactobacilli; the levels of various metabolites fluctuate during the fermentation of sake. For evaluation of the fermentation process, we monitored the concentration of moderate-sized molecules (m/z: 200-1000) dynamically changed during the fermentation process of "yamahai-ginjo-shikomi" Japanese sake. This analysis revealed that six compounds were the main factors with characteristic differences in the fermentation process. Among the six compounds, four were leucine- or isoleucine-containing peptides and the remaining two were predicted to be small molecules. Quantification of these compounds revealed that their quantities changed during the month of fermentation process. Our metabolomic approach revealed the dynamic changes observed in moderate-sized molecules during the fermentation process of sake, and the factors found in this analysis will be candidate molecules that indicate the progress of "yamahai-ginjo-shikomi" sake fermentation.

Ferritin 2 domain-containing protein found in lacquer tree (Toxicodendron vernicifluum) sap has negative effects on laccase and peroxidase reactions.

Lacquer tree sap, a raw material of traditional paints in East Asia, is hardened through laccase-catalyzed oxidation and the following polymerization of phenolic compound urushiol. In the sap's water-insoluble fraction, we found two plantacyanins and a ferritin 2 domain-containing protein (TvFe2D, a homolog of Arabidopsis AT1G47980 and AT3G62730). The recombinant TvFe2D protein suppressed the accumulation of laccase-catalyzed oxidation products of a model substrate syringaldazine without decreasing oxygen consumption, the second substrate of laccase. The suppression was also observed when another substrate guaiacol or another oxidizing enzyme peroxidase was used. The functional domain of the suppression was the C-terminal half, downstream of the ferritin 2 domain. The results suggest that this protein may be involved in regulating the sap polymerization/hardening. We also discuss the possibility that homologous proteins of TvFe2D in other plants might be involved in the laccase- or peroxidase-mediated polymerization of phenolic compounds, such as lignin and flavonoids.

Fatty acid analysis of triacylglycerols: Preparation of fatty acid methyl esters for gas chromatography.

A method to prepare fatty acid methyl esters was developed for fatty acid analysis of triacylglycerols by gas chromatography (GC). Triacylglycerols were mixed with methanolic CH3ONa in hexane containing a mid-polar solvent for 10 s at room temperature. Under these conditions, trioleoylglycerol was converted to methyl oleate with an average yield of 99.3%. This procedure gave reliable and reproducible data on fatty acid compositions determined by GC.

Identification of pyroglutamyl peptides in Japanese rice wine (sake): presence of hepatoprotective pyroGlu-Leu.

Japanese rice wine, sake, is made from steamed rice, water, and lactic acid by "multiple parallel fermentation" with mold (Aspergillus oryzae) and yeast (Saccharomyces cerevisiae). Nineteen pyroglutamyl peptides were identified in commercially available sake. Among them, pyroGlu-Leu and pyroGlu-Gln were the major constituents. PyroGlu-Leu has been demonstrated to attenuate hepatitis and colitis in animal models. Commercial products (n = 5) contained pyroGlu-Leu at concentrations ranging from 40 to 60 µM (10-15 mg/L). The pyroGlu-Leu content in sake mash increased during the fermentation processes. However, no pyroGlu-Leu was produced by yeast inoculated into preheated mash. Furthermore, addition of (13)C-Leu to the mash did not increase the ratio of pyroGlu-(13)C-Leu to pyroGlu-(12)C-Leu. On the other hand, digestion of steamed rice with A. oryzae proteases increased the pyroGlu-Leu content. These results indicate that pyroGlu-Leu in sake is produced from rice proteins by digestion with A. oryzae proteases.

Improved production of isoamyl acetate by a sake yeast mutant resistant to an isoprenoid analog and its dependence on alcohol acetyltransferase activity, but not on isoamyl alcohol production.

1-Farnesylpyridinium (FPy), an analog of isoprenoid farnesol, strongly inhibited the growth of sake yeast at 120 microM in YPD medium, whereas at 30 microM it reduced cellular production of isoamyl acetate to 20% of the control level despite the absence of inhibitory effect on CO2 evolution. The FPy-resistant mutant A1 was characterized by the high production of flavor compounds represented by a nearly threefold increase in the level of isoamyl acetate in YPD medium in which the level of isoamyl alcohol as its precursor remained almost unchanged. The FPy resistance phenotype of strain A1 was not accompanied by cellular resistance to either the L-leucine analog or L-canavanine, which alters yeast amino acid metabolism in favor of isoamyl alcohol production. Alcohol acetyltransferase (AATase) activity was high in strain A1, which further increased in response to isoamyl alcohol accumulation in medium. Flavor compound production in sake brewing could be improved using strain A1, resulting in a 1.4-fold increase in isoamyl acetate production in spite of a limited production of isoamyl alcohol.

Synergistic fungicidal activity of Cu(2+) and allicin, an allyl sulfur compound from garlic, and its relation to the role of alkyl hydroperoxide reductase 1 as a cell surface defense in Saccharomyces cerevisiae.

Cu(2+) showed a dose-dependent fungicidal activity against Saccharomyces cerevisiae cells, and its lethal effect was extremely enhanced in the presence of allicin, an allyl sulfur compound from garlic. The fungicidal activity of Cu(2+) was unaffected or rather attenuated by other sulfur-containing compounds such as N-acetyl-cysteine, l-cysteine or dithiothreitol. Ca(2+) could absolutely protect against the lethal effect of Cu(2+) itself, but showed no protection against the fungicidal activity of Cu(2+) newly generated in combination with allicin. Cu(2+) accelerated an endogenous generation of reactive oxygen species (ROS) in S. cerevisiae cells at a lethal concentration, but such intracellular oxidative stress induction was not observed during cell death progression upon treatment with Cu(2+) and allicin. A surfactant, sodium N-lauroyl sarcosinate (SLS), enhanced the solubilization of a few proteins including alkyl hydroperoxide reductase 1 (AHP1) in intact cells, accounting for the absence of this protein in the extract from allicin-treated cells. Allicin-treated cells were rendered extremely sensitive to the subsequent Cu(2+) treatment as in the case of SLS-treated cells. Allicin-treated cells and SLS-treated cells similarly showed an increased sensitivity to exogenously added tert-butyl hydroperoxide (t-BOOH), an organic peroxide that is detoxified by the action of AHP1. Our study suggests that allicin influences the mode of cell surface localization or the related function of AHP1 as a defense against phospholipid peroxidation by the external action of Cu(2+).

Purification and properties of betaine aldehyde dehydrogenase from Avena sativa.

Betaine aldehyde dehydrogenase (BADH; EC 1.2.1.8) is the enzyme that catalyzes the second step in the synthesis of the osmoprotectant, glycine betaine. NAD-dependent BADH was purified from Avena sativa shoots by DEAE Sephacel, hydroxyapatite, 5'-AMP Sepharose 4B, Mono Q and TSK-GEL column chromatographies to homogeneity by the criterion of native PAGE, and the properties of BADH were compared with those of aminoaldehyde dehydrogenase purified to homogeneity from A. sativa. The molecular mass estimated by both gel filtration using TSK-GEL column and Sephacryl S-200 was 120 and 115, kDa, respectively. The enzyme is a homodimer with a subunit molecular mass of 61 kDa as shown by SDS-PAGE. The pI value of the enzyme was found to be 6.3. The purified enzyme catalyzed not only the oxidation of betaine aldehyde (BAL), but also that of aminoaldehydes, 3-aminopropionaldehyde (APAL), 4-aminobutyraldehyde (ABAL), and 4-guanidinobutyraldehyde (GBAL). The K(m) values for BAL, APAL, ABAL and GBAL were 5x10(-6), 5.4x10(-7), 2.4x10(-5) and 5x10(-5) M, respectively. APAL showed substrate inhibition at a concentration of 0.1 mM. A fragment of BADH cleaved by V8 protease shared homology with other plant BADHs.

Purification and properties of aminoaldehyde dehydrogenase from Avena sativa.

NAD-dependent aminoaldehyde dehydrogenase (AMADH, EC 1.2.1.-) from Avena shoots was purified by DEAE Sephacel, hydroxyapatite, 5'-AMP Sepharose 4B, Mono Q, and TSK-GEL column chromatographies to homogeneity by the criterion of native PAGE. SDS-PAGE yielded a single band at a molecular mass of 55 kDa. IEF studies showed a band with a p I value of 5.3. In contrast to AMADHs from other species, the TSK-GEL chromatography showed that AvenaAMADH exists as a monomer in the native state. The purified enzyme catalyzed the oxidations of 3-aminopropionaldehyde (APAL), 4-aminobutyraldehyde (ABAL) N-(3-aminopropyl)-4-aminobutyraldehyde (APBAL), and 4-guanidinobutyraldehyde (GBAL), but not of betaine aldehyde or indoleacetaldehyde. The K(m) values for APAL, ABAL, and GBAL were 1.5x10(-6), 2.2x10(-6), and 1.3x10(-5) M, respectively. Although N-terminal amino acid sequence of Avena AMADH could not be determined due to a modification of the amino residue, the sequence of the fragment of AMADH cleaved by V8 protease showed greater similarity to the barley BADH than to the pea AMADH.