Effect of S-adenosyl-methionine accumulation on hineka odor in sake brewed with a non-Kyokai yeast.

Hineka is a type of off-flavor of sake and is attributed to the presence of several compounds, including a major one called dimethyl trisulfide (DMTS). The production of the main precursor of DMTS involves yeast methionine salvage pathway. The DMTS-producing potential (DMTS-pp) of sake brewed using the Km67 strain, a non-Kyokai sake yeast, is lower than that of sake brewed using Kyokai yeast; however, the detailed mechanism is unclear. We focused on S-adenosyl-methionine (SAM) and aimed to elucidate the mechanism that prevents DMTS production in sake brewed using the Km67 strain. We revealed that SAM is involved in DMTS production in sake, and that the conversion of SAM to the DMTS precursor occurs through an enzymatic reaction rather than a chemical reaction. Based on previous reports on ADO1 and MDE1 genes, sake brewing tests were performed using the Km67 Δmde1, Δado1, and Δmde1Δado1 strains. A comparison of the SAM content of pressed sake cakes and DMTS-pp of sake produced using the Km67 Δado1 strain showed an increase in both SAM content and DMTS-pp compared to those produced using the parent strain. However, the Km67 Δmde1Δado1 strain showed little increase in DMTS-pp compared to the Km67 Δmde1 strain, despite an increase in SAM content. These results suggest that SAM accumulation in yeast plays a role in the production of DMTS in sake through the methionine salvage pathway. Moreover, the low SAM-accumulation characteristic of the Km67 strain contributes to low DMTS production in sake.

Quantitative stability of the folates highly accumulated in a non-Kyokai sake yeast.

Pressed sake cake, a by-product of sake brewing, is a rich dietary source of folates, which are important vitamins for humans. However, considerable losses of folates occur during storage and cooking. We have previously reported that Km67, the house sake yeast strain of Kiku-masamune sake brewery, can accumulate high folate levels. In this study, we found that the folate content of pressed sake cakes produced with Km67 remained at approximately their maximum level after the fermentation activity stopped. To elucidate the mechanisms of high folate accumulation in Km67, we analyzed the expression of 23 folate-metabolizing genes. The expression of ABZ1 and FOL3 was almost always higher in Km67 than in Kyokai no. 701 yeast (K701), which suggested that enhanced expression of the genes involved in folate biosynthesis was a mechanism of high folate accumulation in Km67. We found that the folates of Km67 pressed sake cakes were quantitatively stable at 4°C under refrigerated storage conditions. In addition, the homocysteine content of Km67 pressed sake cakes was almost always higher than that of K701 pressed sake cakes. This result suggests that a reason for high folate accumulation in Km67 yeast is the need to reduce the intracellular concentration of homocysteine. Our results provide biologically meaningful information on folate metabolism in yeast.

Profiling of volatile compounds in Japanese sake stored in sherry casks using solid phase microextraction/gas chromatography/mass spectrometry analysis.

The combination of solid phase microextraction (SPME) and gas chromatograph-mass spectrometer (GC-MS) is frequently used for comprehensive analysis of aroma components in foods because it can be used to easily analyze volatile components, allowing saving of the amount of solvent used. In this study, SPME-GC-MS analysis of sake samples before and after sherry cask storage was performed to investigate the special flavor derived from sherry cask storage. A GC column with polyethylene glycol as the stationary phase, which is the first choice for volatile component analysis, was used. However, the peak of the acid having a carbonyl group was tailed due to its bond with the hydroxyl group of the stationary phase. In the analysis of sake samples, a large and tailing peak derived from the large amount of fatty acids in Japanese sake was observed. Additionally, it was not possible to analyze other co-eluting components. To overcome this problem, a novel extraction condition was examined using SPME and tris (hydroxymethyl) aminomethane (Tris). By adding Tris solution to sake, the fatty acid peak was removed successfully, thereby facilitating analysis of the peaks of compounds co-eluting with fatty acids and comprehensive analysis of the aroma components in sake. Furthermore, a comparative analysis of sake before and after storage in sherry cask showed that levels of fatty alcohols, organic acid esters, fatty acid esters, and terpenes increased significantly after storage in sherry cask, suggesting that these ingredients might constitute the special flavor of sherry cask-stored sake.

Profiling of taste-related compounds during the fermentation of Japanese sake brewed with or without a traditional seed mash (kimoto).

Japanese sake production involves three processes: rice koji fermentation, seed mash fermentation, and main mash fermentation. Traditional seed mash (kimoto) production utilizes natural lactic acid produced by lactic acid bacteria for pure cultures of only sake yeast, preventing the growth of wild yeast and other unwanted bacteria. Recently, because kimoto production requires substantial time and labor, sake yeast mass-cultured in usual liquid medium has been used as a seed mash alternative. Sake quality is highly similar to that of kimoto, suggesting that they share similar component profiles. However, comparative component analyses of sake brewed with kimoto and sake brewed with cultured yeast are lacking. In this study, a time-course analysis of hydrophilic compounds in the main mash brewed with kimoto and with cultured yeast as well as a sensory evaluation of the products were performed. As a result, differences in various compounds and in umami taste level between sake brewed with kimoto and cultured yeast were detected. This is the first comparative analysis of changes in the component profile during sake main mash brewing using kimoto seed mash and cultured sake yeast; our results clarify the effects of kimoto seed mash on main mash brewing and sake quality.

Mechanism of high folate accumulation in a sake yeast other than Kyokai yeasts.

Folates are important vitamins in human nutrition. Pressed sake cake, a brewing by-product of sake, is a rich dietary source of folates derived from sake yeast (Saccharomyces cerevisiae). The National Research Institute of Brewing investigated 106 samples of pressed sake cake and revealed that three samples containing large amounts of folates were produced by Km67 yeast derived from the house sake yeast strain of Kiku-Masamune sake brewery. In this study, we performed sake brewing tests using Km67 and Kyokai no. 7 group strains and confirmed that Km67 yeast contributed to the production of pressed sake cake containing large amounts of folates. To elucidate the mechanisms of high folate accumulation in Km67, we performed whole-genome sequence analysis in Km67 and then screened 10 folate-metabolizing genes showing different sequences in Km67 and K7 strains. By folate analysis of each gene-disrupted strain derived from strain BY4743, we also selected four genes having significant effects on folate content in yeast from 10 candidate genes. Folate analysis of gene-disrupted yeast strains complemented with either Km67-type genes or K7-type genes revealed that the Km67-type HMT1 gene was related to high folate accumulation not only in laboratory yeast but also in sake yeast. In this gene, Leu63Phe was present in the methyltransferase motif I of Hmt1p, which was essential for the methyltransferase activity of Hmt1p. Our results and previous reports suggested that the methyltransferase activity of Km67-Hmt1p was higher than that of K7-Hmt1p, leading to enhanced production and high accumulation of folates in Km67 yeast.

Characteristic features of the unique house sake yeast strain Saccharomyces cerevisiae Km67 used for industrial sake brewing.

For several decades, almost all sake has been brewed with sake yeast Saccharomyces cerevisiae Kyokai no. 7 (K7) group strains. Although the widespread use of these strains has contributed to sake quality improvement, it may have lessened the diversity of sake gustatory properties brought about by house sake yeast (indigenous yeast of sake brewery). Sake yeast S. cerevisiae strain Km67 derives from the house yeast strain of Kiku-masamune Sake Brewing Co., Ltd., and it has been playing a central role in industrial sake brewing for decades. By using DNA sequencing, we revealed that strain Km67 does not possess specific loss-of-function mutations of stress response-related genes, which are characteristic of K7 group strains. Km67 had higher stress tolerance than K7 group strains likely because of the more efficient function of the stress response and heat shock elements in this strain. Sensory evaluation and taste sensor analysis demonstrated that sake brewed with Km67 had characteristically thicker body than sake brewed with K7 group strains. Chemical analysis suggested that unique sensory properties of the sake brewed with Km67 were due to high citramalic acid concentration. Taken together, these results revealed that strain Km67 differs from K7 group strains by genetic background and confers unique chemical composition and taste qualities upon sake it generates. It is expected that sake quality and gustatory properties will be diversified by utilizing house yeast such as strain Km67.

Metabolic switching of sake yeast by kimoto lactic acid bacteria through the [GAR+] non-genetic element.

In traditional kimoto-type sake production, cells of Saccharomyces cerevisiae sake yeast are grown in a starter mash generated by lactate fermentation by lactic acid bacteria (LAB) such as Leuconostoc mesenteroides and Lactobacillus sakei. However, the microbial interactions between sake yeast and kimoto LAB have not been well analyzed. Since the formation of a prion-like element (designated [GAR+]) in yeast cells is promoted by bacteria, we here examined the associated phenotype (i.e., increased glucosamine resistance) in sake yeast strains K701 (a representative sake strain) and Km67 (a strain isolated from kimoto-type sake mash). Approximately 0.5% of K701 and Km67 cells, as well as 0.2% of laboratory strain X2180 cells, exhibited increased glucosamine resistance under pure culture conditions, and the frequency of this metabolic switching was further enhanced by coculture with kimoto LAB. The LAB-promoted emergence of the glucosamine-resistant cells was the most prominent in Km67, suggesting that this strain possesses an advanced mechanism for response to LAB. While the glucosamine-resistant clones of X2180 and K701 exhibited lower rates of alcoholic fermentation under high-glucose conditions than did the respective naive strains, glucosamine resistance did not severely affect alcoholic fermentation in Km67. The population of dead cells after alcoholic fermentation was decreased in the glucosamine-resistant clones of X2180, K701, and Km67. These results suggested that the formation of [GAR+] in Km67 may be beneficial in kimoto-type sake making, since [GAR+] may increase cell viability in the sake starter mash without impairing alcoholic fermentation performance.

Antifungal properties of Japanese cedar essential oil from waste wood chips made from used sake barrels.

In this study, we prepared essential oil (EO) from waste wood chips made from used sake barrels (USBs) of Japanese cedar (i.e., EO-USB) by steam distillation. We found that EO-USB and three commercially purchased EOs derived from xylem tissue of Japanese woods, such as Japanese cedar (Cryptomeria japonica), Japanese cypress (Chamaecyparis obtusa) and false arborvitae (Thujopsis dolabrata), suppressed fungal growth activity against Trichophyton rubrum, which is the cause of tinea disease. The magnitude of the suppressive effects of the EOs ranked as follows: T. dolabrata > USB = C. japonica > C. obtusa. These EOs also inhibited the activity of DNA polymerase in an extract from T. rubrum mycelia with the following ranking: T. dolabrata > USB = C. japonica > C. obtusa. In addition, 50 µg/ml of EO-USB showed antifungal properties, killing T. rubrum mycelia at 27-42˚C in 20 min. By gas chromatography/mass spectrometry analysis, the main sesquiterpenes in EO-USB were δ-cadinene (25.94%) and epi-cubenol (11.55%), and the composition of EO-USB was approximately the same as that of EO-C. japonica. Three prepared sesquiterpenes, δ-cadinene, epi-cubenol and ß-eudesmol, inhibited the fungal growth and DNA polymerase activities of T. rubrum, and epi-cubenol showed the strongest inhibition among the compounds tested. These sesquiterpenes had no inhibitory effects on the activities of other DNA metabolic enzymes, such as DNA topoisomerase II, IMP dehydrogenase, polynucleotide kinase and deoxyribonuclease from T. rubrum. Taken together, these results suggest that EO-USB containing epi-cubenol may be useful for its anti-tinea disease properties, which are based on DNA polymerase inhibition.

Ionic multilayers at the free surface of an ionic liquid, trioctylmethylammonium bis(nonafluorobutanesulfonyl)amide, probed by x-ray reflectivity measurements.

The presence of ionic multilayers at the free surface of an ionic liquid, trioctylmethylammonium bis(nonafluorobutanesulfonyl)amide ([TOMA(+)][C(4)C(4)N(-)]), extending into the bulk from the surface to the depth of approximately 60 A has been probed by x-ray reflectivity measurements. The reflectivity versus momentum transfer (Q) plot shows a broad peak at Q approximately 0.4 A(-1), implying the presence of ionic layers at the [TOMA(+)][C(4)C(4)N(-)] surface. The analysis using model fittings revealed that at least four layers are formed with the interlayer distance of 16 A. TOMA(+) and C(4)C(4)N(-) are suggested not to be segregated as alternating cationic and anionic layers at the [TOMA(+)][C(4)C(4)N(-)] surface. It is likely that the detection of the ionic multilayers with x-ray reflectivity has been realized by virtue of the greater size of TOMA(+) and C(4)C(4)N(-) and the high critical temperature of [TOMA(+)][C(4)C(4)N(-)].

Mast cells play a critical role in the pathogenesis of viral myocarditis.

BACKGROUND: Mast cells are powerful producers of multiple cytokines and chemical mediators playing a pivotal role in the pathogenesis of various cardiovascular diseases. We examined the role of mast cells in murine models of heart failure due to viral myocarditis, using 2 strains of mast cell-deficient mice. METHODS AND RESULTS: Two strains of mast cell-deficient mice, WBB6F1-Kit(W)/Kit(W-v) (W/W(V)) and WCB6F1-Kitl(Sl)/Kitl(Sl-d) (Sl/Sl(d)), were inoculated with 10 plaque-forming units of the encephalomyocarditis virus intraperitoneally. On day 14 after inoculation, survival of W/W(V) mice was significantly higher than that of their control littermates (77% versus 31%; P=0.03; n=13). On histological examination on day 7, myocardial necrosis and cellular infiltration were significantly less pronounced in W/W(V) and Sl/Sl(d) mice than in their control littermates (area of infiltration, 7.6+/-3.5% versus 29.3+/-15.6%; P=0.002; area of necrosis, 7.6+/-3.5% versus 30.0+/-17.2%; P=0.003; n=10). Histological examination showed more severe changes in mast cell-reconstituted than in -nonreconstituted W/W(V) and Sl/Sl(d) mice. The gene expressions of mast cell proteases were upregulated in the acute phase of viral myocarditis and rose further in the subacute phase of heart failure. Their activation coincided with the development of myocardial necrosis and fibrosis and correlated with the upregulation of gene expression of matrix metalloproteinase-9. The histamine H1-receptor antagonist bepotastine improved encephalomyocarditis viral myocarditis. CONCLUSIONS: These observations suggest that mast cells participate in the acute inflammatory reaction and the onset of ventricular remodeling associated with acute viral myocarditis and that the inhibition of their function may be therapeutic in this disease.

Isolation of copper-tolerant mutants of sake yeast with defective peptide uptake.

Mutants of Saccharomyces cerevisiae that have a decreased peptide uptake ability were isolated from sake yeast. A copper medium containing copper sulfate, histidylleucine and sodium lactate as a carbon source was used for the isolation. One mutant showed a decreased peptide uptake ability due to PTR2 transcriptional repression, but for other mutants, the decrease was not due to the transcriptional repression. The peptide concentration in sake brewed with these mutants increased to about 1.5-fold that of sake brewed with the parental strain. The angiotensin I-converting enzyme (ACE) inhibitory activity of sake brewed with the mutant was about 3.6-fold that of sake brewed with the parental strain.

Effect of amino acids on peptide transport in sake yeast.

The PTR 2 gene of Saccharomyces cerevisiae encodes a major peptide permease responsible for the uptake of low-molecular-weight peptides consisting of two or three amino acids. We show that the PTR 2 gene of sake yeast encodes a major peptide permease in the main mash of sake brewing. The peptide uptake activity in sake yeast is decreased by the addition of certain types of amino acids, particularly asparagine, serine and lysine. Northern blot analysis suggested that asparagine and serine repress the expression of the PTR 2 gene, but lysine decreases the peptide transport activity without repressing PTR 2 gene transcription. The deletion analysis of the PTR 2 promoter region confirmed these suggestions and revealed that the cis-element involved in the regulation of the PTR 2 gene by amino acids is located in the region from residue --400 to the start codon.

High expression of unsaturated fatty acid synthesis gene OLE 1 in sake yeasts.

Gene Filters and Northern blot analysis revealed that the sake yeast strain Kyokai no. 7 (K 7) showed a higher expression level of OLE 1, which encodes a Delta-9 fatty acid desaturase gene, compared with the laboratory yeast strain X 2180-1A. Other sake yeasts also showed a high expression level of OLE 1. Unsaturated fatty acid concentrations in strain K 7 are higher than that in strain X 2180-1A, suggesting that the higher expression level of OLE 1 in sake yeasts increases the unsaturated fatty acid content in the cell membrane. Experiments using OLE 1 promoter:lacZ fusion reporter genes revealed that both the cis element of the OLE 1 promoter and trans factors are involved in the increased expression of OLE 1 in sake yeasts.

Combined measurements of cardiac troponin T and N-terminal pro-brain natriuretic peptide in patients with heart failure.

BACKGROUND: To examine the prognostic contribution of combined cardiac troponin T (cTnT) and N-terminal pro-brain natriuretic peptide (NT-proBNP) in patients with heart failure (CHF) in the absence of acute coronary syndrome. METHODS AND RESULTS: Between July 2001 and March 2002, 71 consecutive patients (mean age = 68.4+/-1.4 years, 37 men), hospitalised for heart failure, were studied during hospitalisation and follow up until December 2002. Serum cTnT and NT-proBNP were measured on admission. Actuarial rates of adverse cardiac events, including sudden or CHF death, or rehospitalisation for CHF during follow up were compared with patients grouped according to initial serum cTnT and/or NT-proBNP concentrations. The adverse cardiac event-free rate among the 20 patients with cTnT > or 0.01 ng/ml was significantly lower than the 51 patients with cTnT <0.01 ng/ml (P<0.05). Similarly, the adverse cardiac event-free rate among the 36 patients with NT-proBNP > or =1,357 pg/ml (median) was significantly lower than the 35 patients with NT-proBNP <1,357 pg/ml (P<0.01). The 16 patients with high concentrations of both cTnT and NT-proBNP had a lower adverse cardiac event-free rate than the 31 patients with low cTnT and low NT-proBNP upon commencement of the study (P<0.005). CONCLUSION: Measurements of serum cTnT and NT-proBNP were reliable prognostic markers of adverse cardiac event in patients with CHF.

A hap1 mutation in a laboratory strain of Saccharomyces cerevisiae results in decreased expression of ergosterol-related genes and cellular ergosterol content compared to sake yeast.

DNA microarray and Northern blot analysis revealed that a sake yeast strain Kyokai no. 7 (K7) showed higher expression of genes encoding proteins involved in ergosterol biosynthesis than a laboratory yeast strain X2180. We hypothesized that these differences in expression levels were caused by a defect of a transcriptional factor Hap1, because strain X2180 contained a Ty1 insertion mutation in the HAP1 gene. To confirm this, we constructed a strain X2180 derivative (strain HX) that contained the wild-type HAP1 genes originating from strain K7. The expression levels of ergosterol-related genes and cellular ergosterol content in strain HX were higher than those in strain X2180 and were almost comparable to those in strain K7. These results suggest that the differences in the expression levels of ergosterol-related genes and ergosterol content between strains K7 and X2180 were largely caused by the hap1 mutation in strain X2180. Involvement of the mutated Hap1 in the differential gene expression between strain K7 and strain X2180 was further confirmed by a lacZ reporter assay of HMG1, one of the Hap1-regulated genes. We also revealed that the HMG1 promoter region between -500 and -376 was important in the transcriptional activation by Hap1.

Increased ethyl caproate production by inositol limitation in Saccharomyces cerevisiae.

Sake mash was prepared using rice with polishing ratios of 70%, 80%, 90% and 98%. At a polishing ratio of 70%, the highest amounts of ethyl caproate were produced in sake mash, and supplementation of inositol caused a decrease in ethyl caproate production. However, at a polishing ratio of over 90%, supplementation of inositol had no effect on ethyl caproate production. These results suggest that the use of rice with a polishing ratio of 70% results in increased ethyl caproate content in sake when limiting the inositol available to yeast. The reduction in ethyl caproate production following inositol addition was due to the decrease in its enzymatic substrate caproic acid, because the concentrations of middle chain fatty acids (MCFA), caproic acid, caprylic acid and capric acid in sake were lowered by inositol. A disruptant of the OPI1 gene, an inositol/choline-mediated negative regulatory gene, produced higher amounts of MCFA than the control strain both in the static culture and in sake mash when a sufficient amount of inositol was supplemented. Therefore, the enhancement of MCFA biosynthesis by inositol limitation was thought to be caused not by a posttranscriptional event, but predominantly by transcriptional enhancement of fatty acid biosynthetic genes. The overexpression of FAS1 considerably stimulated MCFA formation while that of ASC2, ACC1 and FAS2 genes was not effective. Co-overexpression of FAS1 and FAS2 resulted in a maximal stimulation of MCFA formation and substantially abolished the inhibitory effect of inositol on MCFA formation. These results suggest that the repression of FAS1 gene expression by inositol results in the decrease in MCFA formation. Therefore, it is presumed that the removal of inositol by polishing the rice used in sake brewing, increases the production of ethyl esters of MCFA, since high-level production of MCFA is achieved by the derepression of FAS1 transcription.

Increased alcohol acetyltransferase activity by inositol limitation in Saccharomyces cerevisiae in sake mash.

Sake mash was prepared using rice with polishing ratios of 70%, 80%, 90% and 98%. At a polishing ratio of 70%, the highest isoamyl acetate/isoamyl alcohol (E/A) ratio in sake was obtained, and inositol addition caused a decrease in E/A ratio. In several strains tested, inositol addition to the mash decreased isoamyl acetate content and E/A ratio in sake Inositol addition significantly decreased alcohol acetyltransferase (AATase) activity which is responsible for the synthesis of acetate esters from alcohols and acetyl coenzyme A. The results of Northern blot analysis and disruption of the OPII gene, an inositol/choline-mediated negative regulatory gene, showed that the decrease in AATase activity following inositol addition is not due to a transcriptional event. Inositol addition increased phosphatidylinositol (PI) content 3-fold in sake mash yeast cells, while it had no effect on phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidyl-serine (PS) contents. When cell-free extracts prepared from sake mash yeast cells were treated with chloroform or phospholipase C to remove PI, no difference in AATase activity in sake mash between with (Ino+) and without (Ino-) inositol addition was observed. PI prepared from sake mash yeast cells inhibited AATase activity more strongly than PC and PE. Furthermore, when PI, PC, PE and PS at a ratio (1.0:1.28:0.70:0.09) corresponding to the phospholipid composition of Ino+ sake mash yeast cells were added to a reaction mixture, the AATase activity decreased to 26-55% that of yeast cells from the Ino- mash with a phospholipid composition of 0.34:1.28:0.7:0.09. Approximately all of the PI was recovered in the ammonium sulfate precipitate of the cell-free extract, while only half of the PC and PE was recovered. The acidic phospholipid, phosphatidylglycerol, as well as PI inhibited AATase activity more strongly than PC, despite its having the same fatty acid composition as PC. These results suggest that the strong inhibition of AATase activity by PI is due to its high adsorptive capacity for the AATase protein. Therefore, rice polishing can remove inositol from rice leading to an increase in AATase activity, and resulting in a high E/A ratio in sake.