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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.

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.

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.

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.

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.

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

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.

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.

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.

Ionic amphiphilic diblock copolymer poly(hydrogenated isoprene)-b-poly(styrenesulfonate) was synthesized by living anionic polymerization. The nanostructure of its monolayer on the water surface, e...

In the supernatant of sokujo-moto, a high level of acid carboxypeptidase (ACP) activity and a large amount of peptides were observed, however, the amount of free amino acids liberated was small. In order to determine why these peptides were not hydrolyzed to any significant degree by the ACP, the properties of the peptides in sokujo-moto were investigated in this study. Peptides were fractionated from sokujo-moto by ion exchange column chromatography. ACP purified from rice-koji (rice overgrown with Aspergillus oryzae) was allowed to react with the peptides, and it was found that they were not hydrolyzed to any significant degree by the enzyme. Gel filtration chromatography was performed to ascertain the molecular size distribution of the peptides in sokujo-moto, and it was revealed that they were of low molecular sizes; molecular size: mainly in the range of 200-400, and chain length: 2-3. ACP purified from rice-koji was also allowed to react with various synthetic peptides, and it was found that ACP of rice-koji could not rapidly hydrolyze low-molecular-size peptides, such as dipeptides or tripeptides. Acid protease (AP) purified from rice-koji released peptides of molecular sizes mainly in the range of 300-600 or above from rice protein under acidic conditions (pH 3.6; the pH of sokujomoto). When AP and ACP were allowed to act at the same time on rice protein, mainly low-molecular-size peptides (molecular sizes mainly in the range of 200-400) were produced. From these results, it was estimated that AP released peptides with molecular sizes mainly in the range of 300-600 or above from rice protein and ACP degraded the relatively higher molecular size peptides among them to lower molecular size peptides; consequently only low-molecular-size peptides with molecular sizes mainly in the range of 200-400 were released in the supernatant of sokujo-moto.

It was found that a large amount of TCA (trichloroacetic acid)-insoluble peptides were liberated into the supernatant of kimoto on the 7th-10th day of mashing. These TCA-insoluble peptides had five polypeptide groups (12, 21, 31, 38, and 55 kDa) on SDS-PAGE (SDS-polyacrylamide gel electrophoresis), and a large amount of high molecular weight peptides, higher than 10,000, were observed upon gel filtration chromatography using TSKgel G2000swxl (Tosoh Co.). Four of these peptides (12, 21, 31, and 38 kDa on SDS-PAGE) appeared specifically in kimoto, and were not detected at all either in sokujo-moto or in the main mash for sake brewing. These TCA-insoluble peptides were fractionated from the supernatant of kimoto on the 9th day, and it was revealed that free amino acids were produced abundantly from them in the presence of the enzyme of rice-koji. Therefore, it was assumed that the peptides are related to the abundant production of free amino acids in kimoto. For the liberation of these TCA-insoluble peptides from rice protein, the enzyme of rice-koji was indispensable. The enzyme liberating the TCA-insoluble peptides from rice protein was purified from rice-koji, and was presumed to be identical with acid protease (AP) of rice-koji. The presence of a high concentration of glucose (higher than 20%) was also indispensable for the liberation of the TCA-insoluble peptides. Furthermore, it was revealed that the peptides were liberated from rice protein under a limited pH of around 4.5.

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 β-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.

It was found that the peptide content of the main mash in the sake brewing process, seeded with kimoto, was higher than in that seeded with sokujo-moto, although the peptide content in kimoto was lower than in sokujo-moto. We investigated the underlying reasons. As a result, we found that the high concentration of free amino acids originating from kimoto decreased the peptide uptake ability of yeast cells in the main mash seeded with kimoto.

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.

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.

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.

Autologous iris pigment epithelial cell transplantation was performed on patients with exudative age-related macular degeneration (AMD). Autologous IPE cell culture was performed using autologous serum after iridectomy in 7 patients with AMD. The cell suspensions (2 approximately 20 x 10(4) cells) were transplanted into the submacular lesion of individuals after removal of neovascular membranes. Subsequent ophthalmological examinations, including best corrected visual acuity and fluorescein or indocyanine green angiography, were performed. In addition, 15 patients with AMD, who underwent removal of neovascular membrane without transplantation, were evaluated as non randomized controls. Varying degrees of atrophy or defects of choriocapillaris and retinal pigment epithelium were observed in all of the patients. No cystoid macular edema or fluorescein leakage was observed after treatment, but window defects were present. No patient had decreased visual acuity. One treated patient developed mild subretinal fibrosis and an other patient developed mild preretinal fibrosis, however no difference was significant when compared with the control. In conclusion, the treatment resulted in no significant improvement in macular function, as compared with the control; however, no rejection or deterioration in visual acuity occurred up to the 13 month follow up.

Recent transplantation studies indicate that subretinal space is not always an immunologically privileged site and non-autologous cells may be rejected in patients with exudative age-related macular degeneration (AMD). We performed autologous iris pigment epithelial (IPE) cell transplantation by cell suspension after autologous IPE cell culture in 8 patients with AMD. These patients were followed without immunosuppression between 1.5 and 8 months and the retinal function was analyzed. No cystoid macular edema or fluorescein leakage was observed. Six of the 8 patients improved visual acuity of more than two lines and the other two patients retained preoperative visual acuity. Five patients had increased visual field sensitivity, one patient retained pretransplantation sensitivity, and one patient showed a gradual decrease in sensitivity (one patient was not examined). Although 2 of the 8 patients showed decreased amplitude of flicker electroretinography (ERG) (about 60 to 70% as that of preoperative level), the average improvement of each amplitude of a single white flash (a wave), photopic, or flicker ERG was 123, 102, and 107%, respectively. No proliferative change in the submacular lesion or vitreous cavity was observed after transplantation. From this functional analysis, transplanted autologous IPE may have, in part, an alternative function in regard to the retinal pigment epithelium in the subretinal space.

Cationic amphiphilic diblock copolymers containing quaternized poly (2-vinylpyridine) chain as a hydrophilic segment (PIp-b-PNMe2VP) were synthesized by living anionic polymerization. By IR measurement, we confirmed the quaternization of the polymer (PIp-b-PNMe2VP), and determined the degree of quaternization by conductometric titration. The surface tension experiment showed that the polymers are non-surface active in nature. The foam formation of the polymer solutions was also investigated with or without added salt. Almost no foam formation behavior was observed without added salt, while a little foam was observed in the presence of 1M NaCl. The critical micelle concentration (cmc) of the diblock copolymers with 3 different chain lengths was measured by the static light scattering method. The cmc values obtained in this study were much lower than the values obtained for anionic non-surface active diblock polymers studied previously. The hydrodynamic radii of the polymer micelle increased slightly in the presence of 1 M NaCl. The transmission electron microscopic images revealed spherical micelles in pure water. In the presence of salt, the cmc values increased as was the case for anionic polymers, which is unlike conventional surfactant systems but consistent with non-surface active anionic block copolymers. The microviscosity of the micelle core was evaluated using Coumarin-153 as a fluorescent anisotropy probe using steady-sate fluorescence depolarization. Non-surface activity has been proved to be universal for ionic amphiphilic block copolymers both for anionic and cationic. Hence, the origin of non-surface activity is not the charged state of water surface itself, but should be an image charge repulsion at the air/water interface.

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(-)].