2010 Research results

1. Speculation on the ratio of materials derived from C4 plants in Sake and Shochu

Author: Sachie HORII, Tomokazu HASHIGUCHI, Yukari IGI, Hanae IZU, Shigetoshi SUDO
Abstract: We determined the carbon isotope ratio (δ¹³C) of ethanol in Sake and Shochu by isotope ratio mass spectrometry (IR-MS). In ethanol condensation, the vacuum condensation had a lower carbon isotope discrimination ratio than simple distillation. Moreover, vacuum condensation was more effective in comparison to simple distillation. In Sake, there was a positive relationship between the δ¹³C value and the ratio of ethanol of added brewing -alcohol in the ethanol of Sake. It seems that we could speculate on the ratio of added brewing-alcohol. In an analysis of commercial Sake, the δ¹³C value among Junmai-shu, Honjozo-shu, and Futsu-shu was significantly different. In Shochu, a relationship between the δ¹³C value and the ratio of ko-rui in konwa Shochu was observed. It is suggested that the mixing ratio could be presumed by the δ¹³C value. The analysis of commercial Shochu supported the possibility of such speculation.
Source: J. Brew. Soc. Japan, 106(1), 45-49 (2011)

2. Enhancement of the Initial Rate of Ethanol Fermentation Due to Dysfunction of Yeast Stress Response Components Msn2p and/or Msn4p

Author: Daisuke WATANABE, Hong WU, Chiemi NOGUCHI, Yan ZHOU, Takeshi AKAO, Hitoshi SHIMOI
Abstract: Sake yeasts (strains of Saccharomyces cerevisiae) produce high concentrations of ethanol in sake fermentation. To investigate the molecular mechanisms underlying this brewing property, we compared gene expression of sake and laboratory yeasts in sake mash. DNA microarray and reporter gene analyses revealed defects of sake yeasts in environmental stress responses mediated by transcription factors Msn2p and/or Msn4p (Msn2/4p) and stress response elements (STRE). Furthermore, we found that dysfunction of MSN2 and/or MSN4 contributes to the higher initial rate of ethanol fermentation in both sake and laboratory yeasts. These results provide novel insights into yeast stress responses as major impediments of effective ethanol fermentation.
Source: Applied and Environmental Microbiology, 77, 934-941 (2011)

3. Purification and characterization of intracellular lipase from the polyunsaturated fatty acid-producing fungus Mortierella alliacea.

Author: W. Jermsuntiea, T. Aki, R. Toyoura, K. Iwashita, S. Kawamoto, K. Ono.
Abstract: Previous studies on an arachidonic acid-producing fungus, Mortierella alliacea YN-15, suggested that its intracellular lipase plays an important role in the metabolism of exogenous and storage lipids. The lipase purified in this study through acetone precipitation and three-step chromatography was estimated to be about 11 kDa in size by SDS-PAGE and mass spectrometry, and it tended to form large aggregates in aqueous solution. The purified lipase retained its activity over wide ranges of pH (2-12) and temperature (20-80 °C). Its activity was enhanced by the Ca(2+) ion and reduced by some heavy metal ions, such as Zn(2+) and Hg(2+), and diethylpyrocarbonate. Among the various substrates tested, monoacylglycerols containing long-chain unsaturated fatty acids and phosphatidylcholine were preferentially hydrolyzed over triacylglycerols and fatty acid methyl esters. The lipase strongly hydrolyzed the sn-1/3 ester bonds and weakly hydrolyzed the sn-2 ester bonds of triolein, and it also catalyzed the acylglycerol synthesis reaction in a solvent-free two-phase system. The results indicate that triacylglycerol may be formed via 2-monoacylglycerol. Thus, the highly stable M. alliacea lipase may be useful for the synthesis of structured lipids, particularly acylglycerols containing functional unsaturated fatty acids at the sn-2 position.
Source: New Biotechnology. 2011 Feb 28;28(2):158-64. Epub 2010 Oct 12

4. Abscisic acid stimulated ripening and gene expressions in berry skins of the Cabernet Sauvignon grape

Author: Kazuya KOYAMA, Keiko SADAMATSU, Nami GOTO
Abstract: We investigated the effect of exogenous abscisic acid (ABA) application on the transcriptome as well as the phenolic profiles in the skins of Vitis vinifera cv. Cabernet Sauvignon grape berries grown on the vine and cultured in vitro. ABA application rapidly induced the accumulation of anthocyanin and flavonol. Correlatively, the structural genes in the phenylpropanoid and flavonoid pathways, their transcriptional regulators, as well as genes considered to be involved in the acylation and transport of anthocyanin into the vacuole, were upregulated by ABA treatment. The Genechip analysis showed that the ABA treatment significantly up- or downregulated a total of 345 and 1,482 transcripts in the skins of berries grown on the vine and cultured in vitro, respectively. Exogenous ABA modulated the transcripts associated with osmotic responses, stress responses, cell wall modification, auxin and ethylene metabolism and responses, in addition to the induction of anthocyanin biosynthetic genes, and reduced those associated with photosynthesis; approximately half of these transcripts were identical to the previously reported ripening-specific genes.
Source: Functional & Integrative Genomics、10、367-381、(2010)

5. Anxiolytic Effect of Drinking Japanese Sake Assessed by the Elevated Plus-Maze Test in Mice

Author: Hanae IZU, Yasue YAMADA, Kuniyasu GOTO, Shigetoshi SUDO
Abstract: The anxiolytic effect of Japanese sake (futsu-shu and ginjo-shu) ingestion was confirmed in mice. Mice were orally given ethanol, futsu-shu, or ginjo-shu (1.2 g of ethanol/kg BW dosed twice), then tested for the elevated plus-maze. Futsu-shu showed a higher anxiolytic effect than that ethanol, but there was no significant difference. Ginjo-shu had a significantly higher anxiolytic effect than futsu-shu. The effects of isoamyl acetate and ethyl caproate, characteristic flavor components of ginjo-shu, were tested with these flavors added to futsu-shu to reveal the components involved in the higher anxiolytic effect of ginjo-shu. As a result, both enhanced the anxyolitic effect. Moreover, it was found that isoamyl acetate, ethyl caproate, and isoamyl alcohol within a range of the concentration contained in Japanese sake have no inhibitory effect on the ethanol metabolism by alcohol dehydrogenase.
Source: J. Brew. Soc. Japan, 105, 664-671 (2010)

6. The Concentration of Ethyl Carbamate in Commercial Ume (Prunus mume) Liqueur Products and a Method of Reducing It

Author: Tomokazu HASHIGUCHI, Sachie HORII, Hanae IZU, and Shigetoshi SUDO
Abstract: The average ethyl carbamate concentration across 38 ume liqueur products was 0.12 mg/l (0.02-0.33 mg/l). When ume liqueurs were produced under deoxygenated conditions created using an oxygen absorber, the ethyl carbamate concentration was reduced by up to 47% as compared with the control group, probably due mainly to a reduction in free hydrogen cyanide.
Source: Biosci. Biotechnol. Biochem., 74(10), 2060-2066, 2010

7. Simple metabolite extraction method for metabolome analysis of the solid-state fermentation of Aspergillus oryzae

Author: Masafumi Tokuoka, Nobuko Sawamura, Ken Kobayashi, Akihiro Mizuno
Abstract: Solid-state culture of microorganisms is an important style of culture both in the traditional food industry and in the modern fermentation industry. We propose here a simple method for metabolite extraction from the solid-state fermentation of a filamentous fungus, Aspergillus oryzae, which is known as rice-koji. To evaluate the efficiency of metabolite extraction, liquid chromatography-mass spectrometry (LC/MS) was used for simultaneous detection of a wide range of metabolites including amino acids, organic acids and vitamins, which are of interest in rice-koji making. Among eleven different metabolite extraction methods tested, we found that extraction using acetonitrile/water (50:50), followed by boiling at 70 oC for 5 min, was advantageous in terms of both extraction yield and metabolism quenching. We consider that our method provides a technical basis for metabolome analysis of a solid-state fermentation.
Source: Journal of Biosceinece and Bioengineering、Vol. 110, No. 6, p665-669 (2010)

8. Contribution of ethanol-tolerant xylanase G2 from Aspergillus oryzae on Japanese

Author: Yuichiro Sato, Hisashi Fukuda, Yan Zhou, and Shigeaki Mikami
Abstract: We purified three xylanase isozymes (XynF1, XynF3 and XynG2) from a solid-state Aspergillus oryzae RIB128 culture using chromatography. The results of our sake-brewing experiment, in which we used exogenously supplemented enzymes,revealed that only XynG2 improved the alcohol yield and the material utilization. The alcohol yield of the XynG2 batch displayed an increase of 4.4% in comparison to the control, and the amount of sake cake decreased by 4.6%. The contribution of XynG2 was further confirmed through our brewing experiment in which we used the yeast heterogeneously expressing fungal xylanase isozymes. Interestingly XynG1, an enzyme with a XynG2-like sequence that is more vulnerable to ethanol, did not improve the sake-mash fermentation. The stability of XynG2 in ethanol was prominent, and it retained most of its original activity after we exposed it to 80% ethanol for 30 min, whereas the stability of the other isozymes in ethanol, including XynG1, was much lower (20-25% ethanol). We concluded, therefore, that the improvement of material utilization achieved with XynG2 is primarily attributable to its characteristically high stability in ethanol, thereby, effectively degrading rice endosperm cell walls under high-alcohol conditions such as a sake-mash environment.
Source: Journal of Bioscience and Bioengineering、110、679-683、(2010)

9. Effect of temperature and water regimes on flavonoid contents and composition in the skin of red-wine grapes

Author: Nami Goto-Yamamoto, Kentaro Mori, Mineyo Numata, Kazuya Koyama and Masahiko Kitayama
Abstract: This study aimed to determine what effects temperature and water regimes have on flavonoid content, concentration, and composition in grape berry skins. Potted vines of Vitis vinifera cv. Cabernet Sauvignon were grown in high (35℃ in daytime/30℃ in night time) and low (25℃ in daytime /20℃ in night time) temperatures with dry, medium, and wet regimes after veraison. The high temperature significantly reduced the anthocyanin content per berry and concentration in the skin and modified the anthocyanin composition. The lower soil moisture significantly increased anthocyanin concentration, even though the dry regime in this study was rather mild. The high temperature after veraison decreased the content and concentration of skin proanthocyanidin and quercetin; however, the decreased rates were much lower than that of anthocyanin. Also, the water regimes showed little or no effects on the proanthocyanidin and quercetin content and concentration. This study showed that the temperature and water regimes after veraison had different influences on each group of flavonoids in the grape berry skins.
Source: J. Int. Sci. Vigne Vin, Special issue Macrowine, 75-80 (2010)

10. Decolorization and treatment of Kokuto-shochu distillery wastewater by the combination treatment involving biodecolorization and biotreatment by Penicillium oxalicum d, physical decolorization by ozonation and treatment by activated sludge.

Author: Watanabe T, Tanaka M, Masaki K, Fujii T, Iefuji H.
Abstract: Kokuto-shochu is a traditional Japanese distilled liquor made from brown sugar. Kokuto-shochu distillery wastewater (KDW) contains high concentrations of organic compounds and brown pigments (called molasses pigments) which are hardly decolorized by general biological wastewater treatment. A fungus, Penicillium oxalicum d, which we isolated in a previous study, decolorizes 47% of the color from KDW without the addition of any nutrients. P. oxalicum d decolorizes KDW by absorbing the pigments into its mycelia. Here we describe a KDW treatment system that combines biodecolorization and biotreatment by P. oxalicum d with treatment by activated sludge and physical decolorization by ozonation. Adding HClO to suppress bacterial growth and replacing fresh seed sludge at regular intervals helped to maintain the dominance and decolorization ability of P. oxalicum d. In a laboratory-scale demonstration, 48 cycles (12 days) achieved a decolorization ratio of 90% and removed more than 97% of dissolved organic carbon (DOC), dissolved total nitrogen (DTN) and dissolved total phosphorus (DTP). A major feature of our system is that it uses only 6% of the water used in an activated sludge-ozonation system.
Source: Biodegradation, 21(6), 1067-1075, 2010

11. Contribution of 1,2-dihydroxy-5-(methylsulfinyl)pentan-3-one (DMTS-P1) to the formation of dimethyl trisulfide (DMTS) during the storage of Japanese sake.

Author: Atsuko Isogai, Ryoko Kanda, Yoshikazu Hiraga, Hiroshi Iwata, and Shigetoshi Sudo
Abstract: Dimethyl trisulfide (DMTS) is involved in the unpalatable aroma of stale Japanese sake, called "hineka". Recently, we isolated one of the precursor compounds of DMTS in sake and identified it as 1,2-dihydroxy-5-(methylsulfinyl)pentan-3-one (DMTS-P1), a previously unknown compound. In this work, the contribution of DMTS-P1 to the formation of DMTS was investigated. DMTS-P1 was chemically synthesized from methional in three steps, consisting of the Grignard reaction, followed by oxidation by MnO2 and an immobilized osmium oxide catalyst. The formation of synthetic DMTS-P1 was confirmed by a comparison of the liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) data to that of natural DMTS-P1. Quantitative analysis of DMTS-P1 in sake was developed using LC-MS/MS, and a positive correlation was observed between the concentration of DMTS-P1 in sake and the production of DMTS during storage. These results indicate that DMTS-P1 contributes to the formation of DMTS in sake.
Source: Journal of Agricultural and Food Chemistry, 58, 7756-7761(2010)

12. Effects of Components of Sake on GABAA receptor

Author: Yasue YAMADA, Masaya EGUCHI, Hanae IZU, Kuniyasu GOTO, Shigetoshi SUDO
Abstract: We investigated the effects of Japanese sake on the GABAA receptor which plays a crucial role in effecting relaxation in the central nervous system. The activities of the GABAA receptor channel were measured by using a Xenopus oocyte expression system. For removing ethanol and volatile compounds, two kinds of sake (junmai-shu) were fractionated by ion-exchange chromatography and lyophilized. The obtained four fractions consisted mainly of basic amino acids (C Fra.), neutral and acidic amino acids (R Fra.), organic acids (A Fra.) and sugars (N Fra.). The GABAA receptor was activated by all fractions of sake. In particular, GABA-free A Fra. highly activated the GABAA receptor. These results indicated that sake contains components able to activate the GABAA receptor except for ethanol and GABA.
Source: J. Brew. Soc. Japan, 105(9), 609-614 (2010)

13. Investigation of Ochratoxin A in Japanese Wine by LC/MS/MS

Author: Sachie HORII, Tomokazu HASHIGUCHI, Yukari IGI, Shigetoshi SUDOU
Abstract: Ochratoxin A in commercial domestic wine was determined by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS). The method involves the use of disposable non-polar polymeric and aminopropyl solid-phase extraction cartridges to extract ochratoxin A from wine. The extracts were subsequently analyzed by LC/MS/MS. Mass spectral acquisition was performed in the positive ion mode by applying multiple reaction monitoring. The recoveries were 75.5% and 93.5% and the detection limits (S/N≧3) were 0.0058 μｇ/L and 0.0045 μｇ/L for red and white wines, respectively. These methods were found to be useful for the determination of low levels of ochratoxin A. In 59 wines examined, ochratoxin A was detected in 5 red wines and 5 white wines. The concentrations of ochratoxin A in the wines were much lower than the EU regulation level (2 μｇ/L).
Source: J. ASEV Jpn., 21, 3-7 (2010)

14. Determination of Ethyl Carbamate in Sake Using Headspace Solid Phase Microextraction

Author: Sachie HORII, Kuniyasu GOTO
Abstract: An analytical method for the determination of ethyl carbamate in alcoholic beverages has been developed and optimized. A combination of headspace solid phase microextraction (HS-SPME), as the extraction technique, and GC/MS, as the determination technique, was utilized. Analytical grade ethyl carbamate dissolved in ethanol solution was analyzed to determine the optimum analytical conditions. Ethyl carbamate-d5 was added as an internal standard. The following HS-SPME conditions were investigated: type of stationary phase of the fibre, ethanol content, sample volume and extraction time. The optimized procedure showed that the detection limit, relative standard deviation, and recovery were 6.7 μg/L, 0.4-2.0%, and 103.6-107.6%, respectively. The precision of this new method was equivalent to previous analyses. Finally, the developed method was applied to the analysis of ethyl carbamate in sake.
Source: J. Inst. Brew. 116(2)、177-181、(2010)

15. Analysis of Sake Component Presented to the Sake Contests in 2009

Author: S. Sudo, A. Isogai, A. Fujita, H. Iwata, J. Hiramatsu
Source: Report of the Research Institute of Brewing, 182, 1-15 (2010)

16. Results of the Wine and Liqueur Contest in 2009

Author: H. Shimoi, N. Goto, K. Takahashi and J. Hiramatsu
Source: Report of the Research Institute of Brewing, 182, 16-26 (2010)

17. Analysis of Traditional Shochu Presented to the 32nd Contest in 2009

Author: H. Fukuda, N. Mukai, K. Kobayashi, S. Mikami, H. Iefuji, J. Hiramatsu
Source: Report of the Research Institute of Brewing, 182, 27-37 (2010)

18. A Study of Transmission of Technology and Skill of Sake Brewing -Analysis of Technology and Skill of Sake Brewing-

Author: A. Muto, A. Matsumoto, H. Utsunomiya
Source: Report of the Research Institute of Brewing, 182, 38-52 (2010)

19. A Study of Transmission of Technology and Skill of Sake Brewing -Learning Theory of Brewers-

Author: A. Muto, A. Matsumoto, H. Utsunomiya
Source: Report of the Research Institute of Brewing, 182, 53-62 (2010)

20. Research of Relations between Attitudes toward Health and Alcoholic Beverages Consumption of Consumers

Author: H. Utsunomiya, M. Yanagiya, K. Hashizume
Source: Report of the Research Institute of Brewing, 182, 63-78 (2010)

21. Effects of Sulfur Dioxide on Formation of Fishy Off-Odor and Undesirable Taste in Wine Consumed with Seafood

Author: Akiko Fujita, Atsuko Isogai, Michiko Endo, Hitoshi Utsunomiya, Shigeyoshi Nakano, Hiroshi Iwata
Abstract: In order to evaluate sensory compatibility of alcoholic beverages with food, beverages and dried squid, namely, "surume", a common Japanese accompaniment, were consumed together. White wine and dried squid pairings had a more undesirable taste and more fishy off-odor than sake and dried squid pairings. The undesirable taste and fishy off-odor appeared to be caused by degradation of unsaturated fatty acids (e.g., docosahexaenoic acid (DHA)), which are found in fish and squid. Upon addition of DHA to the beverage, bitterness intensity, measured by instrumental taste sensor analysis, and the concentration of certain aldehydes reported to contribute to fishy flavors, increased in white wines, whereas they remained largely the same in sake. Among the major chemical constituents that distinguish wine from sake, only wine-specific sulfite markedly increased bitterness intensity and aldehyde levels upon addition of DHA. These results suggest that sulfur dioxide in wine participated in degradation of unsaturated fatty acids, causing an increase in undesirable taste and fishy off-odor in wine and seafood pairings.
Source: J. Agric. Food Chem., 58, 4414-4420 (2010)

22. Analysis of Sake Component Presented to the Sake Contest in 2007

Author: F. Sakamoto, T. Ohnuki , T. Fujii , H. Iefuji
Abstract: We have examined the responses of Saccharomyces cerevisiae in media containing Pb²⁺ ions and compared it to those in media containing UO₂²⁺ ions to elucidate the stress effects of heavy elements. Cultivation of S. cerevisiae in a medium containing 1.0 × 10^-4 M Pb showed nearly the same growth as that in the control medium while growth was inhibited in the medium containing 1.0 × 10^-3 M Pb. Backscattering electron image analyses of thin sections of cells showed that Pb had accumulated inside the cells. These results were different from those for UO₂²⁺ ions obtained in the previous study, where 1.0 × 10^-4 M U inhibited the growth of S. cerevisiae, and UO22+ ions were sorbed on the cell surface. Two-dimensional (2D) gel electrophoresis analysis of the proteins extracted from the S. cerevisiae exposed to Pb²⁺ ions showed that several specific protein spots were expressed after cultivation with Pb, that did not appear in the control medium. The isoelectric point in the gel strip and molecular sizes of the specific proteins expressed by Pb²⁺ were different from those by UO₂²⁺. Our results suggest that Pb²⁺ more weakly inhibits the growth of S. cerevisiae than UO₂²⁺, and produces different stresses in S. cerevisiae than UO₂²⁺.
Source: Geomicrobiology Journal., 27(3), 240-244, 2010

23. PAD1 and FDC1 are essential for the decarboxylation of phenylacrilic acids in Saccharomyces cerevisiae

Author: N. Mukai, K. Masaki, T. Fujii, M. Kawamukai, H. Iefuji
Abstract: The volatile phenols, which Saccharomyces cerevisiae convert from phenylacrylic acids including ferulic acid, p-coumaric acid, and cinnamic acid, generate off-flavors in alcoholic beverages such as beer and wine. Using gene disruptants, transformants and cell-free extracts of these strains, we have verified that the adjacent PAD1 (phenylacrylic acid decarboxylase, YDR538W) and FDC1 (ferulic acid decarboxylase, YDR539W) genes are essential for the decarboxylation of phenylacrylic acids in S. cerevisiae. Pad1p and Fdc1p are homogenous with UbiX and UbiD, respectively, in the ubiquinone synthetic pathway of Escherichia coli. However, ubiquinone was detected quantitatively in all of the yeast single-deletion mutants , Δpad1, Δfdc1, and double-deletion mutant, Δpad1Δfdc1.
Source: J. Biosci. Bioeng., 109(6), 564-569, 2010

24. Formation of spray-dried powder of S-adenosyl-L-methionine

Author: Vichuta Lauruengtana, Arisa Yanagita, Tze Loon Neoh, Takeshi Furuta, Muneyoshi Kanai, Haruyuki Iefuji and Hidefumi Yoshii
Abstract: S-Adenosyl-L-methionine (SAM) is an essential metabolite in all living organisms. In clinical research, SAM has also been suggested as a chemotherapeutic agent in various diseases. The main problem of SAM is its instability at high temperatures, at neutral and alkaline pH, and in the presence of humidity. SAM retention in spray-dried powder was determined under various conditions of spray-drying. The highest SAM retention was obtained when maltodextrin (dextrose equivalent, DE, of 25) was used as the carrier solid with the SAM feed liquid at pH 4.0. The water content in the powder had a significant effect on the stability of SAM. SAM powder with lower water content exhibited higher stability.
Source: Biotechnol J., 5(5), 470-6, (2010)