平成22年度 研究成果

堀井幸江、橋口知一、伊木由香理、伊豆英恵、須藤茂俊

様々な食品において、安定同位体比の測定が、表示の真偽判定に有効であると報告されていることから、清酒および焼酎中のエタノールの炭素安定同位体比を測定した。炭素安定同位体比を測定する上で必要となるエタノールの濃縮方法として、単蒸留を繰り返す方法と減圧濃縮を比較した場合、減圧濃縮が同位体分別の程度が小さく、作業が効率的であった。清酒で、炭素安定同位体比と醸造アルコールの添加割合との間に相関がみられ、添加した醸造アルコール量が推測できる可能性が示唆された。市販清酒の分析では、純米酒、本醸造酒および普通酒の炭素安定同位体比に有意な差が認められた。モデル混和焼酎で、本格焼酎および連続式蒸留焼酎の混和割合との間に相関がみられ、混和焼酎の混和割合が推測可能であると思われた。また、市販甲類乙類混和焼酎の測定結果からも、混和焼酎における混和割合の推測は可能であることが裏付けられた。

Daisuke WATANABE, Hong WU, Chiemi NOGUCHI, Yan ZHOU, Takeshi AKAO, Hitoshi SHIMOI

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.

W. Jermsuntiea, T. Aki, R. Toyoura, K. Iwashita, S. Kawamoto, K. Ono.

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.

Kazuya KOYAMA, Keiko SADAMATSU, Nami GOTO

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.

伊豆英恵、山田康枝、後藤邦康、須藤茂俊

マウスを用いた高架式十字迷路試験によって,清酒の飲用摂取による抗不安作用を検討した。

1. エタノールまたは普通酒を飲用させた場合（エタノール換算1.2 g/kg体重を2回投与）,対照と比較してオープンアームへの進入回数がそれぞれ2.7倍と3.4倍,滞在時間が3.2倍と3.9倍に増加しており,普通酒もエタノール同様に抗不安作用があり,さらにその作用がエタノールよりも高い傾向にあった。
2. 普通酒または吟醸酒を飲用させた場合（エタノール換算1.2 g/kg体重を2回投与）,普通酒と比較して,吟醸酒でオープンアームへの進入回数が1.7倍,滞在時間が1.6倍に増加しており,普通酒よりも吟醸酒の抗不安作用が有意に高いことがわかった。
3. 吟醸酒に含まれるのとほぼ同程度となるように吟醸酒香気成分であるカプロン酸エチル（10 mg/l）または酢酸イソアミル（2 mg/l）を普通酒に添加してマウスに飲用させた場合（エタノール換算1.2 g/kg体重を2回投与）,普通酒と比較してオープンアームへの進入回数がそれぞれ1.5倍と1.4倍,滞在時間がいずれも1.5倍に増加しており,カプロン酸エチルと酢酸イソアミルが抗不安作用を有意に促進することが明らかになった。
4. 通常,清酒に含まれる濃度範囲では,カプロン酸エチル,酢酸イソアミル,イソアミルアルコールはADHによるエタノール代謝を阻害しないことがわかった。

Tomokazu HASHIGUCHI, Sachie HORII, Hanae IZU, and Shigetoshi SUDO

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.

Masafumi Tokuoka, Nobuko Sawamura, Ken Kobayashi, Akihiro Mizuno

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.

Yuichiro Sato, Hisashi Fukuda, Yan Zhou, and Shigeaki Mikami

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.

Nami Goto-Yamamoto, Kentaro Mori, Mineyo Numata, Kazuya Koyama and Masahiko Kitayama

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 v?raison. 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 v?raison 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 v?raison had different influences on each group of flavonoids in the grape berry skins.

Watanabe T, Tanaka M, Masaki K, Fujii T, Iefuji H.

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.

Atsuko Isogai, Ryoko Kanda, Yoshikazu Hiraga, Hiroshi Iwata, and Shigetoshi Sudo

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.

山田康枝、江口将也、伊豆英恵、後藤邦康、須藤茂俊

本研究では、中枢神経系においてリラックス効果に主要な働きをしているGABA_A受容体に対する日本酒成分の効果を明らかにするため、アフリカツメガエル卵母細胞に発現したGABA_A受容体のチャンネル活性への日本酒成分の影響を検討した。受容体へ効果のある物質を特定しやすくするため、イオン交換クロマトグラフィーで日本酒を分画し、揮発性成分を除き濃縮するために凍結乾燥を行い、塩基性アミノ酸画分、中・酸性アミノ酸画分、有機酸画分、糖画分を得た。これらの日本酒画分存在下で測定を行った結果、得られた全ての画分にGABAA受容体を活性化する成分が存在することが示された。特に、有機酸を主に含む画分において、GABAが含まれないのにも関わらず、GABA活性が存在し、さらに高い活性化率を示すことがわかった。以上のことから、日本酒にエタノールやGABA以外のGABA_A受容体活性化成分が存在することが示唆された。

堀井幸江、橋口知一、伊木由香理、須藤茂俊

LC/MS/MSを用いて国産ワイン中のオクラトキシンAの分析を行った。サンプルは固相抽出法で前処理を行い分析した。LC/MS/MSでは移動相は0.1%ギ酸水および0.1%ギ酸アセトニトリルを用いたグラジエント法を用い、ESIポジティブイオンモードで測定した。添加回収試験の結果、回収率は赤ワインで75.5%、白ワインで93.5%であった。変動係数は、赤ワインで10.2%、白ワインで6.4%と良好であった。

赤ワインで0.0058 μg/L、白ワインで0.0045 μg/Lを検出限界とし国産ワイン59点（赤ワイン31点、白ワイン28点）の分析を行った所、赤ワイン5点、白ワイン5点からオクラトキシンAが検出された。しかし、その検出濃度は、最高値がそれぞれ、赤ワインで0.03 μg/L、白ワインで0.022 μg/Lといずれも極低濃度であった。今回検出されたレベルは全てEUで設定されている規制値（2 μg/L）より低く、人に対する健康被害はないものと考えられる。

Sachie HORII, Kuniyasu GOTO

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.

須藤茂俊、磯谷敦子、藤田晃子、岩田博、平松順一

平成20酒造年度全国新酒鑑評会（第97回鑑評会）は、当該年度生産清酒を全国的に調査研究することにより、製造技術と酒質の現状及び動向を明らかにし、もって清酒の品質及び酒造技術の向上に資するとともに、国民の清酒に対する認識を高めることを目的として、日本酒造組合中央会と共催した。

審査は、酒類総合研究所広島事務所において、平成21年4月22日（水）から24日（金）の3日間に予審を行い、5月12日（火）及び13日（水）に決審を行った。また、5月27日（水）に東広島運動公園体育館で製造技術研究会を、6月17日（水）に東京のサンシャインシティ・ワールドインポートマート展示ホールで公開きき酒会を開催した。

出品の内訳は、第Ⅰ部（山田錦の使用割合が50%以下の部）が139点、第Ⅱ部（山田錦の使用割合が50%超の部）が781点の合計920点であった。審査の結果、優秀と認められた456点を入賞酒とし、さらに、決審において特に優秀と認められた249点に金賞を授与した。また、出品酒を公開する製造技術研究会及び公開きき酒会には、全国から、それぞれ、1,265人及び3,738人が来場した。

出品酒については、酒質の現状及び動向を調査研究するため、調査表の内容を集計するとともに成分分析を行った。

下飯仁、後藤奈美、高橋圭、平松順一

本鑑評会は、国内果実酒・リキュール製造者から任意出品された果実酒・甘味果実酒・リキュールについて官能評価及び化学分析を行い、品質及び技術の動向を全国的な視野で調査するとともに、製造者の参考に資することを目的として実施した。

福田央、向井伸彦、小林健、三上重明、家藤治幸、平松順一

単式蒸留しょうちゅうの品質を全国的な視野でとらえ、現在の製造技術の内容と酒質の傾向を把握するとともに製造業者の参考とするため、第32回本格焼酎鑑評会を日本酒造組合中央会と共催で開催した。出品資格として、単式蒸留しょうちゅう製造免許を有する製造者で、日本酒造組合中央会の組合員に限定した。官能審査は平成21年6月4日（木）及び5日（金）に行い、公開きき酒会は6月26日(金)に当所で開催し、出品関係者の参考に供した。

出品酒の官能審査と成分分析を行ったので、その結果の概要について報告する。

武藤彰宣、松本明、宇都宮仁

人の持つ酒造りの技術・技能を整理するため「洗米・浸漬」から「オリ下げ」までの11工程についてクドバス法による技術・技能分析を行い、各工程に必要な技術・技能を、「準備をする」、「作業をする」、「分析・測定・評価する」、「判断する」、「工程全体を管理する」の5つの仕事に分類し、重要度の水準をまとめたクドバス・チャートを作成した。このチャートをもとに、各人の技術・技能の保有水準を評価する技術・技能チェックシートを作成した。

武藤彰宣、松本明、宇都宮仁

酒造技術者・技能者への聞き取り調査等から、彼らが現場においてどのような過程で酒造り技術・技能を学んできたかを分析した。次に、そこから得られた知見をもとに当研究所の清酒製造技術講習の内容を改良した。その結果、酒造り技能の学び方について効果的なポイントを見出した。

宇都宮仁、栁谷光弘、橋爪克己

飲酒者の飲酒動機あるいは特定の酒類の選択に健康意識がどの程度関係しているか等について5875人にアンケート調査を実施した。酒類選択時に重視するポイントのうち健康関連では、「アルコール度数」、「二日酔いになりにくい」、「糖分」が3割を超えており「製造方法」より高かった。また、酒類に関する健康イメージは、前回（平成16年）調査に比べ大きく低下した。飲酒と健康に関する心がけでは「イッキ飲みはしない」等は概ね意識されているが、飲酒量や頻度などに関する心がけは5割以下であった。

Akiko Fujita, Atsuko Isogai, Michiko Endo, Hitoshi Utsunomiya, Shigeyoshi Nakano, Hiroshi Iwata

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.

F. Sakamoto, T. Ohnuki, T. Fujii, H. Iefuji

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⁺.

N. Mukai, K. Masaki, T. Fujii, M. Kawamukai, H. Iefuji

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.

Vichuta Lauruengtana, Arisa Yanagita, Tze Loon Neoh, Takeshi Furuta, Muneyoshi Kanai, Haruyuki Iefuji and Hidefumi Yoshii

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.