平成21年度 研究成果

奥田将生、橋爪克己、上用みどり、沼田美子代、後藤奈美、三上重明

登熟期の気温と原料米の溶解性に密接に関係するデンプン特性の年次・産地間変動との関係について解析し、以下の結果が得られた。1）同一品種でも産地間でデンプン特性や蒸米消化性が異なるのは、産地の登熟期気温が影響したことが主な原因であると示唆された。2）25品種27産地の生産年度の異なる試料について、出穂後気温とデンプン糊化温度の関係について解析したところ、登熟期気温と糊化温度は高い相関性を示し、登熟期気温が低い年は糊化温度が低く、一方気温が高いと糊化温度も高くなることを確認した。以上、デンプン糊化温度は蒸米消化性と高い相関性を示すので、これまでと今回の研究結果から、原料米ごとの出穂後の気温に注目すれば、かなりの精度で原料米の溶解性に関する酒造適性を予測できると考えられた。

H. Tsuji and A. Mizuno

Volatile compounds in beers brewed with different amounts of malt were analyzed by using the stir bar sorptive extraction-gas chromatography-mass spectrometry method. We identified 90 compounds-25 esters, 17 terpenes, 14 alcohols, 11 acids, 6 furans, 6 aroma compounds, 5 carbonyls, and other compounds. Ananalysis of aged beer suggested that the concentration levels of stale flavor compounds-β-damascenone, γ-nonalactone, ethylcinnamate, and 2-methoxy-4-vinylphenol-in nonmalt beer were different from those in all-malt and standard beer. Additionally, concentrations of these compounds did not increase during storage in mostnonmalt beer analyzed in this study. Nerolidol may be a good marker candidate regardless of the malt content.

宇都宮仁、磯谷敦子、岩田博

18種類の化学物質、酢酸エチル、酢酸イソアミル、カプロン酸エチル、イソアミルアルコール、フェネチルアルコール、アセトアルデヒド、イソバレルアルデヒド、ベンズアルデヒド、エタンチオール、ジメチルスルフィド（DMS）、ジメチルトリスルフィド (DMTS)、酢酸、酪酸、イソ吉草酸、カプロン酸、ソトロン、ジアセチル、2,4,6-トリクロロアニソール (TCA)を清酒の官能評価におけるにおい参照標準候補として選定した。

各物質は、90%認知閾値濃度で、市販清酒に添加した。その溶液について専門家79名による評価を実施した。ベンズアルデヒドを除く17物質については、清酒の匂い参照標準物質として使用可能と考えられた。「老香」をにおい用語リストから選択した61名のうち42.6%がDMTS、23.0%がエタンチオールを「老香」としたがソトロンを「老香」としたのは1名のみであった。この結果から、専門家が「老香」に対して有する印象は、硫黄化合物の匂いに近いと考えられた。

伊豆英恵、後藤邦康

酒類の飲用摂取によって酒類間に酔いの違いの有無があるかどうか、マウスを用いた行動実験によって検討を行った。

1. 清酒、赤ワイン、焼酎、ウイスキーをほぼ同じアルコール度（15%）にそろえ、純アルコール換算で同量飲酒（1.2 g/kg体重を1回または2回投与）させたが、各酒類摂取群間で摂取後の自発運動量に違いは見られなかった。
2. 清酒と焼酎を同アルコール度（15%）にそろえ、純アルコール換算で同量飲酒（2.4 g/kg体重を1回投与）させ、摂取後0.5、3、5時間後の血中及び脳内のエタノール・アセトアルデヒド濃度を調べたが、変動に違いは見られなかった。
3. 清酒、焼酎、ウイスキーを同じアルコール度（15%）にそろえ、純アルコール換算で同量飲酒（1.2 g/kg体重を2回投与）させ、高架式十字迷路試験でリラックス効果を調べたが、各酒類摂取群間で作用の程度に違いは見られなかった。

堀井幸江、伊木由香理、後藤邦康

メタミドホス、アセタミプリド、アフラトキシンB1を10および20 ppb添加して清酒を醸造し、醸造工程中の各成分の消長について調査した。時間経過と共に濃度は低下し、その傾向は成分によって異なった。

液部と固形部における残留割合を比較した結果、留め後20日で、メタミドホスは液部に、アフラトキシンB1は固形部に多く残留し、アセタミプリドは液部と固形部に同じ割合で残留した。

活性炭100 mg/Lの30分処理で、アセタミプリドおよびアフラトキシンB1の濃度は50%以下に低下した。しかし、メタミドホスでは、活性炭処理による影響がみられなかった。

75℃で熱処理を行ったが、いずれの成分も濃度に変化はなかった。

貯蔵試験では、メタミドホスおよびアセタミプリドの濃度は期間を通じて変化しなかったが、アフラトキシンB1は固形部で5か月後に濃度の低下が認められた。

Kazutoshi Sakamoto, Kazuhiro Iwashita, Osamu Yamada, Ken Kobayashi, Akihiro Mizuno, Osamu Akita, Shigeaki Mikami, Hitoshi Shimoi and Katsuya Gomi

We compared atfA and atfB, the genes encoding the respective ATF/CREB-type transcription factors in Aspergillus oryzae. The germination ratio of △atfA conidia was low without any stress, unlike that of △atfB conidia. The △atfA conidia were more sensitive to oxidative stress than the △atfB conidia, which are also sensitive to oxidative stress. We compared the gene expressions of these strains by using a DNA microarray, GeneChip. Almost all the genes regulated by atfB were also regulated by atfA, but atfA also regulated many genes that were not regulated by atfB, including some genes putatively involved in oxidative stress resistance. The level of glutamate, the major amino acid in A. oryzae conidia, was significantly low only in the △atfA conidia, and the glycerol accumulation during germination was not observed only in the △atfA strain. We therefore concluded that atfA is involved in germination via carbon and nitrogen source metabolism.

M. Okuda, A. Isogai, M. Joyo, N. Goto-Yamamoto and S. Mikami

The influence of nitrogen and sulfur compounds in rice grains on changes in flavor in stored sake was investigated using Japanese rice cultivars for sake making. Nitrogen content exhibited a significant positive correlation with sulfur content. Based on the molar ratio of nitrogen to sulfur in the rice grain, the sulfur compounds appeared to be derived from protein-associated sulfur-containing amino acids, as reported previously. The higher the protein content of the rice, the greater the amount of nitrogen and sulfur compounds found in both the digest of steamed rice grains and in the sake. Physicochemical changes were investigated in the stored sake to confirm the influence of total sulfur content. Polysulfides in the stored sake appeared to be higher when made from rice grains of high total sulfur content. Staling of stored sake was affected by levels of protein-associated sulfur-containing amino acids in the rice.

M. Okuda, K. Hashizume, I. Aramaki, M. Numata, M. Joyo, N. Goto-Yamamoto and S. Mikami

Retrogradation of starch in steamed rice grains under sake-making conditions was examined. The enthalpy change derived from retrogradation of amylopectin increased during the koji and sake mash processes. Ethanol in the sake mash accelerated retrogradation of amylopectin. Enzyme digestibility and properties of endosperm starches of 136 Japanese rice samples used for sake production were examined. A digestion test of steamed rice grains was conducted after 24 hr storage at 15℃ to estimate more accurately digestibility during the sake-making process. The ratio of short-to-long chain amylopectin exhibited a significant correlation with enzyme digestibility, suggesting that the structure of amylopectin may be used as a predictor of digestibility. The gelatinization temperature of brown/milled rice and purified starch measured by DSC (Differential Scanning Calorimetry) correlated well with the ratio of short-to-long chain amylopectin and with enzyme digestibility. Furthermore, the pasting temperature of milled rice as measured by RVA (Rapid Visco Analyser) correlated well with the ratio of short-to-long chain amylopectin and with enzyme digestibility. Therefore, DSC and RVA appear to be useful tools for estimating enzyme digestibility of steamed rice grains under sake-processing conditions because the measurements are done rapidly and require only small weights of brown/milled rice flour samples.

奥田将生、橋爪克己、沼田美子代、上用みどり、後藤奈美、三上重明

酒米研究会が収集した気象データの明らかな試料を用いて、気象データから米の溶解性に関する酒造適性を予測できるかどうかを検討する目的で、気象データとデンプン特性及び蒸米消化性の関係について解析した。出穂後1ヶ月の平均気温はアミロペクチン短鎖/長鎖比及びアミロース含量とは高い相関関係を示した。また、出穂後1ヶ月の平均気温は、DSCやRVAの測定値とも高い相関性を示した。酒米統一分析条件での蒸米消化性（デンプンの消化性）は出穂後1ヶ月の平均気温が23℃付近で消化性が高くなるような関係を示した。もろみに近いデンプンの老化を反映させた条件では、出穂後1ヶ月の平均気温と蒸米の酵素消化性（デンプンの消化性）は直線的な負の高い相関性を示した。それぞれの条件で出穂後1ヶ月の平均気温でデンプンの消化性を予測する式を構築し、この式より2008年産の試料について予測できるか検定したところ、予測値と実測値は高い相関性を示し出穂後1ヶ月の平均気温でどちらの条件でもデンプンの消化性を比較的精度良く予測できた。また、タンパク質含量は気温と強い相関がみられなかったものの、タンパク質の消化率はデンプンの消化性と同様な傾向を示し気温と有意な相関性を示すことがわかった。以上の結果から、出穂後の1ヶ月間平均気温によってかなり高い精度で米の溶解性に関する酒造適性を予測できる可能性が示された。

Watanabe T, Ikeda H, Masaki K, Fujii T, Iefuji H.

Phosphohydrolysis of organic phosphorus compounds by acid phosphatases (EC 3.1.3.1 and EC 3.1.3.2) is an important method for efficient removal of phosphorus from high concentration organic wastewater. Another important method is supplementation of animal feed with phytase (EC 3.1.3.8 and EC 3.1.3.26), which improves the availability of phytate-phosphates (phosphate that are hydrolyzed by phytases), making it possible to add less phosphate to animal feed and resulting in the excretion of less phosphorus by the animals. In the present study, we purified a novel phytase from the wastewater treatment yeast Hansenula fabianii J640 (Hfphytase), cloned the 1456 bp open reading frame (ORF) encoding Hfphytase, and characterized Hfphytase. The molecular weight of Hfphytase after deglycosylation by PNGaseF was 49 kDa. The optimal pH and temperature for enzyme activity were 4.5 and 50 °C, respectively. Hfphytase exhibits 40% identity with Debaryomyces castellii phytase, 37% identity with Aspergillus niger PhyB, and 34% identity with Saccharomyces cerevisiae Pho5p. Recombinant Hfphytase was transformed and expressed in Pichia pastoris. The yield was 23 g/l by jar fermenter cultivation. The marked phosphohydrolysis activity exhibited by Hfphytase on six substrates (pNP-P, sodium phytate, glucose-1 phosphate, glucose-6 phosphate, α-glycerophosphate and β-glycerophosphate) indicated that it is a non-specific acid phosphatase.

Kodama Y, Masaki K, Kondo H, Suzuki M, Tsuda S, Nagura T, Shimba N, Suzuki EI, Iefuji H

The structural and enzymatic characteristics of a cutinase-like enzyme (CLE) from Cryptococcus sp. strain S-2, which exhibits remote homology to a lipolytic enzyme and a cutinase from the fungus Fusarium solani (FS cutinase), were compared to investigate the unique substrate specificity of CLE. The crystal structure of CLE was solved to a 1.05 A resolution. Moreover, hydrolysis assays demonstrated the broad specificity of CLE for short and long-chain substrates, as well as the preferred specificity of FS cutinase for short-chain substrates. In addition, site-directed mutagenesis was performed to increase the hydrolysis activity on long-chain substrates, indicating that the hydrophobic aromatic residues are important for the specificity to the long-chain substrate. These results indicate that hydrophobic residues, especially the aromatic ones exposed to solvent, are important for retaining lipase activity.

岩田博、磯谷敦子、藤田晃子、平松順一

平成19酒造年度全国新酒鑑評会（第96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。本年度は、製造技術研究会を広島県立広島産業会館で、公開きき酒会を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、それぞれ開催した。

審査は、平成20年4月22日（火）から24日（木）の3日間に予審を行い、5月8日（木）及び9日（金）に決審を行った。また、5月22日（木）に製造技術研究会を、6月11日（水）に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129点、第Ⅱ部（山田錦の使用割合が50%超の部）が828点の合計957点であった。審査の結果、優秀と認められた487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255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、1,416人及び3,500人が来場した。

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

三上重明、後藤奈美、小山和哉、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。

三上重明、家藤治幸、向井伸彦、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31回本格焼酎鑑評会を開催した。官能審査は平成20年6月5日（木）及び6日（金）に行い、公開きき酒会は6月27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。

後藤邦康

当所で買上げを行った市販酒類中の4‐メチルイミダゾール、セミカルバジド、1,3‐ジクロロプロパノールおよび一般微生物の調査結果について示す。

堀井幸江、伊木由香理、後藤邦康

酒類中に含まれる農薬の一斉分析にQuEChERS法を導入した分析結果について報告する。

Taku Katou, Masahiro Namise, Hiroshi Kitagaki, Takeshi Akao and Hitoshi Shimoi

A haploid sake yeast strain, derived from a commercial diploid sake yeast strain Kyokai no. 7, showed better characteristics for sake brewing compared to the haploid laboratory yeast strain X2180-1B, including higher production of ethanol and aromatic components. A hybrid of these two strains showed intermediate characteristics in most cases. After sporulation of the hybrid strain, we obtained 100 haploid segregants of the hybrid. Small scale sake brewing tests of these segregants showed a smooth continuous distribution of the sake brewing characteristics, suggesting that these traits are determined by multiple quantitative trait loci (QTLs). To examine these sake brewing characteristics at the genomic level, we performed QTL analysis of sake brewing characteristics using 142 DNA markers that showed heterogeneity between the two parental strains. As a result, we identified 25 significant QTLs involved in specification of sake brewing characteristics including ethanol fermentation and the production of aromatic components.

Mamoru Watanabe, Daisuke Watanabe, Takeshi Akao and Hitoshi Shimoi

To improve the ethanol tolerance of sake yeast we constructed a sake yeast strain that overexpresses MSN2, a transcription factor that is activated by several environmental stresses, including ethanol. We showed that this strain is more ethanol tolerant and produced more ethanol in a sake mash than a control strain.

Hong Wu, Tomoko Watanabe, Yoshio Araki, Hiroshi Kitagaki, Takeshi Akao, Hiroshi Takagi and Hitoshi Shimoi

Sake yeast can produce high levels of ethanol in concentrated rice mash. While both sake and laboratory yeast strains belong to the species Saccharomyces cerevisiae, the laboratory strains produce much less ethanol. This disparity in fermentation activity may be due to the strains' different responses to environmental stresses, including ethanol accumulation. To obtain more insight into the stress response of yeast cells under sake brewing conditions, we carried out small-scale sake brewing tests using laboratory yeast strains disrupted in specific stress-related genes. Surprisingly, yeast strains with disrupted ubiquitin-related genes produced more ethanol than the parental strain during sake brewing. The elevated fermentation ability conferred by disruption of the ubiquitin-coding gene UBI4 was confined to laboratory strains, and the ubi4 disruptant of a sake yeast strain did not demonstrate a comparable increase in ethanol production. These findings suggest different roles for ubiquitin in sake and laboratory yeast strains.

Nami GOTO-YAMAMOTO, Mineyo NUMATA, Guang-Hua WAN, Toshi SHIMAMOTO, Katsumi HASHIZUME

Six Oriental cultivars of Vitis vinifera, which are native to Japan or China, as well as 2 cultivars of V. labrusca as references, were characterized by 6 microsatellite markers with coded reference alleles. One allele in Oriental cultivars and 3 in V. labrusca have not been reported for Occidental cultivars. Assignment analysis using these data and reported data with grouping of V. labrusca, Oriental cultivars, and vine-growing regions of Occidental cultivars showed that 4 out of 8 Oriental cultivars, but only 3 out of 162 Occidental cultivars, were assigned to Oriental cultivars. These assignments are in accordance with the genetic distances calculated from microsatellite data. These results show that Oriental cultivars have a certain degree of genetic difference from Occidental cultivars within the species V. vinifera.

Akifumi AZUMA, Shozo KOBAYASHI, Nami GOTO-YAMAMOTO, Mikio SHIRAISHI, Nobuhito MITANI, Hiroshi YAKUSHIJI and Yoshiko KOSHITA

Color mutations in grape berry skin are relatively frequent events, and can be easily seen in the vineyard. Both light-red-skinned 'Ruby Okuyama' and more intense and uniform rosy-skinned 'Benitaka' (Vitis vinifera L.) are bud sports of white-skinned 'Italia'. Previously, we reported that 'Ruby Okuyama' was caused by the recovery of VvmybA1 expression, which may have occurred as a result of intra-LTR (long terminal repeat) recombination within a retrotransposon, Gret1. However, the molecular basis of the color recovery in 'Benitaka' has not been elucidated so far. Here, we found that the VvmybA1 locus of 'Benitaka' is heterozygous for the VvmybA1a allele (non-functional) and a novel VvmybA1^BEN allele, and that VvmybA1^BEN restored VvmybA1 transcripts. We hypothesized that VvmybA1^BEN allele was caused by homologous recombination between VvmybA1a and VvmybA3. In addition, the content and composition of anthocyanins in berry skins differed greatly between 'Ruby Okuyama' and 'Benitaka'. The levels of expression of the genes for flavonoid 3',5'-hydroxylase (F3'5'H), O-methyltransferase (OMT), and glutathione-S-transferase (GST) were associated with differences in the anthocyanin content and composition between the two cultivars.

渡部貴志、田中未来、岡田真実、正木和夫、藤井力、家藤治幸

黒糖焼酎を蒸留する際に生じる黒糖焼酎蒸留粕（BSW）には、難分解性の褐色色素を高濃度含有するが、一般的な生物処理では脱色が困難である。本研究で新たに自然界より単離してきた糸状菌Penicillium oxalicum d株は、栄養源を添加しなくてもBSWを47%脱色した。菌体が褐色になることから、P. oxalicum d株のBSWの主な脱色機構は菌体への吸着関連である。P. oxalicum d株を用いて処理することで、オゾンによる脱色時間が半分程度に短縮された。P. oxalicum d株による脱色、活性汚泥処理、オゾンによる物理的脱色処理の組合せ処理はBSWの処理に有効と思われた。

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

Large quantities of deeply pigmented molasses distillery wastewater (MDW), are discharged during the production of bio-ethanol from molasses. Conventional biological wastewater treatment is not effective in removing the molasses pigments. In the present study, a MDW treatment system was developed with combination treatment involving biodecolorization and biotreatment by Aspergillus tubingensis DCT6, together with physical decolorization by ozonation after treatment by activated sludge. A. tubingensis DCT6, which was isolated from soil, decolorized 44% of the pigments in MDW without adding any nutrients. The combination treatment with A. tubingensis DCT6 and activated sludge method (fungi-activated sludge treatment) removed about 90% of organic compounds from MDW and appears to reduce the amount of space and water required for treatment. The fungi-activated sludge treatment reduced the time needed for decolorization by ozone by 83%. Replacing fresh seed sludge at regular intervals was useful to maintain the dominance and decolorization ability of A. tubingensis DCT6. The entire treatment obtained a decolorization ratio of 89-94% and removed more than 90% of each of DOC, DTN, and DTP.