平成18年度 研究成果

O. Yamada, R .Ikeda, Y. Ohkita, R. Hayashi, K. Sakamoto, and O. Akita

We found the orthologous genes required for RNA interference (RNAi) in the Aspergillus oryzae genome database, and constructed a set of tools for gene silencing using RNAi in A. oryzae. This systI utilizes compatible restriction enzyme sites so that only a single target gene fragment is required to create the hairpin RNA cassette. For ease of handling, we also separated the construction of the hairpin RNA cassette for the target gene from its subsequent introduction into the expression vector. Using the brlA gene as a target for RNAi, we detected decreased mRNA levels and a delayed conidiation phenotype in the transformants. Furthermore, even though A. oryzae possesses three copies of the alpha-amylase gene, a single copy of an alpha-amylase RNAi construct was sufficient to downregulate the mRNA levels and decrease the enzymatic activity to 10% of control levels. Gene silencing by RNAi should provide a powerful genetic tool for post-genomic studies of the industrially important fungus A. oryzae.

M. Okuda, K. Kobayashi, T. Itani, I. Aramaki and K. Hashizume

Flour and endosperm starch prepared from rice grains of three Japanese rice cultivars milled from 90 to 30% of their original weight were subjected to physicochIical/structural analysis in order to examine the relationship between milling ratio and physicochIical/starch structural properties. The peak viscosity of the rice flours was found to increase with decreasing milling ratio, while that of the purified starches did not change significantly. The gelatinization peak tIperature of the rice flours was found to decrease while enthalpy changes increased with decreasing milling ratio. The gelatinization peak tIperature of the purified starches was not found to change significantly with different milling ratios. Differences in structural properties of purified starches were examined by gel-filtration chromatography of isoamylase debranched starch. The FI (percentage of amylose) and FIIb/FIIa (ratio of short-to-long chain amylopectin) were found to be constant for all three cultivars for milling ratios ranging from brown rice to 30%.

Ardiansyah, H. Shirakawa, T. Koseki, K. Hashizume, and M. Komai

The aim of this study is to investigate the effects of dietary supplIentation with the Dreselase-treated fraction (DF) of rice bran and ferulic acid (FA) on hypertension and glucose and lipid metabolism in stroke-prone spontaneously hypertensive rats (SHRSP). Male SHRSP at 4 weeks of age were divided into three groups, and for 8 weeks were fed (1) a control diet based on AIN-93M, (2) a DF of rice bran-supplIented diet at 60g/kg and (3) an FA-supplIented diet at 0.01g/kg. Means and standard errors were calculated and the data were tested by one-way ANOVA followed tolerance, plasma nitric oxide (NOx), urinary 8-hydroxy-2'-deoxyguanosine and other parameters. In particular, compared to the FA diet, the DF diet produced a significant improvIent in urinary NOx, hepatic triacylglycerol and several mRNA expressions of metabolic parameters involved in glucose and lipid metabolism. The results of the metabolic syndrome-related parameters obtained from this study suggest that the DF diet is more effective than the FA diet.

M. Shobayashi, E. Ukena, T. Fijii and H. Iefuji

To identify the genes responsible for characteristics, that are different as between sake brewing yeasts and laboratory yeast strains, we used a DNA microarray to compare the genome-wide gene expression profiles of a sake yeast, Saccharomyces cerevisiae K-9 (kyokai 9), and a laboratory yeast, S. cerevisiae X2180-1A, under shaking and static conditions. The genes overexpressed in K-9 more than in X2180-1A were related to C-metabolism, including the HXT, ATP, and COX genes, ergosterol biosynthesis, ERG genes, and thiamine metabolism, THI genes. These genes may contribute to higher growth rates and fermentation ability and the ethanol tolerance of sake yeast. The genes underexpressed in K-9 more than in X2180-1A were CUP1-1 and CUP1-2, PHO genes, which may explain the low copper tolerance and low acid phosphatase activity of sake yeast. These underexpressed genes agree with the features and the alteration of the genome structure of sake yeast.

H. Izu, M. Shobayashi, Y. Manabe, K. Goto, H. Iefuji

Suppressive effects on acute alcoholic liver injury by S-adenosylmethionine (SAM) and sake yeast, Saccharomyces cerevisiae Kyokai No.9, were shown previously. To enhance the suppression of acute alcoholic liver injury by sake yeast, we prepared SAM-accumulating sake yeast (SAM yeast). Male C57BL/6 mice that had been fed a diet containing 0.25% SAM yeast or sake yeast for two weeks received three doses of ethanol (5 g/kg BW). In the mice fed SAM yeast, ethanol-induced increases in both triglyceride (TG) and alanine aminotransferase (ALT) were significantly repressed. In addition, SAM yeast-fed mice did not show an ethanol-induced decrease in hepatic SAM level, suggesting that a disorder of methionine metabolism in the liver caused by ethanol was relieved by SAM yeast. Theses results suggested that SAM yeast has much effect on the suppression of acute alcoholic liver injury in mice compared with sake yeast.

福田央、周延、三上重明

清酒酵母,焼酎酵母及びブドウ酒酵母間の判別にPCR法が検討された。FLO5由来のプライマーによるPCR産物は, 清酒酵母と焼酎酵母で異なった。更にYHR213W由来のプライマーによりブドウ酒酵母と多くの他の醸造酵母は判別できた。しかし, 焼酎酵母KF1のPCR産物はブドウ酒酵母のものと似ているため判別できなかった。PCR法により醸造酵母を判別する方法は, 従来の生理学的性質を利用する方法に比べて容易であることから, これらのプライマーを用いる清酒酵母,焼酎酵母及びブドウ酒酵母間の識別は有用であった。

K. Mori, N. Goto-Yamamoto, M. Kitayama and K. Hashizume

Anthocyanin accumulation in grape berry skins is influenced by temperature during the maturation period. Although it has been reported that high temperatures result in lower anthocyanin concentrations in berry skins, their effects on anthocyanin composition remain unclear. To study the effect of high temperature on anthocyanin composition, the accumulation of individual anthocyanins in the skin of ‘Pinot noir' berries was investigated in two experiments: Experiment 1, high morning and evening temperature treatment; Experiment 2, high day-time temperature treatment. High temperatures (30°C) in the morning and evening did not the affect the total anthocyanin content in the skin, but decreased the levels of delphinidin 3-glucosides, petunidin 3-glucosides and malvidin 3-glucosides, which are 3',5'- hydroxylated, or methylated following 3',5'-hydroxylation. A high day-time temperature (35°C) also decreased the levels of delphinidin, petunidin and malvidin 3-glucosides. In both Experiments, grape berries grown under high temperature had a low abundance of mRNA for flavonoid 3', 5'-hydroxylase (F3'5'H) in the skin. These results suggest that changes in the accumulation of individual anthocyanins in the skins of ‘Pinot noir' grape berries, due to high temperature, are regulated at the level of transcription of the F3'5'H gene.

K.Hashizume, M.Okuda, S.Sakurao, M.Numata, T.Koseki, I.Aramaki, T.Kumamaru and H.Sato

The digestion of proteins in steamed rice grains by sake koji enzymes under simulated sake mash conditions was analyzed by comparing the hydrolysis of steamed rice grains and heat-treated protein bodies (PBs) isolated from seven rice samples including four endosperm-storage protein mutants. The disappearance of peptides in the digest of isolated PBs was faster than that of steamed rice grains; however, more insoluble proteins formed in the case of isolated PBs. Not all of the hydrolyzed PB proteins were immediately solubilized in the digestion tests. High-molecular-weight peptides were more abundant in the solubilized digest of steamed rice grains than in that of isolated PBs. Variance in Ile, Ser, Glu, and Gly levels in the digest of steamed rice grains was relatively high among the seven samples, but was not found to be high in digests of isolated PBs. These results indicate that factors that may be derived from the steamed rice grains profoundly affect the digestion of proteins in steamed rice grains by sake koji enzymes.

T.Koseki, K.Takahashi, T.Handa, Y.Yamane, S.Fushinobu and K.Hashizume

A unique N-linked glycosylation motif (Asn79-Tyr-Thr) was found in the sequence of type-A feruloyl esterases from Aspergillus spp. To clarify the function of the flap, the role of N-linked oligosaccharides located in the flap region on the biochIical properties of feruloyl esterase (AwFAEA) from Aspergillus awamori expressed in Pichia pastoris was analyzed by rIoving the N-linked glycosylation recognition site by site-directed mutagenesis. N79 was replaced with A or Q. N-glycosylation-free N79A and N79Q mutant enzymes had lower activity than that of the glycosylated recombinant AwFAEA wild-type enzyme toward α-naphthylbutyrate (C4), α-naphthylcaprylate (C8), and phenolic acid methyl esters. Kinetic analysis of the mutant enzymes indicated that the lower catalytic efficiency was due to a combination of increased Km and decreased kcat for N79A, and to a considerably decreased kcat for N79Q. N79A and N79Q mutant enzymes also exhibited considerably reduced thermostability relative to the wild-type.

T.Koseki, Y.Miwa, Y.Mese, A.Miyanaga, S.Fushinobu, T.Wakagic, H.Shoun, H.Matsuzawa and K.Hashizume

A role for N-linked oligosaccharides on the biochIical properties of recombinant α-l-arabinofuranosidase 54 (AkAbf54) defined in glycoside hydrolase family 54 from Aspergillus kawachii expressed in Pichia pastoris was analyzed by site-directed mutagenesis. Two N-linked glycosylation motifs (Asn83- Thr- Thr and Asn202- Ser- Thr) were found in the AkAbf54 sequence. AkAbf54 comprises two domains, a catalytic domain and an arabinose-binding domain classified as carbohydrate-binding module 42. Two N-linked glycosylation sites are located in the catalytic domain. Asn83, Asn202, and the two residues together were replaced with glutamine by site-directed mutagenesis. The biochIical properties and kinetic parameters of the wild-type and mutant enzymes expressed in P. pastoris were examined. The N83Q mutant enzyme had the same catalytic activity and thermostability as the wild-type enzyme. On the other hand, the N202Q and N83Q/N202Q mutant enzymes exhibited a considerable decrease in thermostability compared to the glycosylated wild-type enzyme. The N202Q and N83Q/N202Q mutant enzymes also had slightly less specific activity towards arabinan and debranched arabinan. However, no significant effect on the affinity of the mutant enzymes for the ligands arabinan, debranched arabinan, and wheat and rye arabinoxylans was detected by affinity gel electrophoresis. These observations suggest that the glycosylation at Asn202 may contribute to thermostability and catalysis.

後藤（山本）奈美、Dang Hong Anh

アルコール発酵中の酵母による酢酸生成の機作を検討するため、ACS2, ACS3, ACS6, 及び ACH1の遺伝子破壊、並びに ACS1 及び ACS2の高発現が酢酸生成に及ぼす影響を実験室株を用いて検討した。ACH1 破壊及び ALD2、 ALD3 の二重破壊は酢酸生成に影響を及ぼさなかった。 ACS2 の高発現は酢酸生成を抑制したが、アルコール発酵も阻害した。既に報告されているように、ALD6 破壊は酢酸生成を減少させた。しかし、同時にピルビン酸を高生産し、これは還元力が不足したためと推定された。

S.T. Jeong, N.Goto-Yamamoto, K.Hashizume, S.Kobayashi and M.Esaka

VvmybA1 is known to encode a transcriptional factor that induces the anthocyanin biosynthesis in grape skins. However, no VvmybA1 mRNA was detected in the young leaves of Cabernet Sauvignon, Dornfelder, or Muscat Hamburg, nor in the tendrils or stI epidermis of Muscat Bailey A, although these organs accumulated a little anthocyanin. These results indicate that VvmybA1 in these cultivars was transcribed specifically in the berry skin. On the other hand, VvmybA1 of Bailey Alicant A, a teinturier cultivar, was transcribed in all the organs that accumulate anthocyanin, which shows that the organ specificity of its expression was probably reduced.

H.Wu, X.Zheng, Y.Araki, H.Sahara, H.Takagi and H.Shimoi

During the brewing of Japanese sake, Saccharomyces cerevisiae cells produce a high concentration of ethanol compared with other ethanol fermentation methods. We analyzed the gene expression profiles of yeast cells during sake brewing using DNA microarray analysis. This analysis revealed some characteristics of yeast gene expression during sake brewing and provided a scaffold for a molecular level understanding of the sake brewing.

C.Lin, Y.Hiraga, K.Masaki, H.Iefuji and K.Ohkata

The effect of tIperature on enantioselectivity and desymmetrization in the acetylation of 2-phenyl-1,3-propanediol (1a), 2-benzyl-1,3-propanediol (1b), 2-methyl-2-phenyl-1,3-propanediol (1c) and 2-benzyl-2-methyl-1,3-propanediol (1d)by a novel lipase (CSL) isolated from the yeast Cryptococcus spp. S-2 was studied. Desymmetrization of 1a, 1c and 1d by CSL-catalyzed acetylation was observed in the tIperature range of -20oC to 40oC, while diacetylation of 1b occurred considerably even at 0oC. The kinetic parameters of the selectivity indicated that the acetylation of 1a is an entropy controlled process whereas the reaction of 1c and 1d is mainly controlled by the enthalpy term. In the monoacetylation of the diol 1d, the preferred configuration in the enantiomeric induction by CSL was opposite to that of immobilized porcine pancreatic lipase (PPL).

髙橋美絵、磯谷敦子、宇都宮仁、中野成美、小泉武夫、戸塚昭

本研究では、清酒麹の香りを構成する香気成分の同定を行うとともに、各成分の香りへの寄与を検討した。まず、麹の香りの強度を官能的に評価し、HSSE法による麹のGC-O及びGC-MS分析により、Isobutyraldehyde、Isovaleraldehyde、1-Octen-3-olに加えて、キノコ様の香りを有する1-Octen-3-one、ナッツ様の2-Methyl-2-hepten-6-one、ジャガイモ様のMethional、バラ様のPhenylacetaldehydeやゼラニウム様の(Z)-1,5-Octadien-3-oneの5成分を新たに見出した。1-Octen-3-one、1-Octen-3-ol、Phenylacetaldehydeについては香りの弱い麹より香りの強い麹で含有量が多かった。1-Octen-3-one、MethionalのFlavor Dilution factors (FD) は125と最も高くなり、2-Methyl-2-hepten-6-one、Phenylacetaldehyde及び(Z)-1,5-Octadien-3-oneのFDは25と2番目に高くなった。2-Methyl-2-hepten-6-one、Methional、1-Octen-3-one、1-Octen-3-ol、Phenylacetaldehydeの5成分の試薬を用いて調合することにより麹の香りの大部分を再現することができた。以上の結果から、麹の香りにおいては主にMethional、1-Octen-3-one、Phenylacetaldehydeが重要であることが明らかとなった。

H.Izu, M.Shobayashi, Y.Manabe, K.Goto and H.Iefuji

Brewer's and baker's yeasts appear to have components that protect the liver injury. Whether Sake yeast, Saccharomyces cerevisiae Kyokai No.9, also has a hepatoprotective effect has not been examined. Here we show that Sake yeast suppresses acute alcoholic liver injury in mice. Male C57BL/6 mice that had been fed a diet containing 1% Sake yeast for two weeks received three doses of ethanol (5 g/kg BW). In the mice fed Sake yeast, ethanol-induced increases in triglyceride (TG) and glutamate pyruvate transaminase (GPT) were significantly attenuated and hepatic steatosis was improved. In addition, Sake yeast-fed mice showed a smaller decrease in hepatic S-adenosylmethionine (SAM) level and a smaller increase in plasma homocysteine (Hcy) level after ethanol treatment than the control mice, suggesting that a disorder of methonine metabolism in the liver caused by ethanol was relieved by Sake yeast. These results indicate that Sake yeast protects against alcoholic liver injury through maintenance of methionine metabolism in the liver.

伊豆英恵、後藤邦康、家藤治幸

マウスのD-ガラクトサミン誘発肝障害における酒粕投与の影響について検討を行った。カゼイン食に酒粕を10%添加し、マウスに2週間、混餌投与した。その後、肝障害を誘発させるためにD-ガラクトサミンを腹腔内投与（1200mg/kg）し、24時間後に採血を行った。肝障害の指標として血漿中のGPT及びGOTの値を調べたところ、D-ガラクトサミン投与による血漿GPT、GOTの上昇が酒粕投与によって有意に減少することが明らかとなった。また、D-ガラクトサミン投与で誘導されるDNA断片化が酒粕投与によって減少しており、細胞死の抑制が示された。以上より、酒粕投与がD-ガラクトサミン誘発肝障害を抑制することが示唆された。酒粕投与後、肝臓のSAM含量が増加しており、酒粕が肝臓のメチオニン代謝を強化することによって肝障害抑制に寄与する可能性がある。

Y. Haitania, H. Shimoi, H. Takagi

Rsp5 is an essential E3 ubiquitin ligase in Saccharomyces cerevisiae and is known to ubiquitinate plasma membrane permeases followed by endocytosis and vacuolar degradation. We previously isolated the rsp5 mutant that is hypersensitive to various stresses, suggesting that Rsp5 is involved in degradation of stress-induced abnormal proteins. Here, we analyzed the ability to refold the proteins by stress proteins in the rsp5 mutant. The transcription of stress protein genes in the rsp5 mutant was significantly lower than that in the wild-type strain when exposed to temperature up-shift, ethanol or sorbitol. Interestingly, the amounts of transcription factors Hsf1 and Msn4 were remarkably defective in the rsp5 mutant. These results suggest that expression of stress proteins are mediated by Rsp5 and that Rsp5 primarily regulates post-translational modification of Hsf1 and Msn4.

Yun-Hae LEE, M. Tominaga, R. Hayashi, K. Sakamoto, O. Yamada, O. Akita

Recently we divided Aspergillus oryzae RIB strains into group 1, having seven aflatoxin biosynthesis homologous genes (aflT, nor-1, aflR, norA, avnA, verB, and vbs), and group 2, having three homologues (avnA, verB, and vbs). Here, partial aflatoxin homologous gene cluster of RIB62 from group 2 was sequenced and compared with that of RIB40 from group 1. RIB62 showed a large deletion upstream of ver-1 with more than half of the aflatoxin homologous gene cluster missing including aflR, a positive transcriptional regulatory gene. Adjacent to the deletion of the aflatoxin homologous gene cluster, RIB62 has a unique sequence of about 8 kb and a telomere. Southern analysis of A. oryzae RIB strains with four kinds of probe derived from the unique sequence of RIB62 showed that all group 2 strains have identical hybridizing signals. Polymerase chain reaction with specific primer set designed to amplify the junction between ver-1 and the unique sequence of RIB62 resulted in the same size of DNA fragment only from group 2 strains. Based on these results, we developed a useful genetic tool that distinguishes A. oryzae group 2 strains from the other groups'strains and propose that it might have differentiated from the ancestral strains due to chromosomal breakage.

橋口知一、伊木由香理、後藤邦康

酒類中のカルバミン酸エチルを簡易に分析する方法について検討し、内部標準をカルバミン酸ブチルとし、試料を水で希釈せずに固相抽出を行い、分析機器を高感度のGC/MSとして濃縮倍率を小さくすることとした。酒類に内部標準としてカルバミン酸ブチルを加え、エキストレルートカラムに保持させた後、ヘキサン：酢酸エチル=1：1混合溶液で溶出し、ロータリーエバポレータ又は窒素ガス吹きつけによる濃縮装置により濃縮した。濃縮した試料はDB-WAXカラムを装着したGC/MSをSIMモードとして分析を行った。この分析法の検出限界は2.5μg/l、定量限界は7.5μg/l、100μg/l添加における変動係数は4.8%であった。この分析方法を紹興酒及び梅酒に応用すること試みた。清酒と同様の方法を用いて分析を行ったところ、良好な結果となったため、この分析方法は紹興酒及び梅酒に適用できると考えられた。これまでに示した試料の抽出・濃縮方法を応用し、20倍濃縮とした試料をFID-GCにより分析することにより、おおよそのカルバミン酸エチル含有量が推定できるものと考えられた。

橋口知一、後藤邦康

様々な酒類に含まれるフランの定量をガスクロマトグラフ質量分析計により行った。試料50点のうち、検出限界（0.5ppb）以下であったのは20点であった。最もフラン含有量が多かったのは紹興酒（18ppb）で、次に多かったのは貴醸酒古酒（7.9ppb）で、それ以外の酒類は全て5ppb未満であった。今回の酒類中のフランの分析値については、缶詰食品等の分析値に比べると、全般的に小さい値であった。

中野成美、宇都宮仁、磯谷敦子、平松順一

平成16酒造年度全国新酒鑑評会の出品酒について、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。

成分の平均値は、全体及び上位酒とも、アルコール分、日本酒度及び酸度については17.7%、4.0及び1.3であり、日本酒度が平成12年以前に比べて小さな値となっていた。香気成分については、上位酒で、イソアミルアルコール、酢酸イソアミル及びカプロン酸エチルが、それぞれ、123ppm、2.2ppm及び6.6ppmであった。

正井久美子、丸山潤一、坂本和俊、中島春紫、秋田修、北本勝ひこ

寒天固体培地上で生育させると、糸状菌であるAspergillus oryzae（麹菌）は形態的に異なる3つの領域、先端（T）、白色部分（W）及び基部（B）に区分することができる。本研究で我々は角形シャーレ法という新規な方法で各領域の菌糸からmRNAを抽出し、それらの遺伝子発現の差をマイクロアレイにて解析した。T領域ではタンパク質合成に関与する遺伝子群の発現が高く、6倍の発現量を示していた。WやBの領域では機能未知の遺伝子が高発現していた。さらに、B領域では膜輸送タンパク質等の発現が盛んだった。これら3領域の遺伝子発現の解析により、それぞれの領域で遺伝子発現の制御が大きく異なっていることが明らかとなった。これらのことから、形態形成や細胞分化に関わる領域特異的な遺伝子の単離・解析や工業的に有益な遺伝子の単離・解析に有益な培養方法を確立できたといえる。

M. Kawahata, K. Masaki, T. Fujii and H. Iefuji

We found that the acidic condition affects metal metabolism in this study using two types of genome wide analysis to investigate yeast genes involved in response to lactic acid and acetic acid. One is an expression analysis using DNA microarray to investigate yeast acid shock response as the first step to adapt to an acidic condition and yeast acid adaptation response as the result of maintaining integrity in the acidic condition. The other is a functional screening using the non-essential genes deletion collection of S. cerevisiae. The expression analysis showed that genes involved in stress response such as YGP1, TPS1, and HSP150 were induced under the acid shock response conditions. Genes such as FIT2, ARN1, and ARN2 involved in metal metabolism regulated by Aft1p were induced under the acid adaptation conditions. AFT1 was found to be induced under the acid shock response conditions and under the acid adaptation condition by lactic acid. Moreover, GFP-fused Aft1p was localized to the nucleus in cells grown in media containing lactic acid, acetic acid, or hydrochloric acid. The expression analysis and the functional screening both suggested that the acidic condition affects cell wall architecture. The depletion of cell wall components encoded by SED1, DSE2, CTS1, EGT2, SCW11, SUN4, and YNL300W and histone acetyltransferase complex proteins encoded by YID21, EAF3, EAF5, EAF6, and YAF9 increased resistance to lactic acid media. Depletion of the cell wall mannoprotein Sed1p gave resistance to lactic acid, although the expression of SED1 was induced by exposure to lactic acid. Depletion of V-ATPase and HOG MAPK proteins caused acid sensitivity. Moreover, our quantitative PCR analysis showed that expression of PDR12 increased under the acid shock response condition by lactic acid and decreased under the acid adaptation condition by hydrochloric acid.

橋爪克己、奥田将生、沼田美子代、田尻美加、小関卓也

小規模に清酒を醸造し実製造に近い条件でそれを圧搾したものについて窒素の挙動を分析した。原料米の窒素の約55%は清酒醪で溶解し、30-33%は製成酒に移行し、22-25%は酵母に移行した。清酒の窒素の約44-47%は遊離アミノ酸であり、窒素の残りの大部分はペプチドと推定された。酒粕とオリの窒素の17-20%は帰属が分からなかった。

福田央、周延、三上重明

市販清酒酵母を判別するために,広範囲のゲノム上のミニサテライト配列を含む22種のORFを対象にPCR反応による増幅DNA断片を検討した。YOR009W(TIR4), YDR150W(NUM1), YKR102W(FLO10), YDL037C(BSC1),及びYNL190W由来の5組のプライマーによる増幅DNA断片に差が認められた。清酒酵母でPCR産物に差異が認められないプライマーでも焼酎酵母では多型性が認められたことから,清酒酵母の増幅DNA断片の多型性が低い理由として当該酵母の遺伝子差異が他の醸造酵母と比較して低いことに起因すると推定された。

三上重明、福田央、佐藤雄一郎、平松順一

しょうちゅう乙類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28 回本格焼酎鑑評会を平成17年6月 2日（木）及び3日(金)に独立行政法人酒類総合研究所で開催した。出品酒の官能審査と成分分析の結果の概要について報告した。

橋爪克己、後藤奈美、小山和哉、平松順一

本鑑評会は、国内洋酒・果実酒メーカーから任意出品された果実酒類、ウイスキー、ブランデー、スピリッツ類及びリキュール類について官能審査、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業者の参考に資することを目的として実施している。審査会は、平成17年11月 21、22日の2日間にわたり、酒類総合研究所において開催した。出品酒の官能審査と成分分析の結果の概要について報告した。

宇都宮仁、磯谷敦子、岩田博、中野成美

清酒の官能評価分析における分析形試験法又は記述的試験法の標準用語として使用することを目的に、清酒の官能評価用語に関する過去の検討結果を基本に、清酒に添加した香味物質の閾値測定結果等を考慮して、清酒の香味に関する品質評価用語体系を取りまとめた。

清酒中に個々に確認することのできる香味特性を表す86の用語を、16のクラスに分類しさらにクラスの中を2つに階層化して定義を行った。可能な限りの用語の意味を標準見本（参照標準物質又は標準となる処理等）で説明するため、第1層は一般的な用語又は標準見本のある物質名とし、第2層はより分析的な用語又は標準見本のある物質名とした。43の用語に対して標準見本とその調整方法を定めた。また、用語体系の各用語の位置をわかりやすく表すため清酒のフレーバホイールを作成した。

T. Uryu, M. Sugie, S. Ishida, S. Konoma, H. Kato, K. Katsuraya, K. Okuyama, G. Borjihan, K. Iwashita, H. Iefuji

Ethanol was produced in a considerably high yield by fermenting hydrolyzates from cellulosic materials by means of a recombinant laboratory yeast expressingβ-glucosidases. Tissue paper, cotton, and sawdust were hydrolyzed by two-step sulfuric acid hydrolysis to give mixtures containing glucose, cellobiose, and high cello-oligosaccharides. After the cellulosic material was partially hydrolyzed with 80% sulfuric acid, the hydrolysis was continued with 5% sulfuric acid. Except for non-carbohydrate components, all constituents in the hydrolyzates were fermented by the yeast that was preincubated in the medium that the plasmid encoded by the β-glucosidases gene was kept in the multiplicated yeast. A solution containing 4% hydrolyzates from paper was fermented to give as high as 1.9% maximum ethanol concentration and 70% ethanol conversion. Cotton also gave a similar result. Sawdust was converted into ethanol in approx 22% conversion. Accordingly, it was revealed that the β-glucosidases-expressing yeast can ferment the cello-oligosaccharided obtained by hydrolysis of cellulosic materials into ethanol. In addition, a hydrolyzate cotaining a high glucose proportion gave a high ethanol concentration in a short time.

S. Ando, Y. Nishiguch, K Hayasaka, H. Iefuji, J. Takahashi

Candida utilis can assimilate fatty acids, so it was hypothesized that the treatment of rice by Candida utilis would improve feed quality by reducing fat content and adding the yeast function that would stimulate rumen microbes. In this study, the oil assimilation ability of Candida utilis IFO1086, 0988, 0626 and the effect of treatment of Candida utilis IFO1086, IFO0626 on the nutrient contents of rice bran were examined. The effect of Candida utilis adding on the in vitro degradability of forage was also investigated. It was found that the oil assimilating ability of IFO1086 and IFO0626 was significantly (P＜0.01) higher than that of IFO0988. Candida utilis treatment reduced the EE content and increased the CP, ADF and NDF percentage. The absolute amount of ether extract was decreased by 35.9% in IFO0626 treatment. The absolute amount of crude protein was not changed by yeast treatment. The ADF and NDF amounts were increased. The addition of Candida utilis increased in vitro forage degradability significantly (p＜0.05), Based on these results it can be postulated that treatment of rice bran by Candida utilis may improve feed quality by reducing fat content, increasing the CP content and adding the function of yeast for stimulating rumen microbes.

折原佑輔、和気洋子、宇都宮仁、青島均

樽酒中の、エステル、高級アルコール、セスキテルペン類をガスクロマトグラフで測定した。グルコース及び490nmにおける吸光度も測定した。杉樽へ清酒を貯蔵すると、セスキテルペン類と490nmにおける吸光度が増加した。しかし、エステル、高級アルコール、グルコースは貯蔵による影響はなかった。アフリカツメガエル卵母細胞で発現させたGABAA受容体に清酒は応答を引き起こし、GABAの存在を示した。しかし、ウイスキーとは異なり、樽貯蔵した酒のペンタン抽出物がGABAA受容体の応答を増強することはなかった。杉樽貯蔵した清酒は、DPPHラジカル消去活性とポリフェノールが増加していた。

宇都宮仁、木田信、牧則光、磯谷敦子、岩田博、西谷尚道

焼酎とホワイトスピリッツのフレーバーを類別するため、28焼酎乙類、2甲乙混和焼酎、3焼酎甲類、4中国白酒、3ウオッカ、3ホワイトラム、3ジン及び4その他の蒸留酒について、20揮発成分をヘッドスペースSPME法、7揮発成分を直接ヘッドスペース法で、2揮発成分を酵素法で測定した。酸度、TBA価、pHについても測定した。クラスター解析においてこれら32因子を使用すれば、焼酎乙類は他の蒸留酒と区別された。ステップワイズ型の判別分析手法において、酢酸、酢酸エチル、イソアミルアルコールとドデカノールを使用すれば、焼酎乙類とライトスピリッツ（焼酎甲類、甲乙混和焼酎、ウオッカ、ホワイトラム）、中国白酒の3つのグループに分かれた。また、メタノール、1-プロパノール、イソブタノールとカプリル酸エチルを使用すれば、焼酎乙類を6つの原料別カテゴリーに分類可能であった。

H. Yakushhi, S. Kobayashi, N. Goto-Yamamoto, S.T. Jeong, T. Sueta, N. Mitani, A. Azuma

A white-wine grape, Pinot Blanc, is thought to be a white-skinned mutant of a red-wine grape, Pinot Noir. Pinot Noir was heterozygous for VvmybA1. One allele was the non-functional VvmybA1a, and the other was the functional VvmybA1c. In Pinot Blanc, however, only VvmybA1a was observed, and the amount of VvmybA1 DNA in Pinot Blanc was half that in Pinot Noir. These findings suggest that deletion of VvmybA1c from Pinot Noir resulted in Pinot Blanc.

K. Oda, D. Kakizono, O. Yamada, H. Iefuji, O. Akita, and K. Iwashita

Filamentous fungi are widely used for the production of homologous and heterologous proteins. Recently, there has been increasing interest in Aspergillus oryzae because of its ability to produce heterologous proteins in solid-state culture. To provide an overview of protein secretion by A. oryzae in solid-state culture, we carried out a comparative proteome analysis of extracellular proteins in solid-state and submerged (liquid) cultures. Extracellular proteins prepared from both cultures sequentially from 0 to 40 h were subjected to twodimensional electrophoresis, and protein spots at 40 h were identified by peptide mass fingerprinting using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. We also attIpted to identify cell wall-bound proteins of the submerged culture. We analyzed 85 spots from the solid-state culture and 110 spots from the submerged culture. We identified a total of 29 proteins, which were classified into 4 groups. Group 1 consisted of extracellular proteins specifically produced in the solid-state growth condition, such as glucoamylase B and alanyl dipeptidyl peptidase. Group 2 consisted of extracellular proteins specifically produced in the submerged condition, such as glucoamylase A (GlaA) and xylanase G2 (XynG2). Group 3 consisted of proteins produced in both conditions, such as xylanase G1. Group 4 consisted of proteins that were secreted to the medium in the solid-state growth condition but trapped in the cell wall in the submerged condition, such as α-amylase (TAA) and β-glucosidase (Bgl). A Northern analysis of seven genes from the four groups suggested that the secretion of TAA and Bgl was regulated by trapping these proteins in the cell wall in submerged culture and that secretion of GlaA and XynG2 was regulated at the posttranscriptional level in the solid-state culture.

野村佳司、金子一憲、佐藤敬蔵、木曽邦明、水野昭博

特定の波長より短波長側を遮蔽し、長波長側を透過するシャープカットフィルターをとおして清酒に日光を照射することによって、日光の波長が清酒の劣化へ与える影響を調べた。清酒の劣化を測定するためには、シャープカットフィルターをとおして日光を照射した清酒の着色度、ハルマンの濃度、抗酸化性、TBA反応値そして3DG濃度を分析した。その結果、440nmより短波長の日光が清酒を劣化し、440nmより長波長の日光は清酒を劣化しないことが判明した。したがって、440nmより短波長の光を遮蔽し、440nmより長波長の光の透過率を改善したガラス瓶の製作を試みた。そして、日光に照射しても清酒が劣化せず、現在の茶瓶より透明性を確保したガラス瓶を開発した。

福田央、周延、三上重明

市販醸造酵母を判別するために, AGA1, DAN4, HSP150及びSED1のORF由来のプライマーによるPCRを試みた。焼酎酵母及びブドウ酒酵母では増幅DNA断片の長さに多型性が認められ, 供試したブドウ酒酵母はすべて, 焼酎酵母は8株を判別することができた。しかし、清酒酵母では多型性の程度が低く, K-11及びK-9以外は個別に判別できなかった。PCR法により醸造酵母を判別する方法は, 従来の生理学的性質を利用する方法に比べて容易であることから, これらのプライマーを用いる醸造酵母の識別は有用であった。

渡部貴志、鈴木修、藤井力、尾崎則篤、正木和夫、家藤治幸

排水処理用酵母として排水中から分離された酵母を主とした約50株を用い、エチレンジアミン(以下EDA)、ヘキサメチレンジアミン(以下HMDA)資化酵母のスクリーニングを行った。その結果、高温耐性実用排水処理酵母Hansenula fabianii J640、実用排水処理酵母Hansenula anomala J224-1、飼料用酵母Candida utilis IFO0626がEDA、HMDAを窒素源として資化し、増殖することを見出した。次にこれら3種の酵母を用いてEDA、HMDAを唯一の窒素源とする模擬排水の処理試験を行った。最もジアミン除去能力が優れていたのはC. utilis IFO0626であり、48hr後でEDA、HMDA(二塩酸塩で1000ppm)共に98%以上の除去率を示した。続いて炭素源の添加量を検討したところ、ジアミンニ塩酸塩 1gに対し、glucose 10g（C:N比=100：5）必要であった。また植菌量を増やすことで処理時間を短縮できる可能性が示唆された。

後藤（山本）奈美

DNA多型解析を用いた醸造用ブドウ栽培品種および野生種の類縁関係の推定と品種の同定に関する筆者らの研究と,これに海外の代表的な報告加えて,これらを総説にした。RAPD解析法,並びにさらに検出感度の高いAFLP解析法を用いて非共有率の非加重平均率から樹形図を作成した。その結果,これまで生態や形態で分類されていた品種の違いが遺伝子レベルの差異を反映したものであることが明らかになった。また品種「甲州」の類縁関係をより明らかにした。精度が高く,近縁間の差異検出や連鎖解析等に利用されるマイクロサテライト(SSR)解析について,海外のおける多品種のデータ,原産地アサインに対して,数種の東洋系品種のグループアサイメントテストを行った。その結果,品種特有のSSRデータの偏りが認められた。

T. Kaneko, K. Ito, T. Izumi, A. Matsuyama, N. Mukai, T. Ohuchi, M. Takeuchi, S. Tatematsu, and T. Teramoto

Determination of Alcohol and Real Extract by Anton Paar Alcolyzer was reported. No statistically significant differences were found between the Anton Paar Alcolyzer and SCABA methods for the determination of alcohol in beer, happou-shu, and nonalcoholic beer. The Anton Paar Alcolyzer method resulted in significantly higher real extract results than did the SCABA method for beer, happou-shu, and nonalcoholic beer. The repeatability and reproducibility coefficients of variation were judged acceptable.