平成25年度 研究成果

Nami Goto-Yamamoto, Kazuya Koyama, Kaori Tsukamoto, Hiroshi Kamigakiuchi, Masanori Sumihiro, Masaki Okuda, Tomokazu Hashiguchi, Katsumi Matsumaru, Haruhito Sekizawa, and Hitoshi Shimoi

The food-processing transfer parameters of radioactive and stable cesium, as well as radioactive potassium, were determined from grapes to wine. The concentration of cesium in the pomace was higher than that in juice, as was the case of potassium. During white and blush wine fermentation, cesium concentration did not change significantly, while potassium concentration decreased. These results suggest that the absorbance of cesium by yeast is much lower than that of potassium in the wine-making environment. Generally, the food-processing retention factor (Fr, content in wine/content in grape) of radiocesium and stable cesium for red wine were higher than those for white wine, reflecting the yields of wine and the extraction of cesium during maceration.

Katsumi Hashizume, Toshihiko Ito, Airi Nakayama, and Masaki Okuda

Ferulic and p-coumaric acids were analyzed in 50 rice (Oryza sativa L.) samples from 32 cultivars harvested in Japan. In brown rice, ferulic and p-coumaric acid levels ranged from 309 to 607 mg/kg and from 49 to 100 mg/kg, respectively. In 70% polished rice, ferulic and p-coumaric acid levels ranged from 27 to 103 mg/kg and from 0.4 to 3.5 mg/kg, respectively. Ratios of average phenolic acid levels in the 70% polished rice to the brown rice were 13.9% for ferulic acid and 1.9% for p-coumaric acid. The ferulic acid level was highly correlated between brown and 70% polished rice (R = 0.815; P < 0.01), but there was no clear correlation for p-coumaric acid. Phenolic acid levels in the 70% polished rice did not show any clear correlations between the analytical index measurements for sake brewing suitability (weight of 1,000 grains, water absorption, digestibility, crude protein, and potassium content). Phenolic acid levels in the 70% polished rice directly affected levels in the rice koji enzyme digest. The results indicated that phenolic acid levels in sake were affected by the levels in ingredient rice grains, which may then influence the sensory quality of sake.

Tsunehiro Aki, Masaya Yamamoto, Toshiaki Takahashi, Kohki Tomita, Rieko Toyoura, Kazuhiro Iwashita, Seiji Kawamoto, Masashi Hosokawa, Kazuo Miyashita, Kazuhisa Ono

The effects of a seaweed carotenoid, fucoxanthin, and its physiological metabolite, fucoxanthinol, on the biosynthesis of polyunsaturated fatty acids (PUFA) were investigated using cultured rat hepatoma BRL-3A. The metabolism of α-linolenic acid (18:3n-3) was suppressed by the addition of these carotenoids, resulting in a decrease in the content of eicosapentaenoic acid (20:5n-3), which suggested a down-regulation of metabolic enzymes such as fatty acid desaturase and elongase. An increase in the content of docosahexaenoic acid (22:6n-3), as observed in previous studies in vivo, might be a buffering action to maintain the membrane fluidity. The suppressive effect of fucoxanthinol on Δ6 fatty acid desaturase was not at the level of gene expression but due to specific modifications of the protein via a ubiquitin-proteasome system. A proteomic analysis revealed several factors such as phosphatidylethanolamine-binding protein that might be involved in the observed action of fucoxanthin. These findings will contribute to studies on the elucidation of the precise molecular mechanisms underlying the regulation of PUFA biosynthesis by fucoxanthin.

福田央、韓錦順

本格焼酎のフルフラール、5-ヒドロキシメチル-2-フルアルデヒド及び5-メチル-2-フルアルデヒドを固相マイクロ抽出法(Solid Phase Micro Extraction, 以下SPMEと略す) 及びGC/MSを用いて分析を行い、各々の濃度を定量した。常圧蒸留及び減圧蒸留の酒粕焼酎、減圧蒸留のその他の焼酎の区分では、5-ヒドロキシメチル-2-フルアルデヒド含量の平均値が最も高かったが、それ以外の焼酎ではフルフラール含量の平均値が最も高かった。以上の結果から、一部を除き焼酎に含まれるフルフラール類の一般的な主成分はフルフラールと考えられた。フルフラール含量の平均値は麦焼酎の常圧蒸留で最も高く、次いで泡盛、甘藷焼酎に比較的多く含まれており、常圧蒸留と減圧蒸留では常圧蒸留で高い傾向があった。5-ヒドロキシメチル-2-フルアルデヒドと5-メチル-2-フルアルデヒドは、樽貯蔵で高い割合で認められたことから樽由来の成分の関与が考えられた。

T. Hashimoto, T. Handa, Y. Kakudo, M. Kanai, K. Kaneko, C. Kenjo, M. Nakahara, W. Nakamura, A. Ohuchi, T. Watanabe

1. Relative repeatability standard deviation (RSDr) and repeatability limit (r95) for determination of total purine content using the HPLC-UV method ranged from 0.8 to 4.6% and 1.7 to 7.6 mg/L, respectively, and were judged acceptable.
2. Relative reproducibility standard deviation (RSDR) and reproducibility limit (R95) for determination of total purine content using the HPLC-UV method ranged from 11.6 to 16.8% and 8.0 to 49.5 mg/L, respectively, and were judged acceptable.

Daisuke Watanabe, Naoya Hashimoto, Megumi Mizuno, Yan Zhou, Takeshi Akao, and Hitoshi Shimoi

Sake yeast strains maintain a high fermentation rate even after entering the stationary growth phase. To investigate the molecular mechanisms underlying this advantageous brewing property, we compared the gene expression profiles of sake and laboratory yeast strains of Saccharomyces cerevisiae during the stationary growth phase. DNA microarray analysis revealed that the examined sake yeast strain had defects in expression of the glucose derepression-related genes mediated by the transcription factors Adr1p and Cat8p. Furthermore, deletion of the ADR1 and CAT8 genes slightly, but statistically significantly, improved the fermentation rate of a laboratory yeast strain. We also identified two loss-of-function mutations in the ADR1 gene of existent sake yeast strains. Taken together, these results demonstrate that the glucose derepression-related gene expression program of yeast acts as an impediment to effective alcoholic fermentation in the glucose-rich fermentative conditions.

Tomomi Inai, Daisuke Watanabe, Yan Zhou, Rie Fukada, Takeshi Akao, Jun Shima, Hiroshi Takagi, and Hitoshi Shimoi

Inherited loss-of-function mutations in the Rim15p-mediated stress-response pathway contribute to the high fermentation rate of sake yeast strains. In the present study, we found that disruption of the RIM15 gene in ethanol-producing Saccharomyces cerevisiae strain PE-2 accelerated molasses fermentation through enhanced sucrose utilization following glucose starvation.

Takuya Inaba, Daisuke Watanabe, Yoko Yoshiyama, Koichi Tanaka, Jun Ogawa, Hiroshi Takagi, Hitoshi Shimoi, and Jun Shima

Bacterial contamination is known as a major cause of the reduction in ethanol yield during bioethanol production by Saccharomyces cerevisiae. Acetate is an effective agent for the prevention of bacterial contamination, but it negatively affects the fermentation ability of S. cerevisiae. We have proposed that the combined use of organic acids including acetate and lactate and yeast strains tolerant to organic acids may be effective for the elimination of principally lactic acid bacterial (LAB) contamination. In a previous study employing laboratory S. cerevisiae strains, we showed that overexpression of the HAA1 gene, which encodes a transcriptional activator, could be a useful molecular breeding method for acetate-tolerant yeast strains. In the present study, we constructed a HAA1-overexpressing diploid strain (MATa/α, named ER HAA1-OP) derived from the industrial bioethanol strain Ethanol Red (ER). ER HAA1-OP showed tolerance not only to acetate but also to lactate, and this tolerance was dependent on the increased expression of HAA1 gene. The ethanol production ability of ER HAA1-OP was almost equivalent to that of the parent strain during the bioethanol production process from sugarcane molasses in the absence of acetate. The addition of acetate at 0.5% (w/v, pH 4.5) inhibited the fermentation ability of the parent strain, but such an inhibition was not observed in the ethanol production process using ER HAA1-OP.

Satoshi Wakai, Shun-ichi Kidokoro, Kazuo Masaki, Kaoru Nakasone, Yoshihiro Sambongi

A decrease in water activity was thought to result in smaller enthalpy change values during PPi hydrolysis, indicating the importance of solvation for the reaction. Howeve, the physiological significance of this phenomenon is unknown. Here, we combined biochemistry and calorimetry to solve this problem using NaCl, a physiologically occurring water activity-reducing reagent. The pyrophosphatase activities of extremely halophilic Haloarcula japonica, which can grow at approximately 4 M NaCl, and non-halophilic Escherichia coli and Saccharomyces cerevisiae were maximal at 2.0 and 0.1 M NaCl, respectively. Thus, halophilic and non-halophilic pyrophosphatases exhibit distinct maximal activities at different NaCl concentration ranges. Upon calorimetry, the same exothermic enthalpy change of -35 kJ/mol was obtained for the halophile and non-halophiles at 1.5-4.0 and 0.1-2.0 M NaCl, respectively. These results show that solvation changes caused by up to 4.0 M NaCl (water activity of approximately 0.84) do not affect the enthalpy change in PPi hydrolysis. It has been postulated that PPi is an ATP analog, having a so-called high energy phosphate bond, and that the hydrolysis of both compounds is enthalpically driven. Therefore, our results indicate that the hydrolysis of high energy phosphate compounds, which are responsible for biological energy conversion, is enthalpically driven within the physiological limits of NaCl.

Takashi Watanabe, Haruyuki Iefuji, Hiroko K. Kitamoto

Shochu wastewater (alcoholic distillery wastewater) contains large amounts of organic compounds (25,000 - 60,000 COD mg/L), nitrogen (1,000 - 6,000 T-N mg/L), and phosphorus (500 - 1,000 T-P mg/L). Despite its high nutrient content, shochu wastewater is highly perishable, which limits its utilization for animal feed and fertilizer. Therefore, shochu wastewater is mainly treated by methane fermentation. On the other hand, a feed yeast, Candida utilis, can utilize various organic compounds and be utilized as a yeast extract source and animal feed. We previously bred a mutant, C. utilis UNA1, that accumulates a large amount of nitrogen. Here, we investigated the use of C. utilis UNA1 to treat highly concentrated shoshu wastewater. With fed-batch cultivation using a 5-L jar fermenter, controlling pH at 5.0 with H₂SO₄, 62.9% of DOC, 38.4% of DTN, and 44.5% of DTP were stably removed from non-diluted barley shochu wastewater (BSW), and about 16.7 kg of freeze-dried yeast biomass was obtained. The yeast sludge biomass generated from BSW contains about 60% crude protein. Furthermore, using H₂SO₄ to control pH increased the sulfur content of wastewater, which increased the methionine composition of yeast sludge biomass.

渡部貴志、家藤治幸、北本宏子

本研究では、排水処理酵母の窒素除去能力の強化を目的に検討を行った。先に我々は、酵母Saccharomyces cerevisiae を用い、URE2 変異株の取得を目的にしたメチルアミン耐性変異株のポジティブスクリーニングの方法が、酵母の窒素除去能力の強化にも有効であることを確認している。そこで今回我々は、同じ手法を用いて排水処理酵母での窒素除去能力の強化を試み、その結果H. anomala IFO0963-HNA36を得た。HNA36株は米焼酎蒸留粕排水から、親株の1.2倍量の窒素を除去した。また、我々は別の新たな窒素除去能力が強化された変異株の育種方法の検討も行った。その結果、プロテアーゼ活性恒常化変異株Pseudozyma antarctica GB-4(0)-PNR26株を得ることに成功した。PNR26株は米焼酎蒸留粕排水から、親株の1.14倍量の窒素を除去した。以上の結果より、排水処理酵母の窒素除去能力は、遺伝子組み換えを用いない手法でも強化できることを示せた。

Shingo Tomiyama, Tomokazu Hashiguchi, Akira Ueda, Akiko Kitai, Hanae Izu and Shigetoshi Sudo

Understanding the source and chemical characteristics of groundwater used for sake production is an important strategy for the quality management of sake brewing. Oxygen isotope ratios (δ¹⁸O) of 96 sake samples from 19 prefectures in Japan were analyzed. A good correlation was found between the sake δ¹⁸O values and groundwater δ¹⁸O values taken from previously published data, where the δ¹⁸O of sake is similar to that of the groundwater used in the sake breweries. The combined geochemical and numerical modeling approach provides a good tool for determining the source of groundwater used in sake production.

後藤（山本）奈美、沼田美子代、古川準三

北醇は中国で育種されたVitis amurensis とV. vinifera cv. Muscat Hamburg の交配品種で。北海道でMH-AMとして栽培されている品種は北醇またはその実生であると考えられるが、由来が明らかでなかった。SSR解析の結果、北醇とMH-AMは解析した14のSSR遺伝子座すべてで一致し、同じ品種であることが確認された。さらに、北醇の少なくとも一方のSSRデータは、1遺伝子座を除いてMuscat Hamburgと一致すること、V. amurensis、V. coignetiae、及び未同定の野生ブドウとも共通のアレルが多く検出された。さらに主座標分析を行ったところ、北醇はMuscat Hamburg及びV. amurensisの近傍にプロットされた。

Masaki Okuda, Tomokazu Hashiguchi, Midori Joyo, Kaori Tsukamoto, Michiko Endo, Katsumi Matsumaru, Nami Goto-Yamamoto, Hiroshi Yamaoka, Kenji Suzuki, and Hitoshi Shimoi

Using rice grains contaminated with radioactive cesium (¹³⁴Cs and ¹³⁷Cs) that was released by the Fukushima Daiichi Nuclear Power Plant Accident in March of 2011, we investigated the behaviors of the radioactive cesium and potassium (total K and 40K) during sake brewing. Cesium is a congener of K, and yeast cells have the ability to take up Cs using known K transporters. During rice polishing, the concentrations of radioactive Cs and K in the polished rice grains decreased gradually until a milling ratio (polished rice weight/brown rice weight) of 70% was reached. No significant changes were observed below this milling ratio. Sake was brewed on a small scale using the 70% polished rice. The transfer ratio of radioactive Cs to sake and to the sake cake was significantly different than the ratio of K. Approximately 36% and 23% of radioactive Cs in the polished rice was transferred to the sake and sake cake, respectively; however, 40% was removed by washing and steeping the rice grains. On the other hand, 25% and 40% of K in the polished rice was recovered in the sake and sake cake, respectively, and 35% was removed by washing and steeping the rice grains. From the present results, the concentration of radioactive Cs in sake would be 4 Bq/kg fresh weight, which is well below the regulation values (100 Bq/kg), even using brown rice containing 100 Bq/kg of radioactive Cs.

Kou Wakabayashi, Atsuko Isogai, Daisuke Watanabe1, Akiko Fujita, Shigetoshi Sudo

Dimethyl trisulfide (DMTS) is one of the components responsible for the unpalatable aroma of stale Japanese sake, called "hineka". Recently, a precursor compound of DMTS, 1,2-dihydroxy-5-(methylsulfinyl)pentan-3-one (DMTS-P1), was identified. It was speculated that the yeast methionine salvage pathway (MTA cycle) might participate in the formation of DMTS-P1, because the chemical structure of DMTS-P1 was similar to one of the intermediate compounds of that pathway. Here, we carried out sake brewing tests using laboratory yeast strains with disrupted MTA cycle genes and found that DMTS-P1 was hardly produced by Δmeu1, Δmri1, and Δmde1 strains. Furthermore, the DMTS producing potential (production of DMTS during storage of sake) decreased in sake made with Δmri1 and Δmde1. We constructed sake yeast strains with a disrupted MRI1 or MDE1 gene and confirmed a decline in the DMTS-P1 content and DMTS producing potential of sake made with these disruptants. The results of sake brewing tests using MTA cycle disruptants suggested that SPE2 is responsible for the production of DMTS precursors other than DMTS-P1: although the DMTS-P1 content was higher in Δspe2 sake than in Δmri1 or Δmde1 sake, the DMTS producing potential of Δspe2 sake was as low as that of Δmri1 or Δmde1 sake. Sake brewing tests using BY4743 Δspe2 Δmri1 double disruptants revealed that the DMTS producing potential was further decreased as compared with the Δspe2 or Δmri1 single disruptant. These results suggest that MRI1, MDE1, and SPE2 are promising targets for breeding yeast to suppress the formation of DMTS during storage of sake.

Akihiro Mizuno

The relationships among flavor stability, antioxidant activity, and aging-related indices of beer were examined in traditional all-malt and adjunct beer, Happoshu (a beer-like beverage with less than 67% malt content), and New-genre (a beer-like beverage without malt, or Happoshu diluted with spirit) beverages. Flavor stability was evaluated by assessing flavor-staling degree using sensory analysis after an accelerated storage test at 30℃. Hydroxyl radical, the most reactive oxygen species, is considered a major determinant for flavor stability of beer. However, the staling degree measure exhibited a more significant correlation with 1,1'-diphenyl-2-picrylhydrazyl (DPPH)-scavenging activity than the hydroxyl radical-scavenging activity. The lag time of the hydroxyl radical generation analyzed by electron spin resonance spectroscopy is now used as an index of flavor stability of beer; it is thought that longer lag times indicate higher antioxidant activity of beer. However, a markedly longer lag time was observed in Happoshu and New-genre than in beer, although Happoshu and New-genre showed lower antioxidant activities than beer. Lag time did not appear to be applicable as an index of flavor stability for Happoshu and New-genre. DPPH-scavenging activity was found to be a more suitable and a common index for assessing flavor stability in not only traditional all-malt and adjunct beers but also Happoshu and New-genre, depending mostly on polyphenol-mediated antioxidant activity. In a brewing fermentation test, changes in DPPH-scavenging activity during fermentation were not observed, which suggests that the antioxidant activity of casting wort could be critical to the flavor stability of not only beer but also beer-like beverages.

Katsumi HASHIZUME, Toshihiko ITO, Misako SHIMOHASHI, Takahiro ISHIZUKA, and Masaki OKUDA

Ferulic acid (FA) and ethyl ferulate (EF) in sake and mirin samples were quantified. Concentrations of FA and EF in the sake and mirin samples showed high correlations (r=0.91 and 0.89, respectively). The highest level of EF in the sake samples was ca. 14-fold that of the mirin samples. Thresholds of FA and EF in a sake sample were estimated using a pipette method as 0.075 mg/L and 0.39 mg/L, respectively, by eight assessors in their twenties. The FA threshold was far lower than the highest level of FA in the sake samples, which suggested that FA might affect the sensory quality of sake. FA added to the sake sample showed unpleasant bitter, astringent, "egumi", or irritating taste characteristics. Sensory and instrumental analyses suggested that EF has the ability to mitigate the taste of FA in sake.

Satoshi Tashiro, Taiki Futagami, Shotaro Wada, Yasuhiro Kajiwara, Hideharu Takashita, Toshiro Omori, Toru Takahashi, Osamu Yamada, Kaoru Takegawa, and Masatoshi Goto

Aspergillus kawachii IFO4308 is a commonly used strain for research into shochu-making and enzyme production without the technique of genetic engineering. In the present study, we successfully constructed various A. kawachii host strains with the ability of highly efficient homologous recombination. In addition to the genome information about A. kawachii, these strains are useful for development of genetic study concerning shochu making and industrial productions of proteins and organic acids.

伊豆英恵、橋口知一、橋本知子、松丸克己

本研究では、みりん市販品を試料とし、みりんのエキス分（δ¹³C_Ex）とアルコール分(δ¹³C_Alc)の炭素安定同位体比分析を試み、原料判別の可能性について検討した。アルコールが添加されたみりんのδ¹³C_Alc(-14.6 ± 4.0‰)は焼酎が添加されたみりんのδ¹³C_Alc(-26.0 ± 1.7‰)と比べ、有意に高くなった。また、糖添加で有意にみりんのδ¹³C_Exが高くなった(+糖; -17.1 ± 3.3‰, -糖; -26.1 ± 1.0‰)。米、米麹、焼酎のみで仕込みを行った旧式みりんのδ¹³C_Alcとδ¹³C_Ex(δ¹³C_Alc ; -25.8 ± 1.1‰, δ¹³C_Ex; -26.6 ± 0.9‰)はアルコールや糖類を添加した新式みりん(δ¹³C_Alc ; -16.3 ± 0.8‰, δ¹³C_Ex ; -19.8 ± 0.7‰)よりも低い値を示した。みりん類似調味料であるみりん風調味料及び発酵調味料、灰持酒についても、アルコールや糖添加でδ¹³Cが高くなった。以上から、みりん市販品において、糖やアルコール、焼酎の添加がδ¹³Cによって判別できる可能性が示唆された。

磯谷敦子、神田涼子、須藤茂俊、松丸克己

SIDA法によるDMTS-P1分析方法を設定した。内部標準としてメチル基を3つの重水素で標識したDMTS-P1-d₃を用いた。さまざまな試料について回収率を求めたところ、100±10%程度となった。この方法を用いて市販酒類の分析を行ったところ、清酒はビールやワインに比べてDMTS-P1濃度が高い傾向がみられた。清酒酵母、ビール酵母、ワイン酵母を同一培地で培養すると、清酒酵母は他の酵母に比べてDMTS-P1生成量が多かった。DMTS-P1とDMTS生成ポテンシャル（貯蔵によって生じるDMTS量）との関係については、清酒では相関がみられたが、ワインでは相関がみられず、DMTS-P1以外の成分の関与が示唆された。清酒小仕込み試験を行いDMTS-P1の消長を調べたところ、発酵に伴って増加し、上槽後にもやや増加がみられた。発酵温度が高いとDMTS-P1濃度、DMTS生成ポテンシャルともに増加した。

松丸克己、磯谷敦子、伊豆英恵、神田涼子、木崎康造

平成23酒造年度全国新酒鑑評会（第100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の共催により実施した。

審査は、酒類総合研究所広島事務所において、平成24年4月24日（火）から26 日（木）の3日間に予審を行い、5月8日（火）及び9日（水）に決審を行った。また、5月23日（水）に東広島運動公園体育館で製造技術研究会を、6月15日（金）に東京池袋のサンシャインシティ・文化会館展示ホールで公開きき酒会を開催した。

出品は、876点であった。審査の結果、優秀と認められた427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246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、1,444人及び2,907人が来場した。

出品酒については、審査に加え酒質の現状及び動向を調査研究するため、出品者の記載による「全国新酒鑑評会出品酒調査表」（以下、「調査表」という。）の内容を集計するとともに成分分析を行った。

山田修、水谷治、金井宗良、木崎康造

単式蒸留しょうちゅうの品質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把握するとともに製造業者の参考とするため、第34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24年5月 31日（木）及び6月1日(金)に行い、製造技術研究会は6月22日（金）に当所で開催し、出品関係者の参考に供した。

橋口知一、伊豆英恵、松丸克己

平成23年3月に発生した東京電力株式会社福島第一原子力発電所の事故により、大量の放射性物質が放出された。酒類の安全性の確保のため、酒類製造場内にある出荷前の酒類1,904点、醸造用水502点及び小売店から購入した市販酒類1,558点、計3,964点の放射性セシウムの濃度を分析した。いずれの試料からも、食品衛生法の基準値を超える放射性セシウムは検出されなかった。これは、原子力発電所の事故以降、食品衛生法により一定濃度以上の放射性物質を含む食品については流通しない措置が取られていること、地下水の放射性セシウム汚染は考えにくいこと、酒類の製造工程においては放射性セシウムが減少することなどが、その理由として考えられる。

Moriyuki Kawauchi, Mika Nishiura, Kazuhiro Iwashita.

The sirtuins are members of the NAD+-dependent histone deacetylase family that contributes to various cellular functions that affect aging, disease, and cancer development in metazoans. However, the physiological roles of the fungal-specific sirtuin family are still poorly understood. Here, we determined a novel function of the fungal-specific sirtuin hstD/Aohst4, which is a homolog of yeast hst4 in Aspergillus oryzae. The deletion of all histone deacetylases in A. oryzae demonstrated that fungal-specific sirtuin hstD/Aohst4 is required for the coordination of fungal development and secondary metabolite production. We also showed that the gene expression of laeA, which is the most studied fungal-specific coordinator for the regulation of secondary metabolism and fungal development, was induced in the ΔhstD strain. Genetic interaction analysis of hstD/Aohst4 and laeA clearly indicated that hstD/Aohst4 works upstream of laeA to coordinate secondary metabolism and fungal development. The hstD/Aohst4 and laeA genes are fungal-specific but conserved in the vast family of filamentous fungi. Thus, we concluded that the fungal-specific sirtuin hstD/Aohst4 coordinates fungal development and secondary metabolism via the regulation of laeA in filamentous fungi.

Nami Goto-Yamamoto, Akifumi Azuma, Nobuhito Mitani and Shozo Kobayashi

To determine the relationship among wild grape species and grape cultivars, genotyping with 8 SSR loci was carried out using a wide range of grape species and cultivars, i.e., North American species, East Asian species, cultivars of Vitis vinifera, and those of V. vinifera × V. labrusca. SSR genotyping showed that wild grape species are more highly diverse than cultivated grapes. A principal coordinate analysis based on the distance (1- proportion of shared alleles) among accessions showed a clear separation between wild and cultivated grapes. A native Japanese cultivar, 'Koshu', and a Chinese one, 'Niunai', were plotted among the cultivars of V. vinifera, even though these 2 Far East cultivars have some unique alleles.

Seung-Beom Hong, Mina Lee, Dae-Ho Kim, Janos Varga, Jens C. Frisvad, Giancarlo Perrone, Katsuya Gomi, Osamu Yamada, Masayuki Machida, Jos Houbraken, Robert A. Samson

Aspergilli known as black- and white-koji molds which are used for awamori, shochu, makgeolli and other food and beverage fermentations, are reported in the literature as A. luchuensis, A. awamori, A. kawachii, or A. acidus. In order to elucidate the taxonomic position of these species, available ex-type cultures were compared based on morphology and molecular characters. A. luchuensis, A. kawachii and A. acidus showed the same banding patterns in RAPD, and the three species had the same rDNA-ITS, β-tubulin and calmodulin sequences and these differed from those of the closely related A.niger and A. tubingensis. Morphologically, the three species are not significantly different from each other or from A. niger and A. tubingensis. It is concluded that A. luchuensis, A. kawachii and A. acidus are the same species, and A. luchuensis is selected as the correct name based on priority. Strains of A. awamori which are stored in National Research Institute of Brewing in Japan, represent A. niger (n = 14) and A. luchuensis (n = 6). The neotype of A. awamori (CBS 557.65 = NRRL 4948) does not originate from awamori fermentation and it is shown to be identical with the unknown taxon Aspergillus welwitschiae. Extrolite analysis of strains of A. luchuensis showed that they do not produce mycotoxins and therefore can be considered safe for food and beverage fermentations. A. luchuensis is also frequently isolated from meju and nuruk in Korea and Puerh tea in China and the species is probably common in the fermentation environment of East Asia. A re-descriptionof A. luchuensis is provided because the incomplete data in the original literature

Nahoko Nishibori, Kazuo Masaki, Hiroaki Tsuchioka, Tsutomu Fujii, Haruyuki Iefuji

The heterologous expression of the laccase gene from Trametes versicolor and Gaeumannomyces graminis was evaluated in the yeasts Pichia pastoris and Cryptococcus sp. S-2. The expression levels of both laccase genes in Cryptococcus sp. S-2 were considerably higher than those in P. pastoris. The codon usage of Cryptococcus sp. S-2 as well as the GC content were similar to those of T. versicolor and G. graminis. These results suggest that using a host with a similar codon usage for the expressed gene may improve protein expression. The use of Cryptococcus sp. S-2 as a host may be advantageous for the heterologous expression of genes with high GC content. Moreover, this yeast provides the same advantages as P. pastoris for the production of recombinant proteins, such as growth on minimal medium, capacity for high-density growth during fermentation, and capability for post-translational modifications. Therefore, we propose that Cryptococcus sp. S-2 be used as an expression host to improve enzyme production levels when other hosts have not yielded good results.

Jantaporn Thongekkaew, Hiroko Ikeda, Kazuo Masaki, Haruyuki Iefuji

Cryptococcus sp. S-2 carboxymethyl cellulase (CSCMCase) is active in the acidic pH and lacks a binding domain. The absence of the binding domain makes the enzyme inefficient against insoluble cellulosic substrates. To enhance its binding affinity and its cellulolytic activity to insoluble cellulosic substrates, cellulose binding domain (CBD) of cellobiohydrolase I (CBHI) from Trichoderma reesei belonging to carbohydrate binding module (CBM) family 1 was fused at the C-terminus of CSCMCase. The constructed fusion enzymes (CSCMCase-CBD and CSCMCase-2CBD) were expressed in a newly recombinant expression system of Cryptococcus sp. S-2, purified to homogeneity, and then subject to detailed characterization. The recombinant fusion enzymes displayed optimal pH similar to those of the native enzyme. Compared with rCSCMCase, the recombinant fusion enzymes had acquired an increased binding affinity to insoluble cellulose and the cellulolytic activity toward insoluble cellulosic substrates (SIGMACELL^® and Avicel) was higher than that of native enzyme, confirming the presence of CBDs improve the binding and the cellulolytic activity of CSCMCase on insoluble substrates. This attribute should make CSCMCase an attractive applicant for various application.

Takashi Watanabe, Haruyuki Iefuji, and Hiroko K. Kitamoto.

To remove nitrogen efficiently from high-concentration organic wastewater, we studied breeding methods using Saccharomyces cerevisiae as a model yeast with improved nitrogen accumulation ability. By DNA microarray analysis under various nitrogen concentrations with two nitrogen sources (peptone and L-asparagine), we obtained 295 commonly overexpressed (over 2-fold) genes and 283 commonly underexpressed (under one-half) genes under nitrogen-starvation conditions. We speculated that overexpression or underexpression recombination of some of these genes might enhance nitrogen uptake. Because a complete collection of nonessential gene deletion strains had been created, we investigated the nitrogen accumulation profiles of underexpressed gene deletion strains. From 256 nonessential gene deletion strains, three (URE2, SNO1, and AVT3) were selected. Strain SUD2 (ure2Δ::kanMX4) improved by 1.2-fold total nitrogen per cell (TN/OD₆₆₀) as compared to the parent strain, S288c. Positive selection of methylamine-resistant mutants to obtain URE2 mutants was useful for improving nitrogen accumulation ability without recombinant techniques.

Lahiru N. Jayakody, Keisuke Tsuge, Akihiro Suzuki, Hitoshi Shimoi, Hiroshi Kitagaki

Because of the growing market for sports drinks, prevention of yeast contamination of these beverages is of significant concern. This research was performed to achieve insight into the physiology of yeast growing in sports drinks through a genome-wide approach to prevent microbial spoilage of sports drinks. The genome-wide gene expression profile of Saccharomyces cerevisiae growing in the representative sports drink was investigated. Genes that were relevant to sulphate ion starvation response were upregulated in the yeast cells growing in the drink. These results suggest that yeast cells are suffering from deficiency of extracellular sulphate ions during growth in the sports drink. Indeed, the concentration of sulphate ions was far lower in the sports drink than in a medium that allows the optimal growth of yeast. To prove the starvation of sulphate ions of yeast, several ions were added to the beverage and its effects were investigated. The addition of sulphate ions, but not chloride ions or sodium ions, to the beverage stimulated yeast growth in the beverage in a dose-dependent manner. Moreover, the addition of sulphate ions to the sports drink increased the biosynthesis of sulphur-containing amino acids in yeast cells and hydrogen sulphide in the beverage. These results indicate that sulphate ion concentration should be regulated to prevent microbial spoilage of sports drinks.

Kazunori Kume, Shunsuke Kubota, Takayuki Koyano, Muneyoshi Kanai, Masaki Mizunuma, Takashi Toda, Dai Hirata

In eukaryotes, cell morphogenesis is regulated coordinately with the cell cycle. In fission yeast, the morphogenesis network MOR (morphogenesis Orb6 network) consists of 5 conserved proteins, Pmo25, Nak1, Mor2, Orb6, and Mob2, and is essential for cell polarity control and cell separation following cytokinesis. Here we show that the conserved leucine-rich repeat protein Lrp1 is required for cell morphogenesis as a newly recognized component of MOR. Lrp1 has 4 leucine-rich repeats in its N-terminus and is a homolog of the budding yeast Sog2, which is a component of the RAM network (regulation of Ace2 activity and cellular morphogenesis). Lrp1 was essential for both cell growth and cell morphogenesis as were the other MOR components. Lrp1 was localized to the SPBs (spindle pole bodies, the yeast equivalent of the animal centrosome) throughout the cell cycle and to the medial ring during cytokinesis. Lrp1 interacted with Nak1 and was important for Orb6 kinase activity. Thus Lrp1 proved to function upstream of Orb6 in cell morphogenesis.

奥田将生、上用みどり、高橋圭、後藤奈美、高垣幸男、池上勝、鍋倉義仁

イネ登熟期が全国的に記録的な猛暑となった平成22年産米のデンプンの性質および蒸米の酵素消化性について検討した。これまでの報告から予測されるとおり、猛暑となった平成22年産米はデンプン糊化温度が高く、蒸米酵素消化性は気中放置により低下しやすかった。一方、同一県内で採取された試料でも、糊化温度と酵素消化性にバラツキがみられた。特に出穂時期が遅い試料は酵素消化性の低下がゆるやかであった。米粒のアルカリ崩壊性について観察したところ平成21年産米が大きく崩壊したのに対し平成22年産米はアルカリに抵抗性を示した。以上、平成22年のような記録的な猛暑であっても蒸米の酵素消化性が悪くこれまでの知見が適用できることを確認するとともに、アルカリ崩壊性により簡便にデンプンの性質を明らかにできる可能性が示唆された。

奥田将生、上用みどり、橋爪克己、高橋圭、後藤奈美

平成10年から平成22年酒造年度の全国平均気温平年偏差と清酒製造全体の粕歩合との相関係数を求めた。その結果、8,9月に相関係数が0.7以上と高い正の相関性が見られ、夏場の気温が米の溶解性に影響することが示唆され、前報の結果と傾向が一致した。平成14年から9年間について、7試料の蒸米酵素消化性の平均値と粕歩合は負の相関性を示し、実際に蒸米酵素消化性が低い年は粕歩合が高いことが確認された。さらに同試料について、DSC糊化温度の平均値は粕歩合と正の相関性を示し、DSCの糊化温度が高いすなわちアミロペクチンの長鎖比率の高い年は粕歩合が高いことが確認された。以上より、酒米研究会の定点観測試料の分析結果は、全国的な米の溶解性の傾向を知るのに有効であると考えられた。