令和3年度 研究成果

赤松史一、清水秀明、伊木由香理、鎌田綾、小山和哉、山田修、後藤（山本）奈美

ワインに含まれるエタノールの炭素安定同位体比（δ¹³C値）は、ブドウ果実の糖類のδ¹³C値を反映するが、日本産ブドウの糖類のδ¹³C値の変動は、海外産ブドウに比べて知見が少ない。本研究は、日本産シャルドネブドウの糖類のδ¹³C値は変動が大きく（−27.2 ± 0.9‰、 平均値 ±　標準偏差、 n = 33）、糖類のδ¹³C値が単糖類のδ¹³C値と含有量で決まることを明らかにした。シャルドネブドウを完全発酵した後、果実の糖類とワインのエタノールの同位体分別は、1.5 ± 0.1‰だった。ワインのエタノールのδ¹³C値と同位体分別から、果醪の糖類のδ¹³C値が推定可能になった。輸入ワインは日本ワインよりも原料ブドウの糖類のδ¹³C値が高く、ブドウ栽培環境が海外の方が日本よりも乾燥条件にあることが示唆された。ブドウ果実の糖類のδ¹³C値の決定は、ブドウ栽培とワイン認証に価値ある情報をもたらす。

赤松史一、清水秀明、林さくら、鎌田綾、伊木由香理、小山和哉、山田修、後藤（山本）奈美

ワインの原料原産地の決定は、ワインの認証における大きな課題のひとつであるが、ワインに対するノンパラメトリック統計学的アプローチに関する情報は少ない。本研究では、日本産ブドウを使って小規模醸造した日本産シャルドネワイン33本と、8カ国から輸入された42本のシャルドネワインを収集し、酸素安定同位体比分析及び多元素分析を実施した。ワイン中の酸素安定同位体比（δ¹⁸O）及び18元素濃度に対して、非計量多次元尺度構成法（NMDS）、カーネル主成分分析（KPCA）及び主成分分析（PCA）を適用し、パラメトリック法及びノンパラメトリック法による情報抽出の比較を行った。ノンパラメトリック法であるNMDSとKPCAは、パラメトリック法のPCAよりも日本産シャルドネワインと輸入シャルドネワインの分離が良好であることが確認された。19変数のうち、18変数が原料原産地判別に重要であり、δ¹⁸O値はすべての統計手法で最も寄与度の大きい変数であった。ノンパラメトリック多変量解析は、日本産シャルドネワインと海外産シャルドネワインの判別に有用である。

Yukio Osafune, Kenji Toshida, Jinshun Han, Atsuko Isogai, Munehiro Miyamoto, Johji Aizawa, and Nobuhiko Mukai

Triple distillated Awamori "sho" was developed by a new production method for Japanese hard liquor which has a sweet flavor, cleanliness, and smoothness. It was commercialized by 12 Awamori manufacturers in Okinawa. To determine the unique characteristic of the spirits, multi distillations using small pot stills were performed and the distillation behavior of various aroma compounds were analyzed. As a result, the distillation behavior was mainly classified to five groups. Moreover, the concentrations of the aroma compounds increased and decreased respectively through both the second and the third distillations. In addition, the commercially available three times distillated Awamori was analyzed and compared with normal Awamori (Ippanshu). There was a significant difference in some volatile compounds, and they showed the same tendency as with the small pot still study. It is suggested that these compounds, which were changed through the multi distillation, contribute to the characteristics of the triple distillated Awamori.

Atsuko Isogai, Hanae Izu, Ryoko Kanda, Yuriko Ikeda, Yukari Igi, and Tsutomu Fujii

We carried out analyses of sake stored for 10 years presented for the “100 Year Sake Storage Project”. Comparing the sake before and after storage, absorbance at 280nm and 430nm, b* of CIE color value, and the concentration of 3-deoxyglucosone significantly increased. Acidity and the concentrations of most amino acids tended to decrease, although increases in amino acids were observed in some samples. As for flavor components, major aroma components such as ethyl acetate, isoamyl acetate, and ethyl caproate decreased, while carbonyl compounds, ethyl esters of branched chain fatty acids and organic acids, polysulfides and harman tended to increase, although some aldehydes decreased in some samples. Ethyl lactate and ethyl phenylacetate were shown to exist at concentrations exceeding their sensory threshold, in addition to sotolon, isovaleraldehyde, methional, benzaldehyde, dimethyl trisulfide, dimethyl disulfide and harman, which were previously reported to contribute to the aged sake flavor. Principal component analysis indicated that the more amino acids and organic acids the sake contains, the larger the changes were in the components mentioned above. A high correlation between the increment of ethyl carbamate (EC) during storage and urea concentration before storage suggested that urea is the dominant factor of EC formation in sake.

清水秀明、河野美乃里、小山和哉、岩下和裕、後藤奈美

赤ワインの色の向上に効果があると報告のある高温短期醸しについて、日本国内で栽培されている様々な品種を用いて小規模試験醸造及びワイナリーにおける実証試験を行いその効果を検証した。2017年のマスカット・ベーリーA（MBA）を用いた小規模試験では、発酵中盤から後半にさしかかったColor Intensityが最大付近でプレスした場合、製成後のワインのColor Intensityは対照区の1.1∼1.4倍となった。2018年のブドウ品種5種類を用いた小規模試験では、ブドウ品種の違いによって高温短期醸しの効果が異なり、MBAやメルローではColor Intensityが向上したが、それ以外の品種では対照区とほぼ同じとなった。ワイナリーにおける実証試験では、醪初期に28∼30℃程度の高めの温度で醸し発酵を行い、色素やタンニンを適度に抽出後、比重1.020∼1.000付近で速やかにプレスすることで、Color Intensityが向上し、渋味（収斂味）、厚み（ボディ感）が通常醸しと比べて良いと評価される場合もあった。ただし、プレスのタイミングを判断する難しさ、発酵スケールが大きくなるほど醸し発酵開始時に28∼30℃の温度にしつつ30℃を超えないようにする温度コントロールの難しさもあり、ワイン醸造で実施するには更なる条件設定の見直しが必要と思われた。

森一樹、門岡千尋、織田健、奥津果優、吉崎由美子、高峯和則、田代康介、後藤正利、玉置尚徳、二神泰基

Aspergillus luchuensis mut. kawachiiは、日本の伝統的な醸造酒の焼酎に主に使用されている。本稿では、A. luchuensis mut. kawachii IFO 4308 (NBRC 4308) の染色体レベルのゲノム配列と、A. luchuensis RIB2601の配列との比較について報告する。IFO 4308株のゲノムは、8本の染色体と1本のミトコンドリアDNAからなる9つのコンティグにアセンブルされた。IFO 4308株のほぼ完全なゲノムは37,287,730 bpからなり、GC含量は48.85%、予測されるコード配列は12,664個、tRNAは267個であった。IFO 4308とRIB2601のゲノムを比較すると、高度に保存された構造が明らかになった。しかしながら、IFO 4308のゲノムはRIB2601のゲノムより大きく、これは主に第5染色体に起因するものであった。IFO 4308 のゲノム配列は DDBJ/ENA/GenBank にアクセッション番号 AP024425-AP024433 で登録されている。

Katsumi Hashizume, Junya Ohno, Toshihiko Ito, and Masaki Okuda

Effects of exogenous peptidase addition on the concentration of pyroglutamyl (pGlu) peptides in sake and also on the taste characteristics of sake were investigated. The addition of peptidase agents involving high acid carboxypeptidase (ACP) activity to sake decreased bitter-tasting peptides and increased (pGlu)L-ethyl. The addition of a peptidase agent with high (pGlu)peptide ethyl ester forming activity but low ACP activity increased (pGlu)LF-ethyl and (pGlu)LFNP-ethyl in sake ; however, it did not increase (pGlu)L-ethyl. These effects were also observed in test of adding purified (pGlu) peptide ethyl ester forming enzyme. It was suggested that the increase of (pGlu) oligo peptide ethyl esters except (pGlu)L-ethyl yielded positive effects on the taste of sake. The addition of a peptidase agent with high (pGlu)peptide ethyl ester forming activity but low ACP activity increased (pGlu) oligo peptide ethyl esters in the digestion solution of the rice protein body (PB) by rice koji enzymes, but it did not increase (pGlu)L-ethyl. Addition of the peptidase to the sake mash caused similar effects as the addition to the PB digestion solution, but the effects were quite weak.

Katsumi Hashizume, Junya Ohno, Toshihiko Ito, and Masaki Okuda

Two pyroglutamyl (pGlu) peptide ethyl ester forming activities in rice koji samples made by twelve commercial koji starters and Aspergillus oryzae RIB128 were analyzed. The (pGlu)LFGP-ethyl forming activity, type(A) activity, was found only in the rice koji made by so-called “high glucoamylase starter” and RIB128, while the (pGlu)LFGPNVNPW-ethyl forming activity, type(B) activity, was found in all koji samples. The type(A) activity in koji samples showed high relationship (R2 = 0.90) between glucose forming activity, while the type(B) activity did not show the high relationship.

Kei Sasaki, Nahoko Nishibori, Satoko Yoshida, Muneyoshi Kanai, Atsuko Isogai, Osamu Yamada, Nami Goto-Yamamoto, and Tsutomu Fujii

Dimethyl trisulfide (DMTS) is responsible for hineka, an off-flavor in sake that develops during storage. We analyzed sake samples which were donated by sake companies, and extracted and demonstrated 4 sake making factors which could possibly affect DMTS concentration in sake after 7 days of storage at 70℃ (DMTS-pp): differences in yeast strains, differences in shubo (yeast seed mash called kimoto or sokujomoto) making methods with or without of the addition of alcohol to moromi, and differences in moromi pressing methods. Small scale fermentation tests and comparisons of sake commercially made with similar conditions except for shubo revealed that the differences in yeast strains and the differences in shubo making scarcely affect DMTS-pp. On the other hand, we found that the addition of alcohol to moromi decreased DMTS-pp, probably due to a dilution effect, and that DMTS-pp decreased more than the dilution ratio. Also, the pressing process of sake mash was found to affect DMTS-pp. Seme (later fraction of sake obtained under high pressure) has a higher DMTS-pp compared with arabashiri (free run fraction) and nakadare (middle fraction). In seme, absorbance at 260 nm was higher than that of arabashiri and nakadare. These results suggest that yeast cell leakage may affect DMTS-pp in seme.

Mai Okumura, Kazuo Masaki, Akihiro Yoshimura, Yoshinori Sawai, Kentaro Hisamatsu, Yuto Harada, Satoshi Iwamoto, Masaya Shimada, Takashi Hayakawa, and Tomoyuki Nakagawa

In this study, we aimed to breed strain GY115-a3 by mating with strain Ce41, which is high ethyl caproate-producing mutant strain derived from Gifu prefecture yeast, strain G. Among the obtained mating mutant strains between strain GY115-a3 and strain Ce41, the tetraploid strains showed the production characteristics for the unique organic acid composition like strain GY115-a3, and also had high fermentation and ginjo-ka production abilities, similar to those of strain Ce41. From these results, it was concluded that the expected mating mutant strains, such as a3×Ce41_1 and a3×Ce41_5, which inherited characteristic properties from the both parent strains, could be bred.

岸本徹、寺本聡子、藤田晃子、山田修

苦味価 (IBU値）は、ビールの苦味の強さを表すために使用される。本研究では麦汁およびビール中において、IBUに寄与する成分の挙動を追った。苦味価は「イソα酸濃度を反映する」と言われているが、必ずしも当てはまらない。本研究では47.5 IBUで40ppmのイソα酸を含むビール、一方で、49.9 IBUでありながらイソα酸を 2.2 ppmしか含まないビールを作成した。HPLCクロマトグラフより、麦汁およびビールのIBU値には、イソα酸、非イソ化α酸、α酸の酸化劣化物、および麦芽由来極性物質が寄与していることがわかる。α酸のイソ化は麦汁を高温下に置くことで急速に加速する。α酸のイソ化は60℃から70℃で始まることを初めて本論文において図示した。これは煮沸終了後のワールプール工程でもイソ化が進み、苦味価の調節においては本工程にも注意を払わねばならないことを示す。60℃以下の温度で沸騰した低イソ化率の麦汁においては、非イソ化α酸がIBU値の50％以上に寄与していた。しかし、非イソ化α酸は発酵中に消失するため、IBU値が発酵中に著しく低下することとなる。ホップの劣化に伴って増加する酸化劣化物もまた、ビールのIBU値に寄与する。通常の未劣化のホップを用いた場合、主にイソα酸濃度がビールのIBU値に寄与し、酸化劣化物の寄与は8.4％のみであった。しかし、40℃で90日間保存したホップペレットを用いたビールにおいては、IBU値の83％に酸化劣化物が寄与していた。劣化したホップを用いたビールの苦味強度を味センサを用いて調べた。劣化したホップを用いたビールの苦味強度は、通常の未劣化ホップを用いたビールと同じ40 IBUであるにもかかわらず、苦味強度は低いという結果であった。

Masaki Okuda, Midori Joyo, Bao Hong-Bin, Kei Takahashi, Atsuko Isogai, Nobuhiko Mukai,Toru Kishimoto, Ryoko Kanda, Sachiko Iizuka, Hanae Izu, Akiko Fujita, and Osamu Yamada

In the present study, the relationship between the flavor compounds in sake exhibited at the Annual Japan Sake Awards and the meteorological conditions during grain filling and the properties of rice starch during the 2009-2018 brewing years were investigated. The mean concentration of ethyl caproate in sake exhibited at the Annual Japan Sake Awards showed significant correlations with the maximum Baume, the ratio of sake cake, the gelatinization temperature, the enzyme digestibility of steamed rice grains, and the temperature during one month at grain filling of the Yamadanishiki rice cultivar harvested at Hyogo, but that of isoamlylacetate, isoamylalcohol, and ethylacetate did not. Therefore, when the temperature during grain filling of Yamadanishiki was lower and the enzyme digestibility of steamed rice grain was higher, the mean concentration of ethyl caproate in sake exhibited at the Annual Japan Sake Awards was higher. The present study indicated the possibilities that the meteorological conditions during rice grain filling cause the change in digestibility of sake rice and have considerable influence on the ethyl caproate concentrations in daiginjyo-sake.

柴田裕介、高橋俊成、森本朋子、金井宗良、藤井力、赤尾健、五島徹也、山田翼

清酒醸造の副産物である清酒粕は、葉酸に富んだ食品である。葉酸はヒトにとって重要なビタミンである。しかし、保存や調理の際に、無視できない葉酸の消失が起こる。著者らはこれまでに菊正宗酒造の蔵付き清酒酵母であるKm67株が高レベルの葉酸を蓄積できることを報告した。本研究において著者らは、km67株により生成した酒粕中の葉酸含量が、発酵活動の終了後もほぼ最大値を維持することを見出した。Km67株における葉酸高蓄積機構を明らかにするために、23種の葉酸代謝関連遺伝子の発現を解析した。ABZ1とFOL3の発現は、ほとんどの場合でKm67がきょうかい701号（K701）よりも高かった。このことは、葉酸生合成に関与する遺伝子の発現の亢進が、Km67における高葉酸蓄積をもたらしていることを示唆している。また、Km67を使用した酒粕は、4℃の冷蔵保存条件下で定量的に安定していることを見出した。更に、Km67の酒粕のホモシステイン含有量は、K701の酒粕のそれよりも、ほとんどの場合で高かった。この結果は、ホモシステインの細胞内濃度を減少させる必要があるため、Km67株が葉酸を高蓄積することを示唆している。ここでの結果は、酵母の葉酸代謝に関する生物学的に意味のある情報を提供するものである。

Shun Mitsui, Koji Yamamoto, Akitoshi Ito Akane Ieda, Tetsuya Kondo, Nobuyuki Sugiyama, Takeshi Akao, Akihiro Yoshimura, Yasuaki Sakakibara, Hideya Funai, Naoyuki Haramoto, Motoyuki Shimizu, and Masashi Kato

Synchrotron light with a wide wavelength range has features such as high brightness and high directivity. We examined the possibility of using synchrotron light in a mutation method. Specifically, by synchrotron light irradiation, we attempted to breed non-urea producing sake yeast which makes it possible to reduce ethyl carbamate (ECA) in sake. After synchrotron light irradiation using FIA2 (Aichi sake yeast) as a parent strain, we selected candidate strains of arginase-deficient mutants using the CAO medium (containing canavanine, arginine (Arg) and ornithine (Orn)), the Arg medium, and the Orn medium. We performed a similar selection process for ethyl methane sulfonate (EMS) treatment. We selected non-urea producing sake yeast from these selected strains through a 100 g scale fermentation test. Among the selected non-urea producing sake yeasts, FIA2Arg was selected as a practical yeast because of having the same alcohol and acid producing ability as the parent strain FIA2. FIA2Arg was acquired by synchrotron light irradiation, results showing that synchrotron light was available in the mutation method. We sequenced the CAR1 locus (encoding the arginase) to about 16 (11 acquired mutant strains by synchrotron light irradiation. and 5 acquired mutant strains by EMS treatment) of 21 acquired non- urea producing sake yeasts. Base substitution from G/C to A/T occurred in 5 of 9 acquired mutant strains with base substitution by synchrotron light irradiation. It was found that mutations by synchrotron light irradiation were less biased in base substitution from G/C to A/T as compared with EMS treatment. Deletion also occurred to some extent (in 4 of 11 acquired mutant strains by synchrotron light irradiation). As a result of a pilot scale (100 kg) sake brewing test, it was confirmed that FIA2Arg did not pro- duce urea and had brewing characteristics equivalent to FIA2.

Masaru Ikegami, Yukihiro Isono, Hiroyuki Fujimoto, Masanobu Kato, Takuma Sugimoto,　Kei Takahashi, and Masaki Okuda

遠藤路子、松井千佳、前田菜緒美、上原由梨奈、松田稜哉、藤井雄三、藤田晃子、藤井　力、山田修

清酒製造では製麴中に2,4,6-トリクロロフェノール（TCP）が麴菌Aspergillus oryzaeにより2,4,6-トリクロロアニソール（TCA）にメチル化されると、カビ臭が発生し、清酒の品質が著しく低下することが知られている。従って、我々はTCA生成能の低い麴菌株を育種しようとしている。TCPは菌類の増殖を抑える防黴剤であるが、TCAは増殖を抑えない。TCPが麴菌の生育に及ぼす影響はわかっていない。しかしながら、TCP変換能が弱い株はTCP濃度に対する感受性が高いと考えられる。本研究において、我々は種々のTCP濃度が麴菌の生育抑制に及ぼす影響を調べた。麴菌はTCP濃度が高くなるほど生育や胞子形成が遅れた。液体培養（30℃、1日間）では30 µg/mL、寒天培養（30℃、7日間）では50 µg/mLのTCP濃度で完全に生育が阻害されたが、α米においてはより高濃度のTCP存在下でも生育可能であった。寒天培養の結果はAspergillus属の他種におけるTCPの影響と一致した。

橋本渉、新井開、水谷治、山田修, 新谷尚弘、五味勝也

黒麴菌（Aspergillus luchuensis）はAspergillus section Nigriに属する糸状菌であり、主に日本南部の伝統的蒸留酒（焼酎）製造に利用されている。焼酎製造においてアミロリティック酵素は必須であるが、黃麴菌（Aspergillus oryzae）に比べてその遺伝子発現調節機構の解析は遅れている。我々は、黃麴菌転写制御因子AmyR及び CreAの黒麴菌オルソログ破壊株について遺伝子発現プロファイルを解析した。黒麴菌は非耐酸性α-amylasesと耐酸性α-amylasesを生産する。非耐酸性α-amylases遺伝子は恒常的に発現していたが、耐酸性α-amylases遺伝子発現はAmyR及び CreA遺伝子破壊によりそれぞれ低下及び上昇した。さらに、glucoamylase及びα-glucosidase遺伝子も耐酸性α-amylases遺伝子と同様の挙動を示し、デンプン糖化に重要な3遺伝子はAmyR及び CreAにより調節されていた。また、耐酸性α-amylasesは固体培養特異的に生産されると報告されていたが、黃麴菌において固体培養特異的遺伝子発現に関与するflbCの黒麴菌オルソログ破壊株は、耐酸性α-amylases発現への関与を示さなかった。

山田修、西堀奈穂子、林梨咲、有馬寿英、水谷治

黒麹菌（Aspergillus luchuensis）転写制御因子遺伝子破壊ライブラリを構築するために96遺伝子を選抜し、Agrobacterium-mediated transformation（AMT）によりA. luchuensis RIB 2604 ΔligD株を親株として93遺伝子を破壊した。adaB, anBH1, hacA, hapB, hsf1, metR及びsonC遺伝子破壊株ではヘテロカリオン株のみが得られ、abaA及びmcmA遺伝子破壊株は取得できなかった。ライブラリは酒類総合研究所websiteから入手可能とする予定である。

植田浩一、田端純、勢〆康代、正木和夫、山下（鮫島）結香、北本宏子

酵母Pseudozyma や Cryptococcusは、クチナーゼ様酵素（CLE）といわれる生分解性プラスチック（BP）分解酵素を分泌する。この酵素は、既知のクチナーゼとは、30%以下の配列相同性しかもたない。この研究では、CLEがクチナーゼとしての活性を有することを示す。葉から単離された、15℃でBP分解能を示す17株のCryptococcus magnusのうち１株からCLEのDNA配列を同定した。クチンはトマトの葉から調整され、３種のCryptococcus酵母のCLEとPseudozyma antarctia由来のCLE（PaE）により分解が試みられた。典型的なクチンモノマーである10,16-dihydroxyhexadecanoic acidが質量分析ガスクロマトグラフィーにより検出され、CLEによるクチンの分解が示された。さらにPaEによるクチンの分解では、薄層クロマトグラフィーにより高分子量の分解産物が示され、PaEはエンド型として作用することを示した。

Masaki Okuda, Kei Takahashi, Bao Hong-Bin, Hiromi Kohno, Midori Joyo, Nobuhiko Mukai, Yasuhiro Tsuchida, Eizo Yoshinaka, Akihiko Takami, Yuji Kiriyama, and Yoshihiko Murai

The changes in sake making properties of sake rice were investigated during storage of brown rice grains under 4～30℃ for 24 months in two kinds of moisture contents. Significant changes in sake making properties of sake rice- -except for a small increase of potassium concentrations- -have not been observed when the brown rice grains have been stored under 10℃ at the rice warehouse in Hyogo Prefecture and under 4℃ and 10℃ at NRIB for 24 months. On the other hand, the broken rice ratio of a milling test, potassium concentrations of polished rice, and viscosity at RVA have increased during storage of brown rice under 30℃ at NRIB for 6 months. Although the hexanal concentrations in brown rice have increased during storage even under 4～10℃, those in polished rice have not been detected during storage under 4～10℃ for 24 months. The present study suggested that the sake making properties of sake rice would not deteriorate either by both storing rice grains under 10℃ or maintaining the moisture content of rice grains without changes in moisture.

Takeki Tanaka, Masaki Okuda, Kei Takahashi, and Nobuhiko Mukai

In the production of sake, the enzyme digestibility of the steamed rice grains is a critical factor to realize a good quality of sake and to utilize rice grains efficiently. The retrogradation of rice starch in the steamed rice grains proceeds during sake making, and the enzyme digestibility of the steamed rice grains decreases significantly. However, the influencing factors of the retrogradation of starch in the steamed rice grains have not been elucidated clearly. In the present study, the relationships between the moisture content in steamed rice grains, the retrogradation of rice starch, and the enzyme digestibility were examined to clarify the influencing factors of the retrogradation of the starch. Steamed rice grains with a high moisture content showed a faster retrogradation of starch and lower enzyme digestibility than those with a low moisture content after storage in a plastic bag. Moreover, when the moisture content in the steamed rice grains decreased during storing under lower humidity conditions, the rate of the retrogradation and the decrease of the enzyme digestibility were slight. The present results indicated that the moisture contents of the steamed rice grains have a significant influence on rice starch retrogradation and enzyme digestibility after storing in conditions such as the cooling process of steamed rice grains in Daiginjyo sake production.

Sachiko Iizuka, Ryoko Kanda, Satoko Teramoto, Akiko Fujita, and Osamu Yamada

Fermentation nutrients are widely used in winemaking for the manufacturing soundness, and their use is permitted as an article which is not treated as raw materials of alcoholic beverages by the Liquor Tax Law and Liquor Administrative Law or regulation interpretation notifications in Japan. However, there are few data in the literature for their use in sake brewing. In this study, we have investigated the effects of the use of fermentation nutrients for wine (Stimula Sauvignon Blanc and Optimum White) on sake brewing and considered the possibility of the development of products with new characteristics. It was shown that the addition of Stimula Sauvignon Blanc promoted fermentation and improved the utility efficiency of raw materials in a one-step preparation for fermentation mash. The contents of isoamyl acetate and ethyl caproate of the samples with the addition of Stimula Sauvignon Blanc were higher than that of the control (no addition). In the results of the sensory evaluation of brewed sake from the one-step and three-step preparation for fermentation mash, the scores of the brewed sake with the addition of the fermentation nutrients were the same level as the control (no addition). The characteristic comments such as “lychee”, ‘fruity”, “muscat” and “citrus” in those samples show a possibility in which the use of the fermentation nutrients on sake brewing provides a potent tool for the development of products with new characteristics.

Nobuhiko Mukai, Jinshun Han, and Nahoko Nishibori

The authors　conducted a sensory evaluation and measured the phenolic compounds of 35 samples of sake. The maximum contents of phenolic compounds were as follows: p-coumaric acid: 0.4 mg/l; ferulic acid: 5.4 mg/l; 4-vinylphenol: 204.1 μg/l; 4-vinylguaiacol: 282.7 μg/l and guaiacol: 58.0 μg/l.　The contents of 4-ethylphenol and 4-ethylguaiacol were very low. As a result of a multiple regression analysis, the intensity of phenolic flavor was almost able to be explained by 4-vinyilguaiacol and guaiacol. Using activated carbons, most of the p-coumaric acid, ferulic acid, 4-vinylphenol, and 4-vinylguaiacol in sake was removed, but guaiacol could only be removed up to 35%.

Toru Kishimoto, Akiko Fujita, Sachiko Iizuka, Ryoko Kanda, Satoko Teramoto, Osamu Yamada, and Nami Goto-Yamamoto

Nobuhiko Mukai, Atsuko Isogai, Masaki Okuda, Kazuo Masaki, Kei Takahashi, Yukio Osafune, Takayuki Emura, Hisashi Yazawa, and Nami Goto-Yamamoto

Shinichi Ito, Hisashi Yazawa, Kaori Isobe, Satoko Teramoto and Takayuki Emura

Sharon Marie Bahena-Garrido, Yuko Komatsu, Ryota Saito and Kazuhiro Iwashita

In this report, we investigated the production of 6 candidate mycotoxins in A. oryzae species based on the MAFF list of risk assessment. We evaluated the possible production of these mycotoxins using a tandem approach as reported in the previous paper. Based on the DI-HRMS data and further validation by LC-Q/TOF-MS, our results indicate no significant traces of these candidate mycotoxins. Thus, we conclude that these 6 mycotoxins were not detected in A. oryzae species grown in all 11 culture conditions.

Naoto Okazaki, Masaaki Inahashi, Katsumi Nakahara, Hirokazu Hasuda, Takafumi Mutoh, Hitoshi Shimoii, Yasuzo Kizaki, Dai Hirata and Masaki Okuda

As one feature of the sake production year based on the sake rice analyses, the digestibility Brix shows a high positive correlation with the glucose and a negative correlation with the items/factors related to the fermentation process, alcohol, sake meter values etc. As a result, it was indicated that the glucose content showed a positive correlation (p=10%) with the overall evaluation. In a factor analysis, factor 1 -- solubility of the raw material rice, with respect to the fermentation, high digestibile Brix and glucose, contribute to the evaluation were good ; factor 2 - i-AmOH, al-added amount, low acidity, were interpreted as density and cleanliness. This result could lend credence to the report by Sato et al. who proposed the basic structure of the taste of sake. In the sake group with minimal fragrance defects, we analyzed the effect of the sake components on the overall evaluation for each exhibition year. Et-Cap, glucose, sake degree, amino acid degree, and i-AmOAc were selected as the items/factors with a great contribution. However, it was suggested that the changes in the panel's examination criteria affected the contribution rate of Et-Cap and glucose to the overall evaluation.

Yukio Osafune, Kenji Toshida, Jinshun Han, Atsuko Isogai and Nobuhiko Mukai

The relationship between aroma compounds and flavor terminology is an important step in establishing a sensory evaluation system. The detection and recognition thresholds of 16 compounds were determined to identify the compounds contributing to the aroma characteristics of Honkaku shochu and Awamori, both traditional Japanese spirits. The odor activity values calculated using detection thresholds suggested that 13 compounds affect the quality of Honkaku shochu and Awamori. Further, the sensory attribute terms of these compounds are summarized. Thirty-two compounds were selected as candidates for reference standards from among 55 compounds (threshold determined) based on their contribution to the quality and characteristics in Honkaku shochu and Awamori. The result of classifications of the candidates of the reference standard suggested that these compounds were divided into 8 groups according to their aroma characteristics. These findings help to establish reference standards, terminology, and a flavor wheel for the evaluation of Honkaku shochu and Awamori.

Nobuhiko MUKAI, Akinori MUTO, Toshihiro YOSHIMURA, Kaori ONODERA, Kenji TOSHIDA, Jinshun HAN and Yukio OSAFUNE

The authors investigated the methanol content in potato shochu (137 samples of sweet potato shochu, 6 samples of potato shochu, and 3 samples of Japanese yam shochu. There were no samples which exceeded the regulation level of methanol of the Japanese Food Sanitation Act. In sweet potato shochu made from Koganesengan, the methanol content using sweet potato koji was significantly higher than that using rice koji. The methanol content of sweet potato shochu made from purple lineages was significantly higher than that made from Koganesengan.

Nobuhiko MUKAI, Jinshun HAN and Yukio OSAFUNE

The authors investigated the quantitative scale evaluation of barley shochu and sweet potato shochu. Five kinds of evaluation items (body, sweetness, bitterness, aftertaste and overall evaluation) were used for the scale evaluation. The evaluation of sweetness had a significant correlation with that of the overall evaluation in both barley shochu and sweet potato shochu. As a result of a multiple regression analysis, it was found that sweetness and aftertaste were important to the overall evaluation. Futhermore, the correlation analysis between scale evaluation values, general components, and flavor components was investigated.

Kei Takahashi, Masaki Okuda, Midori Joyo, Mineyo Numata, Bao Hong-Bin, Hiromi Kohno

Sequential earlier-period-rice analysis data gathered from the 2002-2019 crop years, comprised of 707 samples in total, were examined focusing on a comparison within sake rice varieties. We discovered varied rice properties in Japanese sake brewing among different rice varieties. The weight of 1,000 rice grains was heavier across Hanafubuki, Okuhomare, and Hyogokitanishiki, followed by Yukimegami, Homarefuji, Akitasakekomachi, Yamadahishiki, Kitashizuku, Senbon-nishiki, and Hattan-nishiki. Water absorption during 20 minute periods was higher in Kyonohana and Dewanosato, followed by Homarefuji, Gin-noyume, Hyogokitanishiki, Hattan-nishiki, Yamadahishiki, Ipponjime, and Hanafubuki. Brix was particularly high in Dewanosato, followed by Kyonohana, Homarefuji, Akitasakekomachi. The ratio of Brix/folmol nitrogen was higher in Dewanosato, followed by Yukimegami, Yamadanishiki, Gin-noyume, Nakateshinsenbon, and Homarefuji. Crude protein was higher in Okuhomare, Ginpu, Kitashizuku, Fukuhikari, and Hyogokitanishiki, but lower in Yukimegami, Dewanosato, Yamadanishiki, Nakateshinsenbon, Akitasakekomachi, Gin-noyume, and Sanukiyoimai. Brewing properties across all rice varieties analyzed did not considerably vary from the properties of rice harvested within the same production area, indicating that sake rice variety strongly affects rice properties upon sake brewing.

Kei Takahashi, Masaki Okuda, Midori Joyo, Mineyo Numata, Bao Hong-Bin, Hiromi Kohno

In the present study, we investigated the annual variation in the brewing properties of sake rice utilizing earlier-period-rice analysis data gathered from 2002-2019 across all rice varieties analyzed. The annual variation for water absorption during a 20 minute period of table rice was higher than sake rice. Water absorption during both 20 minute and 120 minute periods of a large amount of rice varieties was higher among rice harvested during the year 2010, when the highest mean August temperature was recorded in Japan. The starch digestibility (Brix) of a large amount of rice varieties was lower in the years 2007, 2010, 2012, and 2019, during which higher temperatures from July to September contrasted with other crop years. Brix values of Gohyakumangoku varied widely within a year depending upon the production district, indicating that the variety is likely to be strongly affected by meteorological conditions in regards to its digestibility. The annual variation of crude protein and potassium concentrations within white rice varied depending on rice variety and the production district. Brix values of Yamadanishiki and Gohyakumangoku from 2008 to 2018 were strongly and negatively correlated with the mean data of sake cake ratios for junmai ginjo and ginjo of the corresponding year, suggesting that the Brix value would be available for the estimation and improvement of premium sake brewing practices. Taken altogether, by analyzing the annual variation, we discovered that the brewing properties of sake rice can be considerably altered by meteorological conditions, and that the effects of meteorological conditions upon brewing properties vary depending on the type of rice variety in question.

岸本徹、寺本聡子、藤田晃子、山田修

　79種類のホップを加えたビールを小規模スケールにて製造し、Stir Bar Sorptive Extraction法によって香気成分を分析後、無ホップビールのクロマトグラムと比較して、ホップ由来化合物として127の香気成分に着目した。 127のホップ由来香気成分を使用して主成分分析（PCA）を行いホップ品種との相関を調べた。PCAによって主に5つの方向性に分けられ、柑橘類の特性に寄与することが知られているゲラニオールやシトロネロールが寄与する方向IにはCascade, Amarillo, Citra, Simcoe, Mosaic, Wai-iti, Hallertau Blanc が存在し、リナロールや6-メチル-5-ヘプテン-2-オンなどの水溶性テルペンアルコールが寄与する方向Ⅱには、Bobek, Styrian Gold,　Moutuekaが存在した。 HersbruckerとSaphirを含む方向Ⅲには、セスキテルペンアルコールが寄与した。方向IVには疎水性モノテルペンが寄与し、方向Vには疎水性セスキテルペンが寄与した。
　 また「ホップクリープ効果」が見られた。冷却した麦汁に等量のα酸が添加されるようにホップペレットを添加したために、ホップペレットの添加量は品種によって異なった。ホップペレットの添加量が多いと、発酵後の外観エキス濃度(%)が低くなる傾向がみられ、つまりはホップにデキストリン加水分解酵素が存在し発酵中に糖化が進むことが示唆された。
　今後これらのホップの品種と成分をさらに研究することで、ビールにさまざまなホップの品種を使用してビールに特徴的な香りと味を付与する技術を確立することができると考えられる。

茶谷朋哉、大貫俊輔、磯谷敦子、五島徹也、鹿島舞央、ファーザン・ガネゴルモハマディ、西智之、平田大、渡辺大輔，北本勝ひこ、赤尾健，大矢禎一

清酒酵母はほぼ二倍体であるので、醸造特性の改良のための劣性（潜性）変異の導入には相当の努力を要する。複数の優れた醸造特性を備えた清酒酵母の造成のために著者らはゲノム編集を有効活用した、化学的根拠に基づく方法を取った。ここでは、AWA1、CAR1、MDE1、FAS2の各遺伝子を育種ターゲットとした。標準的な清酒酵母に八重変異を導入し、高泡非形成、発がん性物質及びオフフレーバー非生成、かつ吟醸香高生成の株を造成した。小仕込試験により、これらのゲノム編集株により期待される酒質が得られることが示された。育種過程における想定外の遺伝的摂動の導入は少数であり、ゲノム編集技術は清酒酵母の段階的育種に極めて有効であることがわかった。

塚原正俊、伊勢孝太郎、鼠尾まい子、東春奈、赤尾健，外山博英

酵母Saccharomyces cerevisiae泡盛101号株のドラフトゲノムシーケンスを報告する。泡盛101号は蒸留酒の一種である泡盛製造用の実用菌株である。泡盛101号ゲノム配列を、次世代型シーケンサーで得られたショートリードのアセンブリーにより構築した。315本のコンティグにより、6185個の予測遺伝子をコードする11.5メガ塩基対のゲノム配列が構成された。

プスパ・ラジ・ポウデル、東暁史、小林省藏、小山和哉、後藤（山本）奈美

アントシアニン生合成系におけるMYBA転写制御因子の役割は、ブドウ（Vitis vinifera L.）を含む多くの植物で明らかにされている。しかし、ブドウのMYBAのターゲット遺伝子のすべては明らかにされていない。そこで、本研究では、赤色品種（Pinot Noir）とその白色変異体（Pinot Blanc）、及び白色品種（イタリア及びマスカット・オブ・アレキサンドリア）とその赤色変異体（紅高及びフレーム・マスカット）を用いて、MYBAsのターゲットを明らかにすることを目的とした。まず、マイクロアレイと逆転写-リアルタイムPCRの結果、アントシアニン合成系に特異的な遺伝子（UFGT、OMT）に加え、アントシアニンの液胞輸送に関わる遺伝子（GST）や、F3’5’Hも赤品種で高発現していた。これらの遺伝子はおそらくMYBAsのみで誘導されていると考えられる。一方、上流のフラボノイド合成系遺伝子（PAL、CHS3、F3H、DFR及びLDOX）は白品種でも発現しており、赤品種ではやや高発現していた。これらの遺伝子はMYBAsとそれ以外の転写因子の両方で誘導されると考えられる。ゲルシフト解析の結果、VvMYBA1の両方またはいずれか一方はVvMYBA2タンパク質はUFGT、CHS3及びLDOXのプロモーター領域と相互作用することが示された。これらの結果から、VvMYBAsはアントシアニン合成系に含まれるすべての遺伝子の発現を制御し、VvMYBAタンパク質はUFGTだけではなく、CHS3やLDOXにも直接作用することが示唆された。