令和元年度 研究成果

Narumi NAGAI, Nana SHINDO, Akari WADA, Hanae IZU, Tsutomu FUJII, Takahiro AKASHI, Misato TOKUI, Kiminori MATSUBARA, Yuji WADA, and Naoki SAKANE

BACKGROUND

Rice wine lees (RWL), a Japanese traditional fermented product, is a rich source of one-carbon metabolism related nutrients, which may have beneficial effects on cognitive function.

OBJECTIVES

We aimed to examine the effect of the RWL on cognitive function in community-dwelling physically active older adults.

Design

Double-blind, randomized, placebo-controlled study (clinical trial number: UMIN 000027158).

SETTING

Community-based intervention including assessments conducted at the University of Hyogo and a public liberal arts school in Himeji City, Japan.

PARTICIPANTS

A total of 35 community-dwelling older adults (68-80 years) who performed mild exercise before and during the trial were assigned to either the RWL (n=17) or the placebo group (n=18).

INTERVENTION

Daily consumption of 50 g RWL powder, which contained one-carbon metabolism-related nutrients, or the placebo powder (made from soy protein and dextrin) for 12 weeks. Both supplements included equivalent amounts of energy and protein.

MEASUREMENTS

Montreal Cognitive Assessment, computerized cognitive function test, and measurements of serum predictive biomarkers (transthyretin, apolipoprotein A1, and complement C3) were conducted at baseline and follow-up.

RESULTS

Visual selective attention and serum transthyretin significantly improved in the RWL group, whereas there was no significant change in the placebo group. No significant group difference was observed in the remaining cognitive performance tests.

CONCLUSIONS

RWL supplements seem to have a few effects on cognitive function in community-dwelling physically active older adults. However, the impact was limited; therefore, further studies with sufficient sample size are warranted to elucidate this issue.

Ryousuke Kataoka, Taisuke Watanabe, Shigekazu Yano, Osamu Mizutani, Osamu Yamada, Takafumi Kasumi, Jun Ogihara

Awamori is a distilled spirit produced in Okinawa Prefecture, in southern Japan. Awamori contains the volatile organic compound 1-octen-3-ol, an important flavor component. Here, using solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GCMS), we demonstrate that the black koji mold Aspergillus luchuensis produces 1-octen-3-ol in rice koji. To examine the role of the fatty acid oxygenase genes ppoA and ppoC in 1-octen-3-ol biosynthesis by A. luchuensis, we constructed ppoA and ppoC disruptants, ΔppoA and ΔppoC, respectively, via protoplast-PEG transformation. No clear differences in growth and conidiation were observed between the transformants and the parent strain. Volatile compounds in rice koji prepared using these gene disruptants were analyzed by SPME-GCMS. The amount of 1-octen-3-ol contained in koji produced by the ΔppoA strain was the same as that produced by the parental strain. In contrast, although the ΔppoC strain grew on the rice koji, was not detected. These results indicate that ppoC is involved in 1-octen-3-ol biosynthesis in A. luchuensis.

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

Honkaku shochu and Awamori are traditional Japanese spirits. The detection and recognition thresholds of 16 compounds in these spirits were determined to understand the compounds contributing to the aroma characteristics of Honkaku shochu and Awamori. The odour activity values calculated using detection thresholds suggested that 14 compounds affect the quality of Honkaku shochu and Awamori. The odour activity values of β‐damascenone and rose oxide were much higher in sweet potato shochu and these two compounds are considered to contribute to the characteristics of sweet potato shochu. Similarly, the odour activity value of 1‐octen‐3‐ol was higher in Awamori than in the other categories, suggesting that 1‐octen‐3‐ol produces the characteristic aroma of Awamori. In addition, the sensory attribute terms of 16 compounds are summarised.

Yusuke Shibata, Tasuku Yamada, Tomoko Morimoto, Tsutomu Fujii, Takeshi Akao, Tetsuya Goshima, Toshinari Takahashi, and Nobuchika Tanaka

Folates are important vitamins in human nutrition. Pressed sake cake, a brewing by-product of sake, is a rich dietary source of folates derived from sake yeast (Saccharomyces cerevisiae). The National Research Institute of Brewing investigated 106 samples of pressed sake cake and revealed that three samples containing large amounts of folates were produced by Km67 yeast derived from the house sake yeast strain of Kiku-Masamune sake brewery. In this study, we performed sake brewing tests using Km67 and Kyokai no. 7 group strains and confirmed that Km67 yeast contributed to the production of pressed sake cake containing large amounts of folates. To elucidate the mechanisms of high folate accumulation in Km67, we performed whole-genome sequence analysis in Km67 and then screened 10 folate metabolizing genes showing different sequences in Km67 and K7 strains. By folate analysis of each gene-disrupted strain derived from strain BY4743, we also selected four genes having significant effects on folate content in yeast from 10 candidate genes. Folate analysis of gene-disrupted yeast strains complemented with either Km67-type genes or K7-type genes revealed that the Km67-type HMT1 gene was related to high folate accumulation not only in laboratory yeast but also in sake yeast. In this gene, Leu63Phe was present in the methyltransferase motif I of Hmt1p, which was essential for the methyltransferase activity of Hmt1p. Our results and previous reports suggested that the methyltransferase activity of Km67-Hmt1p was higher than that of K7-Hmt1p, leading to enhanced production and high accumulation of folates in Km67 yeast.

Katsumi Hashizume, Toshihiko Ito, Karin Shirato, Naomi Amano, Tetsuo Tokiwano, Tsuyoshi Ohno,Sho Shindo, Seiei Watanabe and Masaki Okuda

Factors affecting ferulic acid, ethyl ferulate and taste-active pyroglutamyl (pGlu) peptides levels in sake were analyzed using small-scale sake brewing tests on eighteen rice samples with differing cultivar variety, cropped year and area, and polishing rate. Ferulic acid concentration in sake was highly positively correlated with its content in rice (r = 0.782^**, double asterisk indicates 1% significance level), feruloylesterase (FE) activity (r = 0.804^**) and feruloylated saccharide forming activity (FSFA) (r =0.619^**) in the rice koji. The results suggested that ferulic acid in rice induced FE activity and FSFA, and these two enzymes accelerated the formation of ferulic acid in sake mash. The concentration of bitter-tasting peptides in sake was highly positively correlated with crude protein content in rice (r = 0.786^**), and negatively correlated with acid carboxypeptidase (ACP) activity to (pGlu)LFGPNVNPWH (r = -0.612^**), fermentation length (r = -0.820^**), and pyroglutamyl leucine ((pGlu)L) concentration in sake (r = -0.502*; 5% significance level). The observation suggested that bitter-tasting peptides are initially formed in sake mash in accordance with protein content in rice, and are then hydrolyzed to smaller peptides, such as (pGlu)L. An ACP specific to the hydrolysis of bitter-tasting peptides was suggested by the observation that ACP activity to (pGlu)LFGPNVNPWH was significantly correlated (r = -0.612^**) to their formation whereas an ACP to Cbz-Glu-Tyr was not (r = -0.220). It was suggested that (pGlu) oligopeptide ethyl esters were formed during the decomposition of bitter-tasting peptides to which the ACP to (pGlu) LFGPNVNPWH might contribute.

Kazuya TOMIMOTO, Yukio Osafune, Dararat Kakizono, Jinshun HAN, Nobuhiko MUKAI

High glycosidase-producing strains of Aspergillus luchuensis were isolated from 2-deoxyglucose (2-DG) resistant mutants. α-Amylase, exo-α-1,4-glucosidase, β-glucosidase and β-xylosidase activity in the mutants was ~3, ~2, ~4 and ~2.5 times higher than the parental strain RIB2604 on koji-making conditions, respectively. Citric acid production and mycelia growth of the mutants, however, approximately halved to that of the parent. Compared to the parent, the alcohol yield from rice and sweet potato shochu mash of the mutant increased ~5.7% and 3.0%, respectively. The mutant strains showed significantly low glucose assimilability despite the fructose one was almost normal, and they had a single missense or nonsense mutation in the glucokinase gene glkA. The recombinant strain that was introduced at one of the mutations, glkA Q300K, demonstrated similar but not identical phenotypes to the mutant strain. This result indicates that glkA Q300K is one of the major mutations in 2-DG resistant strains.

磯谷敦子、村山美乃、木村萌水、篠﨑貴旭、山本英治、藤井力、飯塚幸子、徳永信

ジメチルトリスルフィド（DMTS）は清酒の老香の主要成分である。我々はこれまでに、シリカに担持した金ナノ粒子（Au/SiO2、以下吸着剤）がDMTSの選択的吸着除去に有効であることを、モデル溶液および清酒中で確認した。本研究では、大吟醸酒、純米酒、長期熟成酒を用い、5L-スケールで吸着剤による脱硫試験を行った。成分分析および官能評価の結果、吟醸香成分およびDMTSをともに多く含む大吟醸酒において、吟醸香を残したままDMTSが除去され、良好な評価が得られた。吸着剤の実用化のため、吸着剤の調製方法を検討し、回転式電気炉を用い焼成方法を工夫することで大量調製が可能となった。また、300°Cでの焼成により吸着剤を再利用できることを確認した。

尾仲宏康、丸山潤一、浅水俊平、黒岩真弓、北本勝ひこ、山田雅人、五島徹也、赤尾健

富山県高岡市産六条大麦麦芽より醸造に適した富山酵母「とやま産まれの酵母」を分離した。本菌株はITS領域解析の結果、Saccharomyces cerevsiae NBRC2114 (Kotobukiya whisky No. 1)に最も近い配列を有することが明らかとなった。清酒小仕込み試験による醸造特性解析の結果、とやま産まれの酵母のもろみ日数はK701よりも3日長くなったものの、両者の到達アルコール濃度はほぼ同じ18%であった。K701と比較し、有機酸組成では酢酸、リンゴ酸、コハク酸の含有量が顕著に異なっており、揮発性物質では高級アルコール含量が高く、エステル類の含量が低かった。アミノ酸度はとやま酵母の方が顕著に高かった。これらの性質が官能特性評価にも現れており、K701に比較して、酸味の違いや、旨み、重い、雑味などの味の多さに特徴がある個性的な酒質であった。

とやま産まれの酵母による実地醸造は成政酒造株式会社にて、これまでに5回行われており、現在では比較的濃醇でキレのある酒質となっている。また、本酵母は野生酵母で特定の酒類醸造に特化した育種もなされていないため、富山県地域のクラフトビール、ワイン、ウイスキーの醸造にも利用されるに至っている。

井上絵梨、岸本宗和、根本学、渡辺晃樹、齋藤浩、小山和哉、後藤奈美、美濃英俊、鈴木智博、奥田徹

本研究は、山梨県内の様々な標高に位置する圃場においてブドウ生育期間の気温および生育ステージを比較するとともに、果実成分の含有量に及ぼす影響について調査することを目的とした。山梨県内においてシャルドネ'および'メルロー'が栽培される標高250mから750mの圃場はウィンクラ-・インデックス（WI）に基づいてregion ⅢからⅤの幅広い気候区分に分類された。標高によるブドウ 生育期間の気温差はブドウの萌芽、開花、ヴェレー ゾンあるいは着色、および収穫の生育ステージを遅らせ、リンゴ酸およびアントシアニンの果実成分含有量に大きな影響を及ぼすことが示された。

藤井力、藤田晃子、飯塚幸子、伊豆英恵、神田涼子、岸本徹、後藤奈美

平成29酒造年度全国新酒鑑評会（第106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、酒類総合研究所において、平成30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30日（水）に東広島運動公園体育館で製造技術研究会を開催するとともに、6月16日（土）に日本酒造組合中央会主催の東京池袋のサンシャインシティ・文化会館展示ホールでの公開きき酒会を後援した。

出品は850点であった。審査の結果、優秀と認められた421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232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、1,545人及び3,583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（「老ねやすさ」の受託分析を含む。）を行った。

向井伸彦、磯谷敦子、金井宗良、髙橋正之、長船行雄、武藤彰宣、芳村俊広、後藤奈美

単式蒸留焼酎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41 回本格焼酎・泡盛鑑評会を日本酒造組合中央会と共催で開催した。審査は平成30 年6月 5日（火）及び6月6日(水)に行い、単式蒸留焼酎の製造に関与している者等が出品酒をきき酒する製造技術研究会は6月26日（火）に当研究所で開催し92名が参加した。

開催にあたり、開催要領について日本酒造組合中央会と事前に検討し、今回は、(1)出品者の資格（中央会と共催化された第32回（平成21年開催）以降、単式蒸留焼酎の製造免許を受けている製造者で日本酒造組合中央会の組合員に限定していたが、今回は組合員に限定しないこととし、また、びん詰め等のための蔵置場を持つ製造者が主となって組織する法人（共同びん詰め法人）も追加した。）、(2)審査、成績上位酒の選定及び製造技術研究会における樽貯蔵に該当する出品酒の取扱い方法（樽貯蔵に該当する出品酒については、種類に関わらず樽貯蔵区分としてまとめた上で審査、成績上位酒の選定及び製造技術研究会での展示を行った。）、を変更した。

なお、出品者の資格の変更に伴い、平成21年から中央会の非組合員を対象に同時期に実施してきた単式蒸留焼酎の品質評価受託は廃止した。
おって、輸出促進等に資する品質確保のために、第37回から分析を希望する出品者に対して有料でメタノールの受託分析を実施している。
出品状況、審査及び分析結果の概要について報告する。

伊藤伸一、日下一尊

全国地ビール品質審査会2019（以下、「審査会」という。）は、地ビールの醸造技術向上及び品質改善を目的として、全国地ビール醸造者協議会（Japan Brewers Association）の主催により、平成31年3月13日(火)及び14日(水)に、日本醸造協会赤レンガ酒造工場（東京都北区滝野川）において実施された。審査会には47社から110点の出品があり、出品酒全点が官能評価による審査に供された。出品酒については、事前に独立行政法人酒類総合研究所において成分分析及び微生物検査が実施された。

奥田将生、上用みどり、福田央、玉村隆子

近年泡盛原料米は輸入されるタイ米のロット間差が大きく安定した原料処理が課題となっている。本研究では、泡盛原料米について吸水性や原料利用率に影響を及ぼすと考えられるデンプン特性とタンパク質組成を、清酒醸造に使われる日本晴を対照に用いて解析した。アルカリ崩壊性試験及びDSC解析を一粒毎に行ったところ、泡盛原料のタイ米は糊化温度及びアルカリ崩壊性の大きく異なる米の混合状態であることがわかった。次にアミロペクチンの側鎖分布について解析した。タイ米について、DSCで分別した高温糊化粒と低温糊化粒を比較するとDP6-8の画分に顕著な差異がみられ、タイ米は異なるSSIIa活性の品種が混合されている可能性が示唆された。デンプンの老化特性を解析したところ、老化は日本晴で遅くタイ米で速かった。タイ米の低温糊化粒と高温糊化粒を比較すると、高温糊化粒で老化が速く進んだ。タンパク質組成の解析ではタイ米は60kDa付近にタイ米でバンド濃いバンドがみられたが、タイ米の高温糊化粒と低温糊化粒には顕著な差異がみられなかった。以上から、タイ米の品種混合は、混合比率や糊化特性の違いが変化すると泡盛製造時の原料処理に影響する可能性が考えられた。

藤田晃子、富原健一、久常有里、濱田由紀雄、藤井力

チーズはワインに合うことが広く知られているが、近頃の日本においては清酒のおつまみとしても食されるようになった。チーズにはアミノ酸やペプチドのような旨味成分が豊富に含まれている。その濃厚な味は清酒と比較してワインに合わせた場合、感覚的にすっきりする。このことを確認するため、酒類と合わせた場合のいくつかの食品の後味の強さを、味覚センサーを用いて測定した。チーズ、味噌汁、めんつゆ、グルタミン酸ナトリウム、こく味調味料の旨味応答は清酒と合わせると強く残り、白ワインで弱かった。清酒に比べてワインに多く含まれる化学的成分のなかで、酒石酸やりんご酸等の有機酸が旨味応答を弱くする効果があった。清酒とそれに酒石酸を添加したものを用い、チーズと組み合わせた官能評価を2点試験法によって実施した結果、酒石酸を添加していない方を旨味が残るとした人数が多かった。これらの結果より、食品と酒類の組み合わせにおいて、酒類の有機酸が食品のアミノ酸やペプチドに起因する旨味後味を弱める可能性が示唆された。

Hanae IZU, Sayo YAMASHITA, Hideyuki ARIMA, Tsutomu FUJII

Sake cake contains rice-derived components, as well as cell components and metabolites of Aspergillus oryzae and Saccharomyces cerevisiae. In this study, the effect of food processing on sake cake (sake-kasu) ingredients was investigated. Sake cake, obtained through brewing liquefied rice, was heat-dried (HD) or freeze-dried (FD) and analyzed. There were no differences in the amounts of proteins, lipids, carbohydrates, vitamin B6, choline, betaine, nicotinic acid, β-glucan and resistant proteins in HD and FD. There was also no difference in the amount of hydrolyzed amino acids in HD and FD, but many free amino acids were observed in HD. S-adenosyl methionine (SAM) was found to be abundant in FD. Meanwhile, nucleic acid-related components were found to be increased in HD, which seems to be due to the degradation of microbial metabolites. When considering the health benefits of sake cake, it is necessary to pay attention to the effects of processing method.

Hanae IZU, Sachi SHIBATA, Tsutomu FUJII, and Kiminori MATSUBARA

To examine metabolic effects of sake cake ingestion, plasma and tissues were analyzed in senescence-accelerated mice prone 8 (SAMP8) fed a sake cake diet. As a result, branched-chain amino acids (BCAA) were found to be significantly higher in the plasma, gastrocnemius muscles and brains of the sake cake group than in the control group. Mice in the sake cake group showed stronger grip strength than the control group. High levels of circulating BCAA have been reported to be associated with pathological states, such as metabolic diseases, but the parameters of glucose and lipid metabolism were not affected between the two groups. Otherwise, pyridoxal was significantly higher and nicotinamide as well as 1-methylnicotinamide showed a tendency to be higher in the plasma of the sake cake group than in the control group. These findings indicate that intake of sake cake increases the levels of BCAA, vitamin B6, and vitamin B3.

Kazuya Tomimoto, Takeshi Akao, Hisashi Fukuda

Yeast histone deacetylases (HDAC) affect the production of alcoholic beverages. In this study, we evaluated the sake fermentation characteristics when using HDAC gene-disrupted yeast strain Kyokai No. 701. Flavor components of the sake product were significantly changed. RPD3 or HDA1 disruption increased twofold the amount of isoamyl acetate, and isoamyl alcohol levels also increased in the rpd3△ strain. To determine the contribution of Rpd3L and Rpd3S complexes to sake characteristics, a gene responsible for Rpd3L and/or Rpd3S formation was also disrupted. Disruption of DEP1 or SDS3 that is an essential component of Rpd3L led to increased isoamyl alcohol production similar to that of the rpd3△ strain, but the efficiency of isoamyl alcohol esterification was not affected. In addition, Rpd3 and Hda1 may regulate the responsiveness to oxygen in isoamyl acetate production. We conclude that HDAC genes regulate the production of flavor components during sake fermentation.

Koji Masumura, Sachi Matsukami, Kumiko Yonekita, Muneyoshi Kanai, Kazunori Kume, Dai Hirata & Masaki Mizunuma

Sko1 plays a key role in the control of gene expression by osmotic and oxidative stress in yeast. We demonstrate that the decrease in chronological lifespan (CLS) of hog1Δ cells was suppressed by SKO1 deletion. sko1Δ single mutant cells were shown to have a longer CLS, thus implicating Sko1 in the regulation of their CLS.

Eri ICHIKAWA, Shougo HIRATA, Yuko HATA, Hisashi YAZAWA, Hiroyasu TAMURA, Mitsuoki KANEOKE, Kazuhiro IWASHITA, Dai HIRATA

In sake brewing, the steamed rice is used in two ways, added to sake-mash (as kake-mai) and making koji. The rice is an important determinant for the quality of sake, as the metabolites in sake affect its taste/aroma. The sake rice Koshitanrei (KOS) was developed in Niigata Prefecture by genetically crossing two sake rice, Gohyakumangoku and Yamadanishiki. However, the metabolites in sake from KOS have not been analyzed. Here, to investigate the characteristic metabolites in sake from KOS, we performed two types of small-scale sake fermentation tests changing only the rice used for kake-mai or total rice (both kake-mai and koji) by these three rice cultivars and examined the effect of KOS on sake metabolites by the metabolome analysis method using UPLC-QTOF-MS. We identified the peaks/metabolites, whose intensity in sake from KOS was higher/lower than those from the other cultivars. The brewing properties of KOS were partially characterized by this analysis.

Shunsuke Onuki, Mao Kashima, Toshikazu Yamada, Farzan Ghanegolmohammadi, Yan Zhou, Tetsuya Goshima, Jun-ichi Maruyama, Katsuhiko Kitamoto, Dai Hirata, Takeshi Akao, Yoshikazu Ohya

Mutations frequently occur during breeding of sake yeasts and result in unexpected phenotypes. Here, genome editing tools were applied to develop an ideal nonfoam-forming sake yeast strain, K7GE01, which had homozygous awa1△/awa1△ deletion alleles that were responsible for nonfoam formation and few off-target mutations. High-dimensional morphological phenotyping revealed no detectable morphological differences between the genome-edited strain and its parent, while the canonical nonfoam-forming strain, K701, showed obvious morphological changes. Small-scale fermentation tests also showed differences between components of sake produced by K7GE01 and K701. The K7GE01 strain produced sake with significant differences in the concentrations of ethyl acetate, malic acid, lactic acid, and acetic acid, while K701 produced sake with more differences. Our results indicated genuine phenotypes of awa1△/awa1△ in sake yeast isolates and showed the usefulness of genome editing tools for sake yeast breeding.

Muneyoshi Kanai, Norito Yasuda, Tomoko Morimoto, Satoko Yoshida, Nahoko Nishibori, Masaki Mizunuma, Tsutomu Fujii & Haruyuki Iefuji

Adenosine kinase (ADO1)-deficient mutants can be obtained from cordycepin-resistant strains, and the disruption of ADO1 causes S-adenosylmethionine (SAM) accumulation. To breed a high-SAM-accumulating yeast strain without genetic manipulation for industrial purposes, we bred a cordycepin-resistant strain using sake yeast kyokai No. 9 as the parent strain with a mutation in adenosine kinase (ADO1) and acquired high-SAM-accumulating strain. In the bred strain (NY9-10), a single mutation (T258I) was present in the ADO1, and this mutation site is an ATP binding site and is highly conserved during evolution. Moreover, it was suggested that high accumulation of SAM and cordycepin resistance in NY9-10 was due to functional deficiency of ADO1 by this mutation. This strain is not a genetically-modified organism and can be employed for use in the food and medicine industry such as mass production and sake making.

Hisashi Yazawa, Masafumi Tokuoka, Hajime Kozato, Yutaro Mori, Miyuki Umeo, Rieko Toyoura, Ken Oda, Hisashi Fukuda, and Kazuhiro Iwashita

We developed a sake metabolome analysis method using liquid chromatography-quadrupole/time-of-flight mass spectrometry to investigate the metabolome of various types of sakes and other alcohol beverages. Our method identified 198 compounds by comparison with standard metabolites. Using this method, we investigated the relationship between several sake-making parameters and sake metabolites by conducting combination experiments of these parameters using small-scale fermentation. The results indicated that all parameters significantly affected sake metabolites (P < 0.005) and most peaks were affected by multiple sake-making parameters. Interestingly, the effect of the rice cultivar on sake metabolites was higher for koji rice than for kake-rice. This result suggests that the rice cultivar used has a greater effect on the characteristics of Aspergillus oryzae compared to sake yeast and affects sake metabolites. In this study, we also evaluated the combined effect of several parameters. We demonstrated the different effects of each parameter on several amino acids. The results showed a new aspect of the science of sake making. For example, the amount of α-ethylglucoside, which can affect the taste of sake, was negatively correlated with α-glucosidase activity in koji (r=-0.84). In this study, various unidentified peaks were observed; detectable peaks can be increased by analyzing additional standard reagents. Investigating these unidentified peaks and accumulating datasets for sake-making parameters will give us insight into how to improve sake taste and quality.

Fumikazu AKAMATSU, Hideaki SHIMIZU, Aya KAMADA,Yukari IGI, Tsutomu FUJII, Nami GOTO-YAMAMOTO

The stable isotopic composition of oxygen (δ¹⁸O) in wine is often analysed to determine the geographic origin of the wine and the amount of water dilution. However, little is known regarding the effects of two major winemaking techniques (the addition of acid (acidification) and sugar (chaptalization)) on theδ¹⁸O value of water in wine. Here we show that acidification and chaptalization have minor direct effects on theδ¹⁸O value but indirect effects based on the ethanol yield, which causes isotopic variation of up to 0.6‰. During fermentation,δ¹⁸O values increase at low ethanol yields, suggesting that yeast release water with a highδ¹⁸O value into wine when consuming sugars. Additionally, the ethanol yield is negatively correlated with the consumption of amino acids by the yeast, indicating that yeast growth decreases the ethanol yield. We therefore identify ethanol yield, which is decreased by the consumption of sugars by yeast for non-alcohol-fermentation processes as a potential factor leading to variations in theδ¹⁸O value of water during the winemaking process.

Toshihiko Ito, Anna Sato, Itsuki Takahashi, Takahito Ito, Kouto Takano, Koji Noge, Masaki Okuda, Katsumi Hashizume

The enzymes responsible for acceleration of ferulic acid and ethyl ferulate formation in sake mash were studied. Ferulic acid and ethyl ferulate are formed during the sake brewing process from feruloylated glucuronoarabinoxylan. Cellulase reagent from genus Trichoderma was used instead of rice koji, because rice koji for sake brewing produces extremely low levels of xylan-degrading enzymes. A combination of the reagent with rice koji enzymes accelerated the formation of ferulic acid from a-rice powder. Addition of the reagent to sake mash increased ferulic acid and ethyl ferulate formation. The enzyme responsible for the accelerated formation was purified using a newly developed assay method and a-rice powder as a substrate. During the assay procedure, feruloylated oligosaccharide was converted to ferulic acid by feruloylesterase for HPLC analysis. Analysis of the N-terminal amino acid sequence of the purified samples was successfully conducted after pyroglutamyl aminopeptidase de-blocking. Purified enzymes were identified as members of the glycoside hydrolase family 10 (GH10) and family 11 (GH11) xylanases by BLASTP database research. The GH10 xylanase showed higher specific activity for a-rice powder and insoluble wheat arabinoxylan compared with GH11 xylanase; the GH11 xylanase showed higher specific activity for the other xylan substrates, especially glucuronoarabinoxylan.The GH10 xylanase showed higher accelerating activity than the GH11 xylanase in the sake mash. The results of this study provides useful knowledge on ferulic acid and ethyl ferulate formation in sake mash, the relative levels of these compounds and their influence on the sensory quality of sake.

Kei Takahashi, Hiromi Kohno, Tomomichi Kanabayashi, and Masaki Okuda

Key message

GluA and GluB-4/5 glutelin subfamilies are mainly localized to outer region of the endosperm, particularly in its ventral side, in rice grain, but GluC is localized to throughout the endosperm.

The major seed storage protein in rice (Oryza sativa) is glutelin, which forms a vacuole-derived protein body type-II. Glutelins are encoded by multiple genes, and generally comprise four protein subfamilies, namely, GluA, GluB, GluC, and GluD: however, the localization pattern of glutelin in rice grains remains obscure. In this study, we investigated the localization pattern of five subtypes of the glutelin protein in rice grains using glutelin-subtype specific antibodies. Immunoblot analysis against sequentially polished rice flour fractions from three crop years and seven japonica rice varieties revealed that GluA was strongly localized in the outer region of the endosperm, including the subaleurone layer, whereas GluC was distributed throughout the endosperm. Among the glutelin subtypes, GluA was mostly found in the outer region of the rice grain, followed by GluB-4/5, GluB-1, GluD, and GluC. Immunofluorescence labeling microscopy analysis using immature rice seeds clearly revealed that the localization pattern of GluC and GluD was completely different from that of GluA and GluB. Expression levels of all glutelins, particularly GluA, GluB-1, and GluB-4/5, were stronger on the ventral than dorsal side in rice grains. These results provide strong and consistent evidence that glutelins localize to the rice grain in a subfamily-dependent manner.

Hanae Izu, Mayumi Okuda, Sachi Shibata, Tsutomu Fujii, and Kiminori Matsubara

Six-week-old male KK-Ay mice received drinking water with S-adenosylmethionine (SAM), α-glycerophosphocholine (GPC), or SAM+GPC for 10 weeks. The serum glucose of SAM+GPC at 15 weeks old, total cholesterol of GPC at 12 weeks old, and triglyceride of GPC at 15 weeks old and of SAM at 16 weeks old were reduced. SAM+GPC reduced serum leptin and food intake.