令和6年度 研究成果

A recent trend in some wineries is the return to using spontaneous fermentation, but it is not clear whether winery flora or vineyard microorganisms drive fermentation. We compared fungal communities during the spontaneous fermentation of wine produced in a winery and in a laboratory with sterilized equipment using three grape cultivars (Chardonnay, Merlot, and Muscat Bailey A) obtained from the same harvest. High-throughput sequencing analysis based on the ITS1 region showed that Saccharomyces cerevisiae was the dominant species in winery batches at the end of fermentation, but it was not always dominant in laboratory batches. The number of laboratory batches where S. cerevisiae reached more than 50% at the end of fermentation was only 10 of 26. Consistent with this, in the grape juice/must before fermentation, S. cerevisiae accounted for 1.71% of fungal species identified in winery batches and 0.04% in laboratory batches. In addition, in laboratory-based winemaking, juice clarification of Chardonnay and cold maceration of Merlot influenced the microbial communities observed during fermentation. Our findings suggest that S. cerevisiae present in the winery environment participates at an early stage of fermentation, leading to its dominance at the end in wine produced by spontaneous fermentation in a winery.

Daki warm treatment (daki-ire) is performed during the process of seed mash preparation in the brewing of Japanese sake in order to promote the saccharification of rice by koji enzyme and to enhance the growth of Saccharomyces cerevisiae. Although it is important to control the growth of lactic acid bacteria in the preparation of kimoto-style seed mash (traditional sake-brewing method), it has not been known whether the transient increase in the temperature and/or appropriate temperature zone produced by daki-ire influences the growth of bacteria. A temperature increase generally helps bacterial growth, but we have found no published investigation of the influence of temperature changes in daki-ire on bacterial growth during the kimoto-style seed mash preparation process. In this first comprehensive evaluation of the effects of the temperature change by daki-ire on bacterial communities, we investigated the bacterial community in three batches that were brewed by the same brewery using identical ingredients. The results demonstrated that the bacterial community or its transition during lactic acid fermentation was diverse despite the use of the same brewing conditions. We observed that (i) some lactic acid bacteria were carried over to the subsequent batches, and (ii) the increase in the initial amount of lactic acid bacteria plays an important role in the formation of the bacterial community. Our analysis of the bacterial growth activity before and after daki-ire indicated that the transient increase in temperature and/or local appropriate temperature by daki-ire, in and of itself, has relatively little direct impact on bacterial growth.

Breeding sake yeast typically involves generating several gene mutants through UV irradiation or mutagen treatment and selecting those with desired traits based on indicators such as analog resistance. However, this approach often alters traits beyond the target trait due to the random and numerous mutations introduced. To address this issue, we used a previously established metabolome analysis, a sake metabolome analysis, to evaluate the selected yeast strain. After screening for target traits, 110 sake yeast candidates were cultured in yeast nitrogen-based liquid medium using test tubes. The contents were extracted and subjected to comprehensive metabolite analysis through sake metabolome analysis. A phylogenetic tree was then constructed using the metabolome analysis data, enabling the selection of candidate yeasts with only the target traits modified and other traits similar to the parental strain. Selected 21 candidate strains underwent fermentation tests, and the resulting sakes were analyzed using liquid chromatography quadrupole/ time-of-flight mass spectrometry (LC-Q/TOF-MS). The findings suggested that the metabolomic data of yeast extracts obtained by simple small-scale culture was similar to the data of resulting sake in the larger-scale fermentation tests. This underscores the utility of metabolome analysis data of yeast extracts in the yeast breeding process, marking the first report proposing the use of the sake metabolome analysis method for yeast breeding.

地域特性を明らかにする目的で、2020から2022年までの３年間にわたり全国８県９～11圃場で栽培したワイン用ぶどう品種マスカット・ベーリーAを使用して同じ条件で小規模試験醸造しワインを試作した。果汁およびワインの一般成分分析に加え、７～８評価項目について10点満点で官能評価を行った。ワインの分析結果と官能評価結果との相関を調べた結果、ワインのフラネオール濃度と評価項目「キャンディ・綿飴」において強い正の相関があった。ブドウ抽出液のタンニンはワインのタンニンと強い正の相関があり、官能評価項目「タンニンの強さ」と正の相関があった。Cool night index (CI、収穫前30日の最低気温の平均値）は、Gloriesの方法による全アントシアニン、ワインのA₅₂₀、ワインのフラネオール、官能評価項目「果実香」「キャンディ・綿飴」と強い負の相関が確認された。主成分分析の結果、圃場によってプロットが偏在する傾向が確認され、有効積算温度およびCIが低い長野岩手、 山形、新潟はワインのA₅₂₀、ワインのフラネオール、官能評価項目「果実香」「タンニンの強さ」「ボディ感・味の厚み」が多い傾向であった。また、有効積算温度およびCIが高く収穫が早い山梨や西日本はこれらが少ない傾向であった。

Why was the work done:
Elevated levels of isovaleraldehyde (3-methylbutanal) in sake gives rise to an unfavourable aroma of ‘mureka’ or ‘stuffy smell. The concentration of isovaleraldehyde is typically higher in unpasteurised than pasteurised sake. Controlling the concentration of isovaleraldehyde in unpasteurised sake remains a major challenge for quality control. As existing methods for the quantification of isovaleraldehyde in sake require specialised sample preparation, there is a need for a simple and precise method.

How was the work done:
High-performance liquid chromatography with fluorescence detection and post-column derivatisation (HPLC-PCD-FLD) for determining the isovaleraldehyde content in sake has been developed with optimisation of the separation of peaks and derivatisation of aldehyde compounds. The new method was compared with the established method of headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS).

What are the main findings:
The limit of quantification of the new method was 87 µg/L, and accordingly, the HPLC-PCD-FLD method could determine the concentration of isovaleraldehyde in sake below the reported threshold level. The precision of the HPLC-PCD-FLD method for the analysis of sake containing isovaleraldehyde (〉 threshold level) either matched or was superior to the HS-SPME-GC-MS method.

Why is the work important:
The new approach requires only particle removal for sample preparation, with a rapid analysis time (〉1 h per sample), and requires a smaller sample volume (≈ 100 µL) than the alternative method (10 mL). These improvements contribute to a simpler and more efficient workflow for routine analysis of isovaleraldehyde in the quality control of sake.

Vanillin is naturally occurring in various food products, including alcoholic beverages; however, its contribution to the aroma of sake is unclear. In this study, an HPLC-MS/MS quantification method was developed and validated by linearity, precision, and recovery, and it was applied to 115 bottles of highly diversified sake. Furthermore, the odor detection threshold of vanillin in sake was determined. Notably, the established method exhibited great linearity (5–1500 µg/L), with a R² 0.99, and the limit of detection and limit of quantification were 1.7 and 5.5 µg/L, respectively. The spiked recoveries of vanillin ranged from 96.2% to 97.8%, with relative standard deviation ˂ 6.22%. Results revealed trace amounts to 29.9 µg/L of vanillin in the premium young sake, below the detection threshold (78.9 µg/L), whereas aged sake (43 bottles, 3–56 years aging) exhibited varied concentrations from trace amounts to 1727.5 µg/L of vanillin, notably peaking in a 20-year oak barrel-aged sake. The concentration of vanillin in most of the ambient-temperature-aged sake exceeded the detection threshold after 11–15 years of aging. The proposed method facilitates accurate vanillin quantification in sake, crucial for evaluating its flavor impact. Moreover, the discoveries provide a theoretical basis for the sensory exploration of sake aromas and equip the brewing industry with insights for modulating vanillin synthesis during sake aging,

赤ワイン用ブドウのアントシアニン色素及びタンニンの抽出・分析法として、Bindon et al. (2014)によって報告された方法を、2021及び2022年に国内各地で栽培された‘メルロ’及び‘ピノノワール’を用いて検討した。２年間のブドウ及び赤ワインの分析値の相関係数を求めると、ブドウ抽出液の総アントシアニン濃度は製成したワインのA₅₂₀及び総アントシアニンの指標であるA₅₂₀ (pH 1)と有意な正の相関を示した。また、ブドウ抽出液のタンニン濃度はワインのタンニン濃度と有意な正の相関を示した。別途報告した‘MBA’でも同様の結果であったことから、本方法が日本のブドウにも有効であることが示された。しかし、各回帰直線の傾きは品種によって異なり、試験醸造条件に加え、品種の影響も示唆された。また、味認識装置によるワインの渋味の測定には、タンニン以外の成分も渋味として認識されている可能性が示唆された。

Glucose, a key component of traditional Japanese fermented foods, is derived from rice starch via saccharification by hydrolytic enzymes produced by Aspergillus oryzae. The δ¹³C value of glucose reflects that of its rice source. However, the influence of saccharification parameters (glucose concentration, degradation temperature, and reaction time) on glucose δ¹³C values is unclear. Here, we investigated the influence of saccharification on the δ¹³C value of glucose. Our experiments showed a significant difference in the δ¹³C value of glucose (-27.0 ± 0.1 ‰) obtained from saccharification compared to the ingredient rice (-27.1 ± 0.1 ‰) and remaining solid residue (-27.1 ± 0.1 ‰); however, it did not differ significantly from those of rice koji (-27.0 ± 0.1 ‰) and steamed rice (-27.1 ± 0.1 ‰), despite all values being within 0.1 ‰. Notably, glucose concentration, degradation temperature, and reaction time did not significantly affect glucose δ¹³C values. These findings demonstrate the remarkable preservation of glucose δ¹³C values. The δ¹³C values remain aligned with the original δ¹³C value of the rice, even with up to 60 % degradation during A. oryzae saccharification. This persistence of the δ¹³C value throughout the process offers a potential tool for authenticating the origin of rice-fermented beverages based on the δ¹³C value of their glucose component.

A large number of recombinant plasmids for the yeast Saccharomyces cerevisiae have been constructed and accumulated over the past four decades. It is desirable to apply the recombinant plasmid resources to Saccharomyces sensu stricto species group, which contains an increasing number of natural isolate and industrial strains. The application to the group encounters a difficulty. Natural isolates and industrial strains are exclusively prototrophic and polyploid, whereas direct application of most conventional plasmid resources imposes a prerequisite in host yeast strains of an auxotrophic mutation (i.e., leu2) that is rescued by a selection gene (e.g., LEU2) on the recombinant plasmids. To solve the difficulty, we aimed to generate leu2leu2 mutants from yeast strains belonging to the yeast Saccharomyces sensu stricto species group by DNA editing. First, we modified an all-in-one type CRISPR-Cas9 plasmid pML104 by adding an antibiotic-resistance gene and designing guide sequences to target the LEU2 gene and to enable wide application in this yeast group. Then, the resulting CRISPR-Cas9 plasmids were exploited to seven strains belonging to five species of the group, including natural isolate, industrial, and allopolyploid strains. Colonies having the designed mutations in the gene appeared successfully by introducing the plasmids and assisting oligonucleotides to the strains. Most of the plasmids and resultant leu2leu2-mutants produced in this study will be deposited in several repository organizations.

KEY POINTS:

• All-in-one type CRISPR-Cas9 plasmids targeting LEU2 gene were designed for broad application to Saccharomyces sensu stricto group species strains.
• Application of the plasmids generated leu2 mutants from strains including natural isolates, industrial, and allopolyploid strains.
• The easy conversion to leu2 mutants permits free access to recombinant plasmids having a LEU2 gene.

令和４酒造年度全国新酒鑑評会（第111回鑑評会）は、当該年度に日本国内で生産された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された。

審査は、酒類総合研究所において、令和５年４月19日（水）から21日（金）の３日間に予審を行い、令和５年５月10日（水）から11日（木）の２日間に決審を行った。また、５月31日（水）から６月１日（木）に東広島運動公園体育館にて製造技術研究会を日本酒造組合中央会との共催で実施するとともに、６月17日（土）に東京池袋のサンシャインシティ・文化会館展示ホールにて日本酒造組合中央会主催の公開きき酒会を共催した。

出品は818点であった。審査の結果、優秀と認められた394点を入賞酒とし、さらに入賞酒の中から決審にて特に優秀と認められた218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、833人及び1413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」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。

単式蒸留焼酎を全国的に調査研究することにより、製造技術と品質の現状及び動向を明らかにし、もって単式蒸留焼酎の製造技術及び品質の向上に資することを目的として、第46回本格焼酎・泡盛鑑評会を独立行政法人酒類総合研究所と日本酒造組合中央会の共催で開催した。

令和５年６月20日（火）及び21日（水）に審査を、７月21日（金）に製造技術研究会を実施した。

出品状況、審査及び化学分析結果の概要について報告する。

全国地ビール品質審査会2024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）の主催により実施しており、本年で８回目となる。今回は59社から152点の出品があり、品質審査会を令和６年３月６日（水）及び７日（木）に、独立行政法人酒類総合研究所（広島県東広島市）（以下、「研究所」という。）で実施した。出品酒については、令和６年２月から３月の期間において、研究所にて成分分析及び微生物検査を実施したので、その結果について報告する。

Cellular organelles maintain areas of close apposition with other organelles at which the cytosolic gap in between them is reduced to a minimum. These membrane contact sites (MCS) are vital for organelle communication and are formed by molecular tethers that physically connect opposing membranes. Although many regulatory pathways are known to converge at MCS, a link between MCS and transcriptional regulation-the primary mechanism through which cells adapt their metabolism to environmental cues-remains largely elusive. In this study, we performed RNA-sequencing on Saccharomyces cerevisiae cells lacking tricalbin proteins (Tcb1, Tcb2, and Tcb3), a family of tethering proteins that connect the endoplasmic reticulum with the plasma membrane and Golgi, to investigate if gene expression is altered when MCS are disrupted. Our results indicate that in the tcb1Δ2Δ3Δ strain, pathways responsive to a high-glucose environment, including glycolysis, fermentation, amino acid synthesis, and low-affinity glucose uptake, are upregulated. Conversely, pathways crucial during glucose depletion, such as the tricarboxylic acid cycle, respiration, high-affinity glucose uptake, and amino acid uptake are downregulated. In addition, we demonstrate that the altered gene expression of tcb1Δ2Δ3Δ in glucose metabolism correlates with increased growth, glucose consumption, CO₂ production, and ethanol generation. In conclusion, our findings reveal that tricalbin protein deletion induces a shift in gene expression patterns mimicking cellular responses to a high-glucose environment. This suggests that MCS play a role in sensing and signaling pathways that modulate gene transcription in response to glucose availability.

Background
The quantity and composition of rice proteins play a crucial role in determining taste quality of sake, Japanese rice wine. However, the spatial distribution of proteins within rice grains, especially in endosperm tissue, and the differences between rice varieties remain unclear.
Results
Here, we analyzed the crude protein contents and composition ratios of table (Nipponbare and Koshihikari) and genuine sake rice varieties (Yamadanishiki, Gohyakumangoku, Dewasansan, Dewanosato, and Yumenokaori) to elucidate their spatial distribution within the Japonica rice grain endosperm. Seven sake rice varieties were polished over five harvest years using a brewer’s rice-polishing machine. We obtained fractions at 90%–70% (the outermost endosperm fraction), 70%–50%, 50%–30%, and 30%–0% (the central region of the endosperm fraction). Yamadanishiki and Dewanosato exhibited considerably lower crude protein contents than the other cultivars. After applying SDS-PAGE, the protein composition, comprising glutelin/total protein (G/TP), prolamin/TP (P/TP), and G/P ratios of these fractions was determined. In white rice (at a 90% rice-polishing ratio), the average ratio of the major protein composition was G/TP 41%, P/TP 21%, and G/P ratios of 1.97. Gohyakumangoku and Yamadanishiki had higher G/TP ratio, while Dewanosato had a lower value. Despite having lower crude protein contents, Yamadanishiki and Dewanosato exhibited significantly varying G/TP ratios. The G/TP ratio markedly varied among rice varieties, particularly in the rice grains’ central region. The 50%–30% fraction had the highest P/TP ratio among all tested rice varieties, suggesting spatial differences in P/TP within rice grains. Koshihikari had the lowest P/TP ratio. In addition, the 50%–30% fraction had the lowest G/P ratio among all tested rice varieties, with Gohyakumangoku having the highest G/P ratio. Dewanosato had the lowest G/P value, and this value significantly differed from that of Yamadanishiki in the 30%–0% fraction.
Conclusions
We found substantial differences in protein composition within distinct spatial regions of rice grains, and larger differences among rice varieties were observed in the rice grain's central region.

アルコール飲料と料理の相性に関する議論では、魚料理と一緒に飲むアルコールの種類によっては、生臭さを感じることがある。しかし、日本酒はこの生臭みを効果的に抑制し、料理との相性を高めることが報告されている。本研究では官能評価を行い、ビールにスルメを合わせると魚由来の金属臭が目立つことを確認した。一方、日本酒とスルメを併用すると、金属臭が効果的に抑制されることが官能評価で確認された。この金属臭の原因物質は、既報の(Z)-1,5-オクタジエン-3-オンと同定された。アルコール飲料中の(Z)-1,5-オクタジエン-3-オンの1ng/L(ppt)以下の定量分析を行った。SBSE抽出、PFBOA誘導体化、NCIイオン化、GC/MS/MS分析を組み合わせた高感度で合理的な定量法を確立した。日本酒、ビール、スルメを混合した場合の(Z)-1,5-オクタジエン-3-オンの生成量を測定したところ、日本酒の方が有意にレベルが低く、平均生成量はビールより7.1倍低かった。本研究は、清酒の生臭さの一因とされる「金属臭」の抑制を成分レベルで初めて実証し、「食品との相性」についての知見を提供するものである。

Hineka is a type of off-flavor of sake and is attributed to the presence of several compounds, including a major one called dimethyl trisulfide (DMTS). The production of the main precursor of DMTS involves yeast methionine salvage pathway. The DMTS-producing potential (DMTS-pp) of sake brewed using the Km67 strain, a non-Kyokai sake yeast, is lower than that of sake brewed using Kyokai yeast; however, the detailed mechanism is unclear. We focused on S-adenosyl-methionine (SAM) and aimed to elucidate the mechanism that prevents DMTS production in sake brewed using the Km67 strain. We revealed that SAM is involved in DMTS production in sake, and that the conversion of SAM to the DMTS precursor occurs through an enzymatic reaction rather than a chemical reaction. Based on previous reports on ADO1 and MDE1 genes, sake brewing tests were performed using the Km67 Δmde1, Δado1, and Δmde1Δado1 strains. A comparison of the SAM content of pressed sake cakes and DMTS-pp of sake produced using the Km67 Δado1 strain showed an increase in both SAM content and DMTS-pp compared to those produced using the parent strain. However, the Km67 Δmde1Δado1 strain showed little increase in DMTS-pp compared to the Km67 Δmde1 strain, despite an increase in SAM content. These results suggest that SAM accumulation in yeast plays a role in the production of DMTS in sake through the methionine salvage pathway. Moreover, the low SAM-accumulation characteristic of the Km67 strain contributes to low DMTS production in sake.

赤ワインの醸し方法のうち、デレスタージュ、デレスタージュと高温短期醸し、デレスタージュと後期高温醸しの組み合わせが赤ワインの色やタンニンなどに及ぼす影響について、メルロを用いた２回の小規模試験醸造を行って検討した。デレスタージュ時に種子を除くと、６回中５回の試験区で総フラバノール（タンニン）を有意に減少させたが、赤ワインの色に対しては若干減少～影響なしであった。デレスタージュと後期高温醸し、及び後期高温醸しのみは赤色に一貫した効果を示さなかった。一方、デレスタージュと高温短期醸しを組み合わせた場合は、安定化した赤色を増加させる効果が認められた。さらなる検討が必要ではあるが、これらの結果は赤ワイン醸造に有益な情報となると考えられる。