令和2年度 研究成果

Masayuki TAKAHASHI, Yasuko KITA, Rui MINAKAMI, Nobuhiko MUKAI

Sake (Japanese rice wine) has been recognized as being low risk in terms of its microbiological safety. However, a confirmation of the food safety aspects of sake based on scientific evidence is important for establishing consumer confidence, in part because consumer concerns regarding food safety have increased. The presence of Bacillus cereus spores in refined rice wine has been reported, and in light of consumers' growing concern over food safety, the establishment of food and beverage safety is important for consumers' reassurance. Herein, to confirm the microbiological safety of sake, we investigated the content and growth of B. cereus. We conducted a spore addition test to determine whether B. cereus spores grow during sake production, and we observed no growth or germination of B. cereus spores during the manufacturing process. We also observed that processes such as solid–liquid separation and filtration help remove the risk posed by B. cereus. We then conducted a survey to assess the density of B. cereus in various commercial sake products. We analyzed 162 samples of commercial sake and observed that 11 of the products had ≥1 CFU of living cells in 1 mL of sake (detection rate, 6.8%). There was no product in which ≥100 CFU of living cells per 1 mL of sake was detected. Our findings confirmed that the density of these bacteria in sake is lower than that in other foods and that the probability of infection is very low. The emetic toxin produced by B. cereus was not detected in any of the sake samples. This is the first study based on experimental data demonstrating that B. cereus is not able to grow in sake or during the sake manufacturing process. We, thus, conclude that the safety risk of B. cereus in sake is negligible. Our findings indicating that B. cereus is not a significant hazard in the sake brewing process will contribute to food hygiene management based on scientific evidence in sake breweries.

Masayuki TAKAHASHI, Kana MORIKAWA, Yasuko KITA, Takamasa SHIMODA, Takeshi AKAO and Nami GOTO-YAMAMOTO

Kimoto-style seed mash is a traditional preparation method for sake that takes advantage of spontaneous lactic acid fermentation before the growth of yeast. Lactic acid helps decrease the pH in seed mash and control the growth of unfavorable microorganisms. In this study, we carried out a comprehensive analysis of the change in the bacterial community and chemical composition during the lactic acid fermentation stage in kimoto-style seed mash preparation. The bacterial transitions were diverse at five sake breweries, but they exhibited three patterns. Lactobacillus sakei was the dominant species in the later stage of lactic acid fermentation in all sake breweries. This species was found to be the most important bacterium for the accumulation of lactic acid, because its average production rate of lactic acid in seed mash reached 4.44 × 10^-11mg cell^-1 h^-1, which is 10 times higher than those of other species. As a result of specific growth rate analysis, it was revealed that the growth rate of L. sakei was influenced by the strain, pH, and temperature. The effects of pH and temperature were explained by the square root model, and the result indicates that the strains isolated in this study were incapable of growth below pH 3.9. The growth curve predicted using the growth model fit the actual cell density in two out of five sake breweries; however, our model did not work well for the remaining three sake breweries, and we presume that the error was caused by the strain or an unknown factor.

村上智子, 渡辺守, 髙木昌美, 末次-佐々木春菜, 渡辺大輔, 五島徹也, 福田央, 下飯仁, 赤尾健

清酒酵母きょうかい6号及び7号より、新規のアルコール耐性自然突然変異体をスクリーニングした。一連のストレス耐性に関連した試験によりK6AT1－K6AT8、K7AT1－K7AT5について評価を行った。K6AT及びK7AT株のすべては、K7AT5を例外として、それぞれの親株よりもアルコール及び熱ストレスに対し高い耐性を有していた。K6AT及びK7ATを用いた清酒小仕込み試験の製成酒について、一般成分、有機酸組成、揮発成分組成を調べた。概して製成酒の酸度は親株よりも高く、特徴的な有機酸組成（高リンゴ酸、高コハク酸、高乳酸）とよく一致した。また、アミノ酸度は親株よりも低くかったが、もろみ終点での酵母の生存率を反映していると考えられた。K6ATの揮発成分組成はK6と違いがなく、K7ATの場合は株によってまちまちだった。味認識センサー解析の結果は、各製成酒の化学成分組成を反映していると考えられた。K7AT5株は、試験全般にわたり他の変異株と全く異なる特性を有していた。この株は、スクリーニング段階ではK7よりも高いアルコール耐性を示したが、その後のいくつかの試験の結果は、この株がアルコール耐性を失ったことを示していた。加えて、この株は他のK7AT株と比べ、特有の醸造特性を有していた。これらのことは、アルコール耐性の抑圧変異がK7AT5株内で生じ、広がったことを示唆する。

Kazuo Masaki, Kanako Fujihara, Dararat Kakizono, Taichi Mizukure, Masaki Okuda, Nobuhiko Mukai

Urethanase (EC 3.5.1.75) catalyzes the hydrolysis of ethyl carbamate (EC) to ethanol, carbon dioxide, and ammonia. From our recent study, we expected that an acetamidase encoded by amdS of Aspergillus oryzae may catalyze the degradation of EC because it is homologous with a Candida parapsilosis urethanase (CPUTNase) recently identified. Urethanase is a prospective candidate to reduce EC in alcoholic beverages, but knowledge of this enzyme is very limited. Recombinant AmdS was expressed to study its enzymatic properties. Purified AmdS was identified as a homo-tetramer consisting of four 60 kDa units and exhibited urethanase activity. In a 20% ethanol solution, AmdS had 65% activity compared with a solution without ethanol. Residual activity after 18 h indicated that AmdS was stable in 0%–40% ethanol solutions. The optimum temperature of AmdS was 40°C. This enzyme showed urethanase activity at pH 6.4–9.6 and exhibited its highest activity at pH 9.6. The Km value of AmdS for EC was 8.2 mM, similar to the Km value (7.6 mM) of CPUTNase. AmdS showed activity not only for EC and acetamide but also other amide compounds. In this study, we investigated the enzymatic properties of AmdS that was identified as acetamidase and showed that an amidase can be an enzymatic candidate that degrades EC.

Jun Makimoto, Kou Wakabayashi, Toyohisa Inoue, Yuriko Ikeda, Ryoko Kanda, Atsuko Isogai, Tsutomu Fujii, Takashi Nakae

Dimethyl trisulfide (DMTS) is one of the main components responsible for “hineka”, the aroma associated with deteriorated Japanese sake during storage. The molecule 1,2-dihydroxy-5-(methylsulfinyl)pentan-3-one (DMTS-P1) has been previously identified as a major precursor compound of DMTS. Furthermore, it had been suggested that the yeast methionine salvage pathway is involved in the production of DMTS-P1. In sake brewing tests, DMTS-P1 and the DMTS producing potential (DMTS-pp; DMTS amount of sake after accelerated storage) were significantly reduced in mde1 or mri1 strain, which lack genes of the methionine salvage pathway. Industrial use of the gene-disrupting strains may not be accepted in the Japanese food industry. In order to obtain mde1 or mri1 mutants, we established a method to screen 5’-methylthioadenosine (MTA) non-utilizing strains using minimum culture medium containing methionine or MTA by ethyl methanesulfonate (EMS) mutagenesis with methionine-auxotrophic sake yeast haploid. As expected, mde1 and mri1 mutants were identified among the obtained mutants by an established screening method. The obtained strains had poor fermentation ability in sake brewing tests, so back-crossing was performed on the mutants to obtain mde1 or mri1 homozygous mutants. These strains had improved brewing characteristics, and DMTS-P1 and the DMTS-pp of the produced sake were significantly lower than those of the parent strains. These strains are expected to contribute to improving the maintenance of sake quality during storage.

Hideaki SHIMIZU，Minori KONO, Kazuya KOYAMA, Kazuhiro IWASHITA, Nami GOTO-YAMAMOTO

The effect of cold soak in pilot-scale and plant-scale vinification processes at various maceration temperatures was examined in nine wineries, and the produced wines were analyzed for chemical and sensory properties. Cold soak tended to increase total anthocyanins, color intensity, and total phenols (A280) of wine regardless of grape variety, when the temperatures for the cold soak and the subsequent maceration were controlled appropriately. The basic chemical components of wines were minimally affected by the cold soak. Total anthocyanins in the cold soak wines were higher than those in control wines, and some cold soak wines received favorable evaluation with regard to flavor, astringency, or body in the sensory analysis compared with control wines. The results indicate that cold soak carried out appropriately resulted in good coloration in many cases and had no deleterious effects.

Churan Fu, Yongshou Yang, Thanutchaporn Kumrungsee, Akiko Kimoto,　Hanae Izu and Norihisa Kato

The effects of low-dose alcohol on experimental animals are unclear. This study examined plasma metabolites in senescence-accelerated mice 8 (SAMP8) given lowdose ethanol, and compared them with aging progress and skeletal muscle strength. Male SAMP8 mice (10-wk-old) were given drinking water containing 0% (control), 1%, 2%, or 5% (v/v) ethanol for 14 wk. Compared with the control group, only mice who consumed 1% ethanol experienced a lower senescence score at 18 and 23 wk, as well as an increased limb grip strength at 21 wk. Plasma metabolites of control, 1% and 2% ethanol groups were analyzed by capillary electrophoresis–time-of-flight mass spectrometry (CE-TOF/MS). Among the 7 metabolites affected by ethanol, notewhorthy is the positive association of the ethanol levels in drinking water with the levels of α-ketoglutarate (antioxidant and anti-inflammatory metabolite) and hippurate (antioxidant and microbial co-metabolite) (p<0.05). Intriguingly, the levels of 2-hydroxyisobutyrate (the biomarker of energy metabolism and microbial co-metabolite) were higher in the 1% ethanol group (p<0.05), but not in the 2% ethanol group as compared to the control. Furthermore, the levels of some of the metabolites affected were correlated with some variables in the grading score of senescence and muscle strength. This study provides a novel insight into how low-dose ethanol in SAMP8 mice modulates the levels of circulating metabolites relating to chronic disease risk.

Nami GOTO-YAMAMOTO, Akiko HIRANO, Mayuko KAWADA, Mineyo NUMATA, Kazuya KOYAMA

The effects of maceration temperature and duration on anthocyanin and proanthocyanidin of red wine were examined using Merlot cultivated in Japan. After an extraction experiment using model wine solutions, a small scale vinification test was carried out. A short (3 days) maceration at a high temperature (30℃) resulted in a stronger red color and lower proanthocyanidin concentration than the control (25℃ for 8 days). Also, a combination of cold maceration (15℃ for 2 days) with the short maceration at a high temperature resulted in a stronger red color and similar proanthocyanidin concentration compared with the control. The difference of the color of finished wine was maintained after 1-year aging in bottles.

山田修、藤田晃子、飯塚幸子、伊豆英恵、岸本徹、神田涼子、寺本聡子、後藤奈美

平成30酒造年度全国新酒鑑評会（第107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31年4月23日（火）から25日（木）の3日間に予審を行い、令和元年5月9日（木）及び10日（金）に決審を行った。また、5月29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15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857点であった。審査の結果、優秀と認められた416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237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、1,551人及び3,286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。

向井伸彦、磯谷敦子、金井宗良、髙橋正之、長船行雄、山根善治、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42 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。今回から開催目的を改訂し、鑑評会が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明確化した。

審査は令和元年6月 4日（火）及び6月5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令和元年6月21日（金）に当研究所で開催し112名が参加した。

なお、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とともに、尺度評価項目間の相関関係についても調べたので併せて報告する。

伊藤伸一、矢澤彌、神本真紀、江村隆幸

全国地ビール品質審査会2020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4回目となる。本年においては45社から106点の出品があり、品質審査会を令和2年3月11日（水）及び12日（木）に、公益財団法人日本醸造協会赤レンガ酒造工場（東京都北区滝野川）で実施する予定であったが、新型コロナウイルス感染症拡大防止の観点から中止となった。出品酒については、令和2年2月から4月の期間において、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を実施したので、その結果について報告する。

藤田晃子、後藤奈美

ワインの特徴香やオフフレーバーを適切に識別し、評価することは、ワインの官能評価を行うために不可欠な能力であり、効果的な官能評価訓練を行うためにはこれらの香気成分を示すサンプル作成が重要である。現在、市販のワインフレーバーサンプルには、小さい瓶から香りを嗅ぐタイプの製品や、カプセルに入った香気成分をワイン1本に添加する製品等がある。しかし、研修会などである程度の人数が一度に使用するには、水、アルコール溶液またはワインに香気成分を添加したサンプルが適しており、さらにサンプル量や濃度が調節できることが望ましい。そこで、試薬として入手できる香気成分を用いて官能評価訓練用の標準的な濃度を決め、その1000倍濃度の原液を、必要な場合に使用したり分譲したりできるようスクリューキャップ付きのバイアルに分注して冷凍保存することとした。また、ワインを用いて酸化臭や日光臭のサンプルを作成する際の標準的な方法を定めた。

Sharon Marie Bahena-Garrido, Ryota Saito, Yuko Komatsu, Shiori Kodama, Ken Oda and Kazuhiro Iwashita

Based on the tandem approach of DI-HRMS and LC-QTOF-MS/SIM（and MS/MS), we could finally conclude that no significant traces of harmful secondary metabolites or mycotoxins found in all of the 13 Aspergillus oryzae industrial strains cultivated under different culture conditions. We further confirmed the safety of these Aspergillus oryzae strains for industrial production of fermented foods and beverages.

Eri lchikawa, Shougo Hirata, Yuko Hata, Hisashi Yazawa, Hiroyasu Tamura, Mitsuoki Kaneoke, Kazuhiro lwashita, and Dai Hirata

In sake brewing, the steamed rice is used in 2 ways, added to sake-mash and making rice-koji. Rice-koji is made from the steamed rice by using koji starter, and its quality is an important determinant of the aroma/taste of sake. The sake rice Koshitanrei (KOS) was developed in Niigata Prefecture by crossing 2 sake rice varieties, Gohyakumangoku and Yamadanishiki. Recently, we reported the characteristic components/metabolites in sake made from KOS by conducting metabolome analysis using UPLC-QTOF-MS. In this study, to investigate the effect of koji starter and sake rice cultivars on the sake metabolites, we performed small-scale sake-making tests using the above 3 rice cultivars and 3 koji starters. Finally, we demonstrated that some of the characteristic components/ metabolites of sake from KOS are affected by the koji starter. Thus, in addition to rice cultivar, koji starter plays an important role for establishment/maintenance of the quality of the final product.

Khanh Dung Pham, Yosuke Shida, Atsushi Miyata, Takeru Takamizawa, Yoshiyuki Suzuki, Satoshi Ara, Harutake Yamazaki, Kazuo Masaki, Kazuki Mori, Sachiyo Aburatani, Hideki Hirakawa, Kosuke Tashiro, Satoru Kuhara, Hiroaki Takaku and Wataru Ogasawara

The oleaginous yeast Rhodosporodium toruloides is receiving widespread attention as an alternative energy source for biofuels due to its unicellular nature, high growth rate and because it can be fermented on a large-scale. In this study, R. toruloides was cultured under both light and dark conditions in order to understand the light response involved in lipid and carotenoid biosynthesis. Our results from phenotype and gene expression analysis showed that R. toruloides responded to light by producing darker pigmentation with an associated increase in carotenoid production. Whilst there was no observable difference in lipid production, slight changes in the fatty acid composition were recorded. Furthermore, a two-step response was found in three genes (GGPSI, CAR1, and CAR2) under light conditions and the expression of the gene encoding the photoreceptor CRY1 was similarly affected. Light effect on carotenoid production in Rhodosporidium toruloides

Kazuo Masaki, Taichi Mizukure, Dararat Kakizono, Kanako Fujihara, Tsutomu Fujii, Nobuhiko Mukai

Urethanase (EC 3.5.1.75) is an effective enzyme for removing ethyl carbamate (EC) present in alcoholic beverages. However, urethanase is not well studied and has not yet been developed for practical use. In this study, we report a new urethanase (CPUTNase) from the yeast Candida parapsilosis. Because C. parapsilosis can assimilate EC as its sole nitrogen source, the enzyme was extracted from yeast cells and purified using ion-exchange chromatography. The CPUTNase was estimated as a homotetramer comprising four units of a 61.7 kDa protein. In a 20% ethanol solution, CPUTNase had 73% activity compared with a solution without ethanol. Residual activity after 18 h indicated that CPUTNase was stable in 0%-40% ethanol solutions. The optimum temperature of CPUTNase was 43℃. This enzyme showed urethanase activity at pH 5.5-10.0 and exhibited its highest activity at pH 10. The gene of CPUTNase was identified, and a recombinant enzyme was expressed in the yeast Saccharomyces cerevisiae. Characteristics of recombinant CPUTNase were identical to the native enzyme. The putative amino acid sequence indicated that CPUTNase was an amidase family protein. Further, substrate specificity supported this sequence analysis because CPUTNase showed higher activities toward amide compounds. These results suggest that amidase could be a candidate for urethanase. We discovered a new enzyme and investigated its enzymatic characteristics, sequence, and recombinant CPUTNase expression. These results contribute to a further understanding of urethanase.

Hideaki Shimizu, Fumikazu Akamatsu, Aya Kamada, Kazuya Koyama, Kazuhiro Iwashita, and Nami Goto-Yamamoto

It has been reported that the concentrations of minerals in wines can be used to discriminate their geographical origin. However, some winemaking techniques may also affect the mineral concentration of the final product. In this study, we examined the effects of various winemaking techniques, including (i) fining, (ii) aging with oak tips, (iii) maceration with grape skins, (iv) chaptalization and acidification, and (v) yeast nutrient addition for alcohol fermentation, on the concentration of 18 minerals (Li, B, Na, Mg, Si, P, S, K, Ca, Mn, Co, Ni, Ga, Rb, Sr, Mo, Ba and Pb) in a total of 154 wine samples using grapes from different production areas. Among the various winemaking techniques, maceration with grape skin had the largest effect on mineral content, significantly changing the concentrations of 17 or 18 elements (B, Na, Mg, Si, P, S, K, Ca, Mn, Co, Ni, Ga, Rb, Sr, Mo, Ba and Pb). Fining treatment using bentonites had the second largest effect, altering the concentrations of 14 elements (Li, B, Na, Si, P, S, Ca, Co, Ga, Rb, Sr, Mo, Ba and Pb). However, in principal component analysis using all data (n=154), the wine samples were clearly clustered according to grapes used in the experiments rather than the winemaking technique. In conclusion, some winemaking techniques significantly altered the concentration of some minerals in wine; however, the principal factor influencing wine mineral composition seems to be its geographical origin.

Puspa Raj POUDEL, Kazuya KOYAMA, Nami GOTO-YAMAMOTO

The variability in grape (Vitis vinifera L.) proanthocyanidin content is largely attributable to viticultural and environmental conditions. However, the particular effect temperature has on proanthocyanidin biosynthesis is poorly understood. The aim of the present study was to ascertain the magnitude of the effect of temperature on proanthocyanidin biosynthesis in Cabernet Sauvignon grape berries cultured in vitro. In addition, the effects of temperature on global gene transcription were evaluated, and the microarray data were later validated by quantitative real-time PCR (qPCR). The grape berries used in this research were sampled 3-4 weeks after full bloom and cultured in vitro either under a low (20 ℃) or high (30 ℃) temperature treatment for 15 days (d) with sampling occurring every five days. The proanthocyanidin content was higher in the skin and seeds of grape berries cultured at a low temperature compared with a high temperature. However, overall proanthocyanidin composition between the treatments was not significantly affected. Microarray data revealed a total of 1298 genes with ≥ 3.5-fold expression differences under high temperature conditions. High temperature also inhibited the expression level of key genes involved in proanthocyanidin biosynthesis, anthocyanidin reductase (ANR) and leucoanthocyanidin reductase-1 (LAR-1) within the berry skin. However, the transcriptomic accumulation of transcription factors, such as VvMybPAs, VvMyb5a and VvMyb5b, was barely influenced during the peak expression of ANR and LAR-1. Thus, the present study revealed that temperature has a significant effect on proanthocyanidin biosynthesis in grape during berry development through enhancing the expression of key biosynthetic genes.

Fumikazu AKAMATSU, Yukari IGI, Akiko FUJITA

An existing procedure for the separation and purification of glucose in sake for compound-specific carbon stable isotope analysis was examined with a view to reducing the sample amount required and process complexity, with a focus on the freeze-drying, solid-phase extraction (SPE), and high-performance liquid chromatography (HPLC) steps. In the revised procedure, the carbon stable isotopic composition, as indicated by δ¹³C values, of glucose was not significantly affected by glucose concentration in the range 0.25-1.00 mg mL^-1, solvent (water or 80% aqueous acetonitrile), or HPLC procedure. Glucose freeze-dried after separation and purification by SPE and HPLC exhibited procedure-induced carbon isotopic discrimination of ≤ 0.1‰. The isolation procedure achieved the same accuracy in glucose δ¹³C values as that achieved with traditional ion-exchange procedures while requiring a sample volume (1 mL) only 4% that of the usual volume. The improved method enables cost-effective, labor-efficient, small-scale, compound-specific carbon stable isotope analyses of glucose in sake.

Ryousuke Kataoka, Taisuke Watanabe, Risa Hayashi, Atsuko Isogai, Osamu Yamada, Jun Ogihara

1-Octen-3-ol is a major aroma component of awamori, a traditional distilled liquor produced in Okinawa Prefecture, Japan. As 1-octen-3-ol is thought to affect the sensory properties of awamori, it is important to fully characterize the compound's biosynthetic pathway and control mechanism. We previously reported that the fatty acid oxygenase ppoC (ppo: psi-produced oxygenase) of Aspergillus luchuensis is directly involved in the production of 1-octen-3-ol in rice koji (Kataoka et al., J. Biosci. Bioeng., 129, 192-198, 2020). In the present study, we constructed A. luchuensis ppoD disruptants to characterize the role of ppo genes in 1-octen-3-ol biosynthesis. A small-scale awamori fermentation test was performed using ppoA, ppoC, and ppoD single disruptants (ΔppoA, ΔppoC, and ΔppoD, respectively), along with the parent strain, ΔligD. 1-Octen-3-ol was not detected in the distillate prepared using the ΔppoC strain. We conclude that A. luchuensis ppoC is the only 1-octen-3-ol-producing factor in the awamori brewing process. Because ΔppoA and ΔppoD slightly enhanced 1-octen-3-ol productivity, these two genes may play a role in negatively controlling 1-octen-3-ol biosynthesis.

Risa Yamasaki, Tetsuya Goshima, Kenji Oba, Atsuko Isogai, Ritsushi Ohdoi, Dai Hirata, Takeshi Akao

General sake yeasts (e.g., Kyokai no.7, K7) show high fermentation ability and low sporulation frequency. Former is related to stress-response defect due to the loss-of-function of MSN4 and RIM15. Later is mainly caused by low IME1 expression, leading to difficulty in breeding and genetic analysis. Sake yeast Hiroshima no.6 (H6), which had been applied for sake fermentation, has sporulation ability. However, its detailed properties have not been unveiled. Here we present that the fermentation ability of H6 is suitable for sake brewing, and the precursor of dimethyl trisulfide in sake from H6 is low. MSN4 but not RIM15 of H6 has the same mutation as K7. Our phylogenetic analysis indicated that H6 is closely related to the K7 group. Unlike K7, H6 showed normal sporulation frequency in a partially RIM15-dependent manner, and IME1 in H6 was expressed. H6 possesses excellent properties as a partner strain for breeding by crossing.

Muneyoshi Kanai, Tomoko Kawata, Tomoko Morimoto, Masaki Mizunuma, Daisuke Watanabe, Takeshi Akao, Tsutomu Fujii, Haruyuki Iefuji

To elucidate the mechanism underlying tetrahydrofolate (THF) accumulation in sake yeast strains compared with that in laboratory yeast strains, we performed a quantitative trait locus (QTL) analysis. The results revealed that the sake yeast ERC1 allele contributes to an increase in the ratio of THF to the total folate content in sake yeast.

Masataka NARUKAWA, Aya KAMIYOSHIHARA, Hanae IZU, Tsutomu FUJII, Kiminori MATSUBARA, and Takumi MISAKA

α-Glycerophosphocholine (GPC) is a natural source of choline. It reportedly prevents aging-related decline in cognitive function, but the underlying mechanism remains unclear. Although it is understood that aging influences taste sensitivity and energy regulation, whether GPC exerts antiaging effects on such phenomena requires further elucidation. Here, we used old C57BL/6J mice that were fed a GPC-containing diet, to investigate the molecular mechanisms underlying the prevention of a decline in cognitive function associated with aging and examine the beneficial effects of GPC intake on aging-related phenomena, such as taste sensitivity and energy regulation. We confirmed that GPC intake reduces the aging-related decline in the expression levels of genes related to long-term potentiation. Although we did not observe an improvement in aging-related decline in taste sensitivity, there was a notable improvement in the expression levels of β-oxidation-associated genes in old mice. Our results suggest that the prevention of aging-related decline in cognitive function by GPC intake may be associated with the improvement of gene expression levels of long-term potentiation. Furthermore, GPC intake may positively influence lipid metabolism.

Fumikazu AKAMATSU, Masaki OKUDA, Tsutomu FUJII

The stable isotopic compositions of carbon (δ¹³C) and oxygen (δ¹⁸O) in rice are often used to confirm its authenticity. The gelatinization temperature is a crucial factor in alcoholic fermentation. However, little is known about the isotopic and thermal responses of rice to climate change. We show that in sake rice grown annually in the same paddy field from 1994 to 2013, the δ¹³C (-27.4‰ to -25.9‰) and δ¹⁸O values (20.4‰ to 27.0‰) correlated negatively with the mean daily minimum air temperature and precipitation, and positively with the hours of sunshine during grain-filling. In contrast, of the air temperatures tested, the gelatinization temperature of the rice grains (63.2 ℃ to 70.0 ℃) correlated positively and most strongly with the mean daily mean air temperature. Thus, we identified the mean daily minimum temperature during grain-filling, a nocturnal temperature, as a major factor affecting the isotopic and thermal variations in rice grains.

Mizuki Yasui, Ken Oda, Shunsuke Masuo, Shuji Hosoda, Takuya Katayama, Jun-ichi Maruyama, Naoki Takaya and Norio Takeshita

Background

'Rice koji' is a solid culture of Aspergillus oryzae on steamed rice grains. Multiple parallel fermentation, wherein saccharification of rice by A. oryzae and alcohol fermentation by the budding yeast occur simultaneously, leads to the formation of a variety of ingredients of Japanese sake. In sake brewing, the degree of mycelial invasive growth into the steamed rice, called 'haze-komi', highly correlates with the digestibility and quality of rice koji, since the hyphae growing into the rice secrete amylases and digest starch.

Results

In this study, we investigated mycelial distribution of GFP-tagged A. oryzae in rice koji made with different types of rice, such as sake rice and eating rice, with 50 or 90% polishing rate to remove abundant proteins and lipids near the surface. In addition, we compared transcriptomes of A. oryzae in the different types of rice koji. Finally, we found that A. oryzae increases the nuclear number and hyphal width in the course of 1-3 days cultivation.

Conclusions

Our imaging analyses indicate that A. oryzae hyphae grew more deeply into 50% polished rice than 90% polished rice. The increases of nuclear number may be a selectively acquired characteristic for the high secretory capacity during the long history of cultivation of this species.

Risa Yamasaki, Tetsuya Goshima, Kenji Oba, Muneyoshi Kanai, Ritsushi Ohdoi, Dai Hirata, Takeshi Akao

Among sake yeast strains, Kyokai no. 7 (K7) and its closely related strains (K7 group) are predominantly used because of their excellent brewing properties. In the sake industrial sector, the need for various types of yeast strains is high. Although crossbreeding is an effective method for generating genetic diversity that should result in diverse characteristics, most K7 group strains lack normal sporulation ability, including the ability to undergo meiotic chromosomal recombination, which leads to difficulties in crossbreeding. Accordingly, the improvement of sake yeast strains primarily depends on mutagenesis and suitable selection in a stepwise manner. Our recent study revealed that the long-preserved sake yeast strain Hiroshima no. 6 (H6) does not belong to the K7 group despite genetically being extremely similar. In addition, H6 exhibited normal sporulation. Thus, we isolated haploid cells from H6 and mated them with previously isolated haploid cells of K7 group strains. The crossbred diploid strains had normal sporulation ability; hence, we performed tetrad analysis. The brewing characteristics of the obtained haploid set were extremely diverse. Principal component analysis based on the volatile and organic acid components measured using small-scale sake brewing tests revealed that the haploid strains derived from each diploid strain displayed a characteristic distribution. Thus, we demonstrated the availability of genetic crossbreeding using H6 with sporulation ability to facilitate both the development of novel sake yeast strains with many desirable characteristics and analyses of the function of sake yeast.