平成20年度 研究成果

後藤（山本）奈美、沼田美子代、島本敏、望岡亮介、細見彰洋

'紫'は日本の古い在来品種であると言われているが、一方で'紫'は'甲州'と同じ品種であるという説もある。現在、'紫'は大阪府環境農林水産総合研究所に保存されている1樹のみが知られている。この'紫'は'甲州'と26のSSR遺伝子座で同じ結果を示した。2006年の'紫'は酒類総合研究所の'甲州'よりも若干マルビジン3-グルコシド含量が高かったが、'紫'のアントシアニン含量は'甲州'の年、栽培地、およびおそらくはクローンが異なる場合のアントシアニン含量の範囲の中にあった。従って、現在保存されている'紫'は'甲州'と同じか、少なくとも非常に近い品種であると言える。

奥田将生、磯谷敦子、上用みどり、後藤奈美、三上重明

鑑評会の出品酒を実験材料として、窒素（N）や硫黄（S）含量と50℃1ヶ月間貯蔵後のポリスルフィド生成量の関係を検討した。清酒中のNとS含量には強い正の相関関係がみられ、清酒中の硫黄化合物はその多くは原料米のタンパク質に由来することが推察された。また、全硫黄化合物の2～5割がアミノ酸であることがわかった。50℃1ヶ月貯蔵により生成するポリスルフィドについて、DMDSは全試料で検知閾値以下であったが、DMTSは約半数の試料において検知閾値を上回った。貯蔵前清酒中の成分との関係において、DMTS、DMDS含量は、全N及びS含量、アミノ酸態のNやS含量に有意な正の相関関係がみられた。各成分間の偏相関分析の結果、着色度の増加には全Nとグルコース含量が深く関係するのに対し、ポリスルフィドの生成にはアミノ酸態のS含量が深く関係することが示唆された。以上から、硫黄化合物の多い清酒は老香が生じやすいというこれまでの推定を成分的に裏付ける結果が得られた。

後藤邦康

酒米47点、市販清酒および酒粕各25点中のカドミウム(Cd)濃度を測定した。試料は硝酸と過酸化水素を用いたマイクロ波分解装置により調整し、誘導結合プラズマ発光分析装置(ICP-AES)により分析を行った。測定した酒造用玄米、70 %精白米および市販酒粕中のCd濃度は平均でそれぞれ29.9,25.3,154.7 μg/kg、市販清酒中のCd濃度は1.29 μg/lであった。70 %精白米中のCd濃度は玄米の約78 %(7試料の平均値)となった。以上の結果から、精米工程によるCd濃度の減少効果は少なく、また原料米中のCdは清酒へはほとんど移行せず、酒粕に移行する可能性が示された。

Yoshio Araki, Hong Wu, Hiroshi Kitagaki, Takeshi Akao, Hiroshi Takagi and Hitoshi Shimoi

Environmental stimuli elicit a stress response, which helps to maintain cell survival. In budding yeast Saccharomyces cerevisiae, environmental cues can activate calcineurin, a highly conserved Ca2⁺- and calmodulin-dependent protein phosphatase. Calcineurin dephosphorylates the transcription factor Crz1, leading to accumulation of Crz1 in the nuclei and expression of stress responsive genes under the control of a calcineurin-dependent response element (CDRE). Ethanol is the final product of sugar fermentation by yeast, and thus a frequently encountered yeast stressor. However, adaptation of yeast to ethanol stress is poorly understood. In this study, we show that ethanol stimulates calcineurin-dependent nuclear localization of Crz1 and CDRE-dependent gene expression. Moreover, cells in which CRZ1 is deleted exhibit defective adaptation to ethanol stress, while a multicopy plasmid of CRZ1 confers an increased level of adaptive stress tolerance to ethanol. Taken together, the results indicate that ethanol activates the calcineurin/Crz1 pathway and that CRZ1 is crucial for cell survival under ethanol-induced stress.

Kenta TOMIMURA, Kazuhiro IWASHITA, Osamu YAMADA, Ken KOBAYASHI

Using a bioinformatics approach, we developed 18 variable number of tandem repeat markers for Aspergillus ozyzae for use in population genetic studies. Repeat sequences in the genome sequences of A. ozyzae were identified by a tandem repeat finding program. Length polymorphisms at 18 loci were examined in 41 strains of A. ozyzae. The total number of alleles per locus ranged from two to 20. Investigation of cross-species amplifications with A. sojae and A. tamarii showed success. The variable number of tandem repeat markers will be used to determine the population structure of these three Aspergillus species used in brewing.

Watanabe T, Masaki K, Iwashita K, Fujii T, Iefuji H.

A flocculent yeast, Hansenula anomala J224 PAWA, bred in this study, accumulated twice as much phosphorus as the wild type. Over a 30-d period, PAWA removed 70-80% of dissolved total phosphorus from sweet-potato and barley shochu wastewaters (alcoholic distillery wastewaters) while the wild type removed only 30%. Waste sludge was easily separated from effluent wastewater because PAWA cells made large flocks that rapidly settled. Component analysis suggested that PAWA sludge could be used as a protein source for feedstuff and as a phosphorus source for fertilizer. Under anaerobic conditions, denitrification was rapid, resulting in the removal of large amounts of nitrogen from barley shochu wastewater. These results suggest that small shochu manufacturers could benefit from using PAWA to remove phosphorus and organic compounds and then by using a combination of the upflow anaerobic sludge blanket and the downflow hanging sponge method (UASB-DHS method) for nitrification/denitrification.

Yoshitake Sakae, Toshiaki Matsubara, Misako Aida, Hidemasa Kondo, Kazuo Masaki and Haruyuki Iefuji

A cutinase-like enzyme (CLE), which is purified experimentally from the yeast Cryptococcus sp. strain S-2, has been recently found to degrade biodegradable plastics very efficiently. In this study, we theoretically examine the mechanism of hydrolysis of biodegradable plastics by the CLE by means of the ONIOM method. We optimize all the intermediates and the transition states involved in the considered enzymatic reaction and determine the energy surface of the entire catalytic cycle. The calculations show that the amino acid residues inside the pocket of the active site, Thr17 and Gln86, which stabilize the tetrahedral intermediates, and Gly115 in addition to Ser85, His180, and Asp165, which compose the catalytic triad, significantly contribute to the catalytic reaction. This is similar to the case of serine protease reported to date. Moreover, we have newly found that the energy barrier of the catalytic cycle is significantly lowered by the electronic effect of the OH group in the side-chain of Thr17 and bound water. The calculated potential energy surface of the reaction shows that the cleavage of the ester bond is a rate-determining step.

Koseki T, Mese Y, Nishibori N, Masaki K, Fujii T, Handa T, Yamane Y, Shiono Y, Murayama T, Iefuji H.

An -L-rhamnosidase was purified by fractionating a culture filtrate of Aspergillus kawachii grown on L-rhamnose as the sole carbon source. The α-l-rhamnosidase had a molecular mass of 90 kDa and a high degree of N-glycosylation of approximately 22%. The enzyme exhibited optimal activity at pH 4.0 and temperature of 50 ℃. Further, it was observed to be thermostable, and it retained more than 80% of its original activity following incubation at 60 ℃ for 1 h. Its T ₅₀ value was determined to be 72 ℃. The enzyme was able to hydrolyze -1,2- and -1,6-glycosidic bonds. The specific activity of the enzyme was higher toward naringin than toward hesperidin. The A. kawachii -L-rhamnosidase-encoding gene (Ak-rhaA) codes for a 655-amino-acid protein. Based on the amino acid sequence deduced from the cDNA, the protein possessed 13 potential N-glycosylation recognition sites and exhibited a high degree of sequence identity (up to 75%) with the -L-rhamnosidases belonging to the glycoside hydrolase family 78 from Aspergillus aculeatus and with hypothetical Aspergillus oryzae and Aspergillus fumigatus proteins.

Atsuko Isogai, Ryoko Kanda, Yoshikazu Hiraga, Toshihide Nishimura, Hiroshi Iwata, and Nami Goto-Yamamoto

Dimethyltrisulfide (DMTS) is involved in the unpalatable aroma of stale sake, called “hineka"; however, the mechanism underlying the formation of DMTS during the storage of sake has not been elucidated. Here we investigated the precursors of DMTS in sake. An experiment using [methyl-d₃]-methionine showed that Strecker degradation of methionine plays a minor role in the formation of DMTS. Separation of components in sake by cation exchange resin revealed that DMTS precursors are present in the acidic/neutral fraction rather than in the basic one. Purification of the DMTS precursor compounds was carried out through several chromatographic steps, measuring DMTS producing potential as an index. High-resolution ESI-MS and 1-D/2-D NMR experiments enabled to identify one of the precursor compounds as 1,2-dihydroxy-5-(methylsulfinyl)pentan-3-one.

Taku Katou, Hiroshi Kitagaki, Takeshi Akao and Hitoshi Shimoi

Sake yeast exhibit various characteristics that make them more suitable for sake brewing compared to other yeast strains. Since sake yeast strains are Saccharomyces cerevisiae heterothallic diploid strains, it is likely that they have heterozygous alleles on homologous chromosomes (heterozygosity) due to spontaneous mutations. If this is the case, segregation of phenotypic traits in haploid strains after sporulation and concomitant meiosis of sake yeast strains would be expected to occur. To examine this hypothesis, we isolated 100 haploid strains from Kyokai no. 7 (K7), a typical sake yeast strain in Japan, and compared their brewing characteristics in small scale sake brewing tests. Analyses of the resultant sake samples showed a smooth and continuous distribution of analytical values for brewing characteristics, suggesting that K7 has multiple heterozygosities that affect brewing characteristics and that these heterozygous alleles do segregate after sporulation. Correlation and principal component analyses suggested that the analytical parameters could be classified into two groups indicating fermentation ability and the taste of sake.

Kazuya KOYAMA, Nami GOTO

The effect of bunch shading during early development (before the onset of ripening) and/or during ripening on the phenolic composition of grape skins (Vitis vinifera L. cv. Cabernet Sauvignon) as well as on the mRNA levels of the biosynthetic genes on the flavonoid pathway was examined. Shading during early development resulted in decreased proanthocyanidin (PA) concentrations. The PA concentrations decreased during ripening, and the decrease of the concentrations was lower in berries shaded during early development than that in the exposed berries. Thus, no significant effect of shading during early development was observed at harvest. Shading during ripening did not influence this decline in the PAs. On the other hand, shading during early development induced changes in the composition such as a decrease of the trihydroxylated subunits within PAs, which agreed with the relative decrease of VvF3'5'H expression. The anthocyanin concentrations were remarkably reduced when the bunches were shaded during ripening, which was in accordance with the decreased transcription of several anthocyanin biosynthetic genes and transcriptional factors. Shading during early development did not influence the anthocyanin concentrations at harvest; however, it decreased the proportion of trihydroxylated anthocyanins. Thus, shading during early development also had an influence on the compounds biosynthesized during ripening.

Puspa Raj POUDEL, Nami GOTO-YAMAMOTO, Ryosuke MOCHIOKA, Ikuo KATAOKA, Kenji BEPPU

Kadainou R-1, an interspecific hybrid grape derived from red (Vitis ficifolia var. ganebu) and white (V. vinifera cv. Muscat of Alexandria) grapes, accumulates high concentrations of anthocyanin in the berry skin. Hence, the expression of uridine 5'-diphosphate (UDP)-glucose:flavonoid 3-O-glucosyltransferase (UFGT), the key enzyme of the anthocyanin pathway, was examined in the berry skin of Kadainou R-1. As information on gene sequences of V. ficifolia var. ganebu and other wild grape species was unavailable, we performed GeneChip hybridization using biotin-labeled genomic deoxyribonucleic acid (DNA) to investigate how the genomic sequences of V. vinifera varieties and that of V. ficifolia var. ganebu differ. The study showed a lower correlation coefficient between V. vinifera cultivars and V. ficifolia var. ganebu than that among V. vinifera cultivars. The sequences of the UFGT gene derived from both parents of the red and white cultivars were sequenced in Kadainou R1 and revealed that both were expressed irrespective of the fact that it was not expressed in the white grape (male parent).

橋爪克己、奥田将生

清酒粕歩合の全国平均値と日本の21観測地点の月別平均気温との間の相関係数は9月に正のピークを示したが、冬期には明確な傾向を示さなかった。この結果は、原料米の登熟期間中の気温条件が清酒醪中の蒸米の消化性に強く影響を及ぼしていることを示唆している。一方、冬期の気温条件の影響は明確に観察されなかった。

伊豆英恵、樋詰和久、後藤邦康、広常正人

我々はマウスの慢性アルコール性肝障害モデルを用い、血漿のGPT活性、血漿・肝臓のTG値を測定して清酒濃縮物（CS）及び清酒特異的な糖の1つであるα-エチルグルコシド（α-EG）の肝保護作用を調べた。雄性C57/BL6マウスにLiber-Decalliのアルコール飼料またはコントロール飼料を4週間投与し、この間、CS（3 ml/kg体重）またはα-EG（200 mg/kg体重）を週5回、経口投与した。この結果、アルコール摂取で誘導される血漿GPT活性及び血漿・肝臓TG値の上昇がCSまたはα-EGによって抑制されており、CSとα-EGが慢性アルコール性肝障害を抑制することが示唆された。以上より、清酒にアルコールによる肝障害を軽減する成分が含まれることが推定される。

S.T. Jeong, N. Goto-Yamamoto, K. Hashizume, M. Esaka

The biosynthetic pathways of main grape flavonoids: anthocyanins, flavonols, and flavan-3-ols, hold in common the early step enzymes of biosynthetic pathway: chalcone synthase (CHS), chalcone isomerase (CHI), and flavanone 3-hydroxylase (F3H), the genes of which are multi-copied in the grape genome. The ratios of mRNA levels of the three Chss (Chs1, Chs2, and Chs3), as well as those of the two Chis (Chi1 and Chi2) and those of the two F3hs (F3h1 and F3h2), were different among organs, even though no organ-specificity was observed in the strictest sense. Multiple regression analysis demonstrated that the transcription of particular genes significantly coincided with the biosynthesis of a particular flavonoid: the transcription of Chi2 coincided with flavan-3-ol; Chs1, Chs2, F3h1 and F3h2 with flavonol; and Chs2, Chs3, and F3h2 with anthocyanin biosynthesis. Thus, the transcription of these multi-copy genes is likely induced differently for the biosyntheses of anthocyanins, flavonols, and flavan-3-ols.

Watanabe T, Ozaki N, Iwashita K, Fujii T, Iefuji H.

Inorganic phosphate is an essential nutrient. In general, microorganisms take up phosphorus when the extracellular phosphorus concentration is low, but not when it is high. In Saccharomyces cerevisiae, the major phosphate transporters, such as Pho84p, and acid phosphatases (APases), such as Pho5p, are regulated in parallel by the phosphate signal transduction pathway (PHO pathway). We found that PHO mutants expressing PHO84 and PHO5, even under high-P conditions, could take up phosphorus at twice the rate of the wild-type strain. The regulatory pathway for phosphorus accumulation in two wastewater treatment yeasts, Hansenula fabianii J640 and Hansenula anomala J224-1, was found to be similar to that in S. cerevisiae. We screened for mutants of these yeasts that constitutively expressed APase. Such mutants formed blue colonies on high phosphorus concentration agar plates containing 5-bromo-4-chloro-3-indolylphosphate (X-phosphate). We found four mutants of H. fabianii J640 and one mutant of H. anomala J224-1 that accumulated from 2.2 to 3.5 times more phosphorus than the parent strains. The growth rates and abilities to remove dissolved total nitrogen and dissolved organic carbon of the mutants were similar to those of the parent strains. In addition, the mutants removed 95% of dissolved total phosphorus from shochu wastewater, while the parent strain removed only 50%.

K. Thirunavukarasu, N.G. Edwinoliver, S. Durai Anbarasan, M.K. Gowthaman, H. Iefuji、 N.R. Kamini

Removal of triglyceride molecules from fabrics could be accomplished in laundries using detergents containing lipases. The yeast, Cryptococcus sp. S-2 (CS2) produces a novel lipase, a cutinase-like enzyme that is compatible with various ionic, cationic and non-ionic surfactants as well as commercial detergents and oxidizing agents. In this study, the efficacy of CS2 lipase was assessed for the removal of triglyceride soil, like olive oil from fabrics in a single wash cycle using a tool of response surface methodology (RSM), so as to use the enzyme as an additive in laundry detergent formulations. A five-level four-factorial central composite rotatable design (CCRD) was used to evaluate the interactive effects of detergent concentration, lipase concentration, buffer pH and washing temperature on the percentage removal of triglyceride soil from cotton fabric. The model suggested that all the factors chosen had a significant impact on oil removal and the optimum conditions derived via RSM were 0.5% detergent, 1000 U of lipase, buffer pH of 8.0 and washing temperature of 37.8℃. Under optimal conditions, the removal of olive oil from cotton fabric was 19.6 and 43.1% higher at 20 and 37.8℃, respectively, in the presence of lipase,when compared to treatment with detergent wash alone. Moreover, the present work will also serve as a baseline of initial studies on cutinases and other related enzymes for its exploitation in laundry detergents.

岩田博、磯谷敦子、中野成美、平松順一

平成18酒造年度全国新酒鑑評会の出品酒について、酒質の現状及び動向の調査研究のため、調査表の内容を集計するとともに成分分析を行った。審査は平成19年4月24日(火)から26日(木)に予審を、5月9日(水)及び10日(木)に決審を行った。また、5月24日(木)に製造技術者研究会を、6月7日(木)に公開きき酒会を開催した。

三上重明、後藤奈美、小山和哉、平松順一

本鑑評会は、国内洋酒・果実酒メーカーから任意出品された果実酒類、ウイスキー、ブランデー、スピリッツ類及びリキュール類について官能審査、化学分析を行い、品質及び技術の動向を全国的な視野で調査するとともに製造業者の参考に資することを目的として実施している。審査会は、平成19年11月19、20日の2日間にわたり、酒類総合研究所において開催した。出品酒の官能審査と成分分析の結果の概要について報告した。

三上重明, 家藤治幸, 向井伸彦, 平松順一

第30回本格焼酎鑑評会出品酒の出品酒について官能審査及び成分分析を行った。原料別の出品状況では,米,麦,甘しょ及び酒粕を原料とした製品が増加し,特に甘しょ製品の増加が著しかった。香り,味及び総合評価の評点は,泡盛の特殊製品が良好であった。成分分析については,低沸点香気成分,中高沸点香気成分,チオバルビツル酸価,pH及び酸度などの分析結果について報告した。

後藤邦康

平成16年に市販酒類等(清酒,焼酎,ビール,ワイン,ウイスキー,酒粕)について外因性内分泌かく乱物質の分析を行った。そのとき,他の成分に比べて比較的高い数値が見られたノニルフェノール(NP),ビスフェノールA(BPA),フタル酸ジ-2-エチルヘキシル及びアジピン酸ジ-2-エチルヘキシルについて再度調査した結果について述べた。また一般微生物及び大腸菌群の測定を行った。酒類についてNP及びBPAの平均値は,平成16→18→19年度に対して,それぞれ1.9→1.4→1.3μg/l,及び0.55→0.13→0.09μg/lとなった。平成17年度以降,最大となった分析値と各物質の耐容一日許容量から,酒類及び酒粕による影響は大きくないと考えられた。酒類中の微生物は若干増加したが,検出された濃度は最大でも3cfu/mlと低いものであった。全酒類製品で大腸菌群は検出されなかった。

倉光潤一、宇都宮仁、橋爪克己

酒類に関する国民のニーズについて4,501名に対し調査を行い（有効回答者数3,294、有効回答率73.2%）、前回調査（14年調査）からの変化等について検討した。酒類全般に関する関心分野では、「成分・品質」、「料理との相性」、「種類」が上位を占めた。14年調査と比較すると、男性では全世代で「料理との相性」、40代及び50代以上では「健康」、30代女性では「価格・酒税・格付」に対する関心の割合が増加した。一方、社会生活を営む上で必要な情報では、「飲酒と健康」、「一般知識」、「公共での飲酒マナー」の関心割合が高かった。14年調査と比較すると、「飲酒マナー」、「公共での飲酒マナー」が低下し、「一般知識」「飲酒と健康」及び「新成人への知識」に対する関心が増加した。

宇都宮仁、橋爪克己

清酒、ビール、ウイスキーの飲酒回数・量が1年前に比べ増えたという者各約1000人に対して、その飲酒動機等に関する調査を行った。清酒について最も頻度の高かった理由は、官能的要因の「おいしい・うまい」であった。清酒のおいしさは嗜好変化や清酒を飲んでみて知ったことが推定された。ビールについて最も頻度の高かった理由は、「特にない・なんとなく」であった。第2位「仕事・つきあい・会合」、第3位「機会・宴会の増加」と合わせて考えると、つきあいなどの飲酒機会の増加がビールの飲酒回数や量の増加に大きく関与していることが考えられた。ウイスキーについて最も頻度の高かった理由は、官能的要因の「おいしい・うまい」であった。ウイスキーのおいしさは、嗜好変化やウイスキーを飲んでみて知ったことが推定された。

伊豆英恵、後藤邦康、坂田省吾

ヒトを用いた酔いの評価は個人差や倫理的な問題から困難が多い。ヒトではエタノールを飲用するが、動物を用いて行動実験で酔いを評価する場合、一般的にはエタノールを腹腔内投与していた。本研究では摂食あるいは絶食条件下で様々な用量のエタノールをマウスに経口投与して高架式十字迷路試験及び運動量測定を行い、エタノール飲用による酔いの評価系の構築を試みた。高架式十字迷路試験ではエタノールの経口投与（1.6～4.0 g/kg（単回）、0.8～1.6 g/kg（2回））によってオープンアームへの進入回数及び滞在時間が増加し、エタノール飲用による抗不安作用が確認された。自発運動量試験では絶食条件下でエタノールの経口投与（1.6または4.0 g/kg（単回）、1.2または2.4 g/kg（2回））によって運動量が有意に減少した。半絶食条件下でエタノールを経口投与（1.6 g/kg（単回））後1時間では運動量が促進されたが、その後、運動量は48時間後まで抑制された。以上の条件を用いてマウスへエタノールを経口投与した場合、高架式十字迷路試験ではエタノールによる運動障害が生じる前の軽度の酔い、自発運動量測定試験では運動量が促進あるいは抑制されるあらゆる段階の酔いを評価可能であることが示唆された。

Kazutoshi Sakamoto, Toshi-hide Arima, Kazuhiro Iwashita, Osamu Yamada, Katsuya Gomi, Osamu Akita

Using an Aspergillus oryzae EST database, we identified a gene encoding a transcription factor (atfB), which is a member of the ATF/CREB family. Expression of atfB was barely detectable during vegetative growth, but was readily detected during conidiation in solid-state culture. Microarray analyses showed that expression of many other genes, including catalase (catA), were downregulated in an atfB-disruptant. The expression of most of these genes was upregulated in the wild-type strain during the conidiation phase in solid-state culture, and the expression pattern was similar to that of atfB itself. In the absence of stress, e.g. heat shock or hydrogen peroxide, the conidial germination ratios for the ΔatfB strain and the wild-type strain were similar, but the stress tolerance of conidia carrying the ΔatfB deletion was less than that of the wild-type conidia. CRE-like DNA motifs, which are bound by ATF/CREB proteins, were found in the promoters of most of the downregulated genes in the ΔatfB strain. Thus, atfB appears to encode a transcription factor required for stress tolerance in conidia.

Ogihara, Hiroshi Kitagaki, Qian Wang, Hitoshi Shimoi

Genomic analysis of industrial yeast strains is important for exploitation of their potential. We analysed the genomic structure of the most widely used sake yeast strain, Kyokai no. 7 (K7), by DNA microarray. Since the analysis suggested that the copy number of the AQY1-ARR3 region in the right arm of chromosome XVI was amplified, we performed Southern blot analysis using the AQY1 gene as a probe. The probe hybridized to three bands in the widely used sake strains derived from K7, but only to one band of 1.4 kb in the laboratory strains. Since the extra two bands were not observed in old sake strains, or in other industrial strains, the amplification of this region appeared to be specific for the widely used sake strains. The copy number of the AQY1-ARR3 region appeared to have increased by chromosomal translocation, since chromosomal Southern blot analysis revealed that the AQY1 probe hybridized to chromosomes IV and XIII, in addition to chromosome XVI, in which AQY1 of the laboratory strain is encoded. The chromosomal translocation was also confirmed by PCR analysis using primers that amplify the region containing the breakpoint. Cloning and sequencing of cosmids that encode the AQY1-ARR3 region revealed that this region is flanked by TG1-3 repeats on the centromere-proximal side in chromosomes IV and XIII, suggesting that amplification of this region occurred by homologous recombination through TG1-3 repeats. These results demonstrate the genomic characteristics of the modern widely used sake strains that discriminate them from other strains.

Takashi Inoue and Akihiro Mizuno

A continuous primary bottom fermentation system for beer brewing with the ability to maintain a stable and high level of yeast activity during its long operation was designed and its performance was evaluated. The basic strategy was to realize the same physiologically or morphologically synchronized changes in yeast and wort constituents during the main fermentation as those realized in traditional bottom beer fermentations. The liquid nonabsorbent carrier particles used to fill the fermenter successfully promoted the fermentation of wort sugars, which were supplied from the top of the fermenter, by preventing their rapid sinking. In addition, the carrier particles displayed other benefits that result in effective operation. The quality of the harvested young beer was satisfactory, but had a low ester content that may be improved by further studies.

Sayuri KITAGAWA, Nobuhiko MUKAI, Yuko FURUKAWA, Kanako ADACHI, Akihiro MIZUNO, and Haruyuki IEFUJI

We examined the effect of soy peptides (SPs) on the fermentation and growth of Yeast Bank Weihenstephan 34/70 (W34/70), a bottom-fermenting yeast. We compared fermentation for SP with that for a free amino acid (FAA) mixture having the same amino acid composition as SP, as a nitrogen source. Maltose syrup was used as a carbon source, and the medium contained excess amounts of essential minerals and vitamins. We observed that SP was better than FAA mixture at promoting fermentation and growth and that much more β-phenylethyl alcohol was produced during fermentation with SP than with FAA mixture. Subsequently, we compared fermentations with the FAA mixture and selected mixtures containing various dipeptides of Phe as a nitrogen source. We found that the rates of Phe metabolism and β-phenylethyl alcohol generation were much higher when Phe was presented as a dipeptide (Phe-Asp, Phe-Leu, or Phe-Phe) than when presented as FAA. These results show that amino acids such as Phe are absorbed more rapidly when presented as a peptide than as FAA, resulting in a more rapid production of β-phenylethyl alcohol.

Jantaporn Thongekkaew, Hiroko Ikeda, Kazuo Masaki, Haruyuki Iefuji

The extracellular carboxymethyl cellulase (CSCMCase) from the yeast, Cryptococcus sp. S-2, was produced when grown on cellobiose. It was purified to homogeneity from the supernatant by ultrafiltration, DEAE- 5PW anion exchange column and TSK-Gel G3000SW gel filtration. The purified enzyme was monomeric protein with molecular mass of approximately 34 kDa. The optimum temperature and pH for the action of the enzyme were at 40-50℃ and 3.5, respectively. It was stable at pH range of 5.5-7.5 and retained approximately 50% of its maximum activity after incubating at 90℃ for 1 h. Moreover, it could able to hydrolyze carboxymethyl cellulose sodium salt higher than insoluble cellulose substrate such as Avicel, SIGMACELL and CM cellulose. Due to its action at acidic pH and moderately stable at high temperature, the gene encoding carboxymethyl cellulase (CSCMCase) was isolated and improved the enzyme yield by high cell-density fermentation of Pichia pastoris. The CSCMCase cDNA contains 1023 nucleotides and encodes a 341-amino acid. It was successfully expressed under the control of alcohol oxidase I promoter using methanol induction of P. pastoris fermentation in a 2L ABLE bioreactor. The production of the recombinant carboxymethyl cellulases was higher than that from Cryptococcus sp. S-2 of 657-fold (2.75 and 4.2 x10_^-3 mg protein L_^-1, respectively) indicating that the leader sequence of CSCMCase has been recognized and processed as efficiently by P. pastoris. Furthermore, the recombinant enzyme was purified in two-step of ultrafiltration and hydrophobic interaction chromatography which would be much more convenient for large-scale purification for successful industrial application.

Hiroshi Kitagaki, Taku Kato, Atsuko Isogai, Shigeaki Mikami, Hitoshi Shimoi

We previously demonstrated the presence and fragmentation of mitochondria during alcohol fermentation. Here, we show that Fis1p induces mitochondrial fragmentation, and inhibition of mitochondrial fragmentation causes higher malate production during sake brewing. These findings indicate that mitochondrial morphology affects the metabolism of constituents, providing a breeding strategy for high-malate-producing yeasts.