平成26年度 研究成果

Atsushi OHUCHI, Mikiko ASAKAWA, Makoto KANAUCHI, Kazutaka KUSAKA, Hiroshi TAKAKUWA, Souichiro TATSU, Fumihiko TSUCHIYA, Yayoi TSUKADA, Takayuki WATANABE

1. Relative repeatability standard deviation (RSDr) and repeatability limit (r95) for determination of ethanol content using the headspace GC-FID method ranged from 0.6 to 2.3% and from 0.0003 to 0.0209 v/v%, respectively, and were judged acceptable.
2. Relative reproducibility standard deviation (RSDR) and reproducibility limit (R95) for determination of ethanol content using the headspace GC-FID method ranged from 1.5 to 4.3% and from 0.0006 to 0.0809 v/v%, respectively, and were judged acceptable.

Masayuki TAKAHASHI, Yasuko KITA, Kazutaka KUSAKA, Akihiro MIZUNO, Nami GOTO-YAMAMOTO

Aims

In the brewing industry, microbial management is very important for stabilizing the quality of the product. We investigated the detailed microbial community of beer during fermentation and maturation, to manage beer microbiology in more detail.

Methods and Results

We brewed a beer (all-malt) and two beerlike beverages (half- and low-malt) in pilot-scale fermentation and investigated the microbial community of them using a next-generation sequencer (454 GS FLX titanium), quantitative PCR, flow cytometry and a culture-dependent method. From 28 to 88 genera of bacteria and from 9 to 38 genera of eukaryotic microorganisms were detected in each sample. Almost all micro-organisms died out during the boiling process. However, bacteria belonging to the genera Acidovorax, Bacillus, Brevundimonas, Caulobacter, Chryseobacterium, Methylobacterium, Paenibacillus, Polaromonas, Pseudomonas, Ralstonia, Sphingomonas, Stenotrophomonas, Tepidimonas and Tissierella were detected at the early and middle stage of fermentation, even though their cell densities were low (below approx. 10³ cells ml^-1) and they were not almost detected at the end of fermentation.

Conclusions

We revealed that the microbial community of beer during fermentation and maturation is very diverse and several bacteria possibly survive during fermentation.
Significance and Impact of the Study: In this study, we revealed the detailed microbial communities of beer using next-generation sequencing. Some of the micro-organisms detected in this study were found in beer brewing process for the first time. Additionally, the possibility of growth of several bacteria at the early and middle stage of fermentation was suggested.

高尾佳史、高橋俊成、藤田晃子、松丸克己、溝口晴彦

樽酒は、杉 (Cryptomeria Japonica)の板材で作った酒樽に入れて、木香を付与した清酒である。我々はこれまでに杉材から清酒に移行する成分として、通常の清酒には含まれないセスキテルペン類やジテルペン類などを同定し、これらの成分が機能性を示すことを報告した。また、通常の清酒に比べて樽酒は食品に由来する口腔内の油脂を洗い流す効果が高いことを明らかにした。

近年、特定の物質に対してのみ応答するのではなく、人間の味覚と同様に幅広い選択性を持った味覚センサーが開発され、食品や酒類の味の評価に用いられている。本研究では、樽酒と食品の相性について味覚の面からアプローチするため、樽酒を様々な食品と同時に摂取した際の味覚への影響について、官能評価と味覚センサーによる分析を行った。官能評価結果から、樽酒にはあさりの酒蒸しの旨味を強く、長く感じさせる効果があることが示された。様々なシーフードの味覚センサー分析による旨味後味の強度は官能評価結果と一致した。さらに、味覚センサー分析により杉樽抽出物と樽酒濃縮液に旨味後味を増強する効果があることが確認され、その効果は、樽酒中の比較的極性が高く、揮発しにくい成分に起因すると考えられた。

山田康枝、増田修一、山本翔太、伊豆英恵

N-メチル-D-アスパラギン酸型グルタミン酸受容体（NMDA受容体）とγ-アミノ酪酸受容体A（GABA_A受容体）は中枢神経系に広く分布する。NMDA受容体はシナプス可塑性で重要な役割を果たす。GABA_A受容体はシナプス後ニューロンの膜を過分極化し、抑制性神経伝達を行う。ハーブのエッセンシャルオイルや酒類の香気成分のヒトの気分や意識に対する機能の分子メカニズムにはまだ不明な点がある。香気成分の神経活動に対する影響を理解するため、清酒や焼酎に含まれる13の香気成分について、アフリカツメガエル卵母細胞に発現させたGluN1/GluN2AとGluN1/GluN2BサブタイプのNMDA受容体、GABA_A受容体に対する影響を調べた。清酒や焼酎に含まれる13のうち12の香気成分がGluN1/GluN2AとGluN1/GluN2B受容体活性を阻害した。また、フェネチルアルコール、ネロールとゲラニオールはGABA_A受容体活性を促進した。マウス高架式十字迷路試験において、フェネチルアルコールとシトロネロール投与で有意な抗不安作用が確認された。これらの結果は清酒や焼酎に含まれる香気成分がNMDA受容体活性阻害、GABA_A受容体活性促進の重要な役割を果たすことを示す。

Kenji UEHARA, Jun WATANABE, Takeshi AKAO, Daisuke WATANABE, Yoshinobu MOGI and Hitoshi SHIMOI

4-Hydroxy-2 (or 5)-ethyl-5 (or 2)-methyl-3(2H)-furanone (HEMF) is an important flavor compound that contributes to the sensory properties of many natural products, particularly soy sauce and soybean paste. The compound exhibits a caramel-like aroma and several important physiological activities, such as strong antioxidant activity. HEMF is produced by yeast species in soy sauce manufacturing; however, the enzymes involved in HEMF production remain unknown, hindering efforts to breed yeasts with high-level HEMF production. In this study, we identified high-level HEMF-producing mutants among a Saccharomyces cerevisiae gene deletion mutant collection. Fourteen deletion mutants were screened as high-level HEMF-producing mutants, and the ADH1 gene deletion mutant (adh1Δ) exhibited the maximum HEMF production capacity. Further investigations of the adh1Δ mutant implied that acetaldehyde accumulation contributes to HEMF production, agreeing with previous findings. Therefore, acetaldehyde might be a precursor for HEMF. The ADH1 gene deletion mutant of Zygosaccharomyces rouxii, which is the dominant strain of yeast found during soy sauce fermentation, also produces HEMF effectively, suggesting that acetaldehyde accumulation might be a benchmark for breeding industrial yeasts with excellent HEMF production abilities.

Taiki Futagami, Kazuki Mori, Shotaro Wada, Hiroko Ida, Yasuhiro Kajiwara, Hideharu Takashita, Kosuke Tashiro, Osamu Yamada, Toshiro Omori, Satoru Kuhara, Masatoshi Goto

The koji mold Aspergillus kawachii is used for making the Japanese distilled spirit shochu. During shochu production, A. kawachii is grown in solid-state culture (koji) on steamed grains, such as rice or barley, to convert the grain starch to glucose and produce citric acid. During this process, the cultivation temperature of A. kawachii is gradually increased to 40°C and is then lowered to 30°C. This temperature modulation is important for stimulating amylase activity and the accumulation of citric acid. However, the effects of temperature on A. kawachii at the gene expression level have not been elucidated. In this study, we investigated the effect of solid-state cultivation temperature on gene expression for A. kawachii grown on barley. The results of DNA microarray and gene ontology analyses showed that the expression of genes involved in the glycerol, trehalose, and pentose phosphate metabolic pathways, which function downstream of glycolysis, was downregulated by shifting the cultivation temperature from 40 to 30°C. In addition, significantly reduced expression of genes related to heat shock responses and increased expression of genes related with amino acid transport were also observed. These results suggest that solid-state cultivation at 40°C is stressful for A. kawachii and that heat adaptation leads to reduced citric acid accumulation through activation of pathways branching from glycolysis. The gene expression profile of A. kawachii elucidated in this study is expected to contribute to the understanding of gene regulation during koji production and optimization of the industrially desirable characteristics of A. kawachii.

Nahoko Nishibori, Kei Sasaki, Yuta Okimori, Atsuko Isogai, Osamu Yamada, Tsutomu Fujii and Nami Goto-Yamamoto

The present study showed that the lysis of yeast cells and subsequent release of cell contents in sake mash accelerated dimethyl trisulfide (DMTS) formation. Among these, heat unstable and relatively high molecular weight compounds were assumed to be enzymes; thus, enzymatic reactions probably contribute to DMTS formation.

Aimi OSAKI, Yukako OKAZAKI, Akioko KIMOTO, Hanae IZU, Norihisa KATO

This study investigated the effects of the consumption of 1% or 2% (v/v) ethanol in drinking water for 12 wk on rats fed a high-fat diet. Body weight gain, food intake, and fluid intake were unaffected by ethanol intake. Adipose tissue weight, and serum glucose and lipids were unaffected. Compared to the control (no ethanol), 1% ethanol intake significantly reduced serum levels of alanine aminotransferase (ALT), lactate dehydrogenase (LDH), and ammonia (p < 0.05), whereas 2% ethanol intake did so to a lesser extent. Serum urate was significantly lower in both the 1% and 2% ethanol groups than that in the control group (p < 0.05). The results suggest a low dose of ethanol has beneficial effects on liver function and serum urate in rats fed a high-fat diet.

Natsuki Mimura, Atsuko Isogai, Kazuhiro Iwashita, Takeshi Bamba, and Eiichiro Fukusaki

Sake is a Japanese traditional alcoholic beverage, which is produced by simultaneous saccharification and alcohol fermentation of polished and steamed rice by Aspergillus oryzae and Saccharomyces cerevisiae. About 300 compounds have been identified in sake, and the contribution of individual components to the sake flavor has been examined at the same time. However, only a few compounds could explain the characteristics alone and most of the attributes still remain unclear. The purpose of this study was to examine the relationship between the component profile and the attributes of sake. Gas chromatography coupled with mass spectrometry (GC/MS)-based non-targeted analysis was employed to obtain the low molecular weight component profile of Japanese sake including both nonvolatile and volatile compounds. Sake attributes and overall quality were assessed by analytical descriptive sensory test and the prediction model of the sensory score from the component profile was constructed by means of orthogonal projections to latent structures (OPLS) regression analysis. Our results showed that 12 sake attributes [ginjo-ka (aroma of premium ginjo sake), grassy/aldehydic odor, sweet aroma/caramel/burnt odor, sulfury odor, sour taste, umami, bitter taste, body, amakara (dryness), aftertaste, pungent/smoothness and appearance] and overall quality were accurately explained by component profiles. In addition, we were able to select statistically significant components according to variable importance on projection (VIP). Our methodology clarified the correlation between sake attribute and 200 low molecular components and presented the importance of each component thus, providing new insights to the flavor study of sake.

福田央・韓錦順

黒糖焼酎及びそれ以外の焼酎の低沸点香気成分及び中高沸点香気成分84成分を分析・比較し、黒糖焼酎の特徴となる成分を把握し、黒糖焼酎で特異的に認められる成分の由来及び黒糖焼酎とそれ以外の焼酎との判別の可能性を検討した。その結果、21成分について黒糖焼酎が他の本格焼酎に比べ有意に差があった。特に、2,5-ジメチルピラジン及び2-エチル-5(6)-メチルピラジンは、その他を原料とする焼酎以外では黒糖焼酎のみで認められ、当該成分の由来を検討したところ、黒糖に由来すると考えられた。また、黒糖焼酎と他の本格焼酎で有意差のある21成分からステップワイズ法で6成分の変数を選択し、判別分析を試みた。また、判別分析の検証を行ったところ、当該6成分により判別可能であった。

奥田将生、水谷治、金井宗良、住広匡謙、上用みどり、後藤奈美、福田央、山田修

非放射性セシウム(¹³³Cs)は放射性セシウムの挙動を予測するのに有効な同位元素である。2011年3月の東日本大震災に伴う福島第一原子力発電所事故により放出された放射性セシウム(Cs)の食品への汚染が心配された。本研究では、非放射性セシウムと、その他18種類の無機元素について、日本の蒸留酒である焼酎製造における挙動を解析した。焼酎製造工程中の濃度について調べたところ、セシウム及びその他18の無機元素は蒸留後の留液にはほとんど移行せず、蒸留残渣にその大半が残存した。従って、焼酎原料に放射性セシウムやその他の金属元素が含まれていても、それらの無機元素は蒸留されるため焼酎には移行しないことが確認された。

奥田将生、上用みどり、福田央、後藤奈美

精米による酒造用原料米の元素濃度の変化について、ICP-AES、ICP-MS、Duma法を用いて調べた。多元素同時分析の分析方法の妥当性を確認し、精白米中の26元素の濃度の定量が可能になった。精米を進めるに従いN、 S、Cd、Cu、Mo及びAsは精米歩合30%まで徐々に低減したが、それ以外の20元素は精米歩合70%まで減少したが以後精米を進めても変化しなかった。多くの無機元素は米の外層部に存在するものの、精米による濃度の減少の度合いは元素によって大きく異なった。

Kei Takahashi, Fumihiko Tsuchiya, Atsuko Isogai

Medium-chain fatty acids (MCFAs) and ethyl esters are considered to contribute to some organoleptic properties, such as fatty odor and bitterness in Japanese sake. However, the relationships between these compounds and the organoleptic properties of sake remain unclear. Here, we quantified MCFAs and ethyl hexanoate in ginjo sake using gas chromatography with a flame ionization detector (GC-FID). The hexanoic acid concentration strongly correlated with fatty odor (p < 0.0001). The octanoic acid/hexanoic acid ratio correlated with butanoic acid concentration, which is likely correlated with inharmonious bitter taste. Multiple comparison analysis revealed that the ethyl hexanoate level was negatively correlated with bitterness. We then identified other chemical compounds correlating with fatty odor and bitterness using comprehensive two-dimensional GC coupled with time-of-flight mass spectrometry. By performing correlation analysis between certain compounds and sensory values following statistical selection for chemical compounds, we identified several candidate compounds correlating with fatty odor and bitterness in sake.

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

Dimethyl trisulfide (DMTS) is known to be responsible for hineka, an off-flavor that develops during storage, in sake. Previous studies have attempted to elucidate the mechanism of DMTS formation during sake storage, but the mechanism underlying DMTS formation remains unclear. In this study, we determined the sake-preparation conditions that affect DMTS formation. We analyzed 76 sake samples immediately after filtration, which were donated by sake-producing companies. We measured the DMTS concentration in sake after 7 days of storage at 70℃ (DMTS-pp) using gas chromatography/mass spectrometry. In the statistical analysis, DMTS-pp was set as the objective variable, whereas the preparation conditions and analytical results for sake were set as the explanatory variables. We used multiple linear regression (MLR) analysis with a stepwise method and partial least squares regression (PLSR) to analyze the data. The statistical analysis showed that the significant factors for DMTS-pp were the average temperature in the moromi mash (Temp ave), the total daily temperature in the moromi mash (Temp sum), the concentration of sulfur-containing amino acids in sake, and the Zn concentration in sake. These factors explained 63.4% of the variance in DMTS-pp according to the MLR analysis and 64.2% according to the PLSR analysis. Further MLR analysis showed that Temp ave in early stage and Temp sum in later stage were important factors for DMTS-pp. This result suggests that the rice dissolution caused by high Temp ave in early stage and yeast cell lysis caused by high Temp sum in later stage contribute to high DMTS-pp.

Kazuya Koyama, Nineyo Numata, Ikuko Nakajima, NamiGoto-Yamamoto, Hideo Matsumura and Nobukazu Tanaka

A new regulator of proanthocyanidin (PA) biosynthesis in grapes was found by screening genes coordinately expressed with PA accumulation under different light conditions using a substantially improved method of serial analysis of gene expression (SuperSAGE). This R2R3-MYB transcription factor, VvMYBPAR, shows high protein sequence similarity with PA biosynthesis-regulating plant MYBs, such as VvMYBPA2 and TRANSPARENT TESTA2. Its transcript levels were relatively high in the skins of young berries, whereas the levels were higher in the seeds and at a maximum around veraison. In addition to its response to modified light conditions, the gene responded to abscisic acid application in the skins of cultured berries. Among the PA-specific branch genes, this transcript profile was not correlated with that of VvANR and VvLAR1 but was closely related to that of VvLAR2, suggesting different regulation of PA-specific branch genes from that of a known PA regulator, VvMYBPA2. The PA-specific regulation of VvMYBPAR was confirmed by VvMYBPAR constitutive expression in Arabidopsis in which the transgene specifically induced PA biosynthetic genes and resulted in PA accumulation in plants grown on sucrose-supplemented media to induce anthocyanin synthesis. A transient reporter assay using grapevine cells showed that VvMYBPAR activated the promoters on PA-specific branch genes and candidate genes associated with modification and transport of monomeric PA precursors, as well as the promoters of VvCHS3 and VvF3′5′Hd in the common flavonoid pathway, but not that of VvUFGT on the anthocyanin-specific branch. This new factor suggests the polygenic regulation of PA biosynthesis in grapes by closely related MYB transcription factors.

Nobuhiko MUKAI, Kazuo MASAKI, Tsutomu FUJII, Haruyuki IEFUJI

Among industrial yeasts used for alcoholic beverage production, most wine and weizen beer yeasts decarboxylate ferulic acid to 4-vinylguaiacol, which has a smoke-like flavor, whereas sake, shochu, top-fermenting, and bottom-fermenting yeast strains lack this ability. However, the factors underlying this difference among industrial yeasts are not clear. We previously confirmed that both PAD1 (phenylacrylic acid decarboxylase gene, YDR538W) and FDC1 (ferulic acid decarboxylase gene, YDR539W) are essential for the decarboxylation of phenylacrylic acids in Saccharomyces cerevisiae. In the present study, single nucleotide polymorphisms (SNPs) of PAD1 and FDC1 in sake, shochu, wine, weizen, top-fermenting, bottom-fermenting, and laboratory yeast strains were examined to clarify the differences in ferulic acid decarboxylation ability between these types of yeast. For PAD1, a nonsense mutation was observed in the gene sequence of standard top-fermenting yeast. Gene sequence analysis of FDC1 revealed that sake, shochu, and standard top-fermenting yeasts contained a nonsense mutation, whereas a frameshift mutation was identified in the FDC1 gene of bottom-fermenting yeast. No nonsense or frameshift mutations were detected in laboratory, wine, or weizen beer yeast strains. When FDC1 was introduced into sake and shochu yeast strains, the transformants exhibited ferulic acid decarboxylation activity. Our findings indicate that a positive relationship exists between SNPs in PAD1 and FDC1 genes and the ferulic acid decarboxylation ability of industrial yeast strains.

Yuu Utashima, Hirotaka Matsumoto, Kazuo Masaki, Haruyuki Iefuji

In the present study, we attempted to improve the production of recombinant horseradish peroxidase C1a (HRP-C1a; a heme-binding protein) by Cryptococcus sp. S-2. Both native and codon-optimized HRP-C1a genes were expressed under the control of a high-level expression promoter. When the HRP-C1a gene with native codons was expressed, poly(A) tails tended to be added within the coding region, producing truncated messenger RNAs (mRNAs) that lacked the 3′ ends. Codon optimization prevented polyadenylation within the coding region and increased both the mRNA and protein levels of active HRP-C1a. To improve secretion of the recombinant protein, we tested five types of N-terminal signal peptide (NTP). These included the native HRP-C1a NTP (C1a-NTP), short and long xylanase secretion signals (X1-NTP and X2-NTP), cutinase signal (C-NTP), and amylase signal (A-NTP), with and without a C-terminal propeptide (CTP). X2-NTP without CTP resulted in the highest HRP-C1a secretion into the culture medium. HRP-C1a secretion was further increased by using xylose fed-batch fermentation. The production of HRP-C1a in this study was 2.7 and 15 times higher than the production reported in previous studies that used insect cell and Pichia expression systems, respectively.

橋口知一、伊豆英恵、松丸克己

梅酒中のカルバミン酸エチルの前駆体であるシアン化水素をソーダ石灰により除去することにより、カルバミン酸エチルの低減を試みた。梅酒の空間部にソーダ石灰を配置し、撹拌を行いながら浸漬を行った。対照と比較して、梅酒中のカルバミン酸エチルは70%、シアン化水素は79%減少した。梅酒のアルコール、比重、pH、総酸及び官能評価結果については、対照と比べて大差は見られなかった。

藤井力、磯谷敦子、伊豆英恵、神田涼子、松丸克己、木崎康造

平成24酒造年度全国新酒鑑評会（第101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、酒類総合研究所広島事務所において、平成25年4月23日（火）から25日（木）の3日間に予審を行い、5月8日（水）及び9日（木）に決審を行った。また、5月22日（水）に東広島運動公園体育館で製造技術研究会を、6月14日（金）に東京池袋のサンシャインシティ・文化会館展示ホールで公開きき酒会を開催した。

出品は、864点であった。審査の結果、優秀と認められた426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233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、1,372人及び2,652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を行った。

Masayuki TAKAHASHI, Tami OHTA, Kazuo MASAKI, Akihiro MIZUNO, Nami GOTO-YAMAMOTO

The difference in microbiota including non-lactic acid bacteria, non-acetic acid bacteria, and wild yeast during winemaking and in the end-products between sulfite-added and sulfite-free wine, was investigated using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and a culture-dependent method. There were differences between the microorganisms detected by PCR-DGGE and those detected by the culture-dependent method, probably because of the selectivity of culture medium and the characteristics of PCR-based method. In both the red wine and white wine, the microbial diversity of the sulfite-added wine was lower than that of the sulfite-free wine during fermentation. Tatumella terrea was detected from the fermenting must by PCR-DGGE and by the culture-dependent method, even though sulfite inhibited its growth to some extent. We confirmed that the addition of sulfite plays an important role in winemaking by inhibiting the growth of unexpected microorganisms, but on the other hand, it was revealed that some microorganisms can survive and grow in sulfite-added fermenting must. We also analyzed 15 samples of commercial wines by the PCR-DGGE method and detected various microorganisms. Among them, Sphingomonas sp., Pseudozyma sp., Ochromonas sp. and Methylophilus sp. were found for the first time in wine as far as we know. We did not identify a specific microorganism that was detected only from wines without sulfite addition. Thus, the microbiota of end-products seemed to be influenced by other factors, such as filtration before bottling, the production equipment and the storage environment.

Masayuki TAKAHASHI, Kazuo MASAKI, Akihiro MIZUNO, Nami GOTO-YAMAMOTO

The detection of low-abundant microorganism is difficult when in a sample in which a specific microorganism represents an overwhelming majority using polymerase chain reaction (PCR)-based methods. A modified CO-amplification at Lower Denaturation temperature PCR (mCOLD-PCR) method was developed to detect low-abundant microorganisms using a double-strand RNA probe to inhibit the amplification of the sequence of a major microorganism. Combining the mCOLD-PCR and downstream application (e.g., denaturing gradient gel electrophoresis (DGGE) and next-generation sequencing (NGS)), low-abundant microorganisms were detected more efficiently, even when a specific microorganism represents an overwhelming majority of the sample. We demonstrated that mCOLD-PCR-DGGE enabled us to detect Schizosaccharomyces pombe in a model sample coexisting with 10,000 times as many Saccharomyces cerevisiae. When mCOLD-PCR-DGGE was applied in the microbiota analysis of a fermenting white wine, Candida sp. and Cladosporium sp., which were not detected by conventional PCR, were detected. According to the NGS analysis after mCOLD-PCR of a fermenting red wine, the detection ratio of Saccharomyces was decreased dramatically, and the detection ratios of other microorganisms and the numbers of genera detected were increased compared with the conventional PCR. Thus, the application of mCOLD-PCR will reveal comprehensive microbiota of fermented foods, beverages, and so on.

Moriyuki Kawauchi, Kazuhiro Iwashita

In the eukaryotic cell, histone deacetylases (HDACs) play key roles in the regulation of fundamental cellular process such as development regulation, stress response, secondary metabolism and genome integrity. Here, we provide a comprehensive phenotypic analysis using HDAC disruptants in Aspergillus oryzae. Our study revealed that four HDACs, hdaA/Aohda1, hdaB/Aorpd3, hdaD/Aohos2 and hst4/AohstD were involved in stress response, cell wall synthesis and chromatin integrity in A. oryzae. Osmotic stress sensitivity of HDAC disruptants differed between plate cultures and liquid cultures, suggesting that HDACs adapt to the difference environmental conditions. Using a common A. oryzae fermentation medium, rice-koji, we also characterized HDACs related to growth and enzyme production to investigate which HDACs will be required for adaptation to environmental conditions and stress resistances. Because HDACs are widely conserved, our study has broad applications and may inform work with filamentous fungi and other eukaryote.