2016 Research results

1. Characteristics of the volatile composition of commercially available Awamori

Author

Hisashi Fukuda, Jinshun Han, Osamu Yamada

Abstract

To classify their compounds of commercially available Awamori, we analyzed 78 volatile compounds by head space solid phase micro extraction (SPME), and 6 volatile compounds by direct head space analysis. There was a significant difference in 42 volatile compounds between aged Awamori and Ippanshu. In a stepwise discriminant analysis procedure, 91 of 95 samples were rightly classified by 7 volatile compounds (diacetyl, ethyl isovalerate, isoamyl acetate, propyl caprate, ethyl crotonate, vanillin, and DMTS). Estimating the accuracy of the discriminant analysis by cross validation, the ratios of appropriate classification were 92.6%. For Awamori and the other Shochu, there was a significant difference in 59 of 84 volatile compounds. A total of 256 of 260 samples were rightly classified by 6 volatile compounds (isobutyl alcohol, 1-octen-3-ol, nerolidol, 2-methylbutyl acetate, methyl salicylate, and ethyl S-methyl thioacetate). When estimating the accuracy of the discriminant analysis, the ratios of appropriate classification were 98.1%. In addition, volatile compounds changing due to aging were investigated.

Source

J. Brew. Soc. Japan, 111, 261-270 (2016) in Japanese

2. Analysis of Sake Components Presented to Sake Contest in 2015

Author

Tsutomu Fujii, Atsuko Isogai, Sachiko Iizuka, Hanae Izu, Ryoko Kanda, Yoshitsugu Iemura

Source

Report of the Research Institute of Brewing, 188, 1-16 (2016) in Japanese

3. Review of the 38^th Honkaku Shochu Contest in 2015

Author

Osamu Yamada, Nobuhiko Mukai, Osamu Mizutani, Nahoko Nishibori, Genki Ono, Takeshi Akao, Hideaki Shimizu, Maki Kamimoto, Yoshitsugu Iemura

Source

Report of the Research Institute of Brewing, 188, 17-27 (2016) in Japanese

4. Analysis of Beer Components Presented to the Annual workshop of Japan Brewers Association in 2014

Author

Kazutaka Kusaka, Masayuki Takahashi, Hisashi Yazawa, Akihiro Mizuno

Source

Report of the Research Institute of Brewing, 188, 28-32 (2016) in Japanese

5. Analysis of Beer Components Presented to the Annual workshop of Japan Brewers Association in 2015

Author

Masayuki Takahashi, Kazutaka Kusaka, Hisashi Yazawa, Akihiro Mizuno

Source

Report of the Research Institute of Brewing, 188, 33-37 (2016) in Japanese

6. Aroma Compounds responsible for the "spicy/4VG" character of sake submitted to Sake Contests

Author

Atsuko Isogai, Ryoko Kanda, Sachiko Iizuka, Tsutomu Fujii

Abstract

"Spicy/4VG" character is perceived in 5-10% of sake submitted to Sake Contests. However, the aroma compounds responsible for the character are not known except for 4-vinylguaiacol (4VG). We investigated the aroma compounds responsible for the "spicy/4VG" character in sake submitted to Sake Contests in 2013 and 2014. Comprehensive volatile compound analysis revealed that sake with a "spicy/4VG" character contained higher levels of guaiacol, 4VG, lactones, and ethyl esters compared with controls, sake without the character. Among those compounds, guaiacol and 4VG were detected as having "medicine/smoke" and "curry/spicy" odors reminiscent of a "spicy/4VG" character by GC-olfactometry. We then analyzed volatile phenol compounds using SBSE-GC-MS. The concentration of guaiacol tended to increase as the number of panelists indicating a "spicy/4VG" character, and it exceeded its detection threshold (22 μg/L) in several sake samples. Although the concentration of 4VG was affected by the degradation of ferulic acid during thermal desorption process, its concentration was estimated to exceed its detection threshold (52 μg/L) in at least 70% of sake with a "spicy/4VG" character. 4-Ethylguaiacol (4EG) was detected only in a few samples, but one sample with "spicy/4VG" character contained a relatively high level of 4EG. These results indicated that not only 4VG but guaiacol and 4EG contributed to the "spicy/4VG" character of sake.

Source

J. Brew. Soc. Japan, 111, 483-492 (2016) in Japanese

7. Characteristics of the volatile composition and simplified discrimination of sweet potato shochu and its application

Author

Hisashi Fukuda, Jinshun Han, Osamu Mizutani, Muneyoshi Kanai, Osamu Yamada

Abstract

To classify the compounds of sweet potato shochu, we analyzed 78 volatile compounds by head space solid phase micro extraction (SPME), and 6 volatile compounds by direct head space analysis. The results revealed a significant difference in 40 volatile compounds between sweet potato shochu and other shochu. In a stepwise discriminant analysis procedure, 177 of 181 samples were rightly classified by 4 volatile compounds (farnesol, α-terpineol, nerol, and guaiacol). Estimating the accuracy of the discriminant analysis by cross validation, the ratios of appropriate classification were 97.8%. In discrimination analysis of sweet potato shochu and the other shochu on the basis of α-terpineol and guaiacol, the standard of judgment based on distance calculated from an approximate formula without centroides provides satisfactorily discrimination. In addition, application of a similar method was examined in Kokuto Shochu and Awamori.

Source

J. Brew. Soc. Japan, 111, 545-555 (2016) in Japanes

8. The breeding of high ethyl caproate shochu yeast and its practical application (Part 1) Breeding of high ethyl caproate shochu yeast

Author

Takafumi Muto, Masaaki Inahashi, Hiroyuki Manzen, Yasuzo Kizaki, Naoto Okazaki, Takeaki Ishikawa, Kazuo Sato, Takeshi Akao

Abstract

Shochu is a traditional Japanese distilled liquor. In this study, cerulenin resistant and ethyl caproate high producing shochu yeast NS2-16 was bred by cell-sore mating method of K1601 and Kyokai shochu yeast No. 2 (S2). NS2-16 was estimated to be triploid by its DNA content. The fermentation power of NS2-16 was stronger than that of one of parent strain S2. The stable properties of NS2-16, for example ethyl caproate productivity, were confirmed to be maintained throughout more than 30 generations. Because NS2-16 was able to grow well at higher temperature (38°C) and in a lower pH a yeast suitable for shochu production.

Source

J. Brew. Soc. Japan, 111, 556-562 (2016) in Japanese

9. Shochu Brewing Tests using Wine and Sake yeasts and comparison of the volatile compounds

Author

Hisashi Fukuda, Yan Zhou, Jinshun Han

Abstract

In the small-scale rice-shochu making tests, wine yeast and sake yeast were studied the ability of fermentation and the volatile composition.14 strains of tested yeast train were showed low fermentation ability. A correlation between volatile compounds was evaluated, the correlation coefficients of 19 data pairs is more than absolute value >0.6. 18 strains were selected on the basis of the volatile composition characteristics. To confirm the the volatile composition of schochu brewed by these strains, they were re-tested to make rice shochu in the small scale. In the each yeast strain, the overall tendency of the volatile composition was not change. From these results, 18 strains selected were useful making shochu in volatile composition characteristics.

Source

J. Brew. Soc. Japan, 111, 611-624 (2016) in Japanese

10. Identification of a mutation causing a defective spindle assembly checkpoint in high ethyl caproate-producing sake yeast strain K1801

Author

Tetsuya Goshima, Ryo Nakamura, Kazunori Kume, Hiroki Okada, Eri Ichikawa, Hiroyasu Tamura, Hirokazu Hasuda, Masaaki Inahashi, Naoto Okazaki, Takeshi Akao, Hitoshi Shimoi, Masaki Mizunuma, Yoshikazu Ohya, Dai Hirata

Abstract

In high-quality sake brewing, the cerulenin-resistant sake yeast K1801 with high ethyl caproate-producing ability has been used widely; however, K1801 has a defective spindle assembly checkpoint (SAC). To identify the mutation causing this defect, we first searched for sake yeasts with a SAC-defect like K1801 and found that K13 had such a defect. Then, we searched for a common SNP in only K1801 and K13 by examining 15 checkpoint-related genes in 23 sake yeasts, and found 1 mutation, R48P of Cdc55, the PP2A regulatory B subunit that is important for the SAC. Furthermore, we confirmed that the Cdc55-R48P mutation was responsible for the SAC-defect in K1801 by molecular genetic analyses. Morphological analysis indicated that this mutation caused a high cell morphological variation. But this mutation did not affect the excellent brewing properties of K1801. Thus, this mutation is a target for breeding of a new risk-free K1801 with normal checkpoint integrity.

Source

Bioscience, Biotechnology, and Biochemistry, 80, 1657-1662 (2016)

11. Substantial decrease in cell wall α-1,3-glucan caused by disruption of the kexB gene encoding a subtilisin-like processing protease in Aspergillus oryzae

Author

Osamu Mizutani, Matsuko Shiina, Akira Yoshimi, Motoaki Sano, Takeshi Watanabe, Youhei Yamagata, Tasuku Nakajima, Katsuya Gomi, Keietsu Abe

Abstract

Disruption of the kexB encoding a subtilisin-like processing protease in Aspergillus oryzae (ΔkexB) leads to substantial morphological defects when the cells are grown on Czapek-Dox agar plates. We previously found that the disruption of kexB causes a constitutive activation of the cell wall integrity pathway. To understand how the disruption of the kexB affects cell wall organization and components, we analyzed the cell wall of ΔkexB grown on the plates. The results revealed that both total N-acetylglucosamine content, which constitutes chitin, and chitin synthase activities were increased. Whereas total glucose content, which constitutes β-1,3-glucan and α-1,3-glucan, was decreased; this decrease was attributed to a remarkable decrease in α-1,3-glucan. Additionally, the β-1,3-glucan in the alkali-insoluble fraction of the ΔkexB showed a high degree of polymerization. These results suggested that the loss of α-1,3-glucan in the ΔkexB was compensated by increases in the chitin content and the average degree of β-1,3-glucan polymerization.

Source

Bioscience, Biotechnology, and Biochemistry, 80, 1781-1791 (2016)

12. Development of promoter shutoff system in Aspergillus oryzae using sorbitol-sensitive promoter

Author

Ken Oda, Shiho Terado, Rieko Toyoura, Hisashi Fukuda, Moriyuki Kawauchi, Kazuhiro Iwashita

Abstract

Promoter shutoff is a general method for analyzing essential genes, but there are no tightly repressed promoters reported in A. oryzae. To overcome the current limitations of conditional promoters, we examined the sorbitol and galactose responsive genes by microarray to identify regulatable genes with only minor physiological and genetic effect. We identified 2 sorbitol-induced genes (designated as sorA and sorB,) and cloned their promoters, and built a regulated EGFP and BrlA expression system. Growth medium-dependent EGFP fluorescence and conidiation were confirmed for egfp and brlA under the control of these promoters, respectively. This shutoff system was also applied to essential rhoA regulation, which showed the expected growth inhibition under the repressed growth condition. The new sorbitol promoter shutoff system reported here can serve as a valuable new tool for essential gene analysis in filamentous fungi.

Source

Bioscience, Biotechnology, and Biochemistry, 80, 1792-1801 (2016)

13. Spatial control of translation repression and polarized growth by conserved NDR kinase Orb6 and RNA-binding protein Sts5

Author

Illyce Nunez, Marbelys Rodriguez Pino, David J Wiley, Maitreyi E Das, Chuan Chen, Tetsuya Goshima, Kazunori Kume, Dai Hirata, Takashi Toda, Fulvia Verde

Abstract

RNA-binding proteins contribute to the formation of ribonucleoprotein (RNP) granules by phase transition, but regulatory mechanisms are not fully understood. Conserved fission yeast NDR (Nuclear Dbf2-Related) kinase Orb6 governs cell morphogenesis in part by spatially controlling Cdc42 GTPase. Here we describe a novel, independent function for Orb6 kinase in negatively regulating the recruitment of RNA-binding protein Sts5 into RNPs to promote polarized cell growth. We find that Orb6 kinase inhibits Sts5 recruitment into granules, its association with processing (P) bodies, and degradation of Sts5-bound mRNAs by promoting Sts5 interaction with 14-3-3 protein Rad24. Many Sts5-bound mRNAs encode essential factors for polarized cell growth, and Orb6 kinase spatially and temporally controls the extent of Sts5 granule formation. Disruption of this control system affects cell morphology and alters the pattern of polarized cell growth, revealing a role for Orb6 kinase in the spatial control of translational repression that enables normal cell morphogenesis.

Source

eLIFE, 5, e14216 (2016)

14. Stimulating S-adenosyl-L-methionine synthesis extends lifespan via activation of AMPK

Author

Takafumi Ogawa, Ryohei Tsubakiyama, Muneyoshi Kanai, Tetsuya Koyama, Tsutomu Fujii, Haruyuki Iefuji, Tomoyoshi Soga, Kazunori Kume, Tokichi Miyakawa, Dai Hirata, Masaki Mizunuma

Abstract

Dietary restriction (DR), such as calorie restriction (CR) or methionine (Met) restriction, extends the lifespan of diverse model organisms. Although studies have identified several metabolites that contribute to the beneficial effects of DR, the molecular mechanism underlying the key metabolites responsible for DR regimens is not fully understood. Here we show that stimulating S-adenosyl-L-methionine (AdoMet) synthesis extended the lifespan of the budding yeast Saccharomyces cerevisiae. The AdoMet synthesis-mediated beneficial metabolic effects, which resulted from consuming both Met and ATP, mimicked CR. Indeed, stimulating AdoMet synthesis activated the universal energy-sensing regulator Snf1, which is the S. cerevisiae ortholog of AMP-activated protein kinase (AMPK), resulting in lifespan extension. Furthermore, our findings revealed that S-adenosyl-L-homocysteine contributed to longevity with a higher accumulation of AdoMet only under the severe CR (0.05% glucose) conditions. Thus, our data uncovered molecular links between Met metabolites and lifespan, suggesting a unique function of AdoMet as a reservoir of Met and ATP for cell survival.

Source

Proceedings of the National Academy of Sciences of the United States of America, 113, 11913-11918 (2016)

15. Defective quiescence entry promotes the fermentation performance of bottom-fermenting brewer's yeast

Author

Mayu Oomuro, Taku Kato, Yan Zhou, Daisuke Watanabe, Yasuo Motoyama, Hiromi Yamagishi, Takeshi Akao, Masayuki Aizawa

Abstract

One of the key processes in making beer is fermentation. In the fermentation process, brewer's yeast plays an essential role in both the production of ethanol and the flavor profile of beer. Therefore, the mechanism of ethanol fermentation by of brewer's yeast is attracting much attention. The high ethanol productivity of sake yeast has provided a good basis from which to investigate the factors that regulate the fermentation rates of brewer's yeast. Recent studies found that the elevated fermentation rate of sake Saccharomyces cerevisiae species is closely related to a defective transition from vegetative growth to the quiescent (G0) state. In the present study, to clarify the relationship between the fermentation rate of brewer's yeast and entry into G0, we constructed two types of mutant of the bottom-fermenting brewer's yeast Saccharomyces pastorianus Weihenstephan 34/70: a RIM15 gene disruptant that was defective in entry into G0; and a CLN3ΔPEST mutant, in which the G1 cyclin Cln3p accumulated at high levels. Both strains exhibited higher fermentation rates under high-maltose medium or high-gravity wort conditions (20^o Plato) as compared with the wild-type strain. Furthermore, G1 arrest and/or G0 entry were defective in both the RIM15 disruptant and the CLN3ΔPEST mutant as compared with the wild-type strain. Taken together, these results indicate that regulation of the G0/G1 transition might govern the fermentation rate of bottom-fermenting brewer's yeast in high-gravity wort.

Source

Journal of Bioscience and Bioengineering, 122, 577-582 (2016)

16. The relationship between rice protein composition and nitrogen compounds in sake

Author

Masaki Okuda, Minami Miyamoto, Midori Joyo, Kei Takahashi, Nami Goto-Yamamoto, Shuichi Iida, Takuro Ishii

Abstract

The relationship between the protein composition of rice and nitrogen compounds (amino acids and oligo-peptides) in the produced sake were investigated using endosperm protein mutant rice (LGC-1, LGC-Jun, Kx433, QA28), sake rice (Yamadanishiki) and cooking rice (Nipponbare, Nihonmasari, Koshihikari). The total nitrogen concentration, amino acid concentration and most peptide peak areas determined by RP-HPLC and gel filtration chromatography of the produced sake were lower when sake was made from a low glutelin content rice mutant compared with other rice varieties. The concentration of nitrogen compounds in the sake positively correlated with the glutelin content of the highly milled rice grains used for sake production. Sake produced using a low glutelin content rice mutant is generally evaluated as having a light taste. Our findings suggest that nitrogen compounds (oligo-peptides and amino acids) derived from rice glutelin significantly contribute to the taste of sake.

Source

Journal of Bioscience and Bioengineering, 122, 70-78 (2016)

17. Transfer of caesium and potassium from Japanese apricot (Prunus mume Sieb.et Zucc.) to Japanese apricot liqueur (Ume liqueur)

Author

Masaki Okuda, Takeshi Akao, Masanori Sumihiro, Megumi Mizuno, Nami Goto-Yamamoto

Abstract

The potential contamination with radionuclides released by the Fukushima Daiichi Nuclear Power Plant Accident following the great east Japan earthquake and tsunami in March 2011 of agricultural products and processed foods derived from them is a matter of on-going concern. The stable ¹³³Cs is a useful indicator for predicting the behaviour of radioactive Cs, and potassium (K) is a congener of Cs. In this study, the distribution of ¹³³Cs and K in Ume fruit, and the transfer of ¹³³Cs and K from Ume fruit to Ume liqueur, were investigated. The concentrations of ¹³³Cs and K in Ume fruit were highest in the skin, followed by the kernel or flesh, and then the pit. About 80% of ¹³³Cs and K in all Ume fruits was distributed in the flesh. The transfer ratios of ¹³³Cs and K from Ume fruit to Ume liqueur were similar among all cultivars after 3months, and they were similar to those of organic acids. The food retention factor (Fr = weight of ¹³³Cs in Ume liqueur/weight of ¹³³Cs in Ume fruit) was ca. 0.8, and the processing factor (Pf = concentration of ¹³³Cs in Ume liqueur/concentration of ¹³³Cs in Ume fruit) was 0.2-0.3. From the present study, the radioactivity of Ume liqueur would be below the legal limit if made using Ume fruits within the legal limit, thereby confirming the safety of Ume liqueur.

Source

Journal of the Institute of Brewing, 122, 473-479 (2016)

18. Candida easanensis strain JK-8 β-glucosidase: a glucose-tolerant enzyme with high specific activity for laminarin

Author

Jantaporn Thongekkaew, Tsutomu Fujii, Kazuo Masaki

Abstract

Thermotolerant yeasts, Candida easanensis strain JK-8 secreted a β-glucosidase when grown in liquid media containing various carbon sources. The greatest enzyme activity was found in medium containing 1% cellobiose as a sole carbon source. An extracellular β-glucosidase was purified to homogeneity by anion exchange chromatography and hydrophobic interaction chromatography. The molecular weight (Mw) of the β-glucosidase was estimated to be 48 kDa by SDS-PAGE. The optimal activity was at pH 4.5-5.5 and 60℃. The purified enzyme was stable in the pH range of 4.0-6.0 and had a 2 h half life at 50°C. The presence of metal ions (Ca²⁺, Mn²⁺, Zn²⁺ and Cu²⁺), mercaptoethanol and EDTA at 5 and 10 mM positively influenced its activity. It showed high specific activity for p-nitrophenyl-β-glucopyranoside (p-β-NPG) and laminarin. The similar Vmax of 10 μmol min-1mg protein-1 was found in both p-β-NPG (Km value of 5 mM) and laminarin (Km value of 5 mg mL^-1). The enzyme displayed high tolerance to glucose with a Ki of 1.3 M and it was tolerant in the presence of methanol and ethanol at concentration up to 15%.

Source

Current Chemical Biology, 10, 117-126 (2016)

19. Ferulic acid decarboxylation ability and single nucleotide polymorphisms of PAD1 and FDC1 among sake yeasts and shochu yeasts

Author

Nobuhiko Mukai, Narumi Fuke, Kazuo Masaki, Osamu Yamada, Haruyuki Iefuji

Abstract

We have previously reported that PAD1 (phenylacrylic acid decarborxylase gene, YDR538W) and FDC1 (ferulic acid decarboxylase gene, YDR539W) genes are essential for decarboxylation of ferulic acid in Saccharomyces cerevisiae and that the single polymorphisms of both genes show a positive relationship with ferulic acid decarboxylation ability among industrial yeasts used in alcoholic beverage production. In this paper, we examined the decarboxylation ability of the ferulic acid of various sake yeasts and shochu yeasts including strains which are not used industrially at the present time. Results showed that all 15 sake yeasts and 14 shochu yeasts did not have a ferulic acid decarboxylation ability. The sequences of PAD1 and FDC1 genes among sake yeasts and shochu yeasts were very similar. Sake yeasts were classified in two types based on single nucleotide polymorphisms of PAD1 and FDC1 genes: a K-1 type (K-1, K-3, K-4, K-5, K-8, RIB0001, RIB0006 and RIB0007) and a K-6 type (K-6, K-7, K-9, K-10, K-12 and K-13). With respect to K-2, plural sequences coexisted and were not able to determine genotype. Shochu yeasts were divided into five types based on single nucleotide polymorphisms: a K-1 type (KS-3, MK021, Heisei Miyazaki and K2), a K-6 type (KS-4), a KS-2 type (KS-2, S-2, SH-4, C4), a BY19901 type (BY19901, BY19902, BY19903 and H5), and an Awamori-101 type (Awamori-101). Our findings indicate that sake yeasts and shochu yeasts have been selected for brewing strains which do not have the ability of decarboxylation of ferulic acid from an early stage of the selection. PAD1 and FDC1 genes are very important as markers to distinguish sake yeasts and shochu yeasts from other yeasts for alcoholic beverage production.

Source

J. Brew. Soc. Japan, 111, 679-685 (2016) in Japanese

20. The origin and distillation behavior of 1-octen-3-ol in Awamori

Author

Hisashi Fukuda, Jinshun Han

Abstract

There is conspicuously different in 1-octen-3-ol and methyl salicylate concentration between Awamori and the other shochu. Especially, the concentration of 1-octen-3-ol in Awamori is higher than that of the other shochu. This compound concentration is higher in distilled solution of moromi made from black koji than that of moromi made from white koji and momori made from white koji and steamed rice. In the distillation process, the behavior of 1-octen-3-ol is similar to that of ethyl caproate. The main reason that the concentration of 1-octen-3-ol is higher in Awamori than in the other shochu is considered that awamori making use for black koji, and 1-octen-3-ol with comparatively high concentration contained in vacuum distillations as well as atmospheric distillations is estimated to depend on its volatility.

Source

J. Brew. Soc. Japan, 111, 750-757 (2016) in Japanese

21. Characteristics of volatile compositions and a correlation analysis of the compounds in rice shochu and barley shochu

Author

Hisashi Fukuda, Jinshun Han, Osamu Mizutani, Muneyoshi Kanai, Osamu Yamada

Abstract

Eighty-four volatile compounds of shochu were analyzed by head space solid phase micro extraction (SPME) and direct head space analysis. There was a significant difference in 64 volatile compounds between the groups of rice shochu and barley shochu as well as other shochu. In a stepwise discriminant analysis procedure using nonanal, isobutyl alcohol, α-terpineol, ethyl valerate, ethyl caprylate, ethyl benzoate, guaiacol, and 2,5-dimethylpyrazine, 171 of 181 samples were rightly classified. Estimating the accuracy of the discriminant analysis by cross validation, the ratios of appropriate classification were 93.9%. In rice shochu and barley shochu, in a stepwise discriminant analysis procedure using benzaldehyde, isoamyl alcohol, geraniol, ethyl 2-methylbutyrate, ethyl valerate and 2-pentylfuran, 80 of 81 samples were rightly classified. On estimating the accuracy of the discriminant analysis by cross validation, the ratios of appropriate classification were 96.3%. In addition, the correlation between the compounds in rice shochu and barley shochu were analyzed. The compounds showing a high correlation were given consideration.

Source

J. Brew. Soc. Japan, 111, 841-873 (2016) in Japanese

22. Genome sequence of Aspergillus luchuensis NBRC 4314

Author

Osamu Yamada, Masayuki Machida, Akira Hosoyama, Masatoshi Goto, Toru Takahashi, Taiki Futagami, Youhei Yamagata, Michio Takeuchi, Tetsuo Kobayashi, Hideaki Koike, Keietsu Abe, Kiyoshi Asai, Masanori Arita, Nobuyuki Fujita, Kazuro Fukuda, Ken-ichi Higa, Hiroshi Horikawa, Takeaki Ishikawa, Koji Jinno, Yumiko Kato, Kohtaro Kirimura, Osamu Mizutani, Kaoru Nakasone, Motoaki Sano, Yohei Shiraishi, Masatoshi Tsukahara, Katsuya Gomi

Abstract

Awamori is a traditional distilled beverage made from steamed Thai-Indica rice in Okinawa, Japan. For brewing the liquor, two microbes, local kuro (black) koji mold Aspergillus luchuensis and awamori yeast Saccharomyces cerevisiae are involved. In contrast, that yeasts are used for ethanol fermentation throughout the world, a characteristic of Japanese fermentation industries is the use of Aspergillus molds as a source of enzymes for the maceration and saccharification of raw materials. Here we report the draft genome of a kuro (black) koji mold, A. luchuensis NBRC 4314 (RIB 2604). The total length of nonredundant sequences was nearly 34.7Mb, comprising approximately 2,300 contigs with 16 telomere-like sequences. In total, 11,691 genes were predicted to encode proteins. Most of the housekeeping genes, such as transcription factors and N-and O-glycosylation system, were conserved with respect to Aspergillus niger and Aspergillus oryzae. An alternative oxidase and acid-stable α-amylase regarding citric acid production and fermentation at a low pH as well as a unique glutamic peptidase were also found in the genome. Furthermore, key biosynthetic gene clusters of ochratoxin A and fumonisin B were absent when compared with A. niger genome, showing the safety of A. luchuensis for food and beverage production. This genome information will facilitate not only comparative genomics with industrial kuro-koji molds, but also molecular breeding of the molds in improvements of awamori fermentation.

Source

DNA Research, 23, 507-515 (2016)

23. Analysis of the MAT1-1 and MAT1-2 Gene Ratio in Black Koji Molds Isolated from Meju

Author

Anbazhagan Mageswari, Jeong-seon Kim, Kyu-Ho Cheon, Soon-Wo Kwon, Osamu Yamada, Seung-Beom Hong

Abstract

Aspergillus luchuensis is known as an industrially important fungal species used for making fermented foods such as awamori and shochu in Japan, makgeolli and Meju in Korea, and Pu-erh tea in China. Nonetheless, this species has not yet been widely studied regarding mating-type genes. In this study, we examined the MAT1-1 and MAT1-2 gene ratio in black koji molds (A. luchuensis, Aspergillus niger, and Aspergillus tubingensis) and in Aspergillus welwitschiae isolated from Meju, a fermented soybean starting material for traditional soy sauce and soybean paste in Korea. The number of strains with the MAT1-1 locus was 2 of 23 (A. luchuensis), 6 of 13 (A. tubingensis), 21 of 28 (A. niger), and 5 of 10 (A. welwitschiae). Fungal species A. tubingensis and A. welwitschiae showed a 1 : 1 ratio of MAT1-1 and MAT1-2 mating-type loci. In contrast, A. luchuensis revealed predominance of MAT1-2 (91.3%) and A. niger of MAT1-1 (75%). We isolated and identified 2 A. luchuensis MAT1-1 strains from Meju, although all strains for making shochu in Japan are of the MAT1-2 type. These strains may be a good resource for breeding of A. luchuensis to be used in the Asian fermented-food industry.

Source

Mycobiology, 44, 269-276 (2016)

24. Sake yeast YHR032W/ERC1 haplotype contributes to high S-adenosylmethionine accumulation in sake yeast strains

Author

Muneyoshi Kanai, Tomoko Kawata, Yoshie Yoshida, Yasuko Kita, Takafumi Ogawa, Masaki Mizunuma, Daisuke Watanabe, Hitoshi Shimoi, Akihiro Mizuno, Osamu Yamada, Tsutomu Fujii, Haruyuki Iefuji

Abstract

Sake yeasts are ideally suited for sake making, producing higher levels of ethanol, proliferating at lower temperatures, and producing greater levels of various aromatic components and nutrients than laboratory yeasts. To elucidate the mechanism underlying S-adenosylmethionine (SAM) accumulation in sake yeast strains compared with that in laboratory yeast strains, we performed quantitative trait locus (QTL) analysis and identified a significant QTL on chromosome VIII. Of the 165 genes mapped at 49.8 cM from the left-end DNA marker of chromosome VIII, we focused on the YHR032W/ERC1 gene, encoding a member of the multi-drug and toxin extrusion family having antiporter activity and involved in SAM accumulation and ethionine resistance. Expression of the sake yeast ERC1 haplotype (K7ERC1) in a low and high-copy number plasmid BYΔerc1 resulted in intracellular SAM accumulation, whereas expression of the laboratory yeast ERC1haplotype (XERC1) did not. Comparison between DNA sequences of K7ERC1 and XERC1revealed three amino acid substitutions: S51N, V263I, and N545I. Site-directed mutagenesis revealed that the N545I frameshift mutation was responsible for the K7ERC1phenotype. These results indicate that K7ERC1 contributes to SAM accumulation in sake yeast strains.

Source

Journal of Bioscience and Bioengineering, 123, 8-14 (2017)

25. Development of a Five Factor Drinking Motive Questionnaire for Japanese (DMQ-J)

Author

Sumio Imada, Isato Furumitsu, Hanae Izu

Abstract

In the present study, we developed a Drinking Motive Questionnaire (DMQ-J) composed of 19 items to clarify the drinking motives of Japanese adults. Exploratory factor analysis revealed the following five reliable factors or subscales of drinking motives: "Coping" (drinking to regulate or reduce internally generated negative emotions), "Enhancement" (drinking to enhance internal positive mood), "Social" (drinking to obtain positive social, and thus external, rewards), "Meal" (drinking to enjoy one's meal), and "Restraint" (reasons to restrain one's drinking). Based on analysis of DMQ-J data from 1000 light-to-moderate drinkers, males scored higher than females on the "Coping" and lower on the "Restraint" subscales, which suggests that males have a relatively unhealthy drinking style. Moreover, females scored marginally higher than males on the "Meal" subscale, which suggests that females enjoy alcohol with their meal, but not to an unhealthy degree. In terms of generational differences, "Social" motive scores decreased but "Meal" motive scores increased with increases in age, suggesting that older people drink to enhance their internal pleasure and enjoy their meals, but not to obtain social rewards.

Source

Studies in the Humanities and Sciences, 57(2), 153-162 (2017)

26. Direct activation of GABAA receptors by substances in the organic acid fraction of Japanese sake

Author

Hanae Izu, Kensuke Shigemori, Masaya Eguchi, Shuhei Kawane, Shouko Fujii, Yuji Kitamura, Hitoshi Aoshima, Yasue Yamada

Abstract

We investigated the effect of substances present in Japanese sake on the response of ionotropic γ-aminobutyric acid (GABA)_A receptors expressed in Xenopus oocytes. Sake was fractionated by ion-exchange chromatography. The fraction containing organic acids (OA fraction) showed agonist activities on the GABA_A receptor. OA fractions from sake were analyzed by capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). Of the 64 compounds identified, 13 compounds showed GABA_A receptor agonist activities. Especially, L-lactic acid showed high agonist activity and its EC₅₀ value was 37μM. Intraperitoneal injections of L-lactic acid, gluconic acid, and pyruvic acid (10, 10, and 5mg/kg BW, respectively), which showed agonistic activity on the GABA_A receptor, led to significant anxiolytic effects during an elevated plus-maze test in mice.

Source

Food Chemistry, 214(1), 354-359 (2017)

27. Identification of 4-mercapto-4-methylpentan-2-one as the characteristic aroma of sake made from low-glutelin rice

Author

Sachiko Iizuka-Furukawa, Atsuko Isogai, Kazutaka Kusaka, Tsutomu Fujii, Yoshinori Wakai

Abstract

The grassy characteristic aroma perceived in brewed sake made from low-glutelin rice (Oryza sativa L. Mizuhonoka) was examined by gas chromatography-olfactometry and gas chromatography-mass spectrometry. By comparing the odor properties and Kovats retention indices to those of standard compounds, 4-mercapto-4-methylpentan-2-one (4MMP) was found to contribute to the characteristic aroma. Sake brewing using Mizuhonoka, low-glutelin rice, and Ginohmi (a control) revealed that 4MMP concentrations in Mizuhonoka sake samples were higher than those in Gin-ohmi samples over the fermentation period. The concentration in final Mizuhonoka sake was twice that of Gin-ohmi. To investigate the mechanism of 4MMP formation, the putative precursors, 4-S-cycteinyl-4-methylpentan-2-one (cys-4MMP) and 4-S-glutathionyl-4-methylpentan-2-one (glut-4MMP), in sake and its materials (rice and koji) were analyzed by ultra-performance liquid chromatography tandem mass-spectrometry. Cys-4MMP was not detected in all samples, while glut-4MMP was present in sake and its materials. The glut-4MMP concentration in sake was lower than that in Gin-ohmi over nearly the entire fermentation period, suggesting that conversion of the precursors to 4MMP was more effective in the mash of low-glutelin rice Mizuhonoka than in Gin-ohmi. The release of 4MMP during alcoholic fermentation from a model medium containing its precursors was examined. While no 4MMP release was observed in the control (no addition), with the addition of its precursors, the release of 4MMP increased as fermentation progressed. It was suggested that 4MMP was generated from both cys- and glut-4MMP by sake yeast. The perception threshold of 4MMP in sake was determined as 1.2 ng/L.

Source

Journal of Bioscience and Bioengineering, 123, 209-215 (2017)

28. Correlation analyses of the compounds in kokuto shochu and rum

Author

Hisashi Fukuda, Jinshun Han

Abstract

The correlation between eighty-four volatile compounds in kokuto shochu and rum were analyzed. In kokuto shochu, the compounds to indicate a correlation with fatty acid esters were acetaldehyde, 2-nonanone, n-propyl alcohol, isoamyl alcohol, and β-phenethyl alcohol; the correlations between fatty acid esters are generally high. In rum, the number of the correlations (r²>|0.5|) in industrial rum was more than that in agricol rum, and all of the correlation signs (r²>|0.5|) in industrial rum were positive. In this report, the overall trend of the compounds showing a high correlation were given consideration, and the compounds showing a higher correlation (r²>|0.9|) in rum are primarily mentioned.

Source

J. Brew. Soc. Japan, 112, 35-76 (2017) in Japanese

29. Cellular responses to the expression of unstable secretory proteins in the filamentous fungus Aspergillus oryzae

Author

Jun-ichi Yokota, Daisuke Shiro, Mizuki Tanaka, Yasumichi Onozaki, Osamu Mizutani, Dararat Kakizono, Sakurako Ichinose, Tomoko Shintani, Katsuya Gomi, Takahiro Shintani

Abstract

Filamentous fungi are often used as cell factories for recombinant protein production because of their ability to secrete large quantities of hydrolytic enzymes. However, even using strong transcriptional promoters, yields of nonfungal proteins are generally much lower than those of fungal proteins. Recent analyses revealed that expression of certain nonfungal secretory proteins induced the unfolded protein response (UPR), suggesting that they are recognized as proteins with folding defects in filamentous fungi. More recently, however, even highly expressed endogenous secretory proteins were found to evoke the UPR. These findings raise the question of whether the unfolded or misfolded state of proteins is selectively recognized by quality control mechanisms in filamentous fungi. In this study, a fungal secretory protein (1,2-α-D-mannosidase; MsdS) with a mutation that decreases its thermostability was expressed at different levels in Aspergillus oryzae. We found that, at moderate expression levels, wild-type MsdS was secreted to the medium, while the mutant was not. In the strain with a deletion for the hrdA gene, which is involved in the endoplasmic reticulum-associated degradation pathway, mutant MsdS had specifically increased levels in the intracellular fraction but was not secreted. When overexpressed, the mutant protein was secreted to the medium to a similar extent as the wild-type protein; however, the mutant underwent hyperglycosylation and induced the UPR. Deletion of α-amylase (the most abundant secretory protein in A. oryzae) alleviated the UPR induction by mutant MsdS overexpression. These findings suggest that misfolded MsdS and unfolded species of α-amylase might act synergistically for UPR induction.

Source

Appl. Microbiol. Biotechnol., 101(6), 2437-2446 (2017)

30. Detailed analysis of targeted gene mutations caused by the Platinum-Fungal TALENs in Aspergillus oryzae RIB40 strain and a ligD disruptant

Author

Osamu Mizutani, Takayuki Arazoe, Kenji Toshida, Risa Hayashi, Shuichi Ohsato, Tetsushi Sakuma, Takashi Yamamoto, Shigeru Kuwata, Osamu Yamada

Abstract

Transcription activator-like effector nucleases (TALENs), which can generate DNA double-strand breaks at specific sites in the desired genome locus, have been used in many organisms as a tool for genome editing. In Aspergilli, including Aspergillus oryzae, however, the use of TALENs has not been validated. In this study, we performed genome editing of A. oryzae wild-type strain via error of nonhomologous end-joining (NHEJ) repair by transient expression of high-efficiency Platinum-Fungal TALENs (PtFg TALENs). Targeted mutations were observed as various mutation patterns. In particular, approximately half of the PtFg TALEN-mediated deletion mutants had deletions larger than 1 kb in the TALEN-targeting region. We also conducted PtFg TALEN-based genome editing in A. oryzae ligD disruptant (ΔligD) lacking the ligD gene involved in the final step of the NHEJ repair and found that mutations were still obtained as well as wild-type. In this case, the ratio of the large deletions reduced compared to PtFg TALEN-based genome editing in the wild-type. In conclusion, we demonstrate that PtFg TALENs are sufficiently functional to cause genome editing via error of NHEJ in A. oryzae. In addition, we reveal that genome editing using TALENs in A. oryzae tends to cause large deletions at the target region, which were partly suppressed by deletion of ligD.

Source

Journal of Bioscience and Bioengineering 123(3), 287-293 (2017)

31. Method for the isolation of citric acid and malic acid in Japanese apricot liqueur for carbon stable isotope analysis

Author

Fumikazu Akamatsu, Tomokazu Hashiguchi, Yuri Hisatsune, Takaaki Oe, Takafumi Kawao, Tsutomu Fujii

Abstract

A method for detecting the undeclared addition of acidicingredients is required to control the authenticity of Japanese apricotliqueur. We developed an analytical procedure that minimizes carbon isotopediscrimination for measurement of the δ¹³C values of citric and malic acid isolated fromJapanese apricot liqueur. Our results demonstrated that freeze-drying ispreferable to nitrogen spray-drying, because it does not significantly affectthe δ¹³C valuesof citric acid and results in smaller isotope discrimination for malic acid.Both 0.1% formic acid and 0.2% phosphoric acid are acceptable HPLC mobile phases for the isolation of citric and malic acid, although the δ¹³C valuesof malic acid exhibited relatively large variation compared with citric acid following isolation using either mobile phase. The developed procedure allows precise δ¹³C measurements of citric and malic acid isolated from Japanese apricot liqueur.

Source

Food Chemistry, 217, 112-116 (2017)

32. Polyphenolic diversity and characterization in the red-purple berries of East Asian wild Vitis species

Author

Kazuya Koyama, Hiroshi Kamigakiuchi, Kazuhiro Iwashita, Ryosuke Mochioka and Nami Goto-Yamamoto

Abstract

Grapes (Vitis spp.) produce diverse polyphenolic compounds, which are phytochemicals that contribute to human health. In this study, the polyphenolic profiles of the red-purple berries of two wild grape species native to Japan, Vitis ficifolia and V. coignetiae, and their interspecific hybrid cultivars were investigated and compared with the profiles of V. vinifera and V. × labruscana cultivars. Proanthocyanidins (PAs) were present at lower concentrations in both skins and seeds of wild grape species and their hybrid cultivars than those in V. vinifera cultivars. They also differed in their composition, consisting mainly of epicatechin in wild grape species, but containing considerable amounts of both epigallocatechin in the skins and epicatechin gallate in the seeds of V. vinifera. In contrast, V. ficifolia varieties and their hybrid cultivars accumulated high concentrations of diverse anthocyanins, and whose compositions of anthocyanins and flavonols differed between species in their degree of modification by glucosylation, acylation, methylation and B-ring hydroxylation. Principal component analysis (PCA) indicated that the polyphenolic constituents clearly separate V. vinifera and V. × labruscana cultivars from the wild grape species as well as between wild grape species, V. coignetiae and V. ficifolia. Intermediate compositions were also observed in the hybrid cultivars between these wild grape species and V. vinifera.

Source

Phytochemistry, 134, 78-86 (2017)